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Sample records for acid polymerizing enzyme

  1. Horseradish peroxidase-catalyzed polymerization of L-DOPA for mono-/bi-enzyme immobilization and amperometric biosensing of H2O2 and uric acid.

    PubMed

    Dai, Mengzhen; Huang, Ting; Chao, Long; Xie, Qingji; Tan, Yueming; Chen, Chao; Meng, Wenhua

    2016-03-01

    Horseradish peroxidase (HRP)-catalyzed polymerization of L-DOPA (vs. dopamine) in the presence of H2O2 (and uricase (UOx)) was exploited to immobilize mono-/bi-enzymes for hydroquinone-mediated amperometric biosensing of H2O2 and uric acid (UA). The relevant polymeric biocomposites (PBCs) were prepared in phosphate buffer solution containing HRP and L-DOPA (or plus UOx) after adding H2O2. The mono-/bi-enzyme amperometric biosensors were prepared simply by casting some of the PBCs on Au-plated Au (Au(plate)/Au) electrodes, followed by coating with an outer-layer chitosan (CS) film for each. UV-vis spectrophotometry, scanning electron microscopy, cyclic voltammetry and electrochemical impedance spectroscopy were used for film characterization and/or process monitoring. The HRP immobilized by enzyme catalysis well preserved its bioactivity, as confirmed by UV-vis spectrophotometry. Under optimized conditions, the monoenzyme CS/HRP-poly(L-DOPA) (PD)/Au(plate)/Au electrode potentiostated at -0.1V responded linearly to H2O2 concentration from 0.001 to 1.25mM with a sensitivity of 700μA mM(-1)cm(-2) and a limit of detection (LOD) of 0.1μM, and the bienzyme CS/UOx-HRP-PD/Au(plate)/Au electrode at -0.1V responded linearly to UA concentration from 0.001 to 0.4mM with a sensitivity of 349μA mM(-1)cm(-2) and a LOD of 0.1μM. The mono-/bi-enzyme biosensors based on biosynthesized PD performed better than many reported analogues and those based on similarly biosynthesized polydopamine. PMID:26717822

  2. Immobilization of enzymes on alginic acid-polyacrylamide copolymers

    SciTech Connect

    Kumaraswamy, M.D.K.; Panduranga R.K.; Thomas J.K.; Santappa, M.

    1981-08-01

    In this report, the authors present initial results and limitations of a polymeric system for the immobilization of enzymes. Enzymes attached to insoluble polymers of natural and synthetic origin are gaining importance in many industrial and biomedical applications. Graft copolymers are used as enzyme supports and in this study a novel polymeric system of alginic acid-polyacrylamide graft copolymer is described which was used for immobilizing enzymes. (Refs. 4).

  3. Polymerization of epoxidized triglycerides with fluorosulfonic acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The use of triglycerides as agri-based renewable raw materials for the development of new products is highly desirable in view of uncertain future petroleum prices. A new method of polymerizing epoxidized soybean oil has been devised with the use of fluorosulfonic acid. Depending on the reaction con...

  4. Biologically produced acid precipitable polymeric lignin

    DOEpatents

    Crawford, Don L.; Pometto, III, Anthony L.

    1984-01-01

    A water soluble, acid precipitable polymeric degraded lignin (APPL), having a molecular weight of at least 12,000 daltons, and comprising, by percentage of total weight, at least three times the number of phenolic hydroxyl groups and carboxylic acid groups present in native lignin. The APPL may be modified by chemical oxidation and reduction to increase its phenolic hydroxyl content and reduce the number of its antioxidant inhibitory side chains, thereby improving antioxidant properties.

  5. A mild strategy to encapsulate enzyme into hydrogel layer grafted on polymeric substrate.

    PubMed

    Zhu, Xing; Ma, Yuhong; Zhao, Changwen; Lin, Zhifeng; Zhang, Lihua; Chen, Ruichao; Yang, Wantai

    2014-12-23

    Although the hydrogel network has been widely investigated as a carrier for enzyme immobilization, to in situ encapsulate enzymes into a hydrogel network in an efficient, practical, and active way is still one of the great challenges in the field of biochemical engineering. Here, we report a new protocol to address this issue by encapsulating enzyme into poly(ethylene glycol) (PEG) hydrogel network grafted on polymeric substrates. In our strategy, isopropyl thioxanthone semipinacol (ITXSP) dormant groups were first planted onto the surface of a plastic matrix with low density polyethylene (LDPE) film as a model by a UV-induced abstracting hydrogen-coupling reaction. As a proof of concept, lipase, which could catalyze esterification of glucose with palmitic acid, then was in situ net-immobilized into a PEG-based hydrogel network layer through a visible light-induced surface controlled/living graft cross-linking polymerization. This strategy demonstrates the following novel significant merits: (1) in comparison with the UV irradiation or high temperature, the visible light and room temperature used provide a friendly condition to maintain activity of enzyme during immobilization; (2) the uniqueness of controlled/living cross-linking polymerization not only makes it easy to form a uniform PEG hydrogel network, which is a benefit to avoid the leakage of net-immobilizing enzyme, but also to tune the net-thickness or capacity to accommodate enzyme; and (3) as compared to systems of nanoparticles and porous matrixes, the flexible/robust end-products of the surface net-immobilizing enzyme with polymer film are more suitable to be applied in a bioreactor due to their features of easier separation and reuse. We confirmed that this catalytic film could retain almost all of its initial activity after seven batches of 24 h esterifications. The proposed strategy provides an extremely simple, effective, and flexible method for enzyme immobilization. PMID:25489918

  6. Polymerization of formic acid under high pressure

    SciTech Connect

    Goncharov, A.F.; Manaa, M.R.; Zaug, J.M.; Gee, R.H.; Fried, L.E.; Montgomery, W.B.

    2010-07-19

    We report Raman, infrared, and x-ray diffraction (XRD) measurements, along with ab initio calculations on formic acid (FA) under pressure up to 50 GPa. We find an infinite chain Pna2{sub 1} structure to be a high-pressure phase at room temperature. Our data indicate the symmetrization and a partially covalent character of the intrachain hydrogen bonds above approximately 20 GPa. Raman spectra and XRD patterns indicate a loss of long-range order at pressures above 40 GPa, with a large hysteresis upon decompression. We attribute this behavior to a three-dimensional polymerization of FA.

  7. Polymerization of Formic Acid under High Pressure

    SciTech Connect

    Goncharov, A F; Manaa, M R; Zaug, J M; Fried, L E; Montgomery, W B

    2004-08-23

    We report combined Raman, infrared (IR) and x-ray diffraction (XRD) measurements, along with ab initio calculations on formic acid under pressure up to 50 GPa. Contrary to the report of Allan and Clark (PRL 82, 3464 (1999)), we find an infinite chain low-temperature Pna2{sub 1} structure consisting of trans molecules to be a high-pressure phase at room temperature. Our data indicate the symmetrization and a partially covalent character of the intra-chain hydrogen bonds above approximately 20 GPa. Raman spectra and XRD patterns indicate a loss of the long-range order at pressures above 40 GPa with a large hysteresis at decompression. We attribute this behavior to a three-dimensional polymerization of formic acid.

  8. Atom Transfer Radical Polymerization of Methacrylic Acid: A Won Challenge.

    PubMed

    Fantin, Marco; Isse, Abdirisak A; Venzo, Alfonso; Gennaro, Armando; Matyjaszewski, Krzysztof

    2016-06-15

    Polymerization of acidic monomers is one of the biggest challenges for atom transfer radical polymerization (ATRP). An intramolecular cyclization reaction leading to the loss of the C-X chain-end functionality was found to be the main reason for the partial termination of the growing polymer chains. Three approaches were used to overcome this problem: using Cl as the chain-end halogen, lowering the pH (to 0.9), and increasing polymerization rate. Methacrylic acid (MAA) was polymerized by both electrochemically mediated ATRP and supplemental activator and reducing agent ATRP up to high conversion (>90%), in t ≤ 4 h at 25 °C, using inexpensive and nontoxic reagents (NaCl, diluted HCl, water). Control over molecular weight (MW) dispersity was satisfactory, and MWs were in agreement with theoretical values. The "livingness" of the process was confirmed by an electrochemical switch, used to repeatedly and periodically deactivate/reactivate growing chains. PMID:27244091

  9. Two active forms of Zymomonas mobilis levansucrase. An ordered microfibril structure of the enzyme promotes levan polymerization.

    PubMed

    Goldman, Dan; Lavid, Noa; Schwartz, Alon; Shoham, Gil; Danino, Dganit; Shoham, Yuval

    2008-11-21

    Fructansucrases, members of glycoside hydrolase family 68, catalyze both sucrose hydrolysis and the polymerization of fructose to beta-d-fructofuranose polymers. The resulting fructan polymers are distinguished by the nature of the glycosidic bond: inulin (beta-(2-1)-fructofuranose) and levan (beta-(2-6)-fructofuranose). In this study we demonstrate that Zymomonas mobilis levansucrase exists in two active forms, depending on the pH and ionic strength. At pH values above 7.0, the enzyme is mainly a dimer, whereas at pH values below 6.0, the protein forms well ordered microfibrils that precipitate out of the solution. These two forms are readily interchangeable simply by changing the pH. Surprisingly the manner in which the enzyme is arranged strongly affects its product specificity and kinetic properties. At pH values above 7.0, the activity of the enzyme as a dimer is mainly sucrose hydrolysis and the synthesis of short fructosaccharides (degree of polymerization, 3). At pH values below 6.0, in its microfibril form, the enzyme catalyzes almost exclusively the synthesis of levan (a degree of polymerization greater than 20,000). This difference in product specificity appears to depend on the form of the enzyme, dimer versus microfibril, and not directly on the pH. Images made by negative stain transmission electron microscopy reveal that the enzyme forms a very ordered structure of long fibrils that appear to be composed of repeating rings of six to eight protein units. A single amino acid replacement of H296R abolished the ability of the enzyme to form microfibrils with organized fibril networks and to synthesize levan at pH 6.0. PMID:18809687

  10. Application of Controlled Radical Polymerization for Nucleic Acid Delivery

    PubMed Central

    CHU, DAVID S.H.; SCHELLINGER, JOAN G.; SHI, JULIE; CONVERTINE, ANTHONY J.; STAYTON, PATRICK S.; PUN, SUZIE H.

    2012-01-01

    CONSPECTUS Nucleic acid-based therapeutics can potentially address otherwise untreatable genetic disorders and have significant potential for a wide range of diseases. Therapeutic gene delivery can restore protein function by replacing defunct genes to restore cellular health while RNA interference (RNAi) can mask mutated and harmful genes. Cationic polymers have been extensively studied for nucleic acid delivery applications due to their self-assembly with nucleic acids into virus-sized nanoparticles and high transfection efficiency in vitro, but toxicity and particle stability have limited their clinical applications. The advent of controlled radical polymerization has improved the quality, control and reproducibility of synthesized materials. Controlled radical polymerization yields well-defined, narrowly disperse materials of designable architectures and molecular weight, allowing study of the effects of polymer architecture and molecular weight on transfection efficiency and cytotoxicity for improved design of next-generation vectors. Robust methods such as atom transfer radical polymerization (ATRP), reverse addition-fragmentation chain transfer polymerization (RAFT), and ring-opening metastasis polymerization (ROMP) have been used to engineer materials that specifically enhance extracellular stability, cellular specificity, and decrease toxicity. This Account reviews findings from structure-function studies that have elucidated key design motifs necessary for the development of effective nucleic acid vectors. In addition, polymers that are biodegradable, form supramolecular structures, target specific cells, or facilitate endosomal release are also discussed. Finally, promising materials with in vivo applications ranging from pulmonary gene delivery to DNA vaccines are described. PMID:22242774

  11. PolyPEGA with predetermined molecular weights from enzyme-mediated radical polymerization in water.

    PubMed

    Ng, Yeap-Hung; di Lena, Fabio; Chai, Christina L L

    2011-06-14

    The preparation of acrylic polymers with predetermined molecular weights using metalloenzymes as catalysts, ascorbic acid as reducing agent and alkyl halides as initiators is reported. The mechanism of polymerization resembles an ARGET ATRP process. PMID:21552589

  12. Enzyme biosensor based on plasma-polymerized film-covered carbon nanotube layer grown directly on a flat substrate.

    PubMed

    Muguruma, Hitoshi; Hoshino, Tatsuya; Matsui, Yasunori

    2011-07-01

    We report a novel approach to fabrication of an amperometric biosensor with an enzyme, a plasma-polymerized film (PPF), and carbon nanotubes (CNTs). The CNTs were grown directly on an island-patterned Co/Ti/Cr layer on a glass substrate by microwave plasma enhanced chemical vapor deposition. The as-grown CNTs were subsequently treated by nitrogen plasma, which changed the surface from hydrophobic to hydrophilic in order to obtain an electrochemical contact between the CNTs and enzymes. A glucose oxidase (GOx) enzyme was then adsorbed onto the CNT surface and directly treated with acetonitrile plasma to overcoat the GOx layer with a PPF. This fabrication process provides a robust design of CNT-based enzyme biosensor, because of all processes are dry except the procedure for enzyme immobilization. The main novelty of the present methodology lies in the PPF and/or plasma processes. The optimized glucose biosensor revealed a high sensitivity of 38 μA mM(-1) cm(-2), a broad linear dynamic range of 0.25-19 mM (correlation coefficient of 0.994), selectivity toward an interferent (ascorbic acid), and a fast response time of 7 s. The background current was much smaller in magnitude than the current due to 10 mM glucose response. The low limit of detection was 34 μM (S/N = 3). All results strongly suggest that a plasma-polymerized process can provide a new platform for CNT-based biosensor design. PMID:21678995

  13. Recent Advances in Polymeric Materials Used as Electron Mediators and Immobilizing Matrices in Developing Enzyme Electrodes

    PubMed Central

    Moyo, Mambo; Okonkwo, Jonathan O.; Agyei, Nana M.

    2012-01-01

    Different classes of polymeric materials such as nanomaterials, sol-gel materials, conducting polymers, functional polymers and biomaterials have been used in the design of sensors and biosensors. Various methods have been used, for example from direct adsorption, covalent bonding, crossing-linking with glutaraldehyde on composites to mixing the enzymes or use of functionalized beads for the design of sensors and biosensors using these polymeric materials in recent years. It is widely acknowledged that analytical sensing at electrodes modified with polymeric materials results in low detection limits, high sensitivities, lower applied potential, good stability, efficient electron transfer and easier immobilization of enzymes on electrodes such that sensing and biosensing of environmental pollutants is made easier. However, there are a number of challenges to be addressed in order to fulfill the applications of polymeric based polymers such as cost and shortening the long laboratory synthetic pathways involved in sensor preparation. Furthermore, the toxicological effects on flora and fauna of some of these polymeric materials have not been well studied. Given these disadvantages, efforts are now geared towards introducing low cost biomaterials that can serve as alternatives for the development of novel electrochemical sensors and biosensors. This review highlights recent contributions in the development of the electrochemical sensors and biosensors based on different polymeric material. The synergistic action of some of these polymeric materials and nanocomposites imposed when combined on electrode during sensing is discussed. PMID:22368503

  14. [A new method of immobilizing proteolytic enzymes in polymeric hydrogels].

    PubMed

    Markvicheva, E A; Bronin, A S; Kudriavtseva, N E; Kuz'kina, I F; Pashkin, I I; Kirsh, Iu E; Rumsh, L D; Zubov, V P

    1994-03-01

    A novel one-step method for enzyme immobilization in composite gels based on the thermally reversible polymer poly (N-vinyl-caprolactam) was developed. Conditions for entrapment of trypsin and carboxypeptidase B in the gels were optimized. After immobilization, 80 and 90% of the original carboxypeptidase B and trypsin activities, respectively, were found to retain. Both immobilizes enzymes were active in a wider pH and temperature range. The granules with entrapped enzymes were successfully applied to obtain human insulin from recombinant proinsulin. PMID:8166751

  15. Polymerization of Pu(IV) in aqueous nitric acid solutions

    SciTech Connect

    Toth, L.M.; Friedman, H.A.; Osborne, M.M.

    1980-10-01

    The polymerization of Pu(IV) in aqueous nitric acid solutions has been studied spectrophotometrically both to establish the influence of large UO{sub 2}(NO{sub 3}){sub 2} concentrations on the polymerization rates and, more generally, to review the influence of the major parameters on the polymer reaction. Typically, experiments have been performed at 50{sup 0}C and with 0.05 M Pu in nitric acid solutions that vary in acidity from 0.07 to 0.4 M. An induction period usually precedes the polymer growth stage during which time nucleation of primary hydrolysis products occurs. Uranyl nitrate retards the polymerization reaction by approximately 35% in spite of the counteracting influence of the nitrate ions associated with this solute. The rate of polymer formation, expressed as d(percent polymer)/dt, has been shown to depend on the total plutonium concentration in reactions where the Pu(IV) concentration remained constant; and it is therefore suggested that the polymer reaction rate is not first order with respect to the concentration of plutonium as was previously thought. It has been shown further that accurate acid determinations on stock reagents are essential in order to obtain reliable polymerization experiments. Satisfactory procedures for these analyses did not exist, so appropriate modifications to the iodate precipitation methods were developed. The most ideal plutonium reagent material has been shown to be crystalline Pu(IV) nitrate because it can be added directly to acid solutions without the occurrence of unintentional hydrolysis reactions.

  16. Uronic Acid products release from enzymically active cell wall from tomato fruit and its dependency on enzyme quantity and distribution.

    PubMed

    Huber, D J; Lee, J H

    1988-07-01

    Isolated cell wall from tomato (Lycopersicon esculentum Mill. cv Rutgers) fruit released polymeric (degree of polymerization [DP] > 8), oligomeric, and monomeric uronic acids in a reaction mediated by bound polygalacturonase (PG) (EC 3.2.1.15). Wall autolytic capacity increased with ripening, reflecting increased levels of bound PG; however, characteristic oligomeric and monomeric products were recovered from all wall isolates exhibiting net pectin release. The capacity of wall from fruit at early ripening (breaker, turning) to generate oligomeric and monomeric uronic acids was attributed to the nonuniform ripening pattern of the tomato fruit and, consequently, a locally dense distribution of enzyme in wall originating from those fruit portions at more temporally advanced stages of ripening. Artificial autolytically active wall, prepared by permitting solubilized PG to bind to enzymically inactive wall from maturegreen fruit, released products which were similar in size characteristics to those recovered from active wall isolates. Extraction of wall-bound PG using high concentrations of NaCl (1.2 molar) did not attenuate subsequent autolytic activity but greatly suppressed the production of oligomeric and monomeric products. An examination of water-soluble uronic acids recovered from ripe pericarp tissue disclosed the presence of polymeric and monomeric uronic acids but only trace quantities of oligomers. The significance in autolytic reactions of enzyme quantity and distribution and their possible relevance to in vivo pectin degradation will be discussed. PMID:16666191

  17. Terrestrial evolution of polymerization of amino acids - Heat to ATP

    NASA Technical Reports Server (NTRS)

    Fox, S. W.; Nakashima, T.

    1981-01-01

    Sets of amino acids containing sufficient trifunctional monomer are thermally polymerized at temperatures such as 65 deg; the amino acids order themselves. Various polymers have diverse catalytic activities. The polymers aggregate, in aqueous solution, to cell-like structures having those activities plus emergent properties, e.g. proliferatability. Polyamino acids containing sufficient lysine catalyze conversion of free amino acids, by ATP, to small peptides and a high molecular weight fraction. The lysine-rich proteinoid is active in solution, within suspensions of cell-like particles, or in other particles composed of lysine-rich proteinoid and homopolyribonucleotide. Selectivities are observed. An archaic polyamino acid prelude to coded protein synthesis is indicated.

  18. Polymeric Nucleic Acid Carriers: Current Issues and Novel Design Approaches

    PubMed Central

    Kang, Han Chang; Huh, Kang Moo; Bae, You Han

    2012-01-01

    To deliver nucleic acids including plasmid DNA (pDNA) and short interfering RNA (siRNA), polymeric gene carriers equipped with various functionalities have been extensively investigated. The functionalities of these polymeric vectors have been designed to overcome various extracellular and intracellular hurdles that nucleic acids and their carriers encounter during their journey from injection site to intracellular target site. This review briefly introduces known extracellular and intracellular issues of nucleic acid delivery and their solution strategies. We examine significant yet overlooked factors affecting nucleic acid delivery (e.g., microenvironmental pH, polymer/siRNA complexation, and pharmaceutical formulation) and highlight our reported approaches to solve these problems. PMID:22771981

  19. Polymerization on the rocks: beta-amino acids and arginine

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    We have studied the accumulation of long oligomers of beta-amino acids on the surface of minerals using the 'polymerization on the rocks' protocol. We find that long oligopeptides of beta-glutamic acid which cannot be formed in homogeneous aqueous solution are accumulated efficiently on the surface of hydroxylapatite using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as condensing agent. The EDAC-induced oligomerization of aspartic acid on hydroxylapatite proceeds even more efficiently. Hydroxylapatite can also facilitate the ligation of the tripeptide (glu)3. The 'polymerization on the rocks' scenario is not restricted to negatively-charged amino acids. Oligoarginines are accumulated on the surface of illite using carbonyldiimidizole (CDI) as condensing agent. We find that FeS2 catalyzes the CDI-induced oligomerization of arginine, although it does not adsorb oligoarginines. These results are relevant to the formation of polypeptides on the primitive earth.

  20. Sulfonic acid catalysts prepared by radiation-induced graft polymerization

    SciTech Connect

    Mizota, Tomotoshi; Tsuneda, Satoshi; Saito, Kyoichi, Saito

    1994-09-01

    In this study, the authors prepared two variations of graft-type acid catalysts with different adjacent groups by radiation-induced graft polymerization (RIGP), and compared the hydrolytic activity of the resultant acid catalysts for methyl acetate with that of commercially available SO{sub 3}H-type ion-exchange beads with different degrees of cross-linking. 8 refs., 3 figs.

  1. Cationic RAFT polymerization using ppm concentrations of organic acid.

    PubMed

    Uchiyama, Mineto; Satoh, Kotaro; Kamigaito, Masami

    2015-02-01

    A metal-free, cationic, reversible addition-fragmentation chain-transfer (RAFT) polymerization was proposed and realized. A series of thiocarbonylthio compounds were used in the presence of a small amount of triflic acid for isobutyl vinyl ether to give polymers with controlled molecular weight of up to 1×10(5) and narrow molecular-weight distributions (Mw /Mn <1.1). This "living" or controlled cationic polymerization is applicable to various electron-rich monomers including vinyl ethers, p-methoxystyrene, and even p-hydroxystyrene that possesses an unprotected phenol group. A transformation from cationic to radical RAFT polymerization enables the synthesis of block copolymers between cationically and radically polymerizable monomers, such as vinyl ether and vinyl acetate or methyl acrylate. PMID:25511364

  2. 40 CFR 721.10568 - Diethanolamine salt of polymeric acid (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 32 2013-07-01 2013-07-01 false Diethanolamine salt of polymeric acid... Specific Chemical Substances § 721.10568 Diethanolamine salt of polymeric acid (generic). (a) Chemical... as diethanolamine salt of polymeric acid (PMN P-08-464) is subject to reporting under this...

  3. 40 CFR 721.10568 - Diethanolamine salt of polymeric acid (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 40 Protection of Environment 31 2014-07-01 2014-07-01 false Diethanolamine salt of polymeric acid... Specific Chemical Substances § 721.10568 Diethanolamine salt of polymeric acid (generic). (a) Chemical... as diethanolamine salt of polymeric acid (PMN P-08-464) is subject to reporting under this...

  4. Enzymatic polymerization of natural anacardic acid and antibiofouling effects of polyanacardic acid coatings.

    PubMed

    Chelikani, Rahul; Kim, Yong Hwan; Yoon, Do-Young; Kim, Dong-Shik

    2009-05-01

    Anacardic acid, separated from cashew nut shell liquid, is well known for its strong antibiotic and antioxidant activities. Recent findings indicate that phenolic compounds from plant sources have an effect on Gram-negative bacteria biofilm formation. In this work, a polyphenolic coating was prepared from anacardic acid using enzymatic synthesis and tested for its effects on biofilm formation of both Gram-negative and Gram-positive bacteria. Natural anacardic acid was enzymatically polymerized using soybean peroxidase. Hydrogen peroxide and phenothiazine-10-propionic acid were used as an oxidizing agent and redox mediator, respectively. Nuclear magnetic resonance and Fourier transform infrared (FTIR) analyses showed the formation of oxyphenylene and phenylene units through the phenol rings. No linkage through the alkyl chain was observed, which proved a high chemo-selectivity of the enzyme. Aqueous solvents turned out to play an important role in the polymer production yield and molecular weight. With 2-propanol, the highest production yield (61%) of polymer (molecular weight = 3,900) was observed, and with methanol, higher-molecular-weight polymers (5,000) were produced with lower production yields (43%). The resulting polyanacardic acid was cross-linked on a solid surface to form a permanent natural polymer coating. The FTIR analysis indicates that the cross-linking between the polymers took place through the unsaturated alkyl side chains. The polyanacardic acid coating was then tested for its antibiofouling effect against Gram-negative and Gram-positive bacteria and compared with the antibiofouling effects of polycardanol coatings reported in the literature. The polyanacardic acid coating showed more reduction in biofilm formation on its surface than polycardanol coatings in the case of Gram-positive bacteria, while in the case of Gram-negative bacteria, it showed a similar reduction in biofilm formation as polycardanol. PMID:18592408

  5. Polymerization of amino acids containing nucleotide bases

    NASA Technical Reports Server (NTRS)

    Ben Cheikh, Azzouz; Orgel, Leslie E.

    1990-01-01

    The nucleoamino acids 1-(3'-amino,3'-carboxypropyl)uracil (3) and 9-(3'-amino,3'-carboxypropyl)adenine (4) have been prepared as (L)-en-antiomers and as racemic mixtures. When 3 or 4 is suspended in water and treated with N,N'-carbon-yldiimidazole, peptides are formed in good yield. The products formed from the (L)-enantiomers are hydrolyzed to the monomeric amino acids by pronase. Attempts to improve the efficiency of these oligomerizations by including a polyuridylate template in the reaction mixture were not successful. Similarly, oligomers derived from the (L)-enantiomer of 3 did not act as templates to facilitate the oligomerization of 4.

  6. Enzyme immunoassay for carminic acid in foods.

    PubMed

    Yoshida, A; Takagaki, Y; Nishimune, T

    1995-01-01

    A competitive enzyme immunoassay (EIA) for carminic acid was investigated. Monoclonal anticarminic acid antibody was obtained from A/J mice immunized with carminic acid-human immunoglobulin G (IgG) conjugate. Carminic acid was extracted with distilled water from beverage, jelly, candy, pasta sauce, yogurt, or ice cream samples. Ham or fish paste samples were digested with pronase, then carminic acid was extracted from samples with sodium hydroxide solution. The extract was diluted more than 10-fold with 1% gelatin in borate buffer solution. Microtiter plates were coated with carminic acid-bovine serum albumin (BSA) conjugate or just BSA. Goat anti-mouse IgG(H+L)-peroxidase complex was used as a second antibody, and 3,3',5,5'-tetramethylbenzidine was used as a substrate for the peroxidase. The working range for quantitative analysis was 0.3-10 ng/mL, and the detection limit was 0.2 micrograms/g original sample. Recoveries of carminic acid by this assay were > 95% for milk beverage and jelly, and > 85% for yogurt and fish paste. Carminic acid was detected in 7 of 26 red-colored commercial food products and ranged from 3.5 to 356 micrograms/g. This EIA system also responded to the structural analogue of carminic acid, laccaic acid. PMID:7756895

  7. Lipid and polymeric carrier-mediated nucleic acid delivery

    PubMed Central

    Zhu, Lin; Mahato, Ram I

    2010-01-01

    Importance of the field Nucleic acids such as plasmid DNA, antisense oligonucleotide, and RNA interference (RNAi) molecules, have a great potential to be used as therapeutics for the treatment of various genetic and acquired diseases. To design a successful nucleic acid delivery system, the pharmacological effect of nucleic acids, the physiological condition of the subjects or sites, and the physicochemical properties of nucleic acid and carriers have to be thoroughly examined. Areas covered in this review The commonly used lipids, polymers and corresponding delivery systems are reviewed in terms of their characteristics, applications, advantages and limitations. What the reader will gain This article aims to provide an overview of biological barriers and strategies to overcome these barriers by properly designing effective synthetic carriers for nucleic acid delivery. Take home message A thorough understanding of biological barriers and the structure–activity relationship of lipid and polymeric carriers is the key for effective nucleic acid therapy. PMID:20836625

  8. An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions.

    PubMed

    Ruan, Guihua; Wu, Zhenwei; Huang, Yipeng; Wei, Meiping; Su, Rihui; Du, Fuyou

    2016-04-22

    A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of Nα-benzoyl-l-arginine ethyl ester to Nα-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas. PMID:26995089

  9. Polymeric complexes of isonicotinic acid hydrazide with antituberculosis effects.

    PubMed

    Slivkin, A I; Lapenko, V L; Bychuk, A I; Suslina, S N; Slivkin, D A; Kornienko, S V; Belenova, A S

    2013-10-01

    We studied the effects of an analogue of isonicotinic acid hydrazide on the treatment course of experimental tuberculosis. Complex analysis has demonstrated the efficiency of isonicotinic acid hydrazide immobilized on a carrier that consisted of water-soluble cation-active analogue of chitosan (N-chlorohydroxypropyl chitosan) in a complex with cobalt ions in the therapy of experimental tuberculosis. Immunostimulating activity of the polymeric metal complex was revealed. The obtained data can be used for the development of highly effective methods for tuberculosis treatment. PMID:24288761

  10. Enzyme-responsive polymeric supra-amphiphiles formed by the complexation of chitosan and ATP.

    PubMed

    Kang, Yuetong; Wang, Chao; Liu, Kai; Wang, Zhiqiang; Zhang, Xi

    2012-10-16

    Chitosan and adenosine-5'-triphosphate (ATP) are employed as building blocks to fabricate polymeric supra-amphiphiles based on electrostatic interactions, which can self-assemble to form spherical aggregates. The spherical aggregates inherit the phosphotase responsiveness of ATP. Compared to our previous work, this enzyme-responsive system can be more biocompatible and block polymers are not needed in preparation, which makes it possible to fabricate the chitosan-based enzyme-responsive assemblies in a large-scale, cheap way. Therefore, the application of the assemblies for nanocontainers and drug delivery is greatly anticipated. PMID:23025557

  11. Organo-Lewis acid as cocatalyst for cationic homogenous metallocene Ziegler-Natta olefin polymerizations

    DOEpatents

    Marks, Tobin J.; Chen, You-Xian

    2000-01-01

    The synthesis of the organo-Lewis acid perfluorobiphenylborane (PBB) and the activation of metallocenes for the formation of a variety of highly active homogeneous Ziegler-Natta metallocene olefin polymerization, copolymerization and ring-opening polymerization catalysts is described.

  12. Amine bearing polymeric particles as acid neutralizers for engine oils

    SciTech Connect

    Theodore, A.N.; Chattha, M.S.

    1986-02-04

    This patent describes a lubricating oil composition consisting of a major proportion of a lubricating base oil and about 0.1 to 15 weight percent of an acid neutralizing additive which consists of polymer particles (a) bearing pendant amine groups, and (b) having a diameter of about 500 A and 10,000 A. The amine functional particles are formed by reacting polymer particles bearing pendant epoxide groups with a secondary amine in an amount so as to react essentially all of the epoxide groups on the epoxide bearing polymer particles with the secondary amine. The polymer particles bearing pendant epoxide groups are formed by the free radical addition polymerization of: (a) between about 50 and about 100 weight percent of an ethylenically unsaturated monomers bearing an epoxide group, and (b) 0 up to about 50 weight percent of other monoethylenically unsaturated monomers; in the presence of: (I) a non-polar organic liquid which is a solvent for the polymerizable monomers, but a non-solvent for the resultant polymer, and (II) polymeric dispersion stabilizer containing at least two segments, with one segment being solvated by the non-polar organic liquid and the second segment being of different polarity than the first segment and relatively insoluble in the non-polar organic liquid. The second segment of the stabilizer is chemically attached to the polymerized particle.

  13. Immobilization of Enzymes by Electrochemical and Chemical Oxidative Polymerization of L-DOPA to Fabricate Amperometric Biosensors and Biofuel Cells.

    PubMed

    Dai, Mengzhen; Sun, Lingen; Chao, Long; Tan, Yueming; Fu, Yingchun; Chen, Chao; Xie, Qingji

    2015-05-27

    Electrochemical/chemical oxidative synthesis and biosensing/biofuel cell applications of poly(L-DOPA) (PD) are studied versus polydopamine (PDA) as a recent hotspot biomaterial. The enzyme electrode developed by coelectrodeposition of PD and glucose oxidase (GOx), uricase, or tyrosinase shows biosensing performance superior to that of the corresponding PDA-based enzyme electrode. The chemical oxidative polymerization of L-DOPA (PDC) by NaAuCl4 in GOx-containing neutral aqueous solution is used to immobilize GOx and gold nanoparticles (AuNPs). The thus-prepared chitosan (CS)/GOx-PDC-AuNPs/Au(plate)/Au electrode working in the first-generation biosensing mode responds linearly to glucose concentration with a sensitivity of 152 μA mM(-1) cm(-2), which is larger than those of the CS/GOx-PDAC-AuNPs/Au(plate)/Au electrode, the CS/GOx-poly(3-anilineboronic acid) (PABA)-AuNPs/Au(plate)/Au electrode, and the most reported GOx-based enzyme electrodes. This PDC-based enzyme electrode also works well in the second-generation biosensing mode and as an excellent bioanode in biofuel cell construction, probably because PD as an amino acid polymer has the higher biocompatibility and the more favorable affinity to the enzyme than PDA. The PD material of great convenience in synthesis, outstanding biocompatibility for preparing high-performance bionanocomposites, and strong capability of multifunctional coatings on many surfaces may find wide applications in diversified fields including biotechnology and surface-coating. PMID:25938891

  14. Synthesis of polymeric microcapsule arrays and their use for enzyme immobilization

    NASA Astrophysics Data System (ADS)

    Parthasarathy, Ranjani V.; Martin, Charles R.

    1994-05-01

    CURRENT methods for immobilizing enzymes for use in bioreactors and biosensors1-20 include adsorption on or covalent attachment to a support2-4, micro-encapsulation5,6, and entrapment within a membrane/film7,8,11-20 or gel9. The ideal immobilization method should employ mild chemical conditions, allow for large quantities of enzyme to be immobilized, provide a large surface area for enzyme-substrate contact within a small total volume, minimize barriers to mass transport of substrate and product, and provide a chemically and mechanically robust system. Here we describe a method for enzyme immobilization that satisfies all of these criteria. We have developed a template-based synthetic method that yields hollow polymeric microcapsules of uniform diameter and length. These microcapsules are arranged in a high-density array in which the individual capsules protrude from a surface like the bristles of a brush. We have developed procedures for filling these microcapsules with high concentrations of enzymes. The enzyme-loaded microcapsule arrays function as enzymatic bioreactors in both aqueous solution and organic solvents.

  15. Paclitaxel isomerisation in polymeric micelles based on hydrophobized hyaluronic acid.

    PubMed

    Smejkalová, Daniela; Nešporová, Kristina; Hermannová, Martina; Huerta-Angeles, Gloria; Cožíková, Dagmar; Vištejnová, Lucie; Safránková, Barbora; Novotný, Jaroslav; Kučerík, Jiří; Velebný, Vladimír

    2014-05-15

    Physical and chemical structure of paclitaxel (PTX) was studied after its incorporation into polymeric micelles made of hyaluronic acid (HA) (Mw=15 kDa) grafted with C6 or C18:1 acyl chains. PTX was physically incorporated into the micellar core by solvent evaporation technique. Maximum loading capacity for HAC6 and HAC18:1 was determined to be 2 and 14 wt.%, respectively. The loading efficiency was higher for HAC18:1 and reached 70%. Independently of the derivative, loaded HA micelles had spherical size of approximately 60-80 nm and demonstrated slow and sustained release of PTX in vitro. PTX largely changed its form from crystalline to amorphous after its incorporation into the micelle's interior. This transformation increased PTX sensitivity towards stressing conditions, mainly to UV light exposure, during which the structure of amorphous PTX isomerized and formed C3C11 bond within its structure. In vitro cytotoxicity assay revealed that polymeric micelles loaded with PTX isomer had higher cytotoxic effect to normal human dermal fibroblasts (NHDF) and human colon carcinoma cells (HCT-116) than the same micelles loaded with non-isomerized PTX. Further observation indicated that PTX isomer influenced in different ways cell morphology and markers of cell cycle. Taken together, PTX isomer loaded in nanocarrier systems may have improved anticancer activity in vivo than pure PTX. PMID:24614580

  16. Bioengineering of the silica-polymerizing enzyme silicatein-alpha for a targeted application to hydroxyapatite.

    PubMed

    Natalio, Filipe; Link, Thorben; Müller, Werner E G; Schröder, Heinz C; Cui, Fu-Zhai; Wang, Xiaohong; Wiens, Matthias

    2010-09-01

    Since its discovery, numerous biotechnological approaches have aimed to explore the silica-polymerizing catalytic activity of the enzyme silicatein. In vivo, silicatein catalyzes polymerization of amorphous silica nanospheres from soluble precursors. In vitro, it directs the formation of nanostructured biosilica. This is of interest for various applications that strive to benefit from both the advantages of the biological system (i.e., silica synthesis under physiological conditions) and the cell mineralization-stimulating effect of biosilica. However, so far immobilization of silicatein has been hampered by the complex multistep procedure required. In addition, the chemical surface modifications involved not only restrict the choice of carrier materials but also render application of silicatein to hydroxyapatite (HA) of mineralized tissue impossible. Here we describe the bioengineering of silicatein, adapted for application in the fields of bone regeneration, tissue engineering, and dental care. Inspired by Glu-rich sequences of mammalian proteins that confer binding affinity to HA, a novel protein-tag was developed, the Glu-tag. Following expression of Glu-tagged silicatein, the HA-binding capacity of the enzyme is demonstrated in combination with synthetic and dental HA. Furthermore, immobilized Glu-tagged silicatein catalyzes synthesis of biosilica coatings on both synthetic HA nanofibrils and dental HA. Hence, Glu-tagged silicatein reveals a considerable biomedical potential with regenerative and prophylactic implementations. PMID:20226280

  17. Self-assembled polymeric micelles based on hyaluronic acid-g-poly(D,L-lactide-co-glycolide) copolymer for tumor targeting.

    PubMed

    Son, Gyung Mo; Kim, Hyun Yul; Ryu, Je Ho; Chu, Chong Woo; Kang, Dae Hwan; Park, Su Bum; Jeong, Young-Il

    2014-01-01

    Graft copolymer composed hyaluronic acid (HA) and poly(D,L-lactide-co-glycolide) (PLGA) (HAgLG) was synthesized for antitumor targeting via CD44 receptor of tumor cells. The carboxylic end of PLGA was conjugated with hexamethylenediamine (HMDA) to have amine end group in the end of chain (PLGA-amine). PLGA-amine was coupled with carboxylic acid of HA. Self-assembled polymeric micelles of HAgLG have spherical morphologies and their sizes were around 50-200 nm. Doxorubicin (DOX)-incorporated polymeric micelles were prepared by dialysis procedure. DOX was released over 4 days and its release rate was accelerated by the tumoric enzyme hyaluronidase. To assess targetability of polymeric micelles, CD44-positive HepG2 cells were employed treated with fluorescein isothiocyanate (FITC)-labeled polymeric micelles. HepG2 cells strongly expressed green fluorescence at the cell membrane and cytosol. However, internalization of polymeric micelles were significantly decreased when free HA was pretreated to block the CD44 receptor. Furthermore, the CD44-specific anticancer activity of HAgLG polymeric micelles was confirmed using CD44-negative CT26 cells and CD44-positive HepG2 cells. These results indicated that polymeric micelles of HaLG polymeric micelles have targetability against CD44 receptor of tumor cells. We suggest HAgLG polymeric micelles as a promising candidate for specific drug targeting. PMID:25216338

  18. Assessment of stability, toxicity and immunogenicity of new polymeric nanoreactors for use in enzyme replacement therapy of MNGIE.

    PubMed

    De Vocht, Caroline; Ranquin, An; Willaert, Ronnie; Van Ginderachter, Jo A; Vanhaecke, Tamara; Rogiers, Vera; Versées, Wim; Van Gelder, Patrick; Steyaert, Jan

    2009-08-01

    The lack of a crucial metabolic enzyme can lead to accumulating substrate concentrations in the bloodstream and severe human enzyme deficiency diseases. Mitochondrial Neurogastrointestinal Encephalomyopathy (MNGIE) is such a fatal genetic disorder, caused by a thymidine phosphorylase deficiency. Enzyme replacement therapy is a strategy where the deficient enzyme is administered intravenously in order to decrease the toxic substrate concentrations. Such a therapy is however not very efficient due to the fast elimination of the native enzyme from the circulation. In this study we evaluate the potential of using polymeric enzyme-loaded nanoparticles to improve the delivery of therapeutic enzymes. We constructed new 200-nanometer PMOXA-PDMS-PMOXA polymeric nanoparticles that encapsulate the enzyme thymidine phosphorylase. These particles are permeabilised for substrates and products by the reconstitution of the nucleoside-specific porin Tsx in their polymeric wall. We show that the obtained 'nanoreactors' are enzymatically active and stable in blood serum at 37 degrees C. Moreover, they do not provoke cytotoxicity when incubated with hepatocytes for 4 days, nor do they induce a macrophage-mediated inflammatory response ex vivo and in vivo. All data highlight the potential of such nanoreactors for their application in enzyme replacement therapy of MNGIE. PMID:19371766

  19. Catalyzed ring-opening polymerization of epoxidized soybean oil by hydrated and anhydrous fluoroantimonic acids

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ring-opening polymerization of epoxidized soybean oil (ESO) catalyzed by the super acid, fluroantimonic acid hexahydrate (HSbF6-6H2O), and the anhydrous form (HSbF6) in ethyl acetate was conducted in an effort to develop useful biodegradable polymers. The resulting polymerized ESO (SA-RPESO and SAA-...

  20. 78 FR 46265 - Complex Polymeric Polyhydroxy Acids; Exemption From the Requirement of a Tolerance

    Federal Register 2010, 2011, 2012, 2013, 2014

    2013-07-31

    ...This regulation establishes an exemption from the requirement of a tolerance for residues of Complex Polymeric Polyhydroxy Acids in or on all food commodities. This regulation eliminates the need to establish a maximum permissible level for residues of Complex Polymeric Polyhydroxy Acids (CPPA) under...

  1. [Influence of extracellular polymeric substance on enzyme hydrolysis of sludge under anaerobic condition].

    PubMed

    Chen, Wei; Jia, Yuan-Yuan; Zheng, Wei; Li, Xiao-Ming; Zhou, Jun; Yang, Qi; Luo, Kun

    2011-08-01

    The effect of extracellular polymeric substance (EPS) on the enzymatic solubilisation of sludge and the changes of chemical components was investigated. Sludge solubilization with and without EPS was studied in the enzymatic system, and in the normal system without enzyme addition, respectively. The result indicated that only EPS could be hydrolyzed when the enzyme addition less than 20 mg/g, while the cell lysis occurred significantly with the doses of enzymes increasing. Treatment with lysozyme for the original sludge was proved to have a higher hydrolysis efficiency, and the SCOD/TCOD rate reached up to 28.14%. And at the enzyme dosage of 60 mg/g, the VSS removal rate increased to 51.66% and the concentration of DNA attained 68.34 mg/g (calculated by VSS) after 48 h reaction, which were 29.01% and 59.63 mg/g higher than the control test, respectively, and were 24.86% and 53.39 mg/g higher than that with EPS removed in advance, respectively. Meanwhile, NH4+ -N, PO4(3-)-P and SCOD showed high dissolution efficiency, and the maximal concentrations achieved to 503 mg/L, 78.9 mg/L and 3171 mg/L, respectively. After removal of extracellular polymers, higher lysis efficiency was also observed by protease and cellulose, by which VSS reduction rate reached to 49.95% and 39.85%, respectively. The concentration of DNA showed a correlation coefficient of more than 0.9 with the concentrations of SCOD, NH4+ -N and PO4(3-)-P. And the highest hydrolysis rate obtained in 6 hours, which was about 3 hours earlier than the control test. Moreover, under those condition, sludge hydrolyzation could be well realized by only small amount of the enzyme addition. PMID:22619959

  2. Endothelial delivery of antioxidant enzymes loaded into non-polymeric magnetic nanoparticles.

    PubMed

    Chorny, Michael; Hood, Elizabeth; Levy, Robert J; Muzykantov, Vladimir R

    2010-08-17

    cell death vs. 10+/-4% under non-magnetic conditions). We conclude that non-polymeric MNP formed using the controlled aggregation/precipitation strategy are a promising carrier for targeted antioxidant enzyme therapy, and in combination with magnetic guidance can be applied to protect endothelial cells from oxidative stress mediated damage. This protective effect of magnetically targeted MNP impregnated with antioxidant enzymes can be highly relevant for the treatment of cardiovascular disease and should be further investigated in animal models. PMID:20483366

  3. Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction

    PubMed Central

    Jee, Elizabeth; Bánsági, Tamás

    2016-01-01

    Abstract Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease‐catalyzed hydrolysis of urea was used to trigger the base‐catalyzed Michael addition of a water‐soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min−1). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel. PMID:26732469

  4. Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction

    PubMed Central

    Jee, Elizabeth; Bánsági, Tamás

    2016-01-01

    Abstract Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease‐catalyzed hydrolysis of urea was used to trigger the base‐catalyzed Michael addition of a water‐soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min−1). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel.

  5. Temporal Control of Gelation and Polymerization Fronts Driven by an Autocatalytic Enzyme Reaction.

    PubMed

    Jee, Elizabeth; Bánsági, Tamás; Taylor, Annette F; Pojman, John A

    2016-02-01

    Chemical systems that remain kinetically dormant until activated have numerous applications in materials science. Herein we present a method for the control of gelation that exploits an inbuilt switch: the increase in pH after an induction period in the urease-catalyzed hydrolysis of urea was used to trigger the base-catalyzed Michael addition of a water-soluble trithiol to a polyethylene glycol diacrylate. The time to gelation (minutes to hours) was either preset through the initial concentrations or the reaction was initiated locally by a base, thus resulting in polymerization fronts that converted the mixture from a liquid into a gel (ca. 0.1 mm min(-1)). The rate of hydrolytic degradation of the hydrogel depended on the initial concentrations, thus resulting in a gel lifetime of hours to months. In this way, temporal programming of gelation was possible under mild conditions by using the output of an autocatalytic enzyme reaction to drive both the polymerization and subsequent degradation of a hydrogel. PMID:26732469

  6. Acid-catalyzed Furfuryl Alcohol Polymerization: Characterizations of Molecular Structure and Thermodynamic Properties

    SciTech Connect

    Kim, Taejin; Assary, Rajeev A.; Marshall, Christopher L.; Gosztola, David J.; Curtiss, Larry A.; Stair, Peter C.

    2011-07-22

    The liquid-phase polymerization of furfuryl alcohol catalyzed by sulfuric acid catalysts and the identities of molecular intermediates were investigated by using Raman spectroscopy and density functional theory calculation. At room temperature, with an acid catalyst, a vigorous furfuryl alcohol polymerization reaction was observed, whereas even at a high water concentration, furfuryl alcohol was very stable in the absence of an acid catalyst. Theoretical studies were carried out to investigate the thermodynamics of protonation of furfuryl alcohol, initiation of polymerization, and formation of conjugated dienes and diketonic species by using the B3LYP level of theory. A strong aliphatic C=C band observed in the calculated and measured Raman spectra provided crucial evidence to understand the polymerization reaction mechanism. It is confirmed that the formation of a conjugated diene structure rather than a diketone structure is involved in the furfuryl alcohol polymerization reaction.

  7. Acid-catalyzed furfurly alcohol polymerization : characterizations of molecular structure and thermodynamic properties.

    SciTech Connect

    Kim, T.; Assary, R. S.; Marshall, C. L.; Gosztola, D. J.; Curtiss, L. A.; Stair, P. C.

    2011-01-01

    The liquid-phase polymerization of furfuryl alcohol catalyzed by sulfuric acid catalysts and the identities of molecular intermediates were investigated by using Raman spectroscopy and density functional theory calculation. At room temperature, with an acid catalyst, a vigorous furfuryl alcohol polymerization reaction was observed, whereas even at a high water concentration, furfuryl alcohol was very stable in the absence of an acid catalyst. Theoretical studies were carried out to investigate the thermodynamics of protonation of furfuryl alcohol, initiation of polymerization, and formation of conjugated dienes and diketonic species by using the B3LYP level of theory. A strong aliphatic C=C band observed in the calculated and measured Raman spectra provided crucial evidence to understand the polymerization reaction mechanism. It is confirmed that the formation of a conjugated diene structure rather than a diketone structure is involved in the furfuryl alcohol polymerization reaction.

  8. Ultrasensitive electrochemical detection of breast cancer cells based on DNA-rolling-circle-amplification-directed enzyme-catalyzed polymerization.

    PubMed

    Sheng, Qinglin; Cheng, Ni; Bai, Wushuang; Zheng, Jianbin

    2015-02-01

    An ultrasensitive cytosensor based on DNA-rolling-circle-amplification-directed enzyme-catalyzed polymerization is demonstrated. As a proof of concept, the cytosensor shows excellent sensitivity for MCF-7 cell detection with a lower detection limit of 12 cells per mL. PMID:25536491

  9. Organo-Lewis acid as cocatalyst for cationic homogeneous metallocene Ziegler-Natta olefin polymerizations

    SciTech Connect

    Marks, T.J.; Chen, Y.X.

    2000-07-11

    The synthesis of the organo-Lewis acid perfluorobiphenylborane (PBB) and the activation of metallocenes are disclosed for the formation of a variety of highly active homogeneous Ziegler-Natta metallocene olefin polymerization, copolymerization and ring-opening polymerization catalysts is described.

  10. Affinity labelling enzymes with esters of aromatic sulfonic acids

    DOEpatents

    Wong, Show-Chu; Shaw, Elliott

    1977-01-01

    Novel esters of aromatic sulfonic acids are disclosed. The specific esters are nitrophenyl p- and m-amidinophenylmethanesulfonate. Also disclosed is a method for specific inactivation of the enzyme, thrombin, employing nitrophenyl p-amidinophenylmethanesulfonate.

  11. Lewis-Acid-Mediated Stereospecific Radical Polymerization of Acrylimides Bearing Chiral Oxazolidinones.

    PubMed

    Fujita, Takehiro; Yamago, Shigeru

    2015-12-14

    Lewis acid (MgBr2)-catalyzed radical polymerization of acrylimides bearing chiral oxazolidinones gave highly isotactic polyacrylimides with up to >99% meso tetrad (mmm) selectivity. Polymerization in the absence of Lewis acid gave atactic polymers with 80% racemo diad (r) selectivity; the selectivity was deliberately tuned from 80% r to >99% mmm by varying the polymerization conditions. The polyacrylimide was quantitatively converted to corresponding polyacrylates while preserving the stereoregularity, thus providing a general method for the synthesis of atactic to isotactic polyacrylates. PMID:26500040

  12. Method for Enzyme Design with Genetically Encoded Unnatural Amino Acids.

    PubMed

    Hu, C; Wang, J

    2016-01-01

    We describe the methodologies for the design of artificial enzymes with genetically encoded unnatural amino acids. Genetically encoded unnatural amino acids offer great promise for constructing artificial enzymes with novel activities. In our studies, the designs of artificial enzyme were divided into two steps. First, we considered the unnatural amino acids and the protein scaffold separately. The scaffold is designed by traditional protein design methods. The unnatural amino acids are inspired by natural structure and organic chemistry methods, and synthesized by either organic chemistry methods or enzymatic conversion. With the increasing number of published unnatural amino acids with various functions, we described an unnatural amino acids toolkit containing metal chelators, redox mediators, and click chemistry reagents. These efforts enable a researcher to search the toolkit for appropriate unnatural amino acids for the study, rather than design and synthesize the unnatural amino acids from the beginning. After the first step, the model enzyme was optimized by computational methods and directed evolution. Lastly, we describe a general method for evolving aminoacyl-tRNA synthetase and expressing unnatural amino acids incorporated into a protein. PMID:27586330

  13. Removal of benzene from wastewater via Fenton pre-treatment followed by enzyme catalyzed polymerization.

    PubMed

    Saha, Beeta; Taylor, K E; Bewtra, J K; Biswas, N

    2011-01-01

    This study investigated the feasibility of a two-step process for the removal of benzene from buffered synthetic wastewater. Benzene is outside the scope of enzymatic removal. In order to remove it from wastewater using enzyme, its pretreatment by modified Fenton reaction was employed to generate the corresponding phenolic compounds. In the first phase, the optimum pH, H2O2 and Fe2+ concentrations and reaction time for the Fenton reaction were determined to maximize the conversion of benzene to phenolic compounds without causing significant mineralization. The pretreatment process was followed by oxidative polymerization of the phenolic compounds catalyzed by a laccase from Trametes villosa. Factors of interest for the three-hour enzymatic treatment were pH and laccase concentration. Under optimum Fenton reaction conditions, 80% conversion of the initial benzene concentration was achieved, giving a mixture containing oxidative dimerization product (biphenyl) and hydroxylation products (phenol, catechol, resorcinol, benzoquinone and hydroquinone). Enzymatic removal of biphenyl and benzoquinone was not possible but 2.5 U/mL laccase was successful in removal of the rest of the phenolic PMID:21866766

  14. The Roles of Acids and Bases in Enzyme Catalysis

    ERIC Educational Resources Information Center

    Weiss, Hilton M.

    2007-01-01

    Many organic reactions are catalyzed by strong acids or bases that protonate or deprotonate neutral reactants leading to reactive cations or anions that proceed to products. In enzyme reactions, only weak acids and bases are available to hydrogen bond to reactants and to transfer protons in response to developing charges. Understanding this…

  15. Nucleic acid programmed polymeric nanomaterials for biological communication

    NASA Astrophysics Data System (ADS)

    Rush, Anthony Michael

    A number of nucleic acid-polymer conjugates were synthesized, resulting in amphiphilic polymer-nucleic acid conjugates with the capability to self-assemble into a range of discrete nanoscale architectures. These nanomaterials, termed DNA-polymer amphiphile nanoparticles (DPA NPs), were studied with respect to their enzymatic processing by both endo- and exonucleases and further deployed as antisense genetic regulatory elements in live cultured human cells. DPA NPs were designed to act as substrates for both non sequence-specific exonucleases and a sequence-specific endonuclease. In all cases, nucleic acids arranged in the corona of spherical nanoparticles exhibited increased resistance to nucleolytic cleavage as compared to native single- or double-stranded analogues. For the exonucleases studied (Exonuclease III from E. Coli and phosphodiesterase I from Crotalus adamanteus), nanoparticle display retarded enzymatic processing by roughly a factor of five. For the endonuclease studied (Nt.CviPII), nanoparticle display prohibited virtually all enzyme activity on oligonucleotides within the nanoparticle shell. To test the ability of these materials to regulate mRNA levels in live cultured human cells, LPA (LNA-polymer amphiphile) NPs were designed to be perfectly complementary to a 20-base region of mRNA encoding the anti-apoptosis protein survivin. In this study two key observations were made. The first observation is that packaging LNA into spherical micellar nanoparticles serves to dramatically enhance cellular uptake of LNA based on flow cytometry and fluorescence microscopy data. The second observation is that LPA NPs are capable of regulating mRNA levels by what is hypothesized to be activation of target mRNA for catalytic RNase H-mediated degradation. These materials represent a unique class of DNA delivery system capable of rendering nucleic acids with natural backbone chemistry resistant to nuclease degradation and further serving to deliver DNA into cells to

  16. Dental resins based on dimer acid dimethacrylates: a route to high conversion with low polymerization shrinkage.

    PubMed

    Lu, Hui; Trujillo-Lemon, Marianela; Ge, Junhao; Stansbury, Jeffrey W

    2010-05-01

    Incomplete polymerization, volumetric shrinkage, and shrinkage stress are among the primary disadvantages of current resin-based dental composites. Generally, any attempt to increase final double bond conversion only exacerbates polymerization shrinkage and stress. The use of dimer acid-derived dimethacrylate (DADMA) monomers in novel dental resin formulations is examined in this article as a potential means to address these disparate goals. A series of high molecular weight DADMA monomers with different functional groups used to connect the C36 dimer acid core and the methacrylates were formulated with urethane dimethacrylate (UDMA) and/or ethoxylated bisphenol A dimethacrylate (Bis-EMA) at various compositions to manipulate comonomer compatibility and polymeric mechanical properties. Along with reaction kinetics, dynamic polymerization shrinkage and shrinkage stress were assessed. Specific DADMA monomers demonstrated limited miscibility with either Bis-EMA or UDMA. Appropriate ternary resin formulations produced homogeneous monomeric mixtures capable of controlled polymerization-induced phase separation (PIPS) to yield heterogeneous final polymers. Reduced polymerization shrinkage and stress along with higher conversion was observed for DADMA ternary systems compared with a bisphenol A glycidyl methacrylate (Bis-GMA)/triethylene glycol dimethacrylate (TEGDMA) resin control. The PIPS process resulted in a modest volume recovery and stress relaxation in the later stages of polymerization. These results indicate that certain dimer acid-derived dimethacrylates possess the potential to replace TEGDMA as a reactive diluent in dental resins that display a favorable and unique combination of properties. PMID:20521567

  17. Electron-beam induced RAFT-graft polymerization of poly(acrylic acid) onto PVDF

    NASA Astrophysics Data System (ADS)

    Grasselli, M.; Betz, N.

    2005-07-01

    This paper explores for the first time the post-radiation-induced-graft polymerization on solid substrate using reversible addition-fragmentation transfer (RAFT) mechanism. Radiation-induced graft polymerization onto polymers is a potentially interesting technique to create easily new materials from highly resistant polymers, e.g. surface graft polymerization of acrylic acid (AA) onto poly(vinylidene difluoride) (PVDF) improves its surface properties without losing its excellent mechanical properties. As a consequence of the radical nature of the polymerization processes it is difficult to control molecular weight of grafted chains, and therefore design and standardize the properties of the final product. RAFT polymerization is a suitable method to obtain monodisperse polymers. The ability of the RAFT agents to control the polymer chain length could be an interesting approach to improve the grafted polymers obtained by post-radiation-induced-graft polymerization technique. In this way, graft polymerization of AA onto electron-beam irradiated α-PVDF was performed using trithiocarbonic acid bis(1-phenylethyl) ester as a RAFT agent to control the radical polymerization. We studied several grafting parameters such as solvent, monomer concentration and grafting time in order to achieve a poly(acrylic acid) (PAA) layer onto PVDF surface. Acetic acid was found to be the best solvent for many reasons, as to drive graft polymerization mainly to the polymer surface, complete solubility and stability of all reactants. Hydrolysis of PAA chains was also studied in order to remove the trithiocarbonate functionality from the grafted polymer. A mild chemical condition was achieved in order to have thiol groups that were detected onto the modified PVDF by specific enzymatic reaction.

  18. Selectivity between lactic acid and glucose during recovery of lactic acid with basic extractants and polymeric sorbents

    SciTech Connect

    Dai, Y.; King, C.J.

    1996-04-01

    During recovery of product carboxylic acids from fermentation broths, it is important to maximize the selectivity for the desired acid, as opposed to substrate sugars. In this work uptakes of glucose and competitive uptakes of lactic acid and glucose have been measured for the extractant Alamine 336 in various diluents and three commercially available basic solid polymeric sorbents. The results show that swelling is the main factor governing the selectivity between lactic acid and glucose for the polymeric sorbents. Because of a high uptake capacity and relatively low swelling, Dowex MWA-1 gives a higher selectivity in the pH 5--6 range than do Amberlite IRA-35 and Reillex 425. Extraction with Alamine 336 provides a much higher selectivity, but a lower capacity, than the polymeric sorbents. The extent of water coextraction depends strongly on the diluent used, and larger amounts of water coextracted correspond to larger uptakes of glucose.

  19. Dissolved Divalent Metal and pH Effects on Amino Acid Polymerization: A Thermodynamic Evaluation

    NASA Astrophysics Data System (ADS)

    Kitadai, Norio

    2016-06-01

    Polymerization of amino acids is a fundamentally important step for the chemical evolution of life. Nevertheless, its response to changing environmental conditions has not yet been well understood because of the lack of reliable quantitative information. For thermodynamics, detailed prediction over diverse combinations of temperature and pH has been made only for a few amino acid-peptide systems. This study used recently reported thermodynamic dataset for the polymerization of the simplest amino acid "glycine (Gly)" to its short peptides (di-glycine and tri-glycine) to examine chemical and structural characteristics of amino acids and peptides that control the temperature and pH dependence of polymerization. Results showed that the dependency is strongly controlled by the intramolecular distance between the amino and carboxyl groups in an amino acid structure, although the side-chain group role is minor. The polymerization behavior of Gly reported earlier in the literature is therefore expected to be a typical feature for those of α-amino acids. Equilibrium calculations were conducted to examine effects of dissolved metals as a function of pH on the monomer-polymer equilibria of Gly. Results showed that metals shift the equilibria toward the monomer side, particularly at neutral and alkaline pH. Metals that form weak interaction with Gly (e.g., Mg2+) have no noticeable influence on the polymerization, although strong interaction engenders significant decrease of the equilibrium concentrations of Gly peptides. Considering chemical and structural characteristics of Gly and Gly peptides that control their interactions with metals, it can be expected that similar responses to the addition of metals are applicable in the polymerization of neutral α-amino acids. Neutral and alkaline aqueous environments with dissolved metals having high affinity with neutral α-amino acids (e.g., Cu2+) are therefore not beneficial places for peptide bond formation on the primitive

  20. Tailoring Enzyme-Like Activities of Gold Nanoclusters by Polymeric Tertiary Amines for Protecting Neurons Against Oxidative Stress.

    PubMed

    Liu, Ching-Ping; Wu, Te-Haw; Lin, Yu-Lung; Liu, Chia-Yeh; Wang, Sabrina; Lin, Shu-Yi

    2016-08-01

    The cytotoxicity of nanozymes has drawn much attention recently because their peroxidase-like activity can decompose hydrogen peroxide (H2 O2 ) to produce highly toxic hydroxyl radicals (•OH) under acidic conditions. Although catalytic activities of nanozymes are highly associated with their surface properties, little is known about the mechanism underlying the surface coating-mediated enzyme-like activities. Herein, it is reported for the first time that amine-terminated PAMAM dendrimer-entrapped gold nanoclusters (AuNCs-NH2 ) unexpectedly lose their peroxidase-like activity while still retaining their catalase-like activity in physiological conditions. Surprisingly, the methylated form of AuNCs-NH2 (i.e., MAuNCs-N(+) R3 , where R = H or CH3 ) results in a dramatic recovery of the intrinsic peroxidase-like activity while blocking most primary and tertiary amines (1°- and 3°-amines) of dendrimers to form quaternary ammonium ions (4°-amines). However, the hidden peroxidase-like activity is also found in hydroxyl-terminated dendrimer-encapsulated AuNCs (AuNCs-OH, inside backbone with 3°-amines), indicating that 3°-amines are dominant in mediating the peroxidase-like activity. The possible mechanism is further confirmed that the enrichment of polymeric 3°-amines on the surface of dendrimer-encapsulated AuNCs provides sufficient suppression of the critical mediator •OH for the peroxidase-like activity. Finally, it is demonstrated that AuNCs-NH2 with diminished cytotoxicity have great potential for use in primary neuronal protection against oxidative damage. PMID:27346719

  1. The Synthesis and Characterization of Substituted Polyaniline Hollow Spheres doped with a Polymeric Acid

    NASA Astrophysics Data System (ADS)

    Sui, Jing; Zhang, Lijuan; Travas-Sejdic, Jadranka; Kilmartin, Paul A.

    2009-07-01

    Self-assembled poly(o-toluidine) (POT) and poly(o-anisidine) (POA) hollow spheres were prepared by oxidative polymerization using ammonium persulfate as the oxidant in the presence of 5% by weight of a polymeric acid, poly(methyl vinyl ether-alt-maleic acid) (PMVEA). The substituent at the ortho position had a significant effect on the size of the hollow nanospheres as determined by SEM and TEM. The nanospheres were of a very uniform size in the presence of the polymeric acid, with average diameters of 338±25 nm for POT and 210±20 nm for POA. The POT and POA hollow spheres were also characterized by FTIR and UV-Vis spectroscopy, which confirmed the chemical identity of the substituted polyanilines.

  2. Self-assembled polyaniline nanotubes grown from a polymeric acid solution

    NASA Astrophysics Data System (ADS)

    Zhang, Lijuan; Peng, Hui; Hsu, Chyong Fang; Kilmartin, Paul A.; Travas-Sejdic, Jadranka

    2007-03-01

    Polyaniline (PANI) nanotubes were obtained by a self-assembly process, from a solution containing poly(methyl vinyl ether-alt-maleic acid) (PMVEA) by oxidative polymerization using ammonium persulfate as the oxidant. The size of the nanotubes was greatly affected by the weight ratio of polymeric acid to aniline as measured by SEM images. The outer diameter of the nanotubes increased from 65 to 160 nm as the weight ratio of PMVEA to aniline increased from 1 to 4 wt/wt%. The structural features of the nanotubes were characterized by FTIR, XPS and EPR spectroscopies, which confirmed the presence of the polymeric acid in the PANI nanotubes and their electronic conductive nature. The electrochemical properties were further analysed using cyclic voltammetry.

  3. Stereo- and Temporally Controlled Coordination Polymerization Triggered by Alternating Addition of a Lewis Acid and Base.

    PubMed

    Liu, Bo; Cui, Dongmei; Tang, Tao

    2016-09-19

    Significant progress has been made with regard to temporally controlled radical and ring-opening polymerizations, for example, by means of chemical reagents, light, and voltage, whereas quantitative switch coordination polymerization is still challenging. Herein, we report the temporally and stereocontrolled 3,4-polymerization of isoprene through allosterically regulating the active metal center by alternating addition of Lewis basic pyridine to "poison" the Lewis acidic active metal species through acid-base interactions and Lewis acidic Al(i) Bu3 to release the original active species through pyridine abstraction. This process is quick, quantitative, and can be repeated multiple times while maintaining high 3,4-selectivity. Moreover, this strategy is also effective for the switch copolymerization of isoprene and styrene with dual 3,4- and syndiotactic selectivity. Tuning the switch cycles and intervals enables the isolation of various copolymers with different distributions of 3,4-polyisoprene and syndiotactic polystyrene sequences. PMID:27539866

  4. Polymerization of MIP-1 chemokine (CCL3 and CCL4) and clearance of MIP-1 by insulin-degrading enzyme

    SciTech Connect

    Ren, Min; Guo, Qing; Guo, Liang; Lenz, Martin; Qian, Feng; Koenen, Rory R.; Xu, Hua; Schilling, Alexander B.; Weber, Christian; Ye, Richard D.; Dinner, Aaron R.; Tang, Wei-Jen

    2010-12-07

    Macrophage inflammatory protein-1 (MIP-1), MIP-1{alpha} (CCL3) and MIP-1{beta} (CCL4) are chemokines crucial for immune responses towards infection and inflammation. Both MIP-1{alpha} and MIP-1{beta} form high-molecular-weight aggregates. Our crystal structures reveal that MIP-1 aggregation is a polymerization process and human MIP-1{alpha} and MIP-1{beta} form rod-shaped, double-helical polymers. Biophysical analyses and mathematical modelling show that MIP-1 reversibly forms a polydisperse distribution of rod-shaped polymers in solution. Polymerization buries receptor-binding sites of MIP-1{alpha}, thus depolymerization mutations enhance MIP-1{alpha} to arrest monocytes onto activated human endothelium. However, same depolymerization mutations render MIP-1{alpha} ineffective in mouse peritoneal cell recruitment. Mathematical modelling reveals that, for a long-range chemotaxis of MIP-1, polymerization could protect MIP-1 from proteases that selectively degrade monomeric MIP-1. Insulin-degrading enzyme (IDE) is identified as such a protease and decreased expression of IDE leads to elevated MIP-1 levels in microglial cells. Our structural and proteomic studies offer a molecular basis for selective degradation of MIP-1. The regulated MIP-1 polymerization and selective inactivation of MIP-1 monomers by IDE could aid in controlling the MIP-1 chemotactic gradient for immune surveillance.

  5. Self-Assembled DNA Hydrogel Based on Enzymatically Polymerized DNA for Protein Encapsulation and Enzyme/DNAzyme Hybrid Cascade Reaction.

    PubMed

    Xiang, Binbin; He, Kaiyu; Zhu, Rong; Liu, Zhuoliang; Zeng, Shu; Huang, Yan; Nie, Zhou; Yao, Shouzhuo

    2016-09-01

    DNA hydrogel is a promising biomaterial for biological and medical applications due to its native biocompatibility and biodegradability. Herein, we provide a novel, versatile, and cost-effective approach for self-assembly of DNA hydrogel using the enzymatically polymerized DNA building blocks. The X-shaped DNA motif was elongated by terminal deoxynucleotidyl transferase (TdT) to form the building blocks, and hybridization between dual building blocks via their complementary TdT-polymerized DNA tails led to gel formation. TdT polymerization dramatically reduced the required amount of original DNA motifs, and the hybridization-mediated cross-linking of building blocks endows the gel with high mechanical strength. The DNA hydrogel can be applied for encapsulation and controllable release of protein cargos (for instance, green fluorescent protein) due to its enzymatic responsive properties. Moreover, this versatile strategy was extended to construct a functional DNAzyme hydrogel by integrating the peroxidase-mimicking DNAzyme into DNA motifs. Furthermore, a hybrid cascade enzymatic reaction system was constructed by coencapsulating glucose oxidase and β-galactosidase into DNAzyme hydrogel. This efficient cascade reaction provides not only a potential method for glucose/lactose detection by naked eye but also a promising modular platform for constructing a multiple enzyme or enzyme/DNAzyme hybrid system. PMID:27526861

  6. The polymerization of amino acid adenylates on sodium-montmorillonite with preadsorbed polypeptides

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, Mella; Eirich, Frederick R.

    1988-01-01

    The spontaneous polymerization of amino acid adenylates on Na-montmorillonite in dilute, neutral suspension, after polypeptides were adsorbed on the clay, is studied. It is found that the degrees of polymerization of the oligopeptides and polypeptides obtained is dependent on the amounts of polypeptides that were preadsorbed. It is concluded that a catalytic activity may derive from c-spacings that offer adsorption sites for the reagent amino acid adenylate within the peripheral recesses of irregularly stacked clay platelets by bringing the anhydride bonds and neutral amino groups into favorable reaction distances.

  7. Polymerization of beta-amino acids in aqueous solution

    NASA Technical Reports Server (NTRS)

    Liu, R.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    We have compared carbonyl diimidazole (CDI) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDAC) as activating agents for the oligomerization of negatively-charged alpha- and beta-amino acids in homogeneous aqueous solution. alpha-Amino acids can be oligomerized efficiently using CDI, but not by EDAC. beta-Amino acids can be oligomerized efficiently using EDAC, but not by CDI. Aspartic acid, an alpha- and beta-dicarboxylic acid is oligomerized efficiently by both reagents. These results are explained in terms of the mechanisms of the reactions, and their relevance to prebiotic chemistry is discussed.

  8. Induction of HoxB Transcription by Retinoic Acid Requires Actin Polymerization

    PubMed Central

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M.; Crippa, Massimo P.; Scita, Giorgio

    2009-01-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of “elongating” RNAPII, Prep1, β-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

  9. Induction of HoxB transcription by retinoic acid requires actin polymerization.

    PubMed

    Ferrai, Carmelo; Naum-Onganía, Gabriela; Longobardi, Elena; Palazzolo, Martina; Disanza, Andrea; Diaz, Victor M; Crippa, Massimo P; Scita, Giorgio; Blasi, Francesco

    2009-08-01

    We have analyzed the role of actin polymerization in retinoic acid (RA)-induced HoxB transcription, which is mediated by the HoxB regulator Prep1. RA induction of the HoxB genes can be prevented by the inhibition of actin polymerization. Importantly, inhibition of actin polymerization specifically affects the transcription of inducible Hox genes, but not that of their transcriptional regulators, the RARs, nor of constitutively expressed, nor of actively transcribed Hox genes. RA treatment induces the recruitment to the HoxB2 gene enhancer of a complex composed of "elongating" RNAPII, Prep1, beta-actin, and N-WASP as well as the accessory splicing components p54Nrb and PSF. We show that inhibition of actin polymerization prevents such recruitment. We conclude that inducible Hox genes are selectively sensitive to the inhibition of actin polymerization and that actin polymerization is required for the assembly of a transcription complex on the regulatory region of the Hox genes. PMID:19477923

  10. A Green Polymerization of Aspartic Acid for the Undergraduate Organic Laboratory

    ERIC Educational Resources Information Center

    Bennett, George D.

    2005-01-01

    The green polymerization of aspartic acid carried out during an organic-inorganic synthesis laboratory course for undergraduate students is described. The procedure is based on work by Donlar Corporation, a Peru, Illinois-based company that won a Green Chemistry Challenge Award in 1996 in the Small Business category for preparing thermal…

  11. Polymerization of euphorbia oil with Lewis acid in carbon dioxide media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Boron trifluoride diethyl etherate (BF3-OEt2) Lewis acid catalyzed ring-opening polymerization of euphorbia oil (EO), a natural epoxy oil, in liquid carbon dioxide was conducted in an effort to develop useful vegetable oil based polymers. The resulting polymers (RPEO) were characterized by FTIR, 1H-...

