Sample records for succinimides

  1. Identification of succinimide sites in proteins by N-terminal sequence analysis after alkaline hydroxylamine cleavage. (United States)

    Kwong, M. Y.; Harris, R. J.


    Under favorable conditions, Asp or Asn residues can undergo rearrangement to a succinimide (cyclic imide), which may also serve as an intermediate for deamidation and/or isoaspartate formation. Direct identification of such succinimides by peptide mapping is hampered by their lability at neutral and alkaline pH. We determined that incubation in 2 M hydroxylamine, 0.2 M Tris buffer, pH 9, for 2 h at 45 degrees C will specifically cleave on the C-terminal side of succinimides without cleavage at Asn-Gly bonds; yields are typically approximately 50%. N-terminal sequence analysis can then be used to identify an internal sequence generated by cleavage of the succinimide, hence identifying the succinimide site. PMID:8142891

  2. New complexes of silver (I) with N-hydroxy-succinimide (United States)

    Sibiescu, Doina; Mîţǎ, Carmen; Vizitiu, Mihaela; Crudu, Andra Manuela


    Over the last period of time silver was considerably studied due to its lower resistivity. In the field of materials science, silver was used in applications such as: microelectronics components of high - temperature superconductiviting materials, bactericidal coatings and others domains. This study presents the process of obtaining and characterization the new complexes of silver (I) with Nhydroxy- succinimide. In the process of obtaining the new complex compounds in aqous solution, first we have to look at conductometry and UV-Vis absorbtion spectroscopy in order to determine the molar ratio silver : N-hydroxysuccinimide and the stability constants. The obtained solid coordination compounds were characterized by elemental analysis, IR spectroscopy and also was investigated of their thermostability. The X-ray powder diffraction reflects that the complexes compounds of silver (I) with N-hydroxysuccinimide are amorphous. In our further studies we want to determine if the new synthetized compounds will present the same or improuved properties as in the above mentioned silver characteristics.

  3. Computational studies on non-succinimide-mediated stereoinversion mechanism of aspartic acid residues assisted by phosphate (United States)

    Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Takahashi, Ohgi; Oda, Akifumi


    Although nearly all of the amino acids that constitute proteins are l-amino acids, d-amino acid residues in human proteins have been recently reported. d-amino acid residues cause a change in the three-dimensional structure of proteins, and d-aspartic acid (Asp) residues are considered to be one of the causes of age-related diseases. The stereoinversion of Asp residues in peptides and proteins is thought to proceed via a succinimide intermediate; however, it has been reported that stereoinversion can occur even under conditions where a succinimide intermediate cannot be formed. In order to elucidate the non-succinimide-mediated stereoinversion pathway, we investigated the stereoinversion of l-Asp to d-Asp catalysed by phosphate and estimated the activation barrier using B3LYP/6-31+G(d,p) density functional theory (DFT) calculations. For the DFT calculations, a model compound in which the Asp residue is capped with acetyl and methyl-amino groups on the N- and C-termini, respectively, was used. The calculated activation barrier was not excessively high for the stereoinversion to occur in vivo. Therefore, this stereoinversion mechanism may compete with the succinimide-mediated mechanism.

  4. A Biosensor for Urea from Succinimide-Modified Acrylic Microspheres Based on Reflectance Transduction

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    Musa Ahmad


    Full Text Available New acrylic microspheres were synthesised by photopolymerisation where the succinimide functional group was incorporated during the microsphere preparation. An optical biosensor for urea based on reflectance transduction with a large linear response range to urea was successfully developed using this material. The biosensor utilized succinimide-modified acrylic microspheres immobilized with a Nile blue chromoionophore (ETH 5294 for optical detection and urease enzyme was immobilized on the surface of the microspheres via the succinimide groups. No leaching of the enzyme or chromoionophore was observed. Hydrolysis of the urea by urease changes the pH and leads to a color change of the immobilized chromoionophore. When the color change was monitored by reflectance spectrophotometry, the linear response range of the biosensor to urea was from 0.01 to 1,000 mM (R2 = 0.97 with a limit of detection of 9.97 mM. The biosensor response showed good reproducibility (relative standard deviation = 1.43%, n = 5 with no interference by major cations such as Na+, K+, NH4+ and Mg2+. The use of reflectance as a transduction method led to a large linear response range that is better than that of many urea biosensors based on other optical transduction methods.

  5. Rapid degradation of D- and L-succinimide-containing peptides by a post-proline endopeptidase from human erythrocytes

    Energy Technology Data Exchange (ETDEWEB)

    Momand, J.; Clarke, S.


    The authors have been interested in the metabolic fate of proteins containing aspartyl succinimide (Asu) residues. These residues can be derived from the spontaneous rearrangement of Asp and Asn residues and from the spontaneous demethylation of enzymatically methylated L-isoAsp and D-Asp residues. Incubation of the synthetic hexapeptide N-Ac-Val-Tyr-Pro-Asu-Gly-Ala with the cytosolic fraction of human erythrocytes resulted in rapid cleavage of the prolyl-aspartyl succinimide bond producing the tripeptide N-Ac-Val-Try-Pro. The rate of this reaction is equal for both L- and D-Asu-containing peptides and is 10-fold greater that the rate of cleavage of a corresponding peptide containing a normal Pro-Asp linkage. When the aspartyl succinimide ring was replaced with an isoaspartyl residue, the cleavage rate was about 5 times that of the normal Pro-Asp peptide. The tripeptide-producing activity copurified on DEAE-cellulose chromatography with an activity that cleaves N-carbobenzoxy-Gly-Pro-4-methylcoumarin-7-amide, a post-proline endopeptidase substrate. These two activities were both inhibited by an antiserum to rat brain post-proline endopeptidase, and it appears that they are catalyzed by the same enzyme. This enzyme has a molecular weight of approximately 80,000 and is covalently labeled and inhibited by (/sup 3/H) diisopropyl fluorophosphate. The facile cleavage of the succinimide- and isoaspartyl-containing peptides by this post-proline endopeptidase suggests that it may play a role in the metabolism of peptides containing altered aspartyl residues.

  6. Acetic Acid Can Catalyze Succinimide Formation from Aspartic Acid Residues by a Concerted Bond Reorganization Mechanism: A Computational Study

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    Ohgi Takahashi


    Full Text Available Succinimide formation from aspartic acid (Asp residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe as a model compound, we propose the possibility that acetic acid (AA, which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

  7. Acetic acid can catalyze succinimide formation from aspartic acid residues by a concerted bond reorganization mechanism: a computational study. (United States)

    Takahashi, Ohgi; Kirikoshi, Ryota; Manabe, Noriyoshi


    Succinimide formation from aspartic acid (Asp) residues is a concern in the formulation of protein drugs. Based on density functional theory calculations using Ace-Asp-Nme (Ace = acetyl, Nme = NHMe) as a model compound, we propose the possibility that acetic acid (AA), which is often used in protein drug formulation for mildly acidic buffer solutions, catalyzes the succinimide formation from Asp residues by acting as a proton-transfer mediator. The proposed mechanism comprises two steps: cyclization (intramolecular addition) to form a gem-diol tetrahedral intermediate and dehydration of the intermediate. Both steps are catalyzed by an AA molecule, and the first step was predicted to be rate-determining. The cyclization results from a bond formation between the amide nitrogen on the C-terminal side and the side-chain carboxyl carbon, which is part of an extensive bond reorganization (formation and breaking of single bonds and the interchange of single and double bonds) occurring concertedly in a cyclic structure formed by the amide NH bond, the AA molecule and the side-chain C=O group and involving a double proton transfer. The second step also involves an AA-mediated bond reorganization. Carboxylic acids other than AA are also expected to catalyze the succinimide formation by a similar mechanism.

  8. Succinimide Formation from an NGR-Containing Cyclic Peptide: Computational Evidence for Catalytic Roles of Phosphate Buffer and the Arginine Side Chain

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    Ryota Kirikoshi


    Full Text Available The Asn-Gly-Arg (NGR motif and its deamidation product isoAsp-Gly-Arg (isoDGR have recently attracted considerable attention as tumor-targeting ligands. Because an NGR-containing peptide and the corresponding isoDGR-containing peptide target different receptors, the spontaneous NGR deamidation can be used in dual targeting strategies. It is well known that the Asn deamidation proceeds via a succinimide derivative. In the present study, we computationally investigated the mechanism of succinimide formation from a cyclic peptide, c[CH2CO-NGRC]-NH2, which has recently been shown to undergo rapid deamidation in a phosphate buffer. An H2PO4− ion was explicitly included in the calculations. We employed the density functional theory using the B3LYP functional. While geometry optimizations were performed in the gas phase, hydration Gibbs energies were calculated by the SM8 (solvation model 8 continuum model. We have found a pathway leading to the five-membered ring tetrahedral intermediate in which both the H2PO4− ion and the Arg side chain act as catalyst. This intermediate, once protonated at the NH2 group on the five-membered ring, was shown to easily undergo NH3 elimination leading to the succinimide formation. This study is the first to propose a possible catalytic role for the Arg side chain in the NGR deamidation.

  9. The study of new complex compounds of Ni (II) and Co (II) with N- hydroxy-succinimide and their potential applications as sensors (United States)

    Sibiescu, Doina; Tutulea, Mihaela-Dana; Mîţă, Carmen; Stan, Corneliu; Roţca, Ioan; Vizitiu, Mihaela


    In this paper, the study of obtaining new coordination compounds of Ni (II) and Co(II) using as ligand, N-hydroxy-succinimide, was presented. Also, the stability constants of these compounds in aqueous medium were determined. The obtaining conditions and the stability of the new compounds were accomplished in aqueous solutions using characteristic methods for coordination compounds: pH-metry, conductometry and UV-VIS absorption spectroscopy. The combination ratios and the stability constants were determined with methods characteristic for studies in solutions. From experimental data resulted that the combination ratio of central metallic atoms with the ligand N-hydroxy-succinimide was: 1:1 and respectively 1:2. In the experiments were used salts of NiCl2.6H2O and CoCl2.6H2O. The optimal domain of pH stability of the studied compounds is limited between 5.74 - 5.86 for Co- N-hydroxy-succinimide (for molar ratio 1:1 and 1:2) and respectively 5.69 - 5.87 for Ni-N-hydroxysuccinimide( for molar ratio 1:1 and 1:2, too). It is important to mention that these compounds were used with very good results in determination of wastewaters from textile, metallurgical, chemical and food industry. Complexion reactions with this ligand are very sensitive for the cations in this paper mentioned. Therefore it is used most often with success in analytical chemistry and also it is posibil to use as sensors. The new complex compounds has electronics transitions at λ = 517 nm for both complexes Co-N-hydroxy-succinimide at molar ratio 1:1 and 1:2 and also at the same λ = 397nm for Ni-N-hydroxysuccinimide at molar ratio 1:1 and 1:2. These complexes compounds was separated and recrystallized from aqueous solution. From the spectrophotometric data it was determined the type and the nature of the electronics transitions by Dq parameters.

  10. The release characteristics of a model protein from self-assembled succinimide-terminated poly(lactide-co-glycolide ethylene oxide fumarate) nanoparticles

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    Mercado, Angel E; He Xuezhong; Xu Weijie; Jabbari, Esmaiel [Biomimetic Materials and Tissue Engineering Laboratories, Department of Chemical Engineering, University of South Carolina, SC 29208, Columbia (United States)], E-mail:


    Lactide-co-glycolide-based functionalized nanoparticles (NPs), because of their high surface areas for conjugation and biodegradability, are attractive as carriers for stabilization and sustained delivery of therapeutic agents and protein drugs. The objective of this work was to compare the release characteristics of model molecules encapsulated in NPs produced from poly(lactide-co-glycolide fumarate) (PLGF) macromer with those of model molecules conjugated to NPs produced from succinimide (NHS)-terminated PLGF-NHS macromer. Poly(lactide fumarate) (PLAF), PLGF and poly(lactide-co-ethylene oxide fumarate) (PLEOF) macromers were synthesized by condensation polymerization. The hydroxyl end-groups of PLAF and PLGF macromers were reacted with N,N{sup '}-disuccinimidyl carbonate (DSC) to produce succinimide-terminated PLAF-NHS and PLGF-NHS macromers. The macromers were self-assembled by dialysis to form NPs. The amphiphilic PLEOF macromer was used as the surfactant to stabilize the NPs in the process of self-assembly. 1-(2-pyridylazo)-2-naphthol (PAN) was used as a model small molecule for encapsulation in PLAF or PLGF NPs and bovine serum albumin (BSA) was used as a model protein for conjugation to PLAF-NHS and PLGF-NHS NPs. The profile of release of the encapsulated PAN from PLAF and PLGF NPs was non-linear and consisted of a burst release followed by a period of sustained release. The release profile for BSA, conjugated to PLAF-NHS and PLGF-NHS NPs, was linear up to complete degradation of the NPs. PLGF and PLAF NPs degraded in 15 and 28 days, respectively, while PLGF-NHS and PLAF-NHS NPs degraded in 25 and 38 days, which demonstrated that the release was dominated by erosion of the matrix. PLAF-NHS and PLGF-NHS NPs are potentially useful as carriers for sustained in situ release of protein drugs.

  11. The electrophilic lodi nation with 125 I/ 131 I of gamma globulin: Comparison between a solid-phase oxidizing agent (Iodogen), chloramine-T, iodine mono chloride and N-Bromo succinimide

    International Nuclear Information System (INIS)

    El-Wetery, A.S.; Ayyoub, S.; El-Mohty, A.A.; Raieh, M.; Ghonaim, A.Kh.


    A new available oxidizing agent, 1, 3, 4, 6-tetra chloro-3α, diphenyl glycoluril(iodogen) was compared with chloramine-T (Ch-T), Iodine-mono chloride (I Cl) and N-Bromo succinimide (NBS) in the radio-iodination of gamma-globulin (γ-G) with 'no-carrier-added' (nca) Na 131 I (T 1/2=8 d). In Phosphate and acetate buffer solution, the optimum reaction conditions with respect to PH, concentration of oxidizing agent, reaction time and concentration of γ-G were determined. The optimum conditions which were found require 100μ l of buffer (pH 7.4, 0.025 M), 500μg γ-G (0.003 μmol), (10-40) μg of oxidizing agent and the desired amount of carrier-free radioiodine. Highest radiochemical yield (>85-90%) were obtained at pH 7.4. Separation and identification of the labelled products were achieved by means of high performance liquid chromatography (HPLC) and thin layer chromatography (TLC). 8 figs., 1 tab

  12. succinimide-n-sulfonic acid as an efficient recyclable catalyst

    African Journals Online (AJOL)

    In view of the pharmaceutical importance of heterocyclic compounds containing coumarin moiety, various approaches toward the synthesis of this class of compounds have been explored. [13-17]. Although these methods are quite satisfactory, most of these methods suffer from extended reaction times, low yields, use of ...

  13. Investigation of the structure of isobutylene oligomers, used in the capacity of stock for succinimide additives, by the method of proton magnetic resonance

    Energy Technology Data Exchange (ETDEWEB)

    Iarmoliuk, V.M.; Garun, Ia.E.; Ostroverkhov, V.G.; Pustovit, V.E.; Tikhonov, V.P.


    By the method of proton magnetic resonance, investigation was made of the structure of hydrocarbon framework of isobutylene oligomers of the Salavatsk petrochemical plant, produced by the cation polymerization of the isobutane isobutylene fraction at a temperature from -10 to +20/sup 0/ with A1C1/sub 3/ and used in the production of succinide additives. Determination was made of the qualitative and quantitative compositions of the various structures in the oligomers. It is shown, that as a rule, oligobutenes are not pure oligomers of isobutylene, but represent, at least, fragments of four structures, which can be formed on the basis of isobutylene. The content of the given structures does not affect the reaction capacity of the oligomers in respect to the maleic anhydride. It was established, that the low molecular fraction, contained in oligomers up to 5%, are not isobutylene oligomers, but represent a low molecular polymer of butene-2 and its copolymer with isobutylene.

  14. Molar extinction coefficients of solutions of some organic compounds

    Indian Academy of Sciences (India)

    (C4H8O2), succinimide (C4H5NO2) as estimated from the measured absorbance of. 7 radiations in their ... species in the solution and ε is called the molar absorptivity or extinction coefficient. (l mol-1cm-1 or ... Integration of eq. (4) leads to.

