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Sample records for membrane targeted horseradish

  1. Living target of Ce(III) action on horseradish cells: proteins on/in cell membrane.

    Science.gov (United States)

    Yang, Guangmei; Sun, Zhaoguo; Lv, Xiaofen; Deng, Yunyun; Zhou, Qing; Huang, Xiaohua

    2012-12-01

    Positive and negative effects of rare earth elements (REEs) in life have been reported in many papers, but the cellular mechanisms have not been answered, especially the action sites of REEs on plasma membrane are unknown. Proteins on/in the plasma membrane perform main functions of the plasma membrane. Cerium (Ce) is the richest REEs in crust. Thus, the interaction between Ce(III) and the proteins on/in the plasma membrane, the morphology of protoplast, and the contents of nutrient elements in protoplast of horseradish were investigated using the optimized combination of the fluorescence microscopy, fluorescence spectroscopy, circular dichroism, scanning electron microscopy, and X-ray energy dispersive spectroscopy. It was found that Ce(III) at the low concentrations (10, 30 μM) could interact with proteins on/in the plasma membrane of horseradish, leading to the improvement in the structure of membrane proteins and the plasma membrane, which accelerated the intra-/extra-cellular substance exchange and further promoted the development of cells. When horseradish was treated with Ce(III) at the high concentrations (60, 80 μM), Ce(III) also could interact with the proteins on/in the plasma membrane of horseradish, leading to the destruction in the structure of membrane proteins and the plasma membrane. These effects decelerated the intra-/extra-cellular substance exchange and further inhibited the development of cells. Thus, the interaction between Ce(III) and proteins on/in the plasma membrane in plants was an important reason of the positive and negative effects of Ce(III) on plants. The results would provide some references for understanding the cellular effect mechanisms of REEs on plants.

  2. Interaction between La(III) and proteins on the plasma membrane of horseradish

    Science.gov (United States)

    Yang, Guang-Mei; Chu, Yun-Xia; Lv, Xiao-Fen; Zhou, Qing; Huang, Xiao-Hua

    2012-06-01

    Lanthanum (La) is an important rare earth element in the ecological environment of plant. The proteins on the plasma membrane control the transport of molecules into and out of cell. It is very important to investigate the effect of La(III) on the proteins on the plasma membrane in the plant cell. In the present work, the interaction between La(III) and proteins on the plasma membrane of horseradish was investigated using optimization of the fluorescence microscopy and fluorescence spectroscopy. It is found that the fluorescence of the complex system of protoplasts and 1-aniline Kenai-8-sulfonic acid in horseradish treated with the low concentration of La(III) is increased compared with that of the control horseradish. The opposite effect is observed in horseradish treated with the high concentration of La(III). These results indicated that the low concentration of La(III) can interact with the proteins on the plasma membrane of horseradish, causing the improvement in the structure of proteins on the plasma membrane. The high concentration of La(III) can also interact with the proteins on the plasma membrane of horseradish, leading to the destruction of the structure of proteins on the plasma membrane. We demonstrate that the proteins on the plasma membrane are the targets of La(III) action on plant cell.

  3. A novel membrane-based process to isolate peroxidase from horseradish roots: optimization of operating parameters.

    Science.gov (United States)

    Liu, Jianguo; Yang, Bo; Chen, Changzhen

    2013-02-01

    The optimization of operating parameters for the isolation of peroxidase from horseradish (Armoracia rusticana) roots with ultrafiltration (UF) technology was systemically studied. The effects of UF operating conditions on the transmission of proteins were quantified using the parameter scanning UF. These conditions included solution pH, ionic strength, stirring speed and permeate flux. Under optimized conditions, the purity of horseradish peroxidase (HRP) obtained was greater than 84 % after a two-stage UF process and the recovery of HRP from the feedstock was close to 90 %. The resulting peroxidase product was then analysed by isoelectric focusing, SDS-PAGE and circular dichroism, to confirm its isoelectric point, molecular weight and molecular secondary structure. The effects of calcium ion on HRP specific activities were also experimentally determined.

  4. Aquaporin-2 membrane targeting

    DEFF Research Database (Denmark)

    Olesen, Emma T B; Fenton, Robert A

    2017-01-01

    The targeting of the water channel aquaporin-2 (AQP2) to the apical plasma membrane of kidney collecting duct principal cells is regulated mainly by the antidiuretic peptide hormone arginine vasopressin (AVP). This process is of crucial importance for the maintenance of body water homeostasis...... of aquaporin-2 (AQP2) to the apical plasma membrane of collecting duct (CD) principal cells (10, 20). This process is mainly regulated by the actions of AVP on the type 2 AVP receptor (V2R), although the V1a receptor may also play a minor role (26). The V2R is classified within the group of 7-transmembrane....... For example, 1) stimulation with the nonspecific AC activator forskolin increases AQP2 membrane accumulation in a mouse cortical collecting duct cell line [e.g., Norregaard et al. (16)]; 2) cAMP increases CD water permeability (15); 3) the cAMP-activated protein kinase A (PKA) can phosphorylate AQP2 on its...

  5. Synthetic membrane-targeted antibiotics.

    Science.gov (United States)

    Vooturi, S K; Firestine, S M

    2010-01-01

    Antimicrobial resistance continues to evolve and presents serious challenges in the therapy of both nosocomial and community-acquired infections. The rise of resistant strains like methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Staphylococcus aureus (VRSA) and vancomycin-resistant enterococci (VRE) suggests that antimicrobial resistance is an inevitable evolutionary response to antimicrobial use. This highlights the tremendous need for antibiotics against new bacterial targets. Agents that target the integrity of bacterial membrane are relatively novel in the clinical armamentarium. Daptomycin, a lipopeptide is a classical example of membrane-bound antibiotic. Nature has also utilized this tactic. Antimicrobial peptides (AMPs), which are found in all kingdoms, function primarily by permeabilizing the bacterial membrane. AMPs have several advantages over existing antibiotics including a broad spectrum of activity, rapid bactericidal activity, no cross-resistance with the existing antibiotics and a low probability for developing resistance. Currently, a small number of peptides have been developed for clinical use but therapeutic applications are limited because of poor bioavailability and high manufacturing cost. However, their broad specificity, potent activity and lower probability for resistance have spurred the search for synthetic mimetics of antimicrobial peptides as membrane-active antibiotics. In this review, we will discuss the different classes of synthetic membrane-bound antibiotics published since 2004.

  6. Cooperative tumour cell membrane targeted phototherapy

    Science.gov (United States)

    Kim, Heegon; Lee, Junsung; Oh, Chanhee; Park, Ji-Ho

    2017-06-01

    The targeted delivery of therapeutics using antibodies or nanomaterials has improved the precision and safety of cancer therapy. However, the paucity and heterogeneity of identified molecular targets within tumours have resulted in poor and uneven distribution of targeted agents, thus compromising treatment outcomes. Here, we construct a cooperative targeting system in which synthetic and biological nanocomponents participate together in the tumour cell membrane-selective localization of synthetic receptor-lipid conjugates (SR-lipids) to amplify the subsequent targeting of therapeutics. The SR-lipids are first delivered selectively to tumour cell membranes in the perivascular region using fusogenic liposomes. By hitchhiking with extracellular vesicles secreted by the cells, the SR-lipids are transferred to neighbouring cells and further spread throughout the tumour tissues where the molecular targets are limited. We show that this tumour cell membrane-targeted delivery of SR-lipids leads to uniform distribution and enhanced phototherapeutic efficacy of the targeted photosensitizer.

  7. Biogenesis and Membrane Targeting of Lipoproteins.

    Science.gov (United States)

    Narita, Shin-Ichiro; Tokuda, Hajime

    2010-09-01

    Bacterial lipoproteins represent a unique class of membrane proteins, which are anchored to membranes through triacyl chains attached to the amino-terminal cysteine. They are involved in various functions localized in cell envelope. Escherichia coli possesses more than 90 species of lipoproteins, most of which are localized in the outer membrane, with others being in the inner membrane. All lipoproteins are synthesized in the cytoplasm with an N-terminal signal peptide, translocated across the inner membrane by the Sec translocon to the periplasmic surface of the inner membrane, and converted to mature lipoproteins through sequential reactions catalyzed by three lipoprotein-processing enzymes: Lgt, LspA, and Lnt. The sorting of lipoproteins to the outer membrane requires a system comprising five Lol proteins. An ATP-binding cassette transporter, LolCDE, initiates the sorting by mediating the detachment of lipoproteins from the inner membrane. Formation of the LolA-lipoprotein complex is coupled to this LolCDE-dependent release reaction. LolA accommodates the amino-terminal acyl chain of lipoproteins in its hydrophobic cavity, thereby generating a hydrophilic complex that can traverse the periplasmic space by diffusion. Lipoproteins are then transferred to LolB on the outer membrane and anchored to the inner leaflet of the outer membrane by the action of LolB. In contrast, since LolCDE does not recognize lipoproteins possessing Asp at position +2, these lipoproteins remain anchored to the inner membrane. Genes for Lol proteins are widely conserved among gram-negative bacteria, and Lol-mediated outer membrane targeting of lipoproteins is considered to be the general lipoprotein localization mechanism.

  8. Distribution and Translocation of 141Ce (III) in Horseradish

    Science.gov (United States)

    Guo, Xiaoshan; Zhou, Qing; Lu, Tianhong; Fang, Min; Huang, Xiaohua

    2007-01-01

    Background and Aims Rare earth elements (REEs) are used in agriculture and a large amount of them contaminate the environment and enter foods. The distribution and translocation of 141Ce (III) in horseradish was investigated in order to help understand the biochemical behaviour and toxic mechanism of REEs in plants. Method The distribution and translocation of 141Ce (III) in horseradish were investigated using autoradiography, liquid scintillation counting (LSC) and electron microscopic autoradiography (EMARG) techniques. The contents of 141Ce (III) and nutrient elements were analysed using an inductively coupled plasma-atomic emission spectrometer (ICP-AES). Results The results from autoradiography and LSC indicated that 141Ce (III) could be absorbed by horseradish and transferred from the leaf to the leaf-stalk and then to the root. The content of 141Ce (III) in different parts of horseradish was as follows: root > leaf-stalk > leaf. The uptake rates of 141Ce (III) in horseradish changed with the different organs and time. The content of 141Ce (III) in developing leaves was greater than that in mature leaves. The results from EMARG indicated that 141Ce (III) could penetrate through the cell membrane and enter the mesophyll cells, being present in both extra- and intra-cellular deposits. The contents of macronutrients in horseradish were decreased by 141Ce (III) treatment. Conclusions 141Ce (III) can be absorbed and transferred between organs of horseradish with time, and the distribution was found to be different at different growth stages. 141Ce (III) can enter the mesophyll cells via apoplast and symplast channels or via plasmodesmata. 141Ce (III) can disturb the metabolism of macronutrients in horseradish. PMID:17921527

  9. The plasma membrane as radiosensitive target

    International Nuclear Information System (INIS)

    Koeteles, Gy.J.

    1986-01-01

    Components and conditions rendering the plasma membrane susceptible for ionizing radiation are discussed. The list of reviews and articles pointing to various aspects of radiation effects on membranes is analyzed. Radiation induced alterations of plasma membrane and energy deposition in cellular microstructures are overviewed. The possible role of membrane alterations in the fate of irradiated cell is also discussed. (author)

  10. Horseradish Peroxidase-Encapsulated Hollow Silica Nanospheres for Intracellular Sensing of Reactive Oxygen Species

    Science.gov (United States)

    Chen, Hsin-Yi; Wu, Si-Han; Chen, Chien-Tsu; Chen, Yi-Ping; Chang, Feng-Peng; Chien, Fan-Ching; Mou, Chung-Yuan

    2018-04-01

    Reactive oxygen species (ROS) have crucial roles in cell signaling and homeostasis. Overproduction of ROS can induce oxidative damage to various biomolecules and cellular structures. Therefore, developing an approach capable of monitoring and quantifying ROS in living cells is significant for physiology and clinical diagnoses. Some cell-permeable fluorogenic probes developed are useful for the detection of ROS while in conjunction with horseradish peroxidase (HRP). Their intracellular scenario is however hindered by the membrane-impermeable property of enzymes. Herein, a new approach for intracellular sensing of ROS by using horseradish peroxidase-encapsulated hollow silica nanospheres (designated HRP@HSNs), with satisfactory catalytic activity, cell membrane permeability, and biocompatibility, was prepared via a microemulsion method. These HRP@HSNs, combined with selective probes or targeting ligands, could be foreseen as ROS-detecting tools in specific organelles or cell types. As such, dihydrorhodamine 123-coupled HRP@HSNs were used for the qualitative and semi-quantitative analysis of physiological H2O2 levels in activated RAW 264.7 macrophages. We envision that this HSNs encapsulating active enzymes can be conjugated with selective probes and targeting ligands to detect ROS in specific organelles or cell types of interest.

  11. Membrane Targeting of P-type ATPases in Plant Cells

    International Nuclear Information System (INIS)

    Harper, Jeffrey F.

    2004-01-01

    How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

  12. Superdiffusive motion of membrane-targeting C2 domains

    Science.gov (United States)

    Campagnola, Grace; Nepal, Kanti; Schroder, Bryce W.; Peersen, Olve B.; Krapf, Diego

    2015-12-01

    Membrane-targeting domains play crucial roles in the recruitment of signalling molecules to the plasma membrane. For most peripheral proteins, the protein-to-membrane interaction is transient. After proteins dissociate from the membrane they have been observed to rebind following brief excursions in the bulk solution. Such membrane hops can have broad implications for the efficiency of reactions on membranes. We study the diffusion of membrane-targeting C2 domains using single-molecule tracking in supported lipid bilayers. The ensemble-averaged mean square displacement (MSD) exhibits superdiffusive behaviour. However, traditional time-averaged MSD analysis of individual trajectories remains linear and does not reveal superdiffusion. Our observations are explained in terms of bulk excursions that introduce jumps with a heavy-tail distribution. These hopping events allow proteins to explore large areas in a short time. The experimental results are shown to be consistent with analytical models of bulk-mediated diffusion and numerical simulations.

  13. Membranes as sensitive targets in thymocyte apoptosis

    International Nuclear Information System (INIS)

    Ramakrishnan, N.; McClain, D.E.; Catravas, G.N.

    1993-01-01

    The role of cellular membranes in thymocyte apoptosis has been examined. Trolox, a water soluble analogue of vitamin E and inhibitor of membrane damage, inhibits DNA fragmentation in thymocytes exposed to γ-radiation, and is most effective in inhibiting DNA fragmentation when added to cells within 30 min post-irradiation. Exposure to trolox only during irradiation did not prevent DNA fragmentation. Incubation of the irradiated cell suspension with trolox for 2h post-irradiation was sufficient to prevent DNA fragmentation measured at 24 h in irradiated cells, suggesting that trolox irreversibly inhibits a cellular lesion required for apoptosis. The induction of DNA fragmentation appears to be related to a concurrent, pronounced flow of Ca 2+ into the cell. At 3 h post-irradiation the amount of Ca 2+ in irradiated thymocytes was more than twice that of unirradiated thymocytes. Trolox treatment completely blocked the radiation-induced influx of Ca 2+ into the thymocytes. These results suggest that membrane damage is a critical lesion involved in DNA fragmentation in thymocyte apoptosis. (author)

  14. Oxidation of NAD dimers by horseradish peroxidase.

    OpenAIRE

    Avigliano, L; Carelli, V; Casini, A; Finazzi-Agrò, A; Liberatore, F

    1985-01-01

    Horseradish peroxidase catalyses the oxidation of NAD dimers, (NAD)2, to NAD+ in accordance with a reaction that is pH-dependent and requires 1 mol of O2 per 2 mol of (NAD)2. Horseradish peroxidase also catalyses the peroxidation of (NAD)2 to NAD+. In contrast, bacterial NADH peroxidase does not catalyse the peroxidation or the oxidation of (NAD)2. A free-radical mechanism is proposed for both horseradish-peroxidase-catalysed oxidation and peroxidation of (NAD)2.

  15. Glycosylation and thermodynamic versus kinetic stability of horseradish peroxidase

    DEFF Research Database (Denmark)

    Tams, J.W.; Welinder, Karen G.

    1998-01-01

    Glycoprotein stability, glycoprotein unfolding, horseradish peroxidase, thermodynamic stability, kinetik stability......Glycoprotein stability, glycoprotein unfolding, horseradish peroxidase, thermodynamic stability, kinetik stability...

  16. Designing block copolymer architectures for targeted membrane performance

    KAUST Repository

    Dorin, Rachel Mika

    2014-01-01

    Using a combination of block copolymer self-assembly and non-solvent induced phase separation, isoporous ultrafiltration membranes were fabricated from four poly(isoprene-b-styrene-b-4-vinylpyridine) triblock terpolymers with similar block volume fractions but varying in total molar mass from 43 kg/mol to 115 kg/mol to systematically study the effect of polymer size on membrane structure. Small-angle X-ray scattering was used to probe terpolymer solution structure in the dope. All four triblocks displayed solution scattering patterns consistent with a body-centered cubic morphology. After membrane formation, structures were characterized using a combination of scanning electron microscopy and filtration performance tests. Membrane pore densities that ranged from 4.53 × 1014 to 1.48 × 1015 pores/m 2 were observed, which are the highest pore densities yet reported for membranes using self-assembly and non-solvent induced phase separation. Hydraulic permeabilities ranging from 24 to 850 L m-2 h-1 bar-1 and pore diameters ranging from 7 to 36 nm were determined from permeation and rejection experiments. Both the hydraulic permeability and pore size increased with increasing molar mass of the parent terpolymer. The combination of polymer characterization and membrane transport tests described here demonstrates the ability to rationally design macromolecular structures to target specific performance characteristics in block copolymer derived ultrafiltration membranes. © 2013 Elsevier Ltd. All rights reserved.

  17. Unravelling important odorants in horseradish (Armoracia rusticana).

    Science.gov (United States)

    Kroener, Eva-Maria; Buettner, Andrea

    2017-10-01

    Horseradish (Armoracia rusticana) is a plant well known for its roots' spicy aroma. The present study investigates the main aroma constituents of horseradish roots in general by analysing the aroma profiles of six different horseradish varieties, with one variety grown in two different soils. Odorants were characterised by means of gas chromatography-olfactometry and identified via their mass spectra, retention indices on two columns with different polarity, and their characteristic odour. A series of new aroma compounds from different substance groups were identified that have hitherto not been described in horseradish. Moreover, several of these constituents were successfully shown to exhibit high odour potency, alongside a high potential to influence the overall aroma of horseradish roots, like (3S,3aS,7aR)-wine lactone and 3-isopropyl-2-methoxypyrazine. Copyright © 2017 Elsevier Ltd. All rights reserved.

  18. Membrane Transporters: Structure, Function and Targets for Drug Design

    Science.gov (United States)

    Ravna, Aina W.; Sager, Georg; Dahl, Svein G.; Sylte, Ingebrigt

    Current therapeutic drugs act on four main types of molecular targets: enzymes, receptors, ion channels and transporters, among which a major part (60-70%) are membrane proteins. This review discusses the molecular structures and potential impact of membrane transporter proteins on new drug discovery. The three-dimensional (3D) molecular structure of a protein contains information about the active site and possible ligand binding, and about evolutionary relationships within the protein family. Transporters have a recognition site for a particular substrate, which may be used as a target for drugs inhibiting the transporter or acting as a false substrate. Three groups of transporters have particular interest as drug targets: the major facilitator superfamily, which includes almost 4000 different proteins transporting sugars, polyols, drugs, neurotransmitters, metabolites, amino acids, peptides, organic and inorganic anions and many other substrates; the ATP-binding cassette superfamily, which plays an important role in multidrug resistance in cancer chemotherapy; and the neurotransmitter:sodium symporter family, which includes the molecular targets for some of the most widely used psychotropic drugs. Recent technical advances have increased the number of known 3D structures of membrane transporters, and demonstrated that they form a divergent group of proteins with large conformational flexibility which facilitates transport of the substrate.

  19. Membrane and inclusion body targeting of lyssavirus matrix proteins.

    Science.gov (United States)

    Pollin, Reiko; Granzow, Harald; Köllner, Bernd; Conzelmann, Karl-Klaus; Finke, Stefan

    2013-02-01

    Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation, differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinctintracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis. © 2012 Blackwell Publishing Ltd.

  20. A Peptidomimetic Antibiotic Targets Outer Membrane Proteins and Disrupts Selectively the Outer Membrane in Escherichia coli.

    Science.gov (United States)

    Urfer, Matthias; Bogdanovic, Jasmina; Lo Monte, Fabio; Moehle, Kerstin; Zerbe, Katja; Omasits, Ulrich; Ahrens, Christian H; Pessi, Gabriella; Eberl, Leo; Robinson, John A

    2016-01-22

    Increasing antibacterial resistance presents a major challenge in antibiotic discovery. One attractive target in Gram-negative bacteria is the unique asymmetric outer membrane (OM), which acts as a permeability barrier that protects the cell from external stresses, such as the presence of antibiotics. We describe a novel β-hairpin macrocyclic peptide JB-95 with potent antimicrobial activity against Escherichia coli. This peptide exhibits no cellular lytic activity, but electron microscopy and fluorescence studies reveal an ability to selectively disrupt the OM but not the inner membrane of E. coli. The selective targeting of the OM probably occurs through interactions of JB-95 with selected β-barrel OM proteins, including BamA and LptD as shown by photolabeling experiments. Membrane proteomic studies reveal rapid depletion of many β-barrel OM proteins from JB-95-treated E. coli, consistent with induction of a membrane stress response and/or direct inhibition of the Bam folding machine. The results suggest that lethal disruption of the OM by JB-95 occurs through a novel mechanism of action at key interaction sites within clusters of β-barrel proteins in the OM. These findings open new avenues for developing antibiotics that specifically target β-barrel proteins and the integrity of the Gram-negative OM. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  1. Expression of membrane targeted aequorin in Xenopus laevis oocytes.