  12. Organo-Lewis acid as cocatalyst for cationic homogeneous Ziegler-Natta olefin polymerizations

    DOEpatents

    Marks, Tobin J.; Chen, You-Xian

    2001-01-01

    Organo-Lewis acids of the formula BR'R".sub.2 wherein B is boron, R' is fluorinated biphenyl, and R" is a fluorinated phenyl, fluorinated biphenyl, or fluorinated polycyclic fused ring group, and cationic metallocene complexes formed therewith. Such complexes are useful as polymerization catalysts.

  13. Organo-Lewis acid as cocatalyst for cationic homogeneous Ziegler-Natta olefin polymerizations

    DOEpatents

    Marks, Tobin J.; Chen, You-Xian

    2002-01-01

    Organo-Lewis acids of the formula BR'R".sub.2 wherein B is boron, R' is fluorinated biphenyl, and R" is a fluorinated phenyl, fluorinated biphenyl, or fluorinated polycyclic fused ring group, and cationic metallocene complexes formed therewith. Such complexes are useful as polymerization catalysts.

  14. 40 CFR 721.9485 - Dimer acid/polymerized rosin amidoamine reaction product (generic).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... reaction product (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as Dimer acid/polymerized rosin amidoamine reaction product (PMN... amidoamine reaction product (generic). 721.9485 Section 721.9485 Protection of Environment...

  15. 40 CFR 721.9485 - Dimer acid/polymerized rosin amidoamine reaction product (generic).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... reaction product (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as Dimer acid/polymerized rosin amidoamine reaction product (PMN... amidoamine reaction product (generic). 721.9485 Section 721.9485 Protection of Environment...

  16. 40 CFR 721.9485 - Dimer acid/polymerized rosin amidoamine reaction product (generic).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... reaction product (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as Dimer acid/polymerized rosin amidoamine reaction product (PMN... amidoamine reaction product (generic). 721.9485 Section 721.9485 Protection of Environment...

  17. 40 CFR 721.9485 - Dimer acid/polymerized rosin amidoamine reaction product (generic).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... reaction product (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as Dimer acid/polymerized rosin amidoamine reaction product (PMN... amidoamine reaction product (generic). 721.9485 Section 721.9485 Protection of Environment...

  18. 40 CFR 721.9485 - Dimer acid/polymerized rosin amidoamine reaction product (generic).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... reaction product (generic). (a) Chemical substance and significant new uses subject to reporting. (1) The chemical substance identified generically as Dimer acid/polymerized rosin amidoamine reaction product (PMN... amidoamine reaction product (generic). 721.9485 Section 721.9485 Protection of Environment...

  19. Guanine- Formation During the Thermal Polymerization of Amino Acids

    NASA Technical Reports Server (NTRS)

    Mc Caw, B. K.; Munoz, E. F.; Ponnamperuma, C.; Young, R. S.

    1964-01-01

    The action of heat on a mixture of amino acids was studied as a possible abiological pathway for the synthesis of purines and pyrimidines. Guanine was detected. This result is significant in the context of chemical evolution.

  20. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities.

    PubMed

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying; Naleway, John Joseph

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  1. Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

    PubMed Central

    Harlan, Fiona Karen; Lusk, Jason Scott; Mohr, Breanna Michelle; Guzikowski, Anthony Peter; Batchelor, Robert Hardy; Jiang, Ying

    2016-01-01

    Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson’s Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and

  2. Ammonium Metabolism Enzymes Aid Helicobacter pylori Acid Resistance

    PubMed Central

    Miller, Erica F.

    2014-01-01

    The gastric pathogen Helicobacter pylori possesses a highly active urease to support acid tolerance. Urea hydrolysis occurs inside the cytoplasm, resulting in the production of NH3 that is immediately protonated to form NH4+. This ammonium must be metabolized or effluxed because its presence within the cell is counterproductive to the goal of raising pH while maintaining a viable proton motive force (PMF). Two compatible hypotheses for mitigating intracellular ammonium toxicity include (i) the exit of protonated ammonium outward via the UreI permease, which was shown to facilitate diffusion of both urea and ammonium, and/or (ii) the assimilation of this ammonium, which is supported by evidence that H. pylori assimilates urea nitrogen into its amino acid pools. We investigated the second hypothesis by constructing strains with altered expression of the ammonium-assimilating enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) and the ammonium-evolving periplasmic enzymes glutaminase (Ggt) and asparaginase (AsnB). H. pylori strains expressing elevated levels of either GS or GDH are more acid tolerant than the wild type, exhibit enhanced ammonium production, and are able to alkalize the medium faster than the wild type. Strains lacking the genes for either Ggt or AsnB are acid sensitive, have 8-fold-lower urea-dependent ammonium production, and are more acid sensitive than the parent. Additionally, we found that purified H. pylori GS produces glutamine in the presence of Mg2+ at a rate similar to that of unadenylated Escherichia coli GS. These data reveal that all four enzymes contribute to whole-cell acid resistance in H. pylori and are likely important for assimilation and/or efflux of urea-derived ammonium. PMID:24936052

  3. Bifunctional Organic Polymeric Catalysts with a Tunable Acid-Base Distance and Framework Flexibility

    NASA Astrophysics Data System (ADS)

    Chen, Huanhui; Wang, Yanan; Wang, Qunlong; Li, Junhui; Yang, Shiqi; Zhu, Zhirong

    2014-09-01

    Acid-base bifunctional organic polymeric catalysts were synthesized with tunable structures. we demonstrated two synthesis approaches for structural fine-tune. In the first case, the framework flexibility was tuned by changing the ratio of rigid blocks to flexible blocks within the polymer framework. In the second case, we precisely adjusted the acid-base distance by distributing basic monomers to be adjacent to acidic monomers, and by changing the chain length of acidic monomers. In a standard test reaction for the aldol condensation of 4-nitrobenzaldehyde with acetone, the catalysts showed good reusability upon recycling and maintained relatively high conversion percentage.

  4. Sterically controlled azomethine ylide cycloaddition polymerization of phenyl-C61-butyric acid methyl ester.

    PubMed

    Stephen, Meera; Ramanitra, Hasina H; Santos Silva, Hugo; Dowland, Simon; Bégué, Didier; Genevičius, Kristijonas; Arlauskas, Kęstutis; Juška, Gytis; Morse, Graham E; Distler, Andreas; Hiorns, Roger C

    2016-05-01

    Phenyl-C61-butyric acid methyl ester (PCBM) is polymerized simply using a one-pot reaction to yield soluble, high molecular weight polymers. The sterically controlled azomethine ylide cycloaddition polymerization (SACAP) is demonstrated to be highly adaptable and yields polymers with probable Mn≈ 24 600 g mol(-1) and Mw≈ 73 800 g mol(-1). Products are metal-free and of possible benefit to organic and hybrid photovoltaics and electronics as they form thin films from solution and have raised LUMOs. The promising electronic properties of this new polymer are discussed. PMID:27066898

  5. Polymerization of amino acids under primitive earth conditions.

    NASA Technical Reports Server (NTRS)

    Flores, J. J.; Ponnamperuma, C.

    1972-01-01

    Small amounts of peptides were obtained when equal amounts of methane and ammonia were reacted with vaporized aqueous solutions of C14-labeled glycine, L-alanine, L-aspartic acid, L-glutamic acid and L-threonine in the presence of a continuous spark discharge in a 24-hr cyclic process. The experiment was designed to demonstrate the possibility of peptide synthesis under simulated primeval earth conditions. It is theorized that some dehydration-condensation processes may have taken place, with ammonium cyanide, the hydrogencyanide tetramer or aminonitriles as intermediate products, during the early chemical evolution of the earth.

  6. Graft Polymerization of Acrylic Acid on a Polytetrafluoroethylene Panel by an Inductively Coupled Plasma

    NASA Astrophysics Data System (ADS)

    Lan, Yan; You, Qingliang; Cheng, Cheng; Zhang, Suzhen; Ni, Guohua; Nagatsu, M.; Meng, Yuedong

    2011-02-01

    Surface modification on a polytetrafluoroethylene (PTFE) panel was performed with sequential nitrogen plasma treatments and surface-initiated polymerization. By introducing COO- groups to the surface of the PTFE panel through grafting polymerization of acrylic acid (AA), a transparent poly (acrylic acid) (PAA) membrane was achieved from acrylic acid solution. Grafting polymerization initiating from the active groups was achieved on the PTFE panel surface after the nitrogen plasma treatment. Utilizing the acrylic acid as monomers, with COO- groups as cross link sites to form reticulation structure, a transparent poly (acrylic acid) membrane with arborescent macromolecular structure was formed on the PTFE panel surface. Analysis methods, such as fourier transform infrared spectroscopy (FTIR), microscopy and X-ray photoelectron spectroscopy (XPS), were utilized to characterize the structures of the macromolecule membrane on the PTFE panel surface. A contact angle measurement was performed to characterize the modified PTFE panels. The surface hydrophilicities of modified PTFE panels were significantly enhanced after the plasma treatment. It was shown that the grafting rate is related to the treating time and the power of plasma.

  7. Modeling three-dimensional network formation with an atomic lattice model: Application to silicic acid polymerization

    NASA Astrophysics Data System (ADS)

    Jin, Lin; Auerbach, Scott M.; Monson, Peter A.

    2011-04-01

    We present an atomic lattice model for studying the polymerization of silicic acid in sol-gel and related processes for synthesizing silica materials. Our model is based on Si and O atoms occupying the sites of a body-centered-cubic lattice, with all atoms arranged in SiO4 tetrahedra. This is the simplest model that allows for variation in the Si-O-Si angle, which is largely responsible for the versatility in silica polymorphs. The model describes the assembly of polymerized silica structures starting from a solution of silicic acid in water at a given concentration and pH. This model can simulate related materials—chalcogenides and clays—by assigning energy penalties to particular ring geometries in the polymerized structures. The simplicity of this approach makes it possible to study the polymerization process to higher degrees of polymerization and larger system sizes than has been possible with previous atomistic models. We have performed Monte Carlo simulations of the model at two concentrations: a low density state similar to that used in the clear solution synthesis of silicalite-1, and a high density state relevant to experiments on silica gel synthesis. For the high concentration system where there are NMR data on the temporal evolution of the Qn distribution, we find that the model gives good agreement with the experimental data. The model captures the basic mechanism of silica polymerization and provides quantitative structural predictions on ring-size distributions in good agreement with x-ray and neutron diffraction data.

  8. Prebiotic thermal polymerization of crystals of amino acids via the diketopiperazine reaction.

    PubMed

    Mosqueira, F G; Ramos-Bernal, S; Negrón-Mendoza, A

    2008-01-01

    In this work, we continue our studies on the thermal prebiotic oligomerization of amino acids. The next step is to consider all four types of electromagnetic interactions that our model may admit. In addition, only the polymerization of amino acids via the formation of diketopiperazine, which arises from the cyclodehydration of two amino acids, will be considered. By assuming that only one residue group of two will predominate in the diketopiperazine molecule, it is possible to reduce the three-body problem to a simpler situation with the two objects that we have already solved. PMID:17980955

  9. Role of antioxidant enzymes in bacterial resistance to organic acids.

    PubMed

    Bruno-Bárcena, Jose M; Azcárate-Peril, M Andrea; Hassan, Hosni M

    2010-05-01

    Growth in aerobic environments has been shown to generate reactive oxygen species (ROS) and to cause oxidative stress in most organisms. Antioxidant enzymes (i.e., superoxide dismutases and hydroperoxidases) and DNA repair mechanisms provide protection against ROS. Acid stress has been shown to be associated with the induction of Mn superoxide dismutase (MnSOD) in Lactococcus lactis and Staphylococcus aureus. However, the relationship between acid stress and oxidative stress is not well understood. In the present study, we showed that mutations in the gene coding for MnSOD (sodA) increased the toxicity of lactic acid at pH 3.5 in Streptococcus thermophilus. The inclusion of the iron chelators 2,2'-dipyridyl (DIP), diethienetriamine-pentaacetic acid (DTPA), and O-phenanthroline (O-Phe) provided partial protection against 330 mM lactic acid at pH 3.5. The results suggested that acid stress triggers an iron-mediated oxidative stress that can be ameliorated by MnSOD and iron chelators. These findings were further validated in Escherichia coli strains lacking both MnSOD and iron SOD (FeSOD) but expressing a heterologous MnSOD from S. thermophilus. We also found that, in E. coli, FeSOD did not provide the same protection afforded by MnSOD and that hydroperoxidases are equally important in protecting the cells against acid stress. These findings may explain the ability of some microorganisms to survive better in acidified environments, as in acid foods, during fermentation and accumulation of lactic acid or during passage through the low pH of the stomach. PMID:20305033

  10. Preparation of poly(acrylic acid) particles by dispersion polymerization in an ionic liquid.

    PubMed

    Minami, Hideto; Kimura, Akira; Kinoshita, Keigo; Okubo, Masayoshi

    2010-05-01

    Poly(acrylic acid) (PAA) particles were successfully prepared by dispersion polymerization of acrylic acid in ionic liquid, N,N-diethyl-N-methyl-N-(2-methoxyethyl)ammonium bis(trifluoro-methanesulfonyl)amide ([DEME][TFSA]) at 70 degrees C with low hydrolysis grade (35.4%) poly(vinyl alcohol) as stabilizer. Interestingly, the PAA particles were easily extracted as particle state with water. Thus, the PAA particles had a cross-linked structure during the polymerization without cross-linker. Moreover, it was also noted that the cross-linking density of the PAA particles could be controlled by thermal treatment at various temperatures in [DEME][TFSA] utilizing the advantages of nonvolatility and high thermal stability of the ionic liquid. PMID:20043688

  11. Dual Enzyme-Responsive Capsules of Hyaluronic Acid-block-Poly(Lactic Acid) for Sensing Bacterial Enzymes.

    PubMed

    Tücking, Katrin-Stephanie; Grützner, Verena; Unger, Ronald E; Schönherr, Holger

    2015-07-01

    The synthesis of novel amphiphilic hyaluronic acid (HYA) and poly(lactic acid) (PLA) block copolymers is reported as the key element of a strategy to detect the presence of pathogenic bacterial enzymes. In addition to the formation of defined HYA-block-PLA assemblies, the encapsulation of fluorescent reporter dyes and the selective enzymatic degradation of the capsules by hyaluronidase and proteinase K are studied. The synthesis of the dual enzyme-responsive HYA-b-PLA is carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition. The resulting copolymers are assembled in water to form vesicular structures, which are characterized by scanning electron microscopy, transmission electron microscopy, dynamic light scattering (DLS), and fluorescence lifetime imaging microscopy (FLIM). DLS measurements show that both enzymes cause a rapid decrease in the hydrodynamic diameter of the nanocapsules. Fluorescence spectroscopy data confirm the liberation of encapsulated dye, which indicates the disintegration of the capsules and validates the concept of enzymatically triggered payload release. Finally, cytotoxicity assays confirm that the HYA-b-PLA nanocapsules are biocompatible with primary human dermal microvascular endothelial cells. PMID:25940300

  12. Precision synthesis of bio-based acrylic thermoplastic elastomer by RAFT polymerization of itaconic acid derivatives.

    PubMed

    Satoh, Kotaro; Lee, Dong-Hyung; Nagai, Kanji; Kamigaito, Masami

    2014-01-01

    Bio-based polymer materials from renewable resources have recently become a growing research focus. Herein, a novel thermoplastic elastomer is developed via controlled/living radical polymerization of plant-derived itaconic acid derivatives, which are some of the most abundant renewable acrylic monomers obtained via the fermentation of starch. The reversible addition-fragmentation chain-transfer (RAFT) polymerizations of itaconic acid imides, such as N-phenylitaconimide and N-(p-tolyl)itaconimide, and itaconic acid esters, such as di-n-butyl itaconate and bis(2-ethylhexyl) itaconate, are examined using a series of RAFT agents to afford well-defined polymers. The number-average molecular weights of these polymers increase with the monomer conversion while retaining relatively narrow molecular weight distributions. Based on the successful controlled/living polymerization, sequential block copolymerization is subsequently investigated using mono- and di-functional RAFT agents to produce block copolymers with soft poly(itaconate) and hard poly(itaconimide) segments. The properties of the obtained triblock copolymer are evaluated as bio-based acrylic thermoplastic elastomers. PMID:24243816

  13. Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays

    NASA Technical Reports Server (NTRS)

    Reinecke, D. M.; Bandurski, R. S.

    1988-01-01

    Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

  14. Preparation of ellagic acid molecularly imprinted polymeric microspheres based on distillation-precipitation polymerization for the efficient purification of a crude extract.

    PubMed

    Zhang, Hua; Zhao, Shangge; Zhang, Lu; Han, Bo; Yao, Xincheng; Chen, Wen; Hu, Yanli

    2016-08-01

    Molecularly imprinted polymeric microspheres with a high recognition ability toward the template molecule, ellagic acid, were synthesized based on distillation-precipitation polymerization. The as-obtained polymers were characterized by scanning electron microscopy, infrared spectroscopy, and thermogravimetric analysis. Static, dynamic, and selective binding tests were adopted to study the binding properties and the molecular recognition ability of the prepared polymers for ellagic acid. The results indicated that the maximum static adsorption capacity of the prepared polymers toward ellagic acid was 37.07 mg/g and the adsorption equilibrium time was about 100 min when the concentration of ellagic acid was 40 mg/mL. Molecularly imprinted polymeric microspheres were also highly selective toward ellagic acid compared with its analogue quercetin. It was found that the content of ellagic acid in the pomegranate peel extract was enhanced from 23 to 86% after such molecularly imprinted solid-phase extraction process. This work provides an efficient way for effective separation and enrichment of ellagic acid from complex matrix, which is especially valuable in industrial production. PMID:27311588

  15. Plasma polymerization of acrylic acid onto polystyrene by cyclonic plasma at atmospheric pressure

    NASA Astrophysics Data System (ADS)

    Chang, Yi-Jan; Lin, Chin-Ho; Huang, Chun

    2016-01-01

    The cyclonic atmospheric-pressure plasma is developed for chamberless deposition of poly(acrylic acid) film from argon/acrylic acid mixtures. The photoemission plasma species in atmospheric-pressure plasma polymerization was identified by optical emission spectroscopy (OES). The OES diagnosis data and deposition results indicated that in glow discharge, the CH and C2 species resulted from low-energy electron-impact dissociation that creates deposition species, but the strong CO emission lines are related to nondeposition species. The acrylic acid flow rate is seen as the key factor affecting the film growth. The film surface analysis results indicate that a smooth, continuous, and uniform surface of poly(acrylic acid) films can be formed at a relatively low plasma power input. This study reveals the potential of chamberless film growth at atmospheric pressure for large-area deposition of poly(acrylic acid) films.

  16. 75 FR 1773 - Notice of Receipt of a Pesticide Petition Filed for Residues of Polymeric Polyhydroxy Acid in or...

    Federal Register 2010, 2011, 2012, 2013, 2014

    2010-01-13

    ...This notice announces the Agency's receipt of an initial filing of a pesticide petition proposing the establishment of a regulation for residues of the plant growth regulator, polymeric polyhydroxy acid, in or on all food...

  17. Indoleacetic Acid and the Synthesis of Glucanases and Pectic Enzymes

    PubMed Central

    Datko, Anne Harmon; Maclachlan, G. A.

    1968-01-01

    Indoleacetic acid (IAA) and/or inhibitors of DNA, RNA or protein synthesis were added to the apex of decapitated seedlings of Pisum sativum L. var. Alaska. At various times up to 4 days, enzymic protein was extracted from a segment of epicotyl immediately below the apex and assayed for its ability to hydrolyse polysaccharides or their derivatives. With the exception of amylase, the total amounts per segment of all of the tested enzymes increased due to IAA treatment. The development of β-1,4-glucanase (cellulase) activity per unit of protein or fresh weight proceeded according to a typical sigmoid induction curve. Pectinase was formed for about 2 days in control segments and IAA treatment resulted in continued synthesis for at least another 2 days provided cell division took place. β-1,3-glucanase and pectinesterase activities were only enhanced by IAA to the extent that total protein levels increased. Reaction mechanisms for these effects and functions for the enzymes during growth are discussed. PMID:16656834

  18. Lewis pair polymerization by classical and frustrated Lewis pairs: acid, base and monomer scope and polymerization mechanism.

    PubMed

    Zhang, Yuetao; Miyake, Garret M; John, Mallory G; Falivene, Laura; Caporaso, Lucia; Cavallo, Luigi; Chen, Eugene Y-X

    2012-08-14

    Classical and frustrated Lewis pairs (LPs) of the strong Lewis acid (LA) Al(C(6)F(5))(3) with several Lewis base (LB) classes have been found to exhibit exceptional activity in the Lewis pair polymerization (LPP) of conjugated polar alkenes such as methyl methacrylate (MMA) as well as renewable α-methylene-γ-butyrolactone (MBL) and γ-methyl-α-methylene-γ-butyrolactone (γ-MMBL), leading to high molecular weight polymers, often with narrow molecular weight distributions. This study has investigated a large number of LPs, consisting of 11 LAs as well as 10 achiral and 4 chiral LBs, for LPP of 12 monomers of several different types. Although some more common LAs can also be utilized for LPP, Al(C(6)F(5))(3)-based LPs are far more active and effective than other LA-based LPs. On the other hand, several classes of LBs, when paired with Al(C(6)F(5))(3), can render highly active and effective LPP of MMA and γ-MMBL; such LBs include phosphines (e.g., P(t)Bu(3)), chiral chelating diphosphines, N-heterocyclic carbenes (NHCs), and phosphazene superbases (e.g., P(4)-(t)Bu). The P(4)-(t)Bu/Al(C(6)F(5))(3) pair exhibits the highest activity of the LP series, with a remarkably high turn-over frequency of 9.6 × 10(4) h(-1) (0.125 mol% catalyst, 100% MMA conversion in 30 s, M(n) = 2.12 × 10(5) g mol(-1), PDI = 1.34). The polymers produced by LPs at RT are typically atactic (P(γ)MMBL with ∼47% mr) or syndio-rich (PMMA with ∼70-75% rr), but highly syndiotactic PMMA with rr ∼91% can be produced by chiral or achiral LPs at -78 °C. Mechanistic studies have identified and structurally characterized zwitterionic phosphonium and imidazolium enolaluminates as the active species of the current LPP system, which are formed by the reaction of the monomer·Al(C(6)F(5))(3) adduct with P(t)Bu(3) and NHC bases, respectively. Kinetic studies have revealed that the MMA polymerization by the (t)Bu(3)P/Al(C(6)F(5))(3) pair is zero-order in monomer concentration after an initial

  19. Boronic Acid functionalized core-shell polymer nanoparticles prepared by distillation precipitation polymerization for glycopeptide enrichment.

    PubMed

    Qu, Yanyan; Liu, Jianxi; Yang, Kaiguang; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2012-07-16

    The boronic acid-functionalized core-shell polymer nanoparticles, poly(N,N-methylenebisacrylamide-co-methacrylic acid)@4-vinylphenylboronic acid (poly(MBA-co-MAA)@VPBA), were successfully synthesized for enriching glycosylated peptides. Such nanoparticles were composed of a hydrophilic polymer core prepared by distillation precipitation polymerization (DPP) and a boronic acid-functionalized shell designed for capturing glycopeptides. Owing to the relatively large amount of residual vinyl groups introduced by DPP on the core surface, the VPBA monomer was coated with high efficiency, working as the shell. Moreover, the overall polymerization route, especially the use of DPP, made the synthesis of nanoparticles facile and time-saving. With the poly(MBA-co-MAA)@VPBA nanoparticles, 18 glycopeptides from horseradish peroxidase (HRP) digest were captured and identified by MALDI-TOF mass spectrometric analysis, relative to eight glycopeptides enriched by using commercially available meta-aminophenylboronic acid agarose under the same conditions. When the concentration of the HRP digest was decreased to as low as 5 nmol, glycopeptides could still be selectively isolated by the prepared nanoparticles. Our results demonstrated that the synthetic poly(MBA-co-MAA)@VPBA nanoparticles might be a promising selective enrichment material for glycoproteome analysis. PMID:22707097

  20. Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa

    PubMed Central

    Huai, Dongxin; Zhang, Yuanyuan; Zhang, Chunyu; Cahoon, Edgar B.; Zhou, Yongming

    2015-01-01

    Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production. PMID:26121034

  1. Combinatorial Effects of Fatty Acid Elongase Enzymes on Nervonic Acid Production in Camelina sativa.

    PubMed

    Huai, Dongxin; Zhang, Yuanyuan; Zhang, Chunyu; Cahoon, Edgar B; Zhou, Yongming

    2015-01-01

    Very long chain fatty acids (VLCFAs) with chain lengths of 20 carbons and longer provide feedstocks for various applications; therefore, improvement of VLCFA contents in seeds has become an important goal for oilseed enhancement. VLCFA biosynthesis is controlled by a multi-enzyme protein complex referred to as fatty acid elongase, which is composed of β-ketoacyl-CoA synthase (KCS), β-ketoacyl-CoA reductase (KCR), β-hydroxyacyl-CoA dehydratase (HCD) and enoyl reductase (ECR). KCS has been identified as the rate-limiting enzyme, but little is known about the involvement of other three enzymes in VLCFA production. Here, the combinatorial effects of fatty acid elongase enzymes on VLCFA production were assessed by evaluating the changes in nervonic acid content. A KCS gene from Lunaria annua (LaKCS) and the other three elongase genes from Arabidopsis thaliana were used for the assessment. Five seed-specific expressing constructs, including LaKCS alone, LaKCS with AtKCR, LaKCS with AtHCD, LaKCS with AtECR, and LaKCS with AtKCR and AtHCD, were transformed into Camelina sativa. The nervonic acid content in seed oil increased from null in wild type camelina to 6-12% in LaKCS-expressing lines. However, compared with that from the LaKCS-expressing lines, nervonic acid content in mature seeds from the co-expressing lines with one or two extra elongase genes did not show further increases. Nervonic acid content from LaKCS, AtKCR and AtHCD co-expressing line was significantly higher than that in LaKCS-expressing line during early seed development stage, while the ultimate nervonic acid content was not significantly altered. The results from this study thus provide useful information for future engineering of oilseed crops for higher VLCFA production. PMID:26121034

  2. Modeling suberization with peroxidase-catalyzed polymerization of hydroxycinnamic acids: cross-coupling and dimerization reactions.

    PubMed

    Arrieta-Baez, Daniel; Stark, Ruth E

    2006-04-01

    An anionic potato peroxidase (EC 1.11.1.7, APP) thought to be involved in suberization after wounding was isolated from slices of Solanum tuberosum in order to elucidate the first steps of dehydrogenative polymerization between pairs of different hydroxycinnamic acids (FA, CafA, CA and SA) present in wound-healing plant tissues. Use of a commercial horseradish peroxidase (HRP)-H2O2 catalytic system gave the identical major products in these coupling reactions, providing sufficient quantities for purification and structural elucidation. Using an equimolar mixture of pairs of hydroxycinnamic acid suberin precursors, only caffeic acid is coupled to ferulic acid and sinapic acid in separate cross-coupling reactions. For the other systems, HRP and APP reacted as follows: (1) preferentially with ferulic acid in a reaction mixture that contained p-coumaric and ferulic acids; (2) with sinapic acid in a mixture of p-coumaric and sinapic acids; (3) with sinapic acid in a mixture of ferulic and sinapic acids; (4) with caffeic acid in a reaction mixture of p-coumaric and caffeic acids. The resulting products, isolated and identified by NMR and MS analysis, had predominantly beta-beta-gamma-lactone and beta-5 benzofuran molecular frameworks. Five cross-coupling products are described for the first time, whereas the beta-O-4 dehydrodimers identified from the caffeic acid and sinapic acid cross-coupling reaction are known materials that are highly abundant in plants. These reactivity trends lead to testable hypotheses regarding the molecular architecture of intractable suberin protective plant materials, complementing prior analysis of monomeric constituents by GC-MS and polymer functional group identification from solid-state NMR, respectively. PMID:16524605

  3. Enzyme-mediated spatial segregation on individual polymeric support beads: application to generation and screening of encoded combinatorial libraries.

    PubMed Central

    Vágner, J; Barany, G; Lam, K S; Krchnák, V; Sepetov, N F; Ostrem, J A; Strop, P; Lebl, M

    1996-01-01

    Proteolysis of short N alpha-protected peptide substrates bound to polyoxyethylene-polystyrene beads releases selectively free amino sites in the enzyme-accessible "surface" area. The substantial majority of functional sites in the "interior" of the polymeric support are not reached by the enzyme and remain uncleaved (protected). Subsequent synthesis with two classes of orthogonal protecting groups-N alpha-tert-butyloxycarbonyl (Boc) and N alpha-9-fluorenylmethyloxy-carbonyl (Fmoc)-allows generation of two structures on the same bead. The surface structure is available for receptor interactions, whereas the corresponding interior structure is used for coding. Coding structures are usually readily sequenceable peptides. This "shaving" methodology was illustrated by the preparation of a peptide-encoded model peptide combinatorial library containing 1.0 x 10(5) members at approximately 6-fold degeneracy. From this single library, good ligands were selected for three different receptors: anti-beta-endorphin anti-body, streptavidin, and thrombin, and the binding structures were deduced correctly by sequencing the coding peptides present on the same beads. PMID:8710846

  4. Features of separation on polymeric reversed phase for two classes of higher saturated fatty acids esters

    NASA Astrophysics Data System (ADS)

    Deineka, V. I.; Lapshova, M. S.; Zakharenko, E. V.; Deineka, L. A.

    2013-11-01

    The principles of sorption on polymeric reversed phase (PRP) YMS C30 for members of the two classes of esters formed by higher saturated fatty acids, i.e., lutein diesters ( I) and triacylglycerols ( II), are investigated. It is shown that the logarithm of the retention factor increases nonlinearly with an increase of the length of the acid radical, although the retention on PRP is higher in the case of I and lower in the case of II, compared to their retention on traditional monomeric reversed phase (MRP) Kromasil-100 5C18; however, the equivalence of the contributions to the retention of I that correspond to an identical change in acids, does not depend on the length of the hydrocarbon radical of the second acid. It is noted that the Van't Hoff plot for PRP contains a curve break, indicating a change in the retention mechanism upon a rise in temperature.

  5. Molecular Analysis of the Polymeric Glutenins with Gliadin-Like Characteristics That Were Produced by Acid Dispersion of Wheat Gluten.

    PubMed

    Murakami, Tetsuya; Nishimura, Takahisa; Kitabatake, Naofumi; Tani, Fumito

    2016-03-01

    We had earlier shown that the dispersion of wheat gluten in acetic acid solution conferred gliadin-like characteristics to the polymeric glutenins. To elucidate the molecular behavior of its polymeric glutenins, the characteristics of gluten powder prepared from dispersions with various types of acid were investigated in this study. Mixograph measurements showed that the acid-treated gluten powders, regardless of the type of acid, had dough properties markedly weakened in both resistance and elasticity properties, as though gliadin was supplemented. The polymeric glutenins extracted with 70% ethanol increased greatly in all acid-treated gluten powders. Size exclusion HPLC and SDS-PAGE indicated that the behavior of polymeric glutenins due to acid treatment was attributed to their subunit composition rich in high molecular weight glutenin subunit (HMW-GS) and not their molecular size. The gluten prepared with the addition of NaCl in acid dispersion had properties similar to those of the control gluten. The results suggest that ionic repulsion induced by acid dispersion made the polymeric glutenins rich in HMW-GS disaggregate, and therefore, act like gliadins. PMID:26865190

  6. Novel biocompatible and biodegradable ultrathin films of poly (L-Lactic acid) by plasma polymerization

    NASA Astrophysics Data System (ADS)

    Chang, Yi-Hsin; Chang, Chun-Chih; Chen, Ying-Chu; Yang, A. C.-M.; Liu, Y. C.

    2006-03-01

    Ultra-thin films (<= 50 nm) of biodegradable poly (L-lactic acid) were prepared through efficient RF plasma synthesis. The surface morphology of deposited films was amorphous and molecularly uniform (Ra = 0.7 nm). The chemical compositions as determined from FTIR and NMR demonstrated extraordinarily high retention of ester groups with a small fraction of chain cross-linking that could be controlled by process parameters. The chemical routes of the polymerization were described and discussed. This versatile thin film coating technique is very useful for surface engineering of general biomedical devices and implants for improved biocompatibility. In addition, PLLA polymerized in the liquid phase by plasma was also explored and will be presented. This work is supported by National Science Council of Taiwan.

  7. A Mechanistic Study of a-Amino Acid-N-Carboxy Anhydride Polymerization: Comparing Initiation and Termination Events in High Vacuum and Traditional Polymerization Techniques

    SciTech Connect

    Pickel, Deanna L; Politakos, Nikolaos; Avgeropoulos, Apostolos; Messman, Jamie M

    2009-01-01

    High-vacuum polymerization of -amino acid-N-carboxyanhydride (NCAs) affords polymers with controlled molecular weights and narrow polydispersities, however a comprehensive study of the end-group composition of the resulting poly(amino acid)s has not yet been performed. This reveals crucial information, as the end-groups are indicative of both the polymerization mechanism (i.e., initiation event) as well as the termination pathways. To this end, poly(O-benzyl-L-tyrosine) initiated by 1,6-diaminohexane was synthesized and subsequently characterized by MALDI-TOF MS, NALDI -TOF MS and 13C NMR spectroscopy to ascertain the end-group structure. Polymers were prepared by both high vacuum and glove box techniques in DMF/THF. Preparation of poly(O-benzyl-L-tyrosine) by high vacuum techniques yielded a polymer initiated exclusively by the normal amine mechanism, and termination by reaction with DMF was observed. In contrast, polymers prepared in the glove box were initiated by the normal amine and activated monomer mechanisms, and several termination products are evident. To our knowledge, this is the first rigorous and comparative analysis of the end-group structure, and it demonstrates the advantage of high vacuum techniques for polymerization of NCAs for the preparation of well-defined poly(amino acid)s with end-group fidelity.

  8. Amersorb: a new high-performance polymeric separator for lead-acid batteries

    NASA Astrophysics Data System (ADS)

    Toniazzo, Valérie

    Given the recent improvements in valve-regulated batteries, lead-acid technology is nowadays considered to be well-suited for stationary power applications. Gel and absorptive glass mat (AGM) valve-regulated lead-acid (VRLA) batteries are complementary technologies and provide reliability and efficiency due to progressive optimization of the design and components. Special attention has been paid to the separation system, as its influence on the battery performance has been fully demonstrated. Polymeric calendered ribbed separators are traditionally used in gel VRLA batteries. For this technology, the separator is required to have high pore volume, optimized pore size, low acid displacement and low electrical resistance. It must also support efficient and controlled oxygen transfer. Glass-microfibre separators are presently the preferred material for AGM batteries. In addition to the properties listed for the polymeric type, glass-microfibre separators must not allow any drainage or stratification of the liquid electrolyte, and be able to retain their initial thickness after filling and during the battery life in order to sustain the initial compression in each cell. The Amersorb separator is well adapted to both technologies, for example: (i) the ribbed and corrugated patterns provide improved porosity (pore volume and pore-size distribution); (ii) the flat membrane is not only able to wick and retain the acid, but has also optimal compression properties (low compressibility and excellent springiness).

  9. Advances in polymeric and inorganic vectors for nonviral nucleic acid delivery

    PubMed Central

    Sunshine, Joel C; Bishop, Corey J; Green, Jordan J

    2014-01-01

    Nonviral systems for nucleic acid delivery offer a host of potential advantages compared with viruses, including reduced toxicity and immunogenicity, increased ease of production and less stringent vector size limitations, but remain far less efficient than their viral counterparts. In this article we review recent advances in the delivery of nucleic acids using polymeric and inorganic vectors. We discuss the wide range of materials being designed and evaluated for these purposes while considering the physical requirements and barriers to entry that these agents face and reviewing recent novel approaches towards improving delivery with respect to each of these barriers. Furthermore, we provide a brief overview of past and ongoing nonviral gene therapy clinical trials. We conclude with a discussion of multifunctional nucleic acid carriers and future directions. PMID:22826857

  10. Polymerization of amino acids under high-pressure conditions: Implication to chemical evolution on the early Earth

    NASA Astrophysics Data System (ADS)

    Kakegawa, T.; Ohara, S.; Ishiguro, T.; Abiko, H.; Nakazawa, H.

    2008-12-01

    Prebiotic polymerization of amino acids is the most fundamental reaction to promote the chemical evolution for origin of life. Polymerization of amino acids is the dehydration reaction. This questions as to if submarine hydrothermal conditions, thus hydrated enironments, were appropreate for peptide formations. Our previous experiments implied that non-aqueous and high-pressure environments (more than 20 MPa) would be suitable for polymerization of amino acids (Ohara et al., 2006). This leads to the hypothesis that the first peptides may have formed in the Hadean oceanic crustal environments, where dehydration proceeded with availability of appropriate temperatures and pressures. In the present study, experiments simulating the crustal conditions were performed with various pressures (1-175 MPa) and temperatures (100- 200 C degree) using autoclaves. Purified powders (100 mg) of alanine, glycine, valine and aspartic acid were used in the experiments without mixing water in order to examine the solid-solid reactions. The products were analyzed using HPLC and LC-MS. Results indicate that: (1) longer time is required to form peptide compared to those of previous aqueous experiments; (2) pressure has a role to limit the production of melanoidine and cyclic amino acids, which are inhibitors for elongation of peptides; (3) glycine was polymerized up to 11-mer, which was not formed in any previous experiments without catalyses; (4) valine was polymerized up to 3-mer; and (5) aspartic acid was polymerized to 4-mer, accompanied with production of other amino acids. It is noteworthy that high-pressure environments favor all examined polymerization reactions. Such situations would have happened inside of deep oceanic crusts of the early Earth.

  11. Activation of glucosidase via stress-induced polymerization rapidly increases active pools of abscisic acid.

    PubMed

    Lee, Kwang Hee; Piao, Hai Lan; Kim, Ho-Youn; Choi, Sang Mi; Jiang, Fan; Hartung, Wolfram; Hwang, Ildoo; Kwak, June M; Lee, In-Jung; Hwang, Inhwan

    2006-09-22

    Abscisic acid (ABA) is a phytohormone critical for plant growth, development, and adaptation to various stress conditions. Plants have to adjust ABA levels constantly to respond to changing physiological and environmental conditions. To date, the mechanisms for fine-tuning ABA levels remain elusive. Here we report that AtBG1, a beta-glucosidase, hydrolyzes glucose-conjugated, biologically inactive ABA to produce active ABA. Loss of AtBG1 causes defective stomatal movement, early germination, abiotic stress-sensitive phenotypes, and lower ABA levels, whereas plants with ectopic AtBG1 accumulate higher ABA levels and display enhanced tolerance to abiotic stress. Dehydration rapidly induces polymerization of AtBG1, resulting in a 4-fold increase in enzymatic activity. Furthermore, diurnal increases in ABA levels are attributable to polymerization-mediated AtBG1 activation. We propose that the activation of inactive ABA pools by polymerized AtBG1 is a mechanism by which plants rapidly adjust ABA levels and respond to changing environmental cues. PMID:16990135

  12. Stability study of polyacrylic acid films plasma-polymerized on polypropylene substrates at medium pressure

    NASA Astrophysics Data System (ADS)

    Morent, Rino; De Geyter, Nathalie; Trentesaux, Martine; Gengembre, Léon; Dubruel, Peter; Leys, Christophe; Payen, Edmond

    2010-11-01

    Plasma polymerization of acrylic acid has become an interesting research subject, since these coatings are expected to be beneficial for biomedical applications due to their high surface density of carboxylic acid functional groups. However, the application of these monomers is counteracted by their low stability in humid environments, since a high stability is a required characteristic for almost any biological application. The present work investigates whether it is possible to obtain stable deposits with a high retention of carboxylic acid functions by performing plasma polymerization on polypropylene substrates with a dielectric barrier discharge operating at medium pressure. In order to obtain coatings with the desired properties, the plasma parameters need to be optimized. Therefore, in this paper, the influence of discharge power and location of the substrate in the discharge chamber is examined in detail. The properties of the deposited films are studied using contact angle measurements, X-ray photoelectron spectroscopy, atomic force microscopy and Fourier transform infrared spectroscopy. Moreover, to determine whether the obtained deposits are soluble in water, the coatings are once again analyzed after rinsing in water. This paper will clearly show that stable COOH-rich surfaces can be obtained at high discharge power and close to the monomer inlet, which might open perspectives for future biomedical applications.

  13. A preliminary investigation of acid-catalyzed polymerization reactions of shale oil distillates

    SciTech Connect

    Netzel, D.A.

    1991-04-01

    Sinor (1989) reported that a major specialty market may exist for shale oil as an asphalt blending material. Shale oil can be converted to an asphalt blending material by acid catalyzed condensation and polymerization reactions of the many molecular species comprising the composition of shale oil. To simplify the investigation, crude shale oil was separated by distillation into three distillates of different hydrocarbon and heteroaromatic compositions. These distillates were then treated with two different types of acids to determine the effect of acid type on the end products. Three western shale oil distillates, a naphtha, a middle distillate, and an atmospheric gas oil, were reacted with anhydrous AlCl{sub 3} and 85% H{sub 2}SO{sub 4} under low-severity conditions. At relatively low temperatures, little change in the hydrocarbon composition was noted for the AlCl{sub 3} reactions. AlCl{sub 3}{center dot} (a polymerized product and/or complex) was formed. However, it is assumed that the sludge was mainly the result of heteroaromatic-AlCl{sub 3} reactions.

  14. A preliminary investigation of acid-catalyzed polymerization reactions of shale oil distillates

    SciTech Connect

    Netzel, D.A.

    1991-04-01

    Sinor (1989) reported that a major specialty market may exist for shale oil as an asphalt blending material. Shale oil can be converted to an asphalt blending material by acid catalyzed condensation and polymerization reactions of the many molecular species comprising the composition of shale oil. To simplify the investigation, crude shale oil was separated by distillation into three distillates of different hydrocarbon and heteroaromatic compositions. These distillates were then treated with two different types of acids to determine the effect of acid type on the end products. Three western shale oil distillates, a naphtha, a middle distillate, and an atmospheric gas oil, were reacted with anhydrous AlCl{sub 3} and 85% H{sub 2}SO{sub 4} under low-severity conditions. At relatively low temperatures, little change in the hydrocarbon composition was noted for the AlCl{sub 3} reactions. AlCl{sub 3}{center_dot} (a polymerized product and/or complex) was formed. However, it is assumed that the sludge was mainly the result of heteroaromatic-AlCl{sub 3} reactions.

  15. Enzyme

    MedlinePlus

    Enzymes are complex proteins that cause a specific chemical change in all parts of the body. For ... use them. Blood clotting is another example of enzymes at work. Enzymes are needed for all body ...

  16. Transcriptional Suppression of Renal Antioxidant Enzyme Systems in Guinea Pigs Exposed to Polymerized Cell-Free Hemoglobin

    PubMed Central

    Rentsendorj, Otgonchimeg; Zhang, Xiaoyuan; Williams, Matthew C.; Buehler, Paul W.; D'Agnillo, Felice

    2016-01-01

    Hemoglobin-based oxygen carriers (HBOCs) are being developed as oxygen and plasma volume-expanding therapeutics though their potential to promote oxidative tissue injury has raised safety concerns. Using a guinea pig exchange transfusion model, we examined the effects of polymerized bovine hemoglobin (HbG) on the transcriptional regulation, activity, and expression of the renal antioxidant enzymes; superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx). HbG infusion downregulated the mRNA levels for genes encoding SOD isoforms 1-3, GPx1, GPx3, GPx4, and CAT. This transcriptional suppression correlated with decreased enzymatic activities for SOD, CAT, and GPx. Immunostaining revealed decreased protein expression of SOD1, CAT, and GPx1 primarily in renal cortical tubules. DNA methylation analyses identified CpG hypermethylation in the gene promoters for SOD1-3, GPx1, GPx3, and GPx4, suggesting an epigenetic-based mechanism underlying the observed gene repression. HbG also induced oxidative stress as evidenced by increased renal lipid peroxidation end-products and 4-HNE immunostaining, which could be the result of the depleted antioxidant defenses and/or serve as a trigger for increased DNA methylation. Together, these findings provide evidence that the renal exposure to HbG suppresses the function of major antioxidant defense systems which may have relevant implications for understanding the safety of hemoglobin-based products. PMID:27471729

  17. Effective immobilization of tyrosinase via enzyme catalytic polymerization of l-DOPA for highly sensitive phenol and atrazine sensing.

    PubMed

    Guan, Yun; Liu, Lanjunzi; Chen, Chao; Kang, Xiuzhi; Xie, Qingji

    2016-11-01

    The facile preparation of poly(l-DOPA)-tyrosinase (PDM-Tyr) composite and its application both in substrate (phenol) and inhibitor (atrazine) sensing is reported here for the first time. Effective immobilization of enzyme is realized via in-situ entrapping Tyr in poly(l-DOPA) (PDM), which is formed by Tyr catalytic polymerization of l-DOPA. The Tyr modified electrode is simply prepared by dipping the PDM-Tyr composite on an Au electrode and then covered by Nafion. The thus-prepared Tyr-immobilized electrode exhibits excellent performance superior to most Tyr-based electrochemical biosensors, the sensitivity to phenol is as high as 5122 μA mM(-1) in the linear range of 10nM~1.25 μM, the apparent Michaelis-Menten constant (KM(app)) determined as low as 3.13μM indicates strong substrate binding and high catalytic activity of the immobilized Tyr. The biosensor also works well in atrazine biosensing, with a linear detection range of 50ppb~30ppm and a low detection limit of 10ppb obtained. In addition, the biosensor shows excellent stability, precision, high sensitivity and fabrication simplicity. PMID:27591595

  18. Thermostable lipoxygenase, a key enzyme in bioconversion of linoleic acid to trihycroxy-octadecenoic acid by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipoxygenases, enzymes that contain non-heme iron, catalyze the oxidation of unsaturated fatty acids with a (1Z,4Z)-pentadiene moiety leading to conjugated (Z,E)-hydroperoxydienoic acids. These enzymes are widely distributed in plants and animals, and a few microorganisms are reported as well. It ...

  19. Synthesis and characterization of polymeric materials derived from 2,5-diketopiperazines and pyroglutamic acid

    NASA Astrophysics Data System (ADS)

    Parrish, Dennis Arch

    The research presented in this dissertation describes the investigation of 2,5-diketopiperazines (DKPs) as property modifiers for addition polymers and the self association behavior of pyroglutamic acid derivatives. The first project involved the copolymerization of methyl methacrylate and styrene with DKP-based methacrylate monomers. Low incorporations of serine- and aspartame-based DKPs in the copolymer resulted in dramatic increases in the glass transition temperature (Ts). The research presented in Chapter II focuses on the ring-opening reactions of pyroglutamic diketopiperazine (pyDKP). The original intent was to synthesize polymers containing backbone DKPs through ring-opening polymerization of the five-membered rings. However, it was discovered that regioselective ring-opening occurs at the six-membered ring to give pyroglutamic acid derivatives. Since this reaction had not been reported previously, the focus of research was altered to investigate the scope and limitations of the new reaction. The ring-opening reactions of pyDKP with diamines to give bispyroglutamides is described in Chapter IV. While these materials are not polymeric, they display polymeric behavior. It was found that multi-functional pyroglutamides display Tgs during thermal analysis, exhibit high thermal stability, and form melt-drawn fibers. In contrast, the materials have low solution viscosities and are freely soluble in water, ethanol, and chloroform. This behavior is attributed to non-covalent supramolecular associations. The final part of this dissertation involved the investigation of thermoreversible organic solvent gelators. The ring-opening reaction of pyDKP with long alkyl amines unexpectedly gelled the reaction solvent. A series of analogous gelators were synthesized, and the minimum concentration required for gelation in various solvents was determined. It was found that the nature of the solvent, alkyl chain length, and optical activity of the gelator determined gelator

  20. Topological characterization of a bacterial cellulose-acrylic acid polymeric matrix.

    PubMed

    Halib, N; Mohd Amin, M C I; Ahmad, I; Abrami, M; Fiorentino, S; Farra, R; Grassi, G; Musiani, F; Lapasin, R; Grassi, M

    2014-10-01

    This paper focuses on the micro- and nano-topological organization of a hydrogel, constituted by a mixture of bacterial cellulose and acrylic acid, and intended for biomedical applications. The presence of acrylic acid promotes the formation of two interpenetrated continuous phases: the primary "pores phase" (PP) containing only water and the secondary "polymeric network phase" (PNP) constituted by the polymeric network swollen by the water. Low field Nuclear Magnetic Resonance (LF NMR), rheology, Scanning Electron Microscopy (SEM) and release tests were used to determine the characteristics of the two phases. In particular, we found that this system is a strong hydrogel constituted by 81% (v/v) of PP phase the remaining part being occupied by the PNP phase. Pores diameters span in the range 10-100 μm, the majority of them (85%) falling in the range 30-90 μm. The high PP phase tortuosity indicates that big pores are not directly connected to each other, but their connection is realized by a series of interconnected small pores that rend the drug path tortuous. The PNP is characterized by a polymer volume fraction around 0.73 while mesh size is around 3 nm. The theoretical interpretation of the experimental data coming from the techniques panel adopted, yielded to the micro- and nano-organization of our hydrogel. PMID:24932712

  1. Homo- and co-polymerization of polysytrene-block-poly(acrylic acid)-coated metal nanoparticles.

    PubMed

    Wang, Hong; Song, Xiaohui; Liu, Cuicui; He, Jiating; Chong, Wen Han; Chen, Hongyu

    2014-08-26

    Amphiphilic block copolymers such as polystyrene-block-poly(acrylic acid) (PSPAA) give micelles that are known to undergo sphere-to-cylinder shape transformation. Exploiting this polymer property, core-shell nanoparticles coated in PSPAA can be "polymerized" into long chains following the chain-growth polymerization mode. This method is now extended to include a variety of different nanoparticles. A case study on the assembly process was carried out to understand the influence of the PAA block length, the surface ligand, and the size and morphology of the monomer nanoparticles. Shortening the PAA block promotes the reorganization of the amphiphilic copolymer in the micelles, which is essential for assembling large Au nanoparticles. Small Au nanoparticles can be directly "copolymerized" with empty PSPAA micelles into chains. The reaction time, acid quantity, and the [Au nanoparticles]/[PSPAA micelles] concentration ratio played important roles in controlling the sphere-cylinder-vesicle conversion of the PSPAA micelles, giving rise to different kinds of random "copolymers". With this knowledge, a general method is then developed to synthesize homo, random, and block "copolymers", where the basic units include small Au nanoparticles (d = 16 nm), large Au nanoparticles (d = 32 nm), Au nanorods, Te nanowires, and carbon nanotubes. Given the lack of means for assembling nanoparticles, advancing synthetic capabilities is of crucial importance. Our work provides convenient routes for combining nanoparticles into long-chain structures, facilitating rational design of complex nanostructures in the future. PMID:25000121

  2. Gallic acid and gallic acid derivatives: effects on drug metabolizing enzymes.

    PubMed

    Ow, Yin-Yin; Stupans, Ieva

    2003-06-01

    Gallic acid and its structurally related compounds are found widely distributed in fruits and plants. Gallic acid, and its catechin derivatives are also present as one of the main phenolic components of both black and green tea. Esters of gallic acid have a diverse range of industrial uses, as antioxidants in food, in cosmetics and in the pharmaceutical industry. In addition, gallic acid is employed as a source material for inks, paints and colour developers. Studies utilising these compounds have found them to possess many potential therapeutic properties including anti-cancer and antimicrobial properties. In this review, studies of the effects of gallic acid, its esters, and gallic acid catechin derivatives on Phase I and Phase II enzymes are examined. Many published reports of the effects of the in vitro effects of gallic acid and its derivatives on drug metabolising enzymes concern effects directly on substrate (generally drug or mutagen) metabolism or indirectly through observed effects in Ames tests. In the case of the Ames test an antimutagenic effect may be observed through inhibition of CYP activation of indirectly acting mutagens and/or by scavenging of metabolically generated mutagenic electrophiles. There has been considerable interest in the in vivo effects of the gallate esters because of their incorporation into foodstuffs as antioxidants and in the catechin gallates with their potential role as chemoprotective agents. Principally an induction of Phase II enzymes has been observed however more recent studies using HepG2 cells and primary cultures of human hepatocytes provide evidence for the overall complexity of actions of individual components versus complex mixtures, such as those in food. Further systematic studies of mechanisms of induction and inhibition of drug metabolising enzymes by this group of compounds are warranted in the light of their distribution and consequent ingestion, current uses and suggested therapeutic potential. However, it

  3. Interfacial Polymerization of Dopamine in a Pickering Emulsion: Synthesis of Cross-Linkable Colloidosomes and Enzyme Immobilization at Oil/Water Interfaces.

    PubMed

    Qu, Yanning; Huang, Renliang; Qi, Wei; Su, Rongxin; He, Zhimin

    2015-07-15

    Colloidosomes are promising carriers for immobilizing enzyme for catalytic purposes in aqueous/organic media. However, they often suffer from one or more problems regarding catalytic performance, stability, and recyclability. Here, we report a novel approach for the synthesis of cross-linkable colloidosomes by the selective polymerization of dopamine at oil/water interfaces in a Pickering emulsion. An efficient enzyme immobilization method was further developed by covalently bonding enzymes to the polydopamine (PDA) layer along with the formation of such colloidosomes with lipase as a model enzyme. In this enzyme system, the PDA layer served as a cross-linking layer and enzyme support for simultaneously enhancing the colloidosomes' stability and improving surface availability of the enzymes for catalytic reaction. It was found that the specific activity of lipases immobilized on the colloidosome shells was 8 and 1.4 times higher than that of free lipase and encapsulated lipase positioned in the aqueous cores of colloidosomes, respectively. Moreover, the immobilized lipases demonstrated excellent operational stability and recyclability, retaining 86.6% of enzyme activity after 15 cycles. It is therefore reasonable to expect that this novel approach for enzyme immobilization has great potential to serve as an important technique for the construction of biocatalytic systems. PMID:26104042

  4. Further work on sodium montmorillonite as catalyst for the polymerization of activated amino acids

    NASA Technical Reports Server (NTRS)

    Eirich, F. R.; Paecht-Horowitz, M.

    1986-01-01

    When the polycondensation of amino acid acylates was catalyzed with Na-montmorillonite, the polypeptides were consistently found to exhibit a distribution of discrete molecular weights, for as yet undiscovered reasons. One possible explanation was connected to the stepwise mode of monomer addition. New experiments have eliminated this possibility, so that there is the general assumption that this discreteness is the result of a preference of shorter oligomers to add to others of the same length, a feature that could be attributed to some structure of the platelet aggregates of the montmorillonite. The production of optical stereoisomers is anticipated when D,L-amino acids are polymerized on montmorillonite. Having used an optically active surface, the essence of the results lies not only in the occurrence of optically active oligomers and polymers, but also in the fact that the latter exhibit the same molecular weight characteristics as the D,L-polymers. Preparatory to work contemplated on a parallel synthesis of amino acid and nucleotide oligomers, studies were continued on the co-adsorption of amino acids, nucleotides, and amino acid-nucleotides on montmorillonite.

  5. On-Surface Domino Reactions: Glaser Coupling and Dehydrogenative Coupling of a Biscarboxylic Acid To Form Polymeric Bisacylperoxides.

    PubMed

    Held, Philipp Alexander; Gao, Hong-Ying; Liu, Lacheng; Mück-Lichtenfeld, Christian; Timmer, Alexander; Mönig, Harry; Barton, Dennis; Neugebauer, Johannes; Fuchs, Harald; Studer, Armido

    2016-08-01

    Herein we report the on-surface oxidative homocoupling of 6,6'-(1,4-buta-1,3-diynyl)bis(2-naphthoic acid) (BDNA) via bisacylperoxide formation on different Au substrates. By using this unprecedented dehydrogenative polymerization of a biscarboxylic acid, linear poly-BDNA with a chain length of over 100 nm was prepared. It is shown that the monomer BDNA can be prepared in situ at the surface via on-surface Glaser coupling of 6-ethynyl-2-naphthoic acid (ENA). Under the Glaser coupling conditions, BDNA directly undergoes polymerization to give the polymeric peroxide (poly-BDNA) representing a first example of an on-surface domino reaction. It is shown that the reaction outcome varies as a function of surface topography (Au(111) or Au(100)) and also of the surface coverage, to give branched polymers, linear polymers, or 2D metal-organic networks. PMID:27410485

  6. Chemiluminometric Immunosensor for High-Sensitivity Cardiac Troponin I Employing a Polymerized Enzyme Conjugate as a Tracer

    PubMed Central

    Lim, Guei-Sam; Seo, Sung-Min; Paek, Sung-Ho; Kim, Seung-Wan; Jeon, Jin-Woo; Kim, Dong-Hyung; Cho, Il-Hoon; Paek, Se-Hwan

    2015-01-01

    To detect high-sensitivity cardiac troponin I (hs-cTnI; <0.01 ng/mL) at points of care, we developed a rapid immunosensor by using horseradish peroxidase polymerized in 20 molecules on average (Poly-HRP) as a tracer conjugated with streptavidin (SA-Poly-HRP). As shown in the conventional system, enhanced sensitivity could be achieved by using a sequential binding scheme for the complex formation to contain the huge molecular tracer. We used a 2-dimensional chromatographic technology to carry out the sequential bindings in cross-flow directions. After the complex formation of antigen-antibody with analyte in a vertical direction, SA-Poly-HRP was horizontally supplied across the membrane strip for additional binding via a biotin-SA linkage. The HRP substrate was subsequently supplied along the same direction to produce a chemiluminometric signal, which was measured by a cooled charge-coupled device. Hs-cTnI analysis was completed in this format within 25 min, and the results showed a high correlation with those of the CentaurXP® reference system (R2 > 0.99). The detection limit of the rapid immunosensor was 0.003 ± 0.001 ng/mL cTnI, corresponding to a 10-fold improvement compared to results using the plain enzyme tracer. This demonstrated the measurement of hs-cTnI in a much more cost-effective manner compared to the automated versions currently available. PMID:26442606

  7. Potassium fulvate as co-interpenetrating agent during graft polymerization of acrylic acid from cellulose.

    PubMed

    Ghazy, Mohamed B M; El-Hai, Farag Abd; Mohamed, Magdy F; Essawy, Hisham A

    2016-10-01

    Grafting polymerization of acrylic acid onto cellulose in presence of potassium fulvate (KF) as a co-interpenetrating agent results enhanced water sorption compared to materials prepared similarly in its absence. The insertion of potassium fulvate (KF) did not affect the grafting process and is thought to proceed in parallel to the graft polymerization via intensive polycondensation reactions of its function groups (-COOH and OH) with COOH of the monomer and OH groups of cellulose. The combination of graft copolymerization and polycondensation reactions is assumed to produce interpenetrating network structure. Fourier transform infrared (FTIR) confirmed successful incorporation within the network structure which is an evidence for formation of interpenetrating network. The obtained structures showed homogeneous uniform surface as revealed by scanning electron microscopy (SEM). The obtained superabsorbent possessed high water absorbency 422 and 48.8g/g in distilled water and saline (0.9wt.% NaCl solution), respectively, and enhanced water retention even at elevated temperatures as revealed by thermogravimetric analysis (TGA). This could be explained by the high content of hydrophilic groups. The new superabsorbents proved to be efficient devices for controlled release of fertilizers which expands their use in agricultural applications. PMID:27370745

  8. Self-assembly of poly(o-methoxyaniline) hollow nanospheres from a polymeric acid solution

    NASA Astrophysics Data System (ADS)

    Sui, Jing; Zhang, Lijuan; Peng, Hui; Travas-Sejdic, Jadranka; Kilmartin, Paul A.

    2009-10-01

    Self-assembled poly(o-methoxyaniline) (POMA) hollow nanospheres were prepared in a solution of poly(methyl vinyl ether-alt-maleic acid) (PMVEA) by oxidative polymerization using ammonium persulfate as the oxidant. The weight ratio of PMVEA to o-methoxyaniline in the solution had a significant effect on the morphology of the poly(o-methoxyaniline) nanospheres as determined by scanning electron microscopy. The diameter of the hollow nanospheres decreased from 440 to 210 nm with an increase in the PMVEA concentration from 1% to 5%. Freeze-fracture transmission electron microscopy results showed the presence of spherical micelles composed of PMVEA/ o-methoxyaniline prior to polymerization, which also decreased in size as more PMVEA was added to the solution, and can act as soft templates for the formation of the hollow POMA nanospheres. The POMA/PMVEA hollow nanospheres were characterized by means of Fourier transform infrared, UV-visible, x-ray photoelectron spectroscopy, elemental analysis and conductivity measurements.

  9. Peracetic acid: a practical agent for sterilizing heat-labile polymeric tissue-engineering scaffolds.

    PubMed

    Yoganarasimha, Suyog; Trahan, William R; Best, Al M; Bowlin, Gary L; Kitten, Todd O; Moon, Peter C; Madurantakam, Parthasarathy A

    2014-09-01

    Advanced biomaterials and sophisticated processing technologies aim at fabricating tissue-engineering scaffolds that can predictably interact within a biological environment at the cellular level. Sterilization of such scaffolds is at the core of patient safety and is an important regulatory issue that needs to be addressed before clinical translation. In addition, it is crucial that meticulously engineered micro- and nano- structures are preserved after sterilization. Conventional sterilization methods involving heat, steam, and radiation are not compatible with engineered polymeric systems because of scaffold degradation and loss of architecture. Using electrospun scaffolds made from polycaprolactone, a low melting polymer, and employing spores of Bacillus atrophaeus as biological indicators, we compared ethylene oxide, autoclaving and 80% ethanol to a known chemical sterilant, peracetic acid (PAA), for their ability to sterilize as well as their effects on scaffold properties. PAA diluted in 20% ethanol to 1000 ppm or above sterilized electrospun scaffolds in 15 min at room temperature while maintaining nano-architecture and mechanical properties. Scaffolds treated with PAA at 5000 ppm were rendered hydrophilic, with contact angles reduced to 0°. Therefore, PAA can provide economical, rapid, and effective sterilization of heat-sensitive polymeric electrospun scaffolds that are used in tissue engineering. PMID:24341350

  10. Peracetic Acid: A Practical Agent for Sterilizing Heat-Labile Polymeric Tissue-Engineering Scaffolds

    PubMed Central

    Yoganarasimha, Suyog; Trahan, William R.; Best, Al M.; Bowlin, Gary L.; Kitten, Todd O.; Moon, Peter C.

    2014-01-01

    Advanced biomaterials and sophisticated processing technologies aim at fabricating tissue-engineering scaffolds that can predictably interact within a biological environment at the cellular level. Sterilization of such scaffolds is at the core of patient safety and is an important regulatory issue that needs to be addressed before clinical translation. In addition, it is crucial that meticulously engineered micro- and nano- structures are preserved after sterilization. Conventional sterilization methods involving heat, steam, and radiation are not compatible with engineered polymeric systems because of scaffold degradation and loss of architecture. Using electrospun scaffolds made from polycaprolactone, a low melting polymer, and employing spores of Bacillus atrophaeus as biological indicators, we compared ethylene oxide, autoclaving and 80% ethanol to a known chemical sterilant, peracetic acid (PAA), for their ability to sterilize as well as their effects on scaffold properties. PAA diluted in 20% ethanol to 1000 ppm or above sterilized electrospun scaffolds in 15 min at room temperature while maintaining nano-architecture and mechanical properties. Scaffolds treated with PAA at 5000 ppm were rendered hydrophilic, with contact angles reduced to 0°. Therefore, PAA can provide economical, rapid, and effective sterilization of heat-sensitive polymeric electrospun scaffolds that are used in tissue engineering. PMID:24341350

  11. Identification of gallic acid based glycoconjugates as a novel tubulin polymerization inhibitors.

    PubMed

    Upadhyaya, Kapil; Hamidullah; Singh, Kartikey; Arun, Ashutosh; Shukla, Mahendra; Srivastava, Neetika; Ashraf, Raghib; Sharma, Abhisheak; Mahar, Rohit; Shukla, Sanjeev K; Sarkar, Jayanta; Ramachandran, Ravishankar; Lal, Jawahar; Konwar, Rituraj; Tripathi, Rama Pati

    2016-01-28

    A novel class of gallic acid based glycoconjugates were designed and synthesized as potential anticancer agents. Among all the compounds screened, compound 2a showed potent anticancer activity against breast cancer cells. The latter resulted in tubulin polymerization inhibition and induced G2/M cell cycle arrest, generation of reactive oxygen species, mitochondrial depolarization and subsequent apoptosis in breast cancer cells. In addition, ultraviolet-visible spectroscopy and fluorescence quenching studies of the compound with tubulin confirmed direct interaction of compounds with tubulin. Molecular modeling studies revealed that it binds at the colchicine binding site in tubulin. Further, 2a also exhibited potent in vivo anticancer activity in LA-7 syngeneic rat mammary tumor model. Current data projects its strong candidature to be developed as anticancer agent. PMID:26659548

  12. Polymeric Cryogel-Based Boronate Affinity Chromatography for Separation of Ribonucleic Acid from Bacterial Extracts.

    PubMed

    Shakya, Akhilesh Kumar; Srivastava, Akshay; Kumar, Ashok

    2015-01-01

    Three-dimensional monolithic columns are preferred stationary phase in column chromatography. Conventional columns based on silica or particles are efficient in bioseparation though associated with limitations of nonspecific interaction and uneven porosity that causes high mass transfer resistance for the movement of big molecules. Cryogels as a monolith column have shown promising application in bioseparation. Cryogels column can be synthesized in the form of a monolith at sub-zero temperature through gelation of pre-synthesized polymers or polymerization of monomers. Cryogels are macroporous and mechanically stable materials. They have open interconnected micron-sized pores with a wide range of porosity (10-200 μm). Current protocol demonstrated the ability of poly(hydroxymethyl methacrylate)-co-vinylphenyl boronic acid p(HEMA-co-VPBA) cryogel matrix for selective separation of RNA from the bacterial crude extract. PMID:26623972

  13. Novel solid state proton-conductors based on polymeric non-oxy acids. Final report

    SciTech Connect

    Appleby, A.J.; Srinivasan, S.; Parthasarathy, A.; Gonzalez, E.R.; DesMarteau, D.; Gillette, M.S.; Ghosh, J.K.; Jalan, V.; Desai, M.

    1992-01-01

    Objectives of this project were to prepare and characterize novel solid state proton-conductors and to evaluate these compounds as fuel cell electrolytes. The thrust was on the synthesis of new proton-conducting ``model`` and ``polymeric`` compounds, based on acid functions of the type (R{sub f}SO{sub 2}){sub 2}NH and (R{sub f}SO{sub 2}){sub 2}CH{sub 2} in appropriate fluorinated carbon structures, their physics-chemical characterization (Infra-red, Nuclear Magnetic Resonance, Differential Scanning Calorimetry, and X-ray Diffraction), and is pro. evaluation as candidate fuel cell electrolytes for use at elevated temperatures. This project consisted of four tasks (i) Synthesis of Proton-Conducting Polymer Electrolytes; (ii) Physical and Chemical Characterization of Proton-Conducting Polymer Electrolytes; (iii) Electrochemical Characterization of Proton-Conducting Polymer Electrolytes; and (iv) Evaluation of Proton-Conducting Polymer Electrolytes for Fuel Cells.

  14. Self-assembled polymeric nanocarriers for the targeted delivery of retinoic acid to the hair follicle.

    PubMed

    Lapteva, Maria; Möller, Michael; Gurny, Robert; Kalia, Yogeshvar N

    2015-11-28

    Acne vulgaris is a highly prevalent dermatological disease of the pilosebaceous unit (PSU). An inability to target drug delivery to the PSU results in poor treatment efficacy and the incidence of local side-effects. Cutaneous application of nanoparticulate systems is reported to induce preferential accumulation in appendageal structures. The aim of this work was to prepare stable polymeric micelles containing retinoic acid (RA) using a biodegradable and biocompatible diblock methoxy-poly(ethylene glycol)-poly(hexylsubstituted lactic acid) copolymer (MPEG-dihexPLA) and to evaluate their ability to deliver RA to skin. An innovative punch biopsy sample preparation method was developed to selectively quantify follicular delivery; the amounts of RA present were compared to those in bulk skin, (i.e. without PSU), which served as the control. RA was successfully incorporated into micelle nanocarriers and protected from photoisomerization by inclusion of Quinoline Yellow. Incorporation into the spherical, homogeneous and nanometer-scale micelles (dn < 20 nm) increased the aqueous solubility of RA by >400-fold. Drug delivery experiments in vitro showed that micelles were able to deliver RA to porcine and human skins more efficiently than Retin-A(®) Micro (0.04%), a marketed gel containing RA loaded microspheres, (7.1 ± 1.1% vs. 0.4 ± 0.1% and 7.5 ± 0.8% vs. 0.8 ± 0.1% of the applied dose, respectively). In contrast to a non-colloidal RA solution, Effederm(®) (0.05%), both the RA loaded MPEG-dihexPLA polymeric micelles (0.005%) and Retin-A(®) Micro (0.04%) displayed selectivity for delivery to the PSU with 2-fold higher delivery to PSU containing samples than to control samples. Moreover, the micelle formulation outperformed Retin-A(®) Micro in terms of delivery efficiency to PSU presenting human skin (10.4 ± 3.2% vs. 0.6 ± 0.2%, respectively). The results indicate that the polymeric micelle formulation enabled an increased and targeted delivery of RA to the PSU

  15. Flow injection analysis of cholic acids in pharmaceutical preparations using a polymeric membrane ISE as detector.

    PubMed

    Arias De Fuentes, O; Campanella, L; Crescentini, G; Falcioni, A; Sammartino, M P; Tomassetti, M

    2000-08-01

    The results reported in this paper regard the setting up of a polymeric membrane ISE that is selective for cholic acids (CA) and able to work in a flow system, especially in flow injection analysis (FIA), based on the exchanger (tetrakisdecylammoniumcholate, TDACh), which has proved effective, is of very simple but suitable structure and is above all easy to synthesise starting from commercially available chemicals. A complete analytical characterisation of the sensor was performed working both in batch conditions and in FIA, using in the latter case a 'wall jet' type of flow cell. The response toward different bile acid sodium salts such as the CA, deoxycholic (DCA), chenodeoxycholic (CDCA), ursodeoxycholic (UDCA), taurocholic (TCA) sodium salts was checked. The application to the analysis of different commercial drugs by FIA was also performed to determine the UDCA or CDCA acid content of several pharmaceutical formulations. Lastly, a preliminary study is presented concerning the use of the investigated electrochemical sensor as high performance liquid chromatography (HPLC) detector. PMID:10898158

  16. Non-enzymic beta-decarboxylation of aspartic acid.

    NASA Technical Reports Server (NTRS)

    Doctor, V. M.; Oro, J.

    1972-01-01

    Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

  17. Lewis acid catalyzed ring-opening polymerization of natural epoxy oil (Euphorbia oil) in carbon dioxide media

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In an attempt to build up useful application of plant oil based polymers, natural epoxy oil (euphorbia oil-EuO) was polymerized in liquid carbon dioxide in the presence of Lewis acid catalyst [Boron trifluoride diethyl etherate (BF3•OEt2)]. The resulting polymers (RPEuO) were characterized by FTIR ...

  18. A facile one pot strategy for the synthesis of well-defined polyacrylates from acrylic acid via RAFT polymerization.

    PubMed

    Li, Qianbiao; Wang, Taisheng; Dai, Jingwen; Ma, Chao; Jin, Bangkun; Bai, Ruke

    2014-03-28

    A facile one pot strategy for the preparation of linear and hyperbranched polyacrylates has been successfully developed by the combination of in situ esterification of acrylic acid with halogenated compounds promoted by 1,1,3,3-tetramethylguanidine (TMG) and RAFT polymerization. PMID:24534953

  19. Polarized fluorescence microscopy analysis of patterned, polymerized perfluorotetradecanoic acid-pentacosadiynoic acid thin films

    NASA Astrophysics Data System (ADS)

    Araghi, Hessamaddin Younesi; Giri, Neeraj K.; Paige, Matthew F.

    2014-08-01

    Photoillumination of mixed films comprised of the photopolymerizable fatty acid 10,12 pentacosadiynoic acid and perfluorotetradecanoic acid deposited onto glass substrates gives rise to the formation of oriented polydiacetylene photopolymer fibers. The degree of polymer fiber orientation was investigated using dual-view, polarized fluorescence microscopy of the polydiacetylene, which allowed for characterization of individual fluorescent polymer fibers after photopolymerization, as well as comparison of the orientation of different fibers within the same sample. Measurements indicated that individual fibers consisted of multiple photopolymer strands with various orientations, and that there was a preferred orientation for fibers in the film as a whole. The fibers were preferentially oriented at an angle of approximately 60° to the direction of film compression during deposition from a Langmuir trough, with orientation being the result of mechanical stress exerted by the compression barriers coupled with rotation of the polymer fibers during film draining. These measurements were complemented with conventional “bulk” fluorescence polarization experiments, and compared with mixed film structures described previously for these systems at the air-water interface using Brewster angle microscopy.

  20. Polarized fluorescence microscopy analysis of patterned, polymerized perfluorotetradecanoic acid-pentacosadiynoic acid thin films.

    PubMed

    Araghi, Hessamaddin Younesi; Giri, Neeraj K; Paige, Matthew F

    2014-08-14

    Photoillumination of mixed films comprised of the photopolymerizable fatty acid 10,12 pentacosadiynoic acid and perfluorotetradecanoic acid deposited onto glass substrates gives rise to the formation of oriented polydiacetylene photopolymer fibers. The degree of polymer fiber orientation was investigated using dual-view, polarized fluorescence microscopy of the polydiacetylene, which allowed for characterization of individual fluorescent polymer fibers after photopolymerization, as well as comparison of the orientation of different fibers within the same sample. Measurements indicated that individual fibers consisted of multiple photopolymer strands with various orientations, and that there was a preferred orientation for fibers in the film as a whole. The fibers were preferentially oriented at an angle of approximately 60° to the direction of film compression during deposition from a Langmuir trough, with orientation being the result of mechanical stress exerted by the compression barriers coupled with rotation of the polymer fibers during film draining. These measurements were complemented with conventional "bulk" fluorescence polarization experiments, and compared with mixed film structures described previously for these systems at the air-water interface using Brewster angle microscopy. PMID:24747858

  1. Self-assembled polymeric nanocarriers for the targeted delivery of retinoic acid to the hair follicle

    NASA Astrophysics Data System (ADS)

    Lapteva, Maria; Möller, Michael; Gurny, Robert; Kalia, Yogeshvar N.