  15. Transition Metal Complex/Polymer Systems as Optical Limiting Materials (United States)


    set of Ru and Os compounds at 800 nm wavelength using a z-scan method. Extinction coefficients for the compounds at 800 nm are also shown...visioned. It lts have bee escribing th ency 2DIR oiety and c E4C shows nds on the e succinimid function of on the gra rgy transpor s provide a fer

  16. American Peptide Symposium Proceedings (13th) Held in Edmonton, Alberta, Canada on June 20-25, 1993. Peptides: Chemistry, Structure and Biology (United States)


    protein . rceloMEIS w-mdeimidobenzol UtMA I,-Nfoer N-hydroxy- SONaY succinimide ester iniraioa units 14CM melanin concentrating luM keyhole limpet MCPSA...cyclic depsipeptide aureobasidin A (1), isolated as a major component from the culture medium of the black yeast Aureobasidium puUukans R 106, exhibits

  17. Synthesis of phosphorylated calix[4]arene derivatives for the design of solid phases immobilizing uranyl cations

    International Nuclear Information System (INIS)

    Maroun, E.B.; Hagege, A.; Asfari, Z.; Basset, CH.; Quemeneur, E.; Vidaud, C.


    With the aim of developing supports for uranyl cations immobilisation, new 1, 3-alternate calix[4]arenes bearing both phosphonic acid functions as chelating sites and N-succinimide-4-oxa-butyrate as the anchoring arm were synthesised in good yields. The coupling of such calixarenes to a gel was performed and a successful immobilisation of uranyl cations was obtained. (authors)

  18. Synthesis of phosphorylated calix[4]arene derivatives for the design of solid phases immobilizing uranyl cations

    Energy Technology Data Exchange (ETDEWEB)

    Maroun, E.B.; Hagege, A.; Asfari, Z. [Laboratoire de Chimie Analytique et Minerale, UMR 7178 ULP/CNRS/IN2P3 LC4, ECPM, Strasbourg Cedex (France); Basset, CH.; Quemeneur, E.; Vidaud, C. [CEA IBEB, SBTN, Centre de Marcoule, Bagnols-sur-Ceze (France)


    With the aim of developing supports for uranyl cations immobilisation, new 1, 3-alternate calix[4]arenes bearing both phosphonic acid functions as chelating sites and N-succinimide-4-oxa-butyrate as the anchoring arm were synthesised in good yields. The coupling of such calixarenes to a gel was performed and a successful immobilisation of uranyl cations was obtained. (authors)

  19. Browse Title Index

    African Journals Online (AJOL)

    Items 801 - 850 of 1007 ... Vol 23, No 3 (2009), Solventless migratory-insertion reactions of substituted cyclopentadienyl iron complexes induced by electron donor ligands, Abstract .... Vol 29, No 3 (2015), Succinimide-N-sulfonic acid as an efficient recyclable catalyst for the synthesis of some fused indolo pyrano pyrimidinone ...

  20. A new technique of tritium labelling of neuraminidase from Vibrio cholerae

    International Nuclear Information System (INIS)

    Keune, D.


    By acylation of the free amino groups of the enzyme neuraminidase from Vibrio cholerae using N-[(2,3 - 3 H)-propionyloxy]-succinimide it was possible to transfer tritium-labelled propionyl groups to free amino groups of the enzyme glycoprotein. It was established by preliminary trials that a certain minimum concentration of protein was necessary to achieve a satisfactory degree of acylation. After the various processing stages, the acylation of neuraminidase with N-[(2,3 - 3 H)-propionyloxy]-succinimide led to the incorporation of 5.26 μCi radioactivity per mg enzymal protein. Comparison with a known method for neuraminidase labelling showed that the new process is more effective in terms of incorporation of radioactivity. Enzyme activity is inhibited by both methods. (orig./MG) [de

  1. N-tritioacetoxyphthalimide: A new high specific activity tritioacetylating reagent

    International Nuclear Information System (INIS)

    Saljoughian, M.; Morimoto, Hiromi; Than, Chit


    The authors' aim was to develop a nonvolatile, stable, and facile tritioacetylating reagent and to demonstrate its use on simple peptides. Accordingly, the authors made the synthesis of high specific activity N-(tritioacetoxy) derivatives of succinimide, phthalimide, and naphthalimide a major focus. As the preferred approach, N-(tritioacetoxy)phthalimide was prepared by radical dehalogenation of N-(iodoacetoxy)phthalimide using high specific activity tributyltin tritide. This tritiated acetylation reagent was characterized by 3 H and 1 H NMR spectroscopy and by radio-HPLC. Efficacy of the reagent was investigated by tritioacetylation of several peptides at their N-terminal amino group. 26 refs., 1 fig

  2. New coordination compounds of Cr(III) used in leather tanning (United States)

    Crudu, Marian; Sibiescu, Doina; Rosca, Ioan; Sutiman, Daniel; Vizitiu, Mihaela; Apostolescu, Gabriela


    Some new coordination compounds of Cr(III) using as ligand N-hydroxy - succinimide, were obtained and studied. The combination ratio, central atom: ligand were 1:1; 1:2 and 1:3. The new complex compounds were studied using UV-Vis spectroscopy, conductance and pH measurements. The studies of obtaining and of stability of the new compounds were accomplished in aqueous solutions using methods characteristic for coordination compounds: conductance and pH measurements. The combination ratios and the stability constants were determined with methods characteristic for studies in solutions.

  3. Chromenoquinoline-based thiol probes: a study on the quencher position for controlling fluorescent Off-On characteristics. (United States)

    Kand, Dnyaneshwar; Kalle, Arunasree Marasanapalli; Talukdar, Pinaki


    The design, synthesis and thiol sensing ability of chromenoquinoline-based fluorescent probes 4, 5 and 6 and are reported here. The relative position of the maleimide moiety was varied along the chromenoquinoline fluorophore to decrease the background fluorescence. Lower background fluorescence in probes 4 and 6 was rationalized by the smaller k(r)/k(nr) values compared to that of probe 5. An intramolecular charge transfer (ICT) mechanism was proposed for quenching and the extent was dependent on the position of the maleimide quencher. Fluorescent Off-On characteristics were evaluated by theoretical calculations. All probes were selective only towards thiol containing amino acids. Thiol sensing by probes 4 and 6 were much better compared to 5. Probe 4 displayed a better fluorescence response for less hindered thiol (185-, 223- and 156-fold for Hcy, Cys and GSH, respectively), while for probe 6, a higher enhancement in fluorescence was observed with more hindered thiols (180-, 205- and 245-fold for Hcy, Cys and GSH, respectively). The better response to bulkier thiol, GSH by probe 6 was attributed to the steric crowding at the C-4 position and bulkiness of the GSH group which force the succinimide unit to be in a nearly orthogonal conformation. This spatial arrangement was important in reducing the fluorescence quenching ability of the succinimide moiety. The application of probes 4, 5 and 6 was demonstrated by naked eye detection thiols using a 96-well plate system as well as by live-cell imaging.

  4. Preparative and mechanistic studies toward the rational development of catalytic, enantioselective selenoetherification reactions. (United States)

    Denmark, Scott E; Kalyani, Dipannita; Collins, William R


    A systematic investigation into the Lewis base catalyzed, asymmetric, intramolecular selenoetherification of olefins is described. A critical challenge for the development of this process was the identification and suppression of racemization pathways available to arylseleniranium ion intermediates. This report details a thorough study of the influences of the steric and electronic modulation of the arylselenenyl group on the configurational stability of enantioenriched seleniranium ions. These studies show that the 2-nitrophenyl group attached to the selenium atom significantly attenuates the racemization of seleniranium ions. A variety of achiral Lewis bases catalyze the intramolecular selenoetherification of alkenes using N-(2-nitrophenylselenenyl)succinimide as the electrophile along with a Brønsted acid. Preliminary mechanistic studies suggest the intermediacy of ionic Lewis base-selenium(II) adducts. Most importantly, a broad survey of chiral Lewis bases revealed that 1,1'-binaphthalene-2,2'-diamine (BINAM)-derived thiophosphoramides catalyze the cyclization of unsaturated alcohols in the presence of N-(2-nitrophenylselenenyl)succinimide and methanesulfonic acid. A variety of cyclic seleno ethers were produced in good chemical yields and in moderate to good enantioselectivities, which constitutes the first catalytic, enantioselective selenofunctionalization of unactivated olefins.

  5. Anti-Candida albicans biofilm effect of novel heterocyclic compounds. (United States)

    Kagan, Sarah; Jabbour, Adel; Sionov, Edward; Alquntar, Abed A; Steinberg, Doron; Srebnik, Morris; Nir-Paz, Ran; Weiss, Aryeh; Polacheck, Itzhack


    The aims of this study were to develop new anti-biofilm drugs, examine their activity against Candida albicans biofilm and investigate their structure-activity relationship and mechanism of action. A series of thiazolidinedione and succinimide derivatives were synthesized and their ability to inhibit C. albicans biofilm formation and destroy pre-formed biofilm was tested. The biofilms' structure, metabolic activity and viability were determined by XTT assay and propidium iodide and SYTO 9 live/dead stains combined with confocal microscopic analysis. The effect of the most active compounds on cell morphology, sterol distribution and cell wall morphology and composition was then determined by specific fluorescent stains and transmission electron microscopy. Most of the compounds were active at sub-MICs. Elongation of the aliphatic side chain resulted in reduced anti-biofilm activity and the sulphur atom contributed to biofilm killing, indicating a structure-activity relationship. The compounds differed in their effects on biofilm viability, yeast-to-hyphal form transition, hyphal morphology, cell wall morphology and composition, and sterol distribution. The most effective anti-biofilm compounds were the thiazolidinedione S8H and the succinimide NA8. We developed novel anti-biofilm agents that both inhibited and destroyed C. albicans biofilm. With some further development, these agents might be suitable for therapeutic purposes.

  6. Influence of Antioxidant Addition in Jatropha Biodiesel on the Performance, Combustion and Emission Characteristics of a DI Diesel Engine (United States)

    Arockiasamy, Prabu; Ramachandran Bhagavathiammal, Anand


    An experimental investigation is conducted on a single-cylinder DI diesel engine, to evaluate the performance, combustion and emission characteristics of Jatropha biodiesel with the addition of antioxidants namely, Succinimide (C4H5NO2), N,N-Dimethyl p-phenylenediamine dihydrochloride (C8H14Cl2N2) and N-Phenyl- p-phenylenediamine (C6H5NHC6H4NH2) at 500, 1000 and 2000 ppm. The performance, combustion and emission characteristic tests are conducted at a constant speed of 1500 rpm, injection pressure of 215 bar, injection timing of 26° before top dead centre for the nine test fuels and the experimental results are compared with neat diesel and neat biodiesel as base fuels. The experimental results show that the addition of antioxidant in biodiesel suppresses the NO emission by quenching the OH radicals that are produced by the reaction of hydrocarbon radicals with molecular nitrogen. The maximum percentage reduction of NO emission by 5, 6 and 7% are observed for N-Phenyl- p-phenylenediamine, N,N-Dimethyl p-phenylenediamine dihydrochloride and Succinimide blended test fuels at 2000 ppm antioxidant addition with biodiesel.

  7. Synthesis and characterization of poly(lactic acid)/ montmorillonite nanocomposites by in situ polycondensation catalyzed by non-metal-based compound. (United States)

    Kaewprapan, Kulwadee; Phattanarudee, Siriwan


    Poly(lactic acid)/montmorillonite nanocomposites were prepared by using non-toxic catalysts, i.e., phthalic acid and succinimide, via in situ polycondensation in presence of silicate. Concentrations of catalysts and clay were varied in a range of 0-3% wt and 0-0.5% wt, respectively. The reaction condition was controlled at 180 degrees C for 24 hr under a reduced pressure. Viscosity average molecular weight of the synthesized polymers and nanocomposites were characterized and compared using an Ubbelohde viscometer. Pattern of silicate distribution in the composites was investigated by X-ray diffraction to correlate with thermal properties evaluated by differential scanning calorimetry and thermogravimetric analysis. The results showed that the addition of catalysts at 2% wt gave the highest product yield (55-60%). The presence of silicate affected on molecular weight reduction, and the diffracted patterns suggested an intercalated structure. With a small amount of added filler, a significant improvement in thermal property and crystallinity of the resultant composites was obtained compared to those of the catalyzed polymers, in which the composites with succinimide exhibited overall better thermal stability and higher crystallinity than the ones prepared with phthalic acid.

  8. Non-Cyanide Electrodeposited Ag–PTFE Composite Coating Using Direct or Pulsed Current Deposition

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    Raymond Sieh


    Full Text Available The effects of FC-4 cationic surfactant on electrodeposited Ag–PTFE composite coating using direct or pulsed currents were studied using scanning electron microscope (SEM, energy dispersive X-ray (EDS, optical microscope, and a linear tribometer. FC-4:PTFE in various ratios were added to a non-cyanide succinimide silver complex bath. Direct or pulsed current method was used at a constant current density to enable comparison between both methods. A high incorporation rate of PTFE was successfully achieved, with pulsed current being highly useful in increasing the amount of PTFE in the composite coating. The study of coating wear under sliding showed that a large majority of the electrodeposited coatings still managed to adhere to the substrate, even after 10 wear cycles of sliding tests. Performance improvements were achieved on all the samples with a coefficient of friction (CoF between 0.06 and 0.12.

  9. Modified Polyacrylic Acid-Zinc Composites: Synthesis, Characterization and Biological Activity

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    Mohammed Rafi Shaik


    Full Text Available Polyacrylic acid (PAA is an important industrial chemical, which has been extensively applied in various fields, including for several biomedical purposes. In this study, we report the synthesis and modification of this polymer with various phenol imides, such as succinimide, phthalimide and 1,8-naphthalimide. The as-synthesized derivatives were used to prepare polymer metal composites by the reaction with Zn+2. These composites were characterized by using various techniques, including NMR, FT-IR, TGA, SEM and DSC. The as-prepared PAA-based composites were further evaluated for their anti-microbial properties against various pathogens, which include both Gram-positive and Gram-negative bacteria and different fungal strains. The synthesized composites have displayed considerable biocidal properties, ranging from mild to moderate activities against different strains tested.

  10. Synthesis and characterization of in situ photogelable polysaccharide derivative for drug delivery. (United States)

    Hu, Rong; Chen, Yu-Yun; Zhang, Li-Ming


    A novel polysaccharide derivative with photoreactivity was prepared by the conjugation of carboxymethylated chitosan with N-hydroxyl succinimide-activated nitrocinnamate in the presence of N,N-dicyclohexylcarbodiimide, and characterized by IR, (1)H NMR, UV-vis and rheological analyses. It was found that such a modified polysaccharide could exhibit an unique photogelation ability in the absence of potentially toxic photoinitiator or catalyst and be suitable particularly for the in situ preparation of photocrosslinked hydrogel biomaterials. By changing the photoirradiation time and incorporated nitrocinnamate content, its photogelation property could be modulated. For the resultant hydrogels incorporated with various nitrocinnamate contents, their properties such as swelling, viscoelasticity, in vitro biodegradation and drug release were investigated. In addition, the photogelation mechanism of this polysaccharide derivative was also discussed. 2010 Elsevier B.V. All rights reserved.

  11. Ballistic energy transport in PEG oligomers

    Directory of Open Access Journals (Sweden)

    Kireev Victor V.


    Full Text Available Energy transport between the terminal groups of the azido-PEG-succinimide ester compounds with a number of repeating PEG units of 0, 4, 8, and 12 was studied using relaxation-assisted two-dimensional infrared spectroscopy. The through-bond energy transport time, evaluated as the waiting time at which the cross peak maximum is reached, Tmax, was found to be linearly dependent on the chain length for chain lengths up to 60 Å suggesting a ballistic energy transport regime. The through-bond energy transport speed is found to be ca. 500 m/s. The cross-peak amplitude at the maximum decays exponentially with the chain length with a characteristic decay distance of 15.7 ± 1 Å. Substantial mode delocalization across the PEG bridge is found, which can support the energy propagation as a wavepacket.

  12. Detection of the in vivo conversion of 2-pyrrolidinone to gamma-aminobutyric acid in mouse brain. (United States)

    Callery, P S; Stogniew, M; Geelhaar, L A


    Labeled gamma-aminobutyric acid was detected in mouse brain following intravenous injections of deuterium labeled 2-pyrrolidinone. [2H6]Pyrrolidinone was prepared by the reduction of [2H4]succinimide with lithium aluminum deuteride. Quantification was accomplished by a gas chromatography mass spectrometry assay method. gamma-Aminobutyric acid and internal standard, 5-aminovaleric acid, were converted to volatile derivatives by treatment with N,N-dimethylformamide dimethyl acetal. Quantitative estimates were derived from peak area measurements obtained from monitoring the parent ions of the gamma-aminobutyric acid and internal standard derivatives by repetitive scanning during the GC run. The conversion of pyrrolidinone to gamma-aminobutyric acid may provide a method for labeling central gamma-aminobutyric acid pools.