    Science.gov (United States)

    Daguzan, C; Nicolas, M T; Mazars, C; Leclerc, C; Moreau, M

    1995-08-01

    We described here a system for high level of expression of the calcium activated photoprotein aequorin. This protein has been targeted to the plasma membrane of Xenopus oocyte by nuclear microinjection of a plasmid containing a construction of a chimeric cDNA encoding a fusion protein composed of the photoprotein aequorin and the 5-HT1A receptor. The expression of this fusion protein is placed under the control of RSV promoter. Functional photoprotein was reconstituted in the oocyte by incubation with coelenterazine. The amount of photoprotein 24 h after nuclear microinjection of the plasmid was sufficient to trigger a detectable light emission following calcium entry. The efficiency of the expression is correlated with the dose of plasmid injected. Intracytoplasmic injection of the plasmid always failed in photoprotein expression. Targeting of the apoprotein was demonstrated by immunolocalization under confocal microscopy. In our experimental conditions, the apoprotein was always localized at the animal pole above the nucleus. We never observed expression and targeting to the plasma membrane of the vegetal pole. WE suggest that such expression might be of great interest for the study of numerous problems of developmental biology, in which calcium-dependent pathways are involved.

  2. Iron Deprivation Affects Drug Susceptibilities of Mycobacteria Targeting Membrane Integrity

    Directory of Open Access Journals (Sweden)

    Rahul Pal

    2015-01-01

    Full Text Available Multidrug resistance (MDR acquired by Mycobacterium tuberculosis (MTB through continuous deployment of antitubercular drugs warrants immediate search for novel targets and mechanisms. The ability of MTB to sense and become accustomed to changes in the host is essential for survival and confers the basis of infection. A crucial condition that MTB must surmount is iron limitation, during the establishment of infection, since iron is required by both bacteria and humans. This study focuses on how iron deprivation affects drug susceptibilities of known anti-TB drugs in Mycobacterium smegmatis, a “surrogate of MTB.” We showed that iron deprivation leads to enhanced potency of most commonly used first line anti-TB drugs that could be reverted upon iron supplementation. We explored that membrane homeostasis is disrupted upon iron deprivation as revealed by enhanced membrane permeability and hypersensitivity to membrane perturbing agent leading to increased passive diffusion of drug and TEM images showing detectable differences in cell envelope thickness. Furthermore, iron seems to be indispensable to sustain genotoxic stress suggesting its possible role in DNA repair machinery. Taken together, we for the first time established a link between cellular iron and drug susceptibility of mycobacteria suggesting iron as novel determinant to combat MDR.

  3. Rachiplusia nu larva as a biofactory to achieve high level expression of horseradish peroxidase.

    Science.gov (United States)

    Romero, Lucía Virginia; Targovnik, Alexandra Marisa; Wolman, Federico Javier; Cascone, Osvaldo; Miranda, María Victoria

    2011-05-01

    A process based on orally-infected Rachiplusia nu larvae as biological factories for expression and one-step purification of horseradish peroxidase isozyme C (HRP-C) is described. The process allows obtaining high levels of pure HRP-C by membrane chromatography purification. The introduction of the partial polyhedrin homology sequence element in the target gene increased HRP-C expression level by 2.8-fold whereas it increased 1.8-fold when the larvae were reared at 27 °C instead of at 24 °C, summing up a 4.6-fold overall increase in the expression level. Additionally, HRP-C purification by membrane chromatography at a high flow rate greatly increase D the productivity without affecting the resolution. The V(max) and K(m) values of the recombinant HRP-C were similar to those of the HRP from Armoracia rusticana roots. © Springer Science+Business Media B.V. 2011

  4. Specificity of Plasma Membrane Targeting by the Rous Sarcoma Virus Gag Protein

    OpenAIRE

    Scheifele, Lisa Z.; Rhoads, Jonathan D.; Parent, Leslie J.

    2003-01-01

    Budding of C-type retroviruses begins when the viral Gag polyprotein is directed to the plasma membrane by an N-terminal membrane-binding (M) domain. While dispersed basic amino acids within the M domain are critical for stable membrane association and consequent particle assembly, additional residues or motifs may be required for specific plasma membrane targeting and binding. We have identified an assembly-defective Rous sarcoma virus (RSV) Gag mutant that retains significant membrane affin...

  5. Henipavirus Mediated Membrane Fusion, Virus Entry and Targeted Therapeutics

    Directory of Open Access Journals (Sweden)

    Dimitar B. Nikolov

    2012-02-01

    Full Text Available The Paramyxoviridae genus Henipavirus is presently represented by the type species Hendra and Nipah viruses which are both recently emerged zoonotic viral pathogens responsible for repeated outbreaks associated with high morbidity and mortality in Australia, Southeast Asia, India and Bangladesh. These enveloped viruses bind and enter host target cells through the coordinated activities of their attachment (G and class I fusion (F envelope glycoproteins. The henipavirus G glycoprotein interacts with host cellular B class ephrins, triggering conformational alterations in G that lead to the activation of the F glycoprotein, which facilitates the membrane fusion process. Using the recently published structures of HeV-G and NiV-G and other paramyxovirus glycoproteins, we review the features of the henipavirus envelope glycoproteins that appear essential for mediating the viral fusion process, including receptor binding, G-F interaction, F activation, with an emphasis on G and the mutations that disrupt viral infectivity. Finally, recent candidate therapeutics for henipavirus-mediated disease are summarized in light of their ability to inhibit HeV and NiV entry by targeting their G and F glycoproteins.

  6. Control of Target Molecular Recognition in a Small Pore Space with Biomolecule-Recognition Gating Membrane.

    Science.gov (United States)

    Okuyama, Hiroto; Oshiba, Yuhei; Ohashi, Hidenori; Yamaguchi, Takeo

    2018-05-01

    A biomolecule-recognition gating membrane, which introduces thermosensitive graft polymer including molecular recognition receptor into porous membrane substrate, can close its pores by recognizing target biomolecule. The present study reports strategies for improving both versatility and sensitivity of the gating membrane. First, the membrane is fabricated by introducing the receptor via a selectively reactive click reaction improving the versatility. Second, the sensitivity of the membrane is enhanced via an active delivering method of the target molecules into the pores. In the method, the tiny signal of the target biomolecule is amplified as obvious pressure change. Furthermore, this offers 15 times higher sensitivity compared to the previously reported passive delivering method (membrane immersion to sample solution) with significantly shorter recognition time. The improvement will aid in applying the gating membrane to membrane sensors in medical fields. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  7. Prostate Specific Membrane Antigen (PSMA) Targeted Bio-orthogonal Therapy for Metastatic Prostate Cancer

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-16-1-0595 TITLE: Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate Cancer...Sep 2016 - 14 Sep 2017 4. TITLE AND SUBTITLE Prostate-Specific Membrane Antigen (PSMA) Targeted Bio -orthogonal Therapy for Metastatic Prostate

  8. Wound-induced expression of horseradish peroxidase.

    Science.gov (United States)

    Kawaoka, A; Kawamoto, T; Ohta, H; Sekine, M; Takano, M; Shinmyo, A

    1994-01-01

    Peroxidases have been implicated in the responses of plants to physiological stress and to pathogens. Wound-induced peroxidase of horseradish (Armoracia rusticana) was studied. Total peroxidase activity was increased by wounding in cell wall fractions extracted from roots, stems and leaves of horseradish. On the other hand, wounding decreased the peroxidase activity in the soluble fraction from roots. The enzyme activities of the basic isozymes were induced by wounding in horseradish leaves based on data obtained by fractionation of crude enzyme in isoelectric focusing gel electrophoresis followed by activity staining. We have previously isolated genomic clones for four peroxidase genes, namely, prxC1a, prxC1b, prxC2 and prxC3. Northern blot analysis using gene-specific probes showed that mRNA of prxC2, which encodes a basic isozyme, accumulated by wounding, while the mRNAs for other peroxidase genes were not induced. Tobacco (Nicotiana tabacum) plants were transformed with four chimeric gene constructs, each consisting of a promoter from one of the peroxidase genes and the β-glucuronidase (GUS) structural gene. High level GUS activity induced in response to wounding was observed in tobacco plants containing the prxC2-GUS construct.

  9. Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

    Science.gov (United States)

    Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

    1995-01-01

    Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

  10. Palmitoylation of POTE family proteins for plasma membrane targeting

    International Nuclear Information System (INIS)

    Das, Sudipto; Ise, Tomoko; Nagata, Satoshi; Maeda, Hiroshi; Bera, Tapan K.; Pastan, Ira

    2007-01-01

    The POTE gene family is composed of 13 paralogs and likely evolved by duplications and remodeling of the human genome. One common property of POTE proteins is their localization on the inner aspect of the plasma membrane. To determine the structural elements required for membrane localization, we expressed mutants of different POTEs in 293T cells as EGFP fusion proteins. We also tested their palmitoylation by a biotin-switch assay. Our data indicate that the membrane localizations of different POTEs are mediated by similar 3-4 short cysteine rich repeats (CRRs) near the amino-terminuses and that palmitoylation on paired cysteine residues in each CRR motif is responsible for the localization. Multiple palmitoylation in the small CRRs can result in the strong association of whole POTEs with plasma membrane

  11. Designing block copolymer architectures for targeted membrane performance

    KAUST Repository

    Dorin, Rachel Mika; Phillip, William A.; Sai, Hiroaki; Werner, Jö rg; Elimelech, Menachem; Wiesner, Ulrich

    2014-01-01

    Using a combination of block copolymer self-assembly and non-solvent induced phase separation, isoporous ultrafiltration membranes were fabricated from four poly(isoprene-b-styrene-b-4-vinylpyridine) triblock terpolymers with similar block volume

  12. Binding and Fusion of Extracellular Vesicles to the Plasma Membrane of Their Cell Targets.

    Science.gov (United States)

    Prada, Ilaria; Meldolesi, Jacopo

    2016-08-09

    Exosomes and ectosomes, extracellular vesicles of two types generated by all cells at multivesicular bodies and the plasma membrane, respectively, play critical roles in physiology and pathology. A key mechanism of their function, analogous for both types of vesicles, is the fusion of their membrane to the plasma membrane of specific target cells, followed by discharge to the cytoplasm of their luminal cargo containing proteins, RNAs, and DNA. Here we summarize the present knowledge about the interactions, binding and fusions of vesicles with the cell plasma membrane. The sequence initiates with dynamic interactions, during which vesicles roll over the plasma membrane, followed by the binding of specific membrane proteins to their cell receptors. Membrane binding is then converted rapidly into fusion by mechanisms analogous to those of retroviruses. Specifically, proteins of the extracellular vesicle membranes are structurally rearranged, and their hydrophobic sequences insert into the target cell plasma membrane which undergoes lipid reorganization, protein restructuring and membrane dimpling. Single fusions are not the only process of vesicle/cell interactions. Upon intracellular reassembly of their luminal cargoes, vesicles can be regenerated, released and fused horizontally to other target cells. Fusions of extracellular vesicles are relevant also for specific therapy processes, now intensely investigated.

  13. Chinese hamster ovary cell lysosomes retain pinocytized horseradish peroxidase and in situ-radioiodinated proteins

    International Nuclear Information System (INIS)

    Storrie, B.; Sachdeva, M.; Viers, V.S.

    1984-01-01

    We used Chinese hamster ovary cells, a cell line of fibroblastic origin, to investigate whether lysosomes are an exocytic compartment. To label lysosomal contents, Chinese hamster ovary cells were incubated with the solute marker horseradish peroxidase. After an 18-h uptake period, horseradish peroxidase was found in lysosomes by cell fractionation in Percoll gradients and by electron microscope cytochemistry. Over a 24-h period, lysosomal horseradish peroxidase was quantitatively retained by Chinese hamster ovary cells and inactivated with a t 1/2 of 6 to 8 h. Lysosomes were radioiodinated in situ by soluble lactoperoxidase internalized over an 18-h uptake period. About 70% of the radioiodine incorporation was pelleted at 100,000 X g under conditions in which greater than 80% of the lysosomal marker enzyme beta-hexosaminidase was released into the supernatant. By one-dimensional electrophoresis, about 18 protein species were present in the lysosomal membrane fraction, with radioiodine incorporation being most pronounced into species of 70,000 to 75,000 daltons. After a 30-min or 2-h chase at 37 degrees C, radioiodine that was incorporated into lysosomal membranes and contents was retained in lysosomes. These observations indicate that lysosomes labeled by fluid-phase pinocytosis are a terminal component of endocytic pathways in fibroblasts

  14. Erythrocyte membrane-coated gold nanocages for targeted photothermal and chemical cancer therapy

    Science.gov (United States)

    Zhu, Dao-Ming; Xie, Wei; Xiao, Yu-Sha; Suo, Meng; Zan, Ming-Hui; Liao, Qing-Quan; Hu, Xue-Jia; Chen, Li-Ben; Chen, Bei; Wu, Wen-Tao; Ji, Li-Wei; Huang, Hui-Ming; Guo, Shi-Shang; Zhao, Xing-Zhong; Liu, Quan-Yan; Liu, Wei

    2018-02-01

    Recently, red blood cell (RBC) membrane-coated nanoparticles have attracted much attention because of their excellent immune escapability; meanwhile, gold nanocages (AuNs) have been extensively used for cancer therapy due to their photothermal effect and drug delivery capability. The combination of the RBC membrane coating and AuNs may provide an effective approach for targeted cancer therapy. However, few reports have shown the utilization of combining these two technologies. Here, we design erythrocyte membrane-coated gold nanocages for targeted photothermal and chemical cancer therapy. First, anti-EpCam antibodies were used to modify the RBC membranes to target 4T1 cancer cells. Second, the antitumor drug paclitaxel (PTX) was encapsulated into AuNs. Then, the AuNs were coated with the modified RBC membranes. These new nanoparticles were termed EpCam-RPAuNs. We characterized the capability of the EpCam-RPAuNs for selective tumor targeting via exposure to near-infrared irradiation. The experimental results demonstrate that EpCam-RPAuNs can effectively generate hyperthermia and precisely deliver the antitumor drug PTX to targeted cells. We also validated the biocompatibility of the EpCam-RAuNs in vitro. By combining the molecularly modified targeting RBC membrane and AuNs, our approach provides a new way to design biomimetic nanoparticles to enhance the surface functionality of nanoparticles. We believe that EpCam-RPAuNs can be potentially applied for cancer diagnoses and therapies.

  15. Enhanced membrane pore formation through high-affinity targeted antimicrobial peptides.

    Directory of Open Access Journals (Sweden)

    Christopher J Arnusch

    Full Text Available Many cationic antimicrobial peptides (AMPs target the unique lipid composition of the prokaryotic cell membrane. However, the micromolar activities common for these peptides are considered weak in comparison to nisin, which follows a targeted, pore-forming mode of action. Here we show that AMPs can be modified with a high-affinity targeting module, which enables membrane permeabilization at low concentration. Magainin 2 and a truncated peptide analog were conjugated to vancomycin using click chemistry, and could be directed towards specific membrane embedded receptors both in model membrane systems and whole cells. Compared with untargeted vesicles, a gain in permeabilization efficacy of two orders of magnitude was reached with large unilamellar vesicles that included lipid II, the target of vancomycin. The truncated vancomycin-peptide conjugate showed an increased activity against vancomycin resistant Enterococci, whereas the full-length conjugate was more active against a targeted eukaryotic cell model: lipid II containing erythrocytes. This study highlights that AMPs can be made more selective and more potent against biological membranes that contain structures that can be targeted.

  16. Radiation inactivation target size of rat adipocyte glucose transporters in the plasma membrane and intracellular pools

    International Nuclear Information System (INIS)

    Jacobs, D.B.; Berenski, C.J.; Spangler, R.A.; Jung, C.Y.

    1987-01-01

    The in situ assembly states of the glucose transport carrier protein in the plasma membrane and in the intracellular (microsomal) storage pool of rat adipocytes were assessed by studying radiation-induced inactivation of the D-glucose-sensitive cytochalasin B binding activities. High energy radiation inactivated the glucose-sensitive cytochalasin B binding of each of these membrane preparations by reducing the total number of the binding sites without affecting the dissociation constant. The reduction in total number of binding sites was analyzed as a function of radiation dose based on target theory, from which a radiation-sensitive mass (target size) was calculated. When the plasma membranes of insulin-treated adipocytes were used, a target size of approximately 58,000 daltons was obtained. For adipocyte microsomal membranes, we obtained target sizes of approximately 112,000 and 109,000 daltons prior to and after insulin treatment, respectively. In the case of microsomal membranes, however, inactivation data showed anomalously low radiation sensitivities at low radiation doses, which may be interpreted as indicating the presence of a radiation-sensitive inhibitor. These results suggest that the adipocyte glucose transporter occurs as a monomer in the plasma membrane while existing in the intracellular reserve pool either as a homodimer or as a stoichiometric complex with a protein of an approximately equal size

  17. Oxidative degradation of alkylphenols by horseradish peroxidase.

    Science.gov (United States)

    Sakuyama, Hisae; Endo, Yasushi; Fujimoto, Kenshiro; Hatana, Yasuhiko

    2003-01-01

    Alkylphenols such as bisphenol A (2,2-bis(4-hydroxyphenyl)propane; BPA), p-nonylphenol (p-NP), and p-octylphenol (p-OP) that are known as endocrine disrupters were oxidized by horseradish (Armoracia rusticana) peroxidase (HRP) with H2O2. The optimal pHs for BPA, p-NP, and p-OP were 8.0, 7.0, and 5.0, respectively. The optimal temperature for BPA was 20 degrees C. Although BPA was rapidly degraded by HRP, its degradation depended on the concentration of HRP. Most of the oxidation products of BPA were polymers, although some 4-isopropenylphenol was produced. When male Japanese medaka (Oryzias latipes) were exposed to BPA, vitellogenin in the blood increased. However, no increased vitellogenin was observed in medaka exposed to HRP-oxidized BPA. The enzymatic oxidation of BPA using HRP was able to eliminate its estrogen-like activity.

  18. Basolateral cholesterol depletion alters Aquaporin-2 post-translational modifications and disrupts apical plasma membrane targeting.

    Science.gov (United States)

    Moeller, Hanne B; Fuglsang, Cecilia Hvitfeldt; Pedersen, Cecilie Nøhr; Fenton, Robert A

    2018-01-01

    Apical plasma membrane accumulation of the water channel Aquaporin-2 (AQP2) in kidney collecting duct principal cells is critical for body water homeostasis. Posttranslational modification (PTM) of AQP2 is important for regulating AQP2 trafficking. The aim of this study was to determine the role of cholesterol in regulation of AQP2 PTM and in apical plasma membrane targeting of AQP2. Cholesterol depletion from the basolateral plasma membrane of a collecting duct cell line (mpkCCD14) using methyl-beta-cyclodextrin (MBCD) increased AQP2 ubiquitylation. Forskolin, cAMP or dDAVP-mediated AQP2 phosphorylation at Ser269 (pS269-AQP2) was prevented by cholesterol depletion from the basolateral membrane. None of these effects on pS269-AQP2 were observed when cholesterol was depleted from the apical side of cells, or when MBCD was applied subsequent to dDAVP stimulation. Basolateral, but not apical, MBCD application prevented cAMP-induced apical plasma membrane accumulation of AQP2. These studies indicate that manipulation of the cholesterol content of the basolateral plasma membrane interferes with AQP2 PTM and subsequently regulated apical plasma membrane targeting of AQP2. Copyright © 2017 Elsevier Inc. All rights reserved.

  19. HIV-1 matrix dependent membrane targeting is regulated by Gag mRNA trafficking.

    Directory of Open Access Journals (Sweden)

    Jing Jin

    Full Text Available Retroviral Gag polyproteins are necessary and sufficient for virus budding. Productive HIV-1 Gag assembly takes place at the plasma membrane. However, little is known about the mechanisms by which thousands of Gag molecules are targeted to the plasma membrane. Using a bimolecular fluorescence complementation (BiFC assay, we recently reported that the cellular sites and efficiency of HIV-1 Gag assembly depend on the precise pathway of Gag mRNA export from the nucleus, known to be mediated by Rev. Here we describe an assembly deficiency in human cells for HIV Gag whose expression depends on hepatitis B virus (HBV post-transcriptional regulatory element (PRE mediated-mRNA nuclear export. PRE-dependent HIV Gag expressed well in human cells, but assembled with slower kinetics, accumulated intracellularly, and failed to associate with a lipid raft compartment where the wild-type Rev-dependent HIV-1 Gag efficiently assembles. Surprisingly, assembly and budding of PRE-dependent HIV Gag in human cells could be rescued in trans by co-expression of Rev-dependent Gag that provides correct membrane targeting signals, or in cis by replacing HIV matrix (MA with other membrane targeting domains. Taken together, our results demonstrate deficient membrane targeting of PRE-dependent HIV-1 Gag and suggest that HIV MA function is regulated by the trafficking pathway of the encoding mRNA.