    2015-11-01

    Acne vulgaris is a highly prevalent dermatological disease of the pilosebaceous unit (PSU). An inability to target drug delivery to the PSU results in poor treatment efficacy and the incidence of local side-effects. Cutaneous application of nanoparticulate systems is reported to induce preferential accumulation in appendageal structures. The aim of this work was to prepare stable polymeric micelles containing retinoic acid (RA) using a biodegradable and biocompatible diblock methoxy-poly(ethylene glycol)-poly(hexylsubstituted lactic acid) copolymer (MPEG-dihexPLA) and to evaluate their ability to deliver RA to skin. An innovative punch biopsy sample preparation method was developed to selectively quantify follicular delivery; the amounts of RA present were compared to those in bulk skin, (i.e. without PSU), which served as the control. RA was successfully incorporated into micelle nanocarriers and protected from photoisomerization by inclusion of Quinoline Yellow. Incorporation into the spherical, homogeneous and nanometer-scale micelles (dn < 20 nm) increased the aqueous solubility of RA by >400-fold. Drug delivery experiments in vitro showed that micelles were able to deliver RA to porcine and human skins more efficiently than Retin-A® Micro (0.04%), a marketed gel containing RA loaded microspheres, (7.1 +/- 1.1% vs. 0.4 +/- 0.1% and 7.5 +/- 0.8% vs. 0.8 +/- 0.1% of the applied dose, respectively). In contrast to a non-colloidal RA solution, Effederm® (0.05%), both the RA loaded MPEG-dihexPLA polymeric micelles (0.005%) and Retin-A® Micro (0.04%) displayed selectivity for delivery to the PSU with 2-fold higher delivery to PSU containing samples than to control samples. Moreover, the micelle formulation outperformed Retin-A® Micro in terms of delivery efficiency to PSU presenting human skin (10.4 +/- 3.2% vs. 0.6 +/- 0.2%, respectively). The results indicate that the polymeric micelle formulation enabled an increased and targeted delivery of RA to the PSU

  2. Self-assembled polymeric nanocarriers for the targeted delivery of retinoic acid to the hair follicle

    NASA Astrophysics Data System (ADS)

    Lapteva, Maria; Möller, Michael; Gurny, Robert; Kalia, Yogeshvar N.

    2015-11-01

    Acne vulgaris is a highly prevalent dermatological disease of the pilosebaceous unit (PSU). An inability to target drug delivery to the PSU results in poor treatment efficacy and the incidence of local side-effects. Cutaneous application of nanoparticulate systems is reported to induce preferential accumulation in appendageal structures. The aim of this work was to prepare stable polymeric micelles containing retinoic acid (RA) using a biodegradable and biocompatible diblock methoxy-poly(ethylene glycol)-poly(hexylsubstituted lactic acid) copolymer (MPEG-dihexPLA) and to evaluate their ability to deliver RA to skin. An innovative punch biopsy sample preparation method was developed to selectively quantify follicular delivery; the amounts of RA present were compared to those in bulk skin, (i.e. without PSU), which served as the control. RA was successfully incorporated into micelle nanocarriers and protected from photoisomerization by inclusion of Quinoline Yellow. Incorporation into the spherical, homogeneous and nanometer-scale micelles (dn < 20 nm) increased the aqueous solubility of RA by >400-fold. Drug delivery experiments in vitro showed that micelles were able to deliver RA to porcine and human skins more efficiently than Retin-A® Micro (0.04%), a marketed gel containing RA loaded microspheres, (7.1 +/- 1.1% vs. 0.4 +/- 0.1% and 7.5 +/- 0.8% vs. 0.8 +/- 0.1% of the applied dose, respectively). In contrast to a non-colloidal RA solution, Effederm® (0.05%), both the RA loaded MPEG-dihexPLA polymeric micelles (0.005%) and Retin-A® Micro (0.04%) displayed selectivity for delivery to the PSU with 2-fold higher delivery to PSU containing samples than to control samples. Moreover, the micelle formulation outperformed Retin-A® Micro in terms of delivery efficiency to PSU presenting human skin (10.4 +/- 3.2% vs. 0.6 +/- 0.2%, respectively). The results indicate that the polymeric micelle formulation enabled an increased and targeted delivery of RA to the PSU

  3. Effect of oleic acid modified polymeric bilayered nanoparticles on percutaneous delivery of spantide II and ketoprofen

    PubMed Central

    Shah, Punit; Desai, Pinaki; Singh, Mandip

    2011-01-01

    The objective of present study was to evaluate the effect of oleic acid modified polymeric bilayered nanoparticles (NPS) on combined delivery of two anti-inflammatory drugs, spantide II (SP) and ketoprofen (KP) on the skin permeation. NPS were prepared using poly(lactic-co-glycolic acid) (PLGA) and chitosan. SP and KP were encapsulated in different layers alone or/and in combination (KP-NPS, SP-NPS and SP+KP-NPS). The surface of NPS was modified with oleic acid (OA) (`Nanoease' technology) using an established procedure in the laboratory (KP-NPS-OA, SP-NPS-OA and SP+KP-NPS-OA). Fluorescent dyes (DiO and DID) containing surface modified (DiO-NPS-OA and DID-NPS-OA) and unmodified NPS (DiO-NPS and DID-NPS) were visualized in lateral rat skin sections using confocal microscopy and Raman confocal spectroscopy after skin permeation. In vitro skin permeation was performed in dermatomed human skin and HPLC was used to analyze the drug levels in different skin layers. Further, allergic contact dermatitis (ACD) model was used to evaluate the response of KP-NPS, SP-NPS, SP+KP-NPS, KP-NPS-OA, SP-NPS-OA and SP+KP-NPS-OA treatment in C57BL/6 mice. The fluorescence from OA modified NPS was observed upto depth of 240 μm and was significantly higher as compared to non-modified NPS. The amount of SP and KP retained in skin layers from OA modified NPS increased by several folds compare to unmodified NPS and control solution. In addition, the combination index value calculated from ACD response for solution suggested additive effect and moderate synergism for NPS-OA. Our results strongly suggest that surface modification of bilayered nanoparticles with oleic acid improved drug delivery to the deeper skin layers. PMID:22134117

  4. Recovery of uranium from phosphoric acid medium by polymeric composite beads encapsulating organophosphorus extractants

    SciTech Connect

    Singh, D.K.; Yadav, K.K.; Varshney, L.; Singh, H.

    2013-07-01

    The present study deals with the preparation and evaluation of the poly-ethersulfone (PES) based composite beads encapsulating synergistic mixture of D2EHPA and Cyanex 923 (at 4:1 mole ratio) for the separation of uranium from phosphoric acid medium. SEM was used for the characterization of the composite materials. Addition of 1% PVA (polyvinyl alcohol) improved the internal morphology and porosity of the beads. Additionally, microscopic examination of the composite bead confirmed central coconut type cavity surrounded by porous polymer layer of the beads through which exchange of metal ions take place. Effect of various experimental variables including aqueous acidity, metal ion concentration in aqueous feed, concentration of organic extractant inside the beads, extractant to polymer ratio, liquid to solid (L/S) ratio and temperature on the extraction of uranium was studied. Increase in acidity (1-6 M), L/S ratio (1- 10), metal ion concentration (0.2-3 g/L U{sub 3}O{sub 8}) and polymer to extractant ratio (1:4 -1:10) led to decrease in extraction of uranium. At 5.5 M (comparable to wet process phosphoric acid concentration) the extraction of uranium was about 85% at L/S ratio 5. Increase in extractant concentration inside the bead resulted in enhanced extraction of metal ion. Increase in temperature in the range of 30 to 50 Celsius degrees increased the extraction, whereas further increase to 70 C degrees led to the decrease in extraction of uranium. Amongst various reagents tested, stripping of uranium was quantitative by 12% Na{sub 2}CO{sub 3} solution. Polymeric beads were found to be stable and reusable up-to 10 cycles of extraction/stripping. (authors)

  5. The stability and controlled release of I-ascorbic acid encapsulated in poly (ethyl-2-cyanoacrylate) nanocapsules prepared by interfacial polymerization of water-in-oil microemulsions.

    PubMed

    Zhang, Su-Ning; Chen, Tao; Guo, Yi-Guang; Zhang, Jian; Song, Xiaoqiu; Zhou, Lei

    2015-01-01

    The L-ascorbic acid (AA) was encapsulated into biodegradable and biocompatible poly(ethyl-2-cyanoacrylate) (PECA) nanocapsules by interfacial polymerization of water-in-oil (W/O) microemulsions. The influences of surfactant concentration, pH value of the dispersed aqueous phase, and W/O ratio on nanocapsule size were discussed. The stability and in vitro release of encapsulated AA were also investigated. The results show that nanocapsules could be obtained under the conditions with low pH value, high fraction of aqueous phase, and appropriate surfactant concentration. The encapsulated AA was protected by nanocapsules from oxidation and presented superior storage stability in aqueous medium than pure AA. Releasing AA from the inner core of nanocapsules could be controlled by adjusting the enzyme hydrolysis extent of the PECA wall. PMID:26665980

  6. Fatty acids of lipid fractions in extracellular polymeric substances of activated sludge flocs.

    PubMed

    Conrad, Arnaud; Suutari, Merja Kontro; Keinänen, Minna M; Cadoret, Aurore; Faure, Pierre; Mansuy-Huault, Laurence; Block, Jean-Claude

    2003-10-01

    Phospholipid (PL), glycolipid (GL), and neutral lipid (NL) FA, and the lipopolysaccharide 2- and 3-hydroxy (LPS 2-OH and 3-OH) FA of activated sludges and extracted extracellular polymeric substances (EPS) were determined on samples collected from two wastewater treatment plants. EPS extracted from sludges by means of sonication and cation exchange contained proteins (43.4%), humic-like substances (11.5%), nucleic acids (10.9%), carbohydrates (9.9%), and lipid-bound FA (1.8%). The lipids associated with EPS were composed of GL, PL, NL, and LPS acids in proportions of 61, 21, 16, and 2%, respectively. The profiles of lipid-bound FA in activated sludges and EPS were similar (around 85 separate FA were identified). The FA signatures observed can be attributed to the likely presence of yeasts, fungi, sulfate-reducing bacteria, gram-positive and gram-negative bacteria, and, in lesser quantities, mycobacteria. Comparison of data from the dates of sampling (January and September) showed that there were more unsaturated PLFA in the EPS extracted from the activated sludges sampled in January. This observation could be partly related to microorganism adaptation to temperature variations. The comparison between two wastewater treatment plants showed that the FA profiles were similar, although differences in microbial community structure were also seen. Most of the FA in sludges had an even number of carbons. PMID:14669975

  7. Characterization of thin-film deposition in a pulsed acrylic acid polymerizing discharge

    SciTech Connect

    Voronin, Sergey A.; Bradley, James W.; Fotea, Catalin; Zelzer, Mischa; Alexander, Morgan R.

    2007-07-15

    In this study, thin-film deposition in a pulsed rf polymerizing discharge (13.56 MHz) struck in acrylic acid has been investigated by mass spectrometry, x-ray photoelectron spectroscopy, and quartz crystal microbalance techniques. The experiment was conducted at a fixed acrylic acid pressure of 1.3 Pa and 'on' pulse duration of 0.1 ms, whereas the 'off' time was varied between 0 and 20 ms. The rf input power in the 'on' time and gas flow rate were varied between 10 and 50 W and 1.5 and 4.8 sccm (sccm denotes cubic centimeter per minute at STP), respectively. These changes of the discharge conditions resulted in large-scale progressive variations in film and gas-phase plasma composition. In particular, the -COOH functionality of the monomer was increasingly retained in the plasma-generated thin films as the duty cycle was lowered (i.e., with lowered time-averaged powers). The monomer retention reached its maximum value of 66% for 'off' times exceeding 5 ms, when the discharge was operating in the power-deficient regime. The results show that the film deposition rate is a strong function of the monomer flow rate, whereas -COOH retention is correlated to the amount of unfragmented monomer in the plasma, controlled by the applied power.

  8. Effects of iron on growth, antioxidant enzyme activity, bound extracellular polymeric substances and microcystin production of Microcystis aeruginosa FACHB-905.

    PubMed

    Wang, Chao; Wang, Xun; Wang, Peifang; Chen, Bin; Hou, Jun; Qian, Jin; Yang, Yangyang

    2016-10-01

    Toxic cyanobacterial blooms have occurred in various water bodies during recent decades and made serious health hazards to plants, animals and humans. Iron is an important micronutrient for algal growth and recently, the concentration of which has increased remarkably in freshwaters. In this paper, the cyanobacterium Microcystis aeruginosa FACHB-905 was cultivated under non-iron (0μM), iron-limited (10μM) and iron-replete (100μM) conditions to investigate the effects of iron on growth, antioxidant enzyme activity, EPS and microcystin production. The results showed that algal cell density and chlorophyll-a content were maximal at the highest iron concentration. Antioxidant enzymes activity increased notably under all three conditions in the early stage of experiment, of which the SOD activity recovered soon from oxidative stress in 10μM group. The productions of some protein-like substances and humic acid-like substances of bound EPS were inhibited in iron-containing groups in the early stage of experiment while promoted after the adaptation period of Microcystis aeruginosa. Iron addition is a factor affecting the formation of cyanobacterial blooms through its impact on the content of LB-EPS and the composition of TB-EPS. The intracellular MC-LR concentration and the productivity potential of MC-LR were the lowest in 0μM group and highest in 10μM group. No obvious extracellular release of MC-LR was observed during the cultivation time. Therefore, iron addition can promote the physiological activities of M. aeruginosa, but a greater harm could be brought into environment under iron-limited (10μM) condition than under iron-replete (100μM) condition. PMID:27337497

  9. Kinetic characteristics of polygalacturonase enzymes hydrolyzing galacturonic acid oligomers using isothermal titration calorimetry

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Polygalacturonase enzymes hydrolyze the polygalacturonic acid chains found in pectin. Interest in polygalacturonase enzymes continues as they are useful in a number of industrial processes and conversely, detrimental, as they are involved in maceration of economically important crops. While a good...

  10. Effects of Non-Natural Amino Acid Incorporation into the Enzyme Core Region on Enzyme Structure and Function

    PubMed Central

    Wong, H. Edward; Kwon, Inchan

    2015-01-01

    Techniques to incorporate non-natural amino acids (NNAAs) have enabled biosynthesis of proteins containing new building blocks with unique structures, chemistry, and reactivity that are not found in natural amino acids. It is crucial to understand how incorporation of NNAAs affects protein function because NNAA incorporation may perturb critical function of a target protein. This study investigates how the site-specific incorporation of NNAAs affects catalytic properties of an enzyme. A NNAA with a hydrophobic and bulky sidechain, 3-(2-naphthyl)-alanine (2Nal), was site-specifically incorporated at six different positions in the hydrophobic core of a model enzyme, murine dihydrofolate reductase (mDHFR). The mDHFR variants with a greater change in van der Waals volume upon 2Nal incorporation exhibited a greater reduction in the catalytic efficiency. Similarly, the steric incompatibility calculated using RosettaDesign, a protein stability calculation program, correlated with the changes in the catalytic efficiency. PMID:26402667

  11. Lipoxygenase, a key enzyme in bioconversion of linoleic acid into trihydroxy-octadecenoic acid by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipoxygenases catalyze the oxidation of unsaturated fatty acids with a (1Z,4Z)-pentadiene structure leading to the formation of conjugated (Z,E)-hydroperoxydienoic acids, which in turn result in production of hydroxy lipid. These enzymes are widely distributed in plants, animals, and microorganisms...

  12. Radical-Mediated Enzymatic Polymerizations.

    PubMed

    Zavada, Scott R; Battsengel, Tsatsral; Scott, Timothy F

    2016-01-01

    Polymerization reactions are commonly effected by exposing monomer formulations to some initiation stimulus such as elevated temperature, light, or a chemical reactant. Increasingly, these polymerization reactions are mediated by enzymes--catalytic proteins--owing to their reaction efficiency under mild conditions as well as their environmental friendliness. The utilization of enzymes, particularly oxidases and peroxidases, for generating radicals via reduction-oxidation mechanisms is especially common for initiating radical-mediated polymerization reactions, including vinyl chain-growth polymerization, atom transfer radical polymerization, thiol-ene step-growth polymerization, and polymerization via oxidative coupling. While enzyme-mediated polymerization is useful for the production of materials intended for subsequent use, it is especially well-suited for in situ polymerizations, where the polymer is formed in the place where it will be utilized. Such polymerizations are especially useful for biomedical adhesives and for sensing applications. PMID:26848652

  13. Horseradish peroxidase-catalyzed synthesis of poly(thiophene-3-boronic acid) biocomposites for mono-/bi-enzyme immobilization and amperometric biosensing.

    PubMed

    Huang, Yi; Wang, Wen; Li, Zou; Qin, Xiaoli; Bu, Lijuan; Tang, Zhiyong; Fu, Yingchun; Ma, Ming; Xie, Qingji; Yao, Shouzhuo; Hu, Jiming

    2013-06-15

    We report here on a facile enzymatic polymerization protocol to prepare enzyme-poly(thiophene-3-boronic acid) (PTBA) polymeric biocomposites (PBCs) for high-performance mono-/bi-enzyme amperometric biosensing. Horseradish peroxidase (HRP)-catalyzed polymerization of thiophene-3-boronic acid (TBA) monomer was conducted in aqueous solution containing HRP (or plus glucose oxidase (GOx)) by either directly added or GOx-glucose generated oxidant H2O2. The mono-/bi-enzyme amperometric biosensors were prepared simply by casting the dialysis-isolated PBCs on Au-plated Au electrode (Auplate/Au), followed by coating with an outer-layer chitosan (CS) film. The boronic acid residues are capable of covalent bonding with enzyme at the glycosyl sites (boronic acid-diols interaction), which should less affect the enzymatic activity as compared with the common cases of covalent bonding at the peptide chains, and UV-vis spectrophotometric tests confirmed that the encapsulated HRP almost possesses its pristine enzymatic specific activity. The enzyme electrodes were studied by cyclic voltammetry, electrochemical impedance spectroscopy and chronoamperometry in the presence of Fe(CN)6(4-) mediator. The CS/HRP-PTBA/Auplate/Au electrode responded linearly to H2O2 concentration from 1 to 300 μM with a sensitivity of 390 μA mM(-1)cm(-2) and a limit of detection (LOD) of 0.1 μM. The bienzyme CS/GOx-HRP-PTBA(H2O2)/Auplate/Au electrode responded linearly to glucose concentration from 5 μM to 0.83 mM with a sensitivity of 75.1 μA mM(-1)cm(-2) and a LOD of 1 μM, and it is found here that the use of Fe(CN)6(4-) that can only efficiently mediate HRP favorably avoids the "unusual amperometric responses" observed when other mediators that can efficiently turn over both HRP and GOx are used. PMID:23391705

  14. Ketol-acid reductoisomerase enzymes and methods of use

    DOEpatents

    Govindarajan, Sridhar; Li, Yougen; Liao, Der-Ing; O'Keefe, Daniel P.; Minshull, Jeremy Stephen; Rothman, Steven Cary; Tobias, Alexander Vincent

    2016-07-12

    Provided herein are polypeptides having ketol-acid reductoisomerase activity as well as microbial host cells comprising such polypeptides. Polypeptides provided herein may be used in biosynthetic pathways, including, but not limited to, isobutanol biosynthetic pathways.

  15. Ion chromatographic analysis of tetracyclines using polymeric column and acidic eluent.

    PubMed

    Ding, X; Mou, S

    2000-11-01

    High-performance ion chromatography (HPIC) is first successfully used to analyze tetracycline antibiotics (TCs) in this work. The TCs are well separated on a solvent compatible polymeric cation-exchange column within 12 min. Isocratic elution with acetonitrile-hydrochloride is very advantageous for routine analysis. HPIC may be seen as a specific variant of the more common high-performance liquid chromatography (HPLC) for water-soluble and polar pharmaceuticals with low hydrophobicity. The detection limits (signal-to-noise ratio=3:1) of oxytetracycline (OTC), tetracycline (TC), chlortetracycline (CTC), doxycycline (DC) are 10, 10, 20 and 20 microg l(-1), respectively. Samples are prepared by vortex mixing with an ethylenediaminetetraacetic acid disodium salt (Na2EDTA)-McIlvaine buffer (pH 4.0) solution and the mixture filtrates through a molecular weight cut-off filter. The method has been successfully applied to monitor the OTC removal rate through every reactor in the process of OTC manufacturing wastewater treatment by bio-chemical technology. It is also applicable to determine the TCs residues in milk and milk powder with satisfying results. PMID:11128204

  16. Antifouling polyethersulfone hemodialysis membranes incorporated with poly (citric acid) polymerized multi-walled carbon nanotubes.

    PubMed

    Abidin, Muhammad Nidzhom Zainol; Goh, Pei Sean; Ismail, Ahmad Fauzi; Othman, Mohd Hafiz Dzarfan; Hasbullah, Hasrinah; Said, Noresah; Kadir, Siti Hamimah Sheikh Abdul; Kamal, Fatmawati; Abdullah, Mohd Sohaimi; Ng, Be Cheer

    2016-11-01

    Poly (citric acid)-grafted-MWCNT (PCA-g-MWCNT) was incorporated as nanofiller in polyethersulfone (PES) to produce hemodialysis mixed matrix membrane (MMM). Citric acid monohydrate was polymerized onto the surface of MWCNTs by polycondensation. Neat PES membrane and PES/MWCNTs MMMs were fabricated by dry-wet spinning technique. The membranes were characterized in terms of morphology, pure water flux (PWF) and bovine serum albumin (BSA) protein rejection. The grafting yield of PCA onto MWCNTs was calculated as 149.2%. The decrease of contact angle from 77.56° to 56.06° for PES/PCA-g-MWCNTs membrane indicated the increase in surface hydrophilicity, which rendered positive impacts on the PWF and BSA rejection of the membrane. The PWF increased from 15.8Lm(-2)h(-1) to 95.36Lm(-2)h(-1) upon the incorporation of PCA-g-MWCNTs due to the attachment of abundant hydrophilic groups that present on the MWCNTs, which have improved the affinity of membrane towards the water molecules. For protein rejection, the PES/PCA-g-MWCNTs MMM rejected 95.2% of BSA whereas neat PES membrane demonstrated protein rejection of 90.2%. Compared to commercial PES hemodialysis membrane, the PES/PCA-g-MWCNTs MMMs showed less flux decline behavior and better PWF recovery ratio, suggesting that the membrane antifouling performance was improved. The incorporation of PCA-g-MWCNTs enhanced the separation features and antifouling capabilities of the PES membrane for hemodialysis application. PMID:27524052

  17. Polymeric Nucleic Acid Vehicles Exploit Active Inter-Organelle Trafficking Mechanisms

    PubMed Central

    Fichter, Katye M.; Ingle, Nilesh. P.; McLendon, Patrick M.; Reineke, Theresa M.

    2013-01-01

    Materials that self-assemble with nucleic acids into nanocomplexes (polyplexes) are widely used in many fundamental biological and biomedical experiments. However, understanding the intracellular transport mechanisms of these vehicles remains a major hurdle in their effective usage. Here, we investigate two polycation models, Glycofect, (which slowly degrades via hydrolysis) and linear PEI, (which does not rapidly hydrolyze) to determine the impact of polymeric structure on intracellular trafficking. Cells transfected using Glycofect underwent increasing transgene expression over the course of 40 h, and remained benign over the course of 7 days. Transgene expression in cells transfected with PEI peaked at 16 h post-transfection and resulted in less than 10% survival after 7 days. While saccharide-containing Glycofect has a higher buffering capacity than PEI, polyplexes created with Glycofect demonstrate more sustained endosomal release, possibly suggesting an additional or alternative delivery mechanism to the classical “proton sponge mechanism”. PEI appeared to promote release of DNA from acidic organelles more than Glycofect. Immunofluorescence images indicate that both Glycofect and linear PEI traffic oligodeoxynucleotides (ODNs) to the Golgi and endoplasmic reticulum, which may be a route taken for nuclear delivery. However, Glycofect polyplexes demonstrated higher colocalization with the ER than PEI polyplexes and colocalization experiments indicate retrograde transport of polyplexes via COP I vesicles from the Golgi to the ER. We conclude that slow release and unique trafficking behaviors of Glycofect polyplexes may be due to the presence of saccharide units and the degradable nature of the polymer, allowing more efficacious and benign delivery. PMID:23234474

  18. Metabolic Transformation of Mevalonic Acid by an Enzyme System from Peas 1

    PubMed Central

    Pollard, C. J.; Bonner, J.; Haagen-Smit, A. J.; Nimmo, C. C.

    1966-01-01

    En enzyme system has been found in peas which converts mevalonic acid to isoprenoid compounds. Among the intermediates in such conversion are mevalonic acid-5-phosphate and pyrophosphate, isopentenyl pyrophosphate and dimethylallylpyrophosphate. Among the products formed by the system are the pyrophosphates of geraniol, farnesol, nerolidol and higher isoprenoid alcohols. PMID:16656233

  19. Molecularly imprinted polymer for caffeic acid by precipitation polymerization and its application to extraction of caffeic acid and chlorogenic acid from Eucommia ulmodies leaves.

    PubMed

    Miura, Chitose; Matsunaga, Hisami; Haginaka, Jun

    2016-08-01

    Molecularly imprinted polymers (MIPs) for caffeic acid (CA) were prepared using 4-vinylpyridine and methacrylamide (MAM) as functional monomers, divinylbenzene as a crosslinker and acetonitrile-toluene (3:1, v/v) as a porogen by precipitation polymerization. The use of MAM as the co-monomer resulted in the formation of microsphere MIPs and non-imprinted polymers (NIPs) with ca. 3- and 5-μm particle diameters, respectively. Binding experiments and Scatchard analyses revealed that the binding capacity and affinity of the MIP to CA are higher than those of the NIP. The retention and molecular-recognition properties of the prepared MIPs were evaluated using water-acetonitrile and sodium phosphate buffer-acetonitrile as mobile phases in hydrophilic interaction chromatography (HILIC) and reversed-phase chromatography, respectively. In HILIC mode, the MIP showed higher molecular-recognition ability for CA than in reversed-phase mode. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CA on the MIP in HILIC mode, while hydrogen bonding and hydrophobic interactions seem to work for the recognition of CA in reversed-phase mode. The MIP had a specific molecular-recognition ability for CA in HILIC mode, while other structurally related compounds, such as chlorogenic acid (CGA), gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP was successfully applied for extraction of CA and CGA in the leaves of Eucommia ulmodies in HILIC mode. PMID:26776340

  20. A Simple and Efficient Synthesis of an Acid-labile Polyphosphoramidate by Organobase-catalyzed Ring-Opening Polymerization and Transformation to Polyphosphoester Ionomers by Acid Treatment

    PubMed Central

    Zhang, Shiyi; Wang, Hai; Shen, Yuefei; Zhang, Fuwu; Seetho, Kellie; Zou, Jiong; Taylor, John-Stephen A.; Dove, Andrew P.; Wooley, Karen L.

    2013-01-01

    The direct synthesis of an acid-labile polyphosphoramidate by organobase-catalyzed ring-opening polymerization and an overall two-step preparation of polyphosphodiester ionomers (PPEI) by acid-assisted cleavage of the phosphoramidate bonds along the backbone of the polyphosphoramidate were developed in this study. The ultrafast organobase-catalyzed ring-opening polymerization of a cyclic phospholane methoxyethyl amidate monomer initiated by benzyl alcohol allowed for the preparation of well-defined polyphosphoramidates (PPA) with predictable molecular weights, narrow molecular weight distributions (PDI<1.10), and well-defined chain ends. Cleavage of the acid-labile phosphoramidate bonds on the polyphosphoramidate repeat units was evaluated under acidic conditions over a pH range of 1–5, and the complete hydrolysis produced polyphosphodiesters. The thermal properties of the resulting polyphosphoester ionomer acid and polyphosphoester ionomer sodium salt exhibited significant thermal stability. The parent PPA and both forms of the PPEIs showed low cytotoxicities toward HeLa cells and RAW 264.7 mouse macrophage cells. The synthetic methodology developed here has enriched the family of water-soluble polymers prepared by rapid and convenient organobase-catalyzed ring-opening polymerizations and straightforward chemical medication reactions, which are designed to be hydrolytically degradable and have promise for numerous biomedical and other applications. PMID:23997276

  1. Cannabidiolic-acid synthase, the chemotype-determining enzyme in the fiber-type Cannabis sativa.

    PubMed

    Taura, Futoshi; Sirikantaramas, Supaart; Shoyama, Yoshinari; Yoshikai, Kazuyoshi; Shoyama, Yukihiro; Morimoto, Satoshi

    2007-06-26

    Cannabidiolic-acid (CBDA) synthase is the enzyme that catalyzes oxidative cyclization of cannabigerolic-acid into CBDA, the dominant cannabinoid constituent of the fiber-type Cannabis sativa. We cloned a novel cDNA encoding CBDA synthase by reverse transcription and polymerase chain reactions with degenerate and gene-specific primers. Biochemical characterization of the recombinant enzyme demonstrated that CBDA synthase is a covalently flavinylated oxidase. The structural and functional properties of CBDA synthase are quite similar to those of tetrahydrocannabinolic-acid (THCA) synthase, which is responsible for the biosynthesis of THCA, the major cannabinoid in drug-type Cannabis plants. PMID:17544411

  2. Discrimination of acidic and alkaline enzyme using Chou's pseudo amino acid composition in conjunction with probabilistic neural network model.

    PubMed

    Khan, Zaheer Ullah; Hayat, Maqsood; Khan, Muazzam Ali

    2015-01-21

    Enzyme catalysis is one of the most essential and striking processes among of all the complex processes that have evolved in living organisms. Enzymes are biological catalysts, which play a significant role in industrial applications as well as in medical areas, due to profound specificity, selectivity and catalytic efficiency. Refining catalytic efficiency of enzymes has become the most challenging job of enzyme engineering, into acidic and alkaline. Discrimination of acidic and alkaline enzymes through experimental approaches is difficult, sometimes impossible due to lack of established structures. Therefore, it is highly desirable to develop a computational model for discriminating acidic and alkaline enzymes from primary sequences. In this study, we have developed a robust, accurate and high throughput computational model using two discrete sample representation methods Pseudo amino acid composition (PseAAC) and split amino acid composition. Various classification algorithms including probabilistic neural network (PNN), K-nearest neighbor, decision tree, multi-layer perceptron and support vector machine are applied to predict acidic and alkaline with high accuracy. 10-fold cross validation test and several statistical measures namely, accuracy, F-measure, and area under ROC are used to evaluate the performance of the proposed model. The performance of the model is examined using two benchmark datasets to demonstrate the effectiveness of the model. The empirical results show that the performance of PNN in conjunction with PseAAC is quite promising compared to existing approaches in the literature so for. It has achieved 96.3% accuracy on dataset1 and 99.2% on dataset2. It is ascertained that the proposed model might be useful for basic research and drug related application areas. PMID:25452135

  3. Electrodialysis of Sulfuric Acid with Cation-Exchange Membranes Prepared by Electron-Beam-Induced Graft Polymerization

    NASA Astrophysics Data System (ADS)

    Asari, Yuki; Shoji, Nobuyoshi; Miyoshi, Kazuyoshi; Umeno, Daisuke; Saito, Kyoichi

    Strongly acidic cation-exchange membranes were prepared by the electron-beam-induced graft polymerization of glycidyl methacrylate onto a high-density polyethylene film with a thickness of 35 μm and the subsequent conversion of the resulting epoxy group into a sulfonic acid group. The resulting cation-exchange membranes with various ion-exchange capacities or sulfonic acid group densities ranging from 1.9 to 2.7 mmol/g were applied to the enrichment of 0.50 mol/L sulfuric acid by electrodialysis. Concentrated sulfuric acids at concentrations of 1.4 to 2.9 mol/L were obtained in the concentrate chamber during the electrodialysis operated at 30 mA/cm2 and 298 K, using a pair of this cation-exchange membrane and a commercially available anion-exchange membrane.

  4. 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.

  5. Clay catalyzed polymerization of amino acid adenylates and its relationship to biochemical reactions

    NASA Technical Reports Server (NTRS)

    Paecht-Horowitz, M.

    1978-01-01

    The adsorption and polymerization of alanine adenylate on montmorillonite at pH 7 when either its interspacial faces or its edger are blocked by an excess of histidine or sodium hexametaphosphate was investigated. Results indicate that alanine adenylate can be adsorbed any place on the interspacial spaces of the clay; however, adsorption of its phosphate part, which is limited to the edges of the clay, is necessary for polymerization to occur. As a result, polymerization takes place only at sites on the interspacial faces bordering the edges.

  6. Radical polymerization of N-vinylpyrrolidone in the presence of syndiotactic poly(methacrylic acid) templates. [Gamma ray

    SciTech Connect

    Koetsier, D.W.; Tan, Y.Y.; Challa, G.

    1980-06-01

    Radical polymerization of N-vinylpyrrolidone along poly(methacrylic acid) templates of high syndiotactic content was followed dilatometrically in dimethylformamide, which was used as solvent. The effects of template concentration, template molar mass, and temperature on polymerization rate and average molar mass of the formed polyvinylpyrrolidone (PVP) were examined. Template concentrations were varied around the critical concentration for homogeneous segmental distribution, C. Below this concentration, template coils can act as separate microreactors wherein growing PVP radicals exhibit maximum rate enhancement, i.e., relative rate upsilon/sub R/ = upsilon/sub R max/. In the free solution, blank polymerization occurs, i.e., upsilon/sub R/ = 1. Consequently, upsilon/sub R/ can be approximated by the equation ..nu../sub R/ = phi..nu../sub R/max/ + (1 - phi), where phi represents the volume fraction occupied by template coils. The slight increase in ..nu../sub R/ and PVP molar mass with the template chain length is supposed to be caused by the influence of translational diffusion on the termination step. Over the investigated temperature range of 50 to 70/sup 0/C, the activation energy and entropy were almost identical for blank and template polymerization. An expected decrease of ..delta..E not equal to and ..delta..S not equal to in template systems is supposed to be compensated by the effects of desolvation of the template macromolecules during the propagation step.

  7. A Condensing Enzyme from the Seeds of Lesquerella fendleri That Specifically Elongates Hydroxy Fatty Acids1

    PubMed Central

    Moon, Hangsik; Smith, Mark A.; Kunst, Ljerka

    2001-01-01

    Lesquerella fendleri seed oil contains up to 60% hydroxy fatty acids, nearly all of which is the 20-carbon hydroxy fatty acid lesquerolic acid (d-14-hydroxyeicos-cis-11-enoic acid). Previous work suggested that lesquerolic acid in L. fendleri was formed by the elongation of the 18-carbon hydroxy fatty acid, ricinoleic acid. To identify a gene encoding the enzyme involved in hydroxy fatty acid elongation, an L. fendleri genomic DNA library was screened using the coding region of the Arabidopsis Fatty Acid Elongation1 gene as a probe. A gene, LfKCS3, with a high sequence similarity to known very long-chain fatty acid condensing enzymes, was isolated. LfKCS3 has a 2,062-bp open reading frame interrupted by two introns, which encodes a polypeptide of 496 amino acids. LfKCS3 transcripts accumulated only in the embryos of L. fendleri and first appeared in the early stages of development. Fusion of the LfKCS3 promoter to the uidA reporter gene and expression in transgenic Arabidopsis resulted in a high level of β-glucuronidase activity exclusively in developing embryos. Seeds of Arabidopsis plants transformed with LfKCS3 showed no change in their very long-chain fatty acid content. However, when these Arabidopsis plants were crossed with the transgenic plants expressing the castor oleate 12-hydroxylase, significant amounts of 20-carbon hydroxy fatty acids accumulated in the seed, indicating that the LfKCS3 condensing enzyme specifically catalyzes elongation of 18-carbon hydroxy fatty acids. PMID:11743108

  8. Insolubilization process increases enzyme stability

    NASA Technical Reports Server (NTRS)

    Billingham, J.; Lyn, J.

    1971-01-01

    Enzymes complexed with polymeric matrices contain properties suggesting application to enzyme-controlled reactions. Stability of insolubilized enzyme derivatives is markedly greater than that of soluble enzymes and physical form of insolubilized enzymes is useful in column and batch processes.

  9. The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

    SciTech Connect

    Viola, Ronald E.; Faehnle, Christopher R.; Blanco, Julio; Moore, Roger A.; Liu, Xuying; Arachea, Buenafe T.; Pavlovsky, Alexander G.

    2013-02-28

    The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate {beta}-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

  10. The Catalytic Machinery of a Key Enzyme in Amino Acid Biosynthesis

    PubMed Central

    Viola, Ronald E.; Faehnle, Christopher R.; Blanco, Julio; Moore, Roger A.; Liu, Xuying; Arachea, Buenafe T.; Pavlovsky, Alexander G.

    2011-01-01

    The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes. PMID:22332000

  11. The catalytic machinery of a key enzyme in amino Acid biosynthesis.

    PubMed

    Viola, Ronald E; Faehnle, Christopher R; Blanco, Julio; Moore, Roger A; Liu, Xuying; Arachea, Buenafe T; Pavlovsky, Alexander G

    2011-01-01

    The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate β-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes. PMID:22332000

  12. Hyaluronic acid-functionalized polymeric nanoparticles for colon cancer-targeted combination chemotherapy

    NASA Astrophysics Data System (ADS)

    Xiao, Bo; Han, Moon Kwon; Viennois, Emilie; Wang, Lixin; Zhang, Mingzhen; Si, Xiaoying; Merlin, Didier

    2015-10-01

    Nanoparticle (NP)-based combination chemotherapy has been proposed as an effective strategy for achieving synergistic effects and targeted drug delivery for colon cancer therapy. Here, we fabricated a series of hyaluronic acid (HA)-functionalized camptothecin (CPT)/curcumin (CUR)-loaded polymeric NPs (HA-CPT/CUR-NPs) with various weight ratios of CPT to CUR (1 : 1, 2 : 1 and 4 : 1). The resultant spherical HA-CPT/CUR-NPs had a desirable particle size (around 289 nm), relative narrow size distribution, and slightly negative zeta potential. These NPs exhibited a simultaneous sustained release profile for both drugs throughout the time frame examined. Subsequent cellular uptake experiments demonstrated that the introduction of HA to the NP surface endowed NPs with colon cancer-targeting capability and markedly increased cellular uptake efficiency compared with chitosan-coated NPs. Importantly, the combined delivery of CPT and CUR in one HA-functionalized NP exerted strong synergistic effects. HA-CPT/CUR-NP (1 : 1) showed the highest antitumor activity among the three HA-CPT/CUR-NPs, resulting in an extremely low combination index. Collectively, our findings indicate that this HA-CPT/CUR-NP can be exploited as an efficient formulation for colon cancer-targeted combination chemotherapy.Nanoparticle (NP)-based combination chemotherapy has been proposed as an effective strategy for achieving synergistic effects and targeted drug delivery for colon cancer therapy. Here, we fabricated a series of hyaluronic acid (HA)-functionalized camptothecin (CPT)/curcumin (CUR)-loaded polymeric NPs (HA-CPT/CUR-NPs) with various weight ratios of CPT to CUR (1 : 1, 2 : 1 and 4 : 1). The resultant spherical HA-CPT/CUR-NPs had a desirable particle size (around 289 nm), relative narrow size distribution, and slightly negative zeta potential. These NPs exhibited a simultaneous sustained release profile for both drugs throughout the time frame examined. Subsequent cellular uptake experiments

  13. Conjugation of Hyaluronic Acid onto Surfaces via the Interfacial Polymerization of Dopamine to Prevent Protein Adsorption.

    PubMed

    Huang, Renliang; Liu, Xia; Ye, Huijun; Su, Rongxin; Qi, Wei; Wang, Libing; He, Zhimin

    2015-11-10

    A versatile, convenient, and cost-effective method that can be used for grafting antifouling materials onto different surfaces is highly desirable in many applications. Here, we report the one-step fabrication of antifouling surfaces via the polymerization of dopamine and the simultaneous deposition of anionic hyaluronic acid (HA) on Au substrates. The water contact angle of the Au surfaces decreased from 84.9° to 24.8° after the attachment of a highly uniform polydopamine (PDA)/HA hybrid film. The results of surface plasmon resonance analysis showed that the Au-PDA/HA surfaces adsorbed proteins from solutions of bovine serum albumin, lysozyme, β-lactoglobulin, fibrinogen, and soybean milk in ultralow or low amounts (4.8-31.7 ng/cm(2)). The hydrophilicity and good antifouling performance of the PDA/HA surfaces is attributable to the HA chains that probably attached onto their upper surface via hydrogen bonding between PDA and HA. At the same time, the electrostatic repulsion between PDA and HA probably prevents the aggregation of PDA, resulting in the formation of a highly uniform PDA/HA hybrid film with the HA chains (with a stretched structure) on the upper surface. We also developed a simple method for removing this PDA/HA film and recycling the Au substrates by using an aqueous solution of NaOH as the hydrolyzing agent. The Au surface remained undamaged, and a PDA/HA film could be redeposited on the surface, with the surface exhibiting good antifouling performance even after 10 such cycles. Finally, it was found that this grafting method is applicable to other substrates, including epoxy resins, polystyrene, glass, and steel, owing to the strong adhesion of PDA with these substrates. PMID:26488547

  14. Polymerization of acrylamide at acid pH using uranyl nitrate

    SciTech Connect

    Deshpande, V.V.; Bodhe, A.M.; Pawar, H.S.; Vartak, H.G.

    1986-03-01

    A new photopolymerizing reagent, uranyl nitrate, is used for the polymerization of acrylamide gels at low pH. The amount of uranyl nitrate (0.2 mg/ml) required for the polymerization of gels at pH 3.0 is considerably less than that of persulfate (7 mg/ml). Use of this reagent obviates the need for the removal of excess of persulfate by preelectrophoresis. The electrophoretic separation of basic proteins in uranium-polymerized gels showed faster movement and better resolution of proteins and proved the gels to be versatile, uniform, and reproducible. Electrophoresis of trypsin in these gels does not affect the enzymatic activity. The catalyst can also be used for the polymerization of gels containing 3 M urea.

  15. Radical-Mediated Enzymatic Polymerizations

    PubMed Central

    Zavada, Scott R.; Battsengel, Tsatsral; Scott, Timothy F.

    2016-01-01

    Polymerization reactions are commonly effected by exposing monomer formulations to some initiation stimulus such as elevated temperature, light, or a chemical reactant. Increasingly, these polymerization reactions are mediated by enzymes―catalytic proteins―owing to their reaction efficiency under mild conditions as well as their environmental friendliness. The utilization of enzymes, particularly oxidases and peroxidases, for generating radicals via reduction-oxidation mechanisms is especially common for initiating radical-mediated polymerization reactions, including vinyl chain-growth polymerization, atom transfer radical polymerization, thiol–ene step-growth polymerization, and polymerization via oxidative coupling. While enzyme-mediated polymerization is useful for the production of materials intended for subsequent use, it is especially well-suited for in situ polymerizations, where the polymer is formed in the place where it will be utilized. Such polymerizations are especially useful for biomedical adhesives and for sensing applications. PMID:26848652

  16. Oxidase-peroxidase enzymes of Datura innoxia. Oxidation of formylphenylacetic acid ethyl ester.

    PubMed Central

    Kalyanaraman, V S; Mahadevan, S; Kumar, S A

    1975-01-01

    An enzyme system from Datura innoxia roots oxidizing formylphenylacetic acid ethyl ester was purified 38-fold by conventional methods such as (NH4)2SO4 fractionation, negative adsorption on alumina Cy gel and chromatography on DEAE-cellulose. The purified enzyme was shown to catalyse the stoicheiometric oxidation of formylphenylacetic acid ethyl ester to benzoylformic acid ethyl ester and formic acid, utilizing molecular O2. Substrate analogues such as phenylacetaldehyde and phenylpyruvate were oxidized at a very low rate, and formylphenylacetonitrile was an inhilating agents, cyanide, thiol compounds and ascorbic acid. This enzyme was identical with an oxidase-peroxidase isoenzyme. Another oxidase-peroxidase isoenzyme which separated on DEAE-chromatography also showed formylphenylacetic acid ethyl ester oxidase activity, albeit to a lesser extent. The properties of the two isoenzymes of the oxidase were compared and shown to differ in their oxidation and peroxidation properties. The oxidation of formylphenylacetic acid ethyl ester was also catalysed by horseradish peroxidase. The Datura isoenzymes exhibited typical haemoprotein spectra. The oxidation of formylphenylacetic acid ethyl ester was different from other peroxidase-catalysed reactions in not being activated by either Mn2+ or monophenols. The oxidation was inhibited by several mono- and poly-phenols and by catalase. A reaction mechanism for the oxidation is proposed. PMID:997

  17. Antioxidant activity and enzyme inhibition of phenolic acids from fermented rice bran with fungus Rizhopus oryzae.

    PubMed

    Schmidt, Cristiano G; Gonçalves, Letícia M; Prietto, Luciana; Hackbart, Helen S; Furlong, Eliana B

    2014-03-01

    The solid-state fermentation (SSF) has been employed as a form making available a higher content of functional compounds from agroindustrial wastes. In this work, the effect of SSF with the Rhizopus oryzae fungus on the phenolic acid content of rice bran was studied. Phenolic extracts derived from rice bran and fermented rice bran were evaluated for their ability to reduce free radical 1,1-diphenyl-2-picrihidrazil (DPPH) and for the ability to inhibit the enzymes peroxidase and polyphenol oxidase. The phenolic compound content increased by more than two times with fermentation. A change in the content of phenolic acids was observed, with ferulic acid presenting the greatest increase with the fermentation, starting from 33μg/g in rice bran and reaching 765μg/g in the fermented bran. [corrected]. The phenolic extracts showed an inhibition potential for DPPH and for the peroxidase enzyme, however did not inhibit the polyphenol oxidase enzyme. PMID:24176356

  18. GFP Reporter Screens for the Engineering of Amino Acid Degrading Enzymes from Libraries Expressed in Bacteria

    PubMed Central

    Paley, Olga; Agnello, Giulia; Cantor, Jason; Yoo, Tae Hyun; Georgiou, George; Stone, Everett

    2014-01-01

    There is significant interest in engineering human amino acid degrading enzymes as non-immunogenic chemotherapeutic agents. We describe a high-throughput fluorescence activated cell sorting (FACS) assay for detecting the catalytic activity of amino acid degrading enzymes in bacteria, at the single cell level. This assay relies on coupling the synthesis of the GFP reporter to the catalytic activity of the desired amino acid degrading enzyme in an appropriate E. coli genetic background. The method described here allows facile screening of much larger libraries (106–107) than was previously possible. We demonstrate the application of this technique in the screening of libraries of bacterial and human asparaginases and also for the catalytic optimization of an engineered human methionine gamma lyase. PMID:23423887

  19. Enzyme-entrapped mesoporous silica for treatment of uric acid disorders.

    PubMed

    Muthukoori, Shanthini; Narayanan, Naagarajan; Chandra, Manuguri Sesha Sarath; Sethuraman, Swaminathan; Krishnan, Uma Maheswari

    2013-05-01

    Gout is an abnormality in the body resulting in the accumulation of uric acid mainly in joints. Dissolution of uric acid crystals into soluble allantoin by the enzyme uricase might provide a better alternative for the treatment of gout. This work aims to investigate the feasibility of a transdermal patch loaded with uricase for better patient compliance. Mesoporous silica (SBA-15) was chosen as the matrix for immobilisation of uricase. Highly oriented mesoporous SBA-15 was synthesized, characterized and uricase was physisorbed in the mesoporous material. The percentage adsorption and release of enzyme in borate buffer was monitored. The release followed linear kinetics and greater than 80% enzyme activity was retained indicating the potential of this system as an effective enzyme immobilization matrix. The enzyme permeability was studied with Wistar rat skin and human cadaver skin. It was found that in case of untreated rat skin 10% of enzyme permeated through skin in 100 h. The permeation increased by adding permeation enhancer (combination of oleic acid in propylene glycol (OA in PG)). The permeation enhancement was studied under two concentrations of OA in PG (1%, 5%) in both rat and human cadaver skin and it was found that 1% OA in PG showed better result in rat skin and 5% OA in PG showed good results in human cadaver skin. PMID:23802423

  20. Enzyme Regulation in Crassulacean Acid Metabolism Photosynthesis : Studies on Thioredoxin-Linked Enzymes of KalanchoE daigremontiana.

    PubMed

    Hutcheson, S W; Buchanan, B B

    1983-07-01

    Fructose-1,6-bisphosphatase (FBPase) and sedoheptulose-1,7-bisphosphatase (SBPase) were identified and purified from the Crassulacean acid metabolism (CAM) plant, Kalanchoë daigremontiana. FBPase and SBPase showed respective molecular weights of 180,000 and 76,000, and exhibited immunological cross-reactivity with their counterparts from chloroplasts of C(3) (spinach) and C(4) (corn) plants. Based on Western blot analysis, FBPase was composed of four identical 45,000-dalton subunits and SBPase of two identical 38,000-dalton subunits. Immunological evidence, together with physical properties, indicated that both enzymes were of chloroplast origin.Kalanchoë FBPase and SBPase could be activated by thioredoxin f reduced chemically by dithiothreitol or photochemically by a reconstituted Kalanchoë ferredoxin/thioredoxin system. Both enzymes were activated synergistically by reduced thioredoxin f and thier respective substrates.Kalanchoë FBPase could be partially activated by Mg(2+) at concentrations greater than 10 millimolar; however, such activation was considerably less than that observed in the presence of reduced thioredoxin and Ca(2+), especially in the pH range between 7.8 and 8.3. In contrast to FBPase, Kalanchoë SBPase exhibited an absolute requirement for a dithiol such as reduced thioredoxin irrespective of Mg(2+) concentration. However, like FBPase, increased Mg(2+) concentrations enhanced the thioredoxin-linked activation of this enzyme.In conjunction with these studies, an NADP-linked malate dehydrogenase (NADP-MDH) was identified in cell-free preparations of Kalanchoë leaves which required reduced thioredoxin m for activity.These results indicate that Kalanchoë FBPase, SBPase, and NADP-MDH share physical and regulatory properties with their equivalents in C(3) and C(4) plants. In contrast to previous evidence, all three enzymes appear to have the capacity to be photoregulated in chloroplasts of CAM plants, thereby providing a means for the

  1. Production of Cell Wall Hydrolyzing Enzymes by Barley Aleurone Layers in Response to Gibberellic Acid 1

    PubMed Central

    Taiz, Lincoln; Honigman, William A.

    1976-01-01

    The cell walls of barley (Hordeum vulgare var. Himalaya) aleurone layers undergo extensive degradation during the tissue's response to gibberellic acid. Previous work had shown that these cell walls consist almost entirely of arabinoxylan. In this study we show that gibberellic acid stimulates endo-β-1,4-xylanase activity in isolated aleurone layers. In addition, gibberellic acid enhances the activity of two glycosidases: β-xylopyranosidase and α-arabinofuranosidase. No gibberellic acid-stimulated cellulase activity was detected. Germination studies showed a similar pattern of enzyme development in intact seeds. Images PMID:16659683

  2. Exploring omega-3 fatty acids, enzymes and biodiesel producing thraustochytrids from Australian and Indian marine biodiversity.

    PubMed

    Gupta, Adarsha; Singh, Dilip; Byreddy, Avinesh R; Thyagarajan, Tamilselvi; Sonkar, Shailendra P; Mathur, Anshu S; Tuli, Deepak K; Barrow, Colin J; Puri, Munish

    2016-03-01

    The marine environment harbours a vast diversity of microorganisms, many of which are unique, and have potential to produce commercially useful materials. Therefore, marine biodiversity from Australian and Indian habitat has been explored to produce novel bioactives, and enzymes. Among these, thraustochytrids collected from Indian habitats were shown to be rich in saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs), together constituting 51-76% of total fatty acids (TFA). Indian and Australian thraustochytrids occupy separate positions in the dendrogram, showing significant differences exist in the fatty acid profiles in these two sets of thraustochytrid strains. In general, Australian strains had a higher docosahexaenoic acid (DHA) content than Indian strains with DHA at 17-31% of TFA. A range of enzyme activities were observed in the strains, with Australian strains showing overall higher levels of enzyme activity, with the exception of one Indian strain (DBTIOC-1). Comparative analysis of the fatty acid profile of 34 strains revealed that Indian thraustochytrids are more suitable for biodiesel production since these strains have higher fatty acids content for biodiesel (FAB, 76%) production than Australian thraustochytrids, while the Australian strains are more suitable for omega-3 (40%) production. PMID:26580151

  3. Role of Malic Enzyme during Fatty Acid Synthesis in the Oleaginous Fungus Mortierella alpina

    PubMed Central

    Hao, Guangfei; Chen, Haiqin; Wang, Lei; Gu, Zhennan; Song, Yuanda; Zhang, Hao

    2014-01-01

    The generation of NADPH by malic enzyme (ME) was postulated to be a rate-limiting step during fatty acid synthesis in oleaginous fungi, based primarily on the results from research focusing on ME in Mucor circinelloides. This hypothesis is challenged by a recent study showing that leucine metabolism, rather than ME, is critical for fatty acid synthesis in M. circinelloides. To clarify this, the gene encoding ME isoform E from Mortierella alpina was homologously expressed. ME overexpression increased the fatty acid content by 30% compared to that for a control. Our results suggest that ME may not be the sole rate-limiting enzyme, but does play a role, during fatty acid synthesis in oleaginous fungi. PMID:24532075

  4. Production of Glucaric Acid from Hemicellulose Substrate by Rosettasome Enzyme Assemblies.

    PubMed

    Lee, Charles C; Kibblewhite, Rena E; Paavola, Chad D; Orts, William J; Wagschal, Kurt

    2016-07-01

    Hemicellulose biomass is a complex polymer with many different chemical constituents that can be utilized as industrial feedstocks. These molecules can be released from the polymer and transformed into value-added chemicals through multistep enzymatic pathways. Some bacteria produce cellulosomes which are assemblies composed of lignocellulolytic enzymes tethered to a large protein scaffold. Rosettasomes are artificial engineered ring scaffolds designed to mimic the bacterial cellulosome. Both cellulosomes and rosettasomes have been shown to facilitate much higher rates of biomass hydrolysis compared to the same enzymes free in solution. We investigated whether tethering enzymes involved in both biomass hydrolysis and oxidative transformation to glucaric acid onto a rosettasome scaffold would result in an analogous production enhancement in a combined hydrolysis and bioconversion metabolic pathway. Three different enzymes were used to hydrolyze birchwood hemicellulose and convert the substituents to glucaric acid, a top-12 DOE value added chemical feedstock derived from biomass. It was demonstrated that colocalizing the three different enzymes to the synthetic scaffold resulted in up to 40 % higher levels of product compared to uncomplexed enzymes. PMID:27198564

  5. Mycolic acid biosynthesis and enzymic characterization of the beta-ketoacyl-ACP synthase A-condensing enzyme from Mycobacterium tuberculosis.

    PubMed

    Kremer, Laurent; Dover, Lynn G; Carrère, Séverine; Nampoothiri, K Madhavan; Lesjean, Sarah; Brown, Alistair K; Brennan, Patrick J; Minnikin, David E; Locht, Camille; Besra, Gurdyal S

    2002-06-01

    Mycolic acids consist of long-chain alpha-alkyl-beta-hydroxy fatty acids that are produced by successive rounds of elongation catalysed by a type II fatty acid synthase (FAS-II). A key feature in the elongation process is the condensation of a two-carbon unit from malonyl-acyl-carrier protein (ACP) to a growing acyl-ACP chain catalysed by a beta-ketoacyl-ACP synthase (Kas). In the present study, we provide evidence that kasA from Mycobacterium tuberculosis encodes an enzyme that elongates in vivo the meromycolate chain, in both Mycobacterium smegmatis and Mycobacterium chelonae. We demonstrate that KasA belongs to the FAS-II system, which utilizes primarily palmitoyl-ACP rather than short-chain acyl-ACP primers. Furthermore, in an in vitro condensing assay using purified recombinant KasA, palmitoyl-AcpM and malonyl-AcpM, KasA was found to express Kas activity. Also, mutated KasA proteins, with mutation of Cys(171), His(311), Lys(340) and His(345) to Ala abrogated the condensation activity of KasA in vitro completely. Finally, purified KasA was highly sensitive to cerulenin, a well-known inhibitor of Kas, which may lead to the development of novel anti-mycobacterial drugs targeting KasA. PMID:12023885

  6. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity.

    PubMed

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-03-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  7. Molecular annotation of ketol-acid reductoisomerases from Streptomyces reveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

    PubMed Central

    Verdel-Aranda, Karina; López-Cortina, Susana T; Hodgson, David A; Barona-Gómez, Francisco

    2015-01-01

    The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketol-acid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications. PMID:25296650

  8. Structure-Function Relationships of Glucansucrase and Fructansucrase Enzymes from Lactic Acid Bacteria

    PubMed Central

    van Hijum, Sacha A. F. T.; Kralj, Slavko; Ozimek, Lukasz K.; Dijkhuizen, Lubbert; van Geel-Schutten, Ineke G. H.

    2006-01-01

    Lactic acid bacteria (LAB) employ sucrase-type enzymes to convert sucrose into homopolysaccharides consisting of either glucosyl units (glucans) or fructosyl units (fructans). The enzymes involved are labeled glucansucrases (GS) and fructansucrases (FS), respectively. The available molecular, biochemical, and structural information on sucrase genes and enzymes from various LAB and their fructan and α-glucan products is reviewed. The GS and FS enzymes are both glycoside hydrolase enzymes that act on the same substrate (sucrose) and catalyze (retaining) transglycosylation reactions that result in polysaccharide formation, but they possess completely different protein structures. GS enzymes (family GH70) are large multidomain proteins that occur exclusively in LAB. Their catalytic domain displays clear secondary-structure similarity with α-amylase enzymes (family GH13), with a predicted permuted (β/α)8 barrel structure for which detailed structural and mechanistic information is available. Emphasis now is on identification of residues and regions important for GS enzyme activity and product specificity (synthesis of α-glucans differing in glycosidic linkage type, degree and type of branching, glucan molecular mass, and solubility). FS enzymes (family GH68) occur in both gram-negative and gram-positive bacteria and synthesize β-fructan polymers with either β-(2→6) (inulin) or β-(2→1) (levan) glycosidic bonds. Recently, the first high-resolution three-dimensional structures have become available for FS (levansucrase) proteins, revealing a rare five-bladed β-propeller structure with a deep, negatively charged central pocket. Although these structures have provided detailed mechanistic insights, the structural features in FS enzymes dictating the synthesis of either β-(2→6) or β-(2→1) linkages, degree and type of branching, and fructan molecular mass remain to be identified. PMID:16524921

  9. Occurrence of Arginine Deiminase Pathway Enzymes in Arginine Catabolism by Wine Lactic Acid Bacteria

    PubMed Central

    Liu, S.; Pritchard, G. G.; Hardman, M. J.; Pilone, G. J.

    1995-01-01

    l-Arginine, an amino acid found in significant quantities in grape juice and wine, is known to be catabolized by some wine lactic acid bacteria. The correlation between the occurrence of arginine deiminase pathway enzymes and the ability to catabolize arginine was examined in this study. The activities of the three arginine deiminase pathway enzymes, arginine deiminase, ornithine transcarbamylase, and carbamate kinase, were measured in cell extracts of 35 strains of wine lactic acid bacteria. These enzymes were present in all heterofermentative lactobacilli and most leuconostocs but were absent in all the homofermentative lactobacilli and pediococci examined. There was a good correlation among arginine degradation, formation of ammonia and citrulline, and the occurrence of arginine deiminase pathway enzymes. Urea was not detected during arginine degradation, suggesting that the catabolism of arginine did not proceed via the arginase-catalyzed reaction, as has been suggested in some earlier studies. Detection of ammonia with Nessler's reagent was shown to be a simple, rapid test to assess the ability of wine lactic acid bacteria to degrade arginine, although in media containing relatively high concentrations (>0.5%) of fructose, ammonia formation is inhibited. PMID:16534912

  10. Biological Monitoring of 3-Phenoxybenzoic Acid in Urine by an Enzyme -Linked Immunosorbent Assay

    EPA Science Inventory

    An enzyme-linked immunosorbent assay (ELISA) method was employed for determination of the pyrethroid biomarker, 3-phenoxybenzoic acid (3-PBA) in human urine samples. The optimized coating antigen concentration was 0.5 ng/mL with a dilution of 1:4000 for the 3-PBA antibody and 1:6...

  11. A Study of Krebs Citric Acid Cycle Enzymes in Rice Larvae (Corcyrace phalonica St) During Mycotoxicosis

    PubMed Central

    Hegde, Umashashi C.; Shanmugasundaram, E. R. B.

    1967-01-01

    Krebs citric acid cycle enzymes have been studied in rice moth larvae (Corcyra cephalonica St) reared in groundnut meal control and contaminated with A. flavus, wheat bran control and wheat bran contaminated with A. flavus and also wheat bran containing aflatoxin. It was observed that the activity of enzymes other than succinic oxidase, succinic dehydrogenase and isocitric dehydrogenase were reduced significantly in larvae reared in contaminated groundnut meal when compared with the control. In the case of larvae reared in contaminated wheat bran all the enzymes except succinic oxidase were inhibited when compared to the control larvae. It was also observed that the inhibition of these enzymes is greater in the case of larvae reared in contaminated wheat bran than in contaminated groundnut meal. The higher toxicity of wheat bran has been discussed. PMID:4229935

  12. Acid ceramidase and the treatment of ceramide diseases: The expanding role of enzyme replacement therapy.

    PubMed

    Schuchman, Edward H

    2016-09-01

    Ceramides are a diverse group of sphingolipids that play important roles in many biological processes. Acid ceramidase (AC) is one key enzyme that regulates ceramide metabolism. Early research on AC focused on the fact that it is the enzyme deficient in the rare genetic disorder, Farber Lipogranulomatosis. Recent research has revealed that deficiency of the same enzyme is responsible for a rare form of spinal muscular atrophy associated with myoclonic epilepsy (SMA-PME). Due to their diverse role in biology, accumulation of ceramides also has been implicated in the pathobiology of many other common diseases, including infectious lung diseases, diabetes, cancers and others. This has revealed the potential of AC as a therapy for many of these diseases. This review will focus on the biology of AC and the potential role of this enzyme in the treatment of human disease. PMID:27155573

  13. Characterization of the first enzyme in 2,4-dichlorophenoxyacetic acid metabolism.

    PubMed Central

    Hausinger, R P; Fukumori, F

    1995-01-01

    This paper reviews the properties of the Alcaligenes eutrophus JMP134 tfdA gene product, the enzyme responsible for the first step in 2,4-dichlorophenoxyacetic acid (2,4-D) biodegradation. The gene was overexpressed in Escherichia coli and several of its enzymatic properties were characterized. Although this enzyme catalyzes a hydroxylation reaction, it is not a monooxygenase. Rather, TfdA is an Fe(II) and alpha-ketoglutarate-dependent dioxygenase that metabolizes the latter cosubstrate to succinate and carbon dioxide. A variety of other phenoxyacetates and alpha-ketoacids can be used by the enzyme, but the greatest catalytic efficiencies were found using 2,4-D and alpha-ketoglutarate. The enzyme possesses multiple essential histidine residues, whereas catalytically essential cysteine and lysine groups do not appear to be present. PMID:8565907

  14. Enzyme degradable polymersomes from hyaluronic acid-block-poly(ε-caprolactone) copolymers for the detection of enzymes of pathogenic bacteria.

    PubMed

    Haas, Simon; Hain, Nicole; Raoufi, Mohammad; Handschuh-Wang, Stephan; Wang, Tao; Jiang, Xin; Schönherr, Holger

    2015-03-01

    We introduce a new hyaluronidase-responsive amphiphilic block copolymer system, based on hyaluronic acid (HYA) and polycaprolactone (PCL), that can be assembled into polymersomes by an inversed solvent shift method. By exploiting the triggered release of encapsulated dye molecules, these HYA-block-PCL polymersomes lend themselves as an autonomous sensing system for the detection of the presence of hyaluronidase, which is produced among others by the pathogenic bacterium Staphylococcus aureus. The synthesis of the enzyme-responsive HYA-block-PCL block copolymers was carried out by copper-catalyzed Huisgen 1,3-dipolar cycloaddition of ω-azide-terminated PCL and ω-alkyne-functionalized HYA. The structure of the HYA-block-PCL assemblies and their enzyme-triggered degradation and concomitant cargo release were investigated by dynamic light scattering, fluorescence spectroscopy, confocal laser-scanning microscopy, scanning and transmission electron, and atomic force microscopy. As shown, a wide range of reporter dye molecules as well as antimicrobials can be encapsulated into the vesicles during formation and are released upon the addition of hyaluronidase. PMID:25654495

  15. Lewis Acid Catalyzed Ring-opening Polymerization of Epoxidized Soybean Oil in Liquid Carbon Dioxide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ring-opening polymerization of epoxidized soybean oil (ESO) catalyzed by boron trifluoride diethyl etherate (BF3•OEt2), in liquid carbon dioxide, was conducted in an effort to develop useful biobased biodegradable polymers. The resulting polymers (RPESO) were characterized by FTIR spectroscopy, diff...

  16. Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

    PubMed

    Lavrukhin, O V; Lloyd, R S

    2000-12-12

    Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates. PMID:11106507

  17. Enhanced therapeutic efficacy of budesonide in experimental colitis with enzyme/pH dual-sensitive polymeric nanoparticles

    PubMed Central

    Naeem, Muhammad; Cao, Jiafu; Choi, Moonjeong; Kim, Woo Seong; Moon, Hyung Ryong; Lee, Bok Luel; Kim, Min-Soo; Jung, Yunjin; Yoo, Jin-Wook

    2015-01-01

    Current colon-targeted drug-delivery approaches for colitis therapy often utilize single pH-triggered systems, which are less reliable due to the variation of gut pH in individuals and in disease conditions. Herein, we prepared budesonide-loaded dual-sensitive nanoparticles using enzyme-sensitive azo-polyurethane and pH-sensitive methacrylate copolymer for the treatment of colitis. The therapeutic potential of the enzyme/pH dual-sensitive nanoparticles was evaluated using a rat colitis model and compared to single pH-triggered nanoparticles. Clinical activity scores, colon/body weight ratios, myeloperoxidase activity, and proinflammatory cytokine levels were markedly decreased by dual-sensitive nanoparticles compared to single pH-triggered nanoparticles and budesonide solution. Moreover, dual-sensitive nanoparticles accumulated selectively in inflamed segments of the colon. In addition, dual-sensitive nanoparticle plasma concentrations were lower than single pH-triggered nanoparticles, and no noticeable in vitro or in vivo toxicity was observed. Our results demonstrate that enzyme/pH dual-sensitive nanoparticles are an effective and safe colon-targeted delivery system for colitis therapy. PMID:26213469

  18. Immobilization of uricase enzyme on self-assembled gold nanoparticles for application in uric acid biosensor.

    PubMed

    Ahuja, T; Tanwar, V K; Mishra, S K; Kumar, D; Biradar, A M; Rajesh

    2011-06-01

    An enzyme immobilization matrix is described by preparing a self-assembly of gold nanoparticles (GNPs) over a self-assembled monolayer (SAM) of 3-aminopropyltriethoxysilane (APTES) on an indium-tin-oxide (ITO) coated glass plate. The surface of the GNPs was modified with a mixed (1:9) SAM of 11-mercaptoundecanoic acid (MUA) and 3-mercapto-propionic acid (MPA). The enzyme, uricase was covalently immobilized to the carboxyl groups of the mixed SAM of MUA/MPA through carbodiimide coupling reaction. The whole assembly was constructed on 1 cm2 area of ITO-glass plate and was tested as an amperometric biosensor for the detection of uric acid in aqueous solution. The biosensor assembly was characterized by atomic force microscopy (AFM) and electrochemical techniques. The AFM of the enzyme biosensor assembly reveals an asymmetrical sharp regular island-like structure with an average roughness parameter value of 2.81 nm. Chronoamperometric response was measured as a function of uric acid concentration in aqueous solution (pH 7.4), which exhibits a linear response over a concentration range of 0.07 to 0.63 mM with a sensitivity of 19.27 microAmM(-1) and a response of 25 s with excellent reproducibility. These results are not influenced by the presence of interfering reagents such as ascorbic acid, urea and glucose. GNPs-biomolecule assemblies constructed using this method may facilitate development of new hybrid biosensing materials. PMID:21770094

  19. A New Hyaluronic Acid Derivative Obtained from Atom Transfer Radical Polymerization as a siRNA Vector for CD44 Receptor Tumor Targeting.

    PubMed

    Palumbo, Fabio Salvatore; Bavuso Volpe, Antonella; Bongiovì, Flavia; Pitarresi, Giovanna; Giammona, Gaetano

    2015-11-01

    Two derivatives of hyaluronic acid (HA) have been synthesized by atom transfer radical polymerization (ATRP), starting from an ethylenediamino HA derivative (HA-EDA) and by using diethylaminoethyl methacrylate (DEAEMA) as a monomer for polymerization. Both samples, indicated as HA-EDA-pDEAEMA a and b, are able to condense siRNA, as determined by gel retardation assay and resulting complexes show a size and a zeta potential value dependent on polymerization number, as determined by dynamic light scattering measurements. In vitro studies performed on HCT 116 cell line, that over express CD44 receptor, demonstrate a receptor mediated uptake of complexes, regardless of their surface charge. PMID:26136372

  20. Preferential polymerization and adsorption of L-optical isomers of amino acids relative to D-optical isomers on kaolinite templates.

    NASA Technical Reports Server (NTRS)

    Jackson, T. A.

    1971-01-01

    Experiments on the polymerization of the L- and D-optical isomers of aspartic acid and serine using kaolinite as a catalyst showed that the L-optical isomers were polymerized at a much higher rate than the D-optical isomers; racemic (DL-) mixtures were polymerized at an intermediate rate. The peptides formed from the L-monomers were preferentially adsorbed by the clay. In the absence of kaolinite, no significant or consistent difference in the behavior of the L- and D-optical isomers was observed. In experiments on the adsorption of L- and D-phenylalanine by kaolinite, the L-optical isomer was preferentially adsorbed.

  1. Non-enzymic phosphorylation of polyphosphoinositides and phosphatidic acid is catalysed by bivalent metal ions.

    PubMed Central

    Gumber, S C; Lowenstein, J M

    1986-01-01

    Phosphatidylinositol 4-phosphate, phosphatidylinositol 4,5-bisphosphate and phosphatidic acid undergo non-enzymic phosphorylation by ATP in the presence of bivalent metal ions. The non-enzymic reaction is more rapid in a mixture of water, chloroform and methanol than in water alone. Chemical evidence indicates that the product formed from phosphatidylinositol 4-phosphate is the corresponding 4-pyrophosphate. This product shows an RF value very close to that of phosphatidylinositol 4,5-bisphosphate on t.l.c. with an acidic solvent commonly used to characterize and measure the latter; however, it can be separated readily with an alkaline solvent. Chemical evidence indicates that the products formed from phosphatidylinositol 4,5-bisphosphate and phosphatidic acid are also pyrophosphates. Images Fig. 1. Fig. 2. PMID:3017309

  2. The acid and enzymic hydrolysis of O-acetylated sialic acid residues from rabbit Tamm–Horsfall glycoprotein

    PubMed Central

    Neuberger, A.; Ratcliffe, Wendy A.

    1972-01-01

    Rabbit Tamm–Horsfall glycoprotein and bovine submaxillary glycoprotein were both found to contain sialic acid residues which are released at a slow rate by the standard conditions of acid hydrolysis. These residues are also resistant to neuraminidases from Vibrio cholerae and Clostridium perfringens. This behaviour was attributed to the presence of O-acetylated sialic acid, since the removal of O-acetyl groups by mild alkaline treatment normalized the subsequent release of sialic acid from rabbit Tamm–Horsfall glycoprotein by acid and by enzymic hydrolysis. Determination of the O-acetyl residues in rabbit Tamm–Horsfall glycoprotein indicated that on average two hydroxyl groups of sialic acid are O-acetylated, and these were located on the polyhydroxy side-chain of sialic acid or on C-4 and C-8. These findings confirm the assumption that certain O-acetylated forms of sialic acid are not substrates for bacterial neuraminidases. Several explanations have been suggested to explain the effect of O-acetylation of the side-chain on the rate of acidcatalysed hydrolysis of sialic acid residues. PMID:4349114

  3. Ring-opening polymerization of epoxidized soybean oil catalyzed by the superacid, Fluroantimonic acid hexahydrate (HSbF6-6H2O)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Ring-opening polymerization of epoxidized soybean oil (ESO) catalyzed by the super acid, fluroantimonic acid hexahydrate (HSbF6-6H2O), in ethyl acetate was conducted. The resulting polymers, SA-RPESO, were characterized using infrared spectroscopy, differential scanning calorimetry, thermogravimetri...

  4. Solvent-Free Polymerization of L-Aspartic Acid in the Presence of D-Sorbitol to Obtain Water Soluble or Network Copolymers

    Technology Transfer Automated Retrieval System (TEKTRAN)

    L-aspartic acid was thermally polymerized in the presence of D-sorbitol with the goal of synthesizing new, higher molecular weight water soluble and absorbent copolymers. No reaction occurred when aspartic acid alone was heated at 170 or 200 degrees C. In contrast, heating sorbitol and aspartic ac...