  13. KH 2PO4 as a novel catalyst for regioselective monobromination of aralkyl ketones using N-bromosuccinimide: a green methodology

    Directory of Open Access Journals (Sweden)

    P. Md. Khaja Mohinuddin


    Full Text Available A simple, regioselective and green method has been developed for the preparation of monobrominated ketones from various aralkyl ketones by using N-bromosuccinimide in presence of KH 2PO 4 in EtOH at reflux temperature. The present method is of short reaction time and simple with excellent isolated yields of products. The use of eco-friendly solvent, reuse of organic waste (succinimide and recyclable catalyst used for 4 times without loss of activity are advantageous. This is the first example of the use of KH 2PO 4 as a useful catalyst in organohalogen chemistry and the present method meets reduce-reuse-recycle (RRR principle towards development of green protocol.

  14. Preparation of deuterated succinic acids

    Energy Technology Data Exchange (ETDEWEB)

    Tashiro, Masashi; Tsuzuki, Hirohisa; Goto, Hideyuki; Ogasahara, Shoji; Mataka, Shuntaro (Kyushu Univ., Fukuoka (Japan)); Isobe, Shin-ichiro; Yonemitsu, Tadashi (Kyushu Sangyo Univ., Fukuoka (Japan). Dept. of Industrial Chemistry)


    Succinic (2,3-{sup 2}H{sub 2})- and (2,2,3,3-{sup 2}H{sub 4})-acids were prepared from maleic anhydride and dimethyl fumarate, and acetylene dicarboxylic acid and its dimethyl ester by treatment with Cu-Al and Ni-Al alloys in 10% NaOD-D{sub 2}0 in 95% to 100% isotopic purity. The succinic {sup 2}H{sub 4} acid having high isotopic purity was also obtained on the hydrolysis of 1,2-ethanedinitrile with alkaline deuterium oxide. Based on the {sup 1}H({sup 2}H) spectra analysis of N-(o-biphenyl)(2,3-{sup 2}H{sub 2})succinimide, it was elucidated that the Raney alloy reduction with alkaline deuterium oxide proceeds stepwise. (author).

  15. Reaction of vanadocene and its monochloride with salts of nitrogen-containing heterocycles

    International Nuclear Information System (INIS)

    Gordetsov, A.S.; Pereshein, V.V.; Lyatyaeva, V.N.; Mar'in, V.P.; Rukevich, OS.; Zakharova, E.S.


    The reaction of bis(eta 5 -cyclopentadienyl) vanadium (Cp 2 V) and its monochloride with diallyl isocyanurates of Ag and alkali metals and with Ag salt of succinimide is used for preparing almost inaccessible Cp 2 V-substituted nitrogen-containing heterocycles. The reactions have been performed in toluene in an evacuated ampoule at room temperature and at 120-130 deg C in case of diallyl isocyanurates of alkali metals. Reaction yields element analysis data, DTA, IR spectra and ESR spectra. Cp 2 V easily reduces Ag from its salts with nitrogen-containing heterocycles oxidizing with production of V(3) sandwich derivatives In exchange reactions of Cp 2 VCl with diallyl isocyanurate salts of alkali metals and silver Cp 2 V-diallyl isocyanurate with an oxo-structure is prosduced as well. The produced AgCl subsequently oxidizers Cp 2 VCl up to Cp 2 VCl 2

  16. Active Immunization and Evaluation Against Luteinizing Hormone for Radioimmunoassay Technique in Human Serum

    International Nuclear Information System (INIS)

    Ebeid, N.H.; Shafik, H.M.; Ayoub, S.M.; Mehany, N.L.


    This study evaluated the antigenicity of luteinizing hormone conjugate with Bovine Serum Albumin (LH-BSA). The conjugation of LH- BSA was carried out by 1-Ethyl-3-(3-Dimethylaminopropyl) Carbodiimide HCl (ECDI). Three rabbits were immunized against LH-BSA. Two rabbits were immunized against nonconjugated LH and two rabbits against BSA only. Immunization was carried out through primary injection and 4 boosters. The preparation of the radioiodinated 125 I-LH was carried out using N- Bromo-Succinimide as oxidizing agent. The preparation of LH standards was carried out. The obtained LH antisera were characterized of titer, immuno response and displacement profile formulation, optimization and validation of the local liquid phase LH- Radioimmunoassay (RIA) system was carried out. The results provide a highly sensitive and accurate RIA system of LH-BSA. This technique could be used in measuring LH in human serum to investigate fertility especially disorders of the hypothalamic / pituitary / gonadal axis

  17. Immobilized enzymes in blood plasma exchangers via radiation grafting (United States)

    Gombotz, Wayne; Hoffman, Allan; Schmer, Gottfried; Uenoyama, Satoshi

    The enzyme asparaginase was immobilized onto a porous hollow polypropylene (PP) fiber blood plasma exchange device for the treatment of acute lymphocytic leukemia. The devices were first radiation grafted with polymethacrylic acid (poly(MAAc)). This introduces carboxyl groups onto the surface of the fibers. Several variables were studied in the grafting reaction including the effects of solvent type and monomer concentration. The carboxyl groups were activated with N-hydroxy succinimide (NHS) using carbodiimide chemistry. Asparaginase was then covalently immobilized on the activated surfaces. Quantitative relationships were found relating the percent graft to the amount of immobilized enzyme which was active. The enzyme reactor was tested both in vitro and in vivo using a sheep as an animal model.

  18. pH-responsive poly(aspartic acid) hydrogel-coated magnetite nanoparticles for biomedical applications. (United States)

    Vega-Chacón, Jaime; Arbeláez, María Isabel Amaya; Jorge, Janaina Habib; Marques, Rodrigo Fernando C; Jafelicci, Miguel


    A novel multifunctional nanosystem formed by magnetite nanoparticles coated with pH-responsive poly(aspartic acid) hydrogel was developed. Magnetite nanoparticles (Fe 3 O 4 ) have been intensively investigated for biomedical applications due to their magnetic properties and dimensions similar to the biostructures. Poly(aspartic acid) is a water-soluble, biodegradable and biocompatible polymer, which features makes it a potential candidate for biomedical applications. The nanoparticles surface modification was carried out by crosslinking polysuccinimide on the magnetite nanoparticles surface and hydrolyzing the succinimide units in mild alkaline medium to obtain the magnetic poly(aspartic acid) hydrogel. The surface modification in each step was confirmed by DRIFTS, TEM and zeta potential measurements. The hydrodynamic diameter of the nanosystems decreases as the pH value decreases. The nanosystems showed high colloidal stability in water and no cytotoxicity was detected, which make these nanosystems suitable for biomedical applications. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Radioimmunoassay of cholecystokinin in human plasma

    International Nuclear Information System (INIS)

    Byrnes, D.J.; Henderson, L.; Borody, T.; Rehfeld, J.F.


    A sensitive radioimmunoassay for cholecystokinin (CCK) has been developed. Porcine CCK-33 was labelled by conjugation with 125 I-hydroxyphenyl-propionic acid succinimide ester. Antibodies were raised against porcine CCK-33 covalently coupled to egg albumin. Plasma samples were extracted with 96% ethanol prior to assay. Free and bound hormone were separated by dextran-coated charcoal. The antibodies bound CCK-8 and CCK-33 with equimolar potency. The assay detection limit was 1 pmol/l plasma. Within and between assay coefficients of variation were +-12.7 and 13.0% at mean plasma CCK concentrations of 13.2 and 13.6 pmol/l. The concentration of CCK in 47 normal fasting subjects ranged from undetectable to 22 pmol/l. Ingestion of a mixed meal in 9 normal subjects increased the plasma concentration from 8.3 +- 2.5 S.E. to 24.4 +- 6.5 pmol/l. (Auth.)

  20. Design of multimodal degradable hydrogels for controlled therapeutic delivery (United States)

    Kharkar, Prathamesh Madhav

    Hydrogels are of growing interest for the delivery of therapeutics to specific sites in the body. For localized drug delivery, hydrophilic polymeric precursors often are laden with bioactive moieties and then directly injected to the site of interest for in situ gel formation. The release of physically entrapped cargo is dictated by Fickian diffusion, degradation of the drug carrier, or a combination of both. The goal of this work was to design and characterize degradable hydrogel formulations that are responsive to multiple biologically relevant stimuli for degradation-mediated delivery of cargo molecules such as therapeutic proteins, growth factors, and immunomodulatory agents. We began by demonstrating the use of cleavable click linkages formed by Michael-type addition reactions in conjunction with hydrolytically cleavable functionalities for the degradation of injectable hydrogels by endogenous stimuli for controlled protein release. Specifically, the reaction between maleimides and thiols was utilized for hydrogel formation, where thiol selection dictates the degradability of the resulting linkage under thiol-rich reducing conditions. Relevant microenvironments where degradation would occur in vivo include those rich in glutathione (GSH), a tripeptide that is found at elevated concentrations in carcinoma tissues. Degradation of the hydrogels was monitored with rheometry and volumetric swelling measurements. Arylthiol-based thioether succinimide linkages underwent degradation via click cleavage and thiol exchange reaction in the presence of GSH and via ester hydrolysis, whereas alkylthiol-based thioether succinimide linkages only undergo degradation by only ester hydrolysis. The resulting control over the degradation rate within a reducing microenvironment resulted in 2.5 fold differences in the release profile of the model protein, a fluorescently-labeled bovine serum albumin, from dually degradable hydrogels compared to non-degradable hydrogels, where the

  1. Acute behavioral symptomatology at disappearance of epileptiform EEG abnormality. Paradoxical or "forced" normalization. (United States)

    Wolf, P


    Paradoxical or "forced" normalization of the EEG of patients with epilepsy was first described by Landolt in 1953. It refers to conditions where disappearance of epileptiform discharge from the routine scalp EEG is accompanied by some kind of behavioral disorder. The best known of these is a paranoid psychotic state in clear consciousness, which is also known as "alternative" psychosis. Thus, the issue is related to much older observations which indicated a "biological antagonism" between productive psychotic symptomatology and epileptic seizures, which led to the therapy of psychoses with artificially induced convulsions. Apart from psychotic episodes, the clinical manifestations of PN comprise dysphoric states, hysterical and hypochondriacal syndromes, affective disorders, and miscellanea. PN can be observed in both generalized and localization-related epilepsies as a rare complication. A subset where it is more frequently seen are in adults with persistent absence seizures when the latter become finally controlled by succinimide therapy. These seem to be the drugs with the highest hazard of precipitation of PN, but all other AEDs have also been suspected. Sleep disturbance by succinimide treatment may play a crucial role, but a variety of other factors are also involved, including psychosocial factors. The pathogenesis of this condition has given rise to some debate but remains still unresolved. Eleven of the most important hypotheses have been discussed and seem to converge into a more comprehensive hypothesis which basically assumes that, during PN, the epilepsy is still active subcortically, perhaps with spread of discharge along unusual pathways. This activity is supposed to provide energy and, possibly, some of the symptoms included in the psychotic syndrome. A critical clinical condition results, usually with a dysphoric symptomatology, where a development towards psychosis is impending but still depends on the presence or absence of a variety of risk

  2. Functionalization of the hydroxyapatite nanoparticles surface: source of new applications

    International Nuclear Information System (INIS)

    Rojas Sanchez, Leonardo


    The surface of the nano-calcium hydroxyapatite, Ca 10 (PO 4 ) 6 (OH) 2 is reacted with stearic acid, succinic anhydride, succinimide and 2 a minoetil dihydrogenphosphate. Introduction of different functional groups is given onto the surface. An ionic interaction is identified by infrared spectroscopy and Raman between the carboxylate groups of the resulting organic molecules and calcium of the hydroxyapatite. The formation of a P-O-P pyrophosphate type bond has been for 2-aminoethyl dihydrogen phosphate with hydroxyapatite groups. Hydroxyapatite phase was remained in all cases after the reaction as demonstrated by diffraction of x-ray in powder. The amount of spiked molecules is quantified by analysis of thermal degradation which together with the determination of the surface area by BET isotherms of nitrogen adsorption. A degree of surface coverage is estimated by the organic molecules. A maximum percentage of 71% is obtained for the functionalization with succinic anhydride, followed by 57% for the reaction with stearic acid. Dilute suspensions of different materials were prepared for which in phosphate buffer solution have presented two populations around 2 and 5 μm in diameter for the modified particles. The ζ-Potential of various materials was determined occurring a variation in the potential of the unmodified hydroxyapatite. The particles with physicochemical properties different of the starting hydroxyapatite were obtained, this has expanded the range of application of the material. (author) [es

  3. FY1995 development of a novel rapid immunoassay technology; 1995 nendo shinkina kosoku men'eki sokutei gijutsu no kaihatsu

    Energy Technology Data Exchange (ETDEWEB)



    By utilizing the phenomenon that the antigen induces association of VH and VL, which are the antigen-recognizing fragments of antibody, develop a novel rapid immunoassay technology which will eliminate the defects of conventional immunoassays, such as long measuring time and requirement of expensive large automated apparatus. In addition, develop a methodology to make Fvs with low VH-VL affinity but that associate tightly in the presence of antigen. Using a model anti-hen egg lysozyme antibody HyHEL-10 Fv and BlAcore, we have clarified the kinetic basis of the Fv stabilization by HEL. By labeling HyHEL-10 VH, VL with succinimide esters of fluorescein and tetramethylrhodamin, respectively, at their N-termini, we could successfully detect VH-VL association by the increment of fluorescence resonance energy transfer. As a result, 1 {mu} g/ml of antigen could be detected homogeneously in less than 5 minutes. Some VH-VL association measurement systems were made by utilizing M13 phage display of VH fragments of HyHEL-10, anti-nitrophenacetyl, anti-digoxin antibodies. (NEDO)

  4. Electrochemical impedance and spectroscopy study of the EDC/NHS activation of the carboxyl groups on poly(ε-caprolactone/poly(m-anthranilic acid nanofibers

    Directory of Open Access Journals (Sweden)

    Z. Guler


    Full Text Available Electrochemical impedance spectroscopy (EIS and spectroscopy was applied to investigate the surface activation of carboxyl group (–COOH containing nanofibers by the reaction of 1-ethyl-3-(dimethyl-aminopropyl carbodiimide hydrochloride (EDC/N-hydroxyl succinimide (NHS in different concentrations. Poly(!-caprolactone/poly(m-anthranilic acid (PCL/P3ANA nanofibers were fabricated by electrospinning and were activated with 5/0.5, 0.5/5, 5/5 and 50/50 mM of EDC/NHS. The surface activation was investigated by Attenuated Total Reflectance Fourier transform infrared spectroscopy (FTIR-ATR and activation yield was estimated. Albumin was immobilized after surface activation and the amount of covalently immobilized protein was determined by bicinchoninic acid (BCA assay. Morphology and composition of albumin immobilized nanofibers were characterized by Scanning Electron Microscopy/Energy-Dispersive X-ray Spectroscopy (SEM/EDX and Atomic force microscope (AFM. EIS measurements indicated that nanofibers become resistant after albumin immobilization. The obtained data revealed that the highest amount of albumin bound to nanofibers activated with 50/50 mM of EDC/NHS which was found to be the optimum concentration for the activation of PCL/P3ANA nanofibers.

  5. (Au/PANA/PVAc) nanofibers as a novel composite matrix for albumin and streptavidin immobilization

    Energy Technology Data Exchange (ETDEWEB)

    Golshaei, Rana [University of Kashan, Institute of Nano Science and Nano Technology, Kashan, P.O. Box 87317-51167, Islamic Republic of Iran (Iran, Islamic Republic of); Guler, Zeliha [Istanbul Technical University, Nanoscience and Nanoengineering, Maslak, Istanbul 34469 (Turkey); Sarac, Sezai A., E-mail: [Istanbul Technical University, Nanoscience and Nanoengineering, Maslak, Istanbul 34469 (Turkey); Istanbul Technical University, Department of Chemistry and Polymer Science and Technology, Maslak, Istanbul 34469 (Turkey)


    A novel electrospun nanofiber mat (Au/PANA/PVAc) consists of (Gold/Poly Anthranilic acid) (Au/PANA) core/shell nanostructures as a support material for protein immobilization that was developed and characterized by electrochemical impedance spectroscopy. In the core/shells, PANA served carboxyl groups (− COOH) for covalent protein immobilization and Au enhanced the electrochemical properties by acting as tiny conduction centers to facilitate electron transfer. Covalent immobilization of albumin and streptavidin as model proteins onto the (Au/PANA/PVAc) nanofibers was carried out by using 1-ethyl-3-(dimethyl-aminopropyl) carbodiimide hydrochloride (EDC)/N-hydroxyl succinimide (NHS) activation. PVAc nanofibers were compared with Au/PANA/PVAc nanofibers before and after protein immobilization. The successful covalent binding of both albumin and streptavidin onto (Au/PANA/PVAc) nanofibers was confirmed by FTIR-ATR, Electron Microscopy/Energy-Dispersive X-ray Spectroscopy SEM/EDX and Electrochemical impedance spectroscopy (EIS). The nanofibers became resistive due to protein immobilization and the higher charge transfer resistance was observed after higher amount of protein was immobilized. - Highlights: • Au/PANA/PVAc nanofibers with (COOH) groups as a suitable supports for covalent immobilization of proteins. • Increasing of the resistivity of the nanofibers after immobilization of the proteins. • Activation of Au/PANA/PVAc nanofibers by using EDC/NHS.