  20. Design, construction, and characterization of high-performance membrane fusion devices with target-selectivity.

    Science.gov (United States)

    Kashiwada, Ayumi; Yamane, Iori; Tsuboi, Mana; Ando, Shun; Matsuda, Kiyomi

    2012-01-31

    Membrane fusion proteins such as the hemagglutinin glycoprotein have target recognition and fusion accelerative domains, where some synergistically working elements are essential for target-selective and highly effective native membrane fusion systems. In this work, novel membrane fusion devices bearing such domains were designed and constructed. We selected a phenylboronic acid derivative as a recognition domain for a sugar-like target and a transmembrane-peptide (Leu-Ala sequence) domain interacting with the target membrane, forming a stable hydrophobic α-helix and accelerating the fusion process. Artificial membrane fusion behavior between the synthetic devices in which pilot and target liposomes were incorporated was characterized by lipid-mixing and inner-leaflet lipid-mixing assays. Consequently, the devices bearing both the recognition and transmembrane domains brought about a remarkable increase in the initial rate for the membrane fusion compared with the devices containing the recognition domain alone. In addition, a weakly acidic pH-responsive device was also constructed by replacing three Leu residues in the transmembrane-peptide domain by Glu residues. The presence of Glu residues made the acidic pH-dependent hydrophobic α-helix formation possible as expected. The target-selective liposome-liposome fusion was accelerated in a weakly acidic pH range when the Glu-substituted device was incorporated in pilot liposomes. The use of this pH-responsive device seems to be a potential strategy for novel applications in a liposome-based delivery system. © 2011 American Chemical Society

  1. Natural material-decorated mesoporous silica nanoparticle container for multifunctional membrane-controlled targeted drug delivery

    Directory of Open Access Journals (Sweden)

    Hu Y

    2017-11-01

    Full Text Available Yan Hu,1 Lei Ke,2 Hao Chen,1 Ma Zhuo,1 Xinzhou Yang,1 Dan Zhao,1 Suying Zeng,1 Xincai Xiao1 1Department of Pharmaceutics, School of Pharmaceutical Science, South-Central University for Nationalities, 2Department of Medicinal Chemistry, School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, People’s Republic of China Abstract: To avoid the side effects caused by nonspecific targeting, premature release, weak selectivity, and poor therapeutic efficacy of current nanoparticle-based systems used for drug delivery, we fabricated natural material-decorated nanoparticles as a multifunctional, membrane-controlled targeted drug delivery system. The nanocomposite material coated with a membrane was biocompatible and integrated both specific tumor targeting and responsiveness to stimulation, which improved transmission efficacy and controlled drug release. Mesoporous silica nanoparticles (MSNs, which are known for their biocompatibility and high drug-loading capacity, were selected as a model drug container and carrier. The membrane was established by the polyelectrolyte composite method from chitosan (CS which was sensitive to the acidic tumor microenvironment, folic acid-modified CS which recognizes the folate receptor expressed on the tumor cell surface, and a CD44 receptor-targeted polysaccharide hyaluronic acid. We characterized the structure of the nanocomposite as well as the drug release behavior under the control of the pH-sensitive membrane switch and evaluated the antitumor efficacy of the system in vitro. Our results provide a basis for the design and fabrication of novel membrane-controlled nanoparticles with improved tumor-targeting therapy. Keywords: multifunctional, membrane-controlled, natural materials, mesoporous silica nanoparticles, targeted drug delivery

  2. Phylogenetic profiles of all membrane transport proteins of the malaria parasite highlight new drug targets

    Directory of Open Access Journals (Sweden)

    January Weiner 3rd

    2016-08-01

    Full Text Available In order to combat the on-going malaria epidemic, discovery of new drug targets remains vital. Proteins that are essential to survival and specific to malaria parasites are key candidates. To survive within host cells, the parasites need to acquire nutrients and dispose of waste products across multiple membranes. Additionally, like all eukaryotes, they must redistribute ions and organic molecules between their various internal membrane bound compartments. Membrane transport proteins mediate all of these processes and are considered important mediators of drug resistance as well as drug targets in their own right. Recently, using advanced experimental genetic approaches and streamlined life cycle profiling, we generated a large collection of Plasmodium berghei gene deletion mutants and assigned essential gene functions, highlighting potential targets for prophylactic, therapeutic, and transmission-blocking anti-malarial drugs. Here, we present a comprehensive orthology assignment of all Plasmodium falciparum putative membrane transport proteins and provide a detailed overview of the associated essential gene functions obtained through experimental genetics studies in human and murine model parasites. Furthermore, we discuss the phylogeny of selected potential drug targets identified in our functional screen. We extensively discuss the results in the context of the functional assignments obtained using gene targeting available to date.

  3. Video Views and Reviews: Golgi Export, Targeting, and Plasma Membrane Caveolae

    Science.gov (United States)

    Watters, Christopher

    2004-01-01

    In this article, the author reviews videos from "Molecular Biology of the Cell (MBC)" depicting various aspects of plasma membrane (PM) dynamics, including the targeting of newly synthesized components and the organization of those PM invaginations called caveolae. The papers accompanying these videos describe, respectively, the constitutive…

  4. Evaluation of an Aqueous Extract from Horseradish Root (Armoracia rusticana Radix against Lipopolysaccharide-Induced Cellular Inflammation Reaction

    Directory of Open Access Journals (Sweden)

    Corinna Herz

    2017-01-01

    Full Text Available Horseradish (Armoracia rusticana is a perennial crop and its root is used in condiments. Traditionally, horseradish root is used to treat bacterial infections of the respiratory tract and urinary bladder. The antiphlogistic activity, determined in activated primary human peripheral blood mononuclear cells (PBMC, was evaluated for an aqueous extract and its subfractions, separated by HPLC. Compound analysis was done by UHPLC-QToF/MS and GC-MS. The aqueous extract concentration-dependently inhibited the anti-inflammatory response to lipopolysaccharide (LPS in terms of TNF-α release at ≥37 μg/mL. Further, the cyclooxygenase as well as lipoxygenase pathway was blocked by the extract as demonstrated by inhibition of COX-2 protein expression and PGE2 synthesis at ≥4 μg/mL and leukotriene LTB4 release. Mechanistic studies revealed that inhibition of ERK1/2 and c-Jun activation preceded COX-2 suppression upon plant extract treatment in the presence of LPS. Chemical analysis identified target compounds with a medium polarity as relevant for the observed bioactivity. Importantly, allyl isothiocyanate, which is quite well known for its anti-inflammatory capacity and as the principal pungent constituent in horseradish roots, was not relevant for the observations. The results suggest that horseradish root exerts an antiphlogistic activity in human immune cells by regulation of the COX and LOX pathway via MAPK signalling.

  5. Evaluation of an Aqueous Extract from Horseradish Root (Armoracia rusticana Radix) against Lipopolysaccharide-Induced Cellular Inflammation Reaction.

    Science.gov (United States)

    Herz, Corinna; Tran, Hoai Thi Thu; Márton, Melinda-Rita; Maul, Ronald; Baldermann, Susanne; Schreiner, Monika; Lamy, Evelyn

    2017-01-01

    Horseradish ( Armoracia rusticana ) is a perennial crop and its root is used in condiments. Traditionally, horseradish root is used to treat bacterial infections of the respiratory tract and urinary bladder. The antiphlogistic activity, determined in activated primary human peripheral blood mononuclear cells (PBMC), was evaluated for an aqueous extract and its subfractions, separated by HPLC. Compound analysis was done by UHPLC-QToF/MS and GC-MS. The aqueous extract concentration-dependently inhibited the anti-inflammatory response to lipopolysaccharide (LPS) in terms of TNF- α release at ≥37  μ g/mL. Further, the cyclooxygenase as well as lipoxygenase pathway was blocked by the extract as demonstrated by inhibition of COX-2 protein expression and PGE 2 synthesis at ≥4  μ g/mL and leukotriene LTB4 release. Mechanistic studies revealed that inhibition of ERK1/2 and c-Jun activation preceded COX-2 suppression upon plant extract treatment in the presence of LPS. Chemical analysis identified target compounds with a medium polarity as relevant for the observed bioactivity. Importantly, allyl isothiocyanate, which is quite well known for its anti-inflammatory capacity and as the principal pungent constituent in horseradish roots, was not relevant for the observations. The results suggest that horseradish root exerts an antiphlogistic activity in human immune cells by regulation of the COX and LOX pathway via MAPK signalling.

  6. Evaluation of Cytochalasin B-Induced Membrane Vesicles Fusion Specificity with Target Cells

    Directory of Open Access Journals (Sweden)

    Marina Gomzikova

    2018-01-01

    Full Text Available Extracellular vesicles (EV represent a promising vector system for biomolecules and drug delivery due to their natural origin and participation in intercellular communication. As the quantity of EVs is limited, it was proposed to induce the release of membrane vesicles from the surface of human cells by treatment with cytochalasin B. Cytochalasin B-induced membrane vesicles (CIMVs were successfully tested as a vector for delivery of dye, nanoparticles, and a chemotherapeutic. However, it remained unclear whether CIMVs possess fusion specificity with target cells and thus might be used for more targeted delivery of therapeutics. To answer this question, CIMVs were obtained from human prostate cancer PC3 cells. The diameter of obtained CIMVs was 962,13 ± 140,6 nm. We found that there is no statistically significant preference in PC3 CIMVs fusion with target cells of the same type. According to our observations, the greatest impact on CIMVs entry into target cells is by the heterophilic interaction of CIMV membrane receptors with the surface proteins of target cells.

  7. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    International Nuclear Information System (INIS)

    Moaddel, Ruin; Wainer, Irving W.

    2006-01-01

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K d values) and non-linear chromatography can be used to assess the association (k on ) and dissociation (k off ) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein

  8. Development of immobilized membrane-based affinity columns for use in the online characterization of membrane bound proteins and for targeted affinity isolations

    Energy Technology Data Exchange (ETDEWEB)

    Moaddel, Ruin [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States); Wainer, Irving W. [Gerontology Research Center, National Institute on Aging, National Institutes of Health, 5600 Nathan Shock Drive, Baltimore, MD 21224-6825 (United States)]. E-mail: Wainerir@grc.nia.nih.gov

    2006-03-30

    Membranes obtained from cell lines that express or do not express a target membrane bound protein have been immobilized on a silica-based liquid chromatographic support or on the surface of an activated glass capillary. The resulting chromatographic columns have been placed in liquid chromatographic systems and used to characterize the target proteins and to identify small molecules that bind to the target. Membranes containing ligand gated ion channels, G-protein coupled receptors and drug transporters have been prepared and characterized. If a marker ligand has been identified for the target protein, frontal or zonal displacement chromatographic techniques can be used to determine binding affinities (K {sub d} values) and non-linear chromatography can be used to assess the association (k {sub on}) and dissociation (k {sub off}) rate constants and the thermodynamics of the binding process. Membrane-based affinity columns have been created using membranes from a cell line that does not express the target protein (control) and the same cell line that expresses the target protein (experimental) after genomic transfection. The resulting columns can be placed in a parallel chromatography system and the differential retention between the control and experimental columns can be used to identify small molecules and protein that bind to the target protein. These applications will be illustrated using columns created using cellular membranes containing nicotinic acetylcholine receptors and the drug transporter P-glycoprotein.

  9. Percutaneous sharp recanalization of a membranous IVC occlusion with an occlusion balloon as a needle target

    Directory of Open Access Journals (Sweden)

    Michael D. Rivers-Bowerman, MD, MSc, FRCPC

    2017-09-01

    Full Text Available A 50-year-old male with right upper quadrant symptoms and hepatic dysfunction was found to have multiple dilated hepatic veins (HVs with intrahepatic collateralization and membranous occlusion of the intrahepatic inferior vena cava (IVC consistent with primary Budd–Chiari syndrome. Venacavograms depicted drainage of the intrahepatic collaterals through a left-sided HV entering the IVC above the level of the occlusion. Sharp recanalization of the membranous IVC occlusion was performed with an occlusion balloon as a needle target under echocardiographic monitoring followed by balloon angioplasty with restoration of IVC patency. Clinical, laboratory, and venographic procedural success has been demonstrated to 9 months with minimal residual stenosis.

  10. Peroxidase gene discovery from the horseradish transcriptome.

    Science.gov (United States)

    Näätsaari, Laura; Krainer, Florian W; Schubert, Michael; Glieder, Anton; Thallinger, Gerhard G

    2014-03-24

    Horseradish peroxidases (HRPs) from Armoracia rusticana have long been utilized as reporters in various diagnostic assays and histochemical stainings. Regardless of their increasing importance in the field of life sciences and suggested uses in medical applications, chemical synthesis and other industrial applications, the HRP isoenzymes, their substrate specificities and enzymatic properties are poorly characterized. Due to lacking sequence information of natural isoenzymes and the low levels of HRP expression in heterologous hosts, commercially available HRP is still extracted as a mixture of isoenzymes from the roots of A. rusticana. In this study, a normalized, size-selected A. rusticana transcriptome library was sequenced using 454 Titanium technology. The resulting reads were assembled into 14871 isotigs with an average length of 1133 bp. Sequence databases, ORF finding and ORF characterization were utilized to identify peroxidase genes from the 14871 isotigs generated by de novo assembly. The sequences were manually reviewed and verified with Sanger sequencing of PCR amplified genomic fragments, resulting in the discovery of 28 secretory peroxidases, 23 of them previously unknown. A total of 22 isoenzymes including allelic variants were successfully expressed in Pichia pastoris and showed peroxidase activity with at least one of the substrates tested, thus enabling their development into commercial pure isoenzymes. This study demonstrates that transcriptome sequencing combined with sequence motif search is a powerful concept for the discovery and quick supply of new enzymes and isoenzymes from any plant or other eukaryotic organisms. Identification and manual verification of the sequences of 28 HRP isoenzymes do not only contribute a set of peroxidases for industrial, biological and biomedical applications, but also provide valuable information on the reliability of the approach in identifying and characterizing a large group of isoenzymes.

  11. Chitosan derivatives targeting lipid bilayers: Synthesis, biological activity and interaction with model membranes.

    Science.gov (United States)

    Martins, Danubia Batista; Nasário, Fábio Domingues; Silva-Gonçalves, Laiz Costa; de Oliveira Tiera, Vera Aparecida; Arcisio-Miranda, Manoel; Tiera, Marcio José; Dos Santos Cabrera, Marcia Perez

    2018-02-01

    The antimicrobial activity of chitosan and derivatives to human and plant pathogens represents a high-valued prospective market. Presently, two low molecular weight derivatives, endowed with hydrophobic and cationic character at different ratios were synthesized and characterized. They exhibit antimicrobial activity and increased performance in relation to the intermediate and starting compounds. However, just the derivative with higher cationic character showed cytotoxicity towards human cervical carcinoma cells. Considering cell membranes as targets, the mode of action was investigated through the interaction with model lipid vesicles mimicking bacterial, tumoral and erythrocyte membranes. Intense lytic activity and binding are demonstrated for both derivatives in anionic bilayers. The less charged compound exhibits slightly improved selectivity towards bacterial model membranes, suggesting that balancing its hydrophobic/hydrophilic character may improve efficiency. Observing the aggregation of vesicles, we hypothesize that the "charge cluster mechanism", ascribed to some antimicrobial peptides, could be applied to these chitosan derivatives. Copyright © 2017 Elsevier Ltd. All rights reserved.

  12. Plasma Membrane Targeting of Protocadherin 15 Is Regulated by the Golgi-Associated Chaperone Protein PIST

    Directory of Open Access Journals (Sweden)

    Hongyun Nie

    2016-01-01

    Full Text Available Protocadherin 15 (PCDH15 is a core component of hair cell tip-links and crucial for proper function of inner ear hair cells. Mutations of PCDH15 gene cause syndromic and nonsyndromic hearing loss. At present, the regulatory mechanisms responsible for the intracellular transportation of PCDH15 largely remain unknown. Here we show that PIST, a Golgi-associated, PDZ domain-containing protein, interacts with PCDH15. The interaction is mediated by the PDZ domain of PIST and the C-terminal PDZ domain-binding interface (PBI of PCDH15. Through this interaction, PIST retains PCDH15 in the trans-Golgi network (TGN and reduces the membrane expression of PCDH15. We have previously showed that PIST regulates the membrane expression of another tip-link component, cadherin 23 (CDH23. Taken together, our finding suggests that PIST regulates the intracellular trafficking and membrane targeting of the tip-link proteins CDH23 and PCDH15.

  13. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    2012-02-01

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  14. Non-genomic actions of aldosterone: From receptors and signals to membrane targets.

    LENUS (Irish Health Repository)

    Dooley, Ruth

    2011-07-26

    In tissues which express the mineralocorticoid receptor (MR), aldosterone modulates the expression of membrane targets such as the subunits of the epithelial Na(+) channel, in combination with important signalling intermediates such as serum and glucocorticoid-regulated kinase-1. In addition, the rapid \\'non-genomic\\' activation of protein kinases and secondary messenger signalling cascades has also been detected in aldosterone-sensitive tissues of the nephron, distal colon and cardiovascular system. These rapid actions are variously described as being coupled to MR or to an as yet unidentified, membrane-associated aldosterone receptor. The rapidly activated signalling cascades add a level of fine-tuning to the activity of aldosterone-responsive membrane transporters and also modulate the aldosterone-induced changes in gene expression through receptor and transcription factor phosphorylation.

  15. Engineering a genetically-encoded SHG chromophore by electrostatic targeting to the membrane

    Directory of Open Access Journals (Sweden)

    Yuka eJinno

    2014-11-01

    Full Text Available Although second harmonic generation (SHG microscopy provides unique imaging advantages for voltage imaging and other biological applications, genetically-encoded SHG chromophores remain relatively unexplored. SHG only arises from non-centrosymmetric media, so an anisotropic arrangement of chromophores is essential to provide strong SHG signals. Here, inspired by the mechanism by which K-Ras4B associates with plasma membranes, we sought to achieve asymmetric arrangements of chromophores at the membrane-cytoplasm interface using the fluorescent protein mVenus. After adding a farnesylation motif to the C-terminus of mVenus, nine amino acids composing its -barrel surface were replaced by lysine, forming an electrostatic patch. This protein (mVe9Knus-CVIM was efficiently targeted to the plasma membrane in a geometrically defined manner and exhibited SHG in HEK293 cells. In agreement with its design, mVe9Knus-CVIM hyperpolarizability was oriented at a small angle (~7.3º from the membrane normal. Genetically-encoded SHG chromophores could serve as a molecular platform for imaging membrane potential.

  16. Benzothiophen-pyrazine scaffold as a potential membrane targeting drug carrier

    International Nuclear Information System (INIS)

    Mazuryk, Olga; Niemiec, Elżbieta; Stochel, Grażyna; Gillaizeau, Isabelle; Brindell, Małgorzata

    2013-01-01

    The fluorescent properties of 2,5-di(benzo[b]thiophen-2-yl)pyrazine as a potential membrane targeting drug carrier were characterized and it was shown that its fluorescence intensity was much higher in organic solvent than in water. The embedding of studied compound by liposomes leads to ca. 2 orders of magnitude increase in its fluorescence intensity, suggesting its preferential accumulation in membranes. Preliminary biological studies showed its ability to accumulate in cells, and the concentration of 10 μM was sufficient for homogeneous staining of cells. The treatment of mouse carcinoma CT26 cells with studied compound up to 200 μM resulted in decreasing of viable cells by ca. 30%. Its reactivity towards albumin was found to be moderate with an association constant of 6×10 4 M −1 , while no interaction with DNA was observed. Our findings encourage for further studies on functionalization of this molecule to obtain a new class of anticancer drugs targeting membrane. Highlights: ► The fluorescence of 2,5-di(benzo[b]thiophen-2-yl)pyrazine is solvent dependent. ► Weak fluorescence is found in water while high in organic solvents (DMSO, chloroform). ► Embedding of compound in liposomes remarkably increased its fluorescence. ► No interaction with DNA is observed but moderate reactivity towards albumin is found. ► Homogeneous staining of cells is feasible using nontoxic dose of compound

  17. The Membrane Steps of Bacterial Cell Wall Synthesis as Antibiotic Targets

    Directory of Open Access Journals (Sweden)

    Yao Liu

    2016-08-01

    Full Text Available Peptidoglycan is the major component of the cell envelope of virtually all bacteria. It has structural roles and acts as a selective sieve for molecules from the outer environment. Peptidoglycan synthesis is therefore one of the most important biogenesis pathways in bacteria and has been studied extensively over the last twenty years. The pathway starts in the cytoplasm, continues in the cytoplasmic membrane and finishes in the periplasmic space, where the precursor is polymerized into the peptidoglycan layer. A number of proteins involved in this pathway, such as the Mur enzymes and the penicillin binding proteins (PBPs, have been studied and regarded as good targets for antibiotics. The present review focuses on the membrane steps of peptidoglycan synthesis that involve two enzymes, MraY and MurG, the inhibitors of these enzymes and the inhibition mechanisms. We also discuss the challenges of targeting these two cytoplasmic membrane (associated proteins in bacterial cells and the perspectives on how to overcome the issues.

  18. Amplified biosensing using the horseradish peroxidase-mimicking DNAzyme as an electrocatalyst.

    Science.gov (United States)

    Pelossof, Gilad; Tel-Vered, Ran; Elbaz, Johann; Willner, Itamar

    2010-06-01

    The hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme is assembled on Au electrodes. It reveals bioelectrocatalytic properties and electrocatalyzes the reduction of H(2)O(2). The bioelectrocatalytic functions of the hemin/G-quadruplex DNAzyme are used to develop electrochemical sensors that follow the activity of glucose oxidase and biosensors for the detection of DNA or low-molecular-weight substrates (adenosine monophosphate, AMP). Hairpin nucleic structures that include the G-quadruplex sequence in a caged configuration and the nucleic acid sequence complementary to the analyte DNA, or the aptamer sequence for AMP, are immobilized on Au-electrode surfaces. In the presence of the DNA analyte, or AMP, the hairpin structures are opened, and the hemin/G-quadruplex horseradish peroxidase-mimicking DNAzyme structures are generated on the electrode surfaces. The bioelectrocatalytic cathodic currents generated by the functionalized electrodes, upon the electrochemical reduction of H(2)O(2), provide a quantitative measure for the detection of the target analytes. The DNA target was analyzed with a detection limit of 1 x 10(-12) M, while the detection limit for analyzing AMP was 1 x 10(-6) M. Methods to regenerate the sensing surfaces are presented.