  5. Impacts of simulated acid rain on soil enzyme activities in a latosol.

    PubMed

    Ling, Da-Jiong; Huang, Qian-Chun; Ouyang, Ying

    2010-11-01

    Acid rain pollution is a serious environmental problem in the world. This study investigated impacts of simulated acid rain (SAR) upon four types of soil enzymes, namely the catalase, acid phosphatase, urease, and amylase, in a latosol. Latosol is an acidic red soil and forms in the tropical rainforest biome. Laboratory experiments were performed by spraying the soil columns with the SAR at pH levels of 2.5, 3.0, 3.5., 4.0, 4.5, 5.0, and 7.0 (control) over a 20-day period. Mixed results were obtained in enzyme activities for different kinds of enzymes under the influences of the SAR. The catalase activities increased rapidly from day 0 to 5, then decreased slightly from day 5 to 15, and finally decreased sharply to the end of the experiments, whereas the acid phosphatase activities decreased rapidly from day 0 to 5, then increased slightly from day 5 to 15, and finally decreased dramatically to the end of the experiments. A decrease in urease activities was observed at all of the SAR pH levels for the entire experimental period, while an increase from day 0 to 5 and then a decrease from day 5 to 20 in amylase activities were observed at all of the SAR pH levels. In general, the catalase, acid phosphatase, and urease activities increased with the SAR pH levels. However, the maximum amylase activity was found at pH 4.0 and decreased as the SAR pH increased from 4.0 to 5.0 or decreased from 4.0 to 2.5. It is apparent that acid rain had adverse environmental impacts on soil enzyme activities in the latosol. Our study further revealed that impacts of the SAR upon soil enzyme activities were in the following order: amylase>catalase>acid phosphatase>urease. These findings provide useful information on better understanding and managing soil biological processes in the nature under the influence of acid rains. PMID:20701974

  6. Hydroxyapatite surface modified by L-lactic acid and its subsequent grafting polymerization of L-lactide.

    PubMed

    Qiu, Xueyu; Hong, Zhongkui; Hu, Junli; Chen, Li; Chen, Xuesi; Jing, Xiabin

    2005-01-01

    A new method of surface modification of hydroxyapatite nanoparticles (n-HA) by surface grafting reaction of l-lactic acid and ring-opening polymerization of l-lactide (LLA) was developed. Two modified HA nanoparticles were obtained: HA modified by l-lactic acid (l-HA) and HA grafting with poly(l-lactide) (PLLA; p-HA). The modified surface of n-HA was attested by Fourier transformation infrared, (31)P MAS NMR, and thermal gravimetric analysis. The results showed that l-lactic acid could be easily grafted onto the n-HA surface by forming a Ca carboxylate bond and initiated by the hydroxyl group of the grafted l-lactic acid and LLA could be graft-polymerized onto the n-HA surface in the presence of stannous octanoate. The highest grafting amounts of l-lactic acid and PLLA were about 33 and 22 wt %, respectively. The modified HA/PLLA composites showed good mechanical properties and uniform microstructure. The tensile strength and modulus of the p-HA/PLLA composite containing 15 wt % of p-HA were 67 MPa and 2.1 GPa, respectively, while those of the n-HA/PLLA composites were 45 MPa and 1.7 GPa, respectively. The elongation at the break of the l-HA/PLLA composite containing 15 wt % l-HA could reach 44%, in comparison with 6.5% of the n-HA/PLLA composites containing 15 wt % n-HA. PMID:15877333

  7. Purification and characterization of cannabidiolic-acid synthase from Cannabis sativa L.. Biochemical analysis of a novel enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid.

    PubMed

    Taura, F; Morimoto, S; Shoyama, Y

    1996-07-19

    We identified a unique enzyme that catalyzes the oxidocyclization of cannabigerolic acid to cannabidiolic acid (CBDA) in Cannabis sativa L. (CBDA strain). The enzyme, named CBDA synthase, was purified to apparent homogeneity by a four-step procedure: ammonium sulfate precipitation followed by chromatography on DEAE-cellulose, phenyl-Sepharose CL-4B, and hydroxylapatite. The active enzyme consists of a single polypeptide with a molecular mass of 74 kDa and a pI of 6.1. The NH2-terminal amino acid sequence of CBDA synthase is similar to that of Delta1-tetrahydrocannabinolic-acid synthase. CBDA synthase does not require coenzymes, molecular oxygen, hydrogen peroxide, and metal ion cofactors for the oxidocyclization reaction. These results indicate that CBDA synthase is neither an oxygenase nor a peroxidase and that the enzymatic cyclization does not proceed via oxygenated intermediates. CBDA synthase catalyzes the formation of CBDA from cannabinerolic acid as well as cannabigerolic acid, although the kcat for the former (0.03 s-1) is lower than that for the latter (0.19 s-1). Therefore, we conclude that CBDA is predominantly biosynthesized from cannabigerolic acid rather than cannabinerolic acid. PMID:8663284

  8. Does single-amino-acid replacement work in favor of or against improvement of the thermostability of immobilized enzyme?

    PubMed Central

    Koizumi, J; Zhang, M; Imanaka, T; Aiba, S

    1990-01-01

    Thermostabilities of kanamycin nucleotidyltransferase and of its mutants that became thermostable, in the free state, because of single-amino-acid replacements were studied after immobilization of the enzymes on cyanogen bromide-activated Sephadex G-200 particles. Lys in place of Gln at position 102 decreased the thermostability of the immobilized enzyme, whereas replacement with other amino acids enhanced it. PMID:2176451

  9. Location and characteristics of enzymes involved in the breakdown of polygalacturonic acid by Bacteroides thetaiotaomicron.

    PubMed Central

    McCarthy, R E; Kotarski, S F; Salyers, A A

    1985-01-01

    When Bacteroides thetaiotaomicron is grown in medium which contains polygalacturonic acid (PGA) as the sole carbon source, two different polygalacturonases are produced: a PGA lyase (EC 4.2.2.2) and a PGA hydrolase (EC 3.2.1.15). Both enzymes are cell associated. The PGA hydrolase appears to be an inner membrane protein. The PGA lyase is a soluble protein that associates with membranes under certain conditions. The PGA lyase was purified to apparent homogeneity. It has a molecular weight (from sodium dodecyl sulfate-polyacrylamide gel electrophoresis) of 74,000, a pH optimum of 8.7, a pI of 7.5, and a Km for PGA of 40 to 70 micrograms/ml. It requires calcium for maximal activity. The main product of this enzyme appears to be a disaccharide that contains a delta 4,5-unsaturated galacturonic acid residue. The PGA hydrolase can be solubilized from membranes with 2% Triton X-100 and has been partially purified. It has a pH optimum of 5.4 to 5.5, a pI of 4.7 to 4.9, and a Km for PGA of 350 to 400 micrograms/ml. The main product of this enzyme appears to be galacturonic acid. The specific activities of both PGA hydrolase and PGA lyase increase at the same rate when bacteria are exposed to PGA. The two enzymes therefore appear to be similarly regulated. Images PMID:3968032

  10. The Impact of Enzyme Characteristics on Corn Stover Fiber Degradation and Acid Production During Ensiled Storage

    NASA Astrophysics Data System (ADS)

    Ren, Haiyu; Richard, Tom L.; Moore, Kenneth J.

    Ensilage can be used to store lignocellulosic biomass before industrial bioprocessing. This study investigated the impacts of seven commerical enzyme mixtures derived from Aspergillus niger, Trichoderma reesei, and T. longibrachiatum. Treatments included three size grades of corn stover, two enzyme levels (1.67 and 5 IU/g dry matter based on hemicellulase), and various ratios of cellulase to hemicellulase (C ∶ H). The highest C ∶ H ratio tested, 2.38, derived from T. reesei, resulted in the most effective fermentation, with lactic acid as the dominant product. Enzymatic activity during storage may complement industrial pretreatment; creating synergies that could reduce total bioconversion costs.

  11. Morphological characteristics, oxidative stability and enzymic hydrolysis of amylose-fatty acid complexes.

    PubMed

    Marinopoulou, Anna; Papastergiadis, Efthimios; Raphaelides, Stylianos N; Kontominas, Michael G

    2016-05-01

    Complexes of amylose with fatty acids varying in carbon chain length and degree of unsaturation were prepared at 30, 50 or 70°C by dissolving amylose in 0.1N KOH and mixing with fatty acid potassium soap solution. The complexes were obtained in solid form as precipitates after neutralization. SEM microscopy revealed that the morphology of the complexes was that of ordered lamellae separated from amorphous regions whereas confocal laser scanning microscopy showed images of the topography of the guest molecules in the complex matrix. FTIR spectroscopy revealed that the absorption peak attributed to carbonyl group of free fatty acid was shifted when the fatty acid was in the form of amylose complex. Thermo-gravimetry showed that the unsaturated fatty acids were effectively protected from oxidation when they were complexed with amylose whereas enzymic hydrolysis experiments showed that the guest molecules were quantitatively released from the amylose complexes. PMID:26877002

  12. Production of L-lactic Acid from Biomass Wastes Using Scallop Crude Enzymes and Novel Lactic Acid Bacterium

    NASA Astrophysics Data System (ADS)

    Yanagisawa, Mitsunori; Nakamura, Kanami; Nakasaki, Kiyohiko

    In the present study, biomass waste raw materials including paper mill sludge, bamboo, sea lettuce, and shochu residue (from a distiller) and crude enzymes derived from inedible and discarded scallop parts were used to produce L-lactic acid for the raw material of biodegradable plastic poly-lactic acid. The activities of cellulase and amylase in the crude enzymes were 22 and 170units/L, respectively, and L-lactic acid was produced from every of the above mentioned biomass wastes, by the method of liquid-state simultaneous saccharification and fermentation (SSF) . The L-lactic acid concentrations produced from sea lettuce and shochu residue, which contain high concentration of starch were 3.6 and 9.3g/L, respectively, and corresponded to greater than 25% of the conversion of glucans contained in these biomass wastes. Furthermore, using the solid state SSF method, concentrations as high as 13g/L of L-lactic acid were obtained from sea lettuce and 26g/L were obtained from shochu residue.

  13. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme

    PubMed Central

    Gallage, Nethaji J.; Hansen, Esben H.; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

    2014-01-01

    Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco. PMID:24941968

  14. Vanillin formation from ferulic acid in Vanilla planifolia is catalysed by a single enzyme.

    PubMed

    Gallage, Nethaji J; Hansen, Esben H; Kannangara, Rubini; Olsen, Carl Erik; Motawia, Mohammed Saddik; Jørgensen, Kirsten; Holme, Inger; Hebelstrup, Kim; Grisoni, Michel; Møller, Birger Lindberg

    2014-01-01

    Vanillin is a popular and valuable flavour compound. It is the key constituent of the natural vanilla flavour obtained from cured vanilla pods. Here we show that a single hydratase/lyase type enzyme designated vanillin synthase (VpVAN) catalyses direct conversion of ferulic acid and its glucoside into vanillin and its glucoside, respectively. The enzyme shows high sequence similarity to cysteine proteinases and is specific to the substitution pattern at the aromatic ring and does not metabolize caffeic acid and p-coumaric acid as demonstrated by coupled transcription/translation assays. VpVAN localizes to the inner part of the vanilla pod and high transcript levels are found in single cells located a few cell layers from the inner epidermis. Transient expression of VpVAN in tobacco and stable expression in barley in combination with the action of endogenous alcohol dehydrogenases and UDP-glucosyltransferases result in vanillyl alcohol glucoside formation from endogenous ferulic acid. A gene encoding an enzyme showing 71% sequence identity to VpVAN was identified in another vanillin-producing plant species Glechoma hederacea and was also shown to be a vanillin synthase as demonstrated by transient expression in tobacco. PMID:24941968

  15. Functional nanocomposites prepared by self-assembly and polymerization of diacetylene surfactants and silicic acid

    NASA Technical Reports Server (NTRS)

    Yang, Yi; Lu, Yunfeng; Lu, Mengcheng; Huang, Jinman; Haddad, Raid; Xomeritakis, George; Liu, Nanguo; Malanoski, Anthony P.; Sturmayr, Dietmar; Fan, Hongyou; Sasaki, Darryl Y.; Assink, Roger A.; Shelnutt, John A.; van Swol, Frank; Lopez, Gabriel P.; Burns, Alan R.; Brinker, C. Jeffrey

    2003-01-01

    Conjugated polymer/silica nanocomposites with hexagonal, cubic, or lamellar mesoscopic order were synthesized by self-assembly using polymerizable amphiphilic diacetylene molecules as both structure-directing agents and monomers. The self-assembly procedure is rapid and incorporates the organic monomers uniformly within a highly ordered, inorganic environment. By tailoring the size of the oligo(ethylene glycol) headgroup of the diacetylene-containing surfactant, we varied the resulting self-assembled mesophases of the composite material. The nanostructured inorganic host altered the diacetylene polymerization behavior, and the resulting nanocomposites show unique thermo-, mechano-, and solvatochromic properties. Polymerization of the incorporated surfactants resulted in polydiacetylene (PDA)/silica nanocomposites that were optically transparent and mechanically robust. Molecular modeling and quantum calculations and (13)C spin-lattice relaxation times (T(1)) of the PDA/silica nanocomposites indicated that the surfactant monomers can be uniformly organized into precise spatial arrangements prior to polymerization. Nanoindentation and gas transport experiments showed that these nanocomposite films have increased hardness and reduced permeability as compared to pure PDA. Our work demonstrates polymerizable surfactant/silica self-assembly to be an efficient, general approach to the formation of nanostructured conjugated polymers. The nanostructured inorganic framework serves to protect, stabilize, and orient the polymer, mediate its performance, and provide sufficient mechanical and chemical stability to enable integration of conjugated polymers into devices and microsystems.

  16. Cytochemical localisation of lysosomal enzymes and acidic mucopolysaccharides in the salivary glands of Aplysia depilans (Opisthobranchia).

    PubMed

    Lobo-da-Cunha, A

    2002-04-01

    Three types of secretory cells were reported in the salivary glands of Aplysia depilans: granular cells, vacuolated cells and mucocytes. To improve the characterisation of these cells, cytochemical methods for the detection of lysosomal enzymes and acidic mucopolysaccharides were applied. In granular cells, acid phosphatase and arylsulphatase were present in small lysosomes and in some secretory granules. The secretory granules could have received these enzymes after fusion with the small lysosomes that were frequently found very close to them. These cells were not stained with colloidal iron because they do not contain acidic mucopolysaccharides. In vacuolated cells, acid phosphatase and arylsulphatase were detected in lysosomes but not in the secretory vacuoles. Colloidal iron staining revealed the presence of acidic mucopolysaccharides in the vacuoles and in the Golgi apparatus of these cells. In mucocytes, lysosomes were very rare, but the secretion of these cells was very rich in acidic mucopolysaccharides. The filamentous network within the secretory vesicles was completely covered with iron particles, but practically no particles were observed over the granular masses attached to the membrane of the vesicles. Iron particles were also found in the trans-face cisternae of the U-shaped Golgi stacks, but were not seen in the cis-face cisternae or in the rough endoplasmic reticulum. PMID:12117284

  17. The Secreted Enzyme PM20D1 Regulates Lipidated Amino Acid Uncouplers of Mitochondria.

    PubMed

    Long, Jonathan Z; Svensson, Katrin J; Bateman, Leslie A; Lin, Hua; Kamenecka, Theodore; Lokurkar, Isha A; Lou, Jesse; Rao, Rajesh R; Chang, Mi Ra; Jedrychowski, Mark P; Paulo, Joao A; Gygi, Steven P; Griffin, Patrick R; Nomura, Daniel K; Spiegelman, Bruce M

    2016-07-14

    Brown and beige adipocytes are specialized cells that express uncoupling protein 1 (UCP1) and dissipate chemical energy as heat. These cells likely possess alternative UCP1-independent thermogenic mechanisms. Here, we identify a secreted enzyme, peptidase M20 domain containing 1 (PM20D1), that is enriched in UCP1(+) versus UCP1(-) adipocytes. We demonstrate that PM20D1 is a bidirectional enzyme in vitro, catalyzing both the condensation of fatty acids and amino acids to generate N-acyl amino acids and also the reverse hydrolytic reaction. N-acyl amino acids directly bind mitochondria and function as endogenous uncouplers of UCP1-independent respiration. Mice with increased circulating PM20D1 have augmented respiration and increased N-acyl amino acids in blood. Lastly, administration of N-acyl amino acids to mice improves glucose homeostasis and increases energy expenditure. These data identify an enzymatic node and a family of metabolites that regulate energy homeostasis. This pathway might be useful for treating obesity and associated disorders. PMID:27374330

  18. CYP4 Enzymes as potential drug targets: focus on enzyme multiplicity, inducers and inhibitors, and therapeutic modulation of 20-hydroxyeicosatetraenoic acid (20-HETE) synthase and fatty acid ω-hydroxylase activities

    PubMed Central

    Edson, Katheryne Z.; Rettie, Allan E.

    2014-01-01

    The Cytochrome P450 4 (CYP4) family of enzymes in humans is comprised of thirteen isozymes that typically catalyze the ω-oxidation of endogenous fatty acids and eicosanoids. Several CYP4 enzymes can biosynthesize 20-hydroxyeicosatetraenoic acid or 20-HETE, an important signaling eicosanoid involved in regulation of vascular tone and kidney reabsorption. Additionally, accumulation of certain fatty acids is a hallmark of the rare genetic disorders, Refsum disease and X-ALD. Therefore, modulation of CYP4 enzyme activity, either by inhibition or induction, is a potential strategy for drug discovery. Here we review the substrate specificities, sites of expression, genetic regulation, and inhibition by exogenous chemicals of the human CYP4 enzymes, and discuss the targeting of CYP4 enzymes in the development of new treatments for hypertension, stroke, certain cancers and the fatty acid-linked orphan diseases. PMID:23688133

  19. Pistagremic acid, a novel β-secretase enzyme (BACE1) inhibitor from Pistacia integerrima Stewart.

    PubMed

    Rauf, Abdur; Uddin, Ghias; Khan, Ajmal; Siddiqui, Bina S; Arfan, Mohammad; Dalvandi, Kourosh; Ben Hadda, Taibi

    2015-01-01

    A new triterpenic compound named pistagremic acid (PA) was once again isolated from Pistaciaintegerrima. The β-secretase inhibition study was carried out. Compound PA was found significantly active against β-secretase enzyme (BACE1) with IC50 value of 350 ± 2 nM in comparison to the standard inhibitors [Asn670, Sta671, Val672]-amyloid-β/A4 precursor protein 770 fragment 662-675 (IC50 = 290.71 ± 1 nM). The selectivity of this compound was also evaluated against the acetylcholinesterase and butyrylcholinesterase enzymes. Interestingly compound PA was found to be inactive against them and showed selectivity towards β-secretase enzyme (BACE1). PMID:25588845

  20. UV-induced graft polymerization of acrylic acid in the sub-micronchannels of oxidized PET track-etched membrane

    NASA Astrophysics Data System (ADS)

    Korolkov, Ilya V.; Mashentseva, Anastassiya A.; Güven, Olgun; Taltenov, Abzal A.

    2015-12-01

    In this article, we report on functionalization of track-etched membrane based on poly(ethylene terephthalate) (PET TeMs) oxidized by advanced oxidation systems and by grafting of acrylic acid using photochemical initiation technique for the purpose of increasing functionality thus expanding its practical application. Among advanced oxidation processes (H2O2/UV) system had been chosen to introduce maximum concentration of carboxylic acid groups. Benzophenone (BP) photo-initiator was first immobilized on the surfaces of cylindrical pores which were later filled with aq. acrylic acid solution. UV-irradiation from both sides of PET TeMs has led to the formation of grafted poly(acrylic acid) (PAA) chains inside the membrane sub-micronchannels. Effect of oxygen-rich surface of PET TeMs on BP adsorption and subsequent process of photo-induced graft polymerization of acrylic acid (AA) were studied by ESR. The surface of oxidized and AA grafted PET TeMs was characterized by UV-vis, ATR-FTIR, XPS spectroscopies and by SEM.

  1. Assembly of Lipoic Acid on Its Cognate Enzymes: an Extraordinary and Essential Biosynthetic Pathway.

    PubMed

    Cronan, John E

    2016-06-01

    Although the structure of lipoic acid and its role in bacterial metabolism were clear over 50 years ago, it is only in the past decade that the pathways of biosynthesis of this universally conserved cofactor have become understood. Unlike most cofactors, lipoic acid must be covalently bound to its cognate enzyme proteins (the 2-oxoacid dehydrogenases and the glycine cleavage system) in order to function in central metabolism. Indeed, the cofactor is assembled on its cognate proteins rather than being assembled and subsequently attached as in the typical pathway, like that of biotin attachment. The first lipoate biosynthetic pathway determined was that of Escherichia coli, which utilizes two enzymes to form the active lipoylated protein from a fatty acid biosynthetic intermediate. Recently, a more complex pathway requiring four proteins was discovered in Bacillus subtilis, which is probably an evolutionary relic. This pathway requires the H protein of the glycine cleavage system of single-carbon metabolism to form active (lipoyl) 2-oxoacid dehydrogenases. The bacterial pathways inform the lipoate pathways of eukaryotic organisms. Plants use the E. coli pathway, whereas mammals and fungi probably use the B. subtilis pathway. The lipoate metabolism enzymes (except those of sulfur insertion) are members of PFAM family PF03099 (the cofactor transferase family). Although these enzymes share some sequence similarity, they catalyze three markedly distinct enzyme reactions, making the usual assignment of function based on alignments prone to frequent mistaken annotations. This state of affairs has possibly clouded the interpretation of one of the disorders of human lipoate metabolism. PMID:27074917

  2. Production of 5-aminolevulinic acid by cell free multi-enzyme catalysis.

    PubMed

    Meng, Qinglong; Zhang, Yanfei; Ju, Xiaozhi; Ma, Chunling; Ma, Hongwu; Chen, Jiuzhou; Zheng, Ping; Sun, Jibin; Zhu, Jun; Ma, Yanhe; Zhao, Xueming; Chen, Tao

    2016-05-20

    5-Aminolevulinic acid (ALA) is the precursor for the biosynthesis of tetrapyrroles and has broad agricultural and medical applications. Currently ALA is mainly produced by chemical synthesis and microbial fermentation. Cell free multi-enzyme catalysis is a promising method for producing high value chemicals. Here we reported our work on developing a cell free process for ALA production using thermostable enzymes. Cheap substrates (succinate and glycine) were used for ALA synthesis by two enzymes: 5-aminolevulinic acid synthase (ALAS) from Laceyella sacchari (LS-ALAS) and succinyl-CoA synthase (Suc) from Escherichia coli. ATP was regenerated by polyphosphate kinase (Ppk) using polyphosphate as the substrate. Succinate was added into the reaction system in a fed-batch mode to avoid its inhibition effect on Suc. After reaction for 160min, ALA concentration was increased to 5.4mM. This is the first reported work on developing the cell free process for ALA production. Through further process and enzyme optimization the cell free process could be an effective and economic way for ALA production. PMID:27012885

  3. Plastid-localized amino acid biosynthetic pathways of Plantae are predominantly composed of non-cyanobacterial enzymes

    PubMed Central

    Reyes-Prieto, Adrian; Moustafa, Ahmed

    2012-01-01

    Studies of photosynthetic eukaryotes have revealed that the evolution of plastids from cyanobacteria involved the recruitment of non-cyanobacterial proteins. Our phylogenetic survey of >100 Arabidopsis nuclear-encoded plastid enzymes involved in amino acid biosynthesis identified only 21 unambiguous cyanobacterial-derived proteins. Some of the several non-cyanobacterial plastid enzymes have a shared phylogenetic origin in the three Plantae lineages. We hypothesize that during the evolution of plastids some enzymes encoded in the host nuclear genome were mistargeted into the plastid. Then, the activity of those foreign enzymes was sustained by both the plastid metabolites and interactions with the native cyanobacterial enzymes. Some of the novel enzymatic activities were favored by selective compartmentation of additional complementary enzymes. The mosaic phylogenetic composition of the plastid amino acid biosynthetic pathways and the reduced number of plastid-encoded proteins of non-cyanobacterial origin suggest that enzyme recruitment underlies the recompartmentation of metabolic routes during the evolution of plastids. PMID:23233874

  4. SYNTHESIS AND IN VITRO CHARACTERIZATION OF HYDROXYPROPYL METHYLCELLULOSE-GRAFT-POLY (ACRYLIC ACID/2-ACRYLAMIDO-2-METHYL-1-PROPANESULFONIC ACID) POLYMERIC NETWORK FOR CONTROLLED RELEASE OF CAPTOPRIL.

    PubMed

    Furqan Muhammad, Iqbal; Mahmood, Ahmad; Aysha, Rashid

    2016-01-01

    A super-absorbent hydrogel was developed by crosslinking of 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) and acrylic acid with hydroxypropyl methylcellulose (HPMC) for controlled release drug delivery of captopril, a well known antihypertensive drug. Acrylic acid and AMPS were polymerized and crosslinked with HPMC by free radical polymerization, a widely used chemical crosslinking method. N,N'-methylenebisacrylamide (MBA) and potassium persulfate (KPS) were added as cross-linker and initiator, respectively. The hydrogel formulation was loaded with captopril (as model drug). The concentration of captopril was monitored at 205 nm using UV spectrophotometer. Equilibrium swelling ratio was determined at pH 2, 4.5 and 7.4 to evaluate the pH responsiveness of the formed hydrogel. The super-absorbent hydrogels were evaluated by FTIR, SEM, XRD, and thermal analysis (DSC and TGA). The formation of new copolymeric network was determined by FTIR, XRD, TGA and DSC analysis. The hydrogel formulations with acrylic acid and AMPS ratio of 4: 1 and lower amounts of crosslinker had shown maximum swelling. Moreover, higher release rate of captopril was observed at pH 7.4 than at pH 2, because of more swelling capacity of copolymer with increasing pH of the aqueous medium. The present research work confirms the development of a stable hydrogel comprising of HPMC with acrylic acid and AMPS. The prepared hydrogels exhibited pH sensitive behav-ior. This superabsorbent composite prepared could be a successful drug carrier for treating hypertension. PMID:27008813

  5. Crystal structure of FadD32, an enzyme essential for mycolic acid biosynthesis in mycobacteria

    PubMed Central

    Li, Wenjuan; Gu, Shoujin; Fleming, Joy; Bi, Lijun

    2015-01-01

    Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 Å and 2.25 Å resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme. PMID:26628098

  6. Crystal structure of FadD32, an enzyme essential for mycolic acid biosynthesis in mycobacteria.

    PubMed

    Li, Wenjuan; Gu, Shoujin; Fleming, Joy; Bi, Lijun

    2015-01-01

    Fatty acid degradation protein D32 (FadD32), an enzyme required for mycolic acid biosynthesis and essential for mycobacterial growth, has recently been identified as a valid and promising target for anti-tuberculosis drug development. Here we report the crystal structures of Mycobacterium smegmatis FadD32 in the apo and ATP-bound states at 2.4 Å and 2.25 Å resolution, respectively. FadD32 consists of two globular domains connected by a flexible linker. ATP binds in a cleft at the interface between the N- and C-terminal domains and its binding induces significant local conformational changes in FadD32. The binding sites of meromycolic acid and phosphopantetheine are identified by structural comparison with other members of the adenylating enzyme superfamily. These results will improve our understanding of the catalytic mechanism of FadD32 and help in the design of inhibitors of this essential enzyme. PMID:26628098

  7. Catalytic nucleic acid enzymes for the study and development of therapies in the central nervous system

    PubMed Central

    Tritz, Richard; Habita, Cellia; Robbins, Joan M.; Gomez, German G.; Kruse, Carol A.

    2005-01-01

    Summary Nucleic acid enzymes have been used with great success for studying natural processes in the central nervous system (CNS). We first provide information on the structural and enzymatic differences of various ribozymes and DNAzymes. We then discuss how they have been used to explore new therapeutic approaches for treating diseases of the CNS. They have been tested in various systems modeling retinitis pigmentosum, proliferative vitreoretinopathy, Alzheimer's disease, and malignant brain tumors. For these models, effective targets for nucleic acid enzymes have been readily identified and the rules for selecting cleavage sites have been well established. The bulk of studies, including those from our laboratory, have emphasized their use for gliomas. With the availability of multiple excellent animal models to test glioma treatments, good progress has been made in the initial testing of nucleic acid enzymes for brain tumor therapy. However, opportunities still exist to significantly improve the delivery and efficacy of ribozymes to achieve effective treatment. The future holds significant potential for the molecular targeting and therapy of eye diseases, neurodegenerative disorders, and brain tumors with these unique treatment agents. PMID:16467915

  8. Molecularly imprinted polymer for chlorogenic acid by modified precipitation polymerization and its application to extraction of chlorogenic acid from Eucommia ulmodies leaves.

    PubMed

    Miura, Chitose; Li, Hui; Matsunaga, Hisami; Haginaka, Jun

    2015-10-10

    Molecularly imprinted polymers (MIPs) for chlorogenic acid (CGA) were prepared by modified precipitation polymerization using methacrylic acid as a functional monomer, divinylbenzene as a crosslinker and methanol or dimethylsulfoxide as a co-solvent. The prepared MIPs were microspheres with a narrow particle size distribution. Binding experiments and Scatchard analyses revealed that two classes of binding sites, high and low affinity sites, were formed on the MIP. The retention and molecular-recognition properties of the prepared MIP were evaluated using a mixture of water and acetonitrile as a mobile phase in hydrophilic interaction chromatography. With an increase of acetonitrile content, the retention factor of CGA was increased on the MIP. In addition to shape recognition, hydrophilic interactions seem to work for the recognition of CGA on the MIP. The MIP had a specific molecular-recognition ability for CGA, while other related compounds, such as caffeic acid, gallic acid, protocatechuic acid and vanillic acid, could not be recognized by the MIP. Furthermore, the MIP for CGA was successfully applied for extraction of CGA in the leaves of Eucommia ulmodies. PMID:26037163

  9. Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities.

    PubMed

    Akhtar, M Kalim; Turner, Nicholas J; Jones, Patrik R

    2013-01-01

    Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C(6)-C(18)) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C(8)-C(16)) or fatty alkanes (C(7)-C(15)) was reconstituted in vitro. This concept was applied in vivo, in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L(-1) was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C(8)-C(18)). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities. PMID:23248280

  10. Carboxylic acid reductase is a versatile enzyme for the conversion of fatty acids into fuels and chemical commodities

    PubMed Central

    Akhtar, M. Kalim; Turner, Nicholas J.; Jones, Patrik R.

    2013-01-01

    Aliphatic hydrocarbons such as fatty alcohols and petroleum-derived alkanes have numerous applications in the chemical industry. In recent years, the renewable synthesis of aliphatic hydrocarbons has been made possible by engineering microbes to overaccumulate fatty acids. However, to generate end products with the desired physicochemical properties (e.g., fatty aldehydes, alkanes, and alcohols), further conversion of the fatty acid is necessary. A carboxylic acid reductase (CAR) from Mycobacterium marinum was found to convert a wide range of aliphatic fatty acids (C6–C18) into corresponding aldehydes. Together with the broad-substrate specificity of an aldehyde reductase or an aldehyde decarbonylase, the catalytic conversion of fatty acids to fatty alcohols (C8–C16) or fatty alkanes (C7–C15) was reconstituted in vitro. This concept was applied in vivo, in combination with a chain-length-specific thioesterase, to engineer Escherichia coli BL21(DE3) strains that were capable of synthesizing fatty alcohols and alkanes. A fatty alcohol titer exceeding 350 mg·L−1 was obtained in minimal media supplemented with glucose. Moreover, by combining the CAR-dependent pathway with an exogenous fatty acid-generating lipase, natural oils (coconut oil, palm oil, and algal oil bodies) were enzymatically converted into fatty alcohols across a broad chain-length range (C8–C18). Together with complementing enzymes, the broad substrate specificity and kinetic characteristics of CAR opens the road for direct and tailored enzyme-catalyzed conversion of lipids into user-ready chemical commodities. PMID:23248280

  11. Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.).

    PubMed

    Tamura, Ken-ihi; Sanada, Yasuharu; Tase, Kazuhiro; Komatsu, Toshinori; Yoshida, Midori

    2011-06-01

    The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of β(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for β(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against β(2,1)-linked fructans, but considerably less than against β(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy. PMID:21317211

  12. Pp6-FEH1 encodes an enzyme for degradation of highly polymerized levan and is transcriptionally induced by defoliation in timothy (Phleum pratense L.)

    PubMed Central

    Tamura, Ken-ichi; Sanada, Yasuharu; Tase, Kazuhiro; Komatsu, Toshinori; Yoshida, Midori

    2011-01-01

    The ability of grasses to regrow after defoliation by cutting or grazing is a vital factor in their survival and an important trait when they are used as forage crops. In temperate grass species accumulating fructans, defoliation induces the activity of a fructan exohydrolase (FEH) that degrades fructans to serve as a carbon source for regrowth. Here, a cDNA from timothy was cloned, named Pp6-FEH1, that showed similarity to wheat fructan 6-exohydrolase (6-FEH). The recombinant enzyme expressed in Pichia pastoris completely degraded fructans that were composed mainly of β(2,6)-linked and linear fructans (levan) with a high degree of polymerization (DP) in the crown tissues of timothy. The substrate specificity of Pp6-FEH1 differed from previously characterized enzymes with 6-FEH activity in fructan-accumulating plants: (i) Pp6-FEH1 showed 6-FEH activity against levan (mean DP 20) that was 4-fold higher than against 6-kestotriose (DP 3), indicating that Pp6-FEH1 has a preference for β(2,6)-linked fructans with high DP; (ii) Pp6-FEH1 had significant activity against β(2,1)-linked fructans, but considerably less than against β(2,6)-linked fructans; (iii) Pp6-FEH1 had weak invertase activity, and its 6-FEH activity was inhibited slightly by sucrose. In the stubble of seedlings and in young haplocorms from adult timothy plants, transcripts of Pp6-FEH1 were significantly increased within 3 h of defoliation, followed by an increase in 6-FEH activity and in the degradation of fructans. These results suggest that Pp6-FEH1 plays a role in the degradation of fructans and the mobilization of carbon sources for regrowth after defoliation in timothy. PMID:21317211

  13. DEVELOPMENT OF ENZYME-LINKED IMMUNOSORBENT ASSAYS FOR ISOCUPRESSIC ACID AND SERUM METABOLITES OF ISOCUPRESSIC ACID

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The consumption of ponderosa pine (Pinus ponderosa), lodgepole pine (Pinus contorta), common juniper (Juniperus communis) and Monterey cypress (Cupressus macrocarpa) causes abortions in pregnant cattle. Recent studies have identified isocupressic acid as the primary abortifacient compound in these ...

  14. The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism*

    PubMed Central

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-01-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp230 residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  15. The crystal structure of the adenylation enzyme VinN reveals a unique β-amino acid recognition mechanism.

    PubMed

    Miyanaga, Akimasa; Cieślak, Jolanta; Shinohara, Yuji; Kudo, Fumitaka; Eguchi, Tadashi

    2014-11-01

    Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp(230) residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes. PMID:25246523

  16. Dextran-5-(4-ethoxycarbonylphenylazo)salicylic acid ester, a polymeric colon-specific prodrug releasing 5-aminosalicylic acid and benzocaine, ameliorates TNBS-induced rat colitis.

    PubMed

    Nam, Joon; Kim, Wooseong; Lee, Sunyoung; Jeong, Seongkeun; Yoo, Jin-Wook; Kim, Min-Soo; Jung, Yunjin

    2016-06-01

    Local anesthetics have beneficial effects on colitis. Dextran-5-(4-ethoxycarbonylphenylazo)salicylic acid ester (Dex-5-ESA), designed as a polymeric colon-specific prodrug liberating 5-ASA and benzocaine in the large intestine, was prepared and its therapeutic activity against colitis was evaluated using a TNBS-induced rat colitis model. Dex-5-ESA liberated 5-ASA and benzocaine in the cecal contents while (bio)chemically stable in the small intestinal contents and mucosa. Oral administration of Dex-5-ESA (equivalent to 10 mg 5-ASA/kg, twice a day) alleviated colonic injury and reduced MPO activity in the inflamed colon. In parallel, pro-inflammatory mediators, COX-2, iNOS and CINC-3, elevated by TNBS-induced colitis, were substantially diminished in the inflamed colon. Dex-5-ESA was much more effective for the treatment of colitis than 5-(4-ethoxycarbonylphenylazo)salicylic acid (5-ESA) that may not deliver benzocaine to the large intestine. Our data suggest that Dex-5-ESA is a polymeric colon-specific prodrug, liberating 5-ASA and benzocaine in the target site (large intestine), probably exerting anti-colitic effects by combined action of 5-ASA and benzocaine. PMID:26377354

  17. Synthesis of novel amphiphilic hyaluronan containing-aromatic fatty acids for fabrication of polymeric micelles.

    PubMed

    Matelová, Alena; Huerta-Angeles, Gloria; Šmejkalová, Daniela; Brůnová, Zdislava; Dušek, Jan; Vícha, Robert; Velebný, Vladimír

    2016-10-20

    Novel hydrophobized hyaluronan (HA) derivatives, containing ω-phenylalkanoic acids (ω-PAA, 4-phenylbutyric acid, 6-phenylhexanoic, 8-phenyloctanoic or 11-tolylundecanoic acids) were prepared by esterification. Mixed anhydrides obtained after reaction of the carboxyl acid moiety and benzoyl chloride were found to be active acylating agents, affording hydrophobized HA in good yield and under mild conditions. The reactivity of the aromatic fatty acids towards esterification has decreased with the increasing length of the aliphatic spacer between the aromatic substituent and carboxylic acid moiety. The novel HA derivatives self-assembled from very low concentrations and were found to be non-cytotoxic. The potential use of ω-phenylalkanoic acids grafted-HA towards drug delivery applications was demonstrated by hydrophobic drugs (resveratrol and retinyl palmitate) encapsulation. The drug loading capacity of the novel HA derivatives was significantly improved most likely because of π⋯π interactions between the micelle core and loaded hydrophobic aromatic compound. PMID:27474668

  18. Polymerization on the rocks: negatively-charged alpha-amino acids

    NASA Technical Reports Server (NTRS)

    Hill, A. R. Jr; Bohler, C.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    1998-01-01

    Oligomers of the negatively-charged amino acids, glutamic acid, aspartic acid, and O-phospho-L-serine are adsorbed by hydroxylapatite and illite with affinities that increase with oligomer length. In the case of oligo-glutamic acids adsorbed on hydroxylapatite, addition of an extra residue results in an approximately four-fold increase in the strength of adsorption. Oligomers much longer than the 7-mer are retained tenaciously by the mineral. Repeated incubation of short oligo-glutamic acids adsorbed on hydroxylapatite or illite with activated monomer leads to the accumulation of oligomers at least 45 units long. The corresponding reactions of aspartic acid and O-phospho-L-serine on hydroxylapatite are less effective in generating long oligomers, while illite fails to accumulate substantial amounts of long oligomers of aspartic acid or of O-phospho-L-serine.

  19. The Effect of 3-Thiopheneacetic Acid in the Polymerization of a Conductive Electrotextile for Use in Biosensor Development

    PubMed Central

    McGraw, Shannon K.; Alocilja, Evangelyn; Senecal, Andre; Senecal, Kris

    2013-01-01

    Investigations were conducted to develop an electrotextile using a nonwoven polypropylene fiber platform conformally coated in a conductive, functionalized copolymer of polypyrrole and 3-thiopheneacetic acid (3TAA). The objectives of this study were to determine: (1) if the inclusion of 3TAA in the polymerization process would have an effect on the availability of binding sites in the high-surface area electrotextile for biorecognition elements and (2) how the increase in the concentration of 3TAA would affect the physical characteristics of the coating, resistivity of the sample and availability of binding sites. It was found that the addition of 3TAA to the polymerization process resulted in an increase in the size of the polypyrrole coating, as well as the material resistivity and available binding sites for biorecognition elements. These factors were used to determine which of the tested concentrations was best for biosensor development. A polymer coated membrane sample containing a concentration within the range of 10–50 mg/mL of 3TAA was selected as the best for future biosensor work. PMID:25586259

  20. Induction of Arabidopsis tryptophan pathway enzymes and camalexin by amino acid starvation, oxidative stress, and an abiotic elicitor.

    PubMed Central

    Zhao, J; Williams, C C; Last, R L

    1998-01-01

    The tryptophan (Trp) biosynthetic pathway leads to the production of many secondary metabolites with diverse functions, and its regulation is predicted to respond to the needs for both protein synthesis and secondary metabolism. We have tested the response of the Trp pathway enzymes and three other amino acid biosynthetic enzymes to starvation for aromatic amino acids, branched-chain amino acids, or methionine. The Trp pathway enzymes and cytosolic glutamine synthetase were induced under all of the amino acid starvation test conditions, whereas methionine synthase and acetolactate synthase were not. The mRNAs for two stress-inducible enzymes unrelated to amino acid biosynthesis and accumulation of the indolic phytoalexin camalexin were also induced by amino acid starvation. These results suggest that regulation of the Trp pathway enzymes under amino acid deprivation conditions is largely a stress response to allow for increased biosynthesis of secondary metabolites. Consistent with this hypothesis, treatments with the oxidative stress-inducing herbicide acifluorfen and the abiotic elicitor alpha-amino butyric acid induced responses similar to those induced by the amino acid starvation treatments. The role of salicylic acid in herbicide-mediated Trp and camalexin induction was investigated. PMID:9501110

  1. Study and comparison of two enzyme membrane reactors for fatty acids and glycerol production

    SciTech Connect

    Molinari, R.; Santoro, M.E.; Drioli, E. . Dept. of Chemical Engineering and Materials Inst. on Membranes and Chemical Reactors-CNR, Arcavacata di Rende )

    1994-11-01

    Two enzyme membrane reactors (EMR), (1) with one substrate (olive oil) in an oil-in-water emulsion (E-EMR) and (2) with two separated liquid phases (oil and water) (TSLP-EMR), have been studied for the conversion of the triglycerides to fatty acids and glycerol. The enzyme was Candida cylindracea lipase confined on the pressurized face or entrapped in the sponge side of capillary ultrafiltration membranes. Two methods for immobilizing the enzyme in the TSLP-EMR were used: ultrafiltration on a virgin membrane and ultrafiltration on glutaraldehyde pretreated membranes. A multiple use of the reactor was obtained immobilizing the enzyme on the membrane preactivated with glutaraldehyde. The TSLP-EMR showed a specific activity of 0.529 mmol/(mg[center dot]h) versus a specific activity of 0.170 mmol/(mg[center dot]h) of the E-EMR. The rate of fatty acid production in the TSLP-EMR was linear with time showing no enzyme deactivation in an operating time of 80 h. The kinetics observed in the two reactors was different: an equilibrium reaction product-inhibited for the E-EMR and an apparent irreversible reaction of zero order for the TSLP-EMR. Taking into account that in the TSLP-EMR, compared to the E-EMR, (1) the specific activity was higher, (2) the specific rate was constant with the time, and (3) the two products were already separated after the reaction, the TSLP-EMR configuration seems the more convenient.

  2. Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme.

    PubMed

    Lederer, Florence; Vignaud, Caroline; North, Paul; Bodevin, Sabrina

    2016-09-01

    A controversy exists with respect to the mechanism of l-2-hydroxy acid oxidation by members of a family of FMN-dependent enzymes. A so-called carbanion mechanism was initially proposed, in which the active site histidine abstracts the substrate α-hydrogen as a proton, followed by electron transfer from the carbanion to the flavin. But an alternative mechanism was not incompatible with some results, a mechanism in which the active site histidine instead picks up the substrate hydroxyl proton and a hydride transfer occurs. Even though more recent experiments ruling out such a mechanism were published (Rao & Lederer (1999) Protein Science 7, 1531-1537), a few authors have subsequently interpreted their results with variant enzymes in terms of a hydride transfer. In the present work, we analyse the reactivity of trifluorolactate, a substrate analogue, with the flavocytochrome b2 (Fcb2) flavodehydrogenase domain, compared to its reactivity with an NAD-dependent lactate dehydrogenase (LDH), for which this compound is known to be an inhibitor (Pogolotti & Rupley (1973) Biochem. Biophys. Res. Commun, 55, 1214-1219). Indeed, electron attraction by the three fluorine atoms should make difficult the removal of the α-H as a hydride. We also analyse the reactivity of trifluoropyruvate with the FMN- and NAD-dependent enzymes. The results substantiate a different effect of the fluorine substituents on the two enzymes compared to their normal substrates. In the discussion we analyse the conclusions of recent papers advocating a hydride transfer mechanism for the family of l-2-hydroxy acid oxidizing FMN-dependent enzymes. PMID:27155230

  3. Ultrasensitive electrochemical detection of nucleic acid by coupling an autonomous cascade target replication and enzyme/gold nanoparticle-based post-amplification.

    PubMed

    Liu, Shufeng; Wei, Wenji; Wang, Yanqun; Fang, Li; Wang, Li; Li, Feng

    2016-06-15

    Owing to the intrinsic importance of nucleic acid as bio-targets, the development of isothermal and ultrasensitive electrochemical DNA biosensor is very essential for biological studies and medical diagnostics. Herein, the autonomous cascade DNA replication strategy was effectively married with the enzyme/gold nanoparticle-based post-amplification strategy to promote the detection performance toward target DNA. A hairpin DNA probe (HP) is designed that consists of an overhang at 3'-end as the recognition unit for target DNA, a recognition site for nicking endonuclease, and an alkane spacer to terminate polymerization reaction. The autonomous DNA replication-scission-displacement reaction operated by the nicking endonuclease/KF polymerase induced the autocatalytic opening of HP, which was then specifically bound by the enzyme/gold nanoparticles for further dual-signal amplification toward target-related sensing events. A low detection limit of 0.065fM with an excellent selectivity toward target DNA could be achieved. The proposed biosensor could be also easily regenerated for target detection. The developed biosensor creates an opportunity for the effective coupling of the target replication with post-amplification strategies and thus opens a promising avenue for the detection of nucleic acid with low abundance in bioanalysis and clinical biomedicine. PMID:26849348

  4. Effects of Omega-3 Fatty Acids Supplement on Antioxidant Enzymes Activity in Type 2 Diabetic Patients

    PubMed Central

    TOORANG, Fatemeh; DJAZAYERY, Abolghassem; DJALALI, Mahmoud

    2016-01-01

    Background: Diabetes is a major cause of death. Oxidative stress mainly caused by hyperglycemia is the primary reason of related complications. Omega-3 fatty acids are prescribed in diabetes but the effect on antioxidant defense is controversial. This study investigated effects of omega-3 supplementation on antioxidant enzymes activity in type 2 diabetic patients. Methods: A randomized, placebo controlled, double blind clinical trial was performed on 90 type2 diabetic patients. The treatment group took, daily, three capsules of omega-3 for two mo, which totally provided 2714mg omega-3 (EPA=1548 mg, DHA=828 mg and 338 mg of other omega=3 fatty acids). Placebo contained 2100 mg sunflower oil (12% SFA, 65% linoleic acid, 23% MUFA), which is the main oil used in the study population. Food intakes, anthropometric and demographic characteristics, and therapeutic regimen data were recorded before and after the intervention. Fasting blood samples were taken before and after the intervention to measure super oxide dismutase, glutathione peroxidase, glutathione reductase, catalase and total antioxidant capacity in erythrocytes. Results: A total of 81 subjects completed the study. Two study groups were similar as regards duration of diabetes, age and the enzymes at baseline. Energy and macro- and micronutrients intakes, weight and hypoglycemic agent consumption were similar in the two groups at baseline and did not change. Supplementation had no effect on antioxidant enzyme status. Glycated hemoglobin showed a significant reduction by supplementation. Conclusion: Daily supplementation of 2714 mg mega-3 for two mo results in a significant reduction in HbA1c level in type2 diabetic patients with no effects on antioxidant enzymes activity. PMID:27141496

  5. Dissecting Proton Delocalization in an Enzyme's Hydrogen Bond Network with Unnatural Amino Acids.

    PubMed

    Wu, Yufan; Fried, Stephen D; Boxer, Steven G

    2015-12-01

    Extended hydrogen bond networks are a common structural motif of enzymes. A recent analysis proposed quantum delocalization of protons as a feature present in the hydrogen bond network spanning a triad of tyrosines (Y(16), Y(32), and Y(57)) in the active site of ketosteroid isomerase (KSI), contributing to its unusual acidity and large isotope shift. In this study, we utilized amber suppression to substitute each tyrosine residue with 3-chlorotyrosine to test the delocalization model and the proton affinity balance in the triad. X-ray crystal structures of each variant demonstrated that the structure, notably the O-O distances within the triad, was unaffected by 3-chlorotyrosine substitutions. The changes in the cluster's acidity and the acidity's isotope dependence in these variants were assessed via UV-vis spectroscopy and the proton sharing pattern among individual residues with (13)C nuclear magnetic resonance. Our data show pKa detuning at each triad residue alters the proton delocalization behavior in the H-bond network. The extra stabilization energy necessary for the unusual acidity mainly comes from the strong interactions between Y(57) and Y(16). This is further enabled by Y(32), which maintains the right geometry and matched proton affinity in the triad. This study provides a rich picture of the energetics of the hydrogen bond network in enzymes for further model refinement. PMID:26571340

  6. Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme.

    PubMed

    Matte, Allan; Grosse, Stephan; Bergeron, Hélène; Abokitse, Kofi; Lau, Peter C K

    2010-11-01

    The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavouring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a β-barrel structure and two α-helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the β-barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site. PMID:21045284

  7. Structural analysis of Bacillus pumilus phenolic acid decarboxylase, a lipocalin-fold enzyme

    SciTech Connect

    Matte, Allan; Grosse, Stephan; Bergeron, Hélène; Abokitse, Kofi; Lau, Peter C.K.

    2012-04-30

    The decarboxylation of phenolic acids, including ferulic and p-coumaric acids, to their corresponding vinyl derivatives is of importance in the flavoring and polymer industries. Here, the crystal structure of phenolic acid decarboxylase (PAD) from Bacillus pumilus strain UI-670 is reported. The enzyme is a 161-residue polypeptide that forms dimers both in the crystal and in solution. The structure of PAD as determined by X-ray crystallography revealed a -barrel structure and two -helices, with a cleft formed at one edge of the barrel. The PAD structure resembles those of the lipocalin-fold proteins, which often bind hydrophobic ligands. Superposition of structurally related proteins bound to their cognate ligands shows that they and PAD bind their ligands in a conserved location within the -barrel. Analysis of the residue-conservation pattern for PAD-related sequences mapped onto the PAD structure reveals that the conservation mainly includes residues found within the hydrophobic core of the protein, defining a common lipocalin-like fold for this enzyme family. A narrow cleft containing several conserved amino acids was observed as a structural feature and a potential ligand-binding site.

  8. Effect of exogenous amylolytic enzymes on the accumulation of chlorogenic acid isomers in wounded potato tubers.

    PubMed

    Torres-Contreras, Ana Mariel; Nair, Vimal; Cisneros-Zevallos, Luis; Jacobo-Velázquez, Daniel A

    2014-08-01

    Potato tubers under wounding stress synthesize chlorogenic acid isomers, which are phenolic compounds that prevent chronic diseases. The biosynthesis of phenolic compounds in plants requires aromatic amino acids that are produced from sugars. Therefore, in this study, we hypothesized that the wound-induced accumulation of chlorogenic acid isomers in potatoes could be enhanced if the availability of sugars is increased by exogenous amylolytic enzymes applied to the surface of the site of wounding. To test this hypothesis, wounded potatoes stored at 20 °C were treated with amylolytic enzymes (pullulanase and amyloglucosidase, 282 units/mL, 10 mL/kg) after being stored for 0 (E0h), 48 (E48h), or 96 h (E96h). The highest level of accumulation of total chlorogenic acid isomers (∼210% higher than that of time 0 h samples) was observed after storage for 120 h for the E96h treatment. The results suggest that increasing the availability of carbon sources needed for the biosynthesis of phenolic compounds would trigger their accumulation in wounded plants. PMID:25032895

  9. Ensemble Methods for Monitoring Enzyme Translocation along Single Stranded Nucleic Acids

    PubMed Central

    Tomko, Eric J.; Fischer, Christopher J.; Lohman, Timothy M.

    2010-01-01

    We review transient kinetic methods developed to study the mechanism of translocation of nucleic acid motor proteins. One useful stopped-flow fluorescence method monitors arrival of the translocase at the end of a fluorescently labeled nucleic acid. When conducted under single-round conditions the time courses can be analyzed quantitatively using n-step sequential models to determine the kinetic parameters for translocation (rate, kinetic step size and processivity). The assay and analysis discussed here can be used to study enzyme translocation along a linear lattice such as ssDNA or ssRNA. We outline the methods for experimental design and two approaches, along with their limitations, that can be used to analyze the time courses. Analysis of the full time courses using n-step sequential models always yields an accurate estimate of the translocation rate. An alternative semi-quantitative “time to peak” analysis yields accurate estimates of translocation rates only if the enzyme initiates translocation from a unique site on the nucleic acid. However, if initiation occurs at random sites along the nucleic acid, then the “time to peak” analysis can yield inaccurate estimates of even the rates of translocation depending on the values of other kinetic parameters, especially the rate of dissociation of the translocase. Thus, in those cases analysis of the full time course is needed to obtain accurate estimates of translocation rates. PMID:20371288

  10. Experiment K-7-21: Effect of Microgravity on 1: Metabolic Enzymes of Type 1 and Type 2 Muscle Fibers, and on 2: Metabolic Enzymes, Neurotransmitter Amino Acids, and Neurotransmitter Associated Enzymes in Selected Regions of the Central Nervous System. Part 2; The Distribution of Selected Enzymes and Amino Acids in the Hippocampal Formation

    NASA Technical Reports Server (NTRS)

    Lowry, O. H.; Krasnov, I.; Ilyina-Kakueva, E. I.; Nemeth, P. M.; McDougal, D. B., Jr.; Choksi, R.; Carter, J. G.; Chi, M. M. Y.; Manchester, J. K.; Pusateri, M. E.

    1994-01-01

    Six key metabolic enzymes plus glutaminase and glutamate decarboxylase, as well as glutamate, aspartate and GABA, were measured in 11 regions of the hippocampal formation of synchronous, flight and tail suspension rats. Major differences were observed in the normal distribution patterns of each enzyme and amino acid, but no substantive effects of either microgravity or tail suspension on these patterns were clearly demonstrated.

  11. Antioxidant enzymes and fatty acid composition as related to disease resistance in postharvest loquat fruit.

    PubMed

    Cao, Shifeng; Yang, Zhenfeng; Cai, Yuting; Zheng, Yonghua

    2014-11-15

    Two cultivars of loquat fruit were stored at 20°C for 10days to investigate the relationship between disease resistance, and fatty acid composition and activities of endogenous antioxidant enzymes. The results showed that decay incidence increased with storage time in both cultivars. A significantly lower disease incidence was observed in 'Qingzhong' fruit than in 'Fuyang', suggesting 'Qingzhong' had increased disease resistance. Meanwhile, 'Qingzhong' fruit also had lower levels of superoxide radical and hydrogen peroxide, and lower lipoxygenase activity, but higher levels of linolenic and linoleic acids and higher activities of catalase (CAT) and ascorbate peroxidase (APX) compared with 'Fuyang'. These results suggest that the higher levels of linolenic and linoleic acids and the higher activity of CAT and APX have a role in disease resistance of postharvest loquat fruit. PMID:24912701

  12. Combined Effects of Lanthanum (III) and Acid Rain on Antioxidant Enzyme System in Soybean Roots

    PubMed Central

    Zhang, Xuanbo; Du, Yuping; Wang, Lihong; Zhou, Qing; Huang, Xiaohua; Sun, Zhaoguo

    2015-01-01

    Rare earth element pollution (REEs) and acid rain (AR) pollution simultaneously occur in many regions, which resulted in a new environmental issue, the combined pollution of REEs and AR. The effects of the combined pollution on the antioxidant enzyme system of plant roots have not been reported. Here, the combined effects of lanthanum ion (La3+), one type of REE, and AR on the antioxidant enzyme system of soybean roots were investigated. In the combined treatment of La3+ (0.08 mM) and AR, the cell membrane permeability and the peroxidation of cell membrane lipid of soybean roots increased, and the superoxide dismutase, catalase, peroxidase and reduced ascorbic acid served as scavengers of reactive oxygen species. In other combined treatments of La3+ (0.40 mM, 1.20 mM) and AR, the membrane permeability, malonyldialdehyde content, superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content increased, while the catalase activity decreased. The increased superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content were inadequate to scavenge the excess hydrogen peroxide and superoxide, leading to the damage of the cell membrane, which was aggravated with the increase in the concentration of La3+ and the level of AR. The deleterious effects of the combined treatment of La3+ and AR were stronger than those of the single treatment of La3+ or AR. Moreover, the activity of antioxidant enzyme system in the combined treatment group was affected directly and indirectly by mineral element content in soybean plants. PMID:26230263

  13. Combined Effects of Lanthanum (III) and Acid Rain on Antioxidant Enzyme System in Soybean Roots.

    PubMed

    Zhang, Xuanbo; Du, Yuping; Wang, Lihong; Zhou, Qing; Huang, Xiaohua; Sun, Zhaoguo

    2015-01-01

    Rare earth element pollution (REEs) and acid rain (AR) pollution simultaneously occur in many regions, which resulted in a new environmental issue, the combined pollution of REEs and AR. The effects of the combined pollution on the antioxidant enzyme system of plant roots have not been reported. Here, the combined effects of lanthanum ion (La3+), one type of REE, and AR on the antioxidant enzyme system of soybean roots were investigated. In the combined treatment of La3+ (0.08 mM) and AR, the cell membrane permeability and the peroxidation of cell membrane lipid of soybean roots increased, and the superoxide dismutase, catalase, peroxidase and reduced ascorbic acid served as scavengers of reactive oxygen species. In other combined treatments of La3+ (0.40 mM, 1.20 mM) and AR, the membrane permeability, malonyldialdehyde content, superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content increased, while the catalase activity decreased. The increased superoxide dismutase activity, peroxidase activity and reduced ascorbic acid content were inadequate to scavenge the excess hydrogen peroxide and superoxide, leading to the damage of the cell membrane, which was aggravated with the increase in the concentration of La3+ and the level of AR. The deleterious effects of the combined treatment of La3+ and AR were stronger than those of the single treatment of La3+ or AR. Moreover, the activity of antioxidant enzyme system in the combined treatment group was affected directly and indirectly by mineral element content in soybean plants. PMID:26230263

  14. Immunohistochemical Localization of Key Arachidonic Acid Metabolism Enzymes during Fracture Healing in Mice

    PubMed Central

    Lin, Hsuan-Ni; O’Connor, J. Patrick

    2014-01-01

    This study investigated the localization of critical enzymes involved in arachidonic acid metabolism during the initial and regenerative phases of mouse femur fracture healing. Previous studies found that loss of cyclooxygenase-2 activity impairs fracture healing while loss of 5-lipoxygenase activity accelerates healing. These diametric results show that arachidonic acid metabolism has an essential function during fracture healing. To better understand the function of arachidonic acid metabolism during fracture healing, expression of cyclooxygenase-1 (COX-1), cyclooxygenase -2 (COX-2), 5-lipoxygenase (5-LO), and leukotriene A4 hydrolase (LTA4H) was localized by immunohistochemistry in time-staged fracture callus specimens. All four enzymes were detected in leukocytes present in the bone marrow and attending inflammatory response that accompanied the fracture. In the tissues surrounding the fracture site, the proportion of leukocytes expressing COX-1, COX-2, or LTA4H decreased while those expressing 5-LO remained high at 4 and 7 days after fracture. This may indicate an inflammation resolution function for 5-LO during fracture healing. Only COX-1 was consistently detected in fracture callus osteoblasts during the later stages of healing (day 14 after fracture). In contrast, callus chondrocytes expressed all four enzymes, though 5-LO appeared to be preferentially expressed in newly differentiated chondrocytes. Most interestingly, osteoclasts consistently and strongly expressed COX-2. In addition to bone surfaces and the growth plate, COX-2 expressing osteoclasts were localized at the chondro-osseous junction of the fracture callus. These observations suggest that arachidonic acid mediated signaling from callus chondrocytes or from callus osteoclasts at the chondro-osseous junction regulate fracture healing. PMID:24516658

  15. A novel enzyme-based acidizing system: Matrix acidizing and drilling fluid damage removal

    SciTech Connect

    Harris, R.E.; McKay, D.M.; Moses, V.

    1995-12-31

    A novel acidizing process is used to increase the permeability of carbonate rock cores in the laboratory and to remove drilling fluid damage from cores and wafers. Field results show the benefits of the technology as applied both to injector and producer wells.

  16. Importance of ALDH1A enzymes in determining human testicular retinoic acid concentrations

    PubMed Central

    Arnold, Samuel L.; Kent, Travis; Hogarth, Cathryn A.; Schlatt, Stefan; Prasad, Bhagwat; Haenisch, Michael; Walsh, Thomas; Muller, Charles H.; Griswold, Michael D.; Amory, John K.; Isoherranen, Nina

    2015-01-01

    Retinoic acid (RA), the active metabolite of vitamin A, is required for spermatogenesis and many other biological processes. RA formation requires irreversible oxidation of retinal to RA by aldehyde dehydrogenase enzymes of the 1A family (ALDH1A). While ALDH1A1, ALDH1A2, and ALDH1A3 all form RA, the expression pattern and relative contribution of these enzymes to RA formation in the testis is unknown. In this study, novel methods to measure ALDH1A protein levels and intrinsic RA formation were used to accurately predict RA formation velocities in individual human testis samples and an association between RA formation and intratesticular RA concentrations was observed. The distinct localization of ALDH1A in the testis suggests a specific role for each enzyme in controlling RA formation. ALDH1A1 was found in Sertoli cells, while only ALDH1A2 was found in spermatogonia, spermatids, and spermatocytes. In the absence of cellular retinol binding protein (CRBP)1, ALDH1A1 was predicted to be the main contributor to intratesticular RA formation, but when CRBP1 was present, ALDH1A2 was predicted to be equally important in RA formation as ALDH1A1. This study provides a comprehensive novel methodology to evaluate RA homeostasis in human tissues and provides insight to how the individual ALDH1A enzymes mediate RA concentrations in specific cell types. PMID:25502770

  17. Are Phragmites australis enzymes involved in the degradation of the textile azo dye acid orange 7?

    PubMed

    Carias, Cátia C; Novais, Júlio M; Martins-Dias, Susete

    2008-01-01

    The role of antioxidant and detoxification enzymes of Phragmites australis, in the degradation of an azo dye, acid orange 7 (AO7), was studied. Activities of several enzymes involved in plant protection against stress were assayed through the activity characterization of superoxide dismutase (SOD), peroxidases (POD), catalase (CAT), ascorbate peroxidase (APOX), dehydroascorbate reductase (DHAR) and glutathione S-transferase (GST), obtained from P. australis crude extracts of leaves, stems and roots. A sub-surface vertical flow constructed wetland, planted with P. australis was used to test the plants response to the AO7 exposure at two different concentrations (130 and 700 mg l(-1)). An activity increase was detected for an AO7 concentration of 130 mg l(-1) for most enzymes studied (SOD, CAT and APOX), especially in leaves, suggesting a response of the reactive oxygen species scavenging enzymes to the chemical stress imposed. GST activity increase in this situation can also be interpreted as an activation of the detoxification pathway and subsequent AO7 conjugation. A totally different behaviour was observed for AO7 at 700 mg l(-1). An evident decrease in activity was observed for SOD, CAT, APOX and GST, probably due to enzymatic inhibition by AO7. Contrarily, DHAR activity augmented drastically in this situation. POD activity was not greatly affected during trial. Altogether these results suggest that P. australis effectively uses the ascorbate-glutathione pathway for the detoxification of AO7. PMID:17336060

  18. Effects of acid/alkaline pretreatment and gamma-ray irradiation on extracellular polymeric substances from sewage sludge

    NASA Astrophysics Data System (ADS)

    Xie, Shuibo; Wu, Yuqi; Wang, Wentao; Wang, Jingsong; Luo, Zhiping; Li, Shiyou

    2014-04-01

    In order to investigate the mechanism of extracellular polymeric substances (EPS) influencing sludge characteristics, variations of extractable EPS from municipal sewage sludge by acid/alkaline pretreatment and gamma-ray irradiation were studied. The changes in constituents of EPS were analyzed by UV-vis spectra and SEM images. The effects of alkaline pretreatment and gamma-ray irradiation on the functional groups in EPS were investigated by Fourier transform infrared (FTIR) spectrometer. Results showed that the extractable EPS increased clearly with increasing irradiation dose from 0 to 15 kGy. UV-vis spectra indicated that a new absorption band from 240 nm to 300 nm existed in all irradiated samples, apart from acid condition. The results of FTIR spectroscopic analysis indicated that, irradiation influenced major functional groups in EPS, such as protein and polysaccharide, and these effects were clearer under alkaline condition. SEM images provided that after alkaline hydrolysis, gamma-ray irradiation was more effective in resulting in the sludge flocs and cells broken, compared with acid pretreatment (pH 2.50).

  19. Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis.

    PubMed Central

    Elkins, Jonathan M; Clifton, Ian J; Hernández, Helena; Doan, Linh X; Robinson, Carol V; Schofield, Christopher J; Hewitson, Kirsty S

    2002-01-01

    During biosynthesis of the clinically used beta-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(L)-arginine and (3R)-hydroxy-N-acetyl-(L)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75 A and 2.45 A resolution, where 1 A=0.1 nm) imply how it binds beta-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of beta-strands surrounded by alpha-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding. PMID:12020346

  20. Reverse reaction of malic enzyme for HCO3- fixation into pyruvic acid to synthesize L-malic acid with enzymatic coenzyme regeneration.

    PubMed

    Ohno, Yoko; Nakamori, Toshihiko; Zheng, Haitao; Suye, Shin-ichiro

    2008-05-01

    Malic enzyme [L-malate: NAD(P)(+) oxidoreductase (EC 1.1.1.39)] catalyzes the oxidative decarboxylation of L-malic acid to produce pyruvic acid using the oxidized form of NAD(P) (NAD(P)(+)). We used a reverse reaction of the malic enzyme of Pseudomonas diminuta IFO 13182 for HCO(3)(-) fixation into pyruvic acid to produce L-malic acid with coenzyme (NADH) generation. Glucose-6-phosphate dehydrogenase (EC1.1.1.49) of Leuconostoc mesenteroides was suitable for coenzyme regeneration. Optimum conditions for the carboxylation of pyruvic acid were examined, including pyruvic acid, NAD(+), and both malic enzyme and glucose-6-phosphate dehydrogenase concentrations. Under optimal conditions, the ratio of HCO(3)(-) and pyruvic acid to malic acid was about 38% after 24 h of incubation at 30 degrees C, and the concentration of the accumulated L-malic acid in the reaction mixture was 38 mM. The malic enzyme reverse reaction was also carried out by the conjugated redox enzyme reaction with water-soluble polymer-bound NAD(+). PMID:18460807

  1. Water-soluble polymeric chemosensor for selective detection of Hg(2+) in aqueous solution using rhodamine-based modified poly(acrylamide-acrylic acid).

    PubMed

    Geng, Tong-Mou; Wu, Da-Yu

    2015-12-01

    We report the fabrication of a novel easily available turn-on fluorescent water-soluble polymeric chemosensor for Hg(2+) ions that was simply prepared by micellar free radical polymerization of a water-insoluble organic rhodamine-based Hg(2+)-recognizing monomer (GR6GH), with hydrophilic monomers acrylamide (AM) and acrylic acid (AA). The chemical structure of the polymeric sensor was characterized by FT-IR and (1)H NMR spectroscopy. The apparent viscosity average molecular weight Mη of poly(acrylamide-acrylic acid) [poly(AM-NaAA)] and the water-soluble polymeric chemosensor poly(AM-NaAA-GR6GH) were 1.76 × 10(6) and 6.84 × 10(4) g/mol, respectively. Because of its amphiphilic property, the water-soluble polymeric chemosensor can be used as a chemosensor in aqueous media. Upon addition of Hg(2+) ions to an aqueous solution of poly(AM-NaAA-GR6GH), fluorescence enhancements were observed instantly. Moreover, other metal ions did not induce obvious changes to the fluorescence spectra. This approach may provide an easily measurable and inherently sensitive method for Hg(2+) ion detection in environmental and biological applications. PMID:25808221

  2. Relationship of lipogenic enzyme activities to the rate of rat liver fatty acid synthesis

    SciTech Connect

    Nelson, G.; Kelley, D.; Schmidt, P.; Virk, S.; Serrato, C.

    1986-05-01

    The mechanism by which diet regulates liver lipogenesis is unclear. Here the authors report how dietary alterations effect the activities of key enzymes of fatty acid (FA) synthesis. Male Sprague-Dawley rats, 400-500 g, were fasted for 48h and then refed a fat-free, high carbohydrate (HC) diet (75% cal. from sucrose) for 0,3,9,24 and 48h, or refed a HC diet for 48h, then fed a high-fat (HF) diet (44% cal. from corn oil) for 3,9,24 and 48h. The FA synthesis rate and the activities of acetyl CoA carboxylase (AC), fatty acid synthase (FAS), ATP citrate lyase (CL), and glucose 6-phosphate dehydrogenase (G6PDH) were determined in the livers. FA synthesis was assayed with /sup 3/H/sub 2/O, enzyme activities were measured spectrophotometrically except for AC which was assayed with /sup 14/C-bicarbonate. There was no change in the activity of AC during fasting or on the HC diet. Fasting decreased the rate of FA synthesis by 25% and the activities of FAS and CL by 50%; refeeding the HC diet induced parallel changes in FA synthesis and the activities of FAS, CL, and G6PDH. After 9h on the HF diet, FA synthesis had decreased sharply, AC activity increased significantly while no changes were detected in the other activities. Subsequently FA synthesis did not change while the activities of the enzymes decreased slowly. These enzymes did not appear to regulate FA synthesis during inhibition of lipogenesis, but FAS, CL or G6PDH may be rate limiting in the induction phase. Other key factors may regulate FA synthesis during dietary alterations.

  3. Jasmonate-inducible plant enzymes degrade essential amino acids in the herbivore midgut

    PubMed Central

    Chen, Hui; Wilkerson, Curtis G.; Kuchar, Jason A.; Phinney, Brett S.; Howe, Gregg A.

    2005-01-01

    The plant hormone jasmonic acid (JA) activates host defense responses against a broad spectrum of herbivores. Although it is well established that JA controls the expression of a large set of target genes in response to tissue damage, very few gene products have been shown to play a direct role in reducing herbivore performance. To test the hypothesis that JA-inducible proteins (JIPs) thwart attack by disrupting digestive processes in the insect gut, we used a MS-based approach to identify host proteins that accumulate in the midgut of Manduca sexta larvae reared on tomato (Solanum lycopersicum) plants. We show that two JIPs, arginase and threonine deaminase (TD), act in the M. sexta midgut to catabolize the essential amino acids Arg and Thr, respectively. Transgenic plants that overexpress arginase were more resistant to M. sexta larvae, and this effect was correlated with reduced levels of midgut Arg. We present evidence indicating that the ability of TD to degrade Thr in the midgut is enhanced by herbivore-induced proteolytic removal of the enzyme's C-terminal regulatory domain, which confers negative feedback regulation by isoleucine in planta. Our results demonstrate that the JA signaling pathway strongly influences the midgut protein content of phytophagous insects and support the hypothesis that catabolism of amino acids in the insect digestive tract by host enzymes plays a role in plant protection against herbivores. PMID:16357201

  4. Crystal structures of a purple acid phosphatase, representing different steps of this enzyme's catalytic cycle

    PubMed Central

    Schenk, Gerhard; Elliott, Tristan W; Leung, Eleanor; Carrington, Lyle E; Mitić, Nataša; Gahan, Lawrence R; Guddat, Luke W

    2008-01-01

    Background Purple acid phosphatases belong to the family of binuclear metallohydrolases and are involved in a multitude of biological functions, ranging from bacterial killing and bone metabolism in animals to phosphate uptake in plants. Due to its role in bone resorption purple acid phosphatase has evolved into a promising target for the development of anti-osteoporotic chemotherapeutics. The design of specific and potent inhibitors for this enzyme is aided by detailed knowledge of its reaction mechanism. However, despite considerable effort in the last 10 years various aspects of the basic molecular mechanism of action are still not fully understood. Results Red kidney bean purple acid phosphatase is a heterovalent enzyme with an Fe(III)Zn(II) center in the active site. Two new structures with bound sulfate (2.4 Å) and fluoride (2.2 Å) provide insight into the pre-catalytic phase of its reaction cycle and phosphorolysis. The sulfate-bound structure illustrates the significance of an extensive hydrogen bonding network in the second coordination sphere in initial substrate binding and orientation prior to hydrolysis. Importantly, both metal ions are five-coordinate in this structure, with only one nucleophilic μ-hydroxide present in the metal-bridging position. The fluoride-bound structure provides visual support for an activation mechanism for this μ-hydroxide whereby substrate binding induces a shift of this bridging ligand towards the divalent metal ion, thus increasing its nucleophilicity. Conclusion In combination with kinetic, crystallographic and spectroscopic data these structures of red kidney bean purple acid phosphatase facilitate the proposal of a comprehensive eight-step model for the catalytic mechanism of purple acid phosphatases in general. PMID:18234116

  5. Characterization of Two Streptomyces Enzymes That Convert Ferulic Acid to Vanillin

    PubMed Central

    Yang, Wenwen; Tang, Hongzhi; Ni, Jun; Wu, Qiulin; Hua, Dongliang; Tao, Fei; Xu, Ping

    2013-01-01

    Production of flavors from natural substrates by microbial transformation has become a growing and expanding field of study over the past decades. Vanillin, a major component of vanilla flavor, is a principal flavoring compound used worldwide. Streptomyces sp. strain V-1 is known to be one of the most promising microbial producers of natural vanillin from ferulic acid. Although identification of the microbial genes involved in the biotransformation of ferulic acid to vanillin has been previously reported, purification and detailed characterization of the corresponding enzymes with important functions have rarely been studied. In this study, we isolated and identified 2 critical genes, fcs and ech, encoding feruloyl-CoA synthetase and enoyl-CoA hydratase/aldolase, respectively, which are involved in the vanillin production from ferulic acid. Both genes were heterologously expressed in Escherichia coli, and the resting cell reactions for converting ferulic acid to vanillin were performed. The corresponding crucial enzymes, Fcs and Ech, were purified for the first time and the enzymatic activity of each purified protein was studied. Furthermore, Fcs was comprehensively characterized, at an optimal pH of 7.0 and temperature of 30°C. Kinetic constants for Fcs revealed the apparent Km, kcat, and Vmax values to be 0.35 mM, 67.7 s−1, and 78.2 U mg−1, respectively. The catalytic efficiency (kcat/Km) value of Fcs was 193.4 mM−1 s−1 for ferulic acid. The characterization of Fcs and Ech may be helpful for further research in the field of enzymatic engineering and metabolic regulation. PMID:23840666

  6. Nucleic acid polymeric properties and electrostatics: Directly comparing theory and simulation with experiment.

    PubMed

    Sim, Adelene Y L

    2016-06-01

    Nucleic acids are biopolymers that carry genetic information and are also involved in various gene regulation functions such as gene silencing and protein translation. Because of their negatively charged backbones, nucleic acids are polyelectrolytes. To adequately understand nucleic acid folding and function, we need to properly describe its i) polymer/polyelectrolyte properties and ii) associating ion atmosphere. While various theories and simulation models have been developed to describe nucleic acids and the ions around them, many of these theories/simulations have not been well evaluated due to complexities in comparison with experiment. In this review, I discuss some recent experiments that have been strategically designed for straightforward comparison with theories and simulation models. Such data serve as excellent benchmarks to identify limitations in prevailing theories and simulation parameters. PMID:26482088

  7. Mechanism of in situ surface polymerization of gallic acid in an environmental-inspired preparation of carboxylated core-shell magnetite nanoparticles.