  6. Label Free QCM Immunobiosensor for AFB1 Detection Using Monoclonal IgA Antibody as Recognition Element

    Directory of Open Access Journals (Sweden)

    Özlem Ertekin


    Full Text Available This study introduces the use of an IgA isotype aflatoxin (AF specific monoclonal antibody for the development of a highly sensitive Quartz Crystal Microbalance (QCM immunobiosensor for the detection of AF in inhibitory immunoassay format. The higher molecular weight of IgA antibodies proved an advantage over commonly used IgG antibodies in label free immunobiosensor measurements. IgA and IgG antibodies with similar affinity for AF were used in the comparative studies. Sensor surface was prepared by covalent immobilization of AFB1, using self assembled monolayer (SAM formed on gold coated Quartz Crystal, with 1-Ethyl-3-(3-dimethylaminopropyl carbodiimide/N-hydroxy succinimide (EDC/NHS method using a diamine linker. Nonspecific binding to the surface was decreased by minimizing the duration of EDC/NHS activation. Sensor surface was chemically blocked after AF immobilization without any need for protein blocking. This protein free sensor chip endured harsh solutions with strong ionic detergent at high pH, which is required for the regeneration of the high affinity antibody-antigen interaction. According to the obtained results, the detection range with IgA antibodies was higher than IgG antibodies in QCM immunosensor developed for AFB1.

  7. A Highly Sensitive Electrochemical DNA Biosensor from Acrylic-Gold Nano-composite for the Determination of Arowana Fish Gender (United States)

    Rahman, Mahbubur; Heng, Lee Yook; Futra, Dedi; Chiang, Chew Poh; Rashid, Zulkafli A.; Ling, Tan Ling


    The present research describes a simple method for the identification of the gender of arowana fish ( Scleropages formosus). The DNA biosensor was able to detect specific DNA sequence at extremely low level down to atto M regimes. An electrochemical DNA biosensor based on acrylic microsphere-gold nanoparticle (AcMP-AuNP) hybrid composite was fabricated. Hydrophobic poly(n-butylacrylate-N-acryloxysuccinimide) microspheres were synthesised with a facile and well-established one-step photopolymerization procedure and physically adsorbed on the AuNPs at the surface of a carbon screen printed electrode (SPE). The DNA biosensor was constructed simply by grafting an aminated DNA probe on the succinimide functionalised AcMPs via a strong covalent attachment. DNA hybridisation response was determined by differential pulse voltammetry (DPV) technique using anthraquinone monosulphonic acid redox probe as an electroactive oligonucleotide label (Table 1). A low detection limit at 1.0 × 10-18 M with a wide linear calibration range of 1.0 × 10-18 to 1.0 × 10-8 M ( R 2 = 0.99) can be achieved by the proposed DNA biosensor under optimal conditions. Electrochemical detection of arowana DNA can be completed within 1 hour. Due to its small size and light weight, the developed DNA biosensor holds high promise for the development of functional kit for fish culture usage.

  8. A technique to prepare boronated B72.3 monoclonal antibody for boron neutron capture therapy

    International Nuclear Information System (INIS)

    Ranadive, G.N.; Rosenzweig, H.S.; Epperly, M.W.


    B72.3 monoclonal antibody has been successfully boronated using mercaptoundecahydro-closo-dodecaborate (boron cage compound). The reagent was incorporated by first reacting the lysine residues of the antibody with m-maleimidobenzoyl succinimide ester (MBS), followed by Michael addition to the maleimido group by the mercapto boron cage compound to form a physiologically stable thioether linkage. Boron content of the antibody was determined by atomic absorption spectroscopy. For biodistribution studies, boronated antibody was radioiodinated with iodogen. 125 I-labeled and boronated B72.3 monoclonal antibody demonstrated clear tumor localization when administered via tail vein injections to athymic nude mice bearing LS174-T tumor xenografts. Boronated antibody was calculated to deliver 10 6 boron atoms per tumor cell. Although this falls short of the specific boron content originally proposed as necessary for boron neutron capture therapy (BNCT), recent calculations suggest that far fewer atoms of 10 B per tumor cell would be necessary to effect successful BNCT when the boron is targeted to the tumor cell membrane. (author)

  9. An Amperometric Biosensor for the Determination of Bacterial Sepsis Biomarker, Secretory Phospholipase Group 2-IIA Using a Tri-Enzyme System

    Directory of Open Access Journals (Sweden)

    Nik Nurhanan Nik Mansor


    Full Text Available A tri-enzyme system consisting of choline kinase/choline oxidase/horseradish peroxidase was used in the rapid and specific determination of the biomarker for bacterial sepsis infection, secretory phospholipase Group 2-IIA (sPLA2-IIA. These enzymes were individually immobilized onto the acrylic microspheres via succinimide groups for the preparation of an electrochemical biosensor. The reaction of sPLA2-IIA with its substrate initiated a cascading enzymatic reaction in the tri-enzyme system that led to the final production of hydrogen peroxide, which presence was indicated by the redox characteristics of potassium ferricyanide, K3Fe(CN6. An amperometric biosensor based on enzyme conjugated acrylic microspheres and gold nanoparticles composite coated onto a carbon-paste screen printed electrode (SPE was fabricated and the current measurement was performed at a low potential of 0.20 V. This enzymatic biosensor gave a linear range 0.01–100 ng/mL (R2 = 0.98304 with a detection limit recorded at 5 × 10−3 ng/mL towards sPLA2-IIA. Moreover, the biosensor showed good reproducibility (relative standard deviation (RSD of 3.04% (n = 5. The biosensor response was reliable up to 25 days of storage at 4 °C. Analysis of human serum samples for sPLA2-IIA indicated that the biosensor has potential for rapid bacterial sepsis diagnosis in hospital emergency department.

  10. Direct determination of N-methyl-2-pyrrolidone metabolites in urine by HPLC-electrospray ionization-MS/MS using deuterium-labeled compounds as internal standard. (United States)

    Suzuki, Yoshihiro; Endo, Yoko; Ogawa, Masanori; Yamamoto, Shinobu; Takeuchi, Akito; Nakagawa, Tomoo; Onda, Nobuhiko


    N-methyl-2-pyrrolidone (NMP) has been used in many industries and biological monitoring of NMP exposure is preferred to atmospheric monitoring in occupational health. We developed an analytical method that did not include solid phase extraction (SPE) but utilized deuterium-labeled compounds as internal standard for high-performance liquid chromatography-electrospray ionization-mass spectrometry using a C30 column. Urinary concentrations of NMP and its known metabolites 5-hydoxy-N-methyl-2-pyrrolidone (5-HNMP), N-methyl-succinimide (MSI), and 2-hydroxy-N-methylsuccinimide (2-HMSI) were determined in a single run. The method provided baseline separation of these compounds. Their limits of detection in 10-fold diluted urine were 0.0001, 0.006, 0.008, and 0.03 mg/L, respectively. Linear calibration covered a biological exposure index (BEI) for urinary concentration. The within-run and total precisions (CV, %) were 5.6% and 9.2% for NMP, 3.4% and 4.2% for 5-HNMP, 3.7% and 6.0% for MSI, and 6.5% and 6.9% for 2-HMSI. The method was evaluated using international external quality assessment samples, and urine samples from workers exposed to NMP in an occupational area.

  11. Autonomic composite hydrogels by reactive printing: materials and oscillatory response. (United States)

    Kramb, R C; Buskohl, P R; Slone, C; Smith, M L; Vaia, R A


    Autonomic materials are those that automatically respond to a change in environmental conditions, such as temperature or chemical composition. While such materials hold incredible potential for a wide range of uses, their implementation is limited by the small number of fully-developed material systems. To broaden the number of available systems, we have developed a post-functionalization technique where a reactive Ru catalyst ink is printed onto a non-responsive polymer substrate. Using a succinimide-amine coupling reaction, patterns are printed onto co-polymer or biomacromolecular films containing primary amine functionality, such as polyacrylamide (PAAm) or poly-N-isopropyl acrylamide (PNIPAAm) copolymerized with poly-N-(3-Aminopropyl)methacrylamide (PAPMAAm). When the films are placed in the Belousov-Zhabotinsky (BZ) solution medium, the reaction takes place only inside the printed nodes. In comparison to alternative BZ systems, where Ru-containing monomers are copolymerized with base monomers, reactive printing provides facile tuning of a range of hydrogel compositions, as well as enabling the formation of mechanically robust composite monoliths. The autonomic response of the printed nodes is similar for all matrices in the BZ solution concentrations examined, where the period of oscillation decreases in response to increasing sodium bromate or nitric acid concentration. A temperature increase reduces the period of oscillations and temperature gradients are shown to function as pace-makers, dictating the direction of the autonomic response (chemical waves).

  12. Electrochemical DNA biosensor for detection of porcine oligonucleotides using ruthenium(II) complex as intercalator label redox

    Energy Technology Data Exchange (ETDEWEB)

    Halid, Nurul Izni Abdullah; Hasbullah, Siti Aishah; Heng, Lee Yook; Karim, Nurul Huda Abd [School of Chemical Sciences and Food Technology, Universiti Kebangsaan Malaysia, 43600 UKM Bangi, Selangor Darul Ehsan (Malaysia); Ahmad, Haslina; Harun, Siti Norain [Chemistry Department, Faculty of Science, Universiti Putra Malaysia, 43400, Serdang, Selangor (Malaysia)


    A DNA biosensor detection of oligonucleotides via the interactions of porcine DNA with redox active complex based on the electrochemical transduction is described. A ruthenium(II) complex, [Ru(bpy){sub 2}(PIP)]{sup 2+}, (bpy = 2,2′bipyridine, PIP = 2-phenylimidazo[4,5-f[[1,10-phenanthroline]) as DNA label has been synthesized and characterized by 1H NMR and mass spectra. The study was carried out by covalent bonding immobilization of porcine aminated DNA probes sequences on screen printed electrode (SPE) modified with succinimide-acrylic microspheres and [Ru(bpy){sub 2}(PIP)]{sup 2+} was used as electrochemical redox intercalator label to detect DNA hybridization event. Electrochemical detection was performed by cyclic voltammetry (CV) and differential pulse voltammetry (DPV) over the potential range where the ruthenium (II) complex was active. The results indicate that the interaction of [Ru(bpy){sub 2}(PIP)]{sup 2+} with hybridization complementary DNA has higher response compared to single-stranded and mismatch complementary DNA.

  13. Chitosan–Collagen Coated Magnetic Nanoparticles for Lipase Immobilization—New Type of “Enzyme Friendly” Polymer Shell Crosslinking with Squaric Acid

    Directory of Open Access Journals (Sweden)

    Marta Ziegler-Borowska


    Full Text Available This article presents a novel route for crosslinking a polysaccharide and polysaccharide/protein shell coated on magnetic nanoparticles (MNPs surface via condensation reaction with squaric acid (SqA. The syntheses of four new types of collagen-, chitosan-, and chitosan–collagen coated magnetic nanoparticles as supports for enzyme immobilization have been done. Structure and morphology of prepared new materials were characterized by attenuated total reflectance Fourier-transform infrared (ATR-FTIR, XRD, and TEM analysis. Next, the immobilization of lipase from Candida rugosa was performed on the nanoparticles surface via N-(3-dimethylaminopropyl-N′-ethylcarbodiimide hydrochloride (EDC/N-hydroxy-succinimide (NHS mechanism. The best results of lipase activity recovery and specific activities were observed for nanoparticles with polymer shell crosslinked via a novel procedure with squaric acid. The specific activity for lipase immobilized on materials crosslinked with SqA (52 U/mg lipase was about 2-fold higher than for enzyme immobilized on MNPs with glutaraldehyde addition (26 U/mg lipase. Moreover, a little hyperactivation of lipase immobilized on nanoparticles with SqA was observed (104% and 112%.

  14. The Effect of Novel Heterocyclic Compounds on Cryptococcal Biofilm (United States)

    Korem, Maya; Kagan, Sarah


    Biofilm formation by microorganisms depends on their communication by quorum sensing, which is mediated by small diffusible signaling molecules that accumulate in the extracellular environment. During human infection, the pathogenic yeast Cryptococcus neoformans can form biofilm on medical devices, which protects the organism and increases its resistance to antifungal agents. The aim of this study was to test two novel heterocyclic compounds, S-8 (thiazolidinedione derivative, TZD) and NA-8 (succinimide derivative, SI), for their anti-biofilm activity against strains of Cryptococcus neoformans and Cryptococcus gattii. Biofilms were formed in a defined medium in 96-well polystyrene plates and 8-well micro-slides. The effect of sub-inhibitory concentrations of S-8 and NA-8 on biofilm formation was measured after 48 h by a metabolic reduction assay and by confocal laser microscopy analysis using fluorescent staining. The formation and development of cryptococcal biofilms was inhibited significantly by these compounds in concentrations below the minimum inhibitory concentration (MIC) values. These compounds may have a potential role in preventing fungal biofilm development on indwelling medical devices or even as a therapeutic measure after the establishment of biofilm. PMID:29371559

  15. Electrochemical Biosensor for Nitrite Based on Polyacrylic-Graphene Composite Film with Covalently Immobilized Hemoglobin

    Directory of Open Access Journals (Sweden)

    Raja Zaidatul Akhmar Raja Jamaluddin


    Full Text Available A new biosensor for the analysis of nitrite in food was developed based on hemoglobin (Hb covalently immobilized on the succinimide functionalized poly(n-butyl acrylate-graphene [poly(nBA-rGO] composite film deposited on a carbon-paste screen-printed electrode (SPE. The immobilized Hb on the poly(nBA-rGO conducting matrix exhibited electrocatalytic ability for the reduction of nitrite with significant enhancement in the reduction peak at −0.6 V versus Ag/AgCl reference electrode. Thus, direct determination of nitrite can be achieved by monitoring the cathodic peak current signal of the proposed polyacrylic-graphene hybrid film-based voltammetric nitrite biosensor. The nitrite biosensor exhibited a reproducible dynamic linear response range from 0.05–5 mg L−1 nitrite and a detection limit of 0.03 mg L−1. No significant interference was observed by potential interfering ions such as Ca2+, Na+, K+, NH4+, Mg2+, and NO3− ions. Analysis of nitrite in both raw and processed edible bird’s nest (EBN samples demonstrated recovery of close to 100%. The covalent immobilization of Hb on poly(nBA-rGO composite film has improved the performance of the electrochemical nitrite biosensor in terms of broader detection range, lower detection limit, and prolonged biosensor stability.

  16. A Potentiometric Formaldehyde Biosensor Based on Immobilization of Alcohol Oxidase on Acryloxysuccinimide-modified Acrylic Microspheres

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    Lee Yook Heng


    Full Text Available A new alcohol oxidase (AOX enzyme-based formaldehyde biosensor based on acrylic microspheres has been developed. Hydrophobic poly(n-butyl acrylate-N-acryloxy-succinimide [poly(nBA-NAS] microspheres, an enzyme immobilization matrix, was synthesized using photopolymerization in an emulsion form. AOX-poly(nBA-NAS microspheres were deposited on a pH transducer made from a layer of photocured and self-plasticized polyacrylate membrane with an entrapped pH ionophore coated on a Ag/AgCl screen printed electrode (SPE. Oxidation of formaldehyde by the immobilized AOX resulted in the production of protons, which can be determined via the pH transducer. Effects of buffer concentrations, pH and different amount of immobilization matrix towards the biosensor’s analytical performance were investigated. The formaldehyde biosensor exhibited a dynamic linear response range to formaldehyde from 0.3–316.2 mM and a sensitivity of 59.41 ± 0.66 mV/decade (R2 = 0.9776, n = 3. The lower detection limit of the biosensor was 0.3 mM, while reproducibility and repeatability were 3.16% RSD (relative standard deviation and 1.11% RSD, respectively (n = 3. The use of acrylic microspheres in the potentiometric formaldehyde biosensor improved the biosensor’s performance in terms of response time, linear response range and long term stability when compared with thick film immobilization methods.