  19. Membrane-lipid therapy: A historical perspective of membrane-targeted therapies - From lipid bilayer structure to the pathophysiological regulation of cells.

    Science.gov (United States)

    Escribá, Pablo V

    2017-09-01

    Our current understanding of membrane lipid composition, structure and functions has led to the investigation of their role in cell signaling, both in healthy and pathological cells. As a consequence, therapies based on the regulation of membrane lipid composition and structure have been recently developed. This novel field, known as Membrane Lipid Therapy, is growing and evolving rapidly, providing treatments that are now in use or that are being studied for their application to oncological disorders, Alzheimer's disease, spinal cord injury, stroke, diabetes, obesity, and neuropathic pain. This field has arisen from relevant discoveries on the behavior of membranes in recent decades, and it paves the way to adopt new approaches in modern pharmacology and nutrition. This innovative area will promote further investigation into membranes and the development of new therapies with molecules that target the cell membrane. Due to the prominent roles of membranes in the cells' physiology and the paucity of therapeutic approaches based on the regulation of the lipids they contain, it is expected that membrane lipid therapy will provide new treatments for numerous pathologies. The first on-purpose rationally designed molecule in this field, minerval, is currently being tested in clinical trials and it is expected to enter the market around 2020. However, it seems feasible that during the next few decades other membrane regulators will also be marketed for the treatment of human pathologies. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017. Published by Elsevier B.V.

  20. The cDNA sequence of a neutral horseradish peroxidase.

    Science.gov (United States)

    Bartonek-Roxå, E; Eriksson, H; Mattiasson, B

    1991-02-16

    A cDNA clone encoding a horseradish (Armoracia rusticana) peroxidase has been isolated and characterized. The cDNA contains 1378 nucleotides excluding the poly(A) tail and the deduced protein contains 327 amino acids which includes a 28 amino acid leader sequence. The predicted amino acid sequence is nine amino acids shorter than the major isoenzyme belonging to the horseradish peroxidase C group (HRP-C) and the sequence shows 53.7% identity with this isoenzyme. The described clone encodes nine cysteines of which eight correspond well with the cysteines found in HRP-C. Five potential N-glycosylation sites with the general sequence Asn-X-Thr/Ser are present in the deduced sequence. Compared to the earlier described HRP-C this is three glycosylation sites less. The shorter sequence and fewer N-glycosylation sites give the native isoenzyme a molecular weight of several thousands less than the horseradish peroxidase C isoenzymes. Comparison with the net charge value of HRP-C indicates that the described cDNA clone encodes a peroxidase which has either the same or a slightly less basic pI value, depending on whether the encoded protein is N-terminally blocked or not. This excludes the possibility that HRP-n could belong to either the HRP-A, -D or -E groups. The low sequence identity (53.7%) with HRP-C indicates that the described clone does not belong to the HRP-C isoenzyme group and comparison of the total amino acid composition with the HRP-B group does not place the described clone within this isoenzyme group. Our conclusion is that the described cDNA clone encodes a neutral horseradish peroxidase which belongs to a new, not earlier described, horseradish peroxidase group.

  1. Activity targets for nanostructured platinum-group-metal-free catalysts in hydroxide exchange membrane fuel cells

    Science.gov (United States)

    Setzler, Brian P.; Zhuang, Zhongbin; Wittkopf, Jarrid A.; Yan, Yushan

    2016-12-01

    Fuel cells are the zero-emission automotive power source that best preserves the advantages of gasoline automobiles: low upfront cost, long driving range and fast refuelling. To make fuel-cell cars a reality, the US Department of Energy has set a fuel cell system cost target of US$30 kW-1 in the long-term, which equates to US$2,400 per vehicle, excluding several major powertrain components (in comparison, a basic, but complete, internal combustion engine system costs approximately US$3,000). To date, most research for automotive applications has focused on proton exchange membrane fuel cells (PEMFCs), because these systems have demonstrated the highest power density. Recently, however, an alternative technology, hydroxide exchange membrane fuel cells (HEMFCs), has gained significant attention, because of the possibility to use stable platinum-group-metal-free catalysts, with inherent, long-term cost advantages. In this Perspective, we discuss the cost profile of PEMFCs and the advantages offered by HEMFCs. In particular, we discuss catalyst development needs for HEMFCs and set catalyst activity targets to achieve performance parity with state-of-the-art automotive PEMFCs. Meeting these targets requires careful optimization of nanostructures to pack high surface areas into a small volume, while maintaining high area-specific activity and favourable pore-transport properties.

  2. Targeting N-acyl-homoserine-lactones to mitigate membrane biofouling based on quorum sensing using a biofouling reducer.

    Science.gov (United States)

    Siddiqui, Muhammad Faisal; Sakinah, Mimi; Singh, Lakhveer; Zularisam, A W

    2012-10-31

    Exploring novel biological anti-quorum sensing (QS) agents to control membrane biofouling is of great worth in order to allow sustainable performance of membrane bioreactors (MBRs) for wastewater treatment. In recent studies, QS inhibitors have provided evidence of alternative route to control membrane biofouling. This study investigated the role of Piper betle extract (PBE) as an anti-QS agent to mitigate membrane biofouling. Results demonstrated the occurrence of the N-acyl-homoserine-lactone (AHL) autoinducers (AIs), correlate QS activity and membrane biofouling mitigation. The AIs production in bioreactor was confirmed using an indicator strain Agrobacterium tumefaciens (NTL4) harboring plasmid pZLR4. Moreover, three different AHLs were found in biocake using thin layer chromatographic analysis. An increase in extracellular polymeric substances (EPS) and transmembrane pressure (TMP) was observed with AHL activity of the biocake during continuous MBR operation, which shows that membrane biofouling was in close relationship with QS activity. PBE was verified to mitigate membrane biofouling via inhibiting AIs production. SEM analysis further confirmed the effect of PBE on EPS and biofilm formation. These results exhibited that PBE could be a novel agent to target AIs for mitigation of membrane biofouling. Further work can be carried out to purify the active compound of Piper betle extract to target the QS to mitigate membrane biofouling. Copyright © 2012 Elsevier B.V. All rights reserved.

  3. Prostate-Specific Membrane Antigen Targeted Therapy of Prostate Cancer Using a DUPA-Paclitaxel Conjugate.

    Science.gov (United States)

    Lv, Qingzhi; Yang, Jincheng; Zhang, Ruoshi; Yang, Zimeng; Yang, Zhengtao; Wang, Yongjun; Xu, Youjun; He, Zhonggui

    2018-05-07

    Prostate cancer (PCa) is the most prevalent cancer among men in the United States and remains the second-leading cause of cancer mortality in men. Paclitaxel (PTX) is the first line chemotherapy for PCa treatment, but its therapeutic efficacy is greatly restricted by the nonspecific distribution in vivo. Prostate-specific membrane antigen (PSMA) is overexpressed on the surface of most PCa cells, and its expression level increases with cancer aggressiveness, while being present at low levels in normal cells. The high expression level of PSMA in PCa cells offers an opportunity for target delivery of nonspecific cytotoxic drugs to PCa cells, thus improving therapeutic efficacy and reducing toxicity. PSMA has high affinity for DUPA, a glutamate urea ligand. Herein, a novel DUPA-PTX conjugate is developed using DUPA as the targeting ligand to deliver PTX specifically for treatment of PSMA expressing PCa. The targeting ligand DUPA enhances the transport capability and selectivity of PTX to tumor cells via PSMA mediated endocytosis. Besides, DUPA is conjugated with PTX via a disulfide bond, which facilitates the rapid and differential drug release in tumor cells. The DUPA-PTX conjugate exhibits potent cytotoxicity in PSMA expressing cell lines and induces a complete cessation of tumor growth with no obvious toxicity. Our findings give new insight into the PSMA-targeted delivery of chemotherapeutics and provide an opportunity for the development of novel active targeting drug delivery systems for PCa therapy.

  4. Prostate specific membrane antigen- a target for imaging and therapy with radionuclides

    DEFF Research Database (Denmark)

    Bouchelouche, Kirsten; Choyke, Peter L; Capala, Jacek

    2010-01-01

    Prostate cancer continues to represent a major health problem, and yet there is no effective treatment available for advanced metastatic disease. Thus, there is an urgent need for the development of more effective treatment modalities that could improve the outcome. Because prostate specific...... membrane antigen (PSMA), a transmembrane protein, is expressed by virtually all prostate cancers, and its expression is further increased in poorly differentiated, metastatic, and hormone-refractory carcinomas, it is a very attractive target. Molecules targeting PSMA can be labelled with radionuclides...... to become both diagnostic and/or therapeutic agents. The use of PSMA binding agents, labelled with diagnostic and therapeutic radio-isotopes, opens up the potential for a new era of personalized management of metastatic prostate cancer....

  5. Single-particle fusion of influenza viruses reveals complex interactions with target membranes

    Science.gov (United States)

    van der Borg, Guus; Braddock, Scarlett; Blijleven, Jelle S.; van Oijen, Antoine M.; Roos, Wouter H.

    2018-05-01

    The first step in infection of influenza A virus is contact with the host cell membrane, with which it later fuses. The composition of the target bilayer exerts a complex influence on both fusion efficiency and time. Here, an in vitro, single-particle approach is used to study this effect. Using total internal reflection fluorescence (TIRF) microscopy and a microfluidic flow cell, the hemifusion of single virions is visualized. Hemifusion efficiency and kinetics are studied while altering target bilayer cholesterol content and sialic-acid donor. Cholesterol ratios tested were 0%, 10%, 20%, and 40%. Sialic-acid donors GD1a and GYPA were used. Both cholesterol ratio and sialic-acid donors proved to have a significant effect on hemifusion efficiency. Furthermore, comparison between GD1a and GYPA conditions shows that the cholesterol dependence of the hemifusion time is severely affected by the sialic-acid donor. Only GD1a shows a clear increasing trend in hemifusion efficiency and time with increasing cholesterol concentration of the target bilayer with maximum rates for GD1A and 40% cholesterol. Overall our results show that sialic acid donor and target bilayer composition should be carefully chosen, depending on the desired hemifusion time and efficiency in the experiment.

  6. Incorporation of carbohydrate residues into peroxidase isoenzymes in horseradish roots.

    Science.gov (United States)

    Lew, J Y; Shannon, L M

    1973-11-01

    Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-(14)C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-(3)H and mannose-U-(14)C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of (14)C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems.

  7. Purification and characterization of myrosinase from horseradish (Armoracia rusticana) roots.

    Science.gov (United States)

    Li, Xian; Kushad, Mosbah M

    2005-06-01

    Myrosinase (beta-thioglucoside glucohydrolase; EC 3.2.3.147) from horseradish (Armoracia rusticana) roots was purified to homogeneity by ammonium sulfate fractionation, Q-sepharose, and concanavalin A sepharose affinity chromatography. The purified protein migrated as a single band with a mass of about 65 kDa on SDS-polyacrylamide gel electrophoresis. Using LC-MS/MS, this band was identified as myrosinase. Western blot analysis, using the anti-myrosinase monoclonal antibody 3D7, showed a single band of about 65 kDa for horseradish crude extract and for the purified myrosinase. The native molecular mass of the purified myrosinase was estimated, using gel filtration, to be about 130 kDa. Based on these data, it appeared that myrosinase from horseradish root consists of two subunits of similar molecular mass of about 65 kDa. The enzyme exhibited high activity at broad pH (pH 5.0-8.0) and temperature (37 and 45 degrees C). The purified enzyme remained stable at 4 degrees C for more than 1 year. Using sinigrin as a substrate, the Km and Vmax values for the purified enzyme were estimated to be 0.128 mM and 0.624 micromol min(-1), respectively. The enzyme was strongly activated by 0.5 mM ascorbic acid and was able to breakdown intact glucosinolates in a crude extract of broccoli.

  8. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer?s-Associated A? Oligomers

    OpenAIRE

    Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein int...

  9. Targeting of OSBP-related protein 3 (ORP3) to endoplasmic reticulum and plasma membrane is controlled by multiple determinants

    International Nuclear Information System (INIS)

    Lehto, Markku; Hynynen, Riikka; Karjalainen, Katja; Kuismanen, Esa; Hyvaerinen, Kati; Olkkonen, Vesa M.

    2005-01-01

    The intracellular targeting determinants of oxysterol binding protein (OSBP)-related protein 3 (ORP3) were studied using a series of truncated and point mutated constructs. The pleckstrin homology (PH) domain of ORP3 binds the phosphoinositide-3-kinase (PI3K) products, PI(3,4)P 2 and PI(3,4,5)P 3 . A functional PH domain and flanking sequences are crucial for the plasma membrane (PM) targeting of ORP3. The endoplasmic reticulum (ER) targeting of ORP3 is regulated the by a FFAT motif (EFFDAxE), which mediates interaction with VAMP-associated protein (VAP)-A. The targeting function of the FFAT motif dominates over that of the PH domain. In addition, the exon 10/11 region modulates interaction of ORP3 with the ER and the nuclear membrane. Analysis of a chimeric ORP3:OSBP protein suggests that ligand binding by the C-terminal domain of OSBP induces allosteric changes that activate the N-terminal targeting modules of ORP3. Notably, over-expression of ORP3 together with VAP-A induces stacked ER membrane structures also known as organized smooth ER (OSER). Moreover, lipid starvation promotes formation of dilated peripheral ER (DPER) structures dependent on the ORP3 protein. Based on the present data, we introduce a model for the inter-relationships of the functional domains of ORP3 in the membrane targeting of the protein

  10. Partitioning the proteome: phase separation for targeted analysis of membrane proteins in human post-mortem brain.

    Directory of Open Access Journals (Sweden)

    Jane A English

    Full Text Available Neuroproteomics is a powerful platform for targeted and hypothesis driven research, providing comprehensive insights into cellular and sub-cellular disease states, Gene × Environmental effects, and cellular response to medication effects in human, animal, and cell culture models. Analysis of sub-proteomes is becoming increasingly important in clinical proteomics, enriching for otherwise undetectable proteins that are possible markers for disease. Membrane proteins are one such sub-proteome class that merit in-depth targeted analysis, particularly in psychiatric disorders. As membrane proteins are notoriously difficult to analyse using traditional proteomics methods, we evaluate a paradigm to enrich for and study membrane proteins from human post-mortem brain tissue. This is the first study to extensively characterise the integral trans-membrane spanning proteins present in human brain. Using Triton X-114 phase separation and LC-MS/MS analysis, we enriched for and identified 494 membrane proteins, with 194 trans-membrane helices present, ranging from 1 to 21 helices per protein. Isolated proteins included glutamate receptors, G proteins, voltage gated and calcium channels, synaptic proteins, and myelin proteins, all of which warrant quantitative proteomic investigation in psychiatric and neurological disorders. Overall, our sub-proteome analysis reduced sample complexity and enriched for integral membrane proteins by 2.3 fold, thus allowing for more manageable, reproducible, and targeted proteomics in case vs. control biomarker studies. This study provides a valuable reference for future neuroproteomic investigations of membrane proteins, and validates the use Triton X-114 detergent phase extraction on human post mortem brain.

  11. An antibody that confers plant disease resistance targets a membrane-bound glyoxal oxidase in Fusarium.

    Science.gov (United States)

    Song, Xiu-Shi; Xing, Shu; Li, He-Ping; Zhang, Jing-Bo; Qu, Bo; Jiang, Jin-He; Fan, Chao; Yang, Peng; Liu, Jin-Long; Hu, Zu-Quan; Xue, Sheng; Liao, Yu-Cai

    2016-05-01

    Plant germplasm resources with natural resistance against globally important toxigenic Fusarium are inadequate. CWP2, a Fusarium genus-specific antibody, confers durable resistance to different Fusarium pathogens that infect cereals and other crops, producing mycotoxins. However, the nature of the CWP2 target is not known. Thus, investigation of the gene coding for the CWP2 antibody target will likely provide critical insights into the mechanism underlying the resistance mediated by this disease-resistance antibody. Immunoblots and mass spectrometry analysis of two-dimensional electrophoresis gels containing cell wall proteins from Fusarium graminearum (Fg) revealed that a glyoxal oxidase (GLX) is the CWP2 antigen. Cellular localization studies showed that GLX is localized to the plasma membrane. This GLX efficiently catalyzes hydrogen peroxide production; this enzymatic activity was specifically inhibited by the CWP2 antibody. GLX-deletion strains of Fg, F. verticillioides (Fv) and F. oxysporum had significantly reduced virulence on plants. The GLX-deletion Fg and Fv strains had markedly reduced mycotoxin accumulation, and the expression of key genes in mycotoxin metabolism was downregulated. This study reveals a single gene-encoded and highly conserved cellular surface antigen that is specifically recognized by the disease-resistance antibody CWP2 and regulates both virulence and mycotoxin biosynthesis in Fusarium species. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

  12. Myosin-1A Targets to Microvilli Using Multiple Membrane Binding Motifs in the Tail Homology 1 (TH1) Domain*

    Science.gov (United States)

    Mazerik, Jessica N.; Tyska, Matthew J.

    2012-01-01

    One of the most abundant components of the enterocyte brush border is the actin-based monomeric motor, myosin-1a (Myo1a). Within brush border microvilli, Myo1a carries out a number of critical functions at the interface between membrane and actin cytoskeleton. Proper physiological function of Myo1a depends on its ability to bind to microvillar membrane, an interaction mediated by a C-terminal tail homology 1 (TH1) domain. However, little is known about the mechanistic details of the Myo1a-TH1/membrane interaction. Structure-function analysis of Myo1a-TH1 targeting in epithelial cells revealed that an N-terminal motif conserved among class I myosins and a C-terminal motif unique to Myo1a-TH1 are both required for steady state microvillar enrichment. Purified Myo1a bound to liposomes composed of phosphatidylserine and phosphoinositol 4,5-bisphosphate, with moderate affinity in a charge-dependent manner. Additionally, peptides of the N- and C-terminal regions required for targeting were able to compete with Myo1a for binding to highly charged liposomes in vitro. Single molecule total internal reflection fluorescence microscopy showed that these motifs are also necessary for slowing the membrane detachment rate in cells. Finally, Myo1a-TH1 co-localized with both lactadherin-C2 (a phosphatidylserine-binding protein) and PLCδ1-PH (a phosphoinositol 4,5-bisphosphate-binding protein) in microvilli, but only lactaderin-C2 expression reduced brush border targeting of Myo1a-TH1. Together, our results suggest that Myo1a targeting to microvilli is driven by membrane binding potential that is distributed throughout TH1 rather than localized to a single motif. These data highlight the diversity of mechanisms that enable different class I myosins to target membranes in distinct biological contexts. PMID:22367206

  13. Prostate-Specific Membrane Antigen Targeted Gold Nanoparticles for Theranostics of Prostate Cancer.

    Science.gov (United States)

    Mangadlao, Joey Dacula; Wang, Xinning; McCleese, Christopher; Escamilla, Maria; Ramamurthy, Gopalakrishnan; Wang, Ziying; Govande, Mukul; Basilion, James P; Burda, Clemens

    2018-04-24

    Prostate cancer is one of the most common cancers and among the leading causes of cancer deaths in the United States. Men diagnosed with the disease typically undergo radical prostatectomy, which often results in incontinence and impotence. Recurrence of the disease is often experienced by most patients with incomplete prostatectomy during surgery. Hence, the development of a technique that will enable surgeons to achieve a more precise prostatectomy remains an open challenge. In this contribution, we report a theranostic agent (AuNP-5kPEG-PSMA-1-Pc4) based on prostate-specific membrane antigen (PSMA-1)-targeted gold nanoparticles (AuNPs) loaded with a fluorescent photodynamic therapy (PDT) drug, Pc4. The fabricated nanoparticles are well-characterized by spectroscopic and imaging techniques and are found to be stable over a wide range of solvents, buffers, and media. In vitro cellular uptake experiments demonstrated significantly higher nanoparticle uptake in PSMA-positive PC3pip cells than in PSMA-negative PC3flu cells. Further, more complete cell killing was observed in Pc3pip than in PC3flu cells upon exposure to light at different doses, demonstrating active targeting followed by Pc4 delivery. Likewise, in vivo studies showed remission on PSMA-expressing tumors 14 days post-PDT. Atomic absorption spectroscopy revealed that targeted AuNPs accumulate 4-fold higher in PC3pip than in PC3flu tumors. The nanoparticle system described herein is envisioned to provide surgical guidance for prostate tumor resection and therapeutic intervention when surgery is insufficient.

  14. Two-step membrane binding by the bacterial SRP receptor enable efficient and accurate Co-translational protein targeting.

    Science.gov (United States)

    Hwang Fu, Yu-Hsien; Huang, William Y C; Shen, Kuang; Groves, Jay T; Miller, Thomas; Shan, Shu-Ou

    2017-07-28

    The signal recognition particle (SRP) delivers ~30% of the proteome to the eukaryotic endoplasmic reticulum, or the bacterial plasma membrane. The precise mechanism by which the bacterial SRP receptor, FtsY, interacts with and is regulated at the target membrane remain unclear. Here, quantitative analysis of FtsY-lipid interactions at single-molecule resolution revealed a two-step mechanism in which FtsY initially contacts membrane via a Dynamic mode, followed by an SRP-induced conformational transition to a Stable mode that activates FtsY for downstream steps. Importantly, mutational analyses revealed extensive auto-inhibitory mechanisms that prevent free FtsY from engaging membrane in the Stable mode; an engineered FtsY pre-organized into the Stable mode led to indiscriminate targeting in vitro and disrupted FtsY function in vivo. Our results show that the two-step lipid-binding mechanism uncouples the membrane association of FtsY from its conformational activation, thus optimizing the balance between the efficiency and fidelity of co-translational protein targeting.