    PubMed

    Tóth, Ildikó Y; Szekeres, Márta; Turcu, Rodica; Sáringer, Szilárd; Illés, Erzsébet; Nesztor, Dániel; Tombácz, Etelka

    2014-12-30

    Magnetite nanoparticles (MNPs) with biocompatible coatings are good candidates for MRI (magnetic resonance imaging) contrasting, magnetic hyperthermia treatments, and drug delivery systems. The spontaneous surface induced polymerization of dissolved organic matter on environmental mineral particles inspired us to prepare carboxylated core-shell MNPs by using a ubiquitous polyphenolic precursor. Through the adsorption and in situ surface polymerization of gallic acid (GA), a polygallate (PGA) coating is formed on the nanoparticles (PGA@MNP) with possible antioxidant capacity. The present work explores the mechanism of polymerization with the help of potentiometric acid-base titration, dynamic light scattering (for particle size and zeta potential determination), UV-vis (UV-visible light spectroscopy), FTIR-ATR (Fourier-transformed infrared spectroscopy by attenuated total reflection), and XPS (X-ray photoelectron spectroscopy) techniques. We observed the formation of ester and ether linkages between gallate monomers both in solution and in the adsorbed state. Higher polymers were formed in the course of several weeks both on the surface of nanoparticles and in the dispersion medium. The ratio of the absorbances of PGA supernatants at 400 and 600 nm (i.e., the E4/E6 ratio commonly used to characterize the degree of polymerization of humic materials) was determined to be 4.3, similar to that of humic acids. Combined XPS, dynamic light scattering, and FTIR-ATR results revealed that, prior to polymerization, the GA monomers became oxidized to poly(carboxylic acid)s due to ring opening while Fe(3+) ions reduced to Fe(2+). Our published results on the colloidal and chemical stability of PGA@MNPs are referenced thoroughly in the present work. Detailed studies on biocompatibility, antioxidant property, and biomedical applicability of the particles will be published. PMID:25517214

  8. Transgenic Production of Epoxy Fatty Acids by Expression of a Cytochrome P450 Enzyme from Euphorbia lagascae Seed

    PubMed Central

    Cahoon, Edgar B.; Ripp, Kevin G.; Hall, Sarah E.; McGonigle, Brian

    2002-01-01

    Seed oils of a number of Asteraceae and Euphorbiaceae species are enriched in 12-epoxyoctadeca-cis-9-enoic acid (vernolic acid), an unusual 18-carbon Δ12-epoxy fatty acid with potential industrial value. It has been previously demonstrated that the epoxy group of vernolic acid is synthesized by the activity of a Δ12-oleic acid desaturase-like enzyme in seeds of the Asteraceae Crepis palaestina and Vernonia galamensis. In contrast, results from metabolic studies have suggested the involvement of a cytochrome P450 enzyme in vernolic acid synthesis in seeds of the Euphorbiaceae species Euphorbia lagascae. To clarify the biosynthetic origin of vernolic acid in E. lagascae seed, an expressed sequence tag analysis was conducted. Among 1,006 randomly sequenced cDNAs from developing E. lagascae seeds, two identical expressed sequence tags were identified that encode a cytochrome P450 enzyme classified as CYP726A1. Consistent with the seed-specific occurrence of vernolic acid in E. lagascae, mRNA corresponding to the CYP726A1 gene was abundant in developing seeds, but was not detected in leaves. In addition, expression of the E. lagascae CYP726A1 cDNA in Saccharomyces cerevisiae was accompanied by production of vernolic acid in cultures supplied with linoleic acid and an epoxy fatty acid tentatively identified as 12-epoxyoctadeca-9,15-dienoic acid (12-epoxy-18:2Δ9,15) in cultures supplied with α-linolenic acid. Consistent with this, expression of CYP726A1 in transgenic tobacco (Nicotiana tabacum) callus or somatic soybean (Glycine max) embryos resulted in the accumulation of vernolic acid and 12-epoxy-18:2Δ9,15. Overall, these results conclusively demonstrate that Asteraceae species and the Euphorbiaceae E. lagascae have evolved structurally unrelated enzymes to generate the Δ12-epoxy group of vernolic acid. PMID:11842164

  9. Improve the Strength of PLA/HA Composite Through the Use of Surface Initiated Polymerization and Phosphonic Acid Coupling Agent

    PubMed Central

    Wang, Tongxin; Chow, Laurence C.; Frukhtbeyn, Stanislav A.; Ting, Andy Hai; Dong, Quanxiao; Yang, Mingshu; Mitchell, James W.

    2011-01-01

    Bioresorbable composite made from degradable polymers, e.g., polylactide (PLA), and bioactive calcium phosphates, e.g., hydroxyapatite (HA), are clinically desirable for bone fixation, repair and tissue engineering because they do not need to be removed by surgery after the bone heals. However, preparation of PLA/HA composite from non-modified HA usually results in mechanical strength reductions due to a weak interface between PLA and HA. In this study, a calcium-phosphate/phosphonate hybrid shell was developed to introduce a greater amount of reactive hydroxyl groups onto the HA particles. Then, PLA was successfully grafted on HA by surface-initiated polymerization through the non-ionic surface hydroxyl groups. Thermogravimetric analysis indiated that the amount of grafted PLA on HA can be up to 7 %, which is about 50 % greater than that from the literature. PLA grafted HA shows significantly different pH dependent ζ-potential and particle size profiles from those of uncoated HA. By combining the phosphonic acid coupling agent and surface initiated polymerization, PLA could directly link to HA through covalent bond so that the interfacial interaction in the PLA/HA composite can be significantly improved. The diametral tensile strength of PLA/HA composite prepared from PLA-grafted HA was found to be over twice that of the composite prepared from the non-modified HA. Moreover, the tensile strength of the improved composite was 23 % higher than that of PLA alone. By varying additional variables, this approach has the potential to produce bioresorbable composites with improved mechanical properties that are in the range of natural bones, and can have wide applications for bone fixation and repair in load-bearing areas. PMID:22399838

  10. The biosynthetic gene cluster for coronamic acid, an ethylcyclopropyl amino acid, contains genes homologous to amino acid-activating enzymes and thioesterases.

    PubMed Central

    Ullrich, M; Bender, C L

    1994-01-01

    Coronamic acid (CMA), an ethylcyclopropyl amino acid derived from isoleucine, functions as an intermediate in the biosynthesis of coronatine, a chlorosis-inducing phytotoxin produced by Pseudomonas syringae pv. glycinea PG4180. The DNA required for CMA biosynthesis (6.9 kb) was sequenced, revealing three distinct open reading frames (ORFs) which share a common orientation for transcription. The deduced amino acid sequence of a 2.7-kb ORF designated cmaA contained six core sequences and two conserved motifs which are present in a variety of amino acid-activating enzymes, including nonribosomal peptide synthetases. Furthermore, CmaA contained a spatial arrangement of histidine, aspartate, and arginine residues which are conserved in the ferrous active site of some nonheme iron(II) enzymes which catalyze oxidative cyclizations. The deduced amino acid sequence of a 1.2-kb ORF designated cmaT was related to thioesterases of both procaryotic and eucaryotic origins. These data suggest that CMA assembly is similar to the thiotemplate mechanism of nonribosomal peptide synthesis. No significant similarities between a 0.9-kb ORF designated cmaU and other database entries were found. The start sites of two transcripts required for CMA biosynthesis were identified in the present study. pRG960sd, a vector containing a promoterless glucuronidase gene, was used to localize and study the promoter regions upstream of the two transcripts. Data obtained in the present study indicate that CMA biosynthesis is regulated at the transcriptional level by temperature. Images PMID:8002582

  11. Label-free electrochemical nucleic acid biosensing by tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme amplification.

    PubMed

    Liu, Shufeng; Gong, Hongwei; Wang, Yanqun; Wang, Li

    2016-03-15

    Owing to the intrinsic importance of nucleic acid as bio-targets, the achievement of its simple and sensitive detection with high confidence is very essential for biological studies and diagnostic purposes. Herein, a label-free, isothermal, and ultrasensitive electrochemical detection of target DNA was developed by using a tandem polymerization and cleavage-mediated cascade target recycling and DNAzyme releasing amplification strategy. Upon sensing of the nucleic acid analyte for the assembled hairpin-like probe DNA on the electrode, the DNA polymerase guided the target recycling and simultaneously triggered the lambda exonuclease cleavage, accompanied by the cascade recycling of the released new complementary strand and the amplified liberation of the G-rich sequence of the HRP-mimicking DNAzyme. The electrocatalytic reduction of H2O2 by the generated hemin/G-quadruplex DNAzyme was used for the signal readout and further amplification toward target response. Such tandem functional operation by DNA polymerase, lambda exonuclease and DNAzyme endows the developed biosensor with a high sensitivity and also a high confidence. A low detection limit of 5 fM with an excellent selectivity toward target DNA could be achieved. It also exhibits the distinct advantages of simplicity in probe design and biosensor fabrication, and label-free electrochemical detection, thus may offer a promising avenue for the applications in disease diagnosis and clinical biomedicine. PMID:26513289

  12. Synthesis and chelation properties of a new polymeric ligand derived from 1-amino-2-naphthol-4-sulfonic Acid.

    PubMed

    Manivannan, Dhanasekaran; Biju, Valsala Madhavan Nair

    2015-01-01

    A novel chelating resin for preconcentration of heavy metals from various seawater samples has been developed by condensing 1-amino-2-hydroxy-7-[(4-hydroxyphenyl)diazenyl] naphthalene-4-sulfonic acid (AHDNS) with formaldehyde (1:2 mole ratio) in the presence of oxalic acid as the catalyst. The resin thus obtained was used as a solid sorbent for the separation of divalent metal ions present at trace levels in seawater. The functionalized phenol (AHDNS) was characterized by spectral studies. The polymeric resin AHDNS-formaldehyde (AHDNS-F) obtained by condensing the functionalized phenol and formaldehyde was characterized by IR and NMR spectral studies. The chelating property of the AHDNS-F resin towards divalent metal ions was studied as a function of pH and in the presence of electrolyte. The metal uptake properties of the resin were determined by using an atomic absorption spectrophotometer. This procedure was validated for recovery of divalent metal ions from seawater samples. The recoveries of cadmium, cobalt, copper, manganese, lead, and zinc were above 92% under the optimum preconcentration conditions. The LOD was <0.73 μg/L and the RSDs were <2%. Thus, the AHDNS-F resin can be widely used as a solid sorbent for the preconcentration of trace metals at ppm levels in seawater samples. PMID:25857896

  13. Guanine nanowire based amplification strategy: Enzyme-free biosensing of nucleic acids and proteins.

    PubMed

    Gao, Zhong Feng; Huang, Yan Li; Ren, Wang; Luo, Hong Qun; Li, Nian Bing

    2016-04-15

    Sensitive and specific detection of nucleic acids and proteins plays a vital role in food, forensic screening, clinical and environmental monitoring. There remains a great challenge in the development of signal amplification method for biomolecules detection. Herein, we describe a novel signal amplification strategy based on the formation of guanine nanowire for quantitative detection of nucleic acids and proteins (thrombin) at room temperature. In the presence of analytes and magnesium ions, the guanine nanowire could be formed within 10 min. Compared to the widely used single G-quadruplex biocatalytic label unit, the detection limits are improved by two orders of magnitude in our assay. The proposed enzyme-free method avoids fussy chemical label-ling process, complex programming task, and sophisticated equipment, which might provide an ideal candidate for the fabrication of selective and sensitive biosensing platform. PMID:26649493

  14. Hyaluronic acid nanogels with enzyme-sensitive cross-linking group for drug delivery.

    PubMed

    Yang, Chenchen; Wang, Xin; Yao, Xikuang; Zhang, Yajun; Wu, Wei; Jiang, Xiqun

    2015-05-10

    A methacrylation strategy was employed to functionalize hyaluronic acid and prepare hyaluronic acid (HA) nanogels. Dynamic light scattering, zeta potential analyzer and electron microscopy were utilized to characterize the nanogels and their enzyme-degradability in vitro. It was found that these nanogels had a spherical morphology with the diameter of about 70nm, and negative surface potential. When doxorubicin (DOX) was loaded into the nanogels, the diameter decreased to approximately 50nm with a drug loading content of 16% and encapsulation efficiency of 62%. Cellular uptake examinations showed that HA nanogels could be preferentially internalized by two-dimensional (2D) cells and three-dimensional (3D) multicellular spheroids (MCs) which both overexpress CD44 receptor. Near-infrared fluorescence imaging, biodistribution and penetration examinations in tumor tissue indicated that the HA nanogels could efficiently accumulate and penetrate the tumor matrix. In vivo antitumor evaluation found that DOX-loaded HA nanogels exhibited a significantly superior antitumor effect. PMID:25665867

  15. Bioconjugation of therapeutic proteins and enzymes using the expanded set of genetically encoded amino acids.

    PubMed

    Lim, Sung In; Kwon, Inchan

    2016-10-01

    The last decade has witnessed striking progress in the development of bioorthogonal reactions that are strictly directed towards intended sites in biomolecules while avoiding interference by a number of physical and chemical factors in biological environment. Efforts to exploit bioorthogonal reactions in protein conjugation have led to the evolution of protein translational machineries and the expansion of genetic codes that systematically incorporate a range of non-natural amino acids containing bioorthogonal groups into recombinant proteins in a site-specific manner. Chemoselective conjugation of proteins has begun to find valuable applications to previously inaccessible problems. In this review, we describe bioorthogonal reactions useful for protein conjugation, and biosynthetic methods that produce proteins amenable to those reactions through an expanded genetic code. We then provide key examples in which novel protein conjugates, generated by the genetic incorporation of a non-natural amino acid and the chemoselective reactions, address unmet needs in protein therapeutics and enzyme engineering. PMID:26036278

  16. Horseradish peroxidase-catalyzed polymerization of cardanol in the presence of redox mediators.

    PubMed

    Won, Keehoon; Kim, Yong Hwan; An, Eun Suk; Lee, Yeon Soo; Song, Bong Keun

    2004-01-01

    Horseradish peroxidase-catalyzed polymerization of cardanol in aqueous organic solvent was investigated in the presence of a redox mediator. Cardanol is a phenol derivative from a renewable resource mainly having a C15 unsaturated hydrocarbon chain with mostly 1-3 double bonds at a meta position. Unlike soybean peroxidase (SBP), it has been shown that horseradish peroxidase (HRP) is not able to perform oxidative polymerization of phenol derivatives having a bulky meta substituent such as cardanol. For the first time, redox mediators have been applied to enable horseradish peroxidase to polymerize cardanol. Veratryl alcohol, N-ethyl phenothiazine, and phenothiazine-10-propionic acid were tested as a mediator. It is surprising that the horseradish peroxidase-catalyzed polymerization of cardanol took place in the presence of N-ethyl phenothiazine or phenothiazine-10-propionic acid. However, veratryl alcohol showed no effect. FT-IR and GPC analysis of the product revealed that the structure and properties of polycardanol formed by HRP with a mediator were similar to those by SBP. This is the first work to apply a redox mediator to enzyme-catalyzed oxidative polymerization. Our new finding that oxidative polymerization of a poor substrate, which the enzyme is not active with, can take place in the presence of an appropriate mediator will present more opportunities for the application of enzyme-catalyzed polymerization. PMID:14715000

  17. Characterization of inulin hydrolyzing enzyme(s) in commercial glucoamylases and its application in lactic acid production from Jerusalem artichoke tubers (Jat).

    PubMed

    Dao, Thai Ha; Zhang, Jian; Bao, Jie

    2013-11-01

    A high inulinase activity was found in three commercially available glucoamylase enzymes. Its origin was investigated and two proteins in the commercial glucoamylases were identified as the potential enzymes showing inulinase activity. One of the commercial glucoamylases, GA-L New from Genencor, was used for Jerusalem artichoke tubers (Jat) hydrolysis and a high hydrolysis yield of fructose was obtained. The simultaneous saccharification and lactic acid fermentation (SSF) of Jat was carried out using GA-L New as the inulinase and Pediococcus acidilactici DQ2 as the fermenting strain. A high lactic acid titer, yield, and productivity of 111.5 g/L, 0.46 g/g DM, and 1.55 g/L/h, respectively, were obtained within 72 h. The enzyme cost using the commercial glucoamylase as inulinase was compared to that using the typical inulinase and a large profit margin was identified. The results provided a practical way of Jat application for lactic acid production using cheap commercial glucoamylase enzyme. PMID:24050923

  18. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    NASA Astrophysics Data System (ADS)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-01-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  19. Ontogenetic changes in digestive enzyme activities and the amino acid profile of starry flounder Platichthys stellatus

    NASA Astrophysics Data System (ADS)

    Song, Zhidong; Wang, Jiying; Qiao, Hongjin; Li, Peiyu; Zhang, Limin; Xia, Bin

    2016-09-01

    Ontogenetic changes in digestive enzyme activities and the amino acid (AA) profile of starry flounder, Platichthys stellatus, were investigated and limiting amino acids were estimated compared with the essential AA profile between larvae and live food to clarify starry flounder larval nutritional requirements. Larvae were collected at the egg stage and 0, 2, 4, 7, 12, 17, 24 days after hatching (DAH) for analysis. Larvae grew from 1.91 mm at hatching to 12.13 mm at 24 DAH. Trypsin and chymotrypsin activities changed slightly by 4 DAH and then increased significantly 4 DAH. Pepsin activity increased sharply beginning 17 DAH. Lipase activity increased significantly 4 DAH and increased progressively with larval growth. Amylase activity was also detected in newly hatched larvae and increased 7 DAH followed by a gradual decrease. High free amino acid (FAA) content was detected in starry flounder eggs (110.72 mg/g dry weight). Total FAA content dropped to 43.29 mg/g in 4-DAH larvae and then decreased gradually to 13.74 mg/g in 24-DAH larvae. Most FAAs (except lysine and methionine) decreased >50% in 4-DAH larvae compared with those in eggs and then decreased to the lowest values in 24-DAH larvae. Changes in the protein amino acid (PAA) profile were much milder than those observed for FAAs. Most PAAs increased gradually during larval development, except lysine and phenylalanine. The percentages of free threonine, valine, isoleucine, and leucine decreased until the end of the trial, whereas the protein forms of these four AAs followed the opposite trend. A comparison of the essential AA composition of live food (rotifers, Artemia nauplii, and Artemia metanauplii) and larvae suggested that methionine was potentially the first limiting AA. These results may help develop starry flounder larviculture methods by solving the AA imbalance in live food. Moreover, the increased digestive enzyme activities indicate the possibility of introducing artificial compound feed.

  20. Characterization of fatty acid modifying enzyme activity in staphylococcal mastitis isolates and other bacteria

    PubMed Central

    2012-01-01

    Background Fatty acid modifying enzyme (FAME) has been shown to modify free fatty acids to alleviate their bactericidal effect by esterifying fatty acids to cholesterol or alcohols. Although it has been shown in previous studies that FAME is required for Staphylococcus aureus survival in skin abscesses, FAME is poorly studied compared to other virulence factors. FAME activity had also been detected in coagulase-negative staphylococci (CNS). However, FAME activity was only surveyed after a bacterial culture was grown for 24 h. Therefore if FAME activity was earlier in the growth phase, it would not have been detected by the assay and those strains would have been labeled as FAME negative. Results Fifty CNS bovine mastitis isolates and several S. aureus, Escherichia coli, and Streptococcus uberis strains were assayed for FAME activity over 24 h. FAME activity was detected in 54% of CNS and 80% S. aureus strains surveyed but none in E. coli or S. uberis. While some CNS strains produced FAME activity comparable to the lab strain of S. aureus, the pattern of FAME activity varied among strains and across species of staphylococci. All CNS that produced FAME activity also exhibited lipase activity. Lipase activity relative to colony forming units of these CNS decreased over the 24 h growth period. No relationship was observed between somatic cell count in the milk and FAME activity in CNS. Conclusions Some staphylococcal species surveyed produced FAME activity, but E. coli and S. uberis strains did not. All FAME producing CNS exhibited lipase activity which may indicate that both these enzymes work in concert to alter fatty acids in the bacterial environment. PMID:22726316

  1. Lactic acid bacteria affect serum cholesterol levels, harmful fecal enzyme activity, and fecal water content

    PubMed Central

    Lee, Do Kyung; Jang, Seok; Baek, Eun Hye; Kim, Mi Jin; Lee, Kyung Soon; Shin, Hea Soon; Chung, Myung Jun; Kim, Jin Eung; Lee, Kang Oh; Ha, Nam Joo

    2009-01-01

    Background Lactic acid bacteria (LAB) are beneficial probiotic organisms that contribute to improved nutrition, microbial balance, and immuno-enhancement of the intestinal tract, as well as lower cholesterol. Although present in many foods, most trials have been in spreads or dairy products. Here we tested whether Bifidobacteria isolates could lower cholesterol, inhibit harmful enzyme activities, and control fecal water content. Methods In vitro culture experiments were performed to evaluate the ability of Bifidobacterium spp. isolated from healthy Koreans (20~30 years old) to reduce cholesterol-levels in MRS broth containing polyoxyethanylcholesterol sebacate. Animal experiments were performed to investigate the effects on lowering cholesterol, inhibiting harmful enzyme activities, and controlling fecal water content. For animal studies, 0.2 ml of the selected strain cultures (108~109 CFU/ml) were orally administered to SD rats (fed a high-cholesterol diet) every day for 2 weeks. Results B. longum SPM1207 reduced serum total cholesterol and LDL levels significantly (p < 0.05), and slightly increased serum HDL. B. longum SPM1207 also increased fecal LAB levels and fecal water content, and reduced body weight and harmful intestinal enzyme activities. Conclusion Daily consumption of B. longum SPM1207 can help in managing mild to moderate hypercholesterolemia, with potential to improve human health by helping to prevent colon cancer and constipation. PMID:19515264

  2. Gluconic acid production from sucrose in an airlift reactor using a multi-enzyme system.

    PubMed

    Mafra, Agnes Cristina Oliveira; Furlan, Felipe Fernando; Badino, Alberto Colli; Tardioli, Paulo Waldir

    2015-04-01

    Sucrose from sugarcane is produced in abundance in Brazil, which provides an opportunity to manufacture other high-value products. Gluconic acid (GA) can be produced by multi-enzyme conversion of sucrose using the enzymes invertase, glucose oxidase, and catalase. In this process, one of the byproducts is fructose, which has many commercial applications. This work concerns the batch mode production of GA in an airlift reactor fed with sucrose as substrate. Evaluation was made of the influence of temperature and pH, as well as the thermal stability of the enzymes. Operational conditions of 40 °C and pH 6.0 were selected, based on the enzymatic activity profiles and the thermal stabilities. Under these conditions, the experimental data could be accurately described by kinetic models. The maximum yield of GA was achieved within 3.8 h, with total conversion of sucrose and glucose and a volumetric productivity of around 7.0 g L(-1) h(-1). PMID:25326720

  3. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS-PKS hybrid enzyme.

    PubMed

    Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

    2015-01-01

    Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS-PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS-PKS hybrid enzyme. PMID:26503170

  4. Biosynthesis of the mycotoxin tenuazonic acid by a fungal NRPS–PKS hybrid enzyme

    PubMed Central

    Yun, Choong-Soo; Motoyama, Takayuki; Osada, Hiroyuki

    2015-01-01

    Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi. However, its biosynthetic gene has been unknown to date. Here we identify the TeA biosynthetic gene from Magnaporthe oryzae by finding two TeA-inducing conditions of a low-producing strain. We demonstrate that TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase (NRPS–PKS) hybrid enzyme that begins with an NRPS module. In contrast to other NRPS/PKS hybrid enzymes, the PKS portion of TAS1 has only a ketosynthase (KS) domain and this domain is indispensable for TAS1 activity. Phylogenetic analysis classifies this KS domain as an independent clade close to type I PKS KS domain. We demonstrate that the TAS1 KS domain conducts the final cyclization step for TeA release. These results indicate that TAS1 is a unique type of NRPS–PKS hybrid enzyme. PMID:26503170

  5. Laboratory studies of the reactive uptake of biogenic species: Evidence for the direct polymerization of isoprene, terpenes and sesquiterpenes on acidic aerosols

    NASA Astrophysics Data System (ADS)

    Li, S.; Liggio, J.; Mihele, C.; Brook, J.

    2006-12-01

    Numerous studies on heterogeneous reactions have shown that polymerization of semi-volatile and volatile organic compounds occurs in aerosols. To date, most evidence suggests that gaseous hydrocarbon oxidation products containing carbonyl functionality are the prime candidates for these processes. Such processes involve primarily hydration, acetal formation, polymerization and aldol-condensation reactions, resulting in oligomer products of potential significance with respect to secondary organic aerosol formation (SOA). However, little information on the heterogeneous reactions of unsaturated hydrocarbons (olefins) is known. Given that biogenic species, many of them unsaturated, make up a considerable portion of hydrocarbons emitted globally, direct reactive uptake of these compounds on aerosols would also potentially be a major source of SOA. In the present study, individual biogenic hydrocarbons were exposed to pre-existing acidic sulfate aerosols within a 2 m3 Teflon reaction chamber under varying relative humidity conditions. An Aerosol Mass Spectrometer was used to quantify any subsequent increase in organic mass as a function of time, and to obtain information regarding the structure of products via aerosol mass spectra. A Proton Transfer Reaction Mass Spectrometer was used to measure the gas-phase concentrations of isoprene, terpenes (?-pinene, ?-pinene, limonene, and carene) and sesquiterpenes (?-caryophylene and humulene) in the reaction chamber. Results from these experiments show that a significant amount of these compounds are taken up by the acidic aerosols rapidly, in a polymerization process which was highly dependent on the particle acidity. This polymerization mechanism likely involves the oxygenation of the resulting polymers via acid catalyzed hydration. The uptake of the unsaturated hydrocarbons suggests that gas-phase oxidation of biogenics to condensable products is not the only route to SOA. Details of the polymerization and hydration

  6. Synthesis and characterization of a pH responsive folic acid functionalized polymeric drug delivery system.

    PubMed

    Li, Xia; McTaggart, Matt; Malardier-Jugroot, Cecile

    2016-01-01

    We report the computational analysis, synthesis and characterization of folate functionalized poly(styrene-alt-maleic anhydride), PSMA for drug delivery purpose. The selection of the proper linker between the polymer and the folic acid group was performed before conducting the synthesis using Density Functional Theory (DFT). The computational results showed the bio-degradable linker 2, 4-diaminobutyric acid, DABA as a good candidate allowing flexibility of the folic acid group while maintaining the pH sensitivity of PSMA, used as a trigger for drug release. The synthesis was subsequently carried out in multi-step experimental procedures. The functionalized polymer was characterized using InfraRed spectroscopy, Nuclear Magnetic Resonance and Dynamic Light Scattering confirming both the chemical structure and the pH responsiveness of PSMA-DABA-Folate polymers. This study provides an excellent example of how computational chemistry can be used in selection process for the functional materials and product characterization. The pH sensitive polymers are expected to be used in delivering anti-cancer drugs to solid tumors with overly expressed folic acid receptors. PMID:27183249

  7. Effects of traditionally used anxiolytic botanicals on enzymes of the gamma-aminobutyric acid (GABA) system.

    PubMed

    Awad, R; Levac, D; Cybulska, P; Merali, Z; Trudeau, V L; Arnason, J T

    2007-09-01

    In Canada, the use of botanical natural health products (NHPs) for anxiety disorders is on the rise, and a critical evaluation of their safety and efficacy is required. The purpose of this study was to determine whether commercially available botanicals directly affect the primary brain enzymes responsible for gamma-aminobutyric acid (GABA) metabolism. Anxiolytic plants may interact with either glutamic acid decarboxylase (GAD) or GABA transaminase (GABA-T) and ultimately influence brain GABA levels and neurotransmission. Two in vitro rat brain homogenate assays were developed to determine the inhibitory concentrations (IC50) of aqueous and ethanolic plant extracts. Approximately 70% of all extracts that were tested showed little or no inhibitory effect (IC50 values greater than 1 mg/mL) and are therefore unlikely to affect GABA metabolism as tested. The aqueous extract of Melissa officinalis (lemon balm) exhibited the greatest inhibition of GABA-T activity (IC50 = 0.35 mg/mL). Extracts from Centella asiatica (gotu kola) and Valeriana officinalis (valerian) stimulated GAD activity by over 40% at a dose of 1 mg/mL. On the other hand, both Matricaria recutita (German chamomile) and Humulus lupulus (hops) showed significant inhibition of GAD activity (0.11-0.65 mg/mL). Several of these species may therefore warrant further pharmacological investigation. The relation between enzyme activity and possible in vivo mode of action is discussed. PMID:18066140

  8. Adding an appropriate amino acid during crosslinking results in more stable crosslinked enzyme aggregates.

    PubMed

    Mukherjee, Joyeeta; Majumder, Abir Baran; Gupta, Munishwar Nath

    2016-08-15

    Carrier free immobilization, especially crosslinked enzyme aggregates (CLEAs), has become an important design for biocatalysis in several areas. Adding amino acids during formation of CLEAs was found to give biocatalysts more stable at 55 °C and in the presence of 60% acetonitrile. The half-lives of CLEAs prepared with and without Arg addition were 21 and 15 h (subtilisin) and 4 and 1.6 h (α-chymotrypsin) at 55 °C, respectively. The corresponding half-lives during acetonitrile presence were 4.1 and 3.0 h (subtilisin) and 39 and 22 min (α-chymotrypsin), respectively. CLEAs made with Arg had higher percentages of alpha helix. CLEAs made by adding Lys, Ala, or Asp also were more stable. In the case of Thermomyces lanuginosus lipase (TLL), CLEA with Ala was even more stable than CLEA with Arg. The addition of a suitable amino acid, thus, enhances CLEA stabilities. The results are discussed in the light of earlier results on chemical modification of proteins and the observation that the Arg/Lys ratio is invariably high in the case of enzymes from thermophiles. PMID:27237371

  9. Rapid and enzyme-free nucleic acid detection based on exponential hairpin assembly in complex biological fluids.

    PubMed

    Ma, Cuiping; Zhang, Menghua; Chen, Shan; Liang, Chao; Shi, Chao

    2016-05-10

    Herein, we have developed a rapid and enzyme-free nucleic acid amplification detection method that combined the exponential self-assembly of four DNA hairpins and the FRET pair Cy3 and Cy5. This strategy was very ingenious and rapid, and could detect nucleic acids at concentrations as low as 10 pM in 15 min in biological fluids. PMID:27138054

  10. An enzymic assay for uric acid in serum and urine compared with HPLC.

    PubMed

    Dubois, H; Delvoux, B; Ehrhardt, V; Greiling, H

    1989-03-01

    We evaluated a colorimetric method for the assay of uric acid in serum or urine, which utilises a Trinder chromogenic system modified by the inclusion of 2,4,6-tribromo-3-hydroxybenzoic acid for oxidative coupling to p-aminophenazone. Colour development (Amax: 512 nm) is complete within five minutes. Measurement of a sample blank is not needed. The procedure involves pre-incubation with ascorbic acid oxidase and detergent to eliminate interference by ascorbic acid and to abolish turbidity due to lipaemia; this pretreatment was effective up to 1.14 mmol/l ascorbate and up to at least 25 mmol/l triacylglycerol. Interference by icteric sera was insignificant up to about 170 mumol/l bilirubin. The method is linear up to at least 1428 mumol/l. In human serum and urine the procedure correlates well with HPLC and the uricase p-aminophenazone method on the SMAC analyser. Within-run and between-run imprecisions of the enzymic test were higher than for HPLC, but did not exceed 1.2% (CV) and 2.5% (CV), respectively. PMID:2708944

  11. The Mycobacterium tuberculosis FAS-II condensing enzymes: their role in mycolic acid biosynthesis, acid-fastness, pathogenesis and in future drug development.

    PubMed

    Bhatt, Apoorva; Molle, Virginie; Besra, Gurdyal S; Jacobs, William R; Kremer, Laurent

    2007-06-01

    Mycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development. PMID:17555433

  12. Inhibitory action of Epilobium hirsutum extract and its constituent ellagic acid on drug-metabolizing enzymes.

    PubMed

    Celik, Gurbet; Semiz, Aslı; Karakurt, Serdar; Gencler-Ozkan, Ayse Mine; Arslan, Sevki; Adali, Orhan; Sen, Alaattin

    2016-04-01

    Epilobium hirsutum (EH) is a medicinal plant for treating various diseases. Despite its wide usage, there is no available information about its potential influences on drug metabolism. The present study was undertaken to determine the in vivo effects of EH on hepatic CYP2B, CYP2C, CYP2D, and CYP3A enzymes that are primarily involved in drug metabolism. Male Wistar rats were injected intraperitoneally with EH water extract (EHWE) and ellagic acid (EA) at a daily dose of 37.5 and 20 mg/kg, respectively, for 9 days and hepatic drug-metabolizing enzymes were assessed at activity, protein and mRNA levels. Erythromycin N-demethylase activity was inhibited by 53 and 21 % in EHWE- and EA-treated rats, respectively. Benzphetamine N-demethylase and 7-benzyloxyresorufin-O-debenzylase activities were decreased by 53 and 43 %, and 57 and 57 % in EHWE-and EA-treated rats, respectively. Moreover, protein levels of CYP2B1, CYP2C6, CYP2D2, and CYP3A1 also decreased by 55, 15, 33, and 82 % as a result of EHWE treatment of rats, respectively. Similarly, CYP2B1, CYP2C6, CYP2D2, and CYP3A1 protein levels decreased by 62, 63, 49, and 37 % with EA treatment, respectively. qRT-PCR analyses also showed that mRNA levels of these enzymes were significantly inhibited with bothEHWE and EA treatments. In conclusion, inhibition of drug clearances leading to drug toxicity because of the lowered activity and expression of drug-metabolizing enzymes might be observed in the people who used EH as complementary herbal remedy that might be contributed by its EA content. PMID:25425117

  13. Prediction of enzyme function based on 3D templates of evolutionarily important amino acids

    PubMed Central

    Kristensen, David M; Ward, R Matthew; Lisewski, Andreas Martin; Erdin, Serkan; Chen, Brian Y; Fofanov, Viacheslav Y; Kimmel, Marek; Kavraki, Lydia E; Lichtarge, Olivier

    2008-01-01

    Background Structural genomics projects such as the Protein Structure Initiative (PSI) yield many new structures, but often these have no known molecular functions. One approach to recover this information is to use 3D templates – structure-function motifs that consist of a few functionally critical amino acids and may suggest functional similarity when geometrically matched to other structures. Since experimentally determined functional sites are not common enough to define 3D templates on a large scale, this work tests a computational strategy to select relevant residues for 3D templates. Results Based on evolutionary information and heuristics, an Evolutionary Trace Annotation (ETA) pipeline built templates for 98 enzymes, half taken from the PSI, and sought matches in a non-redundant structure database. On average each template matched 2.7 distinct proteins, of which 2.0 share the first three Enzyme Commission digits as the template's enzyme of origin. In many cases (61%) a single most likely function could be predicted as the annotation with the most matches, and in these cases such a plurality vote identified the correct function with 87% accuracy. ETA was also found to be complementary to sequence homology-based annotations. When matches are required to both geometrically match the 3D template and to be sequence homologs found by BLAST or PSI-BLAST, the annotation accuracy is greater than either method alone, especially in the region of lower sequence identity where homology-based annotations are least reliable. Conclusion These data suggest that knowledge of evolutionarily important residues improves functional annotation among distant enzyme homologs. Since, unlike other 3D template approaches, the ETA method bypasses the need for experimental knowledge of the catalytic mechanism, it should prove a useful, large scale, and general adjunct to combine with other methods to decipher protein function in the structural proteome. PMID:18190718

  14. Characterization of bioactive RGD peptide immobilized onto poly(acrylic acid) thin films by plasma polymerization

    NASA Astrophysics Data System (ADS)

    Seo, Hyun Suk; Ko, Yeong Mu; Shim, Jae Won; Lim, Yun Kyong; Kook, Joong-Ki; Cho, Dong-Lyun; Kim, Byung Hoon

    2010-11-01

    Plasma surface modification can be used to improve the surface properties of commercial pure Ti by creating functional groups to produce bioactive materials with different surface topography. In this study, a titanium surface was modified with acrylic acid (AA) using a plasma treatment and immobilized with bioactive arginine-glycine-aspartic acid (RGD) peptide, which may accelerate the tissue integration of bone implants. Both terminals containing the -NH2 of RGD peptide sequence and -COOH of poly(acrylic acid) (PAA) thin film were combined with a covalent bond in the presence of 1-ethyl-3-3-dimethylaminopropyl carbodiimide (EDC). The chemical structure and morphology of AA film and RGD immobilized surface were investigated by X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR), atomic force microscopy (AFM), and scanning electron microscopy (SEM). All chemical analysis showed full coverage of the Ti substrate with the PAA thin film containing COOH groups and the RGD peptide. The MC3T3-E1 cells were cultured on each specimen, and the cell alkaline phosphatase (ALP) activity were examined. The surface-immobilized RGD peptide has a significantly increased the ALP activity of MC3T3-E1 cells. These results suggest that the RGD peptide immobilization on the titanium surface has an effect on osteoblastic differentiation of MC3T3-E1 cells and potential use in osteo-conductive bone implants.

  15. Phenylboronic-Acid-Based Polymeric Micelles for Mucoadhesive Anterior Segment Ocular Drug Delivery.

    PubMed

    Prosperi-Porta, Graeme; Kedzior, Stephanie; Muirhead, Benjamin; Sheardown, Heather

    2016-04-11

    Topical drug delivery to the front of the eye is extremely inefficient due to effective natural protection mechanisms such as precorneal tear turnover and the relative impermeability of the cornea and sclera tissues. This causes low ocular drug bioavailability, requiring large frequent doses that result in high systemic exposure and side effects. Mucoadhesive drug delivery systems have the potential to improve topical drug delivery by increasing pharmaceutical bioavailability on the anterior eye surface. We report the synthesis and characterization of a series of poly(l-lactide)-b-poly(methacrylic acid-co-3-acrylamidophenylboronic acid) block copolymer micelles for use as mucoadhesive drug delivery vehicles. Micelle properties, drug release rates, and mucoadhesion were shown to depend on phenylboronic acid content. The micelles showed low in vitro cytotoxicity against human corneal epithelial cells and undetectable acute in vivo ocular irritation in Sprague-Dawley rats, suggesting good biocompatibility with the corneal surface. The micelles show the potential to significantly improve the bioavailability of topically applied ophthalmic drugs, which could reduce dosage, frequency of administration, and unintentional systemic exposure. This would greatly improve the delivery of the ocular drugs such as the potent immunosuppressive cyclosporine A used in the treatment of severe dry eye disease. PMID:26963738

  16. Thermostable Lipoxygenase, a Key Enzyme in the Conversion of Linoleic Acid into Thrihydroxy-octadecenoic Acid by Pseudomonas aeruginosa PR3

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Lipoxygenases (LOX) constitute a family of lipid-peroxidizing enzymes catalyzing the oxidation of unsaturated fatty acid with (1Z,4Z)-pentadiene structural unit, leading to formation of the conjugated (Z,E)-hydroperoxydienoic acid. LOXs have been known to be widely distributed in plants and animals...

  17. Clostridium thermocellum releases coumaric acid during degradation of untreated grasses by the action of an unknown enzyme.

    PubMed

    Herring, Christopher D; Thorne, Philip G; Lynd, Lee R

    2016-03-01

    Clostridium thermocellum is an anaerobic thermophile with the ability to digest lignocellulosic biomass that has not been pretreated with high temperatures. Thermophilic anaerobes have previously been shown to more readily degrade grasses than wood. Part of the explanation for this may be the presence of relatively large amounts of coumaric acid in grasses, with linkages to both hemicellulose and lignin. We found that C. thermocellum and cell-free cellulase preparations both release coumaric acid from bagasse and switchgrass. Cellulase preparations from a mutant strain lacking the scaffoldin cipA still showed activity, though diminished. Deletion of all three proteins in C. thermocellum with ferulic acid esterase domains, either singly or in combination, did not eliminate the activity. Further work will be needed to identify the novel enzyme(s) responsible for the release of coumaric acid from grasses and to determine whether these enzymes are important factors of microbial biomass degradation. PMID:26762388

  18. Characterization of enzymes in the oxidation of 1,2-propanediol to D: -(-)-lactic acid by Gluconobacter oxydans DSM 2003.

    PubMed

    Wei, Liujing; Yang, Xuepeng; Gao, Keliang; Lin, Jinping; Yang, Shengli; Hua, Qiang; Wei, Dongzhi

    2010-09-01

    Although Gluconobacter oxydans can convert 1,2-propanediol to D: -(-)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of D: -(-)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in D: -(-)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to D: -(-)-lactic acid by G. oxydans DSM 2003. PMID:20300886

  19. Photosynthetic Characteristics of Portulaca grandiflora, a Succulent C(4) Dicot : CELLULAR COMPARTMENTATION OF ENZYMES AND ACID METABOLISM.

    PubMed

    Ku, S B; Shieh, Y J; Reger, B J; Black, C C

    1981-11-01

    on enzyme localization, a scheme of C(4) photosynthesis in P. grandiflora is proposed.Well-watered plants of P. grandiflora exhibit a diurnal fluctuation of total titratable acidity, with an amplitude of 61 and 54 microequivalent per gram fresh weight for the leaves and stems, respectively. These changes were in parallel with changes in malic acid concentration in these tissues. Under severe drought conditions, diurnal changes in both titratable acidity and malic acid concentration in both leaves and stems were much reduced. However, another C(4) dicot Amaranthus graecizans (nonsucculent) did not show any diurnal acid fluctuation under the same conditions. These results confirm the suggestion made by Koch and Kennedy (Plant Physiol. 65: 193-197, 1980) that succulent C(4) dicots can exhibit an acid metabolism similar to Crassulacean acid metabolism plants in certain environments. PMID:16662054

  20. Characterization of pH-Responsive Hydrogels of Poly(Itaconic acid-g-Ethylene Glycol) Prepared by UV-Initiated Free Radical Polymerization as Biomaterials for Oral Delivery of Bioactive Agents

    PubMed Central

    Betancourt, Tania; Pardo, Juan; Soo, Ken; Peppas, Nicholas A.

    2009-01-01

    Effective oral delivery of proteins is impeded by steep pH gradients and proteolytic enzymes in the gastrointestinal tract, as well as low absorption of the proteins into the bloodstream due to their size, charge or solubility. In the present work, pH-responsive complexation hydrogels of poly(itaconic acid) with poly(ethylene glycol) grafts were synthesized for applications in oral drug delivery. These hydrogels were expected to be in collapsed configuration at low pH due to hydrogen bonding between poly(itaconic acid) carboxyl groups and poly(ethylene glycol), and to swell with increasing pH because of charge repulsion between deprotonated carboxylic acid groups. Hydrogels were prepared by UV-initiated free radical polymerization using tetraethylene glycol as the crosslinking agent and Irgacure® 2959 as the initiator. The effect of monomer ratios, crosslinking ratio and solvent amount on the properties of the hydrogels were investigated. The composition of the hydrogels was confirmed by FTIR. Equilibrium swelling studies in the pH range of 1.2 to 7 revealed that the extent of swelling increased with increasing pH up to a pH of about 6, when no further carboxylic acid deprotonation occurred. Studies in Caco-2 colorectal carcinoma cells confirmed the cytocompatibility of these materials at concentrations of up to 5 mg/ml. PMID:19536838

  1. Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate characterization of nucleic acid metabolic enzymes

    PubMed Central

    Greenough, Lucia; Schermerhorn, Kelly M.; Mazzola, Laurie; Bybee, Joanna; Rivizzigno, Danielle; Cantin, Elizabeth; Slatko, Barton E.; Gardner, Andrew F.

    2016-01-01

    Detailed biochemical characterization of nucleic acid enzymes is fundamental to understanding nucleic acid metabolism, genome replication and repair. We report the development of a rapid, high-throughput fluorescence capillary gel electrophoresis method as an alternative to traditional polyacrylamide gel electrophoresis to characterize nucleic acid metabolic enzymes. The principles of assay design described here can be applied to nearly any enzyme system that acts on a fluorescently labeled oligonucleotide substrate. Herein, we describe several assays using this core capillary gel electrophoresis methodology to accelerate study of nucleic acid enzymes. First, assays were designed to examine DNA polymerase activities including nucleotide incorporation kinetics, strand displacement synthesis and 3′-5′ exonuclease activity. Next, DNA repair activities of DNA ligase, flap endonuclease and RNase H2 were monitored. In addition, a multicolor assay that uses four different fluorescently labeled substrates in a single reaction was implemented to characterize GAN nuclease specificity. Finally, a dual-color fluorescence assay to monitor coupled enzyme reactions during Okazaki fragment maturation is described. These assays serve as a template to guide further technical development for enzyme characterization or nucleoside and non-nucleoside inhibitor screening in a high-throughput manner. PMID:26365239

  2. Optimization of the enzyme-catalyzed synthesis of amino acid-based surfactants from palm oil fractions.

    PubMed

    Soo, Ee Lin; Salleh, Abu Bakar; Basri, Mahiran; Zaliha Raja Abdul Rahman, Raja Noor; Kamaruddin, Kamarulzaman

    2003-01-01

    The feasibility of using palm oil fractions as cheap and abundant sources of raw material for the synthesis of amino acid surfactants was investigated. Of a number of enzymes screened, the best results were obtained with the immobilized enzyme, Lipozyme. The effects of temperature, solvent, incubation period, fatty substrate/amino acid molar ratio, enzyme amount, and water removal on the reactions were analyzed and compared to those on reactions with free fatty acids and pure triglycerides as fatty substrates. All reactions were most efficient when carried out at high temperatures (70-80 degrees C) in hexane as a solvent. However, while reactions with free fatty acids proceeded better when a slight excess of the free fatty acids over the amino acids was used, reactions with triglycerides and palm oil fractions were best performed at equimolar ratios. Also, the addition of molecular sieves slightly enhanced reactions with free fatty acids but adversely affected reactions with triglycerides and palm oil fractions. Although reactions with palm oil fractions took longer (6 d) to reach equilibrium compared to reactions with free fatty acids (4 d) and pure triglycerides (4 d), better yields were obtained. Such lipase-catalyzed transacylation of palm oil fractions with amino acids is potentially useful in the production of mixed medium- to long-chain surfactants for specific applications. PMID:16233420

  3. Enzyme-mimetic effects of gold@platinum nanorods on the antioxidant activity of ascorbic acid

    NASA Astrophysics Data System (ADS)

    Zhou, Yu-Ting; He, Weiwei; Wamer, Wayne G.; Hu, Xiaona; Wu, Xiaochun; Lo, Y. Martin; Yin, Jun-Jie

    2013-01-01

    Au@Pt nanorods were prepared by growing platinum nanodots on gold nanorods. Using electron spin resonance (ESR), we determined that the mechanisms for oxidation of ascorbic acid (AA) by Au@Pt nanorods and ascorbic acid oxidase (AAO) were kinetically similar and yielded similar products. In addition we observed that Au@Pt nanorods were stable with respect to temperature and pH. Using UV-VIS spectroscopy, the apparent kinetics of enzyme-mimetic activity of Au@Pt nanorods were studied and compared with the activity of AAO. With the help of ESR, we found that Au@Pt nanorods did not scavenge hydroxyl radicals but inhibited the antioxidant ability of AA for scavenging hydroxyl radicals produced by photoirradiating solutions containing titanium dioxide and zinc oxide. Moreover, the Au@Pt nanorods reduced the ability of AA to scavenge DPPH radicals and superoxide radicals. These results demonstrate that Au@Pt nanorods can reduce the antioxidant activity of AA. Therefore, it is necessary to consider the effects of using Pt nanoparticles together with other reducing agents or antioxidants such as AA due to the oxidase-like property of Au@Pt nanorods.Au@Pt nanorods were prepared by growing platinum nanodots on gold nanorods. Using electron spin resonance (ESR), we determined that the mechanisms for oxidation of ascorbic acid (AA) by Au@Pt nanorods and ascorbic acid oxidase (AAO) were kinetically similar and yielded similar products. In addition we observed that Au@Pt nanorods were stable with respect to temperature and pH. Using UV-VIS spectroscopy, the apparent kinetics of enzyme-mimetic activity of Au@Pt nanorods were studied and compared with the activity of AAO. With the help of ESR, we found that Au@Pt nanorods did not scavenge hydroxyl radicals but inhibited the antioxidant ability of AA for scavenging hydroxyl radicals produced by photoirradiating solutions containing titanium dioxide and zinc oxide. Moreover, the Au@Pt nanorods reduced the ability of AA to scavenge

  4. Post-polymerization modification of poly(L-glutamic acid) with D-(+)-glucosamine.

    PubMed

    Perdih, Peter; Cebašek, Sašo; Možir, Alenka; Zagar, Ema

    2014-01-01

    Carboxyl functional groups of poly(L-glutamic acid) (PGlu) were modified with a D-(+)-glucosamine (GlcN) by amidation using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM) as a coupling reagent. The coupling reaction was performed in aqueous medium without protection of hydroxyl functional groups of D-(+)-glucosamine. Poly(L-glutamic acid) and GlcN functionalized polyglutamates (P(Glu-GlcN)) were thoroughly characterized by 1D and 2D NMR spectroscopy and SEC-MALS to gain detailed information on their structure, composition and molar mass characteristics. The results reveal successful functionalization with GlcN through the amide bond and also to a minor extent through ester bond formation in position 1 of GlcN. In addition, a ratio between the α- and β-form of glucosamine substituent coupled to polyglutamate repeating units as well as the content of residual dimethoxy triazinyl active ester moiety in the samples were evaluated. PMID:25438084

  5. Acid Sensitive Polymeric Micelles Combining Folate and Bioreducible Conjugate for Specific Intracellular siRNA Delivery.

    PubMed

    Yang, Yanfang; Xia, Xuejun; Dong, Wujun; Wang, Hongliang; Li, Lin; Ma, Panpan; Sheng, Wei; Xu, Xueqing; Liu, Yuling

    2016-05-01

    An efficiently siRNA transporting nanocarrier still remains to be developed. In this study, utilizing the dual stimulus of acid tumor extracellular environment and redox effect of glutathione in the cytosol, a new siRNA transporting system combining triple effects of folate targeting, acid sensitive polymer micelles, and bio-reducible disulfide bond linked siRNA-cell penetrating peptides (CPPs) conjugate is developed to suppress c-myc gene expression of breast cancer (MCF-7 cells) both in vitro and in vivo. Subsequent research demonstrates that the vesicle has particle size of about 100 nm and siRNA entrapment efficiency of approximately 80%. In vitro studies verified over 90% of encapsulated siRNA-CPPs can be released and the vesicle shows higher cellular uptake in response to the tumorous zone. Determination of gene expression at both mRNA and protein levels indicates the constructed vesicle exhibited enhanced cancer cell apoptosis and improved therapeutic efficacy in vitro and in vivo. PMID:26822264

  6. Radiation-induced graft polymerization of maleic acid and maleic anhydride onto ultra-fine powdered styrene butadiene rubber (UFSBR)

    NASA Astrophysics Data System (ADS)

    Peng, Jing; Xia, Haibing; Zhai, Maolin; Li, Jiuqiang; Qiao, Jinliang; Wei, Genshuan

    2007-11-01

    The functionalization of ultra-fine powdered styrene-butadiene rubber (UFSBR) was carried out using gamma radiation-induced graft polymerization of maleic acid (MA) and maleic anhydride (MAH), respectively. It was found that the graft yield of MA onto UFSBR increased rapidly up to the peak and then decreased with increasing MA content. Moreover, the peak shifted to the direction of lower MA content with increasing absorbed dose. Similarly, there was the peak of graft yield with increasing MAH content for grafting of MAH onto UFSBR, whereas the peak of graft yield was achieved at 10 wt% MAH content at different absorbed doses. On the other hand, increasing absorbed dose and decreasing monomer contents are useful to improve the graft efficiency of MA and MAH. At high dose and low monomer content, the graft yield of MAH onto UFSBR is higher than that of MA. FTIR spectra confirmed that both MA and MAH can be grafted successfully onto the UFSBR under gamma irradiation, respectively. Comparing with maleation of rubber by melt grafting, the graft yield of MAH on UFSBR is higher, which can be attributed to the network structure and nanometer size of UFSBR as well as high energy provided by radiation.

  7. A study on the effect of the polymeric additive HPMC on morphology and polymorphism of ortho-aminobenzoic acid crystals

    NASA Astrophysics Data System (ADS)

    Simone, E.; Cenzato, M. V.; Nagy, Z. K.

    2016-07-01

    In the present study, the effect of Hydroxy Propyl Methyl Cellulose (HPMC) on the crystallization of ortho-aminobenzoic acid (OABA) was investigated by seeded and unseeded cooling crystallization experiments. The influence of HPMC on the induction time, crystal shape of Forms I and II of OABA and the polymorphic transformation time was studied. Furthermore, the capability of HPMC to inhibit growth of Form I was evaluated quantitatively and modeled using population balance equations (PBE) solved with the method of moments. The additive was found to strongly inhibit nucleation and growth of Form I as well as to increase the time for the polymorphic transformation from Form II to I. Solvent was also found to influence the shape of Form I crystals at equal concentrations of HPMC. In situ process analytical technology (PAT) tools, including Raman spectroscopy, focused beam reflectance measurement (FBRM) and attenuated total reflectance (ATR) UV-vis spectroscopy were used in combination with off-line techniques, such as optical microscopy, scanning electron microscopy (SEM), Raman spectroscopy, Malvern Mastersizer and differential scanning calorimetry (DSC) to study the crystals produced. The results illustrate how shape, size and stability of the two polymorphs of OABA can be controlled and tailored using a polymeric additive.

  8. Polymeric nanoparticles based on chitooligosaccharide as drug carriers for co-delivery of all-trans-retinoic acid and paclitaxel.

    PubMed

    Zhang, Jing; Han, Jian; Zhang, Xiuli; Jiang, Jing; Xu, Maolei; Zhang, Daolai; Han, Jingtian

    2015-09-20

    An amphiphilic all-trans-retinoic acid (ATRA)-chitooligosaccharide (RCOS) conjugate was synthesized to form self-assembled polymeric nanoparticles to facilitate the co-delivery of ATRA and paclitaxel (PTX). The blank RCOS nanoparticles possessed low hemolytic activity and cytotoxicity, and could efficiently load PTX with a drug loading of 22.2% and a high encapsulation efficiency of 71.3%. PTX-loaded RCOS nanoparticles displayed a higher cytotoxicity to HepG2 cells compared to PTX plus ATRA solution when corrected by the accumulated drug release. Cellular uptake profiles of RCOS nanoparticles were evaluated via confocal laser scanning microscope and flow cytometry with FITC as a fluorescent mark. The RCOS nanoparticles could be rapidly and continuously taken up by HepG2 cells via endocytosis and transported into the nucleus, and the uptake rates increased with particle concentration. These results revealed the promising potential of RCOS nanoparticles as drug carriers for co-delivery of ATRA and PTX or other hydrophobic therapeutic agents. PMID:26050884

  9. [Reconstitution of polyunsaturated fatty acid synthesis enzymes in mammalian cells to convert LA to DHA].

    PubMed

    Zhu, Guiming; Saleh, Abdulmomen Ali Mohammed; Bahwal, Said Ahmed; Qiu, Lihong; Sun, Jie; Shang, Yu; Jiang, Xudong; Ge, Tangdong; Zhang, Tao

    2015-02-01

    DHA (22:6n-3) is a Ω-3 polyunsaturated fatty acid with 22 carbon atoms and 6 double bonds, which has important biological functions in human body. Human and other mammals synthesize only limited amounts of DHA, more requirements must be satisfied from food resources. However, the natural resources of DHA (Mainly deep-sea fish and other marine products) are prone to depletion. New resources development is still insufficient to satisfy the growing market demand. Previous studies have revealed that the mammals can increase the synthesis of DHA and other long-chain polyunsaturated fatty acids after transgenic procedures. In this study, mammalian cells were transfected with Δ6, Δ5 desaturase, Δ6, Δ5 elongase, Δ15 desaturase (Isolated from nematode Caenorhabditis elegans) and Δ4 desaturase (Isolated from Euglena gracilis), simultaneously. Results show that the expression or overexpression of these 6 enzymes is capable of conversion of the o-6 linoleic acid (LA, 18:2n-6) in DHA (22:6n-3). DHA content has increased from 16.74% in the control group to 25.3% in the experimental group. The strategy and related technology in our research provided important data for future production the valuable DHA (22:6n-3) by using genetically modified animals. PMID:26062349

  10. Single-Cell Measurements of Enzyme Levels as a Predictive Tool for Cellular Fates during Organic Acid Production

    PubMed Central

    Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna

    2013-01-01

    Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as “acidified”). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This “switch-like” relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

  11. Single-cell measurements of enzyme levels as a predictive tool for cellular fates during organic acid production.

    PubMed

    Zdraljevic, Stefan; Wagner, Drew; Cheng, Kevin; Ruohonen, Laura; Jäntti, Jussi; Penttilä, Merja; Resnekov, Orna; Pesce, C Gustavo

    2013-12-01

    Organic acids derived from engineered microbes can replace fossil-derived chemicals in many applications. Fungal hosts are preferred for organic acid production because they tolerate lignocellulosic hydrolysates and low pH, allowing economic production and recovery of the free acid. However, cell death caused by cytosolic acidification constrains productivity. Cytosolic acidification affects cells asynchronously, suggesting that there is an underlying cell-to-cell heterogeneity in acid productivity and/or in resistance to toxicity. We used fluorescence microscopy to investigate the relationship between enzyme concentration, cytosolic pH, and viability at the single-cell level in Saccharomyces cerevisiae engineered to synthesize xylonic acid. We found that cultures producing xylonic acid accumulate cells with cytosolic pH below 5 (referred to here as "acidified"). Using live-cell time courses, we found that the probability of acidification was related to the initial levels of xylose dehydrogenase and sharply increased from 0.2 to 0.8 with just a 60% increase in enzyme abundance (Hill coefficient, >6). This "switch-like" relationship likely results from an enzyme level threshold above which the produced acid overwhelms the cell's pH buffering capacity. Consistent with this hypothesis, we showed that expression of xylose dehydrogenase from a chromosomal locus yields ∼20 times fewer acidified cells and ∼2-fold more xylonic acid relative to expression of the enzyme from a plasmid with variable copy number. These results suggest that strategies that further reduce cell-to-cell heterogeneity in enzyme levels could result in additional gains in xylonic acid productivity. Our results demonstrate a generalizable approach that takes advantage of the cell-to-cell variation of a clonal population to uncover causal relationships in the toxicity of engineered pathways. PMID:24038690

  12. Secretion of three enzymes for fatty acid synthesis into mouse milk in association with fat globules, and rapid decrease of the secreted enzymes by treatment with rapamycin.

    PubMed

    Moriya, Hitomi; Uchida, Kana; Okajima, Tetsuya; Matsuda, Tsukasa; Nadano, Daita

    2011-04-01

    The mammary epithelium produces numerous lipid droplets during lactation and secretes them in plasma membrane-enclosed vesicles known as milk fat globules. The biogenesis of such fat globules is considered to provide a model for clarifying the mechanisms of lipogenesis in mammals. In the present study, we identified acetyl coenzyme A carboxylase, ATP citrate lyase, and fatty acid synthase in mouse milk. Fractionation of milk showed that these three enzymes were located predominantly in milk fat globules. The three enzymes were resistant to trypsin digestion without Triton X-100, indicating that they were not located on the outer surface of the globules and thus associated with the precursors of the globules before secretion. When a low dose of rapamycin, an inhibitor of the mammalian target of rapamycin (mTOR), was injected into lactating mice, the levels of the three enzymes in milk were decreased within 3h after injection. Since the protein levels of the three enzymes in tissues were not obviously altered by this short-term treatment, known transcriptional control by mTOR signaling was unlikely to account for this decrease in their levels in milk. Our findings suggest a new, putatively mTOR-dependent localization of the three enzymes for de novo lipogenesis. PMID:21281598

  13. Vanadate and selenium inhibit the triiodothyronine induced enzyme activity and mRNA level for both fatty acid synthase and malic enzyme

    SciTech Connect

    Zhu, Y.; Mirmiran, R.; Goodridge, A.G.; Stapleton, S.R. Western Michigan Univ., Kalamazoo )

    1991-03-15

    In chick-embryo hepatocytes in culture, triiodothyronine stimulates enzyme activity, mRNA level and transcription rate for both fatty acid synthase (FAS) and malic enzyme (ME). Insulin alone has no effect but amplifies the induction by T3. Recent evidence has demonstrated the insulin-mimicking action of vanadate and selenium on various physiological processes. Little information, however, is available on the affects of vanadate and selenium on the expression of genes that are regulated by insulin. These studies were initiated to test the potential of vanadate and selenium to mimic the amplification affect of insulin on the T3 induction of FAS and ME. In chick-embryo hepatocytes incubated in a chemically defined medium, addition of T3 for 48h causes an increase in the enzyme activity and mRNA level for both FAS and ME. Addition of sodium vanadate or sodium selenate (20 {mu}M) coincident with the T3 almost completely inhibited the stimulation of FAS and ME activity and accumulation of their respective mRNA's. Fifty percent maximal inhibition occurred at about 3-40{mu}M vanadate or 5-10{mu}M selenium. Vanadate and selenium similarity inhibited FAS and ME enzyme activity and mRNA level when the cells were incubated in the presence of insulin and T3. The effect of these metals was selective; isocitrate dehydrogenase activity as well as the level of glyceraldehyde 3-phosphate mRNA were not affected by any of the additions made to the cells in culture. This effect by vanadate and selenium also does not appear to be a generalized effect of metals on lipogenic enzymes as molydate under similar experimental conditions has no effect on either the enzyme activity or mRNA level of FAS or ME. Studies are continuing to determine the mechanism of action of these agents on the regulation of lipogenic enzymes.

  14. Integrin-targeted zwitterionic polymeric nanoparticles with acid-induced disassembly property for enhanced drug accumulation and release in tumor.

    PubMed

    Huang, Pingsheng; Song, Huijuan; Wang, Weiwei; Sun, Yu; Zhou, Junhui; Wang, Xue; Liu, Jinjian; Liu, Jianfeng; Kong, Deling; Dong, Anjie

    2014-08-11

    Reasonably structural design of nanoparticles (NPs) to combine functions of prolonged systemic circulation, enhanced tumor targeting and specific intracellular drug release is crucial for antitumor drug delivery. Combining advantages of Arg-Gly-Asp (RGD) for active tumor targeting, zwitterionic polycarboxybetaine methacrylate (PCB) for prolonged systemic circulation, poly(2-(diisopropylamino) ethyl methacrylate) (PDPA) for acid-triggered intracellular release, novel RGD-PCB-b-PDPA (RGD-PCD) block copolymers were prepared via reversible addition-fragmentation chain transfer (RAFT) polymerization and followed by functionalization with RGD. Doxorubicine (DOX) was encapsulated within the RGD-PCD NPs as model medicine (RGD-PCD/DOX NPs). With ultra pH-sensitivity of PDPA, the drug release was restrained at pH 7.4 for only 24% within 36 h, which was increased to 60% at pH 6.0 within 24 h, and released more rapidly at pH 5.0 for 100% within 5 h, indicating that the RGD-PCD/DOX NPs were able to turn drug release "off" at neutral pH (e.g., systemic circulation) whereas "on" under acidic conditions (e.g., inside endo/lysosomes). Furthermore, the results of fluorescence microscopy and flow cytometry analysis demonstrated improved internalization of RGD-PCD/DOX NPs in HepG2 cells via integrin-mediated endocytosis with rapid DOX release intracellularly. Consequently, the RGD-PCD/DOX NPs showed considerable cytotoxicity against HepG2 and HeLa cells in comparison with free DOX. Importantly, the RGD-PCD/DOX NPs exhibited little protein adsorption property with excellent serum stability, which led to prolonged systemic circulation and enhanced tumor accumulation in tumor-bearing nude mice. Therefore, this multifunctional RGD-PCD NPs, which represented the flexible design approach, showed great potential for the development of novel nanocarriers in tumor-targeted drug delivery. PMID:25054812

  15. Active-site amino acid residues in γ-glutamyltransferase and the nature of the γ-glutamyl-enzyme bond

    PubMed Central

    Elce, John S.

    1980-01-01

    Active-site residues in rat kidney γ-glutamyltransferase (EC 2.3.2.2) were investigated by means of chemical modification. 1. In the presence of maleate, the activity was inhibited by phenylmethanesulphonyl fluoride, and the inhibition was not reversed by β-mercaptoethanol, suggesting that a serine residue is close to the active site, but is shielded except in the presence of maleate. 2. Treatment of the enzyme with N-acetylimidazole modified an amino group, exposed a previously inaccessible cysteine residue and inhibited hydrolysis of the γ-glutamyl-enzyme intermediate, but not its formation. 3. After reaction of the enzyme successively with N-acetylimidazole and with non-radioactive iodoacetamide/serine/borate, two active-site residues reacted with iodo[14C]acetamide. One of these possessed a carboxy group, which formed a [14C]glycollamide ester, and the other was cysteine, shown by isolation of S-[14C]carboxymethylcysteine after acid hydrolysis. When N-acetylimidazole treatment was omitted, only the carboxy group reacted with iodo[14C]acetamide. 4. Isolation of the γ-[14C]glutamyl-enzyme intermediate was made easier by prior treatment of the enzyme with N-acetylimidazole. The γ-glutamyl-enzyme bond was stable to performic acid, and to hydroxylamine/urea at pH10, but was hydrolysed slowly at pH12, indicating attachment of the γ-[14C]glutamyl group in amide linkage to an amino group on the enzyme. Proteolysis of the γ-[14C]glutamyl-enzyme after performic acid oxidation gave rise to a small acidic radioactive peptide that was resistant to further proteolysis and was not identical with γ-glutamyl-ε-lysine. 5. A scheme for the catalytic mechanism is proposed. PMID:6104953

  16. Aptamer- and nucleic acid enzyme-based systems for simultaneous detection of multiple analytes

    DOEpatents

    Lu, Yi; Liu, Juewen

    2011-11-15

    The present invention provides aptamer- and nucleic acid enzyme-based systems for simultaneously determining the presence and optionally the concentration of multiple analytes in a sample. Methods of utilizing the system and kits that include the sensor components are also provided. The system includes a first reactive polynucleotide that reacts to a first analyte; a second reactive polynucleotide that reacts to a second analyte; a third polynucleotide; a fourth polynucleotide; a first particle, coupled to the third polynucleotide; a second particle, coupled to the fourth polynucleotide; and at least one quencher, for quenching emissions of the first and second quantum dots, coupled to the first and second reactive polynucleotides. The first particle includes a quantum dot having a first emission wavelength. The second particle includes a second quantum dot having a second emission wavelength different from the first emission wavelength. The third polynucleotide and the fourth polynucleotide are different.

  17. Lysosomal Acid Phosphatase Biosynthesis and Dysfunction: A Mini Review Focused on Lysosomal Enzyme Dysfunction in Brain.

    PubMed

    Ashtari, N; Jiao, X; Rahimi-Balaei, M; Amiri, S; Mehr, S E; Yeganeh, B; Marzban, H

    2016-01-01

    Lysosomes are membrane-bound organelles that are responsible for degrading and recycling macromolecules. Lysosomal dysfunction occurs in enzymatic and non-enzymatic deficiencies, which result in abnormal accumulation of materials. Although lysosomal storage disorders affect different organs, the central nervous system is the most vulnerable. Evidence shows the role of lysosomal dysfunction in different neurodegenerative diseases, such as Niemann-Pick Type C disease, juvenile neuronal ceroid lipofuscinosis, Alzheimer's disease and Parkinson's disease. Lysosomal enzymes such as lysosomal acid phosphatase 2 (Acp2) play a critical role in mannose-6-phosphate removal and Acp2 controls molecular and cellular functions in the brain during development and adulthood. Acp2 is essential in cerebellar development, and mutations in this gene cause severe cerebellar neurodevelopmental and neurodegenerative disorders. In this mini-review, we highlight lysosomal dysfunctions in the pathogenesis of neurodevelopmental and/or neurodegenerative diseases with special attention to Acp2 dysfunction. PMID:27132795

  18. The role of CYP26 enzymes in defining appropriate retinoic acid exposure during embryogenesis.

    PubMed

    Pennimpede, Tracie; Cameron, Don A; MacLean, Glenn A; Li, Hui; Abu-Abed, Suzan; Petkovich, Martin

    2010-10-01

    Retinoic acid (RA) is a pleiotropic derivative of vitamin A, or retinol, which is responsible for all of the bioactivity associated with this vitamin. The teratogenic influences of vitamin A deficiency and excess RA in rodents were first observed more than 50 years ago. Efforts over the last 15-20 years have refined these observations by defining the molecular mechanisms that control RA availability and signaling during murine embryonic development. This review will discuss our current understanding of the role of RA in teratogenesis, with specific emphasis on the essential function of the RA catabolic CYP26 enzymes in preventing teratogenic consequences caused by uncontrolled distribution of RA. Particular focus will be paid to the RA-sensitive tissues of the caudal and cranial regions, the limb, and the testis, and how genetic mutation of factors controlling RA distribution have revealed important roles for RA during embryogenesis. PMID:20842651

  19. Physical interactions between tricarboxylic acid cycle enzymes in Bacillus subtilis: evidence for a metabolon.

    PubMed

    Meyer, Frederik M; Gerwig, Jan; Hammer, Elke; Herzberg, Christina; Commichau, Fabian M; Völker, Uwe; Stülke, Jörg

    2011-01-01

    The majority of all proteins of a living cell is active in complexes rather than in an isolated way. These protein-protein interactions are of high relevance for many biological functions. In addition to many well established protein complexes an increasing number of protein-protein interactions, which form rather transient complexes has recently been discovered. The formation of such complexes seems to be a common feature especially for metabolic pathways. In the Gram-positive model organism Bacillus subtilis, we identified a protein complex of three citric acid cycle enzymes. This complex consists of the citrate synthase, the isocitrate dehydrogenase, and the malate dehydrogenase. Moreover, fumarase and aconitase interact with malate dehydrogenase and with each other. These five enzymes catalyze sequential reaction of the TCA cycle. Thus, this interaction might be important for a direct transfer of intermediates of the TCA cycle and thus for elevated metabolic fluxes via substrate channeling. In addition, we discovered a link between the TCA cycle and gluconeogenesis through a flexible interaction of two proteins: the association between the malate dehydrogenase and phosphoenolpyruvate carboxykinase is directly controlled by the metabolic flux. The phosphoenolpyruvate carboxykinase links the TCA cycle with gluconeogenesis and is essential for B. subtilis growing on gluconeogenic carbon sources. Only under gluconeogenic growth conditions an interaction of these two proteins is detectable and disappears under glycolytic growth conditions. PMID:20933603

  20. Enzyme-free detection and quantification of double-stranded nucleic acids.

    PubMed

    Feuillie, Cécile; Merheb, Maxime Mohamad; Gillet, Benjamin; Montagnac, Gilles; Hänni, Catherine; Daniel, Isabelle

    2012-08-01

    We have developed a fully enzyme-free SERRS hybridization assay for specific detection of double-stranded DNA sequences. Although all DNA detection methods ranging from PCR to high-throughput sequencing rely on enzymes, this method is unique for being totally non-enzymatic. The efficiency of enzymatic processes is affected by alterations, modifications, and/or quality of DNA. For instance, a limitation of most DNA polymerases is their inability to process DNA damaged by blocking lesions. As a result, enzymatic amplification and sequencing of degraded DNA often fail. In this study we succeeded in detecting and quantifying, within a mixture, relative amounts of closely related double-stranded DNA sequences from Rupicapra rupicapra (chamois) and Capra hircus (goat). The non-enzymatic SERRS assay presented here is the corner stone of a promising approach to overcome the failure of DNA polymerase when DNA is too degraded or when the concentration of polymerase inhibitors is too high. It is the first time double-stranded DNA has been detected with a truly non-enzymatic SERRS-based method. This non-enzymatic, inexpensive, rapid assay is therefore a breakthrough in nucleic acid detection. PMID:22695500

  1. Boronic Acid-Appended Molecular Glues for ATP-Responsive Activity Modulation of Enzymes.

    PubMed

    Okuro, Kou; Sasaki, Mizuki; Aida, Takuzo

    2016-05-01

    Water-soluble linear polymers GumBAn (m/n = 18/6, 12/12, and 6/18) with multiple guanidinium ion (Gu(+)) and boronic acid (BA) pendants in their side chains were synthesized as ATP-responsive modulators for enzyme activity. GumBAn polymers strongly bind to the phosphate ion (PO4(-)) and 1,2-diol units of ATP via the Gu(+) and BA pendants, respectively. As only the Gu(+) pendants can be used for proteins, GumBAn is able to modulate the activity of enzymes in response to ATP. As a proof-of-concept study, we demonstrated that trypsin (Trp) can be deactivated by hybridization with GumBAn. However, upon addition of ATP, Trp was liberated to retrieve its hydrolytic activity due to a higher preference of GumBAn toward ATP than Trp. This event occurred in a much lower range of [ATP] than reported examples. Under cellular conditions, the hydrolytic activity of Trp was likewise modulated. PMID:27087468

  2. Effect of inhibitors of arachidonic acid metabolism on efflux of intracellular enzymes from skeletal muscle following experimental damage.