  17. Preparation of gold nanoparticles by microwave heating and application of spectroscopy to study conjugate of gold nanoparticles with antibody E. coli O157:H7

    International Nuclear Information System (INIS)

    Ngo, Vo Ke Thanh; Nguyen, Hoang Phuong Uyen; Huynh, Trong Phat; Tran, Nguyen Nguyen Pham; Lam, Quang Vinh; Huynh, Thanh Dat


    Gold nanoparticles (AuNPs) of 15–20 nm size range have attracted attention for producing smart sensing devices as diagnostic tools in biomedical sciences. Citrate capped AuNPs are negatively charged, which can be exploited for electrostatic interactions with some positively charged biomolecules like antibodies. In this paper we describe a method for the low cost synthesis of gold nanoparticles using sodium citrate (Na_3Ct) reduction in chloroauric acid (HAuCl_4.3H_2O) by microwave heating (diameter about 13–15 nm). Gold nanoparticles were functionalized with surface activation by 3-mercaptopropionic acid for attaching antibody. These nanoparticles were then reacted with anti-E. coli O157:H7, using N-hydroxy succinimide (NHS) and carbondimide hydrochloride (EDC) coupling chemistry. The product was characterized with UV-visible spectroscopy, Fourier transform infrared (FTIR) spectroscopy and zeta potential. In addition, the binding of antibody-gold nanoparticles conjugates to E. coli O157:H7 was demonstrated using transmission electron microscopy (TEM). (paper)

  18. Specific racemization and isomerization of the aspartyl residue of αA-crystallin due to UV-B irradiation

    International Nuclear Information System (INIS)

    Fujii, Noriko; Momose, Yuko; Ishibasi, Yoshihiro; Uemura, Toshimasa; Takita, Masatoshi; Takehana, Makoto


    We have reported that the aspartyl (Asp)-151 residue in αA-crystallin in human eye lens was inverted to the D-isomer and isomerized to β-Asp residue with age. We report here that ultraviolet (UV)-B irradiation induces the racemization and isomerization of the Asp-151 residue of αA-crystallin from lenses of 6-week-old rats to form D-isomer and β-Asp residue. Simultaneous racemization and isomerization of the specific Asp residue indicate that the reaction proceeds via formation of a succinimide intermediate. This modification was not observed in UV-A irradiated and normal lenses. UV-B irradiation induced the racemization of only the Asp-151 residue and did not affect the other Asp residues in αA-crystallin. On the other hand, the high molecular weight fraction of the lens protein increased upon UV-B irradiation. Modification of the Asp residue would affect the three-dimensional packing array of the lens protein. (author)

  19. Synthesis facilitates an understanding of the structural basis for translation inhibition by the lissoclimides (United States)

    Könst, Zef A.; Szklarski, Anne R.; Pellegrino, Simone; Michalak, Sharon E.; Meyer, Mélanie; Zanette, Camila; Cencic, Regina; Nam, Sangkil; Voora, Vamsee K.; Horne, David A.; Pelletier, Jerry; Mobley, David L.; Yusupova, Gulnara; Yusupov, Marat; Vanderwal, Christopher D.


    The lissoclimides are unusual succinimide-containing labdane diterpenoids that were reported to be potent cytotoxins. Our short semisynthesis and analogue-oriented synthesis approaches provide a series of lissoclimide natural products and analogues that expand the structure-activity relationships (SARs) in this family. The semisynthesis approach yielded significant quantities of chlorolissoclimide (CL) to permit an evaluation against the National Cancer Institute's 60-cell line panel and allowed us to obtain an X-ray co-crystal structure of the synthetic secondary metabolite with the eukaryotic 80S ribosome. Although it shares a binding site with other imide-based natural product translation inhibitors, CL engages in a particularly interesting and novel face-on halogen-π interaction between the ligand's alkyl chloride and a guanine residue. Our analogue-oriented synthesis provides many more lissoclimide compounds, which were tested against aggressive human cancer cell lines and for protein synthesis inhibitory activity. Finally, computational modelling was used to explain the SARs of certain key compounds and set the stage for the structure-guided design of better translation inhibitors.

  20. Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

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    Anika eRiedel


    Full Text Available Ene-reductases are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ene-reductase OYERo2 was found within a set of 14 putative Old Yellow Enzymes (OYEs obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37 °C. OYERo2 showed highest specific activities (4550 U mg-1 on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R-products with excellent optical purity (ee > 99%. OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32 °C from 28 min to 87 min and improved the tolerance towards organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

  1. Influences of conformations of peptides on stereoinversions and/or isomerizations of aspartic acid residues. (United States)

    Oda, Akifumi; Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Kurimoto, Eiji; Takahashi, Ohgi


    Recently, non-enzymatic stereoinversions of aspartic acid (Asp) residues in proteins and peptides have been reported. Here, we performed replica exchange molecular dynamics (REMD) simulations of model peptides (exon 6, 26A-1, and 26A-2) extracted from elastin to investigate their structural features, thereby revealing the factor that influences stereoinversions. For REMD trajectories, we calculated distances between carboxyl carbon in Asp and amide nitrogen in the (n + 1) residue (CN distances). Because bond formation between carbon and nitrogen is indispensable to the formation of a succinimide intermediate the distance between them seems to play an important role in stereoinversion. Moreover, we calculated polar surface areas (PSAs) for the trajectories, finding that CN distances and PSA were different for each peptide, with the longest CN distance and smallest PSA observed for exon 6 peptide, where stereoinversion of Asp is the slowest. Although the average CN distance was shorter for exon 26A-1 peptide than for exon 26A-2 peptide, the number of conformations with CN distances acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca. Copyright © 2018 Elsevier B.V. All rights reserved.

  2. Biodegradation and Osteosarcoma Cell Cultivation on Poly(aspartic acid) Based Hydrogels. (United States)

    Juriga, Dávid; Nagy, Krisztina; Jedlovszky-Hajdú, Angéla; Perczel-Kovách, Katalin; Chen, Yong Mei; Varga, Gábor; Zrínyi, Miklós


    Development of novel biodegradable and biocompatible scaffold materials with optimal characteristics is important for both preclinical and clinical applications. The aim of the present study was to analyze the biodegradability of poly(aspartic acid)-based hydrogels, and to test their usability as scaffolds for MG-63 osteoblast-like cells. Poly(aspartic acid) was fabricated from poly(succinimide) and hydrogels were prepared using natural amines as cross-linkers (diaminobutane and cystamine). Disulfide bridges were cleaved to thiol groups and the polymer backbone was further modified with RGD sequence. Biodegradability of the hydrogels was evaluated by experiments on the base of enzymes and cell culture medium. Poly(aspartic acid) hydrogels possessing only disulfide bridges as cross-links proved to be degradable by collagenase I. The MG-63 cells showed healthy, fibroblast-like morphology on the double cross-linked and RGD modified hydrogels. Thiolated poly(aspartic acid) based hydrogels provide ideal conditions for adhesion, survival, proliferation, and migration of osteoblast-like cells. The highest viability was found on the thiolated PASP gels while the RGD motif had influence on compacted cluster formation of the cells. These biodegradable and biocompatible poly(aspartic acid)-based hydrogels are promising scaffolds for cell cultivation.


    Directory of Open Access Journals (Sweden)

    K. Yu. Mukhin


    Full Text Available Pseudo-Lennox syndrome (PLS, or atypical benign partial epilepsy of childhood, is a disease from a group of age-related epileptic encephalopathies with a phenomenon of continuous spike-wave activity during slow sleep, which manifests itself as frequent polymorphic focal motor and pseudogeneralized seizures, cognitive impairments, as well as regional and diffuse epileptiform activity on electroencephalogram (EEG by the morphology identical to that of benign epileptiform patterns of childhood. The disease was first described by J. Aicardi and J. J. Chevrie in 1982, based on a study of 7 cases. Its diagnostic complexity is the polymorphism of both epileptic seizures and EEG data, as well as low awareness of the syndrome among physicians and its absence in the international classification of epilepsies. The typical triad of seizures, which occurs in nearly 100 % of patients, encompasses focal motor paroxysms (identical to those as observed in Rolandic epilepsy, atypical absences, and atonic seizures. Seizures in PLS in its active period (generally up to 7–8 years are highly resistant to antiepileptic drugs. Only a few agents have been proven to be effective in PLS; these include valproates, succinimides, benzodiazepines, topiramate, and sulthiame. The frequency of seizures are noted to increase in patients with PLS treated with drugs, such as vigabatrin, gabapentin, lamotrigine, phenobarbital, or phenytoin. The author considers in detail the history of studies of the disease, clinical manifestations, diagnostic criteria, therapeutic approaches, and prognosis.

  4. Problems connected with the production of highly specific antisera against prostaglandin E2 (PGE2) and prostaglandin A2 (PGA2) for radioimmunoassay

    International Nuclear Information System (INIS)

    Kopp, H.G.; Vetter, W.; Siegenthaler, W.


    To obtain sensitive and specific antisera against PGE 2 and PGA 2 these substances were coupled to thyroglobulin (Tg). Coupling reactions were performed by using either a hydroxy-succinimide-ester as intermediate step leading to a complex carrying 170mol PGE 2 per mol Tg (''PGE 2 -OSU-Tg'') and 240mol PGA 2 per mol Tg (''PGA 2 -OSU-Tg''), or N, N'-carbonyl-diimidazole resulting in ''PGE 2 -CDI-Tg'' (400mol PGE 2 per mol Tg) and ''PGA 2 -CDI-Tg'' (600mol PGA 2 per mol Tg). Two tracer systems ( 3 H-prostaglandin and 125 I-histamine-prostaglandin) were used for analysis of antibody activity. The PGE 2 -CDI-Tg and PGA 2 -CDI-Tg complexes were both poor immunogens in rabbits. The PGE 2 -OSU-Tg and PGA 2 -OSU-Tg conjugates were injected in rabbits and in guinea-pigs. These two compounds resulted in very high antibody titres in both animal species. However, in guinea-pigs a markedly higher antibody sensitivity and antibody specificity were observed than in rabbits. Our results indicate that the guinea-pig may be the animal of choice for immunization against prostaglandins. Antibody specificity of guinea-pig antisera may be high enough to measure the concentration of PGE 2 and PGA 2 in the presence of other prostaglandins or prostaglandin metabolites. (author)

  5. Anticancer activity of drug conjugates in head and neck cancer cells. (United States)

    Majumdar, Debatosh; Rahman, Mohammad Aminur; Chen, Zhuo Georgia; Shin, Dong M


    Sexually transmitted oral cancer/head and neck cancer is increasing rapidly. Human papilloma virus (HPV) is playing a role in the pathogenesis of a subset of squamous cell carcinoma of head and neck (SCCHN). Paclitaxel is a widely used anticancer drug for breast, ovarian, testicular, cervical, non-small cell lung, head and neck cancer. However, it is water insoluble and orally inactive. We report the synthesis of water soluble nanosize conjugates of paclitaxel, branched PEG, and EGFR-targeting peptide by employing native chemical ligation. We performed a native chemical ligation between the N-hydroxy succinimide (NHS) ester of paclitaxel succinate and cysteine at pH 6.5 to give the cysteine-conjugated paclitaxel derivative. The thiol functionality of cysteine was activated and subsequently conjugated to multiarm thiol-PEG to obtain the paclitaxel branched PEG conjugate. Finally, we conjugated an EGFR-targeting peptide to obtain conjugates of paclitaxel, branched PEG, and EGFR-targeting peptide. These conjugates show anticancer activity against squamous cell carcinoma of head and neck cells (SCCHN, Tu212).

  6. Binding and assembly of actin filaments by plasma membranes from dictyostelium discoideum

    International Nuclear Information System (INIS)

    Schwartz, M.A.; Luna, E.J.


    The binding of native, 125 I-Bolton-Hunter-labeled actin to purified Dictyostelium discoideum plasma membranes was measured using a sedimentation assay. Binding was saturable only in the presence of the actin capping protein, gelsolin. The binding curves were sigmoidal, indicating positive cooperativity at low actin concentrations. This cooperativity appeared to be due to actin-actin associations during polymerization, since phalloidin converted the curve to a hyperbolic shape. This membrane-bound actin stained with rhodamine-phalloidin and was cross-linked by m-maleimidobenzoyl succinimide ester, a bifunctional cross-linker, into multimers with the same pattern observed for cross-linked F-actin. The authors conclude that D. discoideum plasma membranes bind actin specifically and saturably and that these membranes organize actin into filaments below the normal critical concentration for polymerization. This interaction probably occurs between multiple binding sites on the membrane and the side of the actin filament, and may be related to the clustering of membrane proteins

  7. Reducing the photo-bleaching effect of a new europium complex embedded in styrene butadiene copolymer (United States)

    Jiménez, G. Lesly; Reyes-Rodríguez, J. L.; Padilla, Isela; Alarcón-Flores, G.; Falcony, C.


    A highly luminescent europium complex obtained with two different ligands, succinimide (SI) and 2-thenoyltrifluoroacetone (TTA) , was synthetized with different TTA concentrations. The photoluminescence (PL) emission from these materials corresponds to the characteristic inter-electronic energy level transitions of the Eu3+ ions. However, the excitation spectrum is strongly dependent on the presence of TTA, having an optimum response when 0.75 mmol of this compound is added to the EuL3(H2O)3 complex. The quantum yield obtained by these powders were around 72 % ± 1.7 % indicating an optimum sensitization of these complex. The EuL3 TTA complex with the best PL properties was embedded in a styrene butadiene copolymer (SBC) film, produced by the drop casting method, obtaining similar PL behavior at different concentrations, the highest intensity was observed at 1.2% (w/v) of EuL3 TTA complex and the quantum yield of these composite films was 60.5 % ± 2 % . These films were exposed to continuous UV irradiation and after 141 h no photo-bleaching effect was observed in contrast with the EuL3 TTA complex that exhibited a noticeable photoluminescence intensity degradation at much shorter exposure times. Both the Eu-complexes and the composite films were characterized by FT-IR, XRD, SEM and fluorescence spectroscopy.

  8. Impact of quenching failure of Cy dyes in differential gel electrophoresis.

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    Weiqun Wang

    Full Text Available BACKGROUND: Differential gel electrophoresis (DIGE is a technology widely used for protein expression analysis. It is based on labelling with fluorescent Cy dyes. In comparative fluorescence gel electrophoresis experiments, however, unspecific labelling using N-hydroxy-succinimide-ester-based labelling protocols was recently detected. Cross-talk was observed due to failure of the quenching process. Here, the impact of this effect for DIGE experiments was investigated. METHODOLOGY/PRINCIPAL FINDINGS: Experiments to test quenching efficiency were performed in replicate using Escherichia coli lysate. Parameters such as the amount of dye and quencher were varied. Labelling and quenching were reversed in one experiment. Differences in protein spot volumes due to limited quenching were determined. For some spots twice the volume was detected underscoring the importance of proper control of silencing of active dye. CONCLUSIONS/SIGNIFICANCE: It could be demonstrated that uncontrolled labelling increased protein spot volume, even doubling it in some cases. Moreover, proteins responded differently to the protocol. Such unpredictable and unspecific processes are not acceptable in protein regulation studies so that it is necessary to validate the correct amount of quencher for individual samples before the DIGE experiment is performed. Increase of the concentration of lysine, which is used as quencher, from 10 mM to 2500 mM, was sufficient to silence the dye. Alternatively, active dye molecules can be removed by filtration.

  9. Quantitative kinetics of proteolytic enzymes determined by a surface concentration-based assay using peptide arrays. (United States)

    Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo


    Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).