  15. Evaluation of Seasonal Variations in the Glucosinolate Content in Leaves and Roots of Four European Horseradish (Armoracia rusticana Landraces

    Directory of Open Access Journals (Sweden)

    Ciska Ewa

    2017-12-01

    Full Text Available In comparison with other cruciferous vegetables, horseradish has rarely been the object of scientific research, and the knowledge about the composition, content and distribution of glucosinolates (GLS in different organs of horseradish plants is limited. Therefore, the aim of this study was to evaluate changes in the GLS content in leaves and roots of four horseradish landraces during the growing season.

  16. Synergistic co-targeting of prostate-specific membrane antigen and androgen receptor in prostate cancer.

    Science.gov (United States)

    Murga, Jose D; Moorji, Sameer M; Han, Amy Q; Magargal, Wells W; DiPippo, Vincent A; Olson, William C

    2015-02-15

    Antibody-drug conjugates (ADCs) are an emerging class of cancer therapies that have demonstrated favorable activity both as single agents and as components of combination regimens. Phase 2 testing of an ADC targeting prostate-specific membrane antigen (PSMA) in advanced prostate cancer has shown antitumor activity. The present study examined PSMA ADC used in combination with potent antiandrogens (enzalutamide and abiraterone) and other compounds. Antiproliferative activity and expression of PSMA, prostate-specific antigen and androgen receptor were evaluated in the prostate cancer cell lines LNCaP and C4-2. Cells were tested for susceptibility to antiandrogens or other inhibitors, used alone and in combination with PSMA ADC. Potential drug synergy or antagonism was evaluated using the Bliss independence method. Enzalutamide and abiraterone demonstrated robust, statistically significant synergy when combined with PSMA ADC. Largely additive activity was observed between the antiandrogens and the individual components of the ADC (free drug and unmodified antibody). Rapamycin also synergized with PSMA ADC in certain settings. Synergy was linked in part to upregulation of PSMA expression. In androgen-dependent LNCaP cells, enzalutamide and abiraterone each inhibited proliferation, upregulated PSMA expression, and synergized with PSMA ADC. In androgen-independent C4-2 cells, enzalutamide and abiraterone showed no measurable antiproliferative activity on their own but increased PSMA expression and synergized with PSMA ADC nonetheless. PSMA expression increased progressively over 3 weeks with enzalutamide and returned to baseline levels 1 week after enzalutamide removal. The findings support exploration of clinical treatment regimens that combine potent antiandrogens and PSMA-targeted therapies for prostate cancer. © 2014 Wiley Periodicals, Inc.

  17. Small Molecule Membrane Transporters in the Mammalian Podocyte: A Pathogenic and Therapeutic Target

    Directory of Open Access Journals (Sweden)

    Cristina Zennaro

    2014-11-01

    Full Text Available The intriguingly complex glomerular podocyte has been a recent object of intense study. Researchers have sought to understand its role in the pathogenesis of common proteinuric diseases such as minimal change disease and focal segmental glomerular sclerosis. In particular, considerable effort has been directed towards the anatomic and functional barrier to macromolecular filtration provided by the secondary foot processes, but little attention has been paid to the potential of podocytes to handle plasma proteins beyond the specialization of the slit diaphragm. Renal membrane transporters in the proximal tubule have been extensively studied for decades, particularly in relation to drug metabolism and elimination. Recently, uptake and efflux transporters for small organic molecules have also been found in the glomerular podocyte, and we and others have found that these transporters can engage not only common pharmaceuticals but also injurious endogenous and exogenous agents. We have also found that the activity of podocyte transporters can be manipulated to inhibit pathogen uptake and efflux. It is conceivable that podocyte transporters may play a role in disease pathogenesis and may be a target for future drug development.

  18. Models of plasma membrane organization can be applied to mitochondrial membranes to target human health and disease with polyunsaturated fatty acids.

    Science.gov (United States)

    Raza Shaikh, Saame; Brown, David A

    2013-01-01

    Bioactive n-3 polyunsaturated fatty acids (PUFA), abundant in fish oil, have potential for treating symptoms associated with inflammatory and metabolic disorders; therefore, it is essential to determine their fundamental molecular mechanisms. Recently, several labs have demonstrated the n-3 PUFA docosahexaenoic acid (DHA) exerts anti-inflammatory effects by targeting the molecular organization of plasma membrane microdomains. Here we briefly review the evidence that DHA reorganizes the spatial distribution of microdomains in several model systems. We then emphasize how models on DHA and plasma membrane microdomains can be applied to mitochondrial membranes. We discuss the role of DHA acyl chains in regulating mitochondrial lipid-protein clustering, and how these changes alter several aspects of mitochondrial function. In particular, we summarize effects of DHA on mitochondrial respiration, electron leak, permeability transition, and mitochondrial calcium handling. Finally, we conclude by postulating future experiments that will augment our understanding of DHA-dependent membrane organization in health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  19. The hemifusion structure induced by influenza virus haemagglutinin is determined by physical properties of the target membranes.

    Science.gov (United States)

    Chlanda, Petr; Mekhedov, Elena; Waters, Hang; Schwartz, Cindi L; Fischer, Elizabeth R; Ryham, Rolf J; Cohen, Fredric S; Blank, Paul S; Zimmerberg, Joshua

    2016-04-18

    Influenza A virus haemagglutinin conformational change drives the membrane fusion of viral and endosomal membranes at low pH. Membrane fusion proceeds through an intermediate called hemifusion(1,2). For viral fusion, the hemifusion structures are not determined(3). Here, influenza virus-like particles(4) carrying wild-type haemagglutinin or haemagglutinin hemifusion mutant G1S(5) and liposome mixtures were studied at low pH by Volta phase plate cryo-electron tomography, which improves the signal-to-noise ratio close to focus. We determined two distinct hemifusion structures: a hemifusion diaphragm and a novel structure termed a 'lipidic junction'. Liposomes with lipidic junctions were ruptured with membrane edges stabilized by haemagglutinin. The rupture frequency and hemifusion diaphragm diameter were not affected by G1S mutation, but decreased when the cholesterol level in the liposomes was close to physiological concentrations. We propose that haemagglutinin induces a merger between the viral and target membranes by one of two independent pathways: a rupture-insertion pathway leading to the lipidic junction and a hemifusion-stalk pathway leading to a fusion pore. The latter is relevant under the conditions of influenza virus infection of cells. Cholesterol concentration functions as a pathway switch because of its negative spontaneous curvature in the target bilayer, as determined by continuum analysis.

  20. Early and late HIV-1 membrane fusion events are impaired by sphinganine lipidated peptides that target the fusion site.

    Science.gov (United States)

    Klug, Yoel A; Ashkenazi, Avraham; Viard, Mathias; Porat, Ziv; Blumenthal, Robert; Shai, Yechiel

    2014-07-15

    Lipid-conjugated peptides have advanced the understanding of membrane protein functions and the roles of lipids in the membrane milieu. These lipopeptides modulate various biological systems such as viral fusion. A single function has been suggested for the lipid, binding to the membrane and thus elevating the local concentration of the peptide at the target site. In the present paper, we challenged this argument by exploring in-depth the antiviral mechanism of lipopeptides, which comprise sphinganine, the lipid backbone of DHSM (dihydrosphingomyelin), and an HIV-1 envelope-derived peptide. Surprisingly, we discovered a partnership between the lipid and the peptide that impaired early membrane fusion events by reducing CD4 receptor lateral diffusion and HIV-1 fusion peptide-mediated lipid mixing. Moreover, only the joint function of sphinganine and its conjugate peptide disrupted HIV-1 fusion protein assembly and folding at the later fusion steps. Via imaging techniques we revealed for the first time the direct localization of these lipopeptides to the virus-cell and cell-cell contact sites. Overall, the findings of the present study may suggest lipid-protein interactions in various biological systems and may help uncover a role for elevated DHSM in HIV-1 and its target cell membranes.

  1. Horseradish peroxidase-modified porous silicon for phenol monitoring

    Energy Technology Data Exchange (ETDEWEB)

    Kermad, A., E-mail: amina_energetique@yahoo.fr [Unité de Recherche Matériaux et Energies Renouvelables (URMER), Département de Physique, Faculté des Sciences, Université Abou Baker Belkaid, B.P. 119, Tlemcen 13000 (Algeria); Sam, S., E-mail: Sabrina.sam@polytechnique.edu [Centre de Recherche en Technologie des Semi-conducteurs pour l’Energétique (CRTSE), 02 Bd. Frantz-Fanon, B.P. 140, Alger-7 merveilles, Algiers (Algeria); Ghellai, N., E-mail: na_ghellai@yahoo.fr [Unité de Recherche Matériaux et Energies Renouvelables (URMER), Département de Physique, Faculté des Sciences, Université Abou Baker Belkaid, B.P. 119, Tlemcen 13000 (Algeria); Khaldi, K., E-mail: Khadidjaphy@yahoo.fr [Unité de Recherche Matériaux et Energies Renouvelables (URMER), Département de Physique, Faculté des Sciences, Université Abou Baker Belkaid, B.P. 119, Tlemcen 13000 (Algeria); Gabouze, N., E-mail: ngabouze@yahoo.fr [Centre de Recherche en Technologie des Semi-conducteurs pour l’Energétique (CRTSE), 02 Bd. Frantz-Fanon, B.P. 140, Alger-7 merveilles, Algiers (Algeria)

    2013-11-01

    Highlights: • Horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface. • Multistep strategy was used allowing the maintaining of the enzymatic activity of the immobilized enzyme. • Direct electron transfer has occurred between the immobilized enzyme and the surface. • Electrochemical measurements showed a response of HRP-modified PSi toward phenol in the presence of H{sub 2}O{sub 2}. -- Abstract: In this study, horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface using multistep strategy. First, acid terminations were generated on hydrogenated PSi surface by thermal hydrosilylation of undecylenic acid. Then, the carboxyl-terminated monolayer was transformed to active ester (succinimidyl ester) using N-hydroxysuccinimide (NHS) in the presence of the coupling agent N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC). Subsequently, the enzyme was anchored on the surface via an amidation reaction. The structure of the PSi layers was observed by scanning electron microscopy (SEM). Infrared spectroscopy (FTIR) and contact angle measurements confirmed the efficiency of the modification at each step of the functionalization. Cyclic voltammetry was recorded using the HRP-modified PSi as working electrode. The results show that the enzymatic activity of the immobilized HRP is preserved and in the presence of hydrogen peroxide, the enzyme oxidizes phenolic molecules which were subsequently reduced at the modified-PSi electrode.

  2. Horseradish peroxidase-modified porous silicon for phenol monitoring

    International Nuclear Information System (INIS)

    Kermad, A.; Sam, S.; Ghellai, N.; Khaldi, K.; Gabouze, N.

    2013-01-01

    Highlights: • Horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface. • Multistep strategy was used allowing the maintaining of the enzymatic activity of the immobilized enzyme. • Direct electron transfer has occurred between the immobilized enzyme and the surface. • Electrochemical measurements showed a response of HRP-modified PSi toward phenol in the presence of H 2 O 2 . -- Abstract: In this study, horseradish peroxidase enzyme (HRP) was covalently immobilized on porous silicon (PSi) surface using multistep strategy. First, acid terminations were generated on hydrogenated PSi surface by thermal hydrosilylation of undecylenic acid. Then, the carboxyl-terminated monolayer was transformed to active ester (succinimidyl ester) using N-hydroxysuccinimide (NHS) in the presence of the coupling agent N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC). Subsequently, the enzyme was anchored on the surface via an amidation reaction. The structure of the PSi layers was observed by scanning electron microscopy (SEM). Infrared spectroscopy (FTIR) and contact angle measurements confirmed the efficiency of the modification at each step of the functionalization. Cyclic voltammetry was recorded using the HRP-modified PSi as working electrode. The results show that the enzymatic activity of the immobilized HRP is preserved and in the presence of hydrogen peroxide, the enzyme oxidizes phenolic molecules which were subsequently reduced at the modified-PSi electrode

  3. Targeting of a Nicotiana plumbaginifolia H+ -ATPase to the plasma membrane is not by default and requires cytosolic structural determinants.

    Science.gov (United States)

    Lefebvre, Benoit; Batoko, Henri; Duby, Geoffrey; Boutry, Marc

    2004-07-01

    The structural determinants involved in the targeting of multitransmembrane-span proteins to the plasma membrane (PM) remain poorly understood. The plasma membrane H+ -ATPase (PMA) from Nicotiana plumbaginifolia, a well-characterized 10 transmembrane-span enzyme, was used as a model to identify structural elements essential for targeting to the PM. When PMA2 and PMA4, representatives of the two main PMA subfamilies, were fused to green fluorescent protein (GFP), the chimeras were shown to be still functional and to be correctly and rapidly targeted to the PM in transgenic tobacco. By contrast, chimeric proteins containing various combinations of PMA transmembrane spanning domains accumulated in the Golgi apparatus and not in the PM and displayed slow traffic properties through the secretory pathway. Individual deletion of three of the four cytosolic domains did not prevent PM targeting, but deletion of the large loop or of its nucleotide binding domain resulted in GFP fluorescence accumulating exclusively in the endoplasmic reticulum. The results show that, at least for this polytopic protein, the PM is not the default pathway and that, in contrast with single-pass membrane proteins, cytosolic structural determinants are required for correct targeting.

  4. Insulin alters the target size of the peripheral cyclic AMP phosphodiesterase but not the integral cyclic GMP-stimulated cyclic AMP phosphodiesterase in liver plasma membranes

    International Nuclear Information System (INIS)

    Wallace, A.V.; Martin, B.R.; Houslay, M.D.

    1990-01-01

    Radiation inactivation of the two high affinity cyclic AMP phosphodiesterases (PDE) found in liver plasma membranes afforded an estimation of their molecular target sizes in situ. The activity of the peripheral plasma membrane PDE decayed as a single exponential with a target size corresponding to a monomer of circa 54 kDa. The integral, cyclic GMP-stimulated PDE decayed as a dimer of circa 125 kDa. Preincubation of plasma membranes with insulin (10nM), prior to irradiation, caused the target size of only the peripheral plasma membrane PDE to increase. We suggest that insulin addition causes the peripheral plasma membrane PDE to alter its coupling to an integral plasma membrane protein with a target size of circa 90 kDa

  5. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

    Directory of Open Access Journals (Sweden)

    Kyle C Wilcox

    Full Text Available Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs. AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs. This method gives a soluble membrane protein library (SMPL--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can

  6. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer’s-Associated Aβ Oligomers

    Science.gov (United States)

    Wilcox, Kyle C.; Marunde, Matthew R.; Das, Aditi; Velasco, Pauline T.; Kuhns, Benjamin D.; Marty, Michael T.; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G.; Klein, William L.

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer’s dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)—a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer’s model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug

  7. Nanoscale Synaptic Membrane Mimetic Allows Unbiased High Throughput Screen That Targets Binding Sites for Alzheimer's-Associated Aβ Oligomers.

    Science.gov (United States)

    Wilcox, Kyle C; Marunde, Matthew R; Das, Aditi; Velasco, Pauline T; Kuhns, Benjamin D; Marty, Michael T; Jiang, Haoming; Luan, Chi-Hao; Sligar, Stephen G; Klein, William L

    2015-01-01

    Despite their value as sources of therapeutic drug targets, membrane proteomes are largely inaccessible to high-throughput screening (HTS) tools designed for soluble proteins. An important example comprises the membrane proteins that bind amyloid β oligomers (AβOs). AβOs are neurotoxic ligands thought to instigate the synapse damage that leads to Alzheimer's dementia. At present, the identities of initial AβO binding sites are highly uncertain, largely because of extensive protein-protein interactions that occur following attachment of AβOs to surface membranes. Here, we show that AβO binding sites can be obtained in a state suitable for unbiased HTS by encapsulating the solubilized synaptic membrane proteome into nanoscale lipid bilayers (Nanodiscs). This method gives a soluble membrane protein library (SMPL)--a collection of individualized synaptic proteins in a soluble state. Proteins within SMPL Nanodiscs showed enzymatic and ligand binding activity consistent with conformational integrity. AβOs were found to bind SMPL Nanodiscs with high affinity and specificity, with binding dependent on intact synaptic membrane proteins, and selective for the higher molecular weight oligomers known to accumulate at synapses. Combining SMPL Nanodiscs with a mix-incubate-read chemiluminescence assay provided a solution-based HTS platform to discover antagonists of AβO binding. Screening a library of 2700 drug-like compounds and natural products yielded one compound that potently reduced AβO binding to SMPL Nanodiscs, synaptosomes, and synapses in nerve cell cultures. Although not a therapeutic candidate, this small molecule inhibitor of synaptic AβO binding will provide a useful experimental antagonist for future mechanistic studies of AβOs in Alzheimer's model systems. Overall, results provide proof of concept for using SMPLs in high throughput screening for AβO binding antagonists, and illustrate in general how a SMPL Nanodisc system can facilitate drug discovery

  8. Targeting and Assembly of Components of the TOC Protein Import Complex at the Chloroplast Outer Envelope Membrane

    Directory of Open Access Journals (Sweden)

    Lynn G.L. Richardson

    2014-06-01

    Full Text Available The translocon at the outer envelope membrane of chloroplasts (TOC initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β–barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  9. Targeting and assembly of components of the TOC protein import complex at the chloroplast outer envelope membrane.

    Science.gov (United States)

    Richardson, Lynn G L; Paila, Yamuna D; Siman, Steven R; Chen, Yi; Smith, Matthew D; Schnell, Danny J

    2014-01-01

    The translocon at the outer envelope membrane of chloroplasts (TOC) initiates the import of thousands of nuclear encoded preproteins required for chloroplast biogenesis and function. The multimeric TOC complex contains two GTP-regulated receptors, Toc34 and Toc159, which recognize the transit peptides of preproteins and initiate protein import through a β-barrel membrane channel, Toc75. Different isoforms of Toc34 and Toc159 assemble with Toc75 to form structurally and functionally diverse translocons, and the composition and levels of TOC translocons is required for the import of specific subsets of coordinately expressed proteins during plant growth and development. Consequently, the proper assembly of the TOC complexes is key to ensuring organelle homeostasis. This review will focus on our current knowledge of the targeting and assembly of TOC components to form functional translocons at the outer membrane. Our analyses reveal that the targeting of TOC components involves elements common to the targeting of other outer membrane proteins, but also include unique features that appear to have evolved to specifically facilitate assembly of the import apparatus.

  10. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    Energy Technology Data Exchange (ETDEWEB)

    Baker, Lindsay A. [University of Oxford, Oxford Particle Imaging Centre, The Wellcome Trust Centre for Human Genetics, Division of Structural Biology, Nuffield Department of Medicine (United Kingdom); Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc, E-mail: m.baldus@uu.nl [Utrecht University, NMR Spectroscopy, Department of Chemistry, Faculty of Science, Bijvoet Center for Biomolecular Research (Netherlands)

    2015-06-15

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR.

  11. Efficient cellular solid-state NMR of membrane proteins by targeted protein labeling

    International Nuclear Information System (INIS)

    Baker, Lindsay A.; Daniëls, Mark; Cruijsen, Elwin A. W. van der; Folkers, Gert E.; Baldus, Marc

    2015-01-01

    Solid-state NMR spectroscopy (ssNMR) has made significant progress towards the study of membrane proteins in their native cellular membranes. However, reduced spectroscopic sensitivity and high background signal levels can complicate these experiments. Here, we describe a method for ssNMR to specifically label a single protein by repressing endogenous protein expression with rifampicin. Our results demonstrate that treatment of E. coli with rifampicin during induction of recombinant membrane protein expression reduces background signals for different expression levels and improves sensitivity in cellular membrane samples. Further, the method reduces the amount of time and resources needed to produce membrane protein samples, enabling new strategies for studying challenging membrane proteins by ssNMR

  12. Helicobacter pylori VacA toxin/subunit p34: targeting of an anion channel to the inner mitochondrial membrane.

    Directory of Open Access Journals (Sweden)

    Grazyna Domańska

    2010-04-01

    Full Text Available The vacuolating toxin VacA, released by Helicobacter pylori, is an important virulence factor in the pathogenesis of gastritis and gastroduodenal ulcers. VacA contains two subunits: The p58 subunit mediates entry into target cells, and the p34 subunit mediates targeting to mitochondria and is essential for toxicity. In this study we found that targeting to mitochondria is dependent on a unique signal sequence of 32 uncharged amino acid residues at the p34 N-terminus. Mitochondrial import of p34 is mediated by the import receptor Tom20 and the import channel of the outer membrane TOM complex, leading to insertion of p34 into the mitochondrial inner membrane. p34 assembles in homo-hexamers of extraordinary high stability. CD spectra of the purified protein indicate a content of >40% beta-strands, similar to pore-forming beta-barrel proteins. p34 forms an anion channel with a conductivity of about 12 pS in 1.5 M KCl buffer. Oligomerization and channel formation are independent both of the 32 uncharged N-terminal residues and of the p58 subunit of the toxin. The conductivity is efficiently blocked by 5-nitro-2-(3-phenylpropylaminobenzoic acid (NPPB, a reagent known to inhibit VacA-mediated apoptosis. We conclude that p34 essentially acts as a small pore-forming toxin, targeted to the mitochondrial inner membrane by a special hydrophobic N-terminal signal.