    PubMed Central

    Jackson, M J; Wagenmakers, A J; Edwards, R H

    1987-01-01

    The role of arachidonic acid metabolism in the efflux of intracellular enzymes from damaged skeletal muscle has been examined in vitro using inhibitors of cyclo-oxygenase and lipoxygenase enzymes. Damage to skeletal muscle induced by either calcium ionophore A23187 (25 microM) or dinitrophenol (1 mM) caused an increase in the efflux of prostaglandins E2 and F2 alpha together with a large efflux of intracellular creatine kinase. Use of a cyclo-oxygenase inhibitor completely prevented the efflux of prostaglandins, but had no effect on creatine kinase efflux. However, several agents having the ability to inhibit lipoxygenase enzymes dramatically reduced creatine kinase efflux following damage. These data suggest that a product or products of lipoxygenase enzymes may be mediators of the changes in plasma membrane integrity which permit efflux of intracellular enzymes as a consequence of skeletal muscle damage. PMID:3109374

  3. [Effect of trace metals on cell morphology, enzyme activation, and production of citric acid in a strain of Aspergillus wentii].

    PubMed

    Majolli, M V; Aguirre, S N

    1999-01-01

    Data concerning the effect of very low concentrations of metals on citric acid production by microorganisms, as well as on the activity of enzymes presumptively involved in the process, are confuse. The bulk of information was obtained mainly studying selected strains of Aspergillus niger. Information concerning other citric acid producer filamentous fungi, such as A. wentii, is scanty. In the present article we report the effect of different cations on the growth pattern of A. wentii P1 as well as on the related citric acid production and the activity of several enzymes. It was found that without any addition to the culture medium the fungus developed a pelleted form of growth, pellets being about 1.5 mm in diameter. The citric acid yield was about 90%. The addition of Cu2+ impaired the sugar uptake, as well as the production of citric acid and biomass. The uptake of sugar increased in the presence of Zn2+, and there was a marked increase of the biomass production, which could account for the low citric acid production. The addition of Fe2+ impaired the citric acid production and, as sulfate, the sugar uptake. The presence of Fe3+ markedly impaired the citric acid production and increased the sugar uptake. There is no agreement about the enzymes involved in the accumulation of citric acid by microorganisms. In spite of this, aconitase (Ac), isocitrate lyase (IL), isocitrate dehydrogenase NAD(+)-dependent (ICDH- NAD+) and isocitrate dehydrogenase NADP(+)-dependent (ICDH-NADP+) are often postulated as key enzymes. In our case, these enzymes were active during the standard fermentation, although with variations, particularly concerning Ac and IL. The behavior of enzymes might be different when tested in vivo or in vitro, mainly from the quantitative point of view. Nevertheless, the activity determined in vitro might give some indication concerning the effect on fermentation of substances present in the medium. It was found that all the enzymes tested increased their

  4. Preparation of crosslinked enzyme aggregates (CLEAs) of acid urease with urethanase activity and their application.

    PubMed

    Zhang, Qian; Zha, Xiaohong; Zhou, Nandi; Tian, Yaping

    2016-04-01

    An acid urease from Providencia rettgeri JN-B815 was purified via ultrasonication, ethanol precipitation, and DEAE ion-exchange column chromatography. It was found that the enzyme exhibits not only urease activity, but also urethanase activity, which made it possible to reduce EC already existed or would produce and its precursor urea at the same time. Then, crosslinked enzyme aggregates of P. rettgeri urease (PRU-CLEAs) were prepared using genipin as crosslinking agent. The purification process of acid urease, the effects of genipin concentration, and crosslinking time on PRU-CLEAs activity were investigated. The crosslinking was performed at pH 4.5 for 2.5 h, using 0.3% genipin as crosslinking agent, and 0.3 g · L(-1) bovine serum albumin as protein feeder. Using the obtained PRU-CLEAs, the removal rate of urea was up to 9.31 mg · L(-1) · h(-1). The removal rate of urea was still up to 7.56 mg · L(-1) · h(-1) after PRU-CLEAs was re-used for 6 times. When PRU-CLEAs were applied in a batch stirred and membrane reactor, the removal rate of urea in rice wine reached 5.16 mg · L(-1) · h(-1) and the removal rate of EC was 9.21 μg · L(-1) · h(-1). Furthermore, the treatment with PRU-CLEAs revealed no significant change of volatile flavor substances in Chinese rice wine. Thus PRU-CLEAs have great potential in the elimination of EC in Chinese rice wine. PMID:26627914

  5. Production of succinic acid through overexpression of NAD(+)-dependent malic enzyme in an Escherichia coli mutant.

    PubMed Central

    Stols, L; Donnelly, M I

    1997-01-01

    NAD(+)-dependent malic enzyme was cloned from the Escherichia coli genome by PCR based on the published partial sequence of the gene. The enzyme was overexpressed and purified to near homogeneity in two chromatographic steps and was analyzed kinetically in the forward and reverse directions. The Km values determined in the presence of saturating cofactor and manganese ion were 0.26 mM for malate (physiological direction) and 16 mM for pyruvate (reverse direction). When malic enzyme was induced under appropriate culture conditions in a strain of E. coli that was unable to ferment glucose and accumulated pyruvate, fermentative metabolism of glucose was restored. Succinic acid was the major fermentation product formed. When this fermentation was performed in the presence of hydrogen, the yield of succinic acid increased. The constructed pathway represents an alternative metabolic route for the fermentative production of dicarboxylic acids from renewable feedstocks. PMID:9212416

  6. Enzyme-linked immunosorbent assay (ELISA) for the anthropogenic marker isolithocholic acid in water.

    PubMed

    Baldofski, Stefanie; Hoffmann, Holger; Lehmann, Andreas; Breitfeld, Stefan; Garbe, Leif-Alexander; Schneider, Rudolf J

    2016-11-01

    Bile acids are promising chemical markers to assess the pollution of water samples with fecal material. This study describes the optimization and validation of a direct competitive enzyme-linked immunosorbent assay for the bile acid isolithocholic acid (ILA). The quantification range of the optimized assay was between 0.09 and 15 μg/L. The assay was applied to environmental water samples. Most studies until now were focused on bile acid fractions in the particulate phase of water samples. In order to avoid tedious sample preparation, we undertook to evaluate the dynamics and significance of ILA levels in the aqueous phase. Very low concentrations in tap and surface water samples made a pre-concentration step necessary for this matrix as well as for wastewater treatment plant (WWTP) effluent. Mean recoveries for spiked water samples were between 97% and 109% for tap water and WWTP influent samples and between 102% and 136% for WWTP effluent samples. 90th percentiles of intra-plate and inter-plate coefficients of variation were below 10% for influents and below 20% for effluents and surface water. ILA concentrations were quantified in the range of 33-72 μg/L in influent, 21-49 ng/L in effluent and 18-48 ng/L in surface water samples. During wastewater treatment the ILA levels were reduced by more than 99%. ILA concentrations of influents determined by ELISA and LC-MS/MS were in good agreement. However, findings in LC-ELISA experiments suggest that the true ILA levels in concentrated samples are lower due to interfering effects of matrix compounds and/or cross-reactants. Yet, the ELISA will be a valuable tool for the performance check and comparison of WWTPs and the localization of fecal matter input into surface waters. PMID:27544648

  7. Characterization of the branched-chain amino acid aminotransferase enzyme family in tomato.

    PubMed

    Maloney, Gregory S; Kochevenko, Andrej; Tieman, Denise M; Tohge, Takayuki; Krieger, Uri; Zamir, Dani; Taylor, Mark G; Fernie, Alisdair R; Klee, Harry J

    2010-07-01

    Branched-chain amino acids (BCAAs) are synthesized in plants from branched-chain keto acids, but their metabolism is not completely understood. The interface of BCAA metabolism lies with branched-chain aminotransferases (BCAT) that catalyze both the last anabolic step and the first catabolic step. In this study, six BCAT genes from the cultivated tomato (Solanum lycopersicum) were identified and characterized. SlBCAT1, -2, -3, and -4 are expressed in multiple plant tissues, while SlBCAT5 and -6 were undetectable. SlBCAT1 and -2 are located in the mitochondria, SlBCAT3 and -4 are located in chloroplasts, while SlBCAT5 and -6 are located in the cytosol and vacuole, respectively. SlBCAT1, -2, -3, and -4 were able to restore growth of Escherichia coli BCAA auxotrophic cells, but SlBCAT1 and -2 were less effective than SlBCAT3 and -4 in growth restoration. All enzymes were active in the forward (BCAA synthesis) and reverse (branched-chain keto acid synthesis) reactions. SlBCAT3 and -4 exhibited a preference for the forward reaction, while SlBCAT1 and -2 were more active in the reverse reaction. While overexpression of SlBCAT1 or -3 in tomato fruit did not significantly alter amino acid levels, an expression quantitative trait locus on chromosome 3, associated with substantially higher expression of Solanum pennellii BCAT4, did significantly increase BCAA levels. Conversely, antisense-mediated reduction of SlBCAT1 resulted in higher levels of BCAAs. Together, these results support a model in which the mitochondrial SlBCAT1 and -2 function in BCAA catabolism while the chloroplastic SlBCAT3 and -4 function in BCAA synthesis. PMID:20435740

  8. Production of organic acids by periplasmic enzymes present in free and immobilized cells of Zymomonas mobilis.

    PubMed

    Malvessi, Eloane; Carra, Sabrina; Pasquali, Flávia Cristina; Kern, Denise Bizarro; da Silveira, Mauricio Moura; Ayub, Marco Antônio Záchia

    2013-01-01

    In this work the periplasmic enzymatic complex glucose-fructose oxidoreductase (GFOR)/glucono-δ-lactonase (GL) of permeabilized free or immobilized cells of Zymomonas mobilis was evaluated for the bioconversion of mixtures of fructose and different aldoses into organic acids. For all tested pairs of substrates with permeabilized free-cells, the best enzymatic activities were obtained in reactions with pH around 6.4 and temperatures ranging from 39 to 45 °C. Decreasing enzyme/substrate affinities were observed when fructose was in the mixture with glucose, maltose, galactose, and lactose, in this order. In bioconversion runs with 0.7 mol l(-1) of fructose and with aldose, with permeabilized free-cells of Z. mobilis, maximal concentrations of the respective aldonic acids of 0.64, 0.57, 0.51, and 0.51 mol l(-1) were achieved, with conversion yields of 95, 88, 78, and 78 %, respectively. Due to the important applications of lactobionic acid, the formation of this substance by the enzymatic GFOR/GL complex in Ca-alginate-immobilized cells was assessed. The highest GFOR/GL activities were found at pH 7.0-8.0 and temperatures of 47-50 °C. However, when a 24 h bioconversion run was carried out, it was observed that a combination of pH 6.4 and temperature of 47 °C led to the best results. In this case, despite the fact that Ca-alginate acts as a barrier for the diffusion of substrates and products, maximal lactobionic acid concentration, conversion yields and specific productivity similar to those obtained with permeabilized free-cells were achieved. PMID:23053345

  9. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    PubMed

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications. PMID:26394062

  10. Insight into the structural construction of a perfluorosulfonic acid membrane derived from a polymeric dispersion

    NASA Astrophysics Data System (ADS)

    Wang, Zhao; Tang, Haolin; Li, Junrui; Zeng, Yan; Chen, Lutang; Pan, Mu

    2014-06-01

    The effects of the nature of the solvent on the morphology of perfluorosulfonate ionomers in dispersions and the microstructures of the corresponding formed membranes are investigated. The subsequent electrochemical performances of the formed proton exchange membranes are also studied in detail. It is found that the diameters of the Nafion molecular aggregates in variable solutions decrease with an increase in the solvent dielectric constant (ε) and a decrease in the gap of the solubility parameters (δ) between the resin and the solvent. The micromorphology of Nafion is further examined by means of transmission electron microscopy, small-angle X-ray scattering, and X-ray diffraction. It is found that the membrane cast from a Nafion-2-propanol/water dispersion with ε value of 42.38 and a δ gap of 0.01 (cal cm-3)1/2 has a better ion cluster arrangement, smaller ion cluster size (approximately 13 Å), and higher crystallinity (16.7%) than the other samples. The electrochemical properties of the formed membranes are further investigated as a function of temperature, relative humidity, and the solubility parameter of the applied solvents. The results demonstrate that the electrochemical performance is strongly influenced by the solvent-induced microstructure of the backbone and the ionic clusters in the perfluorosulfonic acid membrane.

  11. Synthesis of bioadhesive poly(acrylic acid) nano- and microparticles using an inverse emulsion polymerization method for the entrapment of hydrophilic drug candidates.

    PubMed

    Kriwet, B; Walter, E; Kissel, T

    1998-12-01

    Bioadhesive latices of water-swollen poly(acrylic acid) nano-and microparticles were synthesized using an inverse (W/O) emulsion polymerization method. They are stabilized by a co-emulsifier system consisting of SpanTM 80 and TweenTM 80 dispersed in aliphatic hydrocarbons. The initial polymerization medium contains emulsion droplets and inverse micelles which solubilize a part of the monomer solution. The polymerization is initiated by free radicals, and particle dispersions with a narrow size distribution are obtained. The particle size is dependent on the type of radical initiator used. With water-soluble initiators, for example ammonium persulfate, microparticles were obtained in the size range of 1 to 10 micrometer indicating that these microparticles originate from the emulsion droplets since the droplet sizes of the W/O emulsion show similar distribution. When lipophilic radical initiators, such as azobis-isobutyronitrile, are used, almost exclusively nanoparticles are generated with diameters in the range of 80 to 150 nm, due to the limited solubility of oligomeric poly(acrylic acid) chains in the lipophilic continuous phase. These poly(acrylic acid) micro- and nanoparticles yielded excellent bioadhesive properties in an in-vitro assay and may, therefore, be suitable for the encapsulation of peptides and other hydrophilic drugs. PMID:9801438

  12. The Expression and Prognostic Significance of Retinoic Acid Metabolising Enzymes in Colorectal Cancer

    PubMed Central

    Brown, Gordon T.; Cash, Beatriz Gimenez; Blihoghe, Daniela; Johansson, Petronella; Alnabulsi, Ayham; Murray, Graeme I.

    2014-01-01

    Colorectal cancer is one of the most common types of cancer with over fifty percent of patients presenting at an advanced stage. Retinoic acid is a metabolite of vitamin A and is essential for normal cell growth and aberrant retinoic acid metabolism is implicated in tumourigenesis. This study has profiled the expression of retinoic acid metabolising enzymes using a well characterised colorectal cancer tissue microarray containing 650 primary colorectal cancers, 285 lymph node metastasis and 50 normal colonic mucosal samples. Immunohistochemistry was performed on the tissue microarray using monoclonal antibodies which we have developed to the retinoic acid metabolising enzymes CYP26A1, CYP26B1, CYP26C1 and lecithin retinol acyl transferase (LRAT) using a semi-quantitative scoring scheme to assess expression. Moderate or strong expression of CYP26A1was observed in 32.5% of cancers compared to 10% of normal colonic epithelium samples (p<0.001). CYP26B1 was moderately or strongly expressed in 25.2% of tumours and was significantly less expressed in normal colonic epithelium (p<0.001). CYP26C1 was not expressed in any sample. LRAT also showed significantly increased expression in primary colorectal cancers compared with normal colonic epithelium (p<0.001). Strong CYP26B1 expression was significantly associated with poor prognosis (HR = 1.239, 95%CI = 1.104–1.390, χ2 = 15.063, p = 0.002). Strong LRAT was also associated with poorer outcome (HR = 1.321, 95%CI = 1.034–1.688, χ2 = 5.039, p = 0.025). In mismatch repair proficient tumours strong CYP26B1 (HR = 1.330, 95%CI = 1.173–1.509, χ2 = 21.493, p<0.001) and strong LRAT (HR = 1.464, 95%CI = 1.110–1.930, χ2 = 7.425, p = 0.006) were also associated with poorer prognosis. This study has shown that the retinoic acid metabolising enzymes CYP26A1, CYP26B1 and LRAT are significantly overexpressed in colorectal cancer and that CYP26B1 and LRAT are

  13. Enhanced antimelanoma activity of methotrexate and zoledronic acid within polymeric sandwiches.

    PubMed

    Schilrreff, Priscila; Cervini, Gabriela; Romero, Eder Lilia; Morilla, Maria Jose

    2014-10-01

    New therapies are urgently needed against melanoma, one of the most aggressive tumors. Melanoma cells are resistant to the antifolate methotrexate (MTX), since MTX is taken up by the folate receptor-α (FRα), sequestered in melanosomes and exported out of the cell. The bisphosphonate zoledronic acid (ZOL) is active in several non-skeletal tumors; however, its antitumoral activity is hampered by its long-term accumulation in bones and low cellular permeability. Recently, we showed that core-shell tecto-dendrimers made of amine-terminated polyamidoamine generation 5 dendrimer (G5) as core and carboxyl-terminated G2.5 dendrimer as shell (G5G2.5) had selective cytotoxicity to melanoma cells. We hypothesized here that the activity of MTX and ZOL on melanoma cells could be enhanced when loaded within G5G2.5. MTX and ZOL were loaded within G5 cores, which were coated by a covalently bound shell of G2.5 dendrimers (drug-sandwiches). 12nm mean diameter and -12mV Z potential drug-sandwiches incorporating 6 and 31 molecules of MTX and ZOL, respectively, per G5G2.5, showed higher cytotoxicity (by MTT and apoptosis/necrosis assays) to melanoma (Sk-Mel-28) cells than free drugs and G5G2.5. Only MTX-sandwich was cytotoxic to Sk-Mel-28 cells and harmless to keratinocytes (HaCaT cells). The intracellular pathway of G5G2.5 was followed using chemical inhibitors of endocytosis. The increased cytotoxicity of MTX-sandwich could be due to its uptake by macropinocytosis instead of by FRα, avoiding MTX exocytosis. The increased cytotoxicity of ZOL-sandwich could be due to an increased intracellular accumulation of ZOL, owed by its endocytic uptake instead of diffusing as free drug. PMID:25016541

  14. A novel sensor based on electrochemical polymerization of diglycolic acid for determination of acetaminophen.

    PubMed

    Xu, Fen; Ru, Hui-Ying; Sun, Li-Xian; Zou, Yong-Jin; Jiao, Cheng-Li; Wang, Tao-Yi; Zhang, Jia-Ming; Zheng, Qian; Zhou, Huai-Ying

    2012-01-01

    Diglycolic acid (DA) polymer was coated on glassy carbon (GC) electrode by cyclic voltammetry (CV) technique for the first time. The electrochemical performances of the modified electrode were investigated by CV and electrochemical impedance (EIS). The obtained electrode showed an excellent electrocatalytic activity for the oxidation of acetaminophen (ACOP). A couple of well-defined reversible electrochemical redox peaks were observed on the ploy(DA)/GC electrode in ACOP solution. Compared with bare GC electrode, the oxidation peak potential of ACOP on ploy(DA)/GC electrode moved from 0.289 V to 0.220 V. Meanwhile, the oxidation peak current was much higher on the modified electrode than that on the bare GC electrode, indicating DA polymer modified electrode possessed excellent performance for the oxidation of ACOP. This kind of capability of the modified electrode can be enlisted for the highly sensitive and selective determination of ACOP. Under the optimized conditions, a wide linear range from 2 × 10(-8) to 5.0 × 10(-4)M with a correlation coefficient 0.9995 was obtained. The detection limit was 6.7 × 10(-9)M (at the ratio of signal to noise, S/N=3:1). The modified electrode also exhibited very good stability and reproducibility for the detection of ACOP. The established method was applied to the determination of ACOP in samples. An average recovery of 100.1% was achieved. These results indicated that this method was reliable for determining ACOP. PMID:22651969

  15. Polymeric microspheres

    DOEpatents

    Walt, David R.; Mandal, Tarun K.; Fleming, Michael S.

    2004-04-13

    The invention features core-shell microsphere compositions, hollow polymeric microspheres, and methods for making the microspheres. The microspheres are characterized as having a polymeric shell with consistent shell thickness.

  16. [Development of direct competitive enzyme-linked immunosorbent assay for the determination of domoic acid].

    PubMed

    Wang, Qian; Cheng, Jin-Ping; Gao, Li-Li; Dong, Yu; Xi, Lei

    2012-02-01

    To develop a direct competitive enzyme-linked immunosorbent assay (ELISA) for rapid detection of domoic acid concentrations, HRP (horse radish peroxidase) was successfully linked to DA using EDC. The concentration of DA was quantitatively analyzed on the basic of the specific immune responses between the DA- HRP and the monoclonal antibodies made in advance. Calibration curve were established after the optimization of reaction conditions such as the type of blocking solution, the blocking time and the incubation temperature. The results show that, the best reaction condition of the direct competitive ELISA is 1% gelatin, blocking 1 h at 37 degrees C, incubating 1 h at 37 degrees C after the monoclonal antibodies added. The detect limit is 3.58 ng x mL(-1), the coefficient of variation between the holes is below 15%, and the recovery is 80% - 120%. The whole analysis process could be completed within 1.5 h. It meets the requirements of rapid and batch detection of domoic acid. The method will have broad development prospects. PMID:22509610

  17. Nicotinamide Adenine Dinucleotide-specific "Malic" Enzyme in Kalanchoë daigremontiana and Other Plants Exhibiting Crassulacean Acid Metabolism.

    PubMed

    Dittrich, P

    1976-02-01

    NAD-specific "malic" enzyme (EC 1.1.1.39) has been isolated and purified 1200-fold from leaves of Kalanchoë daigremontiana. Kinetic studies of this enzyme, which is activated 14-fold by CoA, acetyl-CoA, and SO(4) (2-), suggest allosteric properties. Cofactor requirements show an absolute specificity for NAD and for Mn(2+), which cannot be replaced by NADP or Mg(2+). For maintaining enzyme activity in crude leaf extracts a thiol reagent, Mn(2+), and PVP-40 were required. The latter could be omitted from purified preparations. By sucrose density gradient centrifugation NAD-malic enzyme could be localized in mitochondria. A survey of plants with crassulacean acid metabolism revealed the presence of NAD-malic enzyme in all 31 plants tested. Substantial levels of this enzyme (121-186 mumole/hr.mg of Chl) were detected in all members tested of the family Crassulaceae. It is proposed that NAD-malic enzyme in general supplements activity of NADP-malic enzyme present in these plants and may be specifically employed to increase internal concentrations of CO(2) for recycling during cessation of gas exchange in periods of severe drought. PMID:16659473

  18. (E)-4-aryl-4-oxo-2-butenoic acid amides, chalcone–aroylacrylic acid chimeras: Design, antiproliferative activity and inhibition of tubulin polymerization

    PubMed Central

    Vitorović-Todorović, Maja D.; Erić-Nikolić, Aleksandra; Kolundžija, Branka; Hamel, Ernest; Ristić, Slavica; Juranić, Ivan O.; Drakulić, Branko J.

    2013-01-01

    Antiproliferative activity of twenty-nine (E)-4-aryl-4-oxo-2-butenoic acid amides against three human tumor cell lines (HeLa, FemX, and K562) is reported. Compounds showed antiproliferative activity in one-digit micromolar to submicromolar concentrations. The most active derivatives toward all the cell lines tested bear alkyl substituents on the aroyl moiety of the molecules. Fourteen compounds showed tubulin assembly inhibition at concentrations <20 μM. The most potent inhibitor of tubulin assembly was unsubstituted compound 1, with IC50 = 2.9 μM. Compound 23 had an oral LD50 in vivo of 45 mg/kg in mice. Cell cycle analysis on K562 cells showed that compounds 1, 2 and 23 caused accumulation of cells in the G2/M phase, but inhibition of microtubule polymerization is not the principal mode of action of the compounds. Nevertheless, they may be useful leads for the design of a new class of antitubulin agents. PMID:23353745

  19. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2013-07-23

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  20. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N; Apel, William A; Thompson, Vicki S; Ward, Thomas E

    2014-04-08

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  1. Methods of combined bioprocessing and related microorganisms, thermophilic and/or acidophilic enzymes, and nucleic acids encoding said enzymes

    DOEpatents

    Thompson, David N.; Apel, William A.; Thompson, Vicki S.; Ward, Thomas E.

    2016-03-22

    A genetically modified organism comprising: at least one nucleic acid sequence and/or at least one recombinant nucleic acid isolated from Alicyclobacillus acidocaldarius and encoding a polypeptide involved in at least partially degrading, cleaving, transporting, metabolizing, or removing polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, xylan-, glucan-, galactan-, or mannan-decorating groups; and at least one nucleic acid sequence and/or at least one recombinant nucleic acid encoding a polypeptide involved in fermenting sugar molecules to a product. Additionally, enzymatic and/or proteinaceous extracts may be isolated from one or more genetically modified organisms. The extracts are utilized to convert biomass into a product. Further provided are methods of converting biomass into products comprising: placing the genetically modified organism and/or enzymatic extracts thereof in fluid contact with polysaccharides, cellulose, lignocellulose, hemicellulose, lignin, starch, sugars, sugar oligomers, carbohydrates, complex carbohydrates, chitin, heteroxylans, glycosides, and/or xylan-, glucan-, galactan-, or mannan-decorating groups.

  2. Polymerization of Protecting-Group-Free Peptides via ROMP

    PubMed Central

    Kammeyer, Jacquelin K.; Blum, Angela P.; Adamiak, Lisa; Hahn, Michael E.; Gianneschi, Nathan C.

    2013-01-01

    A study was conducted to survey the tolerance of ring-opening metathesis polymerization (ROMP) with respect to amino acid (a.a) identity of pentapeptide-modified norbornene-based monomers. A library of norbornyl-pentapeptides were prepared with the general structure, norbornyl-GX2PLX5, where residue ‘X’ was changed at each of the two positions (2 or 5) alternately to consist of the natural amino acids F, A, V, R, S, K, N, T, M, Q, H, W, C, Y, E, Q, and D. Each peptide monomer, free of protecting groups, was mixed in turn under a standard set of polymerization conditions with the ROMP initiator (IMesH2)C5H5N)2(Cl)2Ru=CHPh. Two sets of polymerization reactions were performed, one with Monomer:Initiator (M:I) ratio of 20:1, and another with M:I of 200:1. For the nucleophilic amino acids cysteine and lysine, polymerization reactions were quantitatively compared to those of their protected analogues. Furthermore, we describe polymerization of macromonomers containing up to 30 a.a. to test for tolerance of ROMP to peptide molecular weight. These reactions were studied via SEC-MALS and NMR. Finally, with knowledge of sequence scope in hand, we prepared a set of enzyme-substrate containing brush polymers and studied them with respect to their bioactivity. PMID:24015154

  3. Should South Africa Be Performing Nucleic Acid Testing on HIV Enzyme-Linked Immunosorbent Assay-Negative Samples?▿

    PubMed Central

    Gous, Natasha; Scott, Lesley; Perovic, Olga; Venter, Francios; Stevens, Wendy

    2010-01-01

    The frequency of acute HIV infection (AHI) among HIV-1 enzyme-linked immunosorbent assay (ELISA)-negative samples received from general hospital patient admissions was assessed. Of 3,005 samples pooled for nucleic acid testing, a prevalence of 0.13% was found. Pooled nucleic acid testing may be feasible for low-cost identification of AHI in high-prevalence settings. PMID:20610671

  4. Structural characterization of the Mycobacterium tuberculosis biotin biosynthesis enzymes 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase .

    PubMed

    Dey, Sanghamitra; Lane, James M; Lee, Richard E; Rubin, Eric J; Sacchettini, James C

    2010-08-10

    Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 and 1.85 A. Superimposition of the DAPAS structures bound either to the SAM analogue sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 A, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb. PMID:20565114

  5. Enzymes involved in a novel anaerobic cyclohexane carboxylic acid degradation pathway.

    PubMed

    Kung, Johannes W; Meier, Anne-Katrin; Mergelsberg, Mario; Boll, Matthias

    2014-10-01

    The anaerobic degradation of cyclohexane carboxylic acid (CHC) has so far been studied only in Rhodopseudomonas palustris, in which CHC is activated to cyclohexanoyl coenzyme A (cyclohexanoyl-CoA [CHCoA]) and then dehydrogenated to cyclohex-1-ene-1-carboxyl-CoA (CHeneCoA). This intermediate is further degraded by reactions of the R. palustris-specific benzoyl-CoA degradation pathway of aromatic compounds. However, CHeneCoA is not an intermediate in the degradation of aromatic compounds in all other known anaerobic bacteria; consequently, degradation of CHC was mostly unknown in anaerobic bacteria. We identified a previously unknown CHC degradation pathway in the Fe(III)-reducing Geobacter metallireducens by determining the following CHC-induced in vitro activities: (i) the activation of CHC to CHCoA by a succinyl-CoA:CHC CoA transferase, (ii) the 1,2-dehydrogenation of CHCoA to CHeneCoA by CHCoA dehydrogenase, and (iii) the unusual 1,4-dehydrogenation of CHeneCoA to cyclohex-1,5-diene-1-carboxyl-CoA. This last represents a previously unknown joint intermediate of the CHC and aromatic compound degradation pathway in bacteria other than R. palustris. The enzymes catalyzing the three reactions were purified and characterized as specific enzymes after heterologous expression of the encoding genes. Quantitative reverse transcription-PCR revealed that expression of these genes was highly induced during growth with CHC but not with benzoate. The newly identified CHC degradation pathway is suggested to be present in nearly all CHC-degrading anaerobic bacteria, including denitrifying, Fe(III)-reducing, sulfate-reducing, and fermenting bacteria. Remarkably, all three CHC degradation pathways always link CHC catabolism to the catabolic pathways of aromatic compounds. We propose that the capacity to use CHC as a carbon source evolved from already-existing aromatic compound degradation pathways. PMID:25112478

  6. Identification of RALDH2 as a Visually Regulated Retinoic Acid Synthesizing Enzyme in the Chick Choroid

    PubMed Central

    Hollaway, Lindsey R.; Lam, Wengtse; Li, Nan; Napoli, Joseph L.

    2012-01-01

    Purpose. All-trans-retinoic acid (atRA) has been implicated in the local regulation of scleral proteoglycan synthesis in vivo. The purpose of the present study was to identify the enzymes involved in the synthesis of atRA during visually guided ocular growth, the cells involved in modulation of atRA biosynthesis in the choroid, and the effect of choroid-derived atRA on scleral proteoglycan synthesis. Methods. Myopia was induced in White leghorn chicks by form deprivation for 10 days, followed by up to 15 days of unrestricted vision (recovery). Expression of atRA synthesizing enzymes was evaluated by semiquantitative qRT-PCR, in situ hybridization, and immunohistochemistry. atRA synthesis was measured in organ cultures of isolated choroids using LC-tandem MS quantification. Scleral proteoglycan synthesis was measured in vitro by the incorporation of 35SO4 in CPC-precipitable glycosaminoglycans. Results. RALDH2 was the predominant RALDH transcript in the choroid (>100-fold that of RALDH3). RALDH2 mRNA was elevated after 12 and 24 hours of recovery (60% and 188%, respectively; P < 0.01). The atRA concentration was significantly higher in cultures of choroids from 24-hour to 15-day recovering eyes than in paired controls (∼195%; P < 0.01). Choroid conditioned medium from recovering choroids inhibited proteoglycan synthesis to 43% of controls (P < 0.02, paired t-test; n = 16) and produced a relative inhibition corresponding to a RA concentration of 7.20 × 10−8 M. Conclusions. The results of this study suggest that RALDH2 is the major retinal dehydrogenase in the chick choroid and is responsible for increased atRA synthesis in response to myopic defocus. PMID:22323456

  7. Iron-Based Redox Polymerization of Acrylic Acid for Direct Synthesis of Hydrogel/Membranes, and Metal Nanoparticles for Water Treatment

    PubMed Central

    Hernández, Sebastián; Papp, Joseph K.; Bhattacharyya, Dibakar

    2014-01-01

    Functionalized polymer materials with ion exchange groups and integration of nano-structured materials is an emerging area for catalytic and water pollution control applications. The polymerization of materials such as acrylic acid often requires persulfate initiator and a high temperature start. However, is generally known that metal ions accelerate such polymerizations starting from room temperature. If the metal is properly selected, it can be used in environmental applications adding two advantages simultaneously. This paper deals with this by polymerizing acrylic acid using iron as accelerant and its subsequent use for nanoparticle synthesis in hydrogel and PVDF membranes. Characterizations of hydrogel, membranes and nanoparticles were carried out with different techniques. Nanoparticles sizes of 30–60 nm were synthesized. Permeability and swelling measurements demonstrate an inverse relationship between hydrogel mesh size (6.30 to 8.34 nm) and membrane pores (222 to 110 nm). Quantitative reduction of trichloroethylene/chloride generation by Fe/Pd nanoparticles in hydrogel/membrane platforms was also performed. PMID:24954975

  8. In Silico Phylogenetic Analysis and Molecular Modelling Study of 2-Haloalkanoic Acid Dehalogenase Enzymes from Bacterial and Fungal Origin

    PubMed Central

    Satpathy, Raghunath; Konkimalla, V. B.; Ratha, Jagnyeswar

    2016-01-01

    2-Haloalkanoic acid dehalogenase enzymes have broad range of applications, starting from bioremediation to chemical synthesis of useful compounds that are widely distributed in fungi and bacteria. In the present study, a total of 81 full-length protein sequences of 2-haloalkanoic acid dehalogenase from bacteria and fungi were retrieved from NCBI database. Sequence analysis such as multiple sequence alignment (MSA), conserved motif identification, computation of amino acid composition, and phylogenetic tree construction were performed on these primary sequences. From MSA analysis, it was observed that the sequences share conserved lysine (K) and aspartate (D) residues in them. Also, phylogenetic tree indicated a subcluster comprised of both fungal and bacterial species. Due to nonavailability of experimental 3D structure for fungal 2-haloalkanoic acid dehalogenase in the PDB, molecular modelling study was performed for both fungal and bacterial sources of enzymes present in the subcluster. Further structural analysis revealed a common evolutionary topology shared between both fungal and bacterial enzymes. Studies on the buried amino acids showed highly conserved Leu and Ser in the core, despite variation in their amino acid percentage. Additionally, a surface exposed tryptophan was conserved in all of these selected models. PMID:26880911

  9. Endophytic Fungi from Frankincense Tree Improves Host Growth and Produces Extracellular Enzymes and Indole Acetic Acid

    PubMed Central

    Khan, Abdul Latif; Al-Harrasi, Ahmed; Al-Rawahi, Ahmed; Al-Farsi, Zainab; Al-Mamari, Aza; Waqas, Muhammad; Asaf, Sajjad; Elyassi, Ali; Mabood, Fazal; Shin, Jae-Ho; Lee, In-Jung

    2016-01-01

    Boswellia sacra, an economically important frankincense-producing tree found in the desert woodlands of Oman, is least known for its endophytic fungal diversity and the potential of these fungi to produce extracellular enzymes and auxins. We isolated various fungal endophytes belonging to Eurotiales (11.8%), Chaetomiaceae (17.6%), Incertae sadis (29.5%), Aureobasidiaceae (17.6%), Nectriaceae (5.9%) and Sporomiaceae (17.6%) from the phylloplane (leaf) and caulosphere (stem) of the tree. Endophytes were identified using genomic DNA extraction, PCR amplification and sequencing the internal transcribed spacer regions, whereas a detailed phylogenetic analysis of the same gene fragment was made with homologous sequences. The endophytic colonization rate was significantly higher in the leaf (5.33%) than the stem (0.262%). The Shannon-Weiner diversity index was H′ 0.8729, while Simpson index was higher in the leaf (0.583) than in the stem (0.416). Regarding the endophytic fungi’s potential for extracellular enzyme production, fluorogenic 4-methylumbelliferone standards and substrates were used to determine the presence of cellulases, phosphatases and glucosidases in the pure culture. Among fungal strains, Penicillum citrinum BSL17 showed significantly higher amounts of glucosidases (62.15±1.8 μM-1min-1mL) and cellulases (62.11±1.6 μM-1min-1mL), whereas Preussia sp. BSL10 showed significantly higher secretion of glucosidases (69.4±0.79 μM-1min-1mL) and phosphatases (3.46±0.31μM-1min-1mL) compared to other strains. Aureobasidium sp. BSS6 and Preussia sp. BSL10 showed significantly higher potential for indole acetic acid production (tryptophan-dependent and independent pathways). Preussia sp. BSL10 was applied to the host B. sacra tree saplings, which exhibited significant improvements in plant growth parameters and accumulation of photosynthetic pigments. The current study concluded that endophytic microbial resources producing extracellular enzymes and auxin

  10. Endophytic Fungi from Frankincense Tree Improves Host Growth and Produces Extracellular Enzymes and Indole Acetic Acid.

    PubMed

    Khan, Abdul Latif; Al-Harrasi, Ahmed; Al-Rawahi, Ahmed; Al-Farsi, Zainab; Al-Mamari, Aza; Waqas, Muhammad; Asaf, Sajjad; Elyassi, Ali; Mabood, Fazal; Shin, Jae-Ho; Lee, In-Jung

    2016-01-01

    Boswellia sacra, an economically important frankincense-producing tree found in the desert woodlands of Oman, is least known for its endophytic fungal diversity and the potential of these fungi to produce extracellular enzymes and auxins. We isolated various fungal endophytes belonging to Eurotiales (11.8%), Chaetomiaceae (17.6%), Incertae sadis (29.5%), Aureobasidiaceae (17.6%), Nectriaceae (5.9%) and Sporomiaceae (17.6%) from the phylloplane (leaf) and caulosphere (stem) of the tree. Endophytes were identified using genomic DNA extraction, PCR amplification and sequencing the internal transcribed spacer regions, whereas a detailed phylogenetic analysis of the same gene fragment was made with homologous sequences. The endophytic colonization rate was significantly higher in the leaf (5.33%) than the stem (0.262%). The Shannon-Weiner diversity index was H' 0.8729, while Simpson index was higher in the leaf (0.583) than in the stem (0.416). Regarding the endophytic fungi's potential for extracellular enzyme production, fluorogenic 4-methylumbelliferone standards and substrates were used to determine the presence of cellulases, phosphatases and glucosidases in the pure culture. Among fungal strains, Penicillum citrinum BSL17 showed significantly higher amounts of glucosidases (62.15±1.8 μM-1min-1mL) and cellulases (62.11±1.6 μM-1min-1mL), whereas Preussia sp. BSL10 showed significantly higher secretion of glucosidases (69.4±0.79 μM-1min-1mL) and phosphatases (3.46±0.31μM-1min-1mL) compared to other strains. Aureobasidium sp. BSS6 and Preussia sp. BSL10 showed significantly higher potential for indole acetic acid production (tryptophan-dependent and independent pathways). Preussia sp. BSL10 was applied to the host B. sacra tree saplings, which exhibited significant improvements in plant growth parameters and accumulation of photosynthetic pigments. The current study concluded that endophytic microbial resources producing extracellular enzymes and auxin could

  11. Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor

    PubMed Central

    2013-01-01

    Background Phosphatidic acid phosphatase (PAP, EC 3.1.3.4) catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor. Results We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production. Conclusions The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single

  12. Identification of enzyme activity that conjugates indole-3-acetic acid to aspartate in immature seeds of pea (Pisum sativum).

    PubMed

    Ostrowski, Maciej; Jakubowska, Anna

    2008-01-01

    This study describes the first identification of plant enzyme activity catalyzing the conjugation of indole-3-acetic acid to amino acids. Enzymatic synthesis of indole-3-acetylaspartate (IAA-Asp) by a crude enzyme preparation from immature seeds of pea (Pisum sativum) was observed. The reaction yielded a product with the same Rf as IAA-Asp standard after thin layer chromatography. The identity of IAA-Asp was verified by HPLC analysis. IAA-Asp formation was dependent on ATP and Mg2+, and was linear during a 60 min period. The enzyme preparation obtained after poly(ethylene glycol) 6000 fractionation showed optimum activity at pH 8.0, and the temperature optimum for IAA-Asp synthesis was 30 degrees C. PMID:17920159

  13. Changes in the Enzymes for Fatty Acid Synthesis and Desaturation during Acclimation of Developing Soybean Seeds to Altered Growth Temperature

    PubMed Central

    Cheesbrough, Thomas M.

    1989-01-01

    Temperature-induced changes in the enzymes for fatty acid synthesis and desaturation were studied in developing soybean seeds (Glycine max L. var Williams 82). Changes were induced by culture of the seed pods for 20 hours in liquid media at 20, 25, or 35°C. Linoleoyl and oleoyl desaturases were 94 and 10 times as active, respectively, in seeds cultured at 20°C as those cultured at 25°C. Both desaturases had negligible activity in seeds cultured at 35°C compared to seeds cultured at 20°C. Though less dramatic, other enzymes also showed differences in activity after 20 hours in culture at 20, 25, or 35°C. Stearoyl-acyl carrier protein (ACP) desaturase and CDP-choline:diacylglycerol phosphorylcholine transferase were most active in preparations from 20°C cultures. Activities were twofold lower at 25°C and a further threefold lower in 35°C cultures. Cultures from 25 and 35°C had 60 and 40%, respectively, of the phosphorylcholine:CTP cytidylyl transferase activity present in cultures grown at 20°C. Fatty acid synthetase, malonyl-coenzyme A:ACP transacylase, palmitoyl-ACP elongation, and choline kinase were not significantly altered by culture temperature. These data suggest that the enzymes for fatty acid desaturation and phosphatidylcholine synthesis can be rapidly modulated in response to altered growth temperatures, while the enzymes for fatty acid synthesis and elongation are not. PMID:16666840

  14. Activation Energies for an Enzyme-Catalyzed and Acid-Catalyzed Hydrolysis: An Introductory Interdisciplinary Experiment for Chemists and Biochemists.

    ERIC Educational Resources Information Center

    Adams, K. R.; Meyers, M. B.

    1985-01-01

    Background information, procedures used, and typical results obtained are provided for an experiment in which students determine and compare the Arrhenius activation energies (Ea) for the hydrolysis of salicin. This reaction is subject to catalysis both by acid and by the enzyme emulsin (beta-d-glucoside glycohydrolase). (JN)

  15. Activities of enzymes related to NADPH generation and amino acid metabolism in the ruminal mucosa of sheep.

    PubMed

    Weekes, T E

    1984-09-01

    Experiments were performed with growing lambs to investigate dietary influences on enzymes involved in the metabolism of propionate, amino acids and NADPH in the ruminal mucosa. Glutamate dehydrogenase (GDH) was the only enzyme assayed that was consistently affected by diet. First, lambs were fed either rolled barley, resulting in epithelial hyperkeratosis, or whole unprocessed barley, resulting in keratin aplasia and reduced GDH activity. Secondly, lambs were fed isonitrogenous diets containing either fish meal or urea. GDH activity was greater when fish meal was fed. NADP-isocitrate dehydrogenase was more active than other NADPH-generating enzymes in ruminal mucosa and several other lamb tissues, but the operation of the isocitrate cycle in rumen epithelium may be restricted by a low activity of aconitate hydratase. These results suggest that enzyme activities in ruminal mucosa are generally unresponsive to diet and that adaptations in GDH are related to changes in rumen morphology, rather than to isocitrate cycle activity or ammonia assimilation. PMID:6470829

  16. Methane emissions from beef cattle: Effects of monensin, sunflower oil, enzymes, yeast, and fumaric acid.

    PubMed

    McGinn, S M; Beauchemin, K A; Coates, T; Colombatto, D

    2004-11-01

    Methane emitted from the livestock sector contributes to greenhouse gas (GHG) emissions. Understanding the effects of diet on enteric methane production can help refine GHG emission inventories and identify viable GHG reduction strategies. Our study focused on measuring methane and carbon dioxide emissions, total-tract digestibility, and ruminal fermentation in growing beef cattle fed a diet supplemented with various additives or ingredients. Two experiments, each designed as a 4 x 4 Latin square with 21-d periods, were conducted using 16 Holstein steers (initial BW 311.6 +/- 12.3 kg). In Exp. 1, treatments were control (no additive), monensin (Rumensin, Elanco Animal Health, Indianapolis, IN; 33 mg/kg DM), sunflower oil (400 g/d, approximately 5% of DMI), and proteolytic enzyme (Protex 6-L, Genencor Int., Inc., CA; 1 mL/kg DM). In Exp. 2, treatments were control (no additive), Procreatin-7 yeast (Prince Agri Products, Inc., Quincy, IL; 4 g/d), Levucell SC yeast (Lallemand, Inc., Rexdale, Ontario, Canada; 1 g/d), and fumaric acid (Bartek Ingredients Inc., Stoney Creek, Ontario, Canada; 80 g/d). The basal diet consisted of 75% barley silage, 19% steam-rolled barley grain, and 6% supplement (DM basis). Four large chambers (two animals per chamber) were equipped with lasers and infrared gas analyzers to measure methane and carbon dioxide, respectively, for 3 d each period. Total-tract digestibility was determined using chromic oxide. Approximately 6.5% of the GE consumed was lost in the form of methane emissions from animals fed the control diet. In Exp. 1, sunflower oil decreased methane emissions by 22% (P = 0.001) compared with the control, whereas monensin (P = 0.44) and enzyme had no effect (P = 0.82). However, oil decreased (P = 0.03) the total-tract digestibility of NDF by 20%. When CH(4) emissions were corrected for differences in energy intake, the loss of GE to methane was decreased by 21% (P = 0.002) using oil and by 9% (P = 0.09) using monensin. In Exp. 2

  17. Hybrid polymeric hydrogels for ocular drug delivery: nanoparticulate systems from copolymers of acrylic acid-functionalized chitosan and N-isopropylacrylamide or 2-hydroxyethyl methacrylate

    NASA Astrophysics Data System (ADS)

    Barbu, Eugen; Verestiuc, Liliana; Iancu, Mihaela; Jatariu, Anca; Lungu, Adriana; Tsibouklis, John

    2009-06-01

    Nanoparticulate hybrid polymeric hydrogels (10-70 nm) have been obtained via the radical-induced co-polymerization of acrylic acid-functionalized chitosan with either N-isopropylacrylamide or 2-hydroxyethyl methacrylate, and the materials have been investigated for their ability to act as controlled release vehicles in ophthalmic drug delivery. Studies on the effects of network structure upon swelling properties, adhesiveness to substrates that mimic mucosal surfaces and biodegradability, coupled with in vitro drug release investigations employing ophthalmic drugs with differing aqueous solubilities, have identified nanoparticle compositions for each of the candidate drug molecules. The hybrid nanoparticles combine the temperature sensitivity of N-isopropylacrylamide or the good swelling characteristics of 2-hydroxyethyl methacrylate with the susceptibility of chitosan to lysozyme-induced biodegradation.

  18. Superabsorbent hydrogels via graft polymerization of acrylic acid from chitosan-cellulose hybrid and their potential in controlled release of soil nutrients.

    PubMed

    Essawy, Hisham A; Ghazy, Mohamed B M; El-Hai, Farag Abd; Mohamed, Magdy F

    2016-08-01

    Superabsorbent polymers fabricated via grafting polymerization of acrylic acid from chitosan (CTS) yields materials that suffer from poor mechanical strength. Hybridization of chitosan with cellulose (Cell) via chemical bonding using thiourea formaldehyde resin increases the flexibility of the produced hybrid (CTS/Cell). The hybridization process and post graft polymerization of acrylic acid was followed using Fourier transform infrared (FTIR). Also, the obtained structures were homogeneous and exhibited uniform surface as could be shown from imaging with scanning electron microscopy (SEM). Thus, the polymers derived from the grafting of polyacrylic acid from (CTS/Cell) gave rise to much more mechanically robust structures ((CTS/Cell)-g-PAA) that bear wide range of pH response due to presence of chitosan and polyacrylic acid in one homogeneous entity. Additionally, the obtained structures possessed greater water absorbency 390, 39.5g/g in distilled water and saline (0.9wt.% NaCl solution), respectively, and enhanced retention potential even at elevated temperatures as revealed by thermogravimetric analysis (TGA). This could be explained by the high grafting efficiency (GE%), 86.4%, and grafting yield (GY%), 750%. The new superabsorbent polymers proved to be very efficient devices for controlled release of fertilizers into the soil which expands their use in agriculture and horticultural applications. PMID:27126169

  19. Inhibition of endocannabinoid-degrading enzyme fatty acid amide hydrolase increases atherosclerotic plaque vulnerability in mice.

    PubMed

    Hoyer, Friedrich Felix; Khoury, Mona; Slomka, Heike; Kebschull, Moritz; Lerner, Raissa; Lutz, Beat; Schott, Hans; Lütjohann, Dieter; Wojtalla, Alexandra; Becker, Astrid; Zimmer, Andreas; Nickenig, Georg

    2014-01-01

    The role of endocannabinoids such as anandamide during atherogenesis remains largely unknown. Fatty acid amide hydrolase (FAAH) represents the key enzyme in anandamide degradation, and its inhibition is associated with subsequent higher levels of anandamide. Here, we tested whether selective inhibition of FAAH influences the progression of atherosclerosis in mice. Selective inhibition of FAAH using URB597 resulted in significantly increased plasma levels of anandamide compared to control, as assessed by mass spectrometry experiments in mice. Apolipoprotein E-deficient (ApoE(-/-)) mice were fed a high-fat, cholesterol-rich diet to induce atherosclerotic conditions. Simultaneously, mice received either the pharmacological FAAH inhibitor URB597 1mg/kg body weight (n=28) or vehicle (n=25) via intraperitoneal injection three times a week. After eight weeks, mice were sacrificed, and experiments were performed. Vascular superoxide generation did not differ between both groups, as measured by L012 assay. To determine whether selective inhibition of FAAH affects atherosclerotic plaque inflammation, immunohistochemical staining of the aortic root was performed. Atherosclerotic plaque formation, vascular macrophage accumulation, as well as vascular T cell infiltration did not differ between both groups. Interestingly, neutrophil cell accumulation was significantly increased in mice receiving URB597 compared to control. Vascular collagen structures in atherosclerotic plaques were significantly diminished in mice treated with URB597 compared to control, as assessed by picro-sirius-red staining. This was accompanied by an increased aortic expression of matrix metalloproteinase-9, as determined by quantitative RT-PCR and western blot analysis. Inhibition of fatty acid amide hydrolase does not influence plaque size but increases plaque vulnerability in mice. PMID:24286707

  20. Expression of the neurotransmitter-synthesizing enzyme glutamic acid decarboxylase in male germ cells.

    PubMed Central

    Persson, H; Pelto-Huikko, M; Metsis, M; Söder, O; Brene, S; Skog, S; Hökfelt, T; Ritzén, E M

    1990-01-01

    The gene encoding glutamic acid decarboxylase (GAD), the key enzyme in the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid, is shown to be expressed in the testis of several different species. Nucleotide sequence analysis of a cDNA clone isolated from the human testis confirmed the presence of GAD mRNA in the testis. The major GAD mRNA in the testis was 2.5 kilobases. Smaller amounts of a 3.7-kilobase mRNA with the same size as GAD mRNA in the brain was also detected in the testis. In situ hybridization using a GAD-specific probe revealed GAD mRNA expressing spermatocytes and spermatids located in the middle part of rat seminiferous tubules. Studies on the ontogeny of GAD mRNA expression showed low levels of GAD mRNA in testes of prepubertal rats, with increasing levels as sexual maturation is reached, compatible with GAD mRNA expression in germ cells. In agreement with this, fractionation of cells from the rat seminiferous epithelium followed by Northern (RNA) blot analysis showed the highest levels of GAD mRNA associated with spermatocytes and spermatids. Evidence for the presence of GAD protein in the rat testis was obtained from the demonstration of GAD-like immunoreactivity in seminiferous tubules, predominantly at a position where spermatids and spermatozoa are found. Furthermore, GAD-like immunoreactivity was seen in the midpiece of ejaculated human spermatozoa, the part that is responsible for generating energy for spermatozoan motility. Images PMID:1697032

  1. Micro- to nanostructured poly(pyrrole-nitrilotriacetic acid) films via nanosphere templates: applications to 3D enzyme attachment by affinity interactions.

    PubMed

    Cernat, Andreea; Le Goff, Alan; Holzinger, Michael; Sandulescu, Robert; Cosnier, Serge

    2014-02-01

    We report the combination of latex nanosphere lithography with electropolymerization of N-substituted pyrrole monomer bearing a nitrilotriacetic acid (NTA) moiety for the template-assisted nanostructuration of poly(pyrrole-NTA) films and their application for biomolecule immobilization. The electrodes were modified by casting latex beads (100 or 900 nm in diameter) on their surface followed by electropolymerization of the pyrrole-NTA monomer and the subsequent chelation of Cu(2+) ions. The dissolution of the nanobeads leads then to a nanostructured polymer film with increased surface. Thanks to the versatile affinity interactions between the (NTA)Cu(2+) complex and histidine- or biotin-tagged proteins, both tyrosinase and glucose oxidase were immobilized on the modified electrode. Nanostructuration of the polypyrrole via nanosphere lithography (NSL) using 900- and 100-nm latex beads allows an increase in surface concentration of enzymes anchored on the functionalized polypyrrole electrode. The nanostructured enzyme electrodes were characterized by fluorescence microscopy, 3D laser scanning confocal microscopy, and scanning electron microscopy. Electrochemical studies demonstrate the increase in the amount of immobilized biomolecules and associated biosensor performances when achieving NSL compared to conventional polymer formation without bead template. In addition, the decrease in nanobead diameter from 900 to 100 nm provides an enhancement in biosensor performance. Between biosensors based on films polymerized without nanobeads and with 100-nm nanobeads, maximum current density values increase from 4 to 56 μA cm(-2) and from 7 to 45 μA cm(-2) for biosensors based on tyrosinase and glucose oxidase, respectively. PMID:23793398

  2. Enzymatic acylation of di- and trisaccharides with fatty acids: choosing the appropriate enzyme, support and solvent.

    PubMed

    Plou, Francisco J; Cruces, M Angeles; Ferrer, Manuel; Fuentes, Gloria; Pastor, Eitel; Bernabé, Manuel; Christensen, Morten; Comelles, Francisco; Parra, José L; Ballesteros, Antonio

    2002-06-13

    Enzymatic synthesis of fatty acid esters of di- and trisaccharides is limited by the fact that most biological catalysts are inactivated by the polar solvents (e.g. dimethylsulfoxide, dimethylformamide) where these carbohydrates are soluble. This article reviews the methodologies developed to overcome this limitation, namely those involving control over the reaction medium, the enzyme and the support. We have proposed the use of mixtures of miscible solvents (e.g. dimethylsulfoxide and 2-methyl-2-butanol) as a general strategy to acylate enzymatically hydrophilic substrates. We observed that decreasing the hydrophobicity of the medium (i.e. lowering the percentage of DMSO) the molar ratio sucrose diesters versus sucrose monoesters can be substantially enhanced. The different regioselectivity exhibited by several lipases and proteases makes feasible to synthesise different positional isomers, whose properties may vary considerably. In particular, the lipase from Thermomyces lanuginosus displays a notable selectivity for only one hydroxyl group in the acylation of sucrose, maltose, leucrose and maltotriose, compared with lipase from Candida antarctica. We have examined three immobilisation methods (adsorption on polypropylene, covalent coupling to Eupergit C, and silica-granulation) for sucrose acylation catalysed by T. lanuginosus lipase. The morphology of the support affected significantly the reaction rate and/or the selectivity of the process. PMID:12142143

  3. Increased fatty acid unsaturation and production of arachidonic acid by homologous over-expression of the mitochondrial malic enzyme in Mortierella alpina

    PubMed Central

    Hao, Guangfei; Du, Kai; Huang, Xiaoyun; Song, Yuanda; Gu, Zhennan; Wang, Lei; Zhang, Hao; Chen, Wei; Chen, Yong Q.

    2015-01-01

    Malic enzyme (ME) catalyses the oxidative decarboxylation of L-malate to pyruvate and provides NADPH for intracellular metabolism, such as fatty acid synthesis. Here, the mitochondrial ME (mME) gene from Mortierella alpina was homologously over-expressed. Compared with controls, fungal arachidonic acid (ARA; 20:4 n-6) content increased by 60 % without affecting the total fatty acid content. Our results suggest that enhancing mME activity may be an effective mean to increase industrial production of ARA in M. alpina. PMID:24863290

  4. Composition for nucleic acid sequencing

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2008-08-26

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  5. Aedes aegypti juvenile hormone acid methyl transferase, the ultimate enzyme in the biosynthetic pathway of juvenile hormone III, exhibits substrate control

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We report on the cloning, sequencing, characterization, 3D modeling and docking of Aedes aegypti juvenile hormone acid methyl transferase (AeaJHAMT), the enzyme that converts juvenile hormone acid (JHA) into juvenile hormone (JH). Purified recombinant AeaJHAMT was extensively characterized for enzym...

  6. Hydroxylation of ent-kaurenoic acid to steviol in Stevia rebaudiana Bertoni--purification and partial characterization of the enzyme.

    PubMed

    Kim, K K; Sawa, Y; Shibata, H

    1996-08-15

    The diterpenoic compound steviol (ent-kaur-16-en-13-ol-19-oic acid) is the aglycone of sweet glycosides accumulated in Stevia rebaudiana Bertoni. This compound is the hydroxylated form of ent-kaurenoic acid (ent-kaur-16-en-19-oic acid; ent-KA). The hydroxylation of ent-KA to form steviol requiring NADPH and molecular oxygen was detected in stroma prepared from S. rebaudiana Bertoni. The enzyme was purified from leaf extract to apparent homogeneity with a molecular mass of 39 kDa. Taken together with the value of 160 kDa estimated for native enzyme, this suggested that the hydroxylating enzyme is a homotetramer. The N-terminal sequence was determined through 20 residues. The pH optimum was 7.5-7.8. Apparent Km values were 11.1 microM for ent-KA and 20.6 microM for NADPH. Its visible absorption spectrum suggested that the enzyme was flavoprotein. The stoichiometric relationship between the formation of steviol and the utilization of ent-KA and cofactors confirmed the equation ent-KA + NADPH + H(+) + O2-->steviol + NADPH(+) + H2O. PMID:8806729

  7. Tetracycline removal and effect on the formation and degradation of extracellular polymeric substances and volatile fatty acids in the process of hydrogen fermentation.

    PubMed

    Hou, Guangying; Hao, Xiaoyan; Zhang, Rui; Wang, Jing; Liu, Rutao; Liu, Chunguang

    2016-07-01

    Many research indicate antibiotics show adverse effect on methane fermentation, while few research focus on their effect on hydrogen fermentation. The present study aimed to gain insight of the effect of antibiotics on hydrogen fermentation with waste sludge and corn straw as substrate. For this purpose, tetracycline, as a model, was investigated with regard to tetracycline removal, hydrogen production, interaction with extracellular polymeric substances (EPSs) of substrate and volatile fatty acids (VFAs) on concentration and composition. Results show that tetracycline could be removed efficiently by hydrogen fermentation, and relative low-dose tetracycline (200mg/l) exposure affects little on hydrogen production. While tetracycline exposure could change hydrogen fermentation from butyric acid-type to propionic acid-type depending on tetracycline level. Based upon three-dimensional excitation-emission matrix fluorescence spectroscopy and UV-vis tetracycline changed the component and content of EPSs, and static quenching was the main mechanism between EPSs with tetracycline. PMID:27070285

  8. Antioxidative Peptides Derived from Enzyme Hydrolysis of Bone Collagen after Microwave Assisted Acid Pre-Treatment and Nitrogen Protection

    PubMed Central

    Lin, Yun-Jian; Le, Guo-Wei; Wang, Jie-Yun; Li, Ya-Xin; Shi, Yong-Hui; Sun, Jin

    2010-01-01

    This study focused on the preparation method of antioxidant peptides by enzymatic hydrolysis of bone collagen after microwave assisted acid pre-treatment and nitrogen protection. Phosphoric acid showed the highest ability of hydrolysis among the four other acids tested (hydrochloric acid, sulfuric acid and/or citric acid). The highest degree of hydrolysis (DH) was 9.5% using 4 mol/L phosphoric acid with a ratio of 1:6 under a microwave intensity of 510 W for 240 s. Neutral proteinase gave higher DH among the four protease tested (Acid protease, neutral protease, Alcalase and papain), with an optimum condition of: (1) ratio of enzyme and substrate, 4760 U/g; (2) concentration of substrate, 4%; (3) reaction temperature, 55 °C and (4) pH 7.0. At 4 h, DH increased significantly (P < 0.01) under nitrogen protection compared with normal microwave assisted acid pre-treatment hydrolysis conditions. The antioxidant ability of the hydrolysate increased and reached its maximum value at 3 h; however DH decreased dramatically after 3 h. Microwave assisted acid pre-treatment and nitrogen protection could be a quick preparatory method for hydrolyzing bone collagen. PMID:21151439

  9. Feasible electrochemical biosensor based on plasma polymerization-assisted composite of polyacrylic acid and hollow TiO2 spheres for sensitively detecting lysozyme.

    PubMed

    Zhang, Zhihong; Zhang, Shuai; He, Linghao; Peng, Donglai; Yan, Fufeng; Wang, Minghua; Zhao, Jihong; Zhang, Hongzhong; Fang, Shaoming

    2015-12-15

    A composite made of polyacrylic acid and hollow TiO2 spheres (TiO2@PPAA) was prepared by the plasma polymerization method and subsequently used as an electrode material for detecting lysozyme. The chemical structure, surface morphology, and electrochemical performance of the TiO2@PPAA composite were mainly affected by the plasma input power used during plasma polymerization. After optimizing plasma conditions, aptamer strands exhibited high adsorption affinity toward the surface of TiO2@PPAA composite via synergistic effects between TiO2 and PPAA. Electrochemical impedance spectroscopy results showed that the developed TiO2@PPAA aptasensor presents highly sensitive detection ability toward lysozyme; the limit of detection of the proposed aptasensor is 0.015 ng mL(-1) (1.04 pM) within the range of 0.05-100 ng mL(-1) in terms of 3σ value. The film further showed excellent selectivity toward lysozyme in the presence of interfering proteins, such as thrombin, bovine serum albumin, and immunoglobulin E. Thus, this aptasensing strategy might broaden the applications of plasma polymerized nanomaterials in the field of biomedical research and early clinical diagnosis. PMID:26164009

  10. Polymeric assembly of hyperbranched building blocks to establish tunable nanoplatforms for lysosome acidity-responsive gene/drug co-delivery.

    PubMed

    Jia, Hui-Zhen; Zhang, Wei; Wang, Xu-Li; Yang, Bin; Chen, Wei-Hai; Chen, Si; Chen, Gang; Zhao, Yi-Fang; Zhuo, Ren-Xi; Feng, Jun; Zhang, Xian-Zheng

    2015-07-01

    This study plans to develop a nanoparticle technology that can assemble different polymeric "building blocks" with various desired functionalities into one nanosystem in a pH-dependent manner. For this purpose, polymeric building blocks were specifically designed with hyperbranched architectures, and orthogonal pH-reversible phenylboronic acid-diols were taken as "joints" to integrate them together. To verify the idea, a corona-core dual-polymer nanoassembly was prepared as the vehicle for lysosomotropic gene/drug co-delivery. Phenylboronic acid modified hyperbranched oligoethylenimine (OEI-PBA) was arranged to cluster around the hydrophobic core composed of hyperbranched polyglycerol, just by mixing two polymers in an appropriate ratio at neutral conditions. Compared with the parent OEI-PBA, this nanoassembly demonstrated better capture of plasmid DNA, highly enhanced activity for cellular transport and gene transfection (up to 100 fold), the ability to further load hydrophobic drugs, lysosome acidity-targeting pH-dependent release of both carried cargoes, and improved cell-biocompatibility. To evaluate its potential for combinational gene/drug therapy, in vitro experiments using the therapeutic p53 gene and antitumor doxorubicin as models were carried out. This intracellular co-delivery led to apparently synergetic anti-cancer effects in cultured cancer cells. This dynamic paradigm shows interesting features including easy manipulation, reversible conjugation, lysosome-targeting pH-responsiveness, high co-delivery efficiency, and functional expandability by further accommodating other building blocks. PMID:26221940

  11. Study of the surface modification with oleic acid of nanosized HfO2 synthesized by the polymerized complex derived sol-gel method

    NASA Astrophysics Data System (ADS)

    Ramos-González, R.; García-Cerda, L. A.; Quevedo-López, M. A.

    2012-06-01

    The synthesis of nanosized hafnium oxide by the polymerized complex derived sol-gel method is reported. The structural and morphological characterization of the HfO2 was carried out by X-ray diffraction and scanning electron microscopy. The surface of hafnium oxide nanoparticles was modified by capping with oleic acid. The nanoparticle surface area was measured by the gas adsorption technique in order to determine the minimal amount of oleic acid needed to obtain a uniform coverage of the hafnium oxide. The existence of organic layer can be confirmed by Fourier transform spectroscopy, solid state nuclear magnetic resonance spectroscopy, thermal gravimetric analysis and transmission electron microscopy. The FTIR and solid state NMR results reveal that oleic acid is chemisorbed as a carboxylate onto the HfO2 nanoparticle surface and confirm the formation of a monomolecular layer of oleic acid surrounding the HfO2. The cover density of oleic acid on the HfO2 increases with the amount of oleic acid used to modify the nanoparticles and the surface properties of HfO2 nanoparticles modified with oleic acid change from hydrophilic to hydrophobic.

  12. Expression of the retinoic acid catabolic enzyme CYP26B1 in the human brain to maintain signaling homeostasis.

    PubMed

    Stoney, Patrick N; Fragoso, Yara D; Saeed, Reem Bu; Ashton, Anna; Goodman, Timothy; Simons, Claire; Gomaa, Mohamed S; Sementilli, Angelo; Sementilli, Leonardo; Ross, Alexander W; Morgan, Peter J; McCaffery, Peter J

    2016-07-01

    Retinoic acid (RA) is a potent regulator of gene transcription via its activation of a set of nuclear receptors controlling transcriptional activation. Precise maintenance of where and when RA is generated is essential and achieved by local expression of synthetic and catabolic enzymes. The catabolic enzymes Cyp26a1 and Cyp26b1 have been studied in detail in the embryo, where they limit gradients of RA that form patterns of gene expression, crucial for morphogenesis. This paracrine role of RA has been assumed to occur in most tissues and that the RA synthetic enzymes release RA at a site distant from the catabolic enzymes. In contrast to the embryonic CNS, relatively little is known about RA metabolism in the adult brain. This study investigated the distribution of Cyp26a1 and Cyp26b1 transcripts in the rat brain, identifying several novel regions of expression, including the cerebral cortex for both enzymes and striatum for Cyp26b1. In vivo use of a new and potent inhibitor of the Cyp26 enzymes, ser 2-7, demonstrated a function for endogenous Cyp26 in the brain and that hippocampal RA levels can be raised by ser 2-7, altering the effect of RA on differential patterning of cell proliferation in the hippocampal region of neurogenesis, the subgranular zone. The expression of CYP26A1 and CYP26B1 was also investigated in the adult human brain and colocalization of CYP26A1 and the RA synthetic enzyme RALDH2 indicated a different, autocrine role for RA in human hippocampal neurons. Studies with the SH-SY5Y human neuroblastoma cell line implied that the co-expression of RA synthetic and catabolic enzymes maintains retinoid homeostasis within neurons. This presents a novel view of RA in human neurons as part of an autocrine, intracellular signaling system. PMID:26374207

  13. Classifying Multifunctional Enzymes by Incorporating Three Different Models into Chou's General Pseudo Amino Acid Composition.

    PubMed

    Zou, Hong-Liang; Xiao, Xuan

    2016-08-01

    With the avalanche of the newly found protein sequences in the post-genomic epoch, there is an increasing trend for annotating a number of newly discovered enzyme sequences. Among the various proteins, enzyme was considered as the one of the largest kind of proteins. It takes part in most of the biochemical reactions and plays a key role in metabolic pathways. Multifunctional enzyme is enzyme that plays multiple physiological roles. Given a multifunctional enzyme sequence, how can we identify its class? Especially, how can we deal with the multi-classes problem since an enzyme may simultaneously belong to two or more functional classes? To address these problems, which are obviously very important both to basic research and drug development, a multi-label classifier was developed via three different prediction models with multi-label K-nearest algorithm. Experimental results obtained on a stringent benchmark dataset of enzymes by jackknife cross-validation test show that the predicting results were exciting, indicating that the current method could be an effective and promising high throughput method in the enzyme research. We hope it could play an important complementary role to the existing predictors in identifying the classes of enzymes. PMID:27113936

  14. Effect of sodium hypochlorite and peracetic acid on the surface roughness of acrylic resin polymerized by heated water for short and long cycles

    PubMed Central

    Sczepanski, Felipe; Sczepanski, Claudia Roberta Brunnquell; Berger, Sandrine Bittencourt; Consani, Rafael Leonardo Xediek; Gonini-Júnior, Alcides; Guiraldo, Ricardo Danil

    2014-01-01

    Objective: To evaluate the surface roughness of acrylic resin submitted to chemical disinfection via 1% sodium hypochlorite (NaClO) or 1% peracetic acid (C2H4O3). Materials and Methods: The disc-shaped resin specimens (30 mm diameter ×4 mm height) were polymerized by heated water using two cycles (short cycle: 1 h at 74°C and 30 min at 100°C; conventional long cycle: 9 h at 74°C). The release of substances by these specimens in water solution was also quantified. Specimens were fabricated, divided into four groups (n = 10) depending on the polymerization time and disinfectant. After polishing, the specimens were stored in distilled deionized water. Specimens were immersed in 1% NaClO or 1% C2H4O3 for 30 min, and then were immersed in distilled deionized water for 20 min. The release of C2H4O3 and NaClO was measured via visual colorimetric analysis. Roughness was measured before and after disinfection. Roughness data were subjected to two-way ANOVA and Tukey's test. Results: There was no interaction between polymerization time and disinfectant in influencing the average surface roughness (Ra, P = 0.957). Considering these factors independently, there were significant differences between short and conventional long cycles (P = 0.012), but no significant difference between the disinfectants hypochlorite and C2H4O3 (P = 0.366). Visual colorimetric analysis did not detect release of substances. Conclusion: It was concluded that there was the difference in surface roughness between short and conventional long cycles, and disinfection at acrylic resins polymerized by heated water using a short cycle modified the properties of roughness. PMID:25512737

  15. Biological denitrification of brine: the effect of compatible solutes on enzyme activities and fatty acid degradation.

    PubMed

    Cyplik, Paweł; Piotrowska-Cyplik, Agnieszka; Marecik, Roman; Czarny, Jakub; Drozdzyńska, Agnieszka; Chrzanowski, Łukasz

    2012-09-01

    The effect of the addition of compatible solutes (ectoine and trehalose) on the denitrification process of saline wastewater was studied. In saline wastewater, it was observed that the initial concentration of nitrates was 500 mg N l⁻¹. A fatty substance isolated from oiled bleaching earth (waste of vegetable oil refining process) was used as a source of carbon.The consortium, which was responsible for the denitrification process originated from the wastewater of the vegetable oil industry. The consortium of microorganisms was identified by the use of restriction fragment length polymorphism of 16S rRNA gene amplicons and sequencing techniques. It was noted that ectoine affects significantly the activity of lipase and nitrate reductase, and resulted in faster denitrification compared to saline wastewater with the addition of trehalose or control saline wastewater (without compatible solutes). It was observed that relative enzyme activities of lipase and nitrate reductase increased by 32 and 35%, respectively, in the presence of 1 mM ectoine. This resulted in an increase in specific nitrate reduction rate in the presence of 1 mM ectoine to 5.7 mg N g⁻¹ VSS h⁻¹, which was higher than in the absence of ectoine (3.2 mg N g⁻¹ VSS h⁻¹). The addition of trehalose did not have an effect on nitrate removals. Moreover, it was found that trehalose was used up completely by bacteria as a source of carbon in the denitrification process. The fatty acids were biodegraded by 74% in the presence of 1 mM ectoine. PMID:22286267

  16. Gallic acid modulates cerebral oxidative stress conditions and activities of enzyme-dependent signaling systems in streptozotocin-treated rats.

    PubMed

    Kade, I J; Rocha, J B T

    2013-04-01

    Redox imbalances and altered signaling processes in the brain are characteristic features of diabetic complications. Hence, the present study therefore sought to evaluate the effect of gallic acid (GA) on disturbed redox systems and activity of neurotransmission signaling dependent enzymes such as sodium pump, purinergic enzymes and acetylcholinesterase in diabetic animal models. We observed that GA markedly improves the antioxidant status of diabetic animals. Furthermore, the diminution of the activity of Na(+)/K(+)-ATPase and increased activities of acetylcholinesterase and the purinergic enzymes associated with diabetes progression were reversed to normalcy with the administration of GA in diabetic animals. Hence, we conclude that GA is a potential candidate in the management of neuronal dysfunction that often accompanied complications associated with diabetic hyperglycemia. PMID:23381106

  17. Application of enzymes, sodium tripolyphosphate and cation exchange resin for the release of extracellular polymeric substances from sewage sludge. Characterization of the extracted polysaccharides/glycoconjugates by a panel of lectins.