  10. Development of polymer-polysaccharide hydrogels for controlling drug delivery (United States)

    Baldwin, Aaron David

    The use of polymers as biomaterials has evolved over the past several decades, encompassing an expanding synthetic toolbox and many bio-mimetic approaches. Both synthetic and natural polymers have been used as components for biomaterials as their unique chemical structures can provide specific functions for desired applications. Of these materials, heparin, a highly sulfated naturally occurring polysaccharide, has been investigated extensively as a core component in drug delivery platforms and tissue engineering. The goal of this work was to further explore the use of heparin via conjugation with synthetic polymers for applications in drug delivery. We begin by investigating low molecular weight heparin (LMWH), a depolymerized heparin that is used medicinally in the prevention of thrombosis by subcutaneous injection or intravenous drip. Certain disease states or disorders require frequent administration with invasive delivery modalities leading to compliance issues for individuals on prolonged therapeutic courses. To address these issues, a long-term delivery method was developed for LMWH via subcutaneous injection of in situ hydrogelators. This therapy was accomplished by chemical modification of LMWH with maleimide functionality so that it may be crosslinked into continuous hydrogel networks with four-arm thiolated polyethylene glycol (PEG-SH). These hydrogels degrade via hydrolysis over a period of weeks and release bioactive LMWH with first-order kinetics as determined by in vitro and in vivo models, thus indicating the possibility of an alternative means of heparin delivery over current accepted methodologies. Evaluation of the maleimide-thiol chemistries applied in the LMWH hydrogels revealed reversibility for some conjugates under reducing conditions. Addition chemistries, such as maleimide-thiol reactions, are widely employed in biological conjugates and are generally accepted as stable. Here we show that the resulting succinimide thioether formed by the

  11. Development of 177Lu-DOTA-anti-CD20 for radioimmunotherapy

    International Nuclear Information System (INIS)

    Hassan Yousefnia; Amir Reza Jalilian; Ali Bahrami-Samani; Simindokht Shirvani-Arani; Mohammad Ghannadi-Maragheh; Azim Arbabi; Edalat Radfar


    Rituximab was successively labeled with 177 Lu-lutetium chloride. 177 Lu chloride was obtained by thermal neutron flux (4 x 1013 n cm -2 s -1 ) of natural Lu 2 O 3 sample with a specific activity of 2.6-3 GBq/mg. The macrocyclic bifunctional chelating agent, N-succinimidyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA-NHS) was prepared at 25 deg C using DOTA, N-hydroxy succinimide (NHS) in CH 2 Cl 2 . DOTA-rituximab was obtained by the addition of 1 mL of a rituximab pharmaceutical solution (5 mg/mL, in phosphate buffer, pH 7.8) to a glass tube pre-coated with DOTA-NHS (0.01-0.1 mg) at 25 deg C with continuous mild stirring for 15 h. Radiolabeling was performed at 37 deg C in 24 h. Radio-thin layer chromatography showed an overall radiochemical purity of >98% at optimized conditions (specific activity = 444 MBq/mg, labeling efficacy; 82%). The final isotonic 177 Lu-DOTA-rituximab complex was checked by gel electrophoresis for structure integrity control. Radio-TLC was performed to ensure that only one species was present after filtration through a 0.22 μm filter. Preliminary biodistribution studies in normal rats were carried out to determine complex distribution of the radioimmunoconjugate up to 168 h. The biodistribution data were in accordance with other antiCD20 radioimmunoconjugates already reported. (author)

  12. Cell Permeating Nano-Complexes of Amphiphilic Polyelectrolytes Enhance Solubility, Stability, and Anti-Cancer Efficacy of Curcumin. (United States)

    Fatima, Munazza T; Chanchal, Abhishek; Yavvari, Prabhu S; Bhagat, Somnath D; Gujrati, Mansi; Mishra, Ram K; Srivastava, Aasheesh


    Many hydrophobic drugs encounter severe bioavailability issues owing to their low aqueous solubility and limited cellular uptake. We have designed a series of amphiphilic polyaspartamide polyelectrolytes (PEs) that solubilize such hydrophobic drugs in aqueous medium and enhance their cellular uptake. These PEs were synthesized through controlled (∼20 mol %) derivatization of polysuccinimide (PSI) precursor polymer with hydrophobic amines (of varying alkyl chain lengths, viz. hexyl, octyl, dodecyl, and oleyl), while the remaining succinimide residues of PSI were opened using a protonable and hydrophilic amine, 2-(2-amino-ethyl amino) ethanol (AE). Curcumin (Cur) was employed as a representative hydrophobic drug to explore the drug-delivery potential of the resulting PEs. Unprecedented enhancement in the aqueous solubility of Cur was achieved by employing these PEs through a rather simple protocol. In the case of PEs containing oleyl/dodecyl residues, up to >65000× increment in the solubility of Cur in aqueous medium could be achieved without requiring any organic solvent at all. The resulting suspensions were physically and chemically stable for at least 2 weeks. Stable nanosized polyelectrolyte complexes (PECs) with average hydrodynamic diameters (DH) of 150-170 nm (without Cur) and 220-270 nm (after Cur loading) were obtained by using submolar sodium polyaspartate (SPA) counter polyelectrolyte. The zeta potential of these PECs ranged from +36 to +43 mV. The PEC-formation significantly improved the cytocompatibility of the PEs while affording reconstitutable nanoformulations having up to 40 wt % drug-loading. The Cur-loaded PECs were readily internalized by mammalian cells (HEK-293T, MDA-MB-231, and U2OS), majorly through clathrin-mediated endocytosis (CME). Cellular uptake of Cur was directly correlated with the length of the alkyl chain present in the PECs. Further, the PECs significantly improved nuclear transport of Cur in cancer cells, resulting in their

  13. Biochemical characterization of the pancreatic cholecystokinin receptor using monofunctional photoactivatable probes

    International Nuclear Information System (INIS)

    Pearson, R.K.; Miller, L.J.; Powers, S.P.; Hadac, E.M.


    Receptor characterization by affinity labeling can be enhanced by taking multiple complementary approaches. To extend our observations on the subunit structure of the rat pancreatic cholecystokinin (CCK) receptor (made using bifunctional cross-linking reagents), we synthesized two monofunctional photoactivatable receptor probes. CCK-8 was acylated with the iodinated aryl azide derivatives, methyl-3-azido-4-hydroxy-5-[ 125 I]iodobenzimidate and N-[4-(4'-azido-3'-[ 125 I]iodophenylazo)benzoyl]-3-aminopropionyl-N- oxy- succinimide. The products were purified by reverse-phase HPLC to a specific radioactivity of 2000 Ci/mmol. Both analogs demonstrated saturable and specific binding to rat pancreatic plasma membranes. Photoaffinity labeling of pancreatic membranes with these monofunctional probes identified an Mr 85,000-95,000 protein that was not part of a larger disulfide-linked complex. High affinity for CCK was demonstrated by the concentration-dependent inhibition of labeling observed with competing CCK-8 (IC50 = 1 nM). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) this protein co-migrates with the major component we identified using a series of cross-linkable, iodinated decapeptide analogs of CCK, and is different from the major protein labeled using 125 I-Bolton Hunter-CCK-33. Thus, these results support the presence of an Mr 85,000-95,000 subunit in the pancreatic CCK receptor, while the small size of these photoaffinity probes and their monovalency suggest that this subunit may contain or be spatially apposed to the active binding site. These probes should be very useful in the further characterization of this and other receptors for this hormone

  14. Synthesis and evaluation of radiolabeled, folic acid-PEG conjugated, amino silane coated magnetic nanoparticles in tumor bearing Balb/C mice

    Directory of Open Access Journals (Sweden)

    Razjouyan Javad


    Full Text Available To design a potent agent for positron emission tomography/magnetic resonance imaging (PET/MRI imaging and targeted magnetic hyperthermia-radioisotope cancer therapy radiolabeled surface modified superparamagnetic iron oxide nanoparticles (SPIONs were used as nanocarriers. Folic acid was conjugated for increasing selective cellular binding and internalization through receptor-mediated endocytosis. SPIONs were synthesized by the thermal decomposition of tris (acetylacetonato iron (III to achieve narrow and uniform nanoparticles. To increase the biocompatibility of SPIONs, they were coated with (3-aminopropyl triethoxysilane (APTES, and then conjugated with synthesized folic acid-polyethylene glycol (FA-PEG through amine group of (3-aminopropyl triethoxysilane. Finally, the particles were labeled with 64Cu (t1/2 = 12.7 h using 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid mono (N-hydroxy succinimide ester DOTA-NHS chelator. After the characterization of SPIONs, their cellular internalization was evaluated in folate receptor (FR overexpressing KB (established from a HeLa cell contamination and mouse fibroblast cell (MFB lines. Eventually, active and passive targeting effects of complex were assessed in KB tumor-bearing Balb/C mice through biodistribution studies. Synthesized bare SPIONs had low toxicity effect on healthy cells, but surface modification increased their biocompatibility. Moreover, KB cells viability was reduced when using folate conjugated SPIONs due to FR-mediated endocytosis, while having little effect on healthy cells (MFB. Moreover, this radiotracer had tolerable in vivo characteristics and tumor uptake. In the receptor blocked case, tumor uptake was decreased, indicating FR-specific uptake in tumor tissue while enhanced permeability and retention effect was major mechanism for tumor uptake.

  15. Preparation and in vivo evaluation of a novel stabilized linker for 211At labeling of protein

    International Nuclear Information System (INIS)

    Talanov, Vladimir S.; Garmestani, Kayhan; Regino, Celeste A.S.; Milenic, Diane E.; Plascjak, Paul S.; Waldmann, Thomas A.; Brechbiel, Martin W.


    Significant improvement of in vivo stability of 211 At-labeled radioimmunoconjugates achieved upon employment of a recently reported new linker, succinimidyl N-2-(4-[ 211 At]astatophenethyl)succinamate (SAPS), prompted additional studies of its chemistry. The 211 At radiolabeling of succinimidyl N-2-(4-tributylstannylphenethyl)succinamate (1) was noted to decline after storage at -15 o C for greater than 6 months. Compound 1 was found to degrade via a ring closure reaction with the formation of N-2-(4-tributylstannylphenethyl)succinimide (3), and a modified procedure for the preparation of 1 was developed. The N-methyl structural analog of 1, succinimidyl N-2-(4-tributylstannylphenethyl)-N-methyl succinamate (SPEMS), was synthesized to investigate the possibility of improving the stability of reagent-protein linkage chemistry. Radiolabeling of SPEMS with 211 At generates succinimidyl N-2-(4-[ 211 At]astatophenethyl)-N-methyl succinamate (Methyl-SAPS), with yields being consistent for greater than 1 year. Radiolabelings of 1 and SPEMS with 125 I generated succinimidyl N-2-(4-[ 125 I]iodophenethyl)succinamate (SIPS) and succinimidyl N-2-(4-[ 125 I]iodophenethyl)-N-methyl succinamate (Methyl-SIPS), respectively, and showed no decline in yields. Methyl-SAPS, SAPS, Methyl-SIPS and SIPS were conjugated to Herceptin for a comparative assessment in LS-174T xenograft-bearing mice. The conjugates of Herceptin with Methyl-SAPS or Methyl-SIPS demonstrated immunoreactivity equivalent to if not superior to the SAPS and SIPS paired analogs. The in vivo studies also revealed that the N-methyl modification resulted in a superior statinated product

  16. Two independent measurements of Debye lengths in doped nonpolar liquids. (United States)

    Prieve, D C; Hoggard, J D; Fu, R; Sides, P J; Bethea, R


    Electric current measurements were performed between 2.5 cm x 7.5 cm parallel-plate electrodes separated by 1.2 mm of heptane doped with 0-15% w/w poly(isobutylene succinimide) (PIBS) having a molecular weight of about 1700. The rapid (microsecond) initial charging of the capacitor can be used to infer the dielectric constant of the solution. The much slower decay of current arising from the polarization of electrodes depends on the differential capacitance of the diffuse clouds of charge carriers accumulating next to each electrode and on the ohmic resistance of the fluid. Using the Gouy-Chapman model for the differential capacitance, Debye lengths of 80-600 nm were deduced that decrease with increasing concentration of PIBS. Values of the Debye lengths were confirmed by performing independent measurements of double-layer repulsion between a 6 microm polystyrene (PS) latex sphere and a PS-coated glass plate using total internal reflection microscopy in the same solutions. The charge carriers appear to be inverted PIBS micelles having apparent Stokes diameters of 20-40 nm. Dynamic light scattering reveals a broad distribution of sizes having an intensity-averaged diameter of 15 nm. This smaller size might arise (1) from overestimating the electrophoretic mobility of micelles by treating them as point charges or (2) because charged micelles are larger on average than uncharged micelles. When Faradaic reactions and zeta potentials on the electrodes can be neglected, such current versus time experiments yield values for the Debye length and ionic strength with less effort than force measurements. To obtain the concentration of charge carriers from measurements of conductivity, the mobility of the charge carriers must be known.

  17. Simple test system for single molecule recognition force microscopy

    International Nuclear Information System (INIS)

    Riener, Christian K.; Stroh, Cordula M.; Ebner, Andreas; Klampfl, Christian; Gall, Alex A.; Romanin, Christoph; Lyubchenko, Yuri L.; Hinterdorfer, Peter; Gruber, Hermann J.


    We have established an easy-to-use test system for detecting receptor-ligand interactions on the single molecule level using atomic force microscopy (AFM). For this, avidin-biotin, probably the best characterized receptor-ligand pair, was chosen. AFM sensors were prepared containing tethered biotin molecules at sufficiently low surface concentrations appropriate for single molecule studies. A biotin tether, consisting of a 6 nm poly(ethylene glycol) (PEG) chain and a functional succinimide group at the other end, was newly synthesized and covalently coupled to amine-functionalized AFM tips. In particular, PEG 800 diamine was glutarylated, the mono-adduct NH 2 -PEG-COOH was isolated by ion exchange chromatography and reacted with biotin succinimidylester to give biotin-PEG-COOH which was then activated as N-hydroxysuccinimide (NHS) ester to give the biotin-PEG-NHS conjugate which was coupled to the aminofunctionalized AFM tip. The motional freedom provided by PEG allows for free rotation of the biotin molecule on the AFM sensor and for specific binding to avidin which had been adsorbed to mica surfaces via electrostatic interactions. Specific avidin-biotin recognition events were discriminated from nonspecific tip-mica adhesion by their typical unbinding force (∼40 pN at 1.4 nN/s loading rate), unbinding length (<13 nm), the characteristic nonlinear force-distance relation of the PEG linker, and by specific block with excess of free d-biotin. The convenience of the test system allowed to evaluate, and compare, different methods and conditions of tip aminofunctionalization with respect to specific binding and nonspecific adhesion. It is concluded that this system is well suited as calibration or start-up kit for single molecule recognition force microscopy

  18. A pH- and temperature-responsive bioresorbable injectable hydrogel based on polypeptide block copolymers for the sustained delivery of proteins in vivo. (United States)

    Turabee, Md Hasan; Thambi, Thavasyappan; Duong, Huu Thuy Trang; Jeong, Ji Hoon; Lee, Doo Sung


    Sustained delivery of protein therapeutics is limited owing to the fragile nature of proteins. Despite its great potential, delivery of proteins without any loss of bioactivity remains a challenge in the use of protein therapeutics in the clinic. To surmount this shortcoming, we report a pH- and temperature-responsive in situ-forming injectable hydrogel based on comb-type polypeptide block copolymers for the controlled delivery of proteins. Polypeptide block copolymers, composed of hydrophilic polyethylene glycol (PEG), temperature-responsive poly(γ-benzyl-l-glutamate) (PBLG), and pH-responsive oligo(sulfamethazine) (OSM), exhibit pH- and temperature-induced sol-to-gel transition behavior in aqueous solutions. Polypeptide block copolymers were synthesized by combining N-carboxyanhydride-based ring-opening polymerization and post-functionalization of the chain-end using N-hydroxy succinimide ester activated OSM. The physical properties of polypeptide-based hydrogels were tuned by varying the composition of temperature- and pH-responsive PBLG and OSM in block copolymers. Polypeptide block copolymers were non-toxic to human embryonic kidney cells at high concentrations (2000 μg mL -1 ). Subcutaneous administration of polypeptide block copolymer sols formed viscoelastic gel instantly at the back of Sprague-Dawley (SD) rats. The in vivo gels exhibited sustained degradation and were found to be bioresorbable in 6 weeks without any noticeable inflammation at the injection site. Anionic characteristics of hydrogels allow efficient loading of a cationic model protein, lysozyme, through electrostatic interaction. Lysozyme-loaded polypeptide block copolymer sols readily formed a viscoelastic gel in vivo and sustained lysozyme release for at least a week. Overall, the results demonstrate an elegant approach to control the release of certain charged proteins and open a myriad of therapeutic possibilities in protein therapeutics.