  13. Pharmacological targeting of membrane rigidity: implications on cancer cell migration and invasion

    International Nuclear Information System (INIS)

    Braig, Simone; Stoiber, Katharina; Zahler, Stefan; Vollmar, Angelika M

    2015-01-01

    The invasive potential of cancer cells strongly depends on cellular stiffness, a physical quantity that is not only regulated by the mechanical impact of the cytoskeleton but also influenced by the membrane rigidity. To analyze the specific role of membrane rigidity in cancer progression, we treated cancer cells with the Acetyl-CoA carboxylase inhibitor Soraphen A and revealed an alteration of the phospholipidome via mass spectrometry. Migration, invasion, and cell death assays were employed to relate this alteration to functional consequences, and a decrease of migration and invasion without significant impact on cell death has been recorded. Fourier fluctuation analysis of giant plasma membrane vesicles showed that Soraphen A increases membrane rigidity of carcinoma cell membranes. Mechanical measurements of the creep deformation response of whole intact cells were performed using the optical stretcher. The increase in membrane rigidity was observed in one cell line without changing the creep deformation response indicating no restructuring of the cytoskeleton. These data indicate that the increase of membrane rigidity alone is sufficient to inhibit invasiveness of cancer cells, thus disclosing the eminent role of membrane rigidity in migratory processes. (paper)

  14. Specific and efficient targeting of cyanobacterial bicarbonate transporters to the inner envelope membrane of chloroplasts in Arabidopsis

    Directory of Open Access Journals (Sweden)

    Susumu eUehara

    2016-02-01

    Full Text Available Installation of cyanobacterial bicarbonate transporters to the inner envelope membrane (IEM of chloroplasts in C3 plants has been thought to improve photosynthetic performance. However, the method to deliver cyanobacterial bicarbonate transporters to the chloroplast IEM remains to be established. In this study, we provide evidence that the cyanobacterial bicarbonate transporters, BicA and SbtA, can be specifically installed into the chloroplast IEM using the chloroplast IEM targeting signal in conjunction with the transit peptide. We fused the transit peptide and the mature portion of Cor413im1, whose targeting mechanism to the IEM has been characterized in detail, to either BicA or SbtA isolated from Synechocystis sp. PCC6803. Among the seven chimeric constructs tested, we confirmed that four chimeric bicarbonate transporters, designated as BicAI, BicAII, SbtAII, and SbtAIII, were expressed in Arabidopsis. Furthermore, these chimeric transporters were specifically targeted to the chloroplast IEM. They were also resistant to alkaline extraction but can be solubilized by Triton X-100, indicating that they are integral membrane proteins in the chloroplast IEM. One of the transporters, BicA, could reside in the chloroplast IEM even after removal of the IEM targeting signal. Taken together, our results indicate that the addition of IEM targeting signal, as well as the transit peptide, to bicarbonate transporters allows us to efficiently target nuclear-encoded chimeric bicarbonate transporters to the chloroplast IEM.

  15. Targeting membrane-bound viral RNA synthesis reveals potent inhibition of diverse coronaviruses including the middle East respiratory syndrome virus.

    Directory of Open Access Journals (Sweden)

    Anna Lundin

    2014-05-01

    Full Text Available Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs, a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6, a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV, and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.

  16. Prostate-specific membrane antigen-directed nanoparticle targeting for extreme nearfield ablation of prostate cancer cells.

    Science.gov (United States)

    Lee, Seung S; Roche, Philip Jr; Giannopoulos, Paresa N; Mitmaker, Elliot J; Tamilia, Michael; Paliouras, Miltiadis; Trifiro, Mark A

    2017-03-01

    Almost all biological therapeutic interventions cannot overcome neoplastic heterogeneity. Physical ablation therapy is immune to tumor heterogeneity, but nearby tissue damage is the limiting factor in delivering lethal doses. Multi-walled carbon nanotubes offer a number of unique properties: chemical stability, photonic properties including efficient light absorption, thermal conductivity, and extensive surface area availability for covalent chemical ligation. When combined together with a targeting moiety such as an antibody or small molecule, one can deliver highly localized temperature increases and cause extensive cellular damage. We have functionalized multi-walled carbon nanotubes by conjugating an antibody against prostate-specific membrane antigen. In our in vitro studies using prostate-specific membrane antigen-positive LNCaP prostate cancer cells, we have effectively demonstrated cell ablation of >80% with a single 30-s exposure to a 2.7-W, 532-nm laser for the first time without bulk heating. We also confirmed the specificity and selectivity of prostate-specific membrane antigen targeting by assessing prostate-specific membrane antigen-null PC3 cell lines under the same conditions (<10% cell ablation). This suggests that we can achieve an extreme nearfield cell ablation effect, thus restricting potential tissue damage when transferred to in vivo clinical applications. Developing this new platform will introduce novel approaches toward current therapeutic modalities and will usher in a new age of effective cancer treatment squarely addressing tumoral heterogeneity.

  17. LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

    Science.gov (United States)

    Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

    2002-01-01

    The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

  18. Caveolin targeting to late endosome/lysosomal membranes is induced by perturbations of lysosomal pH and cholesterol content

    Science.gov (United States)

    Mundy, Dorothy I.; Li, Wei Ping; Luby-Phelps, Katherine; Anderson, Richard G. W.

    2012-01-01

    Caveolin-1 is an integral membrane protein of plasma membrane caveolae. Here we report that caveolin-1 collects at the cytosolic surface of lysosomal membranes when cells are serum starved. This is due to an elevation of the intralysosomal pH, since ionophores and proton pump inhibitors that dissipate the lysosomal pH gradient also trapped caveolin-1 on late endosome/lysosomes. Accumulation is both saturable and reversible. At least a portion of the caveolin-1 goes to the plasma membrane upon reversal. Several studies suggest that caveolin-1 is involved in cholesterol transport within the cell. Strikingly, we find that blocking cholesterol export from lysosomes with progesterone or U18666A or treating cells with low concentrations of cyclodextrin also caused caveolin-1 to accumulate on late endosome/lysosomal membranes. Under these conditions, however, live-cell imaging shows cavicles actively docking with lysosomes, suggesting that these structures might be involved in delivering caveolin-1. Targeting of caveolin-1 to late endosome/lysosomes is not observed normally, and the degradation rate of caveolin-1 is not altered by any of these conditions, indicating that caveolin-1 accumulation is not a consequence of blocked degradation. We conclude that caveolin-1 normally traffics to and from the cytoplasmic surface of lysosomes during intracellular cholesterol trafficking. PMID:22238363

  19. Direct electron transfer of horseradish peroxidase on Nafion-cysteine modified gold electrode

    International Nuclear Information System (INIS)

    Hong, Jun; Moosavi-Movahedi, Ali Akbar; Ghourchian, Hedayatollah; Rad, Ahmad Molaei; Rezaei-Zarchi, Saeed

    2007-01-01

    Direct electron transfer of horseradish peroxidase, immobilized on a functional membrane-modified gold electrode, was studied. The electrode showed a quasi-reversible electrochemical redox behavior with a formal potential of 60mV (versus Ag/AgCl) in 20mM potassium phosphate buffer solution at pH 7.0 and temperature 25 o C. The cathodic transfer coefficient was 0.42 and electron transfer rate constant was evaluated to be 1.6s -1 . Furthermore, the modified electrode was used as a biosensor and exhibited a satisfactory stability and sensitivity to H 2 O 2 . The linear range of this biosensor for H 2 O 2 determination was from 5.0x10 -6 to 1.5x10 -4 M while its detection limit, based on a signal-to-noise ratio of 3, was 1.3x10 -6 M. The apparent Michaelis-Menten constant (K m app ) for immobilized HRP was calculated to be 1.6x10 -4 M

  20. Phytochelatin homologs induced in hairy roots of horseradish.

    Science.gov (United States)

    Kubota, H; Sato, K; Yamada, T; Maitani, T

    2000-01-01

    When exposed to excess heavy metals, plants induce phytochelatins and related peptides (all designated as PCAs). Thus, when hairy roots of horseradish (Armoracia rusticana) were exposed for 3 days to cadmium (1 mM) along with reduced glutathione (2 mM), PCA induction occurred. Moreover, a new family of thiol peptides was detected as well as the previously known PCAs, as revealed by postcolumn-derivatization HPLC. Two were isolated and their structures were identified as (gamma-Glu-Cys)n-Gln (n = 3 and 4) by electrospray ionization-mass spectrometer spectra, this being confirmed by chemical synthesis of the peptides. These new analogs constitute the sixth PCA family identified to date.

  1. Light Requirement for Shoot Regeneration in Horseradish Hairy Roots 1

    Science.gov (United States)

    Saitou, Tsutomu; Kamada, Hiroshi; Harada, Hiroshi

    1992-01-01

    Hairy roots of horseradish (Armoracia rusticana) were induced by inoculation with Agrobacterium rhizogenes harboring Ri plasmid and cultured on phytohormone-free Murashige and Skoog medium after eliminating the bacteria. Hairy roots grew vigorously and sometimes formed yellowish calli under dark conditions. On the other hand, growth of hairy roots stopped after several weeks of culture with light, then shoots were regenerated. Frequency of shoot formation from hairy roots increased as the culture period in light lengthened and the light intensity increased. The shoot regeneration was induced by treatment with white or red light, but not with far-red light. Shoot regeneration by red light was inhibited by following treatment with far-red light. Red and far-red light reversibly affected shoot regeneration. Excised roots of nontransformed plants grew quite slowly on phytohormone-free Murashige and Skoog medium and occasionally formed shoots under white light conditions. PMID:16669041

  2. Light requirement for shoot regeneration in horseradish hairy roots.

    Science.gov (United States)

    Saitou, T; Kamada, H; Harada, H

    1992-08-01

    Hairy roots of horseradish (Armoracia rusticana) were induced by inoculation with Agrobacterium rhizogenes harboring Ri plasmid and cultured on phytohormone-free Murashige and Skoog medium after eliminating the bacteria. Hairy roots grew vigorously and sometimes formed yellowish calli under dark conditions. On the other hand, growth of hairy roots stopped after several weeks of culture with light, then shoots were regenerated. Frequency of shoot formation from hairy roots increased as the culture period in light lengthened and the light intensity increased. The shoot regeneration was induced by treatment with white or red light, but not with far-red light. Shoot regeneration by red light was inhibited by following treatment with far-red light. Red and far-red light reversibly affected shoot regeneration. Excised roots of nontransformed plants grew quite slowly on phytohormone-free Murashige and Skoog medium and occasionally formed shoots under white light conditions.

  3. Polymerization reactivity of sulfomethylated alkali lignin modified with horseradish peroxidase.

    Science.gov (United States)

    Yang, Dongjie; Wu, Xiaolei; Qiu, Xueqing; Chang, Yaqi; Lou, Hongming

    2014-03-01

    Alkali lignin (AL) was employed as raw materials in the present study. Sulfomethylation was conducted to improve the solubility of AL, while sulfomethylated alkali lignin (SAL) was further polymerized by horseradish peroxidase (HRP). HRP modification caused a significant increase in molecular weight of SAL which was over 20 times. It was also found to increase the amount of sulfonic and carboxyl groups while decrease the amount of phenolic and methoxyl groups in SAL. The adsorption quantity of self-assembled SAL film was improved after HRP modification. Sulfonation and HRP modification were mutually promoted. The polymerization reactivity of SAL in HRP modification was increased with its sulfonation degree. Meanwhile, HRP modification facilitated SAL's radical-sulfonation reaction. Copyright © 2014. Published by Elsevier Ltd.

  4. Plasma membrane proteomics of human breast cancer cell lines identifies potential targets for breast cancer diagnosis and treatment.

    Directory of Open Access Journals (Sweden)

    Yvonne S Ziegler

    Full Text Available The use of broad spectrum chemotherapeutic agents to treat breast cancer results in substantial and debilitating side effects, necessitating the development of targeted therapies to limit tumor proliferation and prevent metastasis. In recent years, the list of approved targeted therapies has expanded, and it includes both monoclonal antibodies and small molecule inhibitors that interfere with key proteins involved in the uncontrolled growth and migration of cancer cells. The targeting of plasma membrane proteins has been most successful to date, and this is reflected in the large representation of these proteins as targets of newer therapies. In view of these facts, experiments were designed to investigate the plasma membrane proteome of a variety of human breast cancer cell lines representing hormone-responsive, ErbB2 over-expressing and triple negative cell types, as well as a benign control. Plasma membranes were isolated by using an aqueous two-phase system, and the resulting proteins were subjected to mass spectrometry analysis. Overall, each of the cell lines expressed some unique proteins, and a number of proteins were expressed in multiple cell lines, but in patterns that did not always follow traditional clinical definitions of breast cancer type. From our data, it can be deduced that most cancer cells possess multiple strategies to promote uncontrolled growth, reflected in aberrant expression of tyrosine kinases, cellular adhesion molecules, and structural proteins. Our data set provides a very rich and complex picture of plasma membrane proteins present on breast cancer cells, and the sorting and categorizing of this data provides interesting insights into the biology, classification, and potential treatment of this prevalent and debilitating disease.

  5. Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer.

    Science.gov (United States)

    Peng, Zheng-Hong; Sima, Monika; Salama, Mohamed E; Kopečková, Pavla; Kopeček, Jindřich

    2013-12-01

    Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (-GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer--DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates' in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice.

  6. Purification of peroxidase from Horseradish (Armoracia rusticana) roots.

    Science.gov (United States)

    Lavery, Christopher B; Macinnis, Morgan C; Macdonald, M Jason; Williams, Joanna Bassey; Spencer, Colin A; Burke, Alicia A; Irwin, David J G; D'Cunha, Godwin B

    2010-08-11

    Peroxidase (EC 1.11.1.7) from horseradish ( Armoracia rusticana ) roots was purified using a simple, rapid, three-step procedure: ultrasonication, ammonium sulfate salt precipitation, and hydrophobic interaction chromatography on phenyl Sepharose CL-4B. The preparation gave an overall yield of 71%, 291-fold purification, and a high specific activity of 772 U mg(-1) protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the purified enzyme was homogeneous and had a molecular weight of approximately 40 kDa. The isolated enzyme had an isoelectric point of 8.8 and a Reinheitszahl value of 3.39 and was stable when stored in the presence of glycerol at -20 degrees C, with >95% retention of original enzyme activity for at least 6 months. Maximal activity of purified horseradish peroxidase (HRP) was obtained under different optimized conditions: substrate (guaiacol and H(2)O(2)) concentrations (0.5 and 0.3 mM, respectively), type of buffer (50 mM phosphate buffer), pH (7.0), time (1.0 min), and temperature of incubation (30 degrees C). In addition, the effect of HRP and H(2)O(2) in a neutral-buffered aqueous solution for the oxidation of phenol and 2-chlorophenol substrates was also studied. Different conditions including concentrations of phenol/2-chlorophenol, H(2)O(2), and enzyme, time, pH, and temperature were standardized for the maximal activity of HRP with these substrates; under these optimal conditions 89.6 and 91.4% oxidations of phenol and 2-chlorophenol were obtained, respectively. The data generated from this work could have direct implications in studies on the commercial production of this biotechnologically important enzyme and its stability in different media.

  7. Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries.

    Science.gov (United States)

    Hoang, Hanh H; Nickerson, Nicholas N; Lee, Vincent T; Kazimirova, Anastasia; Chami, Mohamed; Pugsley, Anthony P; Lory, Stephen

    2011-01-01

    In Gram-negative bacteria, the Lol and Bam machineries direct the targeting of lipidated and nonlipidated proteins, respectively, to the outer membrane (OM). Using Pseudomonas aeruginosa strains with depleted levels of specific Bam and Lol proteins, we demonstrated a variable dependence of different OM proteins on these targeting pathways. Reduction in the level of BamA significantly affected the ability of the β-barrel membrane protein OprF to localize to the OM, while the targeting of three secretins that are functionally related OM proteins was less affected (PilQ and PscC) or not at all affected (XcpQ). Depletion of LolB affected all lipoproteins examined and had a variable effect on the nonlipidated proteins. While the levels of OprF, PilQ, and PscC were significantly reduced by LolB depletion, XcpQ was unaffected and was correctly localized to the OM. These results suggest that certain β-barrel proteins such as OprF primarily utilize the complete Bam machinery. The Lol machinery participates in the OM targeting of secretins to variable degrees, likely through its involvement in the assembly of lipidated Bam components. XcpQ, but not PilQ or PscC, was shown to assemble spontaneously into liposomes as multimers. This work raises the possibility that there is a gradient of utilization of Bam and Lol insertion and targeting machineries. Structural features of individual proteins, including their β-barrel content, may determine the propensity of these proteins for folding (or misfolding) during periplasmic transit and OM insertion, thereby influencing the extent of utilization of the Bam targeting machinery, respectively. Targeting of lipidated and nonlipidated proteins to the outer membrane (OM) compartment in Gram-negative bacteria involves the transfer across the periplasm utilizing the Lol and Bam machineries, respectively. We show that depletion of Bam and Lol components in Pseudomonas aeruginosa does not lead to a general OM protein translocation defect

  8. Membrane composition and dynamics: a target of bioactive virgin olive oil constituents.

    Science.gov (United States)

    Lopez, Sergio; Bermudez, Beatriz; Montserrat-de la Paz, Sergio; Jaramillo, Sara; Varela, Lourdes M; Ortega-Gomez, Almudena; Abia, Rocio; Muriana, Francisco J G

    2014-06-01

    The endogenous synthesis of lipids, which requires suitable dietary raw materials, is critical for the formation of membrane bilayers. In eukaryotic cells, phospholipids are the predominant membrane lipids and consist of hydrophobic acyl chains attached to a hydrophilic head group. The relative balance between saturated, monounsaturated, and polyunsaturated acyl chains is required for the organization and normal function of membranes. Virgin olive oil is the richest natural dietary source of the monounsaturated lipid oleic acid and is one of the key components of the healthy Mediterranean diet. Virgin olive oil also contains a unique constellation of many other lipophilic and amphipathic constituents whose health benefits are still being discovered. The focus of this review is the latest evidence regarding the impact of oleic acid and the minor constituents of virgin olive oil on the arrangement and behavior of lipid bilayers. We highlight the relevance of these interactions to the potential use of virgin olive oil in preserving the functional properties of membranes to maintain health and in modulating membrane functions that can be altered in several pathologies. This article is part of a Special Issue entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy. Copyright © 2014 Elsevier B.V. All rights reserved.

  9. Targeting the plasma membrane of neoplastic cells through alkylation: a novel approach to cancer chemotherapy.

    Science.gov (United States)

    Trendowski, Matthew; Fondy, Thomas P

    2015-08-01

    Although DNA-directed alkylating agents and related compounds have been a mainstay in chemotherapeutic protocols due to their ability to readily interfere with the rapid mitotic progression of malignant cells, their clinical utility is limited by DNA repair mechanisms and immunosuppression. However, the same destructive nature of alkylation can be reciprocated at the cell surface using novel plasma membrane alkylating agents. Plasma membrane alkylating agents have elicited long term survival in mammalian models challenged with carcinomas, sarcomas, and leukemias. Further, a specialized group of plasma membrane alkylating agents known as tetra-O-acetate haloacetamido carbohydrate analogs (Tet-OAHCs) potentiates a substantial leukocyte influx at the administration and primary tumor site, indicative of a potent immune response. The effects of plasma membrane alkylating agents may be further potentiated through the use of another novel class of chemotherapeutic agents, known as dihydroxyacetone phosphate (DHAP) inhibitors, since many cancer types are known to rely on the DHAP pathway for lipid synthesis. Despite these compelling data, preliminary clinical trials for plasma membrane-directed agents have yet to be considered. Therefore, this review is intended for academics and clinicians to postulate a novel approach of chemotherapy; altering critical malignant cell signaling at the plasma membrane surface through alkylation, thereby inducing irreversible changes to functions needed for cell survival.

  10. Polyhydroxybutyrate Targets Mammalian Mitochondria and Increases Permeability of Plasmalemmal and Mitochondrial Membranes

    Science.gov (United States)

    Elustondo, Pia A.; Angelova, Plamena R.; Kawalec, Michał; Michalak, Michał; Kurcok, Piotr; Abramov, Andrey Y.; Pavlov, Evgeny V.

    2013-01-01

    Poly(3-hydroxybutyrate) (PHB) is a polyester of 3-hydroxybutyric acid (HB) that is ubiquitously present in all organisms. In higher eukaryotes PHB is found in the length of 10 to 100 HB units and can be present in free form as well as in association with proteins and inorganic polyphosphate. It has been proposed that PHB can mediate ion transport across lipid bilayer membranes. We investigated the ability of PHB to interact with living cells and isolated mitochondria and the effects of these interactions on membrane ion transport. We performed experiments using a fluorescein derivative of PHB (fluo-PHB). We found that fluo-PHB preferentially accumulated inside the mitochondria of HeLa cells. Accumulation of fluo-PHB induced mitochondrial membrane depolarization. This membrane depolarization was significantly delayed by the inhibitor of the mitochondrial permeability transition pore - Cyclosporin A. Further experiments using intact cells as well as isolated mitochondria confirmed that the effects of PHB directly linked to its ability to facilitate ion transport, including calcium, across the membranes. We conclude that PHB demonstrates ionophoretic properties in biological membranes and this effect is most profound in mitochondria due to the selective accumulation of the polymer in this organelle. PMID:24086638

  11. Determinants for membrane association and permeabilization of the coxsackievirus 2B protein and the identification of the Golgi complex as the target organelle.