    PubMed

    Wawrzynczyk, J; Szewczyk, E; Norrlöw, O; Dey, E Szwajcer

    2007-06-30

    The study describes extraction of extracellular polymeric substances (EPS) from sewage sludge by applying enzymes and enzymes combined with sodium tripolyphosphate (STPP). Additionally, a systematic study of two non-enzymatic extraction agents is described. The assessment of the released products is made by colorimetrical methods and polysaccharides/glycoconjugates identification by the interaction with four immobilized lectins. Bio-sludge from Helsingborg (Sweden) and Damhusåen (Denmark) were used as two case studies for testing enzymatic extractability and thereby to make useful prediction of sludge bio-digestibility. From Helsingborg sludge the enzymes extracted about 40% more of EPS than from Damhusåen. The polysaccharides/glycoconjugates in both sludges maintained the same level, and showed substantial different interaction motifs with lectins panel. Damhusåen enzymatic extracted EPS had an enhanced amount of suspended material that was post-hydrolysed by the use of polygalacturonase and lysozyme resulting in pectin like polymers and petiptidoglycans. Petiptidoglycan is a marker from bacterial cell debris. STPP and cation exchange resin (CER) released different quantities of EPS. The CER released polysaccharides/glycoconjugates had higher molecular weight and stronger affinity towards Concanavalin A than the one released by the action of STPP. Independent of the extraction conditions, STPP released elevated amounts of polyvalent cations and humic substances in contrast to the very low amounts of released by CER. PMID:17543410

  18. Unusual Fatty Acids Produced by Microbial Expression of Enzymes from the Tung Tree

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Tung oil, produced by fruit of the tung tree (Aleurites fordii), is a valuable industrial oil used in formulations of inks, dyes, coatings, and resins. The fruit contains all of the genes and enzymes required for synthesis of the oil, and such enzymes could be used for conversion of low-cost veget...

  19. Flavor and other quality factors of enzyme-peeled oranges treated with citric acid

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Oranges can be satisfactorily processed for fresh slices using a process of enzyme infiltration under vacuum. Scored 'Valencia' and 'Hamlin' oranges were placed under 90 kPa vacuum in a 0 ppm (water-infused) or 1000 ppm enzyme solution (Ultrazym) at 30 °C for 2 min, followed by 30 min incubation in ...

  20. 2-Hexadecynoic Acid Inhibits Plasmodial FAS-II Enzymes and Arrest Erythrocytic and Liver Stage Plasmodium Infections

    PubMed Central

    Tasdemir, Deniz; Sanabria, David; Lauinger, Ina L.; Tarun, Alice; Herman, Rob; Perozzo, Remo; Zloh, Mire; Kappe, Stefan H.; Brun, Reto; Carballeira, Néstor M.

    2010-01-01

    Acetylenic fatty acids are known to display several biological activities, but their antimalarial activity has remained unexplored. In this study, we synthesized the 2-, 5-, 6-, and 9-hexadecynoic acids (HDAs) and evaluated their in vitro activity against erythrocytic (blood) stages of Plasmodium falciparum and liver stages of P. yoelii infections. Since the type II fatty acid biosynthesis pathway (PfFAS-II) has recently been shown to be indispensable for liver stage malaria parasites, the inhibitory potential of the HDAs against multiple P. falciparum FAS-II (PfFAS-II) elongation enzymes was also evaluated. The highest antiplasmodial activity against blood stages of P. falciparum was displayed by 5-HDA (IC50 value 6.6. μg/ml), whereas the 2-HDA was the only acid arresting the growth of liver stage P. yoelii infection, in both flow cytometric assay (IC50 value 2-HDA 15.3 μg/ml, control drug atovaquone 2.5 ng/ml) and immunofluorescense analysis (IC50 2-HDA 4.88 μg/ml, control drug atovaquone 0.37 ng/ml). 2-HDA showed the best inhibitory against the PfFAS-II enzymes PfFabI and PfFabZ with IC50 values of 0.38 and 0.58 μg/ml (IC50 control drugs 14 and 30 ng/ml) respectively. Enzyme kinetics and molecular modeling studies revealed valuable insights into the binding mechanism of 2-HDA on the target enzymes. All HDAs showed in vitro activity against Trypanosoma brucei rhodesiense (IC50 values 3.7–31.7 μg/ml), Trypanosoma cruzi (only 2-HDA, IC50 20.2 μg/ml), and Leishmania donovani (IC50 values 4.1–13.4 μg/ml) with generally low or no significant toxicity on mammalian cells. This is the first study to indicate therapeutic potential of HDAs against various parasitic protozoa. It also points out that the malarial liver stage growth inhibitory effect of the 2-HDA may be promoted via PfFAS-II enzymes. The lack of cytotoxicity, lipophilic nature and calculated pharmacokinetic properties suggest that 2-HDA could be a useful compound to study the interaction of fatty

  1. X-ray absorption studies of the purple acid phosphatase from red kidney beans (native enzyme, metal exchanged form)

    NASA Astrophysics Data System (ADS)

    Ahlers, F.; Zippel, F.; Klabunde, T.; Krebs, B.; Löcke, R.; Witzel, H.; Nolting, H.-F.

    1995-02-01

    Purple acid phosphatase from red kidney beans (KBP) catalyzes the hydrolysis of activated phosphoric acid monoesters and contains a heterodinuclear Fe(III)Zn(II) core in its active site. Iron K-edge X-ray absorption data have been obtained for the native enzyme and for a metal exchanged derivative, where Zn(II) was substituted by Fe(III). The environment of the native enzyme consists of 2.5 O/N at 1.91 Å, 3 O/N at 2.09 Å, and 1 Zn at 4.05 Å. For the metal exchanged form we obtained 2.5 O/N at 1.94 Å, 2.5 O/N at 2.09 Å, and 1 Fe at 3.79 Å.

  2. Production of Cellulose-Hydrogen from Corn Stalk based on Acid-enzyme Two-Stage Pretreatment by Mixed Culture

    NASA Astrophysics Data System (ADS)

    Xing, Y.; Fan, Y. T.; Hou, H. W.

    2010-03-01

    Production of cellulose-hydrogen from corn stalk based on acid-enzyme two-stage pretreatment by lesser panda manure was carried out in batch tests. The acid-enzyme two-stage pretreatment of corn stalk was found most effective, in which the yields of soluble saccharides (SS) were 470 mg/g-TS. The maximum cumulative H2 yield (165.8 ml H2/g-TS) and H2 production rate (12.8 ml H2/g-TS h-1) were obtained at pH 5.5, 36 °C by treating a substrate of 15 g/L. The hydrogen content in biogas was 57.0% and there was no significant methane gas observed.

  3. Hypouricemic effect of allopurinol are improved by Pallidifloside D based on the uric acid metabolism enzymes PRPS, HGPRT and PRPPAT.

    PubMed

    Li, Hong-Gang; Hou, Pi-Yong; Zhang, Xi; He, Yi; Zhang, Jun; Wang, Shu-Qing; Anderson, Samantha; Zhang, Yan-Wen; Wu, Xiao-Hui

    2016-09-01

    Allopurinol is a commonly used medication to treat hyperuricemia and its complications. Pallidifloside D, a saponin glycoside constituent from the total saponins of Smilax riparia, had been proved to enhanced hypouricemic effect of allopurinol based on uric acid metabolism enzyme XOD. In this study, we evaluated whether Pallidifloside D (5mg/kg) enhanced hypouricemic effect of allopurinol (5mg/kg) related to others uric acid metabolism enzymes such as PRPS, HGPRT and PRPPAT. We found that, compared with allopurinol alone, the combination of allopurinol and Pallidifloside D significantly up-regulated HGPRT mRNA expression and down-regulated the mRNA expression of PRPS and PRPPAT in PC12 cells (all P<0.01). These results strongly suggest that hypouricemic effect of allopurinol are improved by Pallidifloside D via numerous mechanisms and our data may have a potential value in clinical practice in the treatment of gout and other hyperuricemic conditions. PMID:27370097

  4. Characterization of mouse lysophosphatidic acid acyltransferase 3: an enzyme with dual functions in the testis1s⃞

    PubMed Central

    Yuki, Koichi; Shindou, Hideo; Hishikawa, Daisuke; Shimizu, Takao

    2009-01-01

    Glycerophospholipids are structural and functional components of cellular membranes as well as precursors of various lipid mediators. Using acyl-CoAs as donors, glycerophospholipids are formed by the de novo pathway (Kennedy pathway) and modified in the remodeling pathway (Lands' cycle). Various acyltransferases, including two lysophosphatidic acid acyltransferases (LPAATs), have been discovered from a 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family. Proteins of this family contain putative acyltransferase motifs, but their biochemical properties and physiological roles are not completely understood. Here, we demonstrated that mouse LPAAT3, previously known as mouse AGPAT3, possesses strong LPAAT activity and modest lysophosphatidylinositol acyltransferase activity with a clear preference for arachidonoyl-CoA as a donor. This enzyme is highly expressed in the testis, where CDP-diacylglycerol synthase 1 preferring 1-stearoyl-2-arachidonoyl-phosphatidic acid as a substrate is also highly expressed. Since 1-stearoyl-2-arachidonoyl species are the main components of phosphatidylinositol, mouse LPAAT3 may function in both the de novo and remodeling pathways and contribute to effective biogenesis of 1-stearoyl-2-arachidonoyl-phosphatidylinositol in the testis. Additionally, the expression of this enzyme in the testis increases significantly in an age-dependent manner, and β-estradiol may be an important regulator of this enzyme's induction. Our findings identify this acyltransferase as an alternative important enzyme to produce phosphatidylinositol in the testis. PMID:19114731

  5. Influence of carboxylic acid type on microstructure and magnetic properties of polymeric complex sol-gel driven NiFe2O4

    NASA Astrophysics Data System (ADS)

    Hessien, M. M.; Mostafa, Nasser Y.; Abd-Elkader, Omar H.

    2016-01-01

    Citric, oxalic and tartaric acids were used for synthesis of NiFe2O4 using polymeric complex precursor route. The dry precursor gels were calcined at various temperatures (400-1100 °C) for 2 h. All carboxylic acids produce iron-deficient NiFe2O4 with considerable amount of α-Fe2O3 at 400 °C. Increase in the annealing temperature caused reaction of α-Fe2O3 with iron-deficient ferrite phase. The amount of initially formed α-Fe2O3 is directly correlated with stability constant and inversely correlated with the decomposition temperature of Fe(III) carboxylate precursors. In case of tartaric acid precursor, single phase of the ferrite was obtained at 450 °C. However, in case of oxalic acid and citric acid precursors, single phase ferrite was obtained at 550 °C and 700 °C, respectively. The lattice parameters were increased with increasing annealing temperature and with decreasing the amount of α-Fe2O3. Maximum saturation magnetization (55 emu/g) was achieved using tartaric acid precursor annealed at 1100 °C.

  6. Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms.

    PubMed Central

    Bush, R S; Sauer, F D

    1976-01-01

    The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 1.2.7.1 and EC 1.2.7.2) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively. Images PLATE 1 PMID:962871

  7. Determining soil enzyme activities for the assessment of fungi and citric acid-assisted phytoextraction under cadmium and lead contamination.

    PubMed

    Mao, Liang; Tang, Dong; Feng, Haiwei; Gao, Yang; Zhou, Pei; Xu, Lurong; Wang, Lumei

    2015-12-01

    Microorganism or chelate-assisted phytoextraction is an effective remediation tool for heavy metal polluted soil, but investigations into its impact on soil microbial activity are rarely reported. Consequently, cadmium (Cd)- and lead (Pb)-resistant fungi and citric acid (CA) were introduced to enhance phytoextraction by Solanum nigrum L. under varied Cd and Pb pollution levels in a greenhouse pot experiment. We then determined accumulation of Cd and Pb in S. nigrum and the soil enzyme activities of dehydrogenase, phosphatase, urease, catalase, sucrase, and amylase. Detrended canonical correspondence analysis (DCCA) was applied to assess the interactions between remediation strategies and soil enzyme activities. Results indicated that the addition of fungi, CA, or their combination enhanced the root biomass of S. nigrum, especially at the high-pollution level. The combined treatment of CA and fungi enhanced accumulation of Cd about 22-47 % and of Pb about 13-105 % in S. nigrum compared with the phytoextraction alone. However, S. nigrum was not shown to be a hyperaccumulator for Pb. Most enzyme activities were enhanced after remediation. The DCCA ordination graph showed increasing enzyme activity improvement by remediation in the order of phosphatase, amylase, catalase, dehydrogenase, and urease. Responses of soil enzyme activities were similar for both the addition of fungi and that of CA. In summary, results suggest that fungi and CA-assisted phytoextraction is a promising approach to restoring heavy metal polluted soil. PMID:26286803

  8. Mechanism and inhibition of human UDP-GlcNAc 2-epimerase, the key enzyme in sialic acid biosynthesis

    PubMed Central

    Chen, Sheng-Chia; Huang, Chi-Hung; Lai, Shu-Jung; Yang, Chia Shin; Hsiao, Tzu-Hung; Lin, Ching-Heng; Fu, Pin-Kuei; Ko, Tzu-Ping; Chen, Yeh

    2016-01-01

    The bifunctional enzyme UDP-GlcNAc 2-epimerase/ManNAc kinase (GNE) plays a key role in sialic acid production. It is different from the non-hydrolyzing enzymes for bacterial cell wall biosynthesis, and it is feed-back inhibited by the downstream product CMP-Neu5Ac. Here the complex crystal structure of the N-terminal epimerase part of human GNE shows a tetramer in which UDP binds to the active site and CMP-Neu5Ac binds to the dimer-dimer interface. The enzyme is locked in a tightly closed conformation. By comparing the UDP-binding modes of the non-hydrolyzing and hydrolyzing UDP-GlcNAc epimerases, we propose a possible explanation for the mechanistic difference. While the epimerization reactions of both enzymes are similar, Arg113 and Ser302 of GNE are likely involved in product hydrolysis. On the other hand, the CMP-Neu5Ac binding mode clearly elucidates why mutations in Arg263 and Arg266 can cause sialuria. Moreover, full-length modelling suggests a channel for ManNAc trafficking within the bifunctional enzyme. PMID:26980148

  9. The synthesis of glutamic acid in the absence of enzymes: Implications for biogenesis

    NASA Technical Reports Server (NTRS)

    Morowitz, Harold; Peterson, Eta; Chang, Sherwood

    1995-01-01

    This paper reports on the non-enzymatic aqueous phase synthesis of amino acids from keto acids, ammonia and reducing agents. The facile synthesis of key metabolic intermediates, particularly in the glycolytic pathway, the citric acid cycle, and the first step of amino acid synthesis, lead to new ways of looking at the problem of biogenesis.

  10. Polymerized and functionalized triglycerides

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Plant oils are useful sustainable raw materials for the development of new chemical products. As part of our research emphasis in sustainability and green polymer chemistry, we have explored a new method for polymerizing epoxidized triglycerides with the use of fluorosulfonic acid. Depending on the ...

  11. Hepatic drug-oxidizing enzyme systems and urinary D-glucaric acid excretion in patients with congestive heart failure.

    PubMed Central

    Tokola, O; Pelkonen, O; Karki, N T; Luoma, P

    1975-01-01

    Drug-oxidizing enzyme systems in liver biopsy samples and the urinary excretion of D-glucaric acid were studied in two different groups of patients with cardiac insufficiency. 2. In one group of six patients, the activities of drug-metabolizing enzymes had decreased considerably as compared with the control values, but in four liver samples from patients treated with oral hypoglycaemic agents for their diabetes, activities were higher than in control samples from ten patients. 3. In the other group of seven patients, the urinary excretion of D-glucaric acid (isolated by ion-exchange chromatography) was 60% lower than in the control group of nine humans, whereas in four patients taking antiepileptic agents excretion rate was higher than control values. 4. Because the age distribution was markedly different between cardiac insufficiency and control groups, it is difficult to conclude, if the impairment of drug metabolism was a consequence of the old age or of the disease process. However, drug-oxidizing enzyme systems seem to be inducible also in old age. 5. The results support further the opinion that the urinary excretion of D-glucaric acid may be one useful index in assessing an individual's capacity to metabolize foreign compounds especially in the patients with lowered drug metabolizing capacity. PMID:786355

  12. The Benzyl Ester Group of Amino Acid Monomers Enhances Substrate Affinity and Broadens the Substrate Specificity of the Enzyme Catalyst in Chemoenzymatic Copolymerization.

    PubMed

    Ageitos, Jose Manuel; Yazawa, Kenjiro; Tateishi, Ayaka; Tsuchiya, Kousuke; Numata, Keiji

    2016-01-11

    The chemoenzymatic polymerization of amino acid monomers by proteases involves a two-step reaction: the formation of a covalent acyl-intermediate complex between the protease and the carboxyl ester group of the monomer and the subsequent deacylation of the complex by aminolysis to form a peptide bond. Although the initiation with the ester group of the monomer is an important step, the influence of the ester group on the polymerization has not been studied in detail. Herein, we studied the effect of the ester groups (methyl, ethyl, benzyl, and tert-butyl esters) of alanine and glycine on the synthesis of peptides using papain as the catalyst. Alanine and glycine were selected as monomers because of their substantially different affinities toward papain. The efficiency of the polymerization of alanine and glycine benzyl esters was much greater than that of the other esters. The benzyl ester group therefore allowed papain to equally polymerize alanine and glycine, even though the affinity of alanine toward papain is substantially higher. The characterization of the copolymers of alanine and glycine in terms of the secondary structure and thermal properties revealed that the thermal stability of the peptides depends on the amino acid composition and resultant secondary structure. The current results indicate that the nature of the ester group drastically affects the polymerization efficiency and broadens the substrate specificity of the protease. PMID:26620763

  13. Polymeric micelles from poly(ethylene glycol)–poly(amino acid) block copolymer for drug and gene delivery

    PubMed Central

    Osada, Kensuke; Christie, R. James; Kataoka, Kazunori

    2009-01-01

    Dramatic advances in biological research have revealed the mechanisms underlying many diseases at the molecular level. However, conventional techniques may be inadequate for direct application of this new knowledge to medical treatments. Nanobiotechnology, which integrates biology with the rapidly growing field of nanotechnology, has great potential to overcome many technical problems and lead to the development of effective therapies. The use of nanobiotechnology in drug delivery systems (DDS) is attractive for advanced treatment of conditions such as cancer and genetic diseases. In this review paper for a special issue on biomaterial research in Japan, we discuss the development of DDS based on polymeric micelles mainly in our group for anti-cancer drug and gene delivery, and also address our challenges associated with developing polymeric micelles as super-functionalized nanodevices with intelligent performance. PMID:19364722

  14. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    SciTech Connect

    Gray, Kevin A; Zhao, Lishan; Cayouette, Michelle H

    2015-11-04

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  15. Celluloytic enzymes, nucleic acids encoding them and methods for making and using them

    SciTech Connect

    Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

    2015-09-08

    The invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  16. Regulation of the Tyrosine Biosynthetic Enzymes in Salmonella typhimurium: Analysis of the Involvement of Tyrosyl-Transfer Ribonucleic Acid and Tyrosyl-Transfer Ribonucleic Acid Synthetase1

    PubMed Central

    Heinonen, J.; Artz, S. W.; Zalkin, H.

    1972-01-01

    Mutants of Salmonella typhimurium were isolated that require tyrosine for growth because of an altered tyrosyl-transfer ribonucleic acid (tRNA) synthetase. Extracts of one strain (JK10) contain a labile enzyme with decreased ability to transfer tyrosine to tRNATyr and a higher Km for tyrosine than the wild-type enzyme. Strain JK10 maintains repressed levels of the tyrosine biosynthetic enzymes when the growth rate is restricted due to limitation of charged tRNATyr. Several second-site revertants of strain JK10 exhibit temperature-sensitive growth due to partially repaired, heat-labile tyrosyl-tRNA synthetase. The tyrosine biosynthetic enzymes are not derepressed in thermosensitive strains grown at the restrictive temperature. A class of tyrosine regulatory mutants, designated tyrR, contains normal levels of tyrosyl-tRNA synthetase and tRNATyr. These results suggest that charging of tRNATyr is not necessary for repression. This conclusion is substantiated by the finding that 4-aminophenylalanine, a tyrosine analogue which causes repression of the tyrosine biosynthetic enzymes, is not attached to tRNATyr in vivo, nor does it inhibit the attachment reaction in vitro. A combined regulatory effect due to the simultaneous presence of tyrS and tyrR mutations in the same strain was detected. The possibility of direct participation of tyrosyl-tRNA synthetase in tyrosine regulation is discussed. PMID:4404819

  17. Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products1[OPEN

    PubMed Central

    Yuen, Macaire M.S.; Bohlmann, Jörg

    2016-01-01

    Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I–IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes. PMID:26936895

  18. Modularity of Conifer Diterpene Resin Acid Biosynthesis: P450 Enzymes of Different CYP720B Clades Use Alternative Substrates and Converge on the Same Products.

    PubMed

    Geisler, Katrin; Jensen, Niels Berg; Yuen, Macaire M S; Madilao, Lina; Bohlmann, Jörg

    2016-05-01

    Cytochrome P450 enzymes of the CYP720B subfamily play a central role in the biosynthesis of diterpene resin acids (DRAs), which are a major component of the conifer oleoresin defense system. CYP720Bs exist in families of up to a dozen different members in conifer genomes and fall into four different clades (I-IV). Only two CYP720B members, loblolly pine (Pinus taeda) PtCYP720B1 and Sitka spruce (Picea sitchensis) PsCYP720B4, have been characterized previously. Both are multisubstrate and multifunctional clade III enzymes, which catalyze consecutive three-step oxidations in the conversion of diterpene olefins to DRAs. These reactions resemble the sequential diterpene oxidations affording ent-kaurenoic acid from ent-kaurene in gibberellin biosynthesis. Here, we functionally characterized the CYP720B clade I enzymes CYP720B2 and CYP720B12 in three different conifer species, Sitka spruce, lodgepole pine (Pinus contorta), and jack pine (Pinus banksiana), and compared their activities with those of the clade III enzymes CYP720B1 and CYP720B4 of the same species. Unlike the clade III enzymes, clade I enzymes were ultimately found not to be active with diterpene olefins but converted the recently discovered, unstable diterpene synthase product 13-hydroxy-8(14)-abietene. Through alternative routes, CYP720B enzymes of both clades produce some of the same profiles of conifer oleoresin DRAs (abietic acid, neoabietic acid, levopimaric acid, and palustric acid), while clade III enzymes also function in the formation of pimaric acid, isopimaric acid, and sandaracopimaric acid. These results highlight the modularity of the specialized (i.e. secondary) diterpene metabolism, which produces conifer defense metabolites through variable combinations of different diterpene synthase and CYP720B enzymes. PMID:26936895

  19. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue

    PubMed Central

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio

    2015-01-01

    Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males’ subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole. PMID:26587356

  20. Effects of sex and site on amino acid metabolism enzyme gene expression and activity in rat white adipose tissue.

    PubMed

    Arriarán, Sofía; Agnelli, Silvia; Remesar, Xavier; Fernández-López, José Antonio; Alemany, Marià

    2015-01-01

    Background and Objectives. White adipose tissue (WAT) shows marked sex- and diet-dependent differences. However, our metabolic knowledge of WAT, especially on amino acid metabolism, is considerably limited. In the present study, we compared the influence of sex on the amino acid metabolism profile of the four main WAT sites, focused on the paths related to ammonium handling and the urea cycle, as a way to estimate the extent of WAT implication on body amino-nitrogen metabolism. Experimental Design. Adult female and male rats were maintained, undisturbed, under standard conditions for one month. After killing them under isoflurane anesthesia. WAT sites were dissected and weighed. Subcutaneous, perigonadal, retroperitoneal and mesenteric WAT were analyzed for amino acid metabolism gene expression and enzyme activities. Results. There was a considerable stability of the urea cycle activities and expressions, irrespective of sex, and with only limited influence of site. Urea cycle was more resilient to change than other site-specialized metabolic pathways. The control of WAT urea cycle was probably related to the provision of arginine/citrulline, as deduced from the enzyme activity profiles. These data support a generalized role of WAT in overall amino-N handling. In contrast, sex markedly affected WAT ammonium-centered amino acid metabolism in a site-related way, with relatively higher emphasis in males' subcutaneous WAT. Conclusions. We found that WAT has an active amino acid metabolism. Its gene expressions were lower than those of glucose-lipid interactions, but the differences were quantitatively less important than usually reported. The effects of sex on urea cycle enzymes expression and activity were limited, in contrast with the wider variations observed in other metabolic pathways. The results agree with a centralized control of urea cycle operation affecting the adipose organ as a whole. PMID:26587356

  1. Polymerization catalyst

    SciTech Connect

    Graves, V.

    1987-05-12

    A process is described for polymerizing at least one alpha olefin under conditions characteristic of Ziegler polymerization wherein the polymerization is conducted in the presence of a catalyst system which comprises: a supported catalyst prepared under anhydrous conditions by the sequential steps of: preparing a slurry of inert particulate support material; adding to the slurry a solution of an organomagnesium compound; adding to the slurry and reacting a solution of a zirconium halide compound, hafnium compound or mixtures thereof; adding to the slurry and reacting a halogenator; adding to the slurry and reacting a tetravalent titanium halide compound; and recovering solid catalyst.

  2. Polymerization catalyst

    SciTech Connect

    Graves, V.

    1986-10-21

    A process is described for polymerizing at least one alpha-olefin under conditions characteristic of Ziegler polymerization wherein the polymerization is conducted in the presence of a catalyst comprising: a supported catalyst prepared under anhydrous conditions by the steps of: (1) sequentially; (a) preparing a slurry of inert particulate support material; (b) adding to the slurry a solution of an organomagnesium compound; (c) adding to the slurry and reacting a solution of zirconium compound; and (2) thereafter; (d) adding to the slurry and reacting a halogenator; (e) adding to the slurry and reacting a tetravalent titanium compound; (f) recovering solid catalyst; and an organoaluminum compound.

  3. Analysis of the Changes in Expression Levels of Sialic Acid on Influenza-Virus-Infected Cells Using Lectin-Tagged Polymeric Nanoparticles

    PubMed Central

    Cho, Jaebum; Miyake, Yukari; Honda, Ayae; Kushiro, Keiichiro; Takai, Madoka

    2016-01-01

    Viral infections affect millions around the world, sometimes leading to severe consequences or even epidemics. Understanding the molecular dynamics during viral infections would provide crucial information for preventing or stopping the progress of infections. However, the current methods often involve the disruption of the infected cells or expensive and time-consuming procedures. In this study, fluorescent polymeric nanoparticles were fabricated and used as bioimaging nanoprobes that can monitor the progression of influenza viral infection through the changes in the expression levels of sialic acids expressed on the cell membrane. The nanoparticles were composed of a biocompatible monomer to prevent non-specific interactions, a hydrophobic monomer to form the core, a fluorescent monomer, and a protein-binding monomer to conjugate lectin, which binds sialic acids. It was shown that these lectin-tagged nanoparticles that specifically target sialic acids could track the changes in the expression levels of sialic acids caused by influenza viral infections in human lung epithelial cells. There was a sudden drop in the levels of sialic acid at the initial onset of virus infection (t = 0~1 h) and at approximately 4~5 h post-infection. The latter drop correlated with the production of viral proteins that was confirmed using traditional techniques. Thus, the accuracy, the rapidity and the efficacy of the nanoprobes were demonstrated. Such molecular bioimaging tools, which allow easy-handling and in situ monitoring, would be useful to directly observe and decipher the viral infection mechanisms. PMID:27493646

  4. Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

    PubMed Central

    Houten, Sander M.; Denis, Simone; Argmann, Carmen A.; Jia, Yuzhi; Ferdinandusse, Sacha; Reddy, Janardan K.; Wanders, Ronald J. A.

    2012-01-01

    L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid β-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadh's expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting. PMID:22534643

  5. Heating of vegetable oils influences the activity of enzymes participating in arachidonic acid formation in Wistar rats.

    PubMed

    Stawarska, Agnieszka; Białek, Agnieszka; Tokarz, Andrzej

    2015-10-01

    Dietary intake of lipids and their fatty acids profile influence many aspects of health. Thermal processing changes the properties of edible oils and can also modify their metabolism, for example, eicosanoids formation. The aim of our study was to verify whether the activity of desaturases can be modified by lipids intake, especially by the fatty acids content. The experimental diets contained rapeseed oil, sunflower oil, and olive oil, both unheated and heated (for 10 minutes at 200 °C each time before administration), and influenced the fatty acids composition in serum and the activity of enzymes participating in arachidonic acid (AA) formation. The activity of desaturases was determined by measuring the amounts of AA formed in vitro derived from linoleic acid as determined in liver microsomes of Wistar rats. In addition, the indices of ∆(6)-desaturase (D6D) and ∆(5)-desaturase (D5D) have been determined. To realize this aim, the method of high-performance liquid chromatography has been used with ultraviolet-visible spectrophotometry detection. Diet supplementation with the oils rich in polyunsaturated fatty acids affects the fatty acids profile in blood serum and the activity of D6D and ∆(5)-desaturase in rat liver microsomes, the above activities being dependent on the kind of oil applied. Diet supplementation with heated oils has been found to increase the amount of AA produced in hepatic microsomes; and in the case of rapeseed oil and sunflower oil, it has also increased D6D activity. PMID:26094213

  6. Characterization of a C-5,13-Cleaving Enzyme of 13(S)-Hydroperoxide of Linolenic Acid by Soybean Seed.

    PubMed Central

    Salch, Y. P.; Grove, M. J.; Takamura, H.; Gardner, H. W.

    1995-01-01

    An activity was found in mature soybean seeds (Glycine max L. cv Century) that cleaved 13(S)-hydroperoxy-9(Z),11(E),15(Z)-octadecatrienoic acid (13S-HPOT) into 13-oxo-9(Z),11(E)-tridecadienoic acid and two isomeric pentenols, 2(Z)-penten-1-ol and 1-penten-3-ol. Isomeric pentene dimers were also produced and were presumably derived from the combination of two pentene radicals. 13(S)-Hydroperoxy-9(Z),11(E)-octadecadienoic acid (13S-HPOD) was, by contrast, a poor substrate. Activity with 13S-HPOT increased 24-fold under anaerobic conditions reminiscent of a similar anaerobic promoted reaction of 13S-HPOD catalyzed by lipoxygenase (LOX) in the presence of linoleic acid. However, prior to ion-exchange chromatography, cleavage activity did not require linoleic acid. After separation by gel filtration followed by ion-exchange chromatography, cleavage activity was lost but reappeared in the presence of either linoleic acid or dithiothreitol. Under these conditions cleavage activity was coincident with the activity of types 1 and 2 LOX. LOX inhibitors suppressed the cleavage reaction in a manner similar to inhibition of LOX activity. Heat-generated alkoxyl radicals derived from either 13S-HPOT or 13S-HPOD afforded similar products and yields of 13-oxo-9(Z),11(E)-tridecadienoic acid compared to the enzymic reaction. The product 1-penten-3-ol may be the precursor of the "raw-bean" volatile ethylvinylketone. PMID:12228538

  7. Molecular mapping of genes encoding microsomal omega-6 desaturase enzymes and their cosegregation with QTL affecting oleic acid content in soybean

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The microsomal omega-6 desaturase enzymes, which catalyze the desaturation of oleic acid to linoleic acid during fatty acid biosynthesis, are encoded by the FAD2-1 and FAD2-2 genes in soybeans. Breeders aim to incorporate the high oleate trait into soybean germplasm in order to improve the nutrition...

  8. In vitro and in silico studies of the inhibitory effects of some novel kojic acid derivatives on tyrosinase enzyme

    PubMed Central

    Asadzadeh, Azizeh; Sirous, Hajar; Pourfarzam, Morteza; Yaghmaei, Parichehreh; Afshin, Fassihi

    2016-01-01

    Objective(s): Tyrosinase is a key enzyme in pigment synthesis. Overproduction of melanin in parts of the skin results in hyperpigmentation diseases. This enzyme is also responsible for the enzymatic browning in fruits and vegetables. Thus, its inhibitors are of great importance in the medical, cosmetic and agricultural fields. Materials and Methods: A series of twelve kojic acid derivatives were designed to be evaluated as tyrosinase activity inhibitors. The potential inhibitory activity of these compounds was investigated in silico using molecular docking simulation method. Four compounds with a range of predicted tyrosinase inhibitory activities were prepared and their inhibitory effect on tyrosinase activity was evaluated. The antioxidant properties of these compounds were also investigated by in vitro DPPH (2,2-diphenyl-1-picrylhydrazyl) and hydrogen peroxide scavenging assays. Results: Compound IIId exhibited the highest tyrosinase inhibitory activity with an IC50 value of 0.216 ± 0.009 mM which was in accordance with the in silico ΔGbind results (-13.24 Kcal/mol). Conclusion: Based on the docking studies, from the twelve compounds studied, one (IIId) appeared to have the highest inhibition on tyrosinase activity. This was confirmed by enzyme activity measurements. Compound IIId has an NO2 group which binds to both of Cu2+ ions located inside the active site of the enzyme. This compound appeared to be even stronger than kojic acid in inhibiting tyrosinase activity. The DPPH free radical scavenging ability of all the studied compounds was more than that of BHT. However, they were not as strong as BHT or gallic acid in scavenging hydrogen peroxide. PMID:27081457

  9. Clinical and microbiological effects of subgingival antimicrobial irrigation with citric acid as evaluated by an enzyme immunoassay and culture analysis.

    PubMed

    Renvert, S; Dahlén, G; Snyder, B

    1997-04-01

    The purpose of the present study was to compare an enzyme immunoassay with culture samples from untreated and non-surgically treated periodontal pockets and to assess the clinical and microbiological effects of citric acid irrigation as a supplement to scaling and root planing. The enzyme immunoassay used in this study is a chairside diagnostic tool aimed at identifying the presence of P. gingivalis, P. intermedia, and A. actinomycetemcomitans. Six sites with pocket depths > or = 6 mm in each of 16 patients were monitored for 24 weeks using clinical and microbiological parameters. In two out of the six sites, scaling and root planing was supplemented with subgingival citric acid irrigation of the pocket after completion of the mechanical treatment. The sensitivity of the immunoassay in relation to culture was calculated to 85.5% and the specificity to 90.2%. The immunoassay corresponded to a detection level of 10(4) as estimated by culture. Sites treated with a combination of scaling and irrigation with citric acid demonstrated a similar healing pattern as sites treated with scaling and root planing alone. The profile of the marker bacteria was almost parallel for the two groups. The results of this investigation thus indicated that the immunoassay can be used as a screening tool for selected periodontal pathogens and that adjunctive irrigation with citric acid has no measurable clinical or microbiological effects. PMID:9150039

  10. 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

  11. 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

  12. Lipase-catalyzed process in an anhydrous medium with enzyme reutilization to produce biodiesel with low acid value.

    PubMed

    Azócar, Laura; Ciudad, Gustavo; Heipieper, Hermann J; Muñoz, Robinson; Navia, Rodrigo

    2011-12-01

    One major problem in the lipase-catalyzed production of biodiesel or fatty acid methyl esters (FAME) is the high acidity of the product, mainly caused by water presence, which produces parallel hydrolysis and esterification reactions instead of transesterification to FAME. Therefore, the use of reaction medium in absence of water (anhydrous medium) was investigated in a lipase-catalyzed process to improve FAME yield and final product quality. FAME production catalyzed by Novozym 435 was carried out using waste frying oil (WFO) as raw material, methanol as acyl acceptor, and 3Å molecular sieves to extract the water. The anhydrous conditions allowed the esterification of free fatty acids (FFA) from feedstock at the initial reaction time. However, after the initial esterification process, water absence avoided the consecutives reactions of hydrolysis and esterification, producing FAME mainly by transesterification. Using this anhydrous medium, a decreasing in both the acid value and the diglycerides content in the product were observed, simultaneously improving FAME yield. Enzyme reuse in the anhydrous medium was also studied. The use of the moderate polar solvent tert-butanol as a co-solvent led to a stable catalysis using Novozym 435 even after 17 successive cycles of FAME production under anhydrous conditions. These results indicate that a lipase-catalyzed process in an anhydrous medium coupled with enzyme reuse would be suitable for biodiesel production, promoting the use of oils of different origin as raw materials. PMID:21889401

  13. The preparation of size-controlled functionalized polymeric nanoparticles in micelles

    NASA Astrophysics Data System (ADS)

    Vakurov, Alexander; Pchelintsev, Nikolay A.; Forde, Jessica; Ó'Fágáin, Ciaran; Gibson, Tim; Millner, Paul

    2009-07-01

    The reverse micellar system of dioctyl-sulfosuccinate (AOT)/octane and toluene have been used as a template for polymerization of acrylamide (AA)/bisacrylamide (BAA)-based functionalized polymeric nanoparticles. Such nanoparticles are typically sized between 20 and 90 nm. They can be synthesized with different functional groups according to the monomers added to the polymerization mixture. In our experiments the nanoparticles carried amino and carboxyl groups following incorporation of allylamine (AAm) or methacrylic acid (MAA) monomers, respectively. The available amine or carboxyl groups can then be used for immobilization of enzymes or other biomolecules. These enzymes, subtilisin, laccase and lipase, were immobilized onto polyAA/BAA/MAA nanoparticles covalently after activating the MAA carboxylic groups with Woodward's K reagent. Non-covalent immobilization via electrostatic interaction was also performed.

  14. Effect of high-intensity intermittent swimming training on fatty acid oxidation enzyme activity in rat skeletal muscle.

    PubMed

    Terada, Shin; Tabata, Izumi; Higuchi, Mitsuru

    2004-02-01

    We previously reported that high-intensity exercise training significantly increased citrate synthase (CS) activity, a marker of oxidative enzyme, in rat skeletal muscle to a level equaling that attained after low-intensity prolonged exercise training (Terada et al., J Appl Physiol 90: 2019-2024, 2001). Since mitochondrial oxidative enzymes and fatty acid oxidation (FAO) enzymes are often increased simultaneously, we assessed the effect of high-intensity intermittent swimming training on FAO enzyme activity in rat skeletal muscle. Male Sprague-Dawley rats (3 to 4 weeks old) were assigned to a 10-day period of high-intensity intermittent exercise training (HIT), low-intensity prolonged exercise training (LIT), or sedentary control conditions. In the HIT group, the rats repeated fourteen 20 s swimming sessions with a weight equivalent to 14-16% of their body weight. Between the exercise sessions, a 10 s pause was allowed. Rats in the LIT group swam 6 h/day in two 3 h sessions separated by 45 min of rest. CS activity in the triceps muscle of rats in the HIT and LIT groups was significantly higher than that in the control rats by 36 and 39%, respectively. Furthermore, 3-beta hydroxyacyl-CoA dehydrogenase (HAD) activity, an important enzyme in the FAO pathway in skeletal muscle, was higher in the two training groups than in the control rats (HIT: 100%, LIT: 88%). No significant difference in HAD activity was observed between the two training groups. In conclusion, the present investigation demonstrated that high-intensity intermittent swimming training elevated FAO enzyme activity in rat skeletal muscle to a level similar to that attained after 6 h of low-intensity prolonged swimming exercise training. PMID:15040848

  15. Schistosoma mansoni: possible involvement of protein kinase C in linoleic acid-induced proteolytic enzyme release from cercariae.

    PubMed

    Matsumura, K; Mitsui, Y; Sato, K; Sakamoto, M; Aoki, Y

    1991-04-01

    The possible involvement of protein kinase C and Ca2+ metabolism in the proteolytic enzyme release from schistosome cercariae was studied. Cercariae were placed in dechlorinated tap water containing 0.37 mM calcium in the small glass petri dish and exposed to the stimuli (linoleic acid, phorbol esters, and Ca2+ ionophore) with or without inhibitors of protein kinase C or Ca2+ metabolism. The proteolytic activity of incubation medium of cercariae thus treated was measured by the azocoll assay. The penetration response of cercariae induced by linoleic acid, a physiological stimulus, was mimicked by phorbol esters. When exposed to phorbol esters, 0.02 to 2 microM of 12-O-tetradecanoylphorbol-13-acetate (TPA) and 0.2 to 2 microM of phorbol-12,13-dibutyrate (PDBu), cercariae ceased the swimming movement, began a rhythmic thrusting of the anterior tip of the parasite, and released the proteolytic enzyme, but they did not shed the tails. Lowering Ca2+ in water by addition of 5 mM ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA), phorbol ester-induced release of enzyme was completely inhibited. Phorbol ester-induced release of enzyme was partially inhibited by 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7), an inhibitor of protein kinase C, at a concentration of 100 microM. H-7 alone, at a concentration of 100 microM, did not affect the swimming movement of cercariae. The cercariae were stimulated to release the enzyme by high concentrations (10 and 100 microM) of the Ca2+ ionophore, A23187, but enzyme was not released by low concentrations (0.5 and 1 microM) of this drug. Cercariae exposed to A23187 behaved differently from those exposed to phorbol esters. They ceased swimming, showed strong muscle contraction, and shed their tail. A23187 stimulated cercariae to release the enzyme in the water containing 5 mM EGTA. A23187-induced enzyme release was not inhibited by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), a calmodulin

  16. Increased Biomass Yield of Lactococcus lactis by Reduced Overconsumption of Amino Acids and Increased Catalytic Activities of Enzymes

    PubMed Central

    Adamberg, Kaarel; Seiman, Andrus; Vilu, Raivo

    2012-01-01

    Steady state cultivation and multidimensional data analysis (metabolic fluxes, absolute proteome, and transcriptome) are used to identify parameters that control the increase in biomass yield of Lactococcus lactis from 0.10 to 0.12 C-mol C-mol−1 with an increase in specific growth rate by 5 times from 0.1 to 0.5 h−1. Reorganization of amino acid consumption was expressed by the inactivation of the arginine deiminase pathway at a specific growth rate of 0.35 h−1 followed by reduced over-consumption of pyruvate directed amino acids (asparagine, serine, threonine, alanine and cysteine) until almost all consumed amino acids were used only for protein synthesis at maximal specific growth rate. This balanced growth was characterized by a high glycolytic flux carrying up to 87% of the carbon flow and only amino acids that relate to nucleotide synthesis (glutamine, serine and asparagine) were consumed in higher amounts than required for cellular protein synthesis. Changes in the proteome were minor (mainly increase in the translation apparatus). Instead, the apparent catalytic activities of enzymes and ribosomes increased by 3.5 times (0.1 vs 0.5 h−1). The apparent catalytic activities of glycolytic enzymes and ribosomal proteins were seen to follow this regulation pattern while those of enzymes involved in nucleotide metabolism increased more than the specific growth rate (over 5.5 times). Nucleotide synthesis formed the most abundant biomonomer synthetic pathway in the cells with an expenditure of 6% from the total ATP required for biosynthesis. Due to the increase in apparent catalytic activity, ribosome translation was more efficient at higher growth rates as evidenced by a decrease of protein to mRNA ratios. All these effects resulted in a 30% decrease of calculated ATP spilling (0.1 vs 0.5 h−1). Our results show that bioprocesses can be made more efficient (using a balanced metabolism) by varying the growth conditions. PMID:23133574

  17. Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme).

    PubMed

    Cook, R. M.; Lindsay, J. G.; Wilkins, M. B.; Nimmo, H. G.

    1995-12-01

    The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle. PMID:12228671

  18. Delta-aminolevulinic acid dehydratase enzyme activity in blood, brain, and liver of lead-dosed ducks

    USGS Publications Warehouse

    Dieter, M.P.; Finley, M.T.

    1979-01-01

    Mallard ducks were dosed with a single shotgun pellet (ca. 200 mg lead). After 1 month there was about 1 ppm lead in blood, 2.5 in liver, and 0.5 in brain. Lead-induced inhibition of delta-aminolevulinic acid dehydratase enzyme in blood and cerebellum was much greater than in cerebral hemisphere or liver and was strongly correlated with the lead concentration in these tissues. The cerebellar portion of the brain was more sensitive to delta-aminolevulinic acid dehydratase enzyme inhibition by lead than were the other tissues examined. There was also a greater increase in the glial cell marker enzyme, butyrylcholinesterase, in cerebellum than in cerebral hemisphere, suggesting that nonregenerating neuronal cells were destroyed by lead and replaced by glial cells in that portion of the brain. Even partial loss of cerebellar tissue is severely debilitating in waterfowl, because functions critical to survival such as visual, auditory, motor, and reflex responses are integrated at this brain center.

  19. RALDH2, the enzyme for retinoic acid synthesis, mediates meiosis initiation in germ cells of the female embryonic chickens.

    PubMed

    Yu, Minli; Yu, Ping; Leghari, Imdad H; Ge, Chutian; Mi, Yuling; Zhang, Caiqiao

    2013-02-01

    Meiosis is a process unique to the differentiation of germ cells and exhibits sex-specific in timing. Previous studies showed that retinoic acid (RA) as the vitamin A metabolite is crucial for controlling Stra8 (Stimulated by retinoic acid gene 8) expression in the gonad and to initiate meiosis; however, the mechanism by which retinoid-signaling acts has remained unclear. In the present study, we investigated the role of the enzyme retinaldehyde dehydrogenase 2 (RALDH2) which catalyzes RA synthesizes by initiating meiosis in chicken ovarian germ cells. Meiotic germ cells were first detected at day 15.5 in chicken embryo ovary when the expression of synaptonemal complex protein 3 (Scp3) and disrupted meiotic cDNA 1 homologue (Dmc1) became elevated, while Stra8 expression was specifically up-regulated at day 12.5 before meiosis onset. It was observed from the increase in Raldh2 mRNA expression levels and decreases in Cyp26b1 (the enzyme for RA catabolism) expression levels during meiosis that requirement for RA accumulation is essential to sustain meiosis. This was also revealed by RA stimulation of the cultured ovaries with the initiation of meiosis response, and the knocking down of the Raldh2 expression during meiosis, leading to abolishment of RA-dependent action. Altogether, these studies indicate that RA synthesis by the enzyme RALDH2 and signaling through its receptor is crucial for meiosis initiation in chicken embryonic ovary. PMID:22733143

  20. Decarboxylation of Malate in the Crassulacean Acid Metabolism Plant Bryophyllum (Kalanchoe) fedtschenkoi (Role of NAD-Malic Enzyme).

    PubMed Central

    Cook, R. M.; Lindsay, J. G.; Wilkins, M. B.; Nimmo, H. G.

    1995-01-01

    The role of NAD-malic enzyme (NAD-ME) in the Crassulacean acid metabolism plant Bryophyllum (Kalanchoe) fedtschenkoi was investigated using preparations of intact and solubilized mitochondria from fully expanded leaves. Intact, coupled mitochondria isolated during the day or night did not differ in their ability to take up [14C]malic acid from the surrounding medium or to respire using malate or succinate as substrate. However, intact mitochondria isolated from plants during the day decarboxylated added malate to pyruvate significantly faster than mitochondria isolated from plants at night. NAD-ME activity in solubilized mitochondrial extracts showed hysteretic kinetics and was stimulated by a number of activators, including acetyl-coenzyme A, fructose-1,6-bisphosphate, and sulfate ions. In the absence of these effectors, reaction progress curves were nonlinear, with a pronounced acceleration phase. The lag period before a steady-state rate was reached in assays of mitochondrial extracts decreased during the photoperiod and increased slowly during the period of darkness. However, these changes in the kinetic properties of the enzyme could not account for the changes in the rate of decarboxylation of malate by intact mitochondria. Gel-filtration experiments showed that mitochondrial extracts contained three forms of NAD-ME with different molecular weights. The relative proportions of the three forms varied somewhat throughout the light/dark cycle, but this did not account for the changes in the kinetics behavior of the enzyme during the diurnal cycle. PMID:12228671

  1. AS1411 aptamer and folic acid functionalized pH-responsive ATRP fabricated pPEGMA-PCL-pPEGMA polymeric nanoparticles for targeted drug delivery in cancer therapy.

    PubMed

    Lale, Shantanu V; R G, Aswathy; Aravind, Athulya; Kumar, D Sakthi; Koul, Veena

    2014-05-12

    Nonspecificity and cardiotoxicity are the primary limitations of current doxorubicin chemotherapy. To minimize side effects and to enhance bioavailability of doxorubicin to cancer cells, a dual-targeted pH-sensitive biocompatible polymeric nanosystem was designed and developed. An ATRP-based biodegradable triblock copolymer, poly(poly(ethylene glycol) methacrylate)-poly(caprolactone)-poly(poly(ethylene glycol) methacrylate) (pPEGMA-PCL-pPEGMA), conjugated with doxorubicin via an acid-labile hydrazone bond was synthesized and characterized. Dual targeting was achieved by attaching folic acid and the AS1411 aptamer through EDC-NHS coupling. Nanoparticles of the functionalized triblock copolymer were prepared using the nanoprecipitation method, resulting in an average particle size of ∼140 nm. The biocompatibility of the nanoparticles was evaluated using MTT cytotoxicity assays, blood compatibility studies, and protein adsorption studies. In vitro drug release studies showed a higher cumulative doxorubicin release at pH 5.0 (∼70%) compared to pH 7.4 (∼25%) owing to the presence of the acid-sensitive hydrazone linkage. Dual targeting with folate and the AS1411 aptamer increased the cancer-targeting efficiency of the nanoparticles, resulting in enhanced cellular uptake (10- and 100-fold increase in uptake compared to single-targeted NPs and non-targeted NPs, respectively) and a higher payload of doxorubicin in epithelial cancer cell lines (MCF-7 and PANC-1), with subsequent higher apoptosis, whereas a normal (noncancerous) cell line (L929) was spared from the adverse effects of doxorubicin. The results indicate that the dual-targeted pH-sensitive biocompatible polymeric nanosystem can act as a potential drug delivery vehicle against various epithelial cancers such as those of the breast, ovary, pancreas, lung, and others. PMID:24689987

  2. Lipid peroxidation and cyclooxygenase enzyme inhibitory activities of acidic aqueous extracts of some dietary supplements.

    PubMed

    Raman, Priyadarshini; Dewitt, David L; Nair, Muraleedharan G

    2008-02-01

    The botanical supplement market is growing at a fast pace with more and more people resorting to them for maintaining good health. Echinacea, garlic, ginkgo, ginseng, Siberian ginseng, grape seed extract, kava kava, saw palmetto and St John's wort are some of the popular supplements used for a variety of health benefits. These supplements are associated with various product claims, which suggest that they possess cyclooxygenase (COX) enzyme and lipid s inhibitory activities. COX enzymes are found to be at elevated levels in inflamed and cancerous cells. To test some of the product claims, selected supplements were analysed for their ability to inhibit COX-1 and -2 enzymes and lipid peroxidation in vitro. The supplements were extracted with acidified water (pH 2) at 37 degrees C to simulate the gastric environment. The supplements tested demonstrated varying degrees of COX enzyme inhibition (5-85% for COX-1 and 13-28% for COX-2). Interestingly, extracts of garlic (Meijer), ginkgo (Solaray), ginseng (Nature's Way), Siberian ginseng (GNC, Nutrilite, Solaray, Natrol), kava kava (GNC, Sundown, Solaray) and St John's wort (Nutrilite) selectively inhibited COX-2 enzyme. These supplements also inhibited lipid peroxidation in vitro (5-99%). The results indicated that the consumption of these botanical supplements studied possess health benefits. PMID:17726737

  3. Cloning and manipulation of the Escherichia coli cyclopropane fatty acid synthase gene: physiological aspects of enzyme overproduction.

    PubMed Central

    Grogan, D W; Cronan, J E

    1984-01-01

    Like many other eubacteria, cultures of Escherichia coli accumulate cyclopropane fatty acids (CFAs) at a well-defined stage of growth, due to the action of the cytoplasmic enzyme CFA synthase. We report the isolation of the putative structural gene, cfa, for this enzyme on an E. coli-ColE1 chimeric plasmid by the use of an autoradiographic colony screening technique. When introduced into a variety of E. coli strains, this plasmid, pLC18-11, induced corresponding increases in CFA content and CFA synthase activity. Subsequent manipulation of the cfa locus, facilitated by the insertion of pLC18-11 into a bacteriophage lambda vector, allowed genetic and physiological studies of CFA synthase in E. coli. Overproduction of this enzyme via multicopy cfa plasmids caused abnormally high levels of CFA in membrane phospholipid but no discernable growth perturbation. Infection with phage lambda derivatives bearing cfa caused transient overproduction of the enzyme, although pL-mediated expression of cfa could not be demonstrated in plasmids derived from such phages. CFA synthase specific activities could be raised to very high levels by using cfa runaway-replication plasmids. A variety of physiological factors were found to modulate the levels of CFA synthase in normal and gene-amplified cultures. These studies argue against several possible mechanisms for the temporal regulation of CFA formation. PMID:6325391

  4. [Combined effects of copper and simulated acid rain on copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa].

    PubMed

    He, Shan-Ying; Gao, Yong-Jie; Shentu, Jia-Li; Chen, Kun-Bai

    2011-02-01

    A pot experiment was conducted to study the combined effects of Cu (0-1500 mg x kg(-1)) and simulated acid rain (pH 2.5-5.6) on the copper accumulation, growth, and antioxidant enzyme activities of Rumex acetosa. With the increasing concentration of soil Cu, the Cu accumulation in R. acetosa increased, being higher in root than in stem and leaf. The exposure to low pH acid rain promoted the Cu uptake by R. acetosa. With the increase of soil Cu concentration and/or of acid rain acidity, the biomass of R. acetosa decreased, leaf and root MDA contents increased and had good correlation with soil Cu concentration, and the SOD and POD activities in leaf and root displayed a decreasing trend after an initial increase. This study showed that R. acetosa had a strong adaptive ability to Cu and acid rain stress, exhibiting a high application potential in the remediation of Cu-contaminated soil in acid rain areas. PMID:21608265

  5. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid

    SciTech Connect

    Messing, Simon A.J.; Gabelli, Sandra B.; Echeverria, Ignacia; Vogel, Jonathan T.; Guan, Jiahn Chou; Tan, Bao Cai; Klee, Harry J.; McCarty, Donald R.; Amzel, L. Mario

    2011-09-06

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  6. Structural Insights into Maize Viviparous14, a Key Enzyme in the Biosynthesis of the Phytohormone Abscisic Acid W

    SciTech Connect

    Messing, S.; Gabelli, S; Echeverria, I; Vogel, J; Guan, J; Tan, B; Klee, H; McCarty, D; Amzela, M

    2010-01-01

    The key regulatory step in the biosynthesis of abscisic acid (ABA), a hormone central to the regulation of several important processes in plants, is the oxidative cleavage of the 11,12 double bond of a 9-cis-epoxycarotenoid. The enzyme viviparous14 (VP14) performs this cleavage in maize (Zea mays), making it a target for the rational design of novel chemical agents and genetic modifications that improve plant behavior through the modulation of ABA levels. The structure of VP14, determined to 3.2-{angstrom} resolution, provides both insight into the determinants of regio- and stereospecificity of this enzyme and suggests a possible mechanism for oxidative cleavage. Furthermore, mutagenesis of the distantly related CCD1 of maize shows how the VP14 structure represents a template for all plant carotenoid cleavage dioxygenases (CCDs). In addition, the structure suggests how VP14 associates with the membrane as a way of gaining access to its membrane soluble substrate.

  7. Reshaping an enzyme binding pocket for enhanced and inverted stereoselectivity: use of smallest amino acid alphabets in directed evolution.

    PubMed

    Sun, Zhoutong; Lonsdale, Richard; Kong, Xu-Dong; Xu, Jian-He; Zhou, Jiahai; Reetz, Manfred T

    2015-10-12

    Directed evolution based on saturation mutagenesis at sites lining the binding pocket is a commonly practiced strategy for enhancing or inverting the stereoselectivity of enzymes for use in organic chemistry or biotechnology. However, as the number of residues in a randomization site increases to five or more, the screening effort for 95 % library coverage increases astronomically until it is no longer feasible. We propose the use of a single amino acid for saturation mutagenesis at superlarge randomization sites comprising 10 or more residues. When used to reshape the binding pocket of limonene epoxide hydrolase, this strategy, which drastically reduces the search space and thus the screening effort, resulted in R,R- and S,S-selective mutants for the hydrolytic desymmetrization of cyclohexene oxide and other epoxides. X-ray crystal structures and docking studies of the mutants unveiled the source of stereoselectivity and shed light on the mechanistic intricacies of this enzyme. PMID:25891639

  8. Polymethyl methacrylate-co-methacrylic acid coatings with controllable concentration of surface carboxyl groups: A novel approach in fabrication of polymeric platforms for potential bio-diagnostic devices

    NASA Astrophysics Data System (ADS)

    Hosseini, Samira; Ibrahim, Fatimah; Djordjevic, Ivan; Koole, Leo H.

    2014-05-01

    The generally accepted strategy in development of bio-diagnostic devices is to immobilize proteins on polymeric surfaces as a part of detection process for diseases and viruses through antibody/antigen coupling. In that perspective, polymer surface properties such as concentration of functional groups must be closely controlled in order to preserve the protein activity. In order to improve the surface characteristics of transparent polymethacrylate plastics that are used for diagnostic devices, we have developed an effective fabrication procedure of polymethylmetacrylate-co-metacrylic acid (PMMA-co-MAA) coatings with controlled number of surface carboxyl groups. The polymers were processed effectively with the spin-coating technique and the detailed control over surface properties is here by demonstrated through the variation of a single synthesis reaction parameter. The chemical structure of synthesized and processed co-polymers has been investigated with nuclear magnetic resonance spectroscopy (NMR) and matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-ToF-MS). The surface morphology of polymer coatings have been analyzed with atomic force microscopy (AFM) and scanning electron microscopy (SEM). We demonstrate that the surface morphology and the concentration of surface -COOH groups (determined with UV-vis surface titration) on the processed PMMA-co-MAA coatings can be precisely controlled by variation of initial molar ratio of reactants in the free-radical polymerization reaction. The wettability of developed polymer surfaces also varies with macromolecular structure.

  9. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-05-30

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  10. Method for sequencing nucleic acid molecules

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2006-06-06

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  11. Effects of nitrogen dioxide and its acid mist on reactive oxygen species production and antioxidant enzyme activity in Arabidopsis plants.

    PubMed

    Liu, Xiaofang; Hou, Fen; Li, Guangke; Sang, Nan

    2015-08-01

    Nitrogen dioxide (NO2) is one of the most common and harmful air pollutants. To analyze the response of plants to NO2 stress, we investigated the morphological change, reactive oxygen species (ROS) production and antioxidant enzyme activity in Arabidopsis thaliana (Col-0) exposed to 1.7, 4, 8.5, and 18.8 mg/m(3) NO2. The results indicate that NO2 exposure affected plant growth and chlorophyll (Chl) content, and increased oxygen free radical (O2(-)) production rate in Arabidopsis shoots. Furthermore, NO2 elevated the levels of lipid peroxidation and protein oxidation, accompanied by the induction of antioxidant enzyme activities and change of ascorbate (AsA) and glutathione (GSH) contents. Following this, we mimicked nitric acid mist under experimental conditions, and confirmed the antioxidant mechanism of the plant to the stress. Our results imply that NO2 and its acid mist caused pollution risk to plant systems. During the process, increased ROS acted as a signal to induce a defense response, and antioxidant status played an important role in plant protection against NO2/nitric acid mist-caused oxidative damage. PMID:26257351

  12. Characterization of two key enzymes for aromatic amino acid biosynthesis in symbiotic archaea.

    PubMed

    Shlaifer, Irina; Turnbull, Joanne L

    2016-07-01

    Biosynthesis of L-tyrosine (L-Tyr) and L-phenylalanine (L-Phe) is directed by the interplay of three enzymes. Chorismate mutase (CM) catalyzes the rearrangement of chorismate to prephenate, which can be either converted to hydroxyphenylpyruvate by prephenate dehydrogenase (PD) or to phenylpyruvate by prephenate dehydratase (PDT). This work reports the first characterization of a trifunctional PD-CM-PDT from the smallest hyperthermophilic archaeon Nanoarchaeum equitans and a bifunctional CM-PD from its host, the crenarchaeon Ignicoccus hospitalis. Hexa-histidine tagged proteins were expressed in Escherichia coli and purified by affinity chromatography. Specific activities determined for the trifunctional enzyme were 21, 80, and 30 U/mg for CM, PD, and PDT, respectively, and 47 and 21 U/mg for bifunctional CM and PD, respectively. Unlike most PDs, these two archaeal enzymes were insensitive to regulation by L-Tyr and preferred NADP(+) to NAD(+) as a cofactor. Both the enzymes were highly thermally stable and exhibited maximal activity at 90 °C. N. equitans PDT was feedback inhibited by L-Phe (Ki = 0.8 µM) in a non-competitive fashion consistent with L-Phe's combination at a site separate from that of prephenate. Our results suggest that PD from the unique symbiotic archaeal pair encompass a distinct subfamily of prephenate dehydrogenases with regard to their regulation and co-substrate specificity. PMID:27290727

  13. Cellulolytic enzymes, nucleic acids encoding them and methods for making and using them

    DOEpatents

    Gray, Kevin A.; Zhao, Lishan; Cayouette, Michelle H.

    2012-01-24

    The invention provides polypeptides having any cellulolytic activity, e.g., a cellulase activity, a endoglucanase, a cellobiohydrolase, a beta-glucosidase, a xylanase, a mannanse, a .beta.-xylosidase, an arabinofuranosidase, and/or an oligomerase activity, polynucleotides encoding these polypeptides, and methods of making and using these polynucleotides and polypeptides. In one aspect, the invention is directed to polypeptides having any cellulolytic activity, e.g., a cellulase activity, e.g., endoglucanase, cellobiohydrolase, beta-glucosidase, xylanase, mannanse, .beta.-xylosidase, arabinofuranosidase, and/or oligomerase activity, including thermostable and thermotolerant activity, and polynucleotides encoding these enzymes, and making and using these polynucleotides and polypeptides. In one aspect, the invention provides polypeptides having an oligomerase activity, e.g., enzymes that convert recalcitrant soluble oligomers to fermentable sugars in the saccharification of biomass. The polypeptides of the invention can be used in a variety of pharmaceutical, agricultural, food and feed processing and industrial contexts. The invention also provides compositions or products of manufacture comprising mixtures of enzymes comprising at least one enzyme of this invention.

  14. Fatty acid composition of muscle fat and enzymes of storage lipid synthesis in whole muscle from beef cattle.

    PubMed

    Kazala, E Chris; Lozeman, Fred J; Mir, Priya S; Aalhus, Jennifer L; Schmutz, Sheila M; Weselake, Randall J

    2006-11-01

    Enhanced intramuscular fat content (i.e., marbling) in beef is a desirable trait, which can result in increased product value. This study was undertaken with the aim of revealing biochemical factors associated with the marbling trait in beef cattle. Samples of longissimus lumborum (LL) and pars costalis diaphragmatis (PCD) were taken from a group of intact crossbred males and females at slaughter, lipids extracted, and the resulting FAME examined for relationships with marbling fat deposition. For LL, significant associations were found between degree of marbling and myristic (14:0, r = 0.55, P < 0.01), palmitic (16:0, r = 0.80, P < 0.001), stearic (18:0, r = -0.58, P < 0.01), and oleic (18:1c-9, r = 0.79, P < 0.001) acids. For PCD, significant relationships were found between marbling and palmitic (r = 0.71, P < 0.001) and oleic (r = 0.74, P < 0.001) acids. Microsomal fractions prepared from PCD muscle were assayed for diacylglycerol acyltransferase (DGAT), lysophosphatidic acid acyltransferase (LPAAT), and phosphatidic acid phosphatase-1 (PAP-1) activity, and the results examined for relationships with degree of intramuscular fat deposition. None of the enzyme activities from PCD displayed an association with marbling fat content, but DGAT specific activity showed significant positive associations with LPAAT (r = 0.54, P < 0.01), total PAP (r = 0.66, P < 0.001), and PAP-1 (r = 0.63, P < 0.01) specific activities. The results on FA compositions of whole muscle tissues provide insight into possible enzyme action associated with the production of specific FA. The increased proportion of oleic acid associated with enhanced lipid content of whole muscle is noteworthy given the known health benefits of this FA. PMID:17263304

  15. Protocatechuic acid induces antioxidant/detoxifying enzyme expression through JNK-mediated Nrf2 activation in murine macrophages.

    PubMed

    Varì, Rosaria; D'Archivio, Massimo; Filesi, Carmelina; Carotenuto, Simona; Scazzocchio, Beatrice; Santangelo, Carmela; Giovannini, Claudio; Masella, Roberta

    2011-05-01

    Protocatechuic acid (PCA) is a main metabolite of anthocyanins, whose daily intake is much higher than that of other polyphenols. PCA has biological effects, e.g., it induces the antioxidant/detoxifying enzyme gene expression. This study was aimed at defining the molecular mechanism responsible for PCA-induced over-expression of glutathione (GSH) peroxidase (GPx) and GSH reductase (GR) in J774 A.1 macrophages. New evidence is provided that PCA increases GPx and GR expression by inducing C-JUN NH(2)-terminal kinase (JNK)-mediated phosphorylation of Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2). RNA and proteins were extracted from cells treated with PCA (25 μM) for different time points. Quantitative real-time polymerase chain reaction and immunoblotting analyses showed a rapid increase in mRNA (>60%) and protein (>50%) for both the enzymes. This was preceded by the up-regulation of Nrf2, in terms of mRNA and protein, and by its significant activation as assessed by increased Nrf2 phosphorylation and nuclear translocation (+60%). By using specific kinase inhibitors and detecting the activated form, we showed that JNK was the main upstream kinase responsible for Nrf2 activation. Convincing evidence is provided of a causal link between PCA-induced Nrf2 activation and increased enzyme expression. By silencing Nrf2 and using a JNK inhibitor, enzyme enhancement was counteracted. Finally, with the ChIP assay, we demonstrated that PCA-activated Nrf2 specifically bound ARE sequences in enzyme gene promoters. Our study demonstrates for the first time that PCA improves the macrophage endogenous antioxidant potential by a mechanism in which JNK-mediated Nrf2 activation plays an essential role. This knowledge could contribute to novel diet-based approaches aimed at counteracting oxidative injury by reinforcing endogenous defences. PMID:20621462

  16. Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium.

    PubMed Central

    Epelbaum, S; Chipman, D M; Barak, Z

    1996-01-01

    The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured. The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter. The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition. This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism. The organization of the pathway thus potentiates the inhibitory effect of SMM. IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate. Valine completely protected strain TV105 from IpOHA at the MIC. A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested. IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration. The recovery is apparently due to

  17. Metabolic effects of inhibitors of two enzymes of the branched-chain amino acid pathway in Salmonella typhimurium.

    PubMed

    Epelbaum, S; Chipman, D M; Barak, Z

    1996-02-01

    The metabolic effects of inhibitors of two enzymes in the pathway for biosynthesis of branched-chain amino acids were examined in Salmonella typhimurium mutant strain TV105, expressing a single isozyme of acetohydroxy acid synthase (AHAS), AHAS isozyme II. One inhibitor was the sulfonylurea herbicide sulfometuron methyl (SMM), which inhibits this isozyme and AHAS of other organisms, and the other was N-isopropyl oxalylhydroxamate (IpOHA), which inhibits ketol-acid reductoisomerase (KARI). The effects of the inhibitors on growth, levels of several enzymes of the pathway, and levels of intermediates of the pathway were measured. The intracellular concentration of the AHAS substrate 2-ketobutyrate increased on addition of SMM, but a lack of correlation between increased ketobutyrate and growth inhibition suggests that the former is not the immediate cause of the latter. The levels of the keto acid precursor of valine, but not of the precursor of isoleucine, were drastically decreased by SMM, and valine, but not isoleucine, partially overcame SMM inhibition. This apparent stronger effect of SMM on the flux into the valine arm, as opposed to the isoleucine arm, of the branched-chain amino acid pathway is explained by the kinetics of the AHAS reaction, as well as by the different roles of pyruvate, ketobutyrate, and the valine precursor in metabolism. The organization of the pathway thus potentiates the inhibitory effect of SMM. IpOHA has strong initial effects at lower concentrations than does SMM and leads to increases both in the acetohydroxy acid substrates of KARI and, surprisingly, in ketobutyrate. Valine completely protected strain TV105 from IpOHA at the MIC. A number of explanations for this effect can be ruled out, so that some unknown arrangement of the enzymes involved must be suggested. IpOHA led to initial cessation of growth, with partial recovery after a time whose duration increased with the inhibitor concentration. The recovery is apparently due to

  18. Direct coating for layered double hydroxide/4,4'-diaminostilbene-2,2'-disulfonic acid nanocomposite with silica by seeded polymerization technique

    NASA Astrophysics Data System (ADS)

    El-Toni, Ahmed Mohamed; Yin, Shu; Sato, Tsugio

    2004-09-01

    Organic ultraviolet (UV) ray absorbents have been used as sunscreen materials, but may pose a safety problem when used at high concentration. In order to prevent direct contact of organic UV rays absorbent by the human skin, an organic UV absorbent such as 4,4'-diaminostilbene-2,2'-disulfonic acid (DASDSA) was intercalated into Zn 2Al-layered double hydroxide (Zn 2Al-LDHs) by coprecipiation reaction. The problem of deintercalation of organic molecules from LDHs by the anion exchange reaction with carbonate ion could be greatly depressed by forming a protection film of silica on the surface. Zn 2Al-LDH/DASDSA was directly coated with silica by means of a polymerization technique based on the Stöber method. The deintercalation behavior as well as UV-shielding properties were investigated for coated particles.

  19. Microbial-processing of fruit and vegetable wastes for production of vital enzymes and organic acids: Biotechnology and scopes.

    PubMed

    Panda, Sandeep K; Mishra, Swati S; Kayitesi, Eugenie; Ray, Ramesh C

    2016-04-01

    Wastes generated from fruits and vegetables are organic in nature and contribute a major share in soil and water pollution. Also, green house gas emission caused by fruit and vegetable wastes (FVWs) is a matter of serious environmental concern. This review addresses the developments over the last one decade on microbial processing technologies for production of enzymes and organic acids from FVWs. The advances in genetic engineering for improvement of microbial strains in order to enhance the production of the value added bio-products as well as the concept of zero-waste economy have been briefly discussed. PMID:26761593

  20. Seasonal upregulation of catabolic enzymes and fatty acid transporters in the flight muscle of migrating hoary bats, Lasiurus cinereus.

    PubMed

    McGuire, Liam P; Fenton, M Brock; Guglielmo, Christopher G

    2013-06-01

    The high energy density of fat, and limited capacity for carbohydrate storage suggest that migrating bats should fuel endurance flights with fat, as observed in migrating birds. Yet, cursorial mammals are unable to support high intensity exercise with fat stores. We hypothesized that migratory bats and birds have converged on similar physiological mechanisms to fuel endurance flight with fat. We predicted bats would seasonally upregulate fatty acid transport and oxidation pathways when migration demands were high. We studied seasonal variation in mitochondrial oxidative enzyme activities and fatty acid transport protein expression in the flight muscle of hoary bats (Lasiurus cinereus). Carnitine palmitoyl transferase, 3-hydroxyacyl-CoA dehydrogenase and citrate synthase activity increased during migration. There were no changes in expression of fatty acid translocase or plasma membrane fatty acid binding protein. Heart-type fatty acid binding protein expression increased 5-fold in migrating females, but did not vary seasonally in males. An aerial insectivore lifestyle, and the coincidence of migration and pregnancy may explain differences in transporter expression compared to previously studied birds. Overall, our results are consistent with seasonal upregulation of lipid metabolism and aerobic capacity, and confirm that migration poses distinct physiological challenges for bats. PMID:23545469

  1. A Polymeric Prodrug of 5-Fluorouracil-1-Acetic Acid Using a Multi-Hydroxyl Polyethylene Glycol Derivative as the Drug Carrier

    PubMed Central

    Sun, Xun; Gong, Tao; Zhang, Zhirong

    2014-01-01

    Purpose Macromolecular prodrugs obtained by covalently conjugating small molecular drugs with polymeric carriers were proven to accomplish controlled and sustained release of the therapeutic agents in vitro and in vivo. Polyethylene glycol (PEG) has been extensively used due to its low toxicity, low immunogenicity and high biocompatibility. However, for linear PEG macromolecules, the number of available hydroxyl groups for drug coupling does not change with the length of polymeric chain, which limits the application of PEG for drug conjugation purposes. To increase the drug loading and prolong the retention time of 5-fluorouracil (5-Fu), a macromolecular prodrug of 5-Fu, 5-fluorouracil-1 acid-PAE derivative (5-FA-PAE) was synthesized and tested for the antitumor activity in vivo. Methods PEG with a molecular weight of 38 kDa was selected to synthesize the multi-hydroxyl polyethylene glycol derivative (PAE) through an addition reaction. 5-fluorouracil-1 acetic acid (5-FA), a 5-Fu derivative was coupled with PEG derivatives via ester bond to form a macromolecular prodrug, 5-FA-PAE. The in vitro drug release, pharmacokinetics, in vivo distribution and antitumor effect of the prodrug were investigated, respectively. Results The PEG-based prodrug obtained in this study possessed an exceedingly high 5-FA loading efficiency of 10.58%, much higher than the maximum drug loading efficiency of unmodified PEG with the same molecular weight, which was 0.98% theoretically. Furthermore, 5-FA-PAE exhibited suitable sustained release in tumors. Conclusion This study provides a new approach for the development of the delivery to tumors of anticancer agents with PEG derivatives. PMID:25389968

  2. Polymeric nanoparticles

    PubMed Central

    Bolhassani, Azam; Javanzad, Shabnam; Saleh, Tayebeh; Hashemi, Mehrdad; Aghasadeghi, Mohammad Reza; Sadat, Seyed Mehdi

    2014-01-01

    Nanocarriers with various compositions and biological properties have been extensively applied for in vitro/in vivo drug and gene delivery. The family of nanocarriers includes polymeric nanoparticles, lipid-based carriers (liposomes/micelles), dendrimers, carbon nanotubes, and gold nanoparticles (nanoshells/nanocages). Among different delivery systems, polymeric carriers have several properties such as: easy to synthesize, inexpensive, biocompatible, biodegradable, non-immunogenic, non-toxic, and water soluble. In addition, cationic polymers seem to produce more stable complexes led to a more protection during cellular trafficking than cationic lipids. Nanoparticles often show significant adjuvant effects in vaccine delivery since they may be easily taken up by antigen presenting cells (APCs). Natural polymers such as polysaccharides and synthetic polymers have demonstrated great potential to form vaccine nanoparticles. The development of new adjuvants or delivery systems for DNA and protein immunization is an expanding research field. This review describes polymeric carriers especially PLGA, chitosan, and PEI as vaccine delivery systems. PMID:24128651

  3. Enzyme-substrate complementarity governs access to a cationic reaction manifold in the P450(BM3)-catalysed oxidation of cyclopropyl fatty acids.