  19. Regulating drug release from pH- and temperature-responsive electrospun CTS-g-PNIPAAm/poly(ethylene oxide) hydrogel nanofibers

    International Nuclear Information System (INIS)

    Yuan, Huihua; Li, Biyun; Liang, Kai; Lou, Xiangxin; Zhang, Yanzhong


    Temperature- and pH-responsive polymers have been widely investigated as smart drug release systems. However, dual-sensitive polymers in the form of nanofibers, which is advantageous in achieving rapid transfer of stimulus to the smart polymeric structures for regulating drug release behavior, have rarely been explored. In this study, chitosan-graft-poly(N-isopropylacrylamide) (CTS-g-PNIPAAm) copolymer was synthesized by using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxy succinimide (NHS) as grafting agents to graft carboxyl-terminated PNIPAAm (PNIPAAm-COOH) chains onto the CTS biomacromolecules, and then CTS-g-PNIPAAm with or without bovine serum albumin (BSA) was fabricated into nanofibers through electrospinning using poly(ethylene oxide) (PEO, 10 wt%) as a fiber-forming facilitating additive. The BSA laden CTS-g-PNIPAAm/PEO hydrogel nanofibers were tested to determine their drug release profiles by varying pH and temperature. Finally, cytotoxicity of the CTS-g-PNIPAAm/PEO hydrogel nanofibers was evaluated by assaying the L929 cell proliferation using the MTT method. It was found that the synthesized CTS-g-PNIPAAm possessed a temperature-induced phase transition and lower critical solution temperature (LCST) at 32° C in aqueous solutions. The rate of BSA release could be well modulated by altering the environmental pH and temperature of the hydrogel nanofibers. The CTS-g-PNIPAAm/PEO hydrogel nanofibers supported L929 cell growth, indicative of appropriate cytocompatibility. Our current work could pave the way towards developing multi-stimuli responsive nanofibrous smart materials for potential applications in the fields of drug delivery and tissue engineering. (paper)

  20. Tubular scaffolds of gelatin and poly(ε-caprolactone)-block-poly(γ-glutamic acid) blending hydrogel for the proliferation of the primary intestinal smooth muscle cells of rats

    International Nuclear Information System (INIS)

    Jwo, Shyh-Chuan; Chiu, Chu-Hua; Hsieh, Ming-Fa; Tang, Shye-Jye


    The proper regeneration of intestinal muscle for functional peristalsis is the most challenging aspect of current small intestine tissue engineering. This study aimed to fabricate a hydrogel scaffold for the proliferation of intestinal smooth muscle cells (ISMCs). Tubular porous scaffolds of 10–20 wt% gelatin and 0.05–0.1 wt% poly(ε-caprolactone)-block-poly(γ-glutamic acid) blending hydrogel were cross-linked by carbodiimide and succinimide in an annular space of a glass mold. The scaffolds with higher gelatin contents degraded slower in the phosphate buffer solution. In rheological measurements, the hydrated scaffolds were elastic (all tangent delta <0.45); they responded differentially to frequency, indicating a complete viscoelastic property that is beneficial for soft tissue regeneration. Isolated rat ISMCs, with the characteristic biomarkers α-SMA, calponin and myh11, were loaded into the scaffolds by using either static or centrifugal methods. The average cell density inside the scaffolds increased in a time-dependent manner in most scaffolds of both seeding groups, although at early time points (seven days) the centrifugal seeding method trapped cells more efficiently and yielded a higher cell density than the static seeding method. The static seeding method increased the cell density from 7.5-fold to 16.3-fold after 28 days, whereas the centrifugal procedure produced a maximum increase of only 2.4-fold in the same period. In vitro degradation data showed that 50–80% of the scaffold was degraded by the 14th day. However, the self-secreted extracellular matrix maintained the integrity of the scaffolds for cell proliferation and spreading for up to 28 days. Confocal microscopic images revealed cell–cell contacts with the formation of a 3D network, demonstrating that the fabricated scaffolds were highly biocompatible. Therefore, these polymeric biomaterials hold great promise for in vivo applications of intestinal tissue engineering. (paper)

  1. Preparation and quality control of radiometal-DOTARituximab

    International Nuclear Information System (INIS)

    Jalilian, A.R.; Kamali-dehghan, M.; Moradkhani, S.; Saddadi, F.; Mirsadeghi, L.


    In this work Rituximab has been labeled by Ga-67 using a new one-step method for in-situ preparation of macrocyclic bifunctional chelating agent, N-succinimidyl-1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTANHS). DOTA-NHS was prepared at 25 deg. C using DOTA, Nhydroxy succinimide (NHS) in CH Cl in one step. DOTA- 2 2 Rituximab was obtained by the addition of 1 ml of a Rituximab pharmaceutical solution (5 mg/ml, in phosphate buffer, pH=7.8) to a glass tube pre-coated with DOTA-NHS (0.01-0.1 mg) at 25 deg. C with continuous mild stirring for 15 hours. Radiolabeling was performed at 37 deg. C in 3 hrs using Ga-67 Chlorides or Cu-64 Acetate. Thin layer radiochromatography demonstrated an overall radiochemical purity of 90-95% at optimized conditions (specific activity =30 GBq/mg, labeling efficacy= 82%). The final isotonic radio-metal-DOTA-Rituximab complex was checked by gel electrophoresis for radiolysis. TLC was performed to ensure that only one species was present after filtration through a 0.22 μm filter. Preliminary bio-distribution studies of the Ga-67 immunoconjugate in normal rat model performed to determine complex distribution of the radioimmunoconjugate up to 28hrs by imaging and organ counting after sacrificing the rats. In this study a facile method for incorporation of metal chelating moiety into peptides and antibody structure is presented. (author)

  2. Radioimmunoassay of cholecystokinin in human tissue and plasma

    International Nuclear Information System (INIS)

    Jansen, J.B.M.J.; Lamers, C.B.H.W.


    A highly sensitive radioimmunoassay for cholecystokinin (CCK) without any cross-reactivity with gastrin is described. The antibody was raised in a rabbit by immunisation with 30% CCK and bound to all COOH-terminal CCK-peptides containing at least 14 amino acid residues. The affinity constant of the antibody was 59.4 x 10 10 l/mol. CCK 33 conjugated to [ 125 I]hydroxyphenylpropionic acid-succinimide ester was used as label. The binding between label and antibody was inhibited by 50% (ID 50 ) at a concentration of 2.8 pmol/l cholecystokinin 33. The detection limit of the assay was between 0.5 and 1.0 pmol/l plasma. Concentrations of CCK in aqueous acid extracts of human upper small intestine were 36.5 +- 9.8 pmol/g and of human cerebral cortex 28.2 +- 2.5 pmol/g tissue. Plasma samples were extracted in 96% ethanol prior to assay. No advantage was obtained by adding aprotinin to the tubes. When frozen at -20 0 C plasma CCK was stable for at least 6 months. Basal plasma CCK concentrations in 30 normal subjects were very low, 0.9 +- 0.1 pmol/l, range 0.5 to 3.1 pmol/l. Intraduodenal administration of fat induced significant increases in plasma CCK from 1.1 +- 0.1 to 8.2 +- 1.3 pmol/l (p = 0.01). Infusion of exogenous CCK, resulting in plasma CCK levels slightly lower than those measured during administration of fat, induced pancreatic enzyme secretion and gallbladder contraction. The reliability of this radioimmunoassay for measurements of CCK in human plasma was extensively evaluated. (Auth.)

  3. Novel enhancement of thin-form-factor galvanic cells: Probing halogenated organic oxidizers and metal anodes

    Energy Technology Data Exchange (ETDEWEB)

    Cardenas-Valencia, Andres M.; Adornato, Lori; Short, R. Timothy; Langebrake, Larry [SRI International, Engineering and Systems Division, Marine Technology Program, 140 Seventh Avenue South, St Petersburg, FL 33701 (United States)


    The work reported herein demonstrates a novel method to improve the overall performance of thin-form-factor galvanic cells, fabricated via micro-electromechanical systems (MEMS) processes. Use of solid, low cost, cyclic-halogenated, organic catholyte materials permits water activation of cells consisting of metal anode and catalytic platinum positive electrodes. Similar cells, employing aluminum and zinc anodes, have been activated using sodium hypochlorite (NaClO) solutions, i.e. bleach, in the past. The oxidizers chosen for this study (bromo-, chloro- and iodo-succinimides, and sodium dichloroisocyanuric acid) supply the cathode's oxy-halogenated ions when in contact with water. Zinc, magnesium and aluminum anodes are utilized to fabricate galvanic cells. A comparison between these anodes, coupled with various oxidizers, is included herein. Results using aluminum anode cells show that, even though the utilization efficiency of the catholyte reagents is low (faradic efficiencies between 16 and 19%), the performance of the new water-activated cells (6 cm x 6 cm x 0.25 cm) is superior when compared to those activated with bleach. For instance, operational lives of 6 h (activation with 10% NaClO solution) increase to more than 30 h using the new approach, with a 100-ohm-load. It is also shown that specific energies of 90-110 Wh kg{sup -1} (calculated to include both reagent and packaging mass) could be obtained using the described approach with current draws between 10 and 20 mA. The specific energies obtained suggest that novel MEMS-type cells could have much broader application than low-current, bleach-activated cells. (author)

  4. Azide photochemistry for facile modification of graphitic surfaces: preparation of DNA-coated carbon nanotubes for biosensing

    International Nuclear Information System (INIS)

    Moghaddam, Minoo J; Yang Wenrong; Bojarski, Barbara; Gengenbach, Thomas R; Gao Mei; Zareie, Hadi; McCall, Maxine J


    A facile, two-step method for chemically attaching single-stranded DNA to graphitic surfaces, represented here by carbon nanotubes, is reported. In the first step, an azide-containing compound, N-5-azido-nitrobenzoyloxy succinimide (ANB-NOS), is used to form photo-adducts on the graphitic surfaces in a solid-state photochemical reaction, resulting in active ester groups being oriented for the subsequent reactions. In the second step, pre-synthesized DNA strands bearing a terminal amine group are coupled in an aqueous solution with the active esters on the photo-adducts. The versatility of the method is demonstrated by attaching pre-synthesized DNA to surfaces of carbon nanotubes in two platforms—as vertically-aligned multi-walled carbon nanotubes on a solid support and as tangled single-walled carbon nanotubes in mats. The reaction products at various stages were characterized by x-ray photoelectron spectroscopy. Two different assays were used to check that the DNA strands attached to the carbon nanotubes were able to bind their partner strands with complementary base sequences. The first assay, using partner DNA strands tethered to gold nanoparticles, enabled the sites of DNA attachment to the carbon nanotubes to be identified in TEM images. The second assay, using radioactively labelled partner DNA strands, quantified the density of functional DNA strands attached to the carbon nanotubes. The diversity of potential applications for these DNA-modified carbon-nanotube platforms is exemplified here by the successful use of a DNA-modified single-walled carbon-nanotube mat as an electrode for the specific detection of metal ions. (paper)

  5. Use of natural gas, methanol, and ethanol fuel emulsions as environmentally friendly energy carriers for mobile heat power plants (United States)

    Likhanov, V. A.; Lopatin, O. P.


    The need for using environmentally friendly energy carriers for mobile heat power plants (HPPs) is grounded. Ecologically friendly sources of energy, such as natural gas as well as renewable methyl and ethyl alcohols, are investigated. In order to develop, determine, and optimize the composition of environmentally friendly energy carriers for an HPP, the latter has been tested when working on diesel fuel (DF), compressed natural gas (CNG), and methanol and ethanol fuel emulsions (MFE, EFE). It has been experimentally established that, for the application of environmentally friendly energy carriers for a 4Ch 11.0/12.5 diesel engine of a mobile fuel and power plant, it is necessary to maintain the following ratio of components when working on CNG: 80% gas and 20% DF primer portion. When working on an alcohol mixture, emulsions of the following composition were used: 25% alcohol (methanol or ethanol), 0.5% detergent-dispersant additive succinimide C-5A, 7% water, and 67.5% DF. When this diesel passed from oil DF to environmentally friendly energy sources, it allowed for the reduction of the content of exhaust gases (EG) (1) when working on CNG with recirculation of exhaust gases (EGR) (recirculation was used to eliminate the increased amount of nitric oxides by using CNG): carbon black by 5.8 times, carbon dioxide by 45.9%, and carbon monoxide by 23.8%; (2) when working on MFE: carbon black by 6.4 times, nitrogen oxides by 29.6%, carbon dioxide by 10.1%, and carbon oxide by 47.6%; (3) when working on EFE: carbon black by 4.8 times; nitrogen oxides by 40.3%, carbon dioxide by 26.6%, and carbon monoxide by 28.6%. The prospects of use of environmentally friendly energy carriers in diesels of mobile HPPs, such as natural gas, ethanol, and methanol, has been determined.

  6. Synthesis and characterization of functionalized methacrylates for coatings and biomedical applications (United States)

    Shemper, Bianca Sadicoff

    photopolymerization kinetics and coatings properties of MHMA systems is then shown in Chapter VII. This chapter also includes the effect of the presence of synthetic clay in these systems and the preparation of nanocomposite-based films. The final chapter of this dissertation involves the design of reactive coatings for biomedical applications. The syntheses and characterization of novel functionalized methacrylates containing succinimide ester groups susceptible to derivatization with amine-containing species were accomplished. Photopolymerization of these monomers led to formation of hydrogels and derivatization of the hydrogel surfaces with the tripeptide RGD (arginine-glycine-aspartic acid) was successfully achieved.

  7. Elastin Based Cell-laden Injectable Hydrogels with Tunable Gelation, Mechanical and Biodegradation Properties (United States)

    Fathi, Ali; Mithieux, Suzanne M.; Wei, Hua; Chrzanowski, Wojciech; Valtchev, Peter; Weiss, Anthony S.; Dehghani, Fariba


    Injectable hydrogels made from extracellular matrix proteins such as elastin show great promise for various biomedical applications. Use of cytotoxic reagents, fixed gelling behavior, and lack of mechanical strength in these hydrogels are the main associated drawbacks. The aim of this study was to develop highly cytocompatible and injectable elastin-based hydrogels with alterable gelation characteristics, favorable mechanical properties and structural stability for load bearing applications. A thermoresponsive copolymer, poly(N-isopropylacrylamide-co-polylactide-2-hydroxyethyl methacrylate-co-oligo(ethylene glycol)monomethyl ether methacrylate, was functionalized with succinimide ester groups by incorporating N-acryloxysuccinimide monomer. These ester groups were exploited to covalently bond this polymer, denoted as PNPHO, to different proteins with primary amine groups such as α-elastin in aqueous media. The incorporation of elastin through covalent bond formation with PNPHO promotes the structural stability, mechanical properties and live cell proliferation within the structure of hydrogels. Our results demonstrated that elastin-co-PNPHO solutions were injectable through fine gauge needles and converted to hydrogels in situ at 37 °C in the absence of any crosslinking reagent. By altering PNPHO content, the gelling time of these hydrogels can be finely tuned within the range of 2 to 15 min to ensure compatibility with surgical requirements. In addition, these hydrogels exhibited compression moduli in the range of 40 to 145 kPa, which are substantially higher than those of previously developed elastin-based hydrogels. These hydrogels were highly stable in the physiological environment with the evidence of 10 wt% mass loss in 30 days of incubation in a simulated environment. This class of hydrogels is in vivo bioabsorbable due to the gradual increase of the lower critical solution temperature of the copolymer to above 37 °C due to the cleavage of polylactide from

  8. Kinetics of isomerization and inversion of aspartate 58 of αA-crystallin peptide mimics under physiological conditions.

    Directory of Open Access Journals (Sweden)

    Kenzo Aki

    Full Text Available Although proteins consist exclusively of L-amino acids, we have reported that aspartyl (Asp 58 and Asp 151 residues of αA-crystallin of eye lenses from elderly cataract donors are highly inverted and isomerized to D-β, D-α and L-β-Asp residues through succinimide intermediates. Of these Asp isomers, large amounts of D-β- and L-β-isomers are present but the amount of D-α-isomer is not significant. The difference in abundance of the Asp isomers in the protein may be due to the rate constants for the formation of the isomers. However, the kinetics have not been well defined. Therefore, in this study, we synthesized a peptide corresponding to human αA-crystallin residues 55 to 65 (T(55VLD(58SGISEVR(65 and its isomers in which L-α-Asp at position 58 was replaced with L-β-, D-β- and D-α-Asp and determined the rate of isomerization and inversion of Asp residues under physiological conditions (37°C, pH7.4. The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 3 times higher than the rate constant for dehydration from L-β-Asp peptide to L-succinimidyl peptide. The rate constant for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide was about 5 times higher than the rate constant for hydrolysis from L-succinimidyl peptide to L-α-Asp peptide. The rate constant for dehydration from L-α-Asp peptide to L-succinimidyl peptide was 2 times higher than the rate constant for dehydration from D-α-Asp peptide to D-succinimidyl peptide. The rate constants for hydrolysis from L-succinimidyl peptide to L-β-Asp peptide and for hydrolysis from D-succinimidyl peptide to D-β-Asp peptide were almost equal. Using these rate constants, we calculated the change in the abundance ratios of the 4 Asp isomers during a human lifespan. This result is consistent with the fact that isomerized Asp residues accumulate in proteins during the ageing process.

  9. Dispersants in an organic medium: synthesis and physicochemical study of dispersants for fuels and lubricants; Dispersants en milieu organique: synthese et etude physicochimique de dispersants pour carburants et lubrifiants

    Energy Technology Data Exchange (ETDEWEB)

    Dubois-Clochard, M.C.