    Science.gov (United States)

    de Jong, Arjan S; Wessels, Els; Dijkman, Henri B P M; Galama, Jochem M D; Melchers, Willem J G; Willems, Peter H G M; van Kuppeveld, Frank J M

    2003-01-10

    The 2B protein of enterovirus is responsible for the alterations in the permeability of secretory membranes and the plasma membrane in infected cells. The structural requirements for the membrane association and the subcellular localization of this essential virus protein, however, have not been defined. Here, we provide evidence that the 2B protein is an integral membrane protein in vivo that is predominantly localized at the Golgi complex upon individual expression. Addition of organelle-specific targeting signals to the 2B protein revealed that the Golgi localization is an absolute prerequisite for the ability of the protein to modify plasma membrane permeability. Expression of deletion mutants and heterologous proteins containing specific domains of the 2B protein demonstrated that each of the two hydrophobic regions could mediate membrane binding individually. However, the presence of both hydrophobic regions was required for the correct membrane association, efficient Golgi targeting, and the membrane-permeabilizing activity of the 2B protein, suggesting that the two hydrophobic regions are cooperatively involved in the formation of a membrane-integral complex. The formation of membrane-integral pores by the 2B protein in the Golgi complex and the possible mechanism by which a Golgi-localized virus protein modifies plasma membrane permeability are discussed.

  12. Self-Assembled Complexes of Horseradish Peroxidase with Magnetic Nanoparticles Showing Enhanced Peroxidase Activity

    KAUST Repository

    Corgié , Sté phane C.; Kahawong, Patarawan; Duan, Xiaonan; Bowser, Daniel; Edward, Joseph B.; Walker, Larry P.; Giannelis, Emmanuel P.

    2012-01-01

    Bio-nanocatalysts (BNCs) consisting of horseradish peroxidase (HRP) self-assembled with magnetic nanoparticles (MNPs) enhance enzymatic activity due to the faster turnover and lower inhibition of the enzyme. The size and magnetization of the MNPs

  13. Ruthenium complexes with phenylterpyridine derivatives target cell membrane and trigger death receptors-mediated apoptosis in cancer cells.

    Science.gov (United States)

    Deng, Zhiqin; Gao, Pan; Yu, Lianling; Ma, Bin; You, Yuanyuan; Chan, Leung; Mei, Chaoming; Chen, Tianfeng

    2017-06-01

    Elucidation of the communication between metal complexes and cell membrane may provide useful information for rational design of metal-based anticancer drugs. Herein we synthesized a novel class of ruthenium (Ru) complexes containing phtpy derivatives (phtpy = phenylterpyridine), analyzed their structure-activity relationship and revealed their action mechanisms. The result showed that, the increase in the planarity of hydrophobic Ru complexes significantly enhanced their lipophilicity and cellular uptake. Meanwhile, the introduction of nitro group effectively improved their anticancer efficacy. Further mechanism studies revealed that, complex (2c), firstly accumulated on cell membrane and interacted with death receptors to activate extrinsic apoptosis signaling pathway. The complex was then transported into cell cytoplasm through transferrin receptor-mediated endocytosis. Most of the intracellular 2c accumulated in cell plasma, decreasing the level of cellular ROS, inducing the activation of caspase-9 and thus intensifying the apoptosis. At the same time, the residual 2c can translocate into cell nucleus to interact with DNA, induce DNA damage, activate p53 pathway and enhance apoptosis. Comparing with cisplatin, 2c possesses prolonged circulation time in blood, comparable antitumor ability and importantly, much lower toxicity in vivo. Taken together, this study uncovers the role of membrane receptors in the anticancer actions of Ru complexes, and provides fundamental information for rational design of membrane receptor targeting anticancer drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. SecA is required for membrane targeting of the cell division protein DivIVA in vivo

    Directory of Open Access Journals (Sweden)

    Sven eHalbedel

    2014-02-01

    Full Text Available The conserved protein DivIVA is involved in different morphogenetic processes in Gram-positive bacteria. In Bacillus subtilis, the protein localises to the cell division site and cell poles, and functions as a scaffold for proteins that regulate division site selection, and for proteins that are required for sporulation. To identify other proteins that bind to DivIVA, we performed an in vivo cross-linking experiment. A possible candidate that emerged was the secretion motor ATPase SecA. SecA mutants have been described that inhibit sporulation, and since DivIVA is necessary for sporulation, we examined the localisation of DivIVA in these mutants. Surprisingly, DivIVA was delocalised, suggesting that SecA is required for DivIVA targeting. To further corroborate this, we performed SecA depletion and inhibition experiments, which provided further indications that DivIVA localisation depends on SecA. Cell fractionation experiments showed that SecA is important for binding of DivIVA to the cell membrane. This was unexpected since DivIVA does not contain a signal sequence, and is able to bind to artificial lipid membranes in vitro without support of other proteins. SecA is required for protein secretion and membrane insertion, and therefore its role in DivIVA localisation is likely indirect. Possible alternative roles of SecA in DivIVA folding and/or targeting are discussed.

  15. Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane.

    Science.gov (United States)

    Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M P; Albano, E; Bianchi, F B

    2000-04-01

    Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack. The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum. Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes. AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.

  16. Contribution of cubilin and amnionless to processing and membrane targeting of cubilin-amnionless complex

    DEFF Research Database (Denmark)

    Coudroy, Gwénaëlle; Gburek, Jakub; Kozyraki, Renata

    2005-01-01

    Cubilin is a peripheral apical membrane receptor for multiple ligands that are taken up in several absorptive epithelia. Recently, amnionless (AMN) was identified to form a functional receptor complex with cubilin. By expression in transfected polarized MDCK cells of AMN and several cubilin fragm...

  17. Evaluation of plasma membrane calcium/calmodulin-dependent ATPase isoform 4 as a potential target for fertility control.

    Science.gov (United States)

    Cartwright, Elizabeth J; Neyses, Ludwig

    2010-01-01

    The array of contraceptives currently available is clearly inadequate and does not meet consumer demands since it is estimated that up to a quarter of all pregnancies worldwide are unintended. There is, therefore, an overwhelming global need to develop new effective, safe, ideally non-hormonal contraceptives for both male and female use. The contraceptive field, unlike other areas such as cancer, has a dearth of new targets. We have addressed this issue and propose that isoform 4 of the plasma membrane calcium ATPase is a potentially exciting novel target for fertility control. The plasma membrane calcium ATPase is a ubiquitously expressed calcium pump whose primary function in the majority of cells is to extrude calcium to the extracellular milieu. Two isoforms of this gene family, PMCA1 and PMCA4, are expressed in spermatozoa, with PMCA4 being the predominant isoform. Although this gene is ubiquitously expressed, its function is highly tissue-specific. Genetic deletion of PMCA4, in PMCA4 knockout mice, led to 100% infertility specifically in the male mutant mice due to a selective defect in sperm motility. It is important to note that the gene deletion did not affect normal mating characteristics in these mice. This phenotype was mimicked in wild-type sperm treated with the non-specific PMCA inhibitor 5-(and 6-) carboxyeosin diacetate succinimidyl ester; a proof-of-principle that inhibition of PMCA4 has potential importance in the control of fertility. This review outlines the potential for PMCA4 to be a novel target for fertility control by acting to inhibit sperm motility. It will outline the characteristics that make this target drugable and will describe methodologies to identify and validate novel inhibitors of this target.

  18. Comparison of Different Solvents and Extraction Methods for Isolation of Phenolic Compounds from Horseradish Roots (Armoracia rusticana)

    OpenAIRE

    Lolita Tomsone; Zanda Kruma; Ruta Galoburda

    2012-01-01

    Horseradish (Armoracia rusticana) is a perennial herb belonging to the Brassicaceae family and contains biologically active substances. The aim of the current research was to determine best method for extraction of phenolic compounds from horseradish roots showing high antiradical activity. Three genotypes (No. 105; No. 106 and variety ‘Turku’) of horseradish roots were extracted with eight different solvents: n-hexane, ethyl acetate, diethyl ether, 2-propanol, acetone, ethanol (95%), ethanol...

  19. Direct Electrochemistry of Horseradish Peroxidase-Gold Nanoparticles Conjugate

    Directory of Open Access Journals (Sweden)

    Chanchal K. Mitra

    2009-02-01

    Full Text Available We have studied the direct electrochemistry of horseradish peroxidase (HRP coupled to gold nanoparticles (AuNP using electrochemical techniques, which provide some insight in the application of biosensors as tools for diagnostics because HRP is widely used in clinical diagnostics kits. AuNP capped with (i glutathione and (ii lipoic acid was covalently linked to HRP. The immobilized HRP/AuNP conjugate showed characteristic redox peaks at a gold electrode. It displayed good electrocatalytic response to the reduction of H2O2, with good sensitivity and without any electron mediator. The covalent linking of HRP and AuNP did not affect the activity of the enzyme significantly. The response of the electrode towards the different concentrations of H2O2 showed the characteristics of Michaelis Menten enzyme kinetics with an optimum pH between 7.0 to 8.0. The preparation of the sensor involves single layer of enzyme, which can be carried out efficiently and is also highly reproducible when compared to other systems involving the layer-by-layer assembly, adsorption or encapsulation of the enzyme. The immobilized AuNP-HRP can be used for immunosensor applications

  20. Antibacterial activity of polyphenolic fraction of Kombucha against Vibrio cholerae: targeting cell membrane.

    Science.gov (United States)

    Bhattacharya, D; Ghosh, D; Bhattacharya, S; Sarkar, S; Karmakar, P; Koley, H; Gachhui, R

    2018-02-01

    The present study was undertaken to determine the mechanism of antibacterial activity of a polyphenolic fraction, composed of mainly catechin and isorhamnetin, previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against the enteropathogen Vibrio cholerae N16961. Bacterial growth was found to be seriously impaired by the polyphenolic fraction in a dose-dependent manner. Scanning Electron Microscopy demonstrated morphological alterations in bacterial cells when exposed to the polyphenolic fraction in a concentration-dependent manner. Permeabilization assays confirmed that the fraction disrupted bacterial membrane integrity in both time- and dose-dependent manners, which were proportional to the production of intracellular reactive oxygen species (ROS). Furthermore, each of the polyphenols catechin and isorhamnetin showed the ability to permeate bacterial cell membranes by generating oxidative stress, thereby suggesting their role in the antibacterial potential of Kombucha. Thus, the basic mechanism of antibacterial activity of the Kombucha polyphenolic fraction against V. cholerae involved bacterial membrane permeabilization and morphological changes, which might be due to the generation of intracellular ROS. To the best of our knowledge, this is the first report on the investigation of antibacterial mechanism of Kombucha, which is mostly attributed to its polyphenolic content. The emergence of multidrug-resistant Vibrio cholerae strains has hindered an efficient anti-Vibrio therapy. This study has demonstrated the membrane damage-mediated antibacterial mechanism of Kombucha, a popular fermented beverage of sugared tea, which is mostly attributed to its polyphenolic content. This study also implies the exploitation of Kombucha as a potential new source of bioactive polyphenols against V. cholerae. © 2017 The Society for Applied Microbiology.

  1. Cancer Patient T Cells Genetically Targeted to Prostate-Specific Membrane Antigen Specifically Lyse Prostate Cancer Cells and Release Cytokines in Response to Prostate-Specific Membrane Antigen

    Directory of Open Access Journals (Sweden)

    Michael C. Gong

    1999-06-01

    Full Text Available The expression of immunoglobulin-based artificial receptors in normal T lymphocytes provides a means to target lymphocytes to cell surface antigens independently of major histocompatibility complex restriction. Such artificial receptors have been previously shown to confer antigen-specific tumoricidal properties in murine T cells. We constructed a novel ζ chain fusion receptor specific for prostate-specific membrane antigen (PSMA termed Pz-1. PSMA is a cell-surface glycoprotein expressed on prostate cancer cells and the neovascular endothelium of multiple carcinomas. We show that primary T cells harvested from five of five patients with different stages of prostate cancer and transduced with the Pz-1 receptor readily lyse prostate cancer cells. Having established a culture system using fibroblasts that express PSMA, we next show that T cells expressing the Pz-1 receptor release cytokines in response to cell-bound PSMA. Furthermore, we show that the cytokine release is greatly augmented by B7.1-mediated costimulation. Thus, our findings support the feasibility of adoptive cell therapy by using genetically engineered T cells in prostate cancer patients and suggest that both CD4+ and CD8+ T lymphocyte functions can be synergistically targeted against tumor cells.

  2. Assessing the Mitochondrial Membrane Potential in Cells and In Vivo using Targeted Click Chemistry and Mass Spectrometry.

    Science.gov (United States)

    Logan, Angela; Pell, Victoria R; Shaffer, Karl J; Evans, Cameron; Stanley, Nathan J; Robb, Ellen L; Prime, Tracy A; Chouchani, Edward T; Cochemé, Helena M; Fearnley, Ian M; Vidoni, Sara; James, Andrew M; Porteous, Carolyn M; Partridge, Linda; Krieg, Thomas; Smith, Robin A J; Murphy, Michael P

    2016-02-09

    The mitochondrial membrane potential (Δψm) is a major determinant and indicator of cell fate, but it is not possible to assess small changes in Δψm within cells or in vivo. To overcome this, we developed an approach that utilizes two mitochondria-targeted probes each containing a triphenylphosphonium (TPP) lipophilic cation that drives their accumulation in response to Δψm and the plasma membrane potential (Δψp). One probe contains an azido moiety and the other a cyclooctyne, which react together in a concentration-dependent manner by "click" chemistry to form MitoClick. As the mitochondrial accumulation of both probes depends exponentially on Δψm and Δψp, the rate of MitoClick formation is exquisitely sensitive to small changes in these potentials. MitoClick accumulation can then be quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This approach enables assessment of subtle changes in membrane potentials within cells and in the mouse heart in vivo. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  3. Nef decreases HIV-1 sensitivity to neutralizing antibodies that target the membrane-proximal external region of TMgp41.

    Directory of Open Access Journals (Sweden)

    Rachel P J Lai

    2011-12-01

    Full Text Available Primate lentivirus nef is required for sustained virus replication in vivo and accelerated progression to AIDS. While exploring the mechanism by which Nef increases the infectivity of cell-free virions, we investigated a functional link between Nef and Env. Since we failed to detect an effect of Nef on the quantity of virion-associated Env, we searched for qualitative changes by examining whether Nef alters HIV-1 sensitivity to agents that target distinct features of Env. Nef conferred as much as 50-fold resistance to 2F5 and 4E10, two potent neutralizing monoclonal antibodies (nAbs that target the membrane proximal external region (MPER of TMgp41. In contrast, Nef had no effect on HIV-1 neutralization by MPER-specific nAb Z13e1, by the peptide inhibitor T20, nor by a panel of nAbs and other reagents targeting gp120. Resistance to neutralization by 2F5 and 4E10 was observed with Nef from a diverse range of HIV-1 and SIV isolates, as well as with HIV-1 virions bearing Env from CCR5- and CXCR4-tropic viruses, clade B and C viruses, or primary isolates. Functional analysis of a panel of Nef mutants revealed that this activity requires Nef myristoylation but that it is genetically separable from other Nef functions such as the ability to enhance virus infectivity and to downregulate CD4. Glycosylated-Gag from MoMLV substituted for Nef in conferring resistance to 2F5 and 4E10, indicating that this activity is conserved in a retrovirus that does not encode Nef. Given the reported membrane-dependence of MPER-recognition by 2F5 and 4E10, in contrast to the membrane-independence of Z13e1, the data here is consistent with a model in which Nef alters MPER recognition in the context of the virion membrane. Indeed, Nef and Glycosylated-Gag decreased the efficiency of virion capture by 2F5 and 4E10, but not by other nAbs. These studies demonstrate that Nef protects lentiviruses from one of the most broadly-acting classes of neutralizing antibodies. This newly

  4. An N-Terminal ER Export Signal Facilitates the Plasma Membrane Targeting of HCN1 Channels in Photoreceptors.

    Science.gov (United States)

    Pan, Yuan; Laird, Joseph G; Yamaguchi, David M; Baker, Sheila A

    2015-06-01

    Hyperpolarization-activated cyclic nucleotide-gated 1 (HCN1) channels are widely expressed in the retina. In photoreceptors, the hyperpolarization-activated current (Ih) carried by HCN1 is important for shaping the light response. It has been shown in multiple systems that trafficking HCN1 channels to specific compartments is key to their function. The localization of HCN1 in photoreceptors is concentrated in the plasma membrane of the inner segment (IS). The mechanisms controlling this localization are not understood. We previously identified a di-arginine endoplasmic reticulum (ER) retention motif that negatively regulates the surface targeting of HCN1. In this study, we sought to identify a forward trafficking signal that could counter the function of the ER retention signal. We studied trafficking of HCN1 and several mutants by imaging their subcellular localization in transgenic X. laevis photoreceptors. Velocity sedimentation was used to assay the assembly state of HCN1 channels. We found the HCN1 N-terminus can redirect a membrane reporter from outer segments (OS) to the plasma membrane of the IS. The sequence necessary for this behavior was mapped to a 20 amino acid region containing a leucine-based ER export motif. The ER export signal is necessary for forward trafficking but not channel oligomerization. Moreover, this ER export signal alone counteracted the di-arginine ER retention signal. We identified an ER export signal in HCN1 that functions with the ER retention signal to maintain equilibrium of HCN1 between the endomembrane system and the plasma membrane.

  5. Glycan specificity of the Vibrio vulnificus hemolysin lectin outlines evolutionary history of membrane targeting by a toxin family.

    Science.gov (United States)

    Kaus, Katherine; Lary, Jeffrey W; Cole, James L; Olson, Rich

    2014-07-29

    Pore-forming toxins (PFTs) are a class of pathogen-secreted molecules that oligomerize to form transmembrane channels in cellular membranes. Determining the mechanism for how PFTs bind membranes is important in understanding their role in disease and for developing possible ways to block their action. Vibrio vulnificus, an aquatic pathogen responsible for severe food poisoning and septicemia in humans, secretes a PFT called V. vulnificus hemolysin (VVH), which contains a single C-terminal targeting domain predicted to resemble a β-trefoil lectin fold. In order to understand the selectivity of the lectin for glycan motifs, we expressed the isolated VVH β-trefoil domain and used glycan-chip screening to identify that VVH displays a preference for terminal galactosyl groups including N-acetyl-d-galactosamine and N-acetyl-d-lactosamine. The X-ray crystal structure of the VVH lectin domain solved to 2.0Å resolution reveals a heptameric ring arrangement similar to the oligomeric form of the related, but inactive, lectin from Vibrio cholerae cytolysin. Structures bound to glycerol, N-acetyl-d-galactosamine, and N-acetyl-d-lactosamine outline a common and versatile mode of recognition allowing VVH to target a wide variety of cell-surface ligands. Sequence analysis in light of our structural and functional data suggests that VVH may represent an earlier step in the evolution of Vibrio PFTs. Copyright © 2014 Elsevier Ltd. All rights reserved.

  6. New Potent Membrane-Targeting Antibacterial Peptides from Viral Capsid Proteins

    Science.gov (United States)

    Dias, Susana A.; Freire, João M.; Pérez-Peinado, Clara; Domingues, Marco M.; Gaspar, Diana; Vale, Nuno; Gomes, Paula; Andreu, David; Henriques, Sónia T.; Castanho, Miguel A. R. B.; Veiga, Ana S.

    2017-01-01

    The increasing prevalence of multidrug-resistant bacteria urges the development of new antibacterial agents. With a broad spectrum activity, antimicrobial peptides have been considered potential antibacterial drug leads. Using bioinformatic tools we have previously shown that viral structural proteins are a rich source for new bioactive peptide sequences, namely antimicrobial and cell-penetrating peptides. Here, we test the efficacy and mechanism of action of the most promising peptides among those previously identified against both Gram-positive and Gram-negative bacteria. Two cell-penetrating peptides, vCPP 0769 and vCPP 2319, have high antibacterial activity against Staphylococcus aureus, MRSA, Escherichia coli, and Pseudomonas aeruginosa, being thus multifunctional. The antibacterial mechanism of action of the two most active viral protein-derived peptides, vAMP 059 and vCPP 2319, was studied in detail. Both peptides act on both Gram-positive S. aureus and Gram-negative P. aeruginosa, with bacterial cell death occurring within minutes. Also, these peptides cause bacterial membrane permeabilization and damage of the bacterial envelope of P. aeruginosa cells. Overall, the results show that structural viral proteins are an abundant source for membrane-active peptides sequences with strong antibacterial properties. PMID:28522994

  7. Antimicrobial activity of isothiocyanates (ITCs) extracted from horseradish (Armoracia rusticana) root against oral microorganisms.

    Science.gov (United States)

    Park, Ho-Won; Choi, Kyu-Duck; Shin, Il-Shik

    2013-01-01

    The antimicrobial activity of isothiocyanates (ITCs) extracted from horseradish root was investigated against oral microorganisms: 6 strains of facultative anaerobic bacteria, Streptococcus mutans, Streptococcus sobrinus, Lactobacillus casei, Staphylococcus aureus, Enterococcus faecalis and Aggregatibacter actinomycetemcomitans; one strain of yeast, Candida albicans, and 3 strains of anaerobic bacteria, Fusobacterium nucleatum, Prevotella nigrescens, and Clostridium perfringens. The ITCs extracted from horseradish root showed antimicrobial activity against all oral microorganisms by the paper disk method. The minimum bactericidal concentration (MBC) of the ITCs extracted from horseradish root ranged from 1.25 to 5.00 mg/ml against 6 strains of facultative anaerobic bacteria and one strain of yeast, and 4.17 to 16.67 mg/ml against 3 strains of anaerobic bacteria. The ITCs extracted from horseradish root showed the strongest antimicrobial activity, with a MBC of 1.25 mg/ml, against C. albicans among facultative microorganisms, and 4.17 mg/ml against F. nucleatum among anaerobic bacteria. These results suggest that the ITCs extracted from horseradish root may be a candidate for use as an antimicrobial agent against oral microorganisms.