    PubMed

    Cryle, Max J; Hayes, Patricia Y; De Voss, James J

    2012-12-01

    The products of cytochrome P450(BM3)-catalysed oxidation of cyclopropyl-containing dodecanoic acids are consistent with the presence of a cationic reaction intermediate, which results in efficient dehydrogenation of the rearranged probes by the enzyme. These results highlight the importance of enzyme-substrate complementarity, with a cationic intermediate occurring only when the probes used begin to diverge from ideal substrates for this enzyme. This also aids in reconciling literature reports supporting the presence of cationic intermediates with certain cytochrome P450 enzyme/substrate pairs. PMID:23109039

  4. Chemically modified polymeric resins for separation of cations, organic acids, and small polar moleculea by high performance liquid chromatography

    SciTech Connect

    Morris, J.B.

    1993-07-01

    This thesis is divided into 4 parts: a review, ion chromatography of metal cations on carboxylic resins, separation of hydrophilic organic acids and small polar compounds on macroporous resin columns, and use of eluent modifiers for liquid chromatographic separation of carboxylic acids using conductivity detection.

  5. Analysis of the key enzymes of butyric and acetic acid fermentation in biogas reactors

    PubMed Central

    Gabris, Christina; Bengelsdorf, Frank R; Dürre, Peter

    2015-01-01

    This study aimed at the investigation of the mechanisms of acidogenesis, which is a key process during anaerobic digestion. To expose possible bottlenecks, specific activities of the key enzymes of acidification, such as acetate kinase (Ack, 0.23–0.99 U mg−1 protein), butyrate kinase (Buk, < 0.03 U mg−1 protein) and butyryl-CoA:acetate-CoA transferase (But, 3.24–7.64 U mg−1 protein), were determined in cell free extracts of biogas reactor content from three different biogas reactors. Furthermore, the detection of Ack was successful via Western blot analysis. Quantification of corresponding functional genes encoding Buk (buk) and But (but) was not feasible, although an amplification was possible. Thus, phylogenetic trees were constructed based on respective gene fragments. Four new clades of possible butyrate-producing bacteria were postulated, as well as bacteria of the genera Roseburia or Clostridium identified. The low Buk activity was in contrast to the high specific But activity in the analysed samples. Butyrate formation via Buk activity does barely occur in the investigated biogas reactor. Specific enzyme activities (Ack, Buk and But) in samples drawn from three different biogas reactors correlated with ammonia and ammonium concentrations (NH3 and NH4+-N), and a negative dependency can be postulated. Thus, high concentrations of NH3 and NH4+-N may lead to a bottleneck in acidogenesis due to decreased specific acidogenic enzyme activities. PMID:26086956

  6. Analysis of the key enzymes of butyric and acetic acid fermentation in biogas reactors.

    PubMed

    Gabris, Christina; Bengelsdorf, Frank R; Dürre, Peter

    2015-09-01

    This study aimed at the investigation of the mechanisms of acidogenesis, which is a key process during anaerobic digestion. To expose possible bottlenecks, specific activities of the key enzymes of acidification, such as acetate kinase (Ack, 0.23-0.99 U mg(-1) protein), butyrate kinase (Buk, < 0.03 U mg(-1) protein) and butyryl-CoA:acetate-CoA transferase (But, 3.24-7.64 U mg(-1) protein), were determined in cell free extracts of biogas reactor content from three different biogas reactors. Furthermore, the detection of Ack was successful via Western blot analysis. Quantification of corresponding functional genes encoding Buk (buk) and But (but) was not feasible, although an amplification was possible. Thus, phylogenetic trees were constructed based on respective gene fragments. Four new clades of possible butyrate-producing bacteria were postulated, as well as bacteria of the genera Roseburia or Clostridium identified. The low Buk activity was in contrast to the high specific But activity in the analysed samples. Butyrate formation via Buk activity does barely occur in the investigated biogas reactor. Specific enzyme activities (Ack, Buk and But) in samples drawn from three different biogas reactors correlated with ammonia and ammonium concentrations (NH₃ and NH₄(+)-N), and a negative dependency can be postulated. Thus, high concentrations of NH₃ and NH₄(+)-N may lead to a bottleneck in acidogenesis due to decreased specific acidogenic enzyme activities. PMID:26086956

  7. Differential effects of valproic acid and enzyme-inducing anticonvulsants on nimodipine pharmacokinetics in epileptic patients

    PubMed Central

    Tartara, A.; Galimberti, C.A.; Manni, R.; Parietti, L.; Zucca, C.; Baasch, H.; Caresia, L.; Mück, W.; Barzaghi, N.; Gatti, G.; Perucca, E.

    1991-01-01

    1 The single dose pharmacokinetics of orally administered nimodipine (60 mg) were investigated in normal subjects and in two groups of epileptic patients receiving chronic treatment with hepatic microsomal enzyme-inducing anticonvulsants (carbamazepine, phenobarbitone or phenytoin) and sodium valproate, respectively. 2 Compared with the values found in the control group, mean areas under the plasma nimodipine concentration curve were lowered by about seven-fold (P < 0.01) in patients taking enzyme-inducing anticonvulsants and increased by about 50% (P < 0.05) in patients taking sodium valproate. 3 Nimodipine half-lives were shorter in enzyme-induced patients than in controls (3.9 ± 2.0 h vs 9.1 ± 3.4 h, means ± s.d., P < 0.01), but this difference could be artifactual since in the patients drug concentrations declined rapidly below the limit of assay, thus preventing identification of a possible slower terminal phase. In valproate-treated patients, half-lives (8.2 ± 1.8 h) were similar to those found in controls. PMID:1777370

  8. Robust enzyme-silica composites made from enzyme nanocapsules.

    PubMed

    Li, Jie; Jin, Xin; Liu, Yang; Li, Fan; Zhang, Linlin; Zhu, Xianyuan; Lu, Yunfeng

    2015-06-14

    Novel enzyme composites are synthesized first by in situ polymerization around enzymes and a subsequent sol-gel process. Both the polymer shell and the silica shell with desired functional moieties provide not only great enzyme protection but also a favorable microenvironment, resulting in significantly enhanced activity and stability. PMID:25971337

  9. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    PubMed

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions. PMID:26156238

  10. Function of heterologous Mycobacterium tuberculosis InhA, a type 2 fatty acid synthase enzyme involved in extending C20 fatty acids to C60-to-C90 mycolic acids, during de novo lipoic acid synthesis in Saccharomyces cerevisiae.

    PubMed

    Gurvitz, Aner; Hiltunen, J Kalervo; Kastaniotis, Alexander J

    2008-08-01

    We describe the physiological function of heterologously expressed Mycobacterium tuberculosis InhA during de novo lipoic acid synthesis in yeast (Saccharomyces cerevisiae) mitochondria. InhA, representing 2-trans-enoyl-acyl carrier protein reductase and the target for the front-line antituberculous drug isoniazid, is involved in the activity of dissociative type 2 fatty acid synthase (FASII) that extends associative type 1 fatty acid synthase (FASI)-derived C(20) fatty acids to form C(60)-to-C(90) mycolic acids. Mycolic acids are major constituents of the protective layer around the pathogen that contribute to virulence and resistance to certain antimicrobials. Unlike FASI, FASII is thought to be incapable of de novo biosynthesis of fatty acids. Here, the genes for InhA (Rv1484) and four similar proteins (Rv0927c, Rv3485c, Rv3530c, and Rv3559c) were expressed in S. cerevisiae etr1Delta cells lacking mitochondrial 2-trans-enoyl-thioester reductase activity. The phenotype of the yeast mutants includes the inability to produce sufficient levels of lipoic acid, form mitochondrial cytochromes, respire, or grow on nonfermentable carbon sources. Yeast etr1Delta cells expressing mitochondrial InhA were able to respire, grow on glycerol, and produce lipoic acid. Commensurate with a role in mitochondrial de novo fatty acid biosynthesis, InhA could accept in vivo much shorter acyl-thioesters (C(4) to C(8)) than was previously thought (>C(12)). Moreover, InhA functioned in the absence of AcpM or protein-protein interactions with its native FASII partners KasA, KasB, FabD, and FabH. None of the four proteins similar to InhA complemented the yeast mutant phenotype. We discuss the implications of our findings with reference to lipoic acid synthesis in M. tuberculosis and the potential use of yeast FASII mutants for investigating the physiological function of drug-targeted pathogen enzymes involved in fatty acid biosynthesis. PMID:18552191

  11. Biocatalyzed approach for the surface functionalization of poly(L-lactic acid) films using hydrolytic enzymes.

    PubMed

    Pellis, Alessandro; Acero, Enrique Herrero; Weber, Hansjoerg; Obersriebnig, Michael; Breinbauer, Rolf; Srebotnik, Ewald; Guebitz, Georg M

    2015-09-01

    Poly(lactic acid) as a biodegradable thermoplastic polyester has received increasing attention. This renewable polyester has found applications in a wide range of products such as food packaging, textiles and biomedical devices. Its major drawbacks are poor toughness, slow degradation rate and lack of reactive side-chain groups. An enzymatic process for the grafting of carboxylic acids onto the surface of poly(L-lactic acid) (PLLA) films was developed using Candida antarctica lipase B as a catalyst. Enzymatic hydrolysis of the PLLA film using Humicola insolens cutinase in order to increase the number of hydroxyl and carboxylic groups on the outer polymer chains for grafting was also assessed and showed a change of water contact angle from 74.6 to 33.1° while the roughness and waviness were an order of magnitude higher in comparison to the blank. Surface functionalization was demonstrated using two different techniques, (14) C-radiochemical analysis and X-ray photoelectron spectroscopy (XPS) using (14) C-butyric acid sodium salt and 4,4,4-trifluorobutyric acid as model molecules, respectively. XPS analysis showed that 4,4,4-trifluorobutyric acid was enzymatically coupled based on an increase of the fluor content from 0.19 to 0.40%. The presented (14) C-radiochemical analyses are consistent with the XPS data indicating the potential of enzymatic functionalization in different reaction conditions. PMID:25963883

  12. Radiation-induced polymerization for the immobilization of penicillin acylase

    SciTech Connect

    Boccu, E.; Carenza, M.; Lora, S.; Palma, G.; Veronese, F.M.

    1987-06-01

    The immobilization of Escherichia coli penicillin acylase was investigated by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperature. A leak-proof composite that does not swell in water was obtained by adding the cross-linking agent trimethylolpropane trimethacrylate to the monomer-aqueous enzyme mixture. Penicillin acylase, which was immobilized with greater than 70% yield, possessed a higher Km value toward the substrate 6-nitro-3-phenylacetamidobenzoic acid than the free enzyme form (Km = 1.7 X 10(-5) and 1 X 10(-5) M, respectively). The structural stability of immobilized penicillin acylase, as assessed by heat, guanidinium chloride, and pH denaturation profiles, was very similar to that of the free-enzyme form, thus suggesting that penicillin acylase was entrapped in its native state into aqueous free spaces of the polymer matrix.

  13. Amino acids flanking the central core of Cu,Zn superoxide dismutase are important in retaining enzyme activity after autoclaving.

    PubMed

    Kumar, Arun; Randhawa, Vinay; Acharya, Vishal; Singh, Kashmir; Kumar, Sanjay

    2016-01-01

    Enzymes are known to be denatured upon boiling, although Cu,Zn superoxide dismutase of Potentilla atrosanguinea (Pot-SOD) retains significant catalytic activity even after autoclaving (heating at 121 °C at a pressure of 1.1 kg per square cm for 20 min). The polypeptide backbone of Pot-SOD consists of 152 amino acids with a central core spanning His45 to Cys145 that is involved in coordination of Cu(2+) and Zn(2+) ions. While the central core is essential for imparting catalytic activity and structural stability to the enzyme, the role of sequences flanking the central core was not understood. Experiments with deletion mutants showed that the amino acid sequences flanking the central core were important in retaining activity of Pot-SOD after autoclaving. Molecular dynamics simulations demonstrated the unfavorable structure of mutants due to increased size of binding pocket and enhanced negative charge on the electrostatic surface, resulting in unavailability of the substrate superoxide radical ([Formula: see text]) to the catalytic pocket. Deletion caused destabilization of structural elements and reduced solvent accessibility that further produced unfavorable structural geometry of the protein. PMID:25990646

  14. Relationship between extracellular enzymes and cell growth during the cell cycle of the fission yeast Schizosaccharomyces pombe: acid phosphatase.

    PubMed Central

    Miyata, M; Miyata, H

    1978-01-01

    By using the intact cells of the fission yeast Schizosaccharomyces pombe, the activity of acid phosphatase (EC 3.1.3.2) was compared through the cell cycle with the growth in cell length as a measure of cell growth. The cells of a growing asynchronous culture increased exponentially in number and in total enzyme activity, but remained constant in average length and in specific activity, In a synchronous culture prepared by selection or by induction, the specific activity was periodic in parallel with the increase in average cell length. When hydroxyurea was added to an asynchronous or a synchronous culture by selection, both specific and total activity followed the same continuous pattern as the growth in cell length after the stoppage of cell division. When oversized cells produced by a hydroxyurea pulse treatment to the culture previously syndronized by selection were transferred to a poor medium, they divided synchronously but could hardly grow in the total cell length. In this experimental situation, the total enzyme activity also scarcely increased through three division cycles. These results suggested that the increase in acid phosphatase in dependent on cell elongation. PMID:711673

  15. The Catalytic Scaffold fo the Haloalkanoic Acid Dehalogenase Enzyme Superfamily Acts as a Mold for the Trigonal Bipyramidal Transition State

    SciTech Connect

    Lu,Z.; Dunaway-Mariano, D.; Allen, K.

    2008-01-01

    The evolution of new catalytic activities and specificities within an enzyme superfamily requires the exploration of sequence space for adaptation to a new substrate with retention of those elements required to stabilize key intermediates/transition states. Here, we propose that core residues in the large enzyme family, the haloalkanoic acid dehalogenase enzyme superfamily (HADSF) form a 'mold' in which the trigonal bipyramidal transition states formed during phosphoryl transfer are stabilized by electrostatic forces. The vanadate complex of the hexose phosphate phosphatase BT4131 from Bacteroides thetaiotaomicron VPI-5482 (HPP) determined at 1.00 Angstroms resolution via X-ray crystallography assumes a trigonal bipyramidal coordination geometry with the nucleophilic Asp-8 and one oxygen ligand at the apical position. Remarkably, the tungstate in the complex determined to 1.03 Angstroms resolution assumes the same coordination geometry. The contribution of the general acid/base residue Asp-10 in the stabilization of the trigonal bipyramidal species via hydrogen-bond formation with the apical oxygen atom is evidenced by the 1.52 Angstroms structure of the D10A mutant bound to vanadate. This structure shows a collapse of the trigonal bipyramidal geometry with displacement of the water molecule formerly occupying the apical position. Furthermore, the 1.07 Angstroms resolution structure of the D10A mutant complexed with tungstate shows the tungstate to be in a typical 'phosphate-like' tetrahedral configuration. The analysis of 12 liganded HADSF structures deposited in the protein data bank (PDB) identified stringently conserved elements that stabilize the trigonal bipyramidal transition states by engaging in favorable electrostatic interactions with the axial and equatorial atoms of the transferring phosphoryl group.

  16. A cryptic role of a glycolytic-gluconeogenic enzyme (aldolase) in amino acid transporter turnover in Aspergillus nidulans.

    PubMed

    Roumelioti, Katerina; Vangelatos, Ioannis; Sophianopoulou, Vicky

    2010-03-01

    In Aspergillus nidulans the fbaA1013 mutation results in reduced or total loss of growth on glycolytic and gluconeogenic carbon sources, respectively. It also negatively affects growth on several amino acids (including L-proline, L-glutamate or L-aspartate) that the fungus can use as nitrogen source on glycolytic carbon sources. Complementation of the fbaA1013 mutation using an A. nidulans genomic library resulted in cloning of the fbaA gene, which encodes a putative fructose 1,6-biphosphate aldolase (FBA), an enzyme involved in both glycolysis and gluconeogenesis. The fbaA1013 mutation is a chromosome rearrangement in the 5' regulatory region of the fbaA gene resulting in reduced or total loss of transcription in response to glycolytic and gluconeogenic carbon sources respectively. The fbaA gene is essential for growth. A functional FbaA protein is necessary for plasma membrane localization of the AgtA acidic amino acid (L-glutamate/L-aspartate) transporter, as the fbaA1013 mutation results in targeting to and presumably subsequent degradation of AgtA in the vacuole. Our results support a novel role of the FbaA protein that is, involvement in the regulation of amino acids transporters. PMID:20026236

  17. Two separate key enzymes and two pathway-specific transcription factors are involved in fusaric acid biosynthesis in Fusarium fujikuroi.

    PubMed

    Studt, Lena; Janevska, Slavica; Niehaus, Eva-Maria; Burkhardt, Immo; Arndt, Birgit; Sieber, Christian M K; Humpf, Hans-Ulrich; Dickschat, Jeroen S; Tudzynski, Bettina

    2016-03-01

    Fusaric acid (FSA) is a mycotoxin produced by several fusaria, including the rice pathogen Fusarium fujikuroi. Genes involved in FSA biosynthesis were previously identified as a cluster containing a polyketide synthase (PKS)-encoding (FUB1) and four additional genes (FUB2-FUB5). However, the biosynthetic steps leading to FSA as well as the origin of the nitrogen atom, which is incorporated into the polyketide backbone, remained unknown. In this study, seven additional cluster genes (FUB6-FUB12) were identified via manipulation of the global regulator FfSge1. The extended FUB gene cluster encodes two Zn(II)2 Cys6 transcription factors: Fub10 positively regulates expression of all FUB genes, whereas Fub12 is involved in the formation of the two FSA derivatives, i.e. dehydrofusaric acid and fusarinolic acid, serving as a detoxification mechanism. The major facilitator superfamily transporter Fub11 functions in the export of FSA out of the cell and is essential when FSA levels become critical. Next to Fub1, a second key enzyme was identified, the non-canonical non-ribosomal peptide synthetase Fub8. Chemical analyses of generated mutant strains allowed for the identification of a triketide as PKS product and the proposition of an FSA biosynthetic pathway, thereby unravelling the unique formation of a hybrid metabolite consisting of this triketide and an amino acid moiety. PMID:26662839

  18. In-Water and Neat Batch and Continuous-Flow Direct Esterification and Transesterification by a Porous Polymeric Acid Catalyst.

    PubMed

    Baek, Heeyoel; Minakawa, Maki; Yamada, Yoichi M A; Han, Jin Wook; Uozumi, Yasuhiro

    2016-01-01

    A porous phenolsulphonic acid-formaldehyde resin (PAFR) was developed. The heterogeneous catalyst PAFR was applied to the esterification of carboxylic acids and alcohols, affording the carboxylic acid esters in a yield of up to 95% where water was not removed from the reaction mixture. Surprisingly, the esterification in water as a solvent proceeded to afford the desired esters in high yield. PAFR provided the corresponding esters in higher yield than other homogeneous and heterogeneous catalysts. The transesterification of alcohols and esters was also investigated by using PAFR, giving the corresponding esters. PAFR was applied to the batch-wise and continuous-flow production of biodiesel fuel FAME. The PAFR-packed flow reactor that was developed for the synthesis of carboxylic acids and FAME worked for four days without loss of its catalytic activity. PMID:27189631

  19. Omega-3 fatty acid production from enzyme saccharified hemp hydrolysate using a novel marine thraustochytrid strain.

    PubMed

    Gupta, Adarsha; Abraham, Reinu E; Barrow, Colin J; Puri, Munish

    2015-05-01

    In this work, a newly isolated marine thraustochytrid strain, Schizochytrium sp. DT3, was used for omega-3 fatty acid production by growing on lignocellulose biomass obtained from local hemp hurd (Cannabis sativa) biomass. Prior to enzymatic hydrolysis, hemp was pretreated with sodium hydroxide to open the biomass structure for the production of sugar hydrolysate. The thraustochytrid strain was able to grow on the sugar hydrolysate and accumulated polyunsaturated fatty acids (PUFAs). At the lowest carbon concentration of 2%, the PUFAs productivity was 71% in glucose and 59% in the sugars hydrolysate, as a percentage of total fatty acids. Saturated fatty acids (SFAs) levels were highest at about 49% of TFA using 6% glucose as the carbon source. SFAs of 41% were produced using 2% of SH. This study demonstrates that SH produced from lignocellulose biomass is a potentially useful carbon source for the production of omega-3 fatty acids in thraustochytrids, as demonstrated using the new strain, Schizochytrium sp. DT3. PMID:25497057

  20. Comparative genomics of citric-acid producing Aspergillus niger ATCC 1015 versus enzyme-producing CBS 513.88

    SciTech Connect

    Andersen, Mikael R.; Salazar, Margarita; Schaap, Peter; van de Vondervoort, Peter; Culley, David E.; Thykaer, Jette; Frisvad, Jens C.; Nielsen, Kristian F.; Albang, Richard; Albermann, Kaj; Berka, Randy; Braus, Gerhard; Braus-Stromeyer, Susanna A.; Corrochano, Luis; Dai, Ziyu; van Dijck, Piet; Hofmann, Gerald; Lasure, Linda L.; Magnuson, Jon K.; Menke, Hildegard; Meijer, Martin; Meijer, Susan; Nielsen, Jakob B.; Nielsen, Michael L.; van Ooyen, Albert; Pel, Herman J.; Poulsen, Lars; Samson, Rob; Stam, Hein; Tsang, Adrian; van den Brink, Johannes M.; ATkins, Alex; Aerts, Andrea; Shapiro, Harris; Pangilinan, Jasmyn; Salamov, Asaf; Lou, Yigong; Lindquist, Erika; Lucas, Susan; Grimwood, Jane; Grigoriev, Igor V.; Kubicek, Christian P.; Martinez, Diego; van Peij, Noel; Roubos, Johannes A.; Nielsen, Jens B.; Baker, Scott E.

    2011-06-01

    The filamentous fungus Aspergillus niger exhibits great diversity in its phenotype. It is found globally, both as marine and terrestrial strains, produces both organic acids and hydrolytic enzymes in high amounts, and some isolates exhibit pathogenicity. Although the genome of an industrial enzyme-producing A. niger strain (CBS 513.88) has already been sequenced, the versatility and diversity of this species compels additional exploration. We therefore undertook whole genome sequencing of the acidogenic A. niger wild type strain (ATCC 1015), and produced a genome sequence of very high quality. Only 15 gaps are present in the sequence and half the telomeric regions have been elucidated. Moreover, sequence information from ATCC 1015 was utilized to improve the genome sequence of CBS 513.88. Chromosome-level comparisons uncovered several genome rearrangements, deletions, a clear case of strain-specific horizontal gene transfer, and identification of 0.8 megabase of novel sequence. Single nucleotide polymorphisms per kilobase (SNPs/kb) between the two strains were found to be exceptionally high (average: 7.8, maximum: 160 SNPs/kb). High variation within the species was confirmed with exo-metabolite profiling and phylogenetics. Detailed lists of alleles were generated, and genotypic differences were observed to accumulate in metabolic pathways essential to acid production and protein synthesis. A transcriptome analysis revealed up-regulation of the electron transport chain, specifically the alternative oxidative pathway in ATCC 1015, while CBS 513.88 showed significant up regulation of genes associated with biosynthesis of amino acids that are abundant in glucoamylase A, tRNA-synthases and protein transporters.

  1. Novel pH-sensitive polysialic acid based polymeric micelles for triggered intracellular release of hydrophobic drug.

    PubMed

    Zhang, Wuxia; Dong, Dongqi; Li, Peng; Wang, Dongdong; Mu, Haibo; Niu, Hong; Duan, Jinyou

    2016-03-30

    Polysialic acid (PSA), a non-immunogenic and biodegradable natural polymer, is prone to hydrolysis under endo-lysosomal pH conditions. Here, we synthesized an intracellular pH-sensitive polysialic acid-ursolic acid conjugate by a condensation reaction. To further test the drug loading capability, we prepared paclitaxel-loaded polysialic acid-based amphiphilic copolymer micelle (PTX-loaded-PSAU) by a nanoprecipitation method. Results showed PTX-loaded-PSAU exhibited well-defined spherical shape and homogeneous distribution. The drug-loading was 4.5% with an entrapment efficiency of 67.5%. PTX released from PTX-loaded-PSAU was 15% and 42% in 72 h under simulated physiological condition (pH 7.4) and mild acidic conditions (pH 5.0), respectively. In addition, In vitro cytotoxicity assay showed that PTX-loaded-PSAU retained anti-tumor (SGC-7901) activity with a cell viability of 53.8% following 72 h incubation, indicating PTX-loaded-PSAU could efficiently release PTX into the tumor cells. These results indicated that the pH-responsive biodegradable PTX-loaded-PSAU possess superior extracellular stability and intracellular drug release ability. PMID:26794949

  2. A localized and propagating SPR, and molecular imprinting based fiber-optic ascorbic acid sensor using an in situ polymerized polyaniline-Ag nanocomposite.

    PubMed

    Shrivastav, Anand M; Usha, Sruthi P; Gupta, Banshi D

    2016-08-26

    We report a successful approach for the fabrication and characterization of a fiber-optic sensor for ascorbic acid (AA) detection, using a molecularly imprinted polyaniline-Ag (PANI-Ag) nanocomposite layer based on the combined phenomena of surface plasmon resonance (SPR) and localized SPR (LSPR). The PANI-Ag nanocomposite is synthesized by an in situ polymerization process and AA imprints are prepared on the polymeric composite. The confirmation of the PANI-Ag nanocomposite and AA imprinting is performed using various characterization methods such as x-ray diffraction (XRD), UV-vis, Fourier transform infrared spectroscopy and scanning electron microscopy. From XRD, the size of Ag nanoparticles is analyzed. The absorbance spectra are recorded for samples of different concentrations of AA around the sensing region of the probe. An increase in peak absorbance wavelength with the increase in AA concentration is observed with a linear response for the concentration range from 10(-8) M to 10(-6) M. The sensor possesses a high sensitivity of 45.1 nm log(-1) M near an AA concentration of 10(-8) M. The limit of detection (LOD) and limit of quantification of the sensor are found to be 7.383 × 10(-11) M and 4.16 × 10(-10) M, respectively. The LOD of the sensor is compared to studies reported in the literature and is found to be the lowest. The sensor possesses several other advantages such as cost effectiveness, selectivity, and low response time (<5 s), along with abilities of remote sensing and online monitoring. PMID:27405256

  3. The Effect of Limited Diffusion and Wet-Dry Cycling on Reversible Polymerization Reactions: Implications for Prebiotic Synthesis of Nucleic Acids.

    PubMed

    Higgs, Paul G

    2016-01-01

    A long-standing problem for the origins of life is that polymerization of many biopolymers, including nucleic acids and peptides, is thermodynamically unfavourable in aqueous solution. If bond making and breaking is reversible, monomers and very short oligomers predominate. Recent experiments have shown that wetting and drying cycles can overcome this problem and drive the formation of longer polymers. In the dry phase, bond formation is favourable, but diffusion is restricted, and bonds only form between monomers that are initially close together. In the wet phase, some of the bonds are hydrolyzed. However, repositioning of the molecules allows new bonds to form in the next dry phase, leading to an increase in mean polymer length. Here, we consider a simple theoretical model that explains the effect of cycling. There is an equilibrium length distribution with a high mean length that could be achieved if diffusion occurred freely in the dry phase. This equilibrium is inaccessible without diffusion. A single dry cycle without diffusion leads to mean lengths much shorter than this. Repeated cycling leads to a significant increase in polymerization relative to a single cycle. In the most favourable case, cycling leads to the same equilibrium length distribution as would be achieved if free diffusion were possible in the dry phase. These results support the RNA World scenario by explaining a potential route to synthesis of long RNAs; however, they also imply that cycling would be beneficial to the synthesis of other kinds of polymers, including peptides, where bond formation involves a condensation reaction. PMID:27338479

  4. A localized and propagating SPR, and molecular imprinting based fiber-optic ascorbic acid sensor using an in situ polymerized polyaniline–Ag nanocomposite

    NASA Astrophysics Data System (ADS)

    Shrivastav, Anand M.; Usha, Sruthi P.; Gupta, Banshi D.

    2016-08-01

    We report a successful approach for the fabrication and characterization of a fiber-optic sensor for ascorbic acid (AA) detection, using a molecularly imprinted polyaniline–Ag (PANI–Ag) nanocomposite layer based on the combined phenomena of surface plasmon resonance (SPR) and localized SPR (LSPR). The PANI–Ag nanocomposite is synthesized by an in situ polymerization process and AA imprints are prepared on the polymeric composite. The confirmation of the PANI–Ag nanocomposite and AA imprinting is performed using various characterization methods such as x-ray diffraction (XRD), UV–vis, Fourier transform infrared spectroscopy and scanning electron microscopy. From XRD, the size of Ag nanoparticles is analyzed. The absorbance spectra are recorded for samples of different concentrations of AA around the sensing region of the probe. An increase in peak absorbance wavelength with the increase in AA concentration is observed with a linear response for the concentration range from 10‑8 M to 10‑6 M. The sensor possesses a high sensitivity of 45.1 nm log‑1 M near an AA concentration of 10‑8 M. The limit of detection (LOD) and limit of quantification of the sensor are found to be 7.383 × 10‑11 M and 4.16 × 10‑10 M, respectively. The LOD of the sensor is compared to studies reported in the literature and is found to be the lowest. The sensor possesses several other advantages such as cost effectiveness, selectivity, and low response time (<5 s), along with abilities of remote sensing and online monitoring.

  5. The Effect of Limited Diffusion and Wet–Dry Cycling on Reversible Polymerization Reactions: Implications for Prebiotic Synthesis of Nucleic Acids

    PubMed Central

    Higgs, Paul G.

    2016-01-01

    A long-standing problem for the origins of life is that polymerization of many biopolymers, including nucleic acids and peptides, is thermodynamically unfavourable in aqueous solution. If bond making and breaking is reversible, monomers and very short oligomers predominate. Recent experiments have shown that wetting and drying cycles can overcome this problem and drive the formation of longer polymers. In the dry phase, bond formation is favourable, but diffusion is restricted, and bonds only form between monomers that are initially close together. In the wet phase, some of the bonds are hydrolyzed. However, repositioning of the molecules allows new bonds to form in the next dry phase, leading to an increase in mean polymer length. Here, we consider a simple theoretical model that explains the effect of cycling. There is an equilibrium length distribution with a high mean length that could be achieved if diffusion occurred freely in the dry phase. This equilibrium is inaccessible without diffusion. A single dry cycle without diffusion leads to mean lengths much shorter than this. Repeated cycling leads to a significant increase in polymerization relative to a single cycle. In the most favourable case, cycling leads to the same equilibrium length distribution as would be achieved if free diffusion were possible in the dry phase. These results support the RNA World scenario by explaining a potential route to synthesis of long RNAs; however, they also imply that cycling would be beneficial to the synthesis of other kinds of polymers, including peptides, where bond formation involves a condensation reaction. PMID:27338479

  6. Influence of wastewater sludge treatment using combined peroxyacetic acid oxidation and inorganic coagulants re-flocculation on characteristics of extracellular polymeric substances (EPS).

    PubMed

    Zhang, Weijun; Cao, Bingdi; Wang, Dongsheng; Ma, Teng; Xia, Hua; Yu, Dehong

    2016-01-01

    Extracellular polymeric substances (EPS) are highly hydrated biopolymers and play important roles in bioflocculation, floc stability, and solid-water separation processes. Destroying EPS structure will result in sludge reduction and release of trapped water. In this study, the effects of combined process of peracetic acid (PAA) pre-oxidation and chemical re-flocculation on morphological properties and distribution and composition of EPS of the resultant sludge flocs were investigated in detail to gain insights into the mechanism involved in sludge treatment. It was found that sludge particles were effectively solubilized and protein-like substances were degraded into small molecules after PAA oxidation. A higher degradation of protein-like substances was observed at acid environments under PAA oxidation. Microscopic analysis revealed that no integral sludge floc was observed after oxidation with PAA at high doses. The floc was reconstructed with addition of inorganic coagulants (polyaluminium chloride (PACl) and ferric chloride (FeCl3)) and PACl performed better in flocculation due to its higher charge neutralization and bridging ability. Combined oxidative lysis and chemical re-flocculation provide a novel solution for sludge treatment. PMID:26584344

  7. In-Water and Neat Batch and Continuous-Flow Direct Esterification and Transesterification by a Porous Polymeric Acid Catalyst

    PubMed Central

    Baek, Heeyoel; Minakawa, Maki; Yamada, Yoichi M. A.; Han, Jin Wook; Uozumi, Yasuhiro

    2016-01-01

    A porous phenolsulphonic acid—formaldehyde resin (PAFR) was developed. The heterogeneous catalyst PAFR was applied to the esterification of carboxylic acids and alcohols, affording the carboxylic acid esters in a yield of up to 95% where water was not removed from the reaction mixture. Surprisingly, the esterification in water as a solvent proceeded to afford the desired esters in high yield. PAFR provided the corresponding esters in higher yield than other homogeneous and heterogeneous catalysts. The transesterification of alcohols and esters was also investigated by using PAFR, giving the corresponding esters. PAFR was applied to the batch-wise and continuous-flow production of biodiesel fuel FAME. The PAFR-packed flow reactor that was developed for the synthesis of carboxylic acids and FAME worked for four days without loss of its catalytic activity. PMID:27189631

  8. In-Water and Neat Batch and Continuous-Flow Direct Esterification and Transesterification by a Porous Polymeric Acid Catalyst

    NASA Astrophysics Data System (ADS)

    Baek, Heeyoel; Minakawa, Maki; Yamada, Yoichi M. A.; Han, Jin Wook; Uozumi, Yasuhiro

    2016-05-01

    A porous phenolsulphonic acid—formaldehyde resin (PAFR) was developed. The heterogeneous catalyst PAFR was applied to the esterification of carboxylic acids and alcohols, affording the carboxylic acid esters in a yield of up to 95% where water was not removed from the reaction mixture. Surprisingly, the esterification in water as a solvent proceeded to afford the desired esters in high yield. PAFR provided the corresponding esters in higher yield than other homogeneous and heterogeneous catalysts. The transesterification of alcohols and esters was also investigated by using PAFR, giving the corresponding esters. PAFR was applied to the batch-wise and continuous-flow production of biodiesel fuel FAME. The PAFR-packed flow reactor that was developed for the synthesis of carboxylic acids and FAME worked for four days without loss of its catalytic activity.

  9. Oligonucleotide-modified screen-printed gold electrodes for enzyme-amplified sensing of nucleic acids.

    PubMed

    Carpini, Guido; Lucarelli, Fausto; Marrazza, Giovanna; Mascini, Marco

    2004-09-15

    An electrochemical genosensor for the detection of specific sequences of DNA has been developed using disposable screen-printed gold electrodes. Screen-printed gold electrodes were firstly modified with a mixed monolayer of a 25-mer thiol-tethered DNA probe and a spacer thiol, 6-mercapto-1-hexanol (MCH). The DNA probe sequence was internal to the sequence of the 35S promoter, which sequence is inserted in the genome of GMOs regulating the transgene expression. An enzyme-amplified detection scheme, based on the coupling of a streptavidin-alkaline phosphatase conjugate and biotinylated target sequences was then applied. The enzyme catalysed the hydrolysis of the electroinactive alpha-naphthyl phosphate to alpha-naphthol; this product is electroactive and has been detected by means of differential pulse voltammetry. The assay was, firstly, characterised using synthetic oligonucleotides. Relevant parameters, such as the probe concentration and the immobilisation time, the use of the MCH and different enzymatic conjugates, were investigated and optimised. The genosensor response was found to be linearly related to the target concentration between 0 and 25 nmol/L; the detection limit was 0.25 nmol/L. The analytical procedure was then applied for the detection of the 35S promoter sequence, which was amplified from the pBI121 plasmid by polymerase chain reaction (PCR). Hybridisation conditions (i.e., hybridisation buffer and hybridisation time) were further optimised. The selectivity of the assay was confirmed using biotinylated non-complementary amplicons and PCR blanks. The results showed that the genosensor enabled sensitive (detection limit: 1 nmol/L) and specific detection of GMO-related sequences, thus providing a useful tool for the screening analysis of bioengineered food samples. PMID:15308218

  10. Annotating enzymes of uncertain function: the deacylation of D-amino acids by members of the amidohydrolase superfamily.

    PubMed

    Cummings, Jennifer A; Fedorov, Alexander A; Xu, Chengfu; Brown, Shoshana; Fedorov, Elena; Babbitt, Patricia C; Almo, Steven C; Raushel, Frank M

    2009-07-14

    The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k(cat)/K(m) = 5.8 x 10(6) M(-1) s(-1)), N-acetyl-d-glutamate (k(cat)/K(m) = 5.2 x 10(6) M(-1) s(-1)), and l-methionine-d-glutamate (k(cat)/K(m) = 3.4 x 10(5) M(-1) s(-1)). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k(cat)/K(m) = 3.2 x 10(4) M(-1) s(-1)), N-acetyl-d-tryptophan (k(cat)/K(m) = 4.1 x 10(4) M(-1) s(-1)), and l-tyrosine-d-leucine (k(cat)/K(m) = 1.5 x 10(4) M(-1) s(-1)). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the alpha-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional approximately 250 sequences identified as members of this group

  11. Annotating Enzymes of Uncertain Function: The Deacylation of d-Amino Acids by Members of the Amidohydrolase Superfamily

    SciTech Connect

    Cummings, J.; Fedorov, A; Xu, C; Brown, S; Fedorov, E; Babbitt, P; Almo, S; Raushel, F

    2009-01-01

    The catalytic activities of three members of the amidohydrolase superfamily were discovered using amino acid substrate libraries. Bb3285 from Bordetella bronchiseptica, Gox1177 from Gluconobacter oxidans, and Sco4986 from Streptomyces coelicolor are currently annotated as d-aminoacylases or N-acetyl-d-glutamate deacetylases. These three enzymes are 22-34% identical to one another in amino acid sequence. Substrate libraries containing nearly all combinations of N-formyl-d-Xaa, N-acetyl-d-Xaa, N-succinyl-d-Xaa, and l-Xaa-d-Xaa were used to establish the substrate profiles for these enzymes. It was demonstrated that Bb3285 is restricted to the hydrolysis of N-acyl-substituted derivatives of d-glutamate. The best substrates for this enzyme are N-formyl-d-glutamate (k{sub cat}/K{sub m} = 5.8 x 10{sup 6} M{sup -1} s{sup -1}), N-acetyl-d-glutamate (k{sub cat}/K{sub m} = 5.2 x 10{sup 6} M{sup -1} s{sup -1}), and l-methionine-d-glutamate (k{sub cat}/K{sub m} = 3.4 x 10{sup 5} M{sup -1} s{sup -1}). Gox1177 and Sco4986 preferentially hydrolyze N-acyl-substituted derivatives of hydrophobic d-amino acids. The best substrates for Gox1177 are N-acetyl-d-leucine (k{sub cat}/K{sub m} = 3.2 x 104 M{sup -1} s-1), N-acetyl-d-tryptophan (kcat/Km = 4.1 x 104 M-1 s-1), and l-tyrosine-d-leucine (kcat/Km = 1.5 x 104 M-1 s-1). A fourth protein, Bb2785 from B. bronchiseptica, did not have d-aminoacylase activity. The best substrates for Sco4986 are N-acetyl-d-phenylalanine and N-acetyl-d-tryptophan. The three-dimensional structures of Bb3285 in the presence of the product acetate or a potent mimic of the tetrahedral intermediate were determined by X-ray diffraction methods. The side chain of the d-glutamate moiety of the inhibitor is ion-paired to Arg-295, while the {alpha}-carboxylate is ion-paired with Lys-250 and Arg-376. These results have revealed the chemical and structural determinants for substrate specificity in this protein. Bioinformatic analyses of an additional {approx}250

  12. Enzymes of the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins

    PubMed Central

    Starcevic, Antonio; Akthar, Shamima; Dunlap, Walter C.; Shick, J. Malcolm; Hranueli, Daslav; Cullum, John; Long, Paul F.

    2008-01-01

    The shikimic acid pathway is responsible for the biosynthesis of many aromatic compounds by a broad range of organisms, including bacteria, fungi, plants, and some protozoans. Animals are considered to lack this pathway, as evinced by their dietary requirement for shikimate-derived aromatic amino acids. We challenge the universality of this traditional view in this report of genes encoding enzymes for the shikimate pathway in an animal, the starlet sea anemone Nematostella vectensis. Molecular evidence establishes horizontal transfer of ancestral genes of the shikimic acid pathway into the N. vectensis genome from both bacterial and eukaryotic (dinoflagellate) donors. Bioinformatic analysis also reveals four genes that are closely related to those of Tenacibaculum sp. MED152, raising speculation for the existence of a previously unsuspected bacterial symbiont. Indeed, the genome of the holobiont (i.e., the entity consisting of the host and its symbionts) comprises a high content of Tenacibaculum-like gene orthologs, including a 16S rRNA sequence that establishes the phylogenetic position of this associate to be within the family Flavobacteriaceae. These results provide a complementary view for the biogenesis of shikimate-related metabolites in marine Cnidaria as a “shared metabolic adaptation” between the partners. PMID:18268342

  13. Enzymes of the shikimic acid pathway encoded in the genome of a basal metazoan, Nematostella vectensis, have microbial origins.

    PubMed

    Starcevic, Antonio; Akthar, Shamima; Dunlap, Walter C; Shick, J Malcolm; Hranueli, Daslav; Cullum, John; Long, Paul F

    2008-02-19

    The shikimic acid pathway is responsible for the biosynthesis of many aromatic compounds by a broad range of organisms, including bacteria, fungi, plants, and some protozoans. Animals are considered to lack this pathway, as evinced by their dietary requirement for shikimate-derived aromatic amino acids. We challenge the universality of this traditional view in this report of genes encoding enzymes for the shikimate pathway in an animal, the starlet sea anemone Nematostella vectensis. Molecular evidence establishes horizontal transfer of ancestral genes of the shikimic acid pathway into the N. vectensis genome from both bacterial and eukaryotic (dinoflagellate) donors. Bioinformatic analysis also reveals four genes that are closely related to those of Tenacibaculum sp. MED152, raising speculation for the existence of a previously unsuspected bacterial symbiont. Indeed, the genome of the holobiont (i.e., the entity consisting of the host and its symbionts) comprises a high content of Tenacibaculum-like gene orthologs, including a 16S rRNA sequence that establishes the phylogenetic position of this associate to be within the family Flavobacteriaceae. These results provide a complementary view for the biogenesis of shikimate-related metabolites in marine Cnidaria as a "shared metabolic adaptation" between the partners. PMID:18268342

  14. The effect of chaya (Cnidoscolus aconitifolius) leaf meal and of exogenous enzymes on amino acid digestibility in broilers.

    PubMed

    Sarmiento-Franco, L; McNab, J M; Pearson, A; Belmar-Casso, R

    2003-07-01

    1. The apparent ileal nitrogen (N) and amino acid digestibilities in chaya leaf meal (CLM) (Cnidoscolus aconitifolius) with added enzymes, and the same variables in diets containing different amounts of CLM were studied in chickens. 2. In the first experiment pectinase, beta-glucanase, and pectinase + beta-glucanase were added to CLM. In the second experiment, there were three diets based on maize and soybean: 0, 150 and 250 g/kg CLM. 3. Pectinase significantly increased both lysine and overall amino acid digestibilities in CLM. 4. In experiment 2, the amino acid digestibility in birds fed on CLM250 was lower than that from birds fed on either control or CLM150. Only the digestibilities of alanine, arginine and proline were lower in birds fed on CLM150 than in those fed on the control diet. Nitrogen digestibility was lower in birds fed on the CLM250 diet than on either control or CLM150 diets. These findings were attributed to the increasing concentration of fibre with increasing dietary CLM. PMID:12964630

  15. Δ9-Tetrahydrocannabinolic acid synthase: The application of a plant secondary metabolite enzyme in biocatalytic chemical synthesis.

    PubMed

    Lange, Kerstin; Schmid, Andreas; Julsing, Mattijs K

    2016-09-10

    Δ(9)-Tetrahydrocannabinolic acid synthase (THCAS) from the secondary metabolism of Cannabis sativa L. catalyzes the oxidative formation of an intramolecular CC bond in cannabigerolic acid (CBGA) to synthesize Δ(9)-tetrahydrocannabinolic acid (THCA), which is the direct precursor of Δ(9)-tetrahydrocannabinol (Δ(9)-THC). Aiming on a biotechnological production of cannabinoids, we investigated the potential of the heterologously produced plant oxidase in a cell-free system on preparative scale. THCAS was characterized in an aqueous/organic two-liquid phase setup in order to solubilize the hydrophobic substrate and to allow in situ product removal. Compared to the single phase aqueous setup the specific activity decreased by a factor of approximately 2 pointing to a substrate limitation of CBGA in the two-liquid phase system. However, the specific activity remained stable for at least 3h illustrating the benefit of the two-liquid phase setup. In a repeated-batch setup, THCAS showed only a minor loss of specific activity in the third batch pointing to a high intrinsic stability and high solvent tolerance of the enzyme. Maximal space-time-yields of 0.121gL(-1)h(-1) were reached proving the two-liquid phase concept suitable for biotechnological production of cannabinoids. PMID:27369551

  16. Synthesis of 2-monoacylglycerols and structured triacylglycerols rich in polyunsaturated fatty acids by enzyme catalyzed reactions.

    PubMed

    Rodríguez, Alicia; Esteban, Luis; Martín, Lorena; Jiménez, María José; Hita, Estrella; Castillo, Beatriz; González, Pedro A; Robles, Alfonso

    2012-08-10

    This paper studies the synthesis of structured triacylglycerols (STAGs) by a four-step process: (i) obtaining 2-monoacylglycerols (2-MAGs) by alcoholysis of cod liver oil with several alcohols, catalyzed by lipases Novozym 435, from Candida antartica and DF, from Rhizopus oryzae, (ii) purification of 2-MAGs, (iii) formation of STAGs by esterification of 2-MAGs with caprylic acid catalyzed by lipase DF, from R. oryzae, and (iv) purification of these STAGs. For the alcoholysis of cod liver oil, absolute ethanol, ethanol 96% (v/v) and 1-butanol were compared; the conditions with ethanol 96% were then optimized and 2-MAG yields of around 54-57% were attained using Novozym 435. In these 2-MAGs, DHA accounted for 24-31% of total fatty acids. In the operational conditions this lipase maintained a stable level of activity over at least 11 uses. These results were compared with those obtained with lipase DF, which deactivated after only three uses. The alcoholysis of cod liver oil and ethanol 96% catalyzed by Novozym 435 was scaled up by multiplying the reactant amounts 100-fold and maintaining the intensity of treatment constant (IOT=3g lipase h/g oil). In these conditions, the 2-MAG yield attained was about 67%; these 2-MAGs contained 36.6% DHA. The synthesized 2-MAGs were separated and purified from the alcoholysis reaction products by solvent extraction using solvents of low toxicity (ethanol and hexane); 2-MAG recovery yield and purity of the target product were approximately 96.4% and 83.9%, respectively. These 2-MAGs were transformed to STAGs using the optimal conditions obtained in a previous work. After synthesis and purification, 93% pure STAGs were obtained, containing 38% DHA at sn-2 position and 60% caprylic acid (CA) at sn-1,3 positions (of total fatty acids at these positions), i.e. the major TAG is the STAG with the structure CA-DHA-CA. PMID:22759534

  17. New metabolically stabilized analogues of lysophosphatidic acid: agonists, antagonists and enzyme inhibitors.

    PubMed

    Prestwich, G D; Xu, Y; Qian, L; Gajewiak, J; Jiang, G

    2005-12-01

    Lysophosphatidic acid (LPA) is a metabolically labile natural phospholipid with a bewildering array of physiological effects. We describe herein a variety of long-lived receptor-specific agonists and antagonists for LPA receptors. Several LPA and PA (phosphatidic acid) analogues also inhibit LPP (lipid phosphate phosphatase). The sn-1 or sn-2 hydroxy groups have been replaced by fluorine, difluoromethyl, difluoroethyl, O-methyl or O-hydroxyethoxy groups to give non-migrating LPA analogues that resist acyltransferases. Alkyl ether replacement of acyl esters produced lipase and acyltransferase-resistant analogues. Replacement of the bridging oxygen in the monophosphate by an alpha-monofluoromethylene-, alpha-bromomethylene- or alpha,alpha-difluoromethylenephosphonate gave phosphatase-resistant analogues. Phosphorothioate analogues with O-acyl and O-alkyl chains are potent, long-lived agonists for LPA1 and LPA3 receptors. Most recently, we have (i) prepared stabilized O-alkyl analogues of lysobisphosphatidic acid, (ii) explored the structure-activity relationship of stabilized cyclic LPA analogues and (iii) synthesized neutral head group trifluoromethylsulphonamide analogues of LPA. Through collaborative studies, we have collected data for these stabilized analogues as selective LPA receptor (ant)agonists, LPP inhibitors, TREK (transmembrane calcium channel) K+ channel agonists, activators of the nuclear transcription factor PPAR-gamma (peroxisome-proliferator-activated receptor-gamma), promoters of cell motility and survival, and radioprotectants for human B-cells. PMID:16246118

  18. Effect of gallic acid on xenobiotic metabolizing enzymes in 1,2-dimethyl hydrazine induced colon carcinogenesis in Wistar rats--a chemopreventive approach.

    PubMed

    Giftson Senapathy, J; Jayanthi, S; Viswanathan, P; Umadevi, P; Nalini, N

    2011-04-01

    Colon cancer risk may be influenced by phase I and II xenobiotic-metabolizing enzyme systems. The chemopreventive agent gallic acid (GA), a plant polyphenol, is found in various natural products. Our aim was to evaluate the potential role of GA on drug-metabolizing enzymes in 1,2-dimethyl hydrazine (DMH) induced rat colon carcinogenesis. The total experimental duration was 30 weeks. The effect of GA (50 mg/kg b.w.) on the activities of phase I enzymes (cytochrome P450 and cytochrome b5) and phase II enzymes (glutathione S-transferase, DT-diaphorase and gamma glutamyl transpeptidase) were assessed in the liver and colonic mucosa and the colons were also examined visually. In DMH induced rats, there was a decrease in the activities of phase II enzymes and an increase in the activities of phase I enzymes. On GA supplementation, there was a significant increase in the activities of phase II enzymes and a significant decrease in the activities of phase I enzymes, in addition to the decreased tumor incidence. Histopathological changes also confirm this. Thus, the marked potential of GA in modulating the phase I and II xenobiotic-metabolizing enzymes suggests that GA may have a major impact on colon cancer chemoprevention. PMID:21172399

  19. Protective effects of ascorbic acid and vitamin E on antioxidant enzyme activity of freeze-thawed semen of Qinchuan bulls.

    PubMed

    Zhao, X L; Li, Y K; Cao, S J; Hu, J H; Wang, W H; Hao, R J; Gui, L S; Zan, L S

    2015-01-01

    The aim of this study was to determine the protective effects of the combination of ascorbic acid (Vc) and vitamin E (VE) on antioxidant enzyme activity, sperm motility, viability, and acrosome integrity of Qinchuan bulls after freeze-thaw. In this study, we determined the effects of Vc and VE on the activity of the antioxidant enzyme defense system comprising glutathione peroxidase (GSH-Px), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD). The combination of Vc and VE had protective effects on sperm motility and viability. With respect to acrosome integrity and the activity of GR and SOD, differences were observed between the experimental groups with added Vc (7 mg/mL) and VE (0.12 IU/mL) and the control group. The activity of GSH-Px in the experimental group (1400 IU/mL Vc and 0.12 IU/mL VE) was not different (P > 0.05) compared with that in the control group, while the activity of CAT showed a significant difference between the 2 groups (P < 0.05). Therefore, we inferred that the combination of Vc (1400 IU/mL) and VE (0.12 IU/mL) protected the sperm quality in the freeze-thaw process. PMID:25867404

  20. Acute Carnosine Administration Increases Respiratory Chain Complexes and Citric Acid Cycle Enzyme Activities in Cerebral Cortex of Young Rats.

    PubMed

    Macedo, Levy W; Cararo, José H; Maravai, Soliany G; Gonçalves, Cinara L; Oliveira, Giovanna M T; Kist, Luiza W; Guerra Martinez, Camila; Kurtenbach, Eleonora; Bogo, Maurício R; Hipkiss, Alan R; Streck, Emilio L; Schuck, Patrícia F; Ferreira, Gustavo C

    2016-10-01

    Carnosine (β-alanyl-L-histidine) is an imidazole dipeptide synthesized in excitable tissues of many animals, whose biochemical properties include carbonyl scavenger, anti-oxidant, bivalent metal ion chelator, proton buffer, and immunomodulating agent, although its precise physiological role(s) in skeletal muscle and brain tissues in vivo remain unclear. The aim of the present study was to investigate the in vivo effects of acute carnosine administration on various aspects of brain bioenergetics of young Wistar rats. The activity of mitochondrial enzymes in cerebral cortex was assessed using a spectrophotometer, and it was found that there was an increase in the activities of complexes I-III and II-III and succinate dehydrogenase in carnosine-treated rats, as compared to vehicle-treated animals. However, quantitative real-time RT-PCR (RT-qPCR) data on mRNA levels of mitochondrial biogenesis-related proteins (nuclear respiratory factor 1 (Nrf1), peroxisome proliferator-activated receptor-γ coactivator 1-α (Ppargc1α), and mitochondrial transcription factor A (Tfam)) were not altered significantly and therefore suggest that short-term carnosine administration does not affect mitochondrial biogenesis. It was in agreement with the finding that immunocontent of respiratory chain complexes was not altered in animals receiving carnosine. These observations indicate that acute carnosine administration increases the respiratory chain and citric acid cycle enzyme activities in cerebral cortex of young rats, substantiating, at least in part, a neuroprotector effect assigned to carnosine against oxidative-driven disorders. PMID:26476839

  1. The outer-coordination sphere: incorporating amino acids and peptides as ligands for homogeneous catalysts to mimic enzyme function

    SciTech Connect

    Shaw, Wendy J.

    2012-10-09

    Great progress has been achieved in the field of homogeneous transition metal-based catalysis, however, as a general rule these solution based catalysts are still easily outperformed, both in terms of rates and selectivity, by their analogous enzyme counterparts, including structural mimics of the active site. This observation suggests that the features of the enzyme beyond the active site, i.e. the outer-coordination sphere, are important for their exceptional function. Directly mimicking the outer-coordination sphere requires the incorporation of amino acids and peptides as ligands for homogeneous catalysts. This effort has been attempted for many homogeneous catalysts which span the manifold of catalytic reactions and often require careful thought regarding solvent type, pH and characterization to avoid unwanted side reactions or catalyst decomposition. This article reviews the current capability of synthesizing and characterizing this often difficult category of metal-based catalysts. This work was funded by the DOE Office of Science Early Career Research Program through the Office of Basic Energy Sciences. Pacific Northwest National Laboratory is operated by Battelle for the U.S. Department of Energy.

  2. pH-Sensitive Polymeric Micelle-based pH Probe for Detecting and Imaging Acidic Biological Environments

    PubMed Central

    Lee, Young Ju; Kang, Han Chang; Hu, Jun; Nichols, Joseph W.; Jeon, Yong Sun; Bae, You Han

    2012-01-01

    To overcome the limitations of monomeric pH probes for acidic tumor environments, this study designed a mixed micelle pH probe composed of polyethylene glycol (PEG)-b- poly(L-histidine) (PHis) and PEG-b-poly(L-lactic acid) (PLLA), which is well-known as an effective antitumor drug carrier. Unlike monomeric histidine and PHis derivatives, the mixed micelles can be structurally destabilized by changes in pH, leading to a better pH sensing system in nuclear magnetic resonance (NMR) techniques. The acidic pH-induced transformation of the mixed micelles allowed pH detection and pH mapping of 0.2–0.3 pH unit differences by pH-induced “on/off”-like sensing of NMR and magnetic resonance spectroscopy (MRS). The micellar pH probes sensed pH differences in non-biological phosphate buffer and biological buffers such as cell culture medium and rat whole blood. In addition, the pH-sensing ability of the mixed micelles was not compromised by loaded doxorubicin. In conclusion, PHis-based micelles could have potential as a tool to simultaneously treat and map the pH of solid tumors in vivo. PMID:22861824

  3. Association with Amino Acids Does Not Enhance Efficacy of Polymerized Liposomes As a System for Lung Gene Delivery.

    PubMed

    Bandeira, Elga; Lopes-Pacheco, Miquéias; Chiaramoni, Nadia; Ferreira, Débora; Fernandez-Ruocco, Maria J; Prieto, Maria J; Maron-Gutierrez, Tatiana; Perrotta, Ramiro M; de Castro-Faria-Neto, Hugo C; Rocco, Patricia R M; Alonso, Silvia Del Valle; Morales, Marcelo M

    2016-01-01

    Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids [1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine] associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with l-arginine, l-tryptophan, or l-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. l-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases. PMID:27199766

  4. Association with Amino Acids Does Not Enhance Efficacy of Polymerized Liposomes As a System for Lung Gene Delivery

    PubMed Central

    Bandeira, Elga; Lopes-Pacheco, Miquéias; Chiaramoni, Nadia; Ferreira, Débora; Fernandez-Ruocco, Maria J.; Prieto, Maria J.; Maron-Gutierrez, Tatiana; Perrotta, Ramiro M.; de Castro-Faria-Neto, Hugo C.; Rocco, Patricia R. M.; Alonso, Silvia del Valle; Morales, Marcelo M.

    2016-01-01

    Development of improved drug and gene delivery systems directly into the lungs is highly desirable given the important burden of respiratory diseases. We aimed to evaluate the safety and efficacy of liposomes composed of photopolymerized lipids [1,2-bis-(tricosa-10,12-diynoyl)-sn-glycero-3-phosphocholine] associated with amino acids as vectors for gene delivery into the lungs of healthy animals. Lipopolymer vesicles, in particular, are more stable than other types of liposomes. In this study, lipopolymers were associated with l-arginine, l-tryptophan, or l-cysteine. We hypothesized that the addition of these amino acids would enhance the efficacy of gene delivery to the lungs by the lipopolymers. l-Arginine showed the highest association efficiency due to its positive charge and better surface interactions. None of the formulations caused inflammation or altered lung mechanics, suggesting that these lipopolymers can be safely administered as aerosols. All formulations were able to induce eGFP mRNA expression in lung tissue, but the addition of amino acids reduced delivery efficacy when compared with the simple lipopolymer particle. These results indicate that this system could be further explored for gene or drug delivery targeting lung diseases. PMID:27199766

  5. Conditional depletion of KasA, a key enzyme of mycolic acid biosynthesis, leads to mycobacterial cell lysis.

    PubMed

    Bhatt, Apoorva; Kremer, Laurent; Dai, Annie Z; Sacchettini, James C; Jacobs, William R

    2005-11-01

    Inhibition or inactivation of InhA, a fatty acid synthase II (FASII) enzyme, leads to mycobacterial cell lysis. To determine whether inactivation of other enzymes of the mycolic acid-synthesizing FASII complex also leads to lysis, we characterized the essentiality of two beta-ketoacyl-acyl carrier protein synthases, KasA and KasB, in Mycobacterium smegmatis. Using specialized transduction for allelic exchange, null kasB mutants, but not kasA mutants, could be generated in Mycobacterium smegmatis, suggesting that unlike kasB, kasA is essential. To confirm the essentiality of kasA, and to detail the molecular events that occur following depletion of KasA, we developed CESTET (conditional expression specialized transduction essentiality test), a genetic tool that combines conditional gene expression and specialized transduction. Using CESTET, we were able to generate conditional null inhA and kasA mutants. We studied the effects of depletion of KasA in M. smegmatis using the former strain as a reference. Depletion of either InhA or KasA led to cell lysis, but with different biochemical and morphological events prior to lysis. While InhA depletion led to the induction of an 80-kDa complex containing both KasA and AcpM, the mycobacterial acyl carrier protein, KasA depletion did not induce the same complex. Depletion of either InhA or KasA led to inhibition of alpha and epoxy mycolate biosynthesis and to accumulation of alpha'-mycolates. Furthermore, scanning electron micrographs revealed that KasA depletion resulted in the cell surface having a "crumpled" appearance, in contrast to the blebs observed on InhA depletion. Thus, our studies support the further exploration of KasA as a target for mycobacterial-drug development. PMID:16267284

  6. Enzymes of type II fatty acid synthesis and apicoplast differentiation and division in Eimeria tenella⋆

    PubMed Central

    Ferguson, D.J.P.; Campbell, S.A.; Henriquez, F.L.; Phan, L.; Mui, E.; Richards, T.A.; Muench, S.P.; Allary, M.; Lu, J.Z.; Prigge, S.T.; Tomley, F.; Shirley, M.W.; Rice, D.W.; McLeod, R.; Roberts, C.W.

    2009-01-01

    Apicomplexan parasites, Eimeria tenella, Plasmodium spp. and Toxoplasma gondii, possess a homologous plastid-like organelle termed the apicoplast, derived from the endosymbiotic enslavement of a photosynthetic alga. However, currently no eimerian nuclear encoded apicoplast targeted proteins have been identified, unlike in Plasmodium spp. and T. gondii. In this study, we demonstrate that nuclear encoded enoyl reductase of E. tenella (EtENR) has a predicted N-terminal bipartite transit sequence, typical of apicoplast-targeted proteins. Using a combination of immunocytochemistry and EM we demonstrate that this fatty acid biosynthesis protein is located in the apicoplast of E. tenella. Using the EtENR as a tool to mark apicoplast development during the Eimeria lifecycle, we demonstrate that nuclear and apicoplast division appear to be independent events, both organelles dividing prior to daughter cell formation, with each daughter cell possessing one to four apicoplasts. We believe this is the first report of multiple apicoplasts present in the infectious stage of an apicomplexan parasite. Furthermore, the microgametes lacked an identifiable apicoplast consistent with maternal inheritance via the macrogamete. It was found that the size of the organelle and the abundance of EtENR varied with developmental stage of the E. tenella lifecycle. The high levels of EtENR protein observed during asexual development and macrogametogony is potentially associated with the increased synthesis of fatty acids required for the rapid formation of numerous merozoites and for the extracellular development and survival of the oocyst. Taken together the data demonstrate that the E. tenella apicoplast participates in type II fatty acid biosynthesis with increased expression of ENR during parasite growth. Apicoplast division results in the simultaneous formation of multiple fragments. The division mechanism is unknown, but is independent of nuclear division and occurs prior to daughter

  7. Expression of a human proprotein processing enzyme: correct cleavage of the von Willebrand factor precursor at a paired basic amino acid site.

    PubMed Central

    Wise, R J; Barr, P J; Wong, P A; Kiefer, M C; Brake, A J; Kaufman, R J

    1990-01-01

    Intracellular proteolytic processing of precursor polypeptides is an essential step in the maturation of many proteins, including plasma proteins, hormones, neuropeptides, and growth factors. Most frequently, propeptide cleavage occurs after paired basic amino acid residues. To date, no mammalian propeptide processing enzyme with such specificity has been purified or cloned and functionally characterized. We report the isolation and functional expression of a cDNA encoding a propeptide-cleaving enzyme from a human liver cell line. The encoded protein, called PACE (paired basic amino acid cleaving enzyme), has structural homology to the well-characterized subtilisin-like protease Kex2 from yeast. The functional specificity of PACE for mediating propeptide cleavage at paired basic amino acid residues was demonstrated by the enhancement of propeptide processing of human von Willebrand factor when coexpressed with PACE in COS-1 cells. Images PMID:2251280

  8. Characterization of the Branched-Chain Amino Acid Aminotransferase Enzyme Family in Tomato1[W][OA

    PubMed Central

    Maloney, Gregory S.; Kochevenko, Andrej; Tieman, Denise M.; Tohge, Takayuki; Krieger, Uri; Zamir, Dani; Taylor, Mark G.; Fernie, Alisdair R.; Klee, Harry J.

    2010-01-01

    Branched-chain amino acids (BCAAs) are synthesized in plants from branched-chain keto acids, but their metabolism is not completely understood. The interface of BCAA metabolism lies with branched-chain aminotransferases (BCAT) that catalyze both the last anabolic step and the first catabolic step. In this study, six BCAT genes from the cultivated tomato (Solanum lycopersicum) were identified and characterized. SlBCAT1, -2, -3, and -4 are expressed in multiple plant tissues, while SlBCAT5 and -6 were undetectable. SlBCAT1 and -2 are located in the mitochondria, SlBCAT3 and -4 are located in chloroplasts, while SlBCAT5 and -6 are located in the cytosol and vacuole, respectively. SlBCAT1, -2, -3, and -4 were able to restore growth of Escherichia coli BCAA auxotrophic cells, but SlBCAT1 and -2 were less effective than SlBCAT3 and -4 in growth restoration. All enzymes were active in the forward (BCAA synthesis) and reverse (branched-chain keto acid synthesis) reactions. SlBCAT3 and -4 exhibited a preference for the forward reaction, while SlBCAT1 and -2 were more active in the reverse reaction. While overexpression of SlBCAT1 or -3 in tomato fruit did not significantly alter amino acid levels, an expression quantitative trait locus on chromosome 3, associated with substantially higher expression of Solanum pennellii BCAT4, did significantly increase BCAA levels. Conversely, antisense-mediated reduction of SlBCAT1 resulted in higher levels of BCAAs. Together, these results support a model in which the mitochondrial SlBCAT1 and -2 function in BCAA catabolism while the chloroplastic SlBCAT3 and -4 function in BCAA synthesis. PMID:20435740

  9. Development of a scintillation proximity assay for the Mycobacterium tuberculosis KasA and KasB enzymes involved in mycolic acid biosynthesis.

    PubMed

    Schaeffer, M L Merrill L; Carson, J D Jeffrey D; Kallender, Howard; Lonsdale, J T John T

    2004-01-01

    Tuberculosis remains a global health problem, and programs dedicated to discovery of novel compounds against Mycobacterium tuberculosis require robust assays for high-throughput screening of chemical and natural product libraries. Enzymes involved in the biosynthesis of mycolic acids, vital components of the mycobacterial cell wall, have received much attention as potential drug targets. KasA and KasB, examples of the beta-ketoacyl-acyl carrier protein synthase I/II (KASI/II) class of condensing enzymes of the M. tuberculosis fatty acid synthase II system have been the focus of several studies designed to biochemically characterize these enzymes. Whilst robust methods have been developed for FabH-like proteins, fast and sensitive assays for high-throughput screening of KASI/II enzymes have not been available. Here we report the development of a direct scintillation proximity assay (SPA) for the KASI/II enzymes, KasA and KasB. The SPA was more sensitive than existing assays, as shown by its ability to measure activity using less enzyme than other assay formats, and the SPA was validated using the known KAS inhibitor thiolactomycin. In addition, the KasA and KasB SPA was adapted for use with Staphylococcus aureus FabF to show the versatility of this assay format to KAS enzymes from other pathogenic organisms. PMID:15525558

  10. Maternal obesity upregulates fatty acid and glucose transporters and increases expression of enzymes mediating fatty acid biosynthesis in fetal adipose tissue depots.

    PubMed

    Long, N M; Rule, D C; Zhu, M J; Nathanielsz, P W; Ford, S P

    2012-07-01

    perirenal depot of OB versus CON fetuses, and specific fatty acid concentrations were altered (P < 0.05) in subcutaneous and pericardial adipose tissue because of maternal obesity. In conclusion, maternal obesity was associated with increased fetal adiposity, increased fatty acid and glucose transporters, and increased expression of enzymes mediating fatty acid biosynthesis in adipose depots. These alterations, if maintained into the postnatal period, could predispose the offspring to later obesity and metabolic disease. PMID:22266999

  11. Metals control activity and expression of the heme biosynthesis enzyme delta-aminolevulinic acid dehydratase in Bradyrhizobium japonicum.

    PubMed Central

    Chauhan, S; Titus, D E; O'Brian, M R

    1997-01-01

    The heme biosynthesis enzyme delta-aminolevulinic acid dehydratase (ALAD) requires magnesium or zinc for activity, depending on the organism, and the heme moiety contains iron. Thus, metals are important for heme formation in at least two different ways. Bradyrhizobium japonicum ALAD* is an engineered derivative of wild-type ALAD that requires Zn2+ for activity rather than Mg2+ (S. Chauhan and M. R. O'Brian, J. Biol. Chem. 270:19823-19827, 1995). The pH optimum for ALAD* activity was over 3.5 units lower than for that of the wild-type enzyme, and ALAD* activity was inhibited by lead and cadmium, as reported for the zinc-containing dehydratases of animals. In addition, ALAD* was significantly more thermostable than ALAD; the temperature optima are 50 and 37 degrees C, respectively. These observations strongly suggest that the metal contributes to both catalysis and structure, and this conclusion may be extrapolated to ALADs in general. Although iron did not affect the activity of the preformed protein, enzyme assays and immunoblot analysis demonstrated that the iron concentration in which the cells were grown had a strong positive effect on ALAD activity and the protein level. RNase protection analysis showed that the transcript quantity of hemB, the gene encoding ALAD, was iron dependent; thus, iron regulates hemB at the mRNA level. Induction of hemB mRNA in response to iron was rapid, suggesting that the factor(s) needed to mediate iron control was present in iron-limited cells and did not need to be synthesized de novo. ALAD protein levels and enzyme activities were similar in cells of the wild type and a heme-defective strain, indicating that control by iron is not an indirect effect of the cellular heme status. We conclude that the heme biosynthetic pathway is coordinated with cellular iron levels and that this control may prevent the accumulation of toxic porphyrin intermediates. PMID:9287008

  12. Synthesis of 5-Substituted Derivatives of Isophthalic Acid as Non-Polymeric Amphiphilic Coating for Metal Oxide Nanoparticles

    PubMed Central

    Nilov, Denis; Kucheryavy, Pavel; Walker, Verina; Kidd, Clayton; Kolesnichenko, Vladimir L.; Goloverda, Galina Z.

    2014-01-01

    In the course of development of novel capping ligands with variable steric factor, which will be used as an organic coating for metal oxide nanoparticles, a base-catalyzed nucleophilic oxirane ring-opening addition reaction between dimethyl 5-hydroxyisophthalate and allyl glycidyl ether was studied. The allyl-terminated 1-1, 1-2 and 1-3 adducts and dihydroxylated derivative of the 1-1 adduct, 5-diglyceroxy isophthalic acid, were synthesized. The latter binds to the surface of 5 nm γ-Fe2O3 nanoparticles in reaction with their surfactant-free diethylene glycol colloids. PMID:25152545

  13. Nucleic acid analysis using terminal-phosphate-labeled nucleotides

    DOEpatents

    Korlach, Jonas; Webb, Watt W.; Levene, Michael; Turner, Stephen; Craighead, Harold G.; Foquet, Mathieu

    2008-04-22

    The present invention is directed to a method of sequencing a target nucleic acid molecule having a plurality of bases. In its principle, the temporal order of base additions during the polymerization reaction is measured on a molecule of nucleic acid, i.e. the activity of a nucleic acid polymerizing enzyme on the template nucleic acid molecule to be sequenced is followed in real time. The sequence is deduced by identifying which base is being incorporated into the growing complementary strand of the target nucleic acid by the catalytic activity of the nucleic acid polymerizing enzyme at each step in the sequence of base additions. A polymerase on the target nucleic acid molecule complex is provided in a position suitable to move along the target nucleic acid molecule and extend the oligonucleotide primer at an active site. A plurality of labelled types of nucleotide analogs are provided proximate to the active site, with each distinguishable type of nucleotide analog being complementary to a different nucleotide in the target nucleic acid sequence. The growing nucleic acid strand is extended by using the polymerase to add a nucleotide analog to the nucleic acid strand at the active site, where the nucleotide analog being added is complementary to the nucleotide of the target nucleic acid at the active site. The nucleotide analog added to the oligonucleotide primer as a result of the polymerizing step is identified. The steps of providing labelled nucleotide analogs, polymerizing the growing nucleic acid strand, and identifying the added nucleotide analog are repeated so that the nucleic acid strand is further extended and the sequence of the target nucleic acid is determined.

  14. Composite polymeric beads containing N,N,N',N'-tetraoctyldiglycolamide for actinide ion uptake from nitric acid feeds: Batch uptake, kinetic modelling and column studies.

    PubMed

    Gujar, R B; Mohapatra, P K; Lakshmi, D Shanthana; Figoli, A

    2015-11-27

    Polyethersulphone (PES) based composite polymeric beads (CPB) containing TODGA (N,N,N',N'-tetraoctyldiglycolamide) as the extractant were prepared by conventional phase inversion technique and were tested for the uptake of actinide ions such as Am(3+), UO2(2+), Pu(4+), Np(4+) and fission product ions such as Eu(3+) and Sr(2+). The CPBs containing 2.5-10wt.% TODGA were characterized by various physical methods and their porosity, size, surface morphology, surface area and the degradation profile by thermogravimetry were analyzed. The batch uptake studies involved kinetics of metal ion sorption, uptake as a function of nitric acid co