    Carbonaceous deposits coming from the fuel and the lubricant are known to form over time at critical locations in an engine. In general, the deposits have an adverse effect on four functional areas which are the fuel metering system, the intake system, the lubrication system and the combustion chambers. These deposits can degrade vehicle performance and drive-ability, reduce fuel economy, increase fuel consumption and pollutant emissions and may lead to the destruction of the engine. In order to remedy these problems, detergent-dispersant additives are used in fuels and lubricants to avoid or decrease deposit adhesion on metallic surfaces and prevent from deposit aggregation. These products are mainly polymer surfactants and in this work, poly-iso-butenyl-succinimide of different structures have been studied. Firstly, 'comb like' polymers have been synthesized. Then they have been compared to classical di-bloc additives in terms of performance and action mechanism. These additives are adsorbed from their hydrophilic polyamine part on the acidic functions of the carbon black surface chosen as an engine deposit model and on the aluminium oxide function of an aluminium powder chosen as an engine wall model. The adsorption increases with temperature on the two solids. Their affinity with the solid surface increases with the length of the hydrophilic part. In the same way, changing the di-bloc structure for a comb like one lead to a better adsorption. At low concentration, it has been shown that the adsorption phenomenon was irreversible, due to the polymer structure of the polar part. Depending on the space required by the hydrophilic part on the solid surface, a more of less dense monolayer is formed. At higher concentrations, an important increase of the adsorbed amount appears. This phenomenon is totally reversible showing that the interactions additive / additive are weak. The dispersing efficiency of a comb like structure is better than a di-bloc one as

  10. Comparative pharmacokinetics and biodistribution studies of {sup 99m}Tc-annexin V produced by different radiolabeling methods

    Energy Technology Data Exchange (ETDEWEB)

    Santos, Josefina da Silva; Pujatti, Priscilla Brunelli; Couto, Renata Martinussi; Mengatti, Jair; Araujo, Elaine Bortoleti de, E-mail: jssantos@usp.b, E-mail:, E-mail:, E-mail: jmengatti@ipen.b, E-mail: ebaraujo@ipen.b [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil)


    The use of radiolabeled annexin A5 (ANXA5) to detect cell death in vivo has increased in the last years. Several {sup 99m}Tc-labeling techniques were reported using different cores, such as [{sup 99m}Tc=O]{sup +3}, [{sup 99m}Tc]HYNIC, [{sup 99m}Tcident toN]{sup +2} and [Tc(CO{sub 3})]{sup +1}. The goal of the present work was to evaluate the influence of {sup 99m}Tc cores in the biological behavior of radiolabeled ANXA5 in Swiss mice using [{sup 99m}Tc=O]{sup +3}, [{sup 99m}Tc]HYNIC cores. Ethylenedicysteine (EC) was applied to obtain [Tc=O]{sup +3} core, N,N,N',N'-tetramethyl(succinimide) uranium tetrafluoroborate (TSTU) was employed to transfer the carboxyl group to their corresponding hydroxysuccinimide ester and HYNIC-ANXA5 was provided by National Cancer Institute-Frederick. ITLC-SG and HPLC analysis were applied to determine non-desirable products and the stability of preparations was evaluated after incubation at room temperature, 4 deg C and in human serum at 37 deg C. In vivo biodistribution and kinetics studies were performed after the intravenous injection of {sup 99m}Tc-HYNIC-ANXA5 and {sup 99m}Tc-EC-ANXA5 and pharmacokinetic parameters were calculated using Biexp software. ANXA5 was radiolabeled at room temperature with high yield (> 95%). The results of biodistribution in mice showed, as expected, higher renal uptake of {sup 99m}Tc-HYNICANXA5 and higher liver uptake of {sup 99m}Tc-EC-ANXA5. The percent injected activity per gram (% IA/g) in liver at 0.5 hours were 6.52 and 1.09 and in kidneys were 1.59 and 32.2 for {sup 99m}Tc-EC-ANXA5 and {sup 99m}Tc-HYNICANXA5, respectively. The results of radioactivity in blood showed that both HYNIC- and EC- radiolabeled ANXA5 presented fast blood clearance. In this study two {sup 99m}Tc-ANXA5 obtained from three different available radiolabeling methods presently were investigated. Each labeling method possesses unique advantages and disadvantages. (author)

  11. Tunable Enzymatic Activity and Enhanced Stability of Cellulase Immobilized in Biohybrid Nanogels. (United States)

    Peng, Huan; Rübsam, Kristin; Jakob, Felix; Schwaneberg, Ulrich; Pich, Andrij


    This paper reports a facile approach for encapsulation of enzymes in nanogels. Our approach is based on the use of reactive copolymers able to get conjugated with enzyme and build 3D colloidal networks or biohybrid nanogels. In a systematic study, we address the following question: how the chemical structure of nanogel network influences the biocatalytic activity of entrapped enzyme? The developed method allows precise control of the enzyme activity and improvement of enzyme resistance against harsh store conditions, chaotropic agents, and organic solvents. The nanogels were constructed via direct chemical cross-linking of water-soluble reactive copolymers poly(N-vinylpyrrolidone-co-N-methacryloxysuccinimide) with proteins such as enhanced green fluorescent protein (EGFP) and cellulase in water-in-oil emulsion. The water-soluble reactive copolymers with controlled amount of reactive succinimide groups and narrow dispersity were synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. Poly(ethylene glycol) bis(3-aminopropyl) and branched polyethylenimine were utilized as model cross-linkers to optimize synthesis of nanogels with different architectures in the preliminary experiments. Biofluorescent nanogels with different loading amount of EGFP and varying cross-linking densities were obtained. We demonstrate that the biocatalytic activity of cellulase-conjugated nanogels (CNG) can be elegantly tuned by control of their cross-linking degrees. Circular dichroism (CD) spectra demonstrated that the secondary structures of the immobilized cellulase were changed in the aspect of α-helix contents. The secondary structures of cellulase in highly cross-linked nanogels were strongly altered compared with loosely cross-linked nanogels. The fluorescence resonance energy transfer (FRET) based study further revealed that nanogels with lower cross-linking degree enable higher substrate transport rate, providing easier access to the active site of

  12. Poly(aspartic acid) with adjustable pH-dependent solubility. (United States)

    Németh, Csaba; Gyarmati, Benjámin; Abdullin, Timur; László, Krisztina; Szilágyi, András


    Poly(aspartic acid) (PASP) derivatives with adjustable pH-dependent solubility were synthesized and characterized to establish the relationship between their structure and solubility in order to predict their applicability as a basic material for enteric coatings. Polysuccinimide, the precursor of PASP, was modified with short chain alkylamines, and the residual succinimide rings were subsequently opened to prepare the corresponding PASP derivatives. Study of the effect of the type and concentration of the side groups on the pH-dependent solubility of PASP showed that solubility can be adjusted by proper selection of the chemical structure. The Henderson-Hasselbalch (HH) and the extended HH equations were used to describe the pH-dependent solubility of the polymers quantitatively. The estimate provided by the HH equation is poor, but an accurate description of the pH-dependent solubility can be found with the extended HH equation. The dissolution rate of a polymer film prepared from a selected PASP derivative was determined by fluorescence marking. The film dissolved rapidly when the pH was increased above its pK a . Cellular viability tests show that PASP derivatives are non-toxic to a human cell line. These polymers are thus of great interest as starting materials for enteric coatings. Poly(amino acid) type biocompatible polymers were synthesized for future use as pharmaceutical film coatings. To this end, we tailored the pH-dependent solubility of poly(aspartic acid) (PASP). It was found that both the solubility and the pK a values of the modified PASP depended strongly on composition. Fluorescent marking was used to characterize the dissolution of a chosen PASP derivative. In acidic media only a negligible amount of the polymer dissolved, but dissolution was very fast and complete at the pH values that prevail in the small intestine. As a consequence, enteric coatings based on such PASP derivatives may be used for drug delivery in the gastrointestinal tract

  13. Synthesis and characterization of carboxylic acid functionalized silicon nanoparticles (United States)

    Shaner, Ted V.

    Silicon nanoparticles are of great interest in a great number of fields. Silicon nanoparticles show great promise particularly in the field of bioimaging. Carboxylic acid functionalized silicon nanoparticles have the ability to covalently bond to biomolecules through the conjugation of the carboxylic acid to an amine functionalized biomolecule. This thesis explores the synthesis of silicon nanoparticles functionalized by both carboxylic acids and alkenes and their carboxylic acid functionality. Also discussed is the characterization of the silicon nanoparticles by the use of x-ray spectroscopy. Finally, the nature of the Si-H bond that is observed on the surface of the silicon nanoparticles will be investigated using photoassisted exciton mediated hydrosilation reactions. The silicon nanoparticles are synthesized from both carboxylic acids and alkenes. However, the lack of solubility of diacids is a significant barrier to carboxylic acid functionalization by a mixture of monoacids and diacids. A synthesis route to overcome this obstacle is to synthesize silicon nanoparticles with terminal vinyl group. This terminal vinyl group is distal to the surface of the silicon nanoparticle. The conversion of the vinyl group to a carboxylic acid is accomplished by oxidative cleavage using ozonolysis. The carboxylic acid functionalized silicon nanoparticles were then successfully conjugated to amine functionalized DNA strand through an n-hydroxy succinimide ester activation step, which promotes the formation of the amide bond. Conjugation was characterized by TEM and polyacrylamide gel electrophoresis (PAGE). The PAGE results show that the silicon nanoparticle conjugates move slower through the polyacrylamide gel, resulting in a significant separation from the nonconjugated DNA. The silicon nanoparticles were then characterized by the use of x-ray absorption near edge spectroscopy (Xanes) and x-ray photoelectron spectroscopy (XPS) to investigate the bonding and chemical

  14. Comparative pharmacokinetics and biodistribution studies of 99mTc-annexin V produced by different radiolabeling methods

    International Nuclear Information System (INIS)

    Santos, Josefina da Silva; Pujatti, Priscilla Brunelli; Couto, Renata Martinussi; Mengatti, Jair; Araujo, Elaine Bortoleti de


    The use of radiolabeled annexin A5 (ANXA5) to detect cell death in vivo has increased in the last years. Several 99m Tc-labeling techniques were reported using different cores, such as [ 99m Tc=O] +3 , [ 99m Tc]HYNIC, [ 99m Tc≡N] +2 and [Tc(CO 3 )] +1 . The goal of the present work was to evaluate the influence of 99m Tc cores in the biological behavior of radiolabeled ANXA5 in Swiss mice using [ 99m Tc=O] +3 , [ 99m Tc]HYNIC cores. Ethylenedicysteine (EC) was applied to obtain [Tc=O] +3 core, N,N,N',N'-tetramethyl(succinimide) uranium tetrafluoroborate (TSTU) was employed to transfer the carboxyl group to their corresponding hydroxysuccinimide ester and HYNIC-ANXA5 was provided by National Cancer Institute-Frederick. ITLC-SG and HPLC analysis were applied to determine non-desirable products and the stability of preparations was evaluated after incubation at room temperature, 4 deg C and in human serum at 37 deg C. In vivo biodistribution and kinetics studies were performed after the intravenous injection of 99m Tc-HYNIC-ANXA5 and 99m Tc-EC-ANXA5 and pharmacokinetic parameters were calculated using Biexp software. ANXA5 was radiolabeled at room temperature with high yield (> 95%). The results of biodistribution in mice showed, as expected, higher renal uptake of 99m Tc-HYNICANXA5 and higher liver uptake of 99m Tc-EC-ANXA5. The percent injected activity per gram (% IA/g) in liver at 0.5 hours were 6.52 and 1.09 and in kidneys were 1.59 and 32.2 for 99m Tc-EC-ANXA5 and 99m Tc-HYNICANXA5, respectively. The results of radioactivity in blood showed that both HYNIC- and EC- radiolabeled ANXA5 presented fast blood clearance. In this study two 99m Tc-ANXA5 obtained from three different available radiolabeling methods presently were investigated. Each labeling method possesses unique advantages and disadvantages. (author)

  15. Catalytic phosphonation of high performance polymers and POSS. Novel components for polymer blend and nanocomposite fuel cell membranes

    Energy Technology Data Exchange (ETDEWEB)

    Bock, T.R.


    Aim of this thesis was the preparation and evaluation of phosphonated high performance (HP) polyelectrolytes and polyhedral oligomeric silsesquioxanes (POSS) for polyelectrolyte membrane fuel cell (PEMFC) application. Brominated derivatives of the commercial high performance (HP) polymers poly(ethersulfone) (PES), poly(etheretherketone) (PEEK), poly(phenylsulfone) (PPSu), poly(sulfone) (PSU) and of octaphenyl-POSS of own production were phosphonated by Ni-catalysed Arbuzov reaction. Phosphonated PSU was cast into pure and blend films with sulfonated PEEK (s-PEEK) to investigate H+-conductivity, water uptake and film morphology. Blend films' properties were referenced to films containing unmodified blend partners. Solution-compounding of phosphonated octaphenyl-POSS and s-PEEK was used to produce novel nanocomposite films. An in-situ zirconisation method was assessed as convenient strategy for novel ionically crosslinked membranes of enhanced swelling resistance. Dibromo isocyanuric acid (DBI) and N-bromo succinimide (NBS) as brominating agents allowed polymer analogous preparation of the novel brominated PES and PEEK with precise reaction control. A random distribution of functional groups, i.e. polyelectrolytes' microstructural homogeneity was revealed as decisive factor concerning solubility of phosphonated PSU. Brominated phT8 was prepared with Br2 by a high temperature approach in tetrachloroethane (TCE). Brominated polymers were phosphonated by Ni-catalysis in non-coordinating high temperature solvents, such as diphenylether, benzophenone and diphenylsulfone without notable solvent influence. The lack of solvent - catalyst complexes and high reaction temperatures of 180-200 C led to halogen-free phosphonates with unprecedented high functionalities. Polymer analogous application of P(OSiMe3)3 offered a novel direct access to easily cleavable disilyl ester derivatives. These were obtained from PEEK and PSU in near quantitative yields at NiCl2-loads as

  16. Synthesis and characterization of tethered lipid assemblies for membrane protein reconstitution (Review). (United States)

    Veneziano, Rémi; Rossi, Claire; Chenal, Alexandre; Brenner, Catherine; Ladant, Daniel; Chopineau, Joël


    Biological membranes and their related molecular mechanisms are essential for all living organisms. Membranes host numerous proteins and are responsible for the exchange of molecules and ions, cell signaling, and cell compartmentation. Indeed, the plasma membrane delimits the intracellular compartment from the extracellular environment and intracellular membranes. Biological membranes also play a major role in metabolism regulation and cellular physiology (e.g., mitochondrial membranes). The elaboration of membrane based biomimetic systems allows us to reconstitute and investigate, in controlled conditions, biological events occurring at the membrane interface. A whole variety of model membrane systems have been developed in the last few decades. Among these models, supported membranes were developed on various hydrophilic supports. The use of solid supports enables the direct use of surface sensitive techniques (e.g., surface plasmon resonance, quartz crystal microbalance, and atomic force microscopy) to monitor and quantify events occurring at the membrane surface. Tethered bilayer membranes (tBLMs) could be considered as an achievement of the first solid supported membranes described by the McConnell group. Tethered bilayers on solid supports were designed to delimit an inside compartment from an outside one. They were used for measuring interactions with ligands or incorporating large membrane proteins or complexes without interference with the support. In this context, the authors developed an easy concept of versatile tBLMs assembled on amino coated substrates that are formed upon the vesicle fusion rupture process applicable to protein-free vesicles as well as proteoliposomes. The phospholipid bilayer (natural or synthetic lipids) incorporated 5% of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly ethylene glycol-N-hydroxy succinimide to ensure the anchorage of the bilayer to the amino coated surface. The conditions for the formation of tBLMs on amino

  17. Acute toxicity induced by 2-aryl-N-methylsuccinimides. (United States)

    Rankin, G O; Shih, H C; Teets, V J; Nicoll, D W; Brown, P I


    Phensuximide (PSX) is a 2-arylsuccinimide useful in the treatment of absence seizures. PSX is a mild urotoxicant and is structurally related to N-phenylsuccinimide (NPS) and its antifungal derivatives. Since substitution of the phenyl ring of NPS with chloro or tert-butyl groups can produce compounds with enhanced nephrotoxic potential, it was felt that similar substitutions on the phenyl ring of PSX also might produce derivatives with enhanced nephrotoxic potential. Three derivatives of PSX were prepared and tested: 2-(3-chlorophenyl)-N-methylsuccinimide (CPMS); 2-(4-tert-butylphenyl)-N-methylsuccinimide (BPMS) and 2-(3,5-dichlorophenyl)-N-methylsuccinimide (DPMS). In one set of experiments, male Fischer 344 rats were administered a single intraperitoneal (i.p.) injection of a succinimide (0.4 or 1.0 mmol kg-1) or vehicle (sesame oil, 2.5 ml kg-1) and renal function monitored at 24 and 48 h. Only minor changes in renal function were noted with the PSX derivatives. BPMS and DPMS (1.0 mmol kg-1) treatment induced mild renal tubular necrosis and thickening of the glomerular membranes. However, no significant morphological changes were noted in ureters, bladder or liver in any treatment group. In a second set of experiments, rats were pretreated with phenobarbital (75 mg kg-1 day-1, i.p., 3 days) followed by a single i.p. injection of DPMS (0.4 or 1.0 mmol kg-1) or DPMS vehicle. Renal function was monitored as before. Phenobarbital pretreatment did not markedly enhance the functional nephrotoxicity induced by DPMS (0.4 mmol), but tubular necrosis was greater than observed in non-phenobarbital-pretreated rats receiving DPMS (1.0 mmol kg-1). In addition, hepatotoxicity was observed as the appearance of numerous non-staining vacuoles in hypertrophied hepatocytes. In the phenobarbital plus DPMS (1.0 mmol kg-1) treatment group, all rats died by 48 h. Prior to death, rats exhibited increased proteinuria (+3), hematuria (+3) and blood urea nitrogen concentration. At 24 h