  8. An updated view on horseradish peroxidases: recombinant production and biotechnological applications.

    Science.gov (United States)

    Krainer, Florian W; Glieder, Anton

    2015-02-01

    Horseradish peroxidase has been the subject of scientific research for centuries. It has been used exhaustively as reporter enzyme in diagnostics and histochemistry and still plays a major role in these applications. Numerous studies have been conducted on the role of horseradish peroxidase in the plant and its catalytic mechanism. However, little progress has been made in its recombinant production. Until now, commercial preparations of horseradish peroxidase are still isolated from plant roots. These preparations are commonly mixtures of various isoenzymes of which only a small fraction has been described so far. The composition of isoenzymes in these mixed isolates is subjected to uncontrollable environmental conditions. Nowadays, horseradish peroxidase regains interest due to its broad applicability in the fields of medicine, life sciences, and biotechnology in cancer therapy, biosensor systems, bioremediation, and biocatalysis. These medically and commercially relevant applications, the recent discovery of new natural isoenzymes with different biochemical properties, as well as the challenges in recombinant production render this enzyme particularly interesting for future biotechnological solutions. Therefore, we reviewed previous studies as well as current developments with biotechnological emphasis on new applications and the major remaining biotechnological challenge-the efficient recombinant production of horseradish peroxidase enzymes.

  9. Pearls and pitfalls in clinical interpretation of prostate-specific membrane antigen (PSMA)-targeted PET imaging

    Energy Technology Data Exchange (ETDEWEB)

    Sheikhbahaei, Sara; Solnes, Lilja B.; Javadi, Mehrbod S.; Pomper, Martin G.; Rowe, Steven P. [Johns Hopkins University School of Medicine, Division of Nuclear Medicine and Molecular Imaging, The Russell H. Morgan Department of Radiology and Radiological Science, Baltimore, MD (United States); Afshar-Oromieh, Ali; Haberkorn, Uwe [Heidelberg University Hospital, Department of Nuclear Medicine, Heidelberg (Germany); Eiber, Matthias [David Geffen School of Medicine at UCLA, Department of Molecular and Medical Pharmacology, Los Angeles, CA (United States); Technical University of Munich, Department of Nuclear Medicine, Klinikum rechts der Isar, Munich (Germany); Ross, Ashley E.; Pienta, Kenneth J.; Allaf, Mohamad E.; Gorin, Michael A. [Johns Hopkins University School of Medicine, The James Buchanan Brady Urological Institute and Department of Urology, Baltimore, MD (United States)

    2017-11-15

    The rapidly expanding clinical adaptation of prostate-specific membrane antigen (PSMA)-targeted PET imaging in the evaluation of patients with prostate cancer has placed an increasing onus on understanding both the potential pearls of interpretation as well as limitations of this new technique. As with any new molecular imaging modality, accurate characterization of abnormalities on PSMA-targeted PET imaging can be accomplished only if one is aware of the normal distribution pattern, physiological variants of radiotracer uptake, and potential sources of false-positive and false-negative imaging findings. In recent years, a growing number of reports have come to light describing incidental non-prostatic benign or malignant pathologies with high uptake on PSMA-targeted PET imaging. In this review, we have summarized the published literature regarding the potential pearls and technical and interpretive pitfalls of this imaging modality. Knowledge of these limitations can increase the confidence of interpreting physicians and thus improve patient care. As PSMA-targeted PET is expected to be evaluated in larger prospective trials, the dissemination of potential diagnostic pitfalls and the biologic underpinning of those findings will be of increased importance. (orig.)

  10. Pearls and pitfalls in clinical interpretation of prostate-specific membrane antigen (PSMA)-targeted PET imaging

    International Nuclear Information System (INIS)

    Sheikhbahaei, Sara; Solnes, Lilja B.; Javadi, Mehrbod S.; Pomper, Martin G.; Rowe, Steven P.; Afshar-Oromieh, Ali; Haberkorn, Uwe; Eiber, Matthias; Ross, Ashley E.; Pienta, Kenneth J.; Allaf, Mohamad E.; Gorin, Michael A.

    2017-01-01

    The rapidly expanding clinical adaptation of prostate-specific membrane antigen (PSMA)-targeted PET imaging in the evaluation of patients with prostate cancer has placed an increasing onus on understanding both the potential pearls of interpretation as well as limitations of this new technique. As with any new molecular imaging modality, accurate characterization of abnormalities on PSMA-targeted PET imaging can be accomplished only if one is aware of the normal distribution pattern, physiological variants of radiotracer uptake, and potential sources of false-positive and false-negative imaging findings. In recent years, a growing number of reports have come to light describing incidental non-prostatic benign or malignant pathologies with high uptake on PSMA-targeted PET imaging. In this review, we have summarized the published literature regarding the potential pearls and technical and interpretive pitfalls of this imaging modality. Knowledge of these limitations can increase the confidence of interpreting physicians and thus improve patient care. As PSMA-targeted PET is expected to be evaluated in larger prospective trials, the dissemination of potential diagnostic pitfalls and the biologic underpinning of those findings will be of increased importance. (orig.)

  11. Structure of the horseradish peroxidase isozyme C genes.

    Science.gov (United States)

    Fujiyama, K; Takemura, H; Shibayama, S; Kobayashi, K; Choi, J K; Shinmyo, A; Takano, M; Yamada, Y; Okada, H

    1988-05-02

    We have isolated, cloned and characterized three cDNAs and two genomic DNAs corresponding to the mRNAs and genes for the horseradish (Armoracia rusticana) peroxidase isoenzyme C (HPR C). The amino acid sequence of HRP C1, deduced from the nucleotide sequence of one of the cDNA clone, pSK1, contained the same primary sequence as that of the purified enzyme established by Welinder [FEBS Lett. 72, 19-23 (1976)] with additional sequences at the N and C terminal. All three inserts in the cDNA clones, pSK1, pSK2 and pSK3, coded the same size of peptide (308 amino acid residues) if these are processed in the same way, and the amino acid sequence were homologous to each other by 91-94%. Functional amino acids, including His40, His170, Tyr185 and Arg183 and S-S-bond-forming Cys, were conserved in the three isozymes, but a few N-glycosylation sites were not the same. Two HRP C isoenzyme genomic genes, prxC1 and prxC2, were tandem on the chromosomal DNA and each gene consisted of four exons and three introns. The positions in the exons interrupted by introns were the same in two genes. We observed a putative promoter sequence 5' upstream and a poly(A) signal 3' downstream in both genes. The gene product of prxC1 might be processed with a signal sequence of 30 amino acid residues at the N terminus and a peptide consisting of 15 amino acid residues at the C terminus.

  12. Vesicle-associated membrane protein 7 (VAMP-7) is essential for target cell killing in a natural killer cell line

    International Nuclear Information System (INIS)

    Marcet-Palacios, Marcelo; Odemuyiwa, Solomon O.; Coughlin, Jason J.; Garofoli, Daniella; Ewen, Catherine; Davidson, Courtney E.; Ghaffari, Mazyar; Kane, Kevin P.; Lacy, Paige; Logan, Michael R.; Befus, A. Dean; Bleackley, R. Chris; Moqbel, Redwan

    2008-01-01

    Natural killer cells recognize and induce apoptosis in foreign, transformed or virus-infected cells through the release of perforin and granzymes from secretory lysosomes. Clinically, NK-cell mediated killing is a major limitation to successful allo- and xenotransplantation. The molecular mechanisms that regulate the fusion of granzyme B-containing secretory lysosomes to the plasma membrane in activated NK cells, prior to target cell killing, are not fully understood. Using the NK cell line YT-Indy as a model, we have investigated the expression of SNAP REceptors (SNAREs), both target (t-) and vesicular (v-) SNAREs, and their function in granzyme B-mediated target cell killing. Our data showed that YT-Indy cells express VAMP-7 and SNAP-23, but not VAMP-2. VAMP-7 was associated with granzyme B-containing lysosomal granules. Using VAMP-7 small interfering RNA (siRNA), we successfully knocked down the expression of VAMP-7 protein in YT-Indy to less than 10% of untreated cells in 24 h. VAMP7-deficient YT-Indy cells activated via co-culture with Jurkat cells released <1 ng/mL of granzyme B, compared to 1.5-2.5 μg/mL from controls. Using Jurkat cells as targets, we showed a 7-fold reduction in NK cell-mediated killing by VAMP-7 deficient YT-Indy cells. Our results show that VAMP-7 is a crucial component of granzyme B release and target cell killing in the NK cell line YT-Indy. Thus, targeting VAMP-7 expression specifically with siRNA, following transplantation, may be a viable strategy for preventing NK cell-mediated transplant rejection, in vivo

  13. Vascular targeted therapy with anti-prostate-specific membrane antigen monoclonal antibody J591 in advanced solid tumors.

    Science.gov (United States)

    Milowsky, Matthew I; Nanus, David M; Kostakoglu, Lale; Sheehan, Christine E; Vallabhajosula, Shankar; Goldsmith, Stanley J; Ross, Jeffrey S; Bander, Neil H

    2007-02-10

    Based on prostate-specific membrane antigen (PSMA) expression on the vasculature of solid tumors, we performed a phase I trial of antibody J591, targeting the extracellular domain of PSMA, in patients with advanced solid tumor malignancies. This was a proof-of-principle evaluation of PSMA as a potential neovascular target. The primary end points were targeting,toxicity, maximum-tolerated dose, pharmacokinetics (PK), and human antihuman antibody (HAHA) response. Patients had advanced solid tumors previously shown to express PSMA on the neovasculature. They received 111Indium (111ln)-J591 for scintigraphy and PK, followed 2 weeks later by J591 with a reduced amount of 111In for additional PK measurements. J591 dose levels were 5, 10, 20, 40, and 80 mg. The protocol was amended for six weekly administrations of unchelated J591. Patients with a response or stable disease were eligible for re-treatment. Immunohistochemistry assessed PSMA expression in tumor tissues. Twenty-seven patients received monoclonal antibody (mAb) J591. Treatment was well tolerated. Twenty (74%) of 27 patients had at least one area of known metastatic disease targeted by 111In-J591, with positive imaging seen in patients with kidney, bladder, lung, breast, colorectal, and pancreatic cancers, and melanoma. Seven of 10 patient specimens available for immunohistochemical assessment of PSMA expression in tumor-associated vasculature demonstrated PSMA staining. No HAHA response was seen. Three patients of 27 with stable disease received re-treatment. Acceptable toxicity and excellent targeting of known sites of metastases were demonstrated in patients with multiple solid tumor types, highlighting a potential role for the anti-PSMA antibody J591 as a vascular-targeting agent.

  14. Limbic system associated membrane protein as a potential target for neuropsychiatric disorders

    Directory of Open Access Journals (Sweden)

    Eero eVasar

    2013-03-01

    Full Text Available The studies performed in laboratory animals and psychiatric patients suggest a possible role of limbic system associated membrane protein (LAMP in the mechanisms of psychiatric disorders. Stressful manipulations and genetic invalidation have revealed a role of the Lsamp gene in the regulation of anxiety in rodents. Besides that, Lsamp deficient mice display reduced aggressiveness and impaired adaptation in novel and stressful environments. The behavioural effects of amphetamine were blunted in genetically modified mice. Recent pharmacological and biochemical studies point towards altered function of GABA-, 5-hydroxytryptamine- and dopaminergic systems in Lsamp deficient mice. Moreover, we found an association between the gene polymorphisms of LSAMP and major depressive disorder. Patients suffering from major depressive disorder had significantly increased ratio between risk and protective haplotypes of the LSAMP gene compared to healthy volunteers. However, the impact of these haplotypes for the function of LAMP is not clear and remains to be elucidated in future studies.

  15. Anti-inflammatory activity of horseradish (Armoracia rusticana) root extracts in LPS-stimulated macrophages.

    Science.gov (United States)

    Marzocco, Stefania; Calabrone, Luana; Adesso, Simona; Larocca, Marilena; Franceschelli, Silvia; Autore, Giuseppina; Martelli, Giuseppe; Rossano, Rocco

    2015-12-01

    Horseradish (Armoracia rusticana) is a perennial crop belonging to the Brassicaceae family. Horseradish root is used as a condiment due to its extremely pungent flavour, deriving from the high content of glucosinolates and their breakdown products such as isothiocyanates and other sulfur compounds. Horseradish also has a long history in ethnomedicine. In this study the anti-inflammatory potential of three accessions of Armoracia rusticana on lipopolysaccharide from E. coli treated J774A.1 murine macrophages was evaluated. Our results demonstrate that Armoracia rusticana reduced nitric oxide, tumor necrosis factor-α and interleukin-6 release and nitric oxide synthase and cyclooxygenase-2 expression in macrophages, acting on nuclear transcription factor NF-κB p65 activation. Moreover Armoracia rusticana reduced reactive oxygen species release and increased heme-oxygenase-1 expression, thus contributing to the cytoprotective cellular effect during inflammation.

  16. Effects of light and growth regulators on adventitious bud formation in horseradish (Armoracia rusticana).

    Science.gov (United States)

    Kamada, H; Tachikawa, Y; Saitou, T; Harada, H

    1995-07-01

    To clarify that the presence of Ri T-DNA genes are not prerequisite for the light-induced bud formation in horseradish (Armoracia rusticana) hairy roots, leaf and root segments of nontransformed horseradish plants were used as explants. Bud formation from nontransformed tissues was observed in hormone-free medium under 16 h daylight conditions, but not under continuous darkness. To investigate the effects of growth regulators on bud formation, leaf and root explants were treated with auxin (1-naphthaleneacetic acid; NAA) and / or cytokinin (6-benzyl-aminopurine; BA). The most effective treatment in the dark to stimulate bud formation was BA at 1 mg·1(-1). These results show that adventitious bud formation in horseradish can be induced by light and growth regulators, and especially cytokinin, may be involved in bud formation, irrespective of whether the tissues were transformed with Ri T-DNA.

  17. Horseradish peroxidase-nanoclay hybrid particles of high functional and colloidal stability.

    Science.gov (United States)

    Pavlovic, Marko; Rouster, Paul; Somosi, Zoltan; Szilagyi, Istvan

    2018-08-15

    Highly stable dispersions of enzyme-clay nanohybrids of excellent horseradish peroxidase activity were developed. Layered double hydroxide nanoclay was synthesized and functionalized with heparin polyelectrolyte to immobilize the horseradish peroxidase enzyme. The formation of a saturated heparin layer on the platelets led to charge inversion of the positively charged bare nanoclay and to highly stable aqueous dispersions. Great affinity of the enzyme to the surface modified platelets resulted in strong horseradish peroxidase adsorption through electrostatic and hydrophobic interactions as well as hydrogen bonding network and prevented enzyme leakage from the obtained material. The enzyme kept its functional integrity upon immobilization and showed excellent activity in decomposition of hydrogen peroxide and oxidation of an aromatic compound in the test reactions. In addition, remarkable long term functional stability of the enzyme-nanoclay hybrid was observed making the developed colloidal system a promising antioxidant candidate in biomedical treatments and industrial processes. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Human lactoferricin derived di-peptides deploying loop structures induce apoptosis specifically in cancer cells through targeting membranous phosphatidylserine.

    Science.gov (United States)

    Riedl, Sabrina; Leber, Regina; Rinner, Beate; Schaider, Helmut; Lohner, Karl; Zweytick, Dagmar

    2015-11-01

    Host defense-derived peptides have emerged as a novel strategy for the development of alternative anticancer therapies. In this study we report on characteristic features of human lactoferricin (hLFcin) derivatives which facilitate specific killing of cancer cells of melanoma, glioblastoma and rhabdomyosarcoma compared with non-specific derivatives and the synthetic peptide RW-AH. Changes in amino acid sequence of hLFcin providing 9-11 amino acids stretched derivatives LF11-316, -318 and -322 only yielded low antitumor activity. However, the addition of the repeat (di-peptide) and the retro-repeat (di-retro-peptide) sequences highly improved cancer cell toxicity up to 100% at 20 μM peptide concentration. Compared to the complete parent sequence hLFcin the derivatives showed toxicity on the melanoma cell line A375 increased by 10-fold and on the glioblastoma cell line U-87mg by 2-3-fold. Reduced killing velocity, apoptotic blebbing, activation of caspase 3/7 and formation of apoptotic DNA fragments proved that the active and cancer selective peptides, e.g. R-DIM-P-LF11-322, trigger apoptosis, whereas highly active, though non-selective peptides, such as DIM-LF11-318 and RW-AH seem to kill rapidly via necrosis inducing membrane lyses. Structural studies revealed specific toxicity on cancer cells by peptide derivatives with loop structures, whereas non-specific peptides comprised α-helical structures without loop. Model studies with the cancer membrane mimic phosphatidylserine (PS) gave strong evidence that PS only exposed by cancer cells is an important target for specific hLFcin derivatives. Other negatively charged membrane exposed molecules as sialic acid, heparan and chondroitin sulfate were shown to have minor impact on peptide activity. Copyright © 2015. Published by Elsevier B.V.

  19. Membrane docking geometry of GRP1 PH domain bound to a target lipid bilayer: an EPR site-directed spin-labeling and relaxation study.

    Directory of Open Access Journals (Sweden)

    Huai-Chun Chen

    Full Text Available The second messenger lipid PIP(3 (phosphatidylinositol-3,4,5-trisphosphate is generated by the lipid kinase PI3K (phosphoinositide-3-kinase in the inner leaflet of the plasma membrane, where it regulates a broad array of cell processes by recruiting multiple signaling proteins containing PIP(3-specific pleckstrin homology (PH domains to the membrane surface. Despite the broad importance of PIP(3-specific PH domains, the membrane docking geometry of a PH domain bound to its target PIP(3 lipid on a bilayer surface has not yet been experimentally determined. The present study employs EPR site-directed spin labeling and relaxation methods to elucidate the membrane docking geometry of GRP1 PH domain bound to bilayer-embedded PIP(3. The model target bilayer contains the neutral background lipid PC and both essential targeting lipids: (i PIP(3 target lipid that provides specificity and affinity, and (ii PS facilitator lipid that enhances the PIP(3 on-rate via an electrostatic search mechanism. The EPR approach measures membrane depth parameters for 18 function-retaining spin labels coupled to the PH domain, and for calibration spin labels coupled to phospholipids. The resulting depth parameters, together with the known high resolution structure of the co-complex between GRP1 PH domain and the PIP(3 headgroup, provide sufficient constraints to define an optimized, self-consistent membrane docking geometry. In this optimized geometry the PH domain engulfs the PIP(3 headgroup with minimal bilayer penetration, yielding the shallowest membrane position yet described for a lipid binding domain. This binding interaction displaces the PIP(3 headgroup from its lowest energy position and orientation in the bilayer, but the headgroup remains within its energetically accessible depth and angular ranges. Finally, the optimized docking geometry explains previous biophysical findings including mutations observed to disrupt membrane binding, and the rapid lateral

  20. Activity ranking of synthetic analogs targeting vascular endothelial growth factor receptor 2 by an integrated cell membrane chromatography system.

    Science.gov (United States)

    Wang, Dongyao; Lv, Diya; Chen, Xiaofei; Liu, Yue; Ding, Xuan; Jia, Dan; Chen, Langdong; Zhu, Zhenyu; Cao, Yan; Chai, Yifeng

    2015-12-01

    Evaluating the biological activities of small molecules represents an important part of the drug discovery process. Cell membrane chromatography (CMC) is a well-developed biological chromatographic technique. In this study, we have developed combined SMMC-7721/CMC and HepG2/CMC with high-performance liquid chromatography and time-of-flight mass spectrometry to establish an integrated screening platform. These systems was subsequently validated and used for evaluating the activity of quinazoline compounds, which were designed and synthesized to target vascular endothelial growth factor receptor 2. The inhibitory activities of these compounds towards this receptor were also tested using a classical caliper mobility shift assay. The results revealed a significant correlation between these two methods (R(2) = 0.9565 or 0.9420) for evaluating the activities of these compounds. Compared with traditional methods of evaluating the activities analogous compounds, this integrated cell membrane chromatography screening system took less time and was more cost effective, indicating that it could be used as a practical method in drug discovery. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  1. Targeting of a chimeric human histone fusion mRNA to membrane-bound polysomes in HeLa cells

    International Nuclear Information System (INIS)

    Zambetti, G.; Stein, J.; Stein, G.

    1987-01-01

    The subcellular location of histone mRNA-containing polysomes may play a key role in the posttranscriptional events that mediate histone mRNA turnover following inhibition of DNA synthesis. Previously, it has been shown that histone mRNA is found primarily on free polysomes that are associated with the cytoskeleton. The authors report here the construction of an Escherichia coli pBR322 β-lactamase signal peptide-human H3 histone fusion gene. The fusion transcript is targeted to membrane-bound polysomes and remains stable following interruption of DNA replication. Relocating mRNA within the cell may provide a procedure for studying the posttranscriptional regulation of gene expression

  2. Transcription factor Nrf1 is topologically repartitioned across membranes to enable target gene transactivation through its acidic glucose-responsive domains.

    Science.gov (United States)

    Zhang, Yiguo; Ren, Yonggang; Li, Shaojun; Hayes, John D

    2014-01-01

    The membrane-bound Nrf1 transcription factor regulates critical homeostatic and developmental genes. The conserved N-terminal homology box 1 (NHB1) sequence in Nrf1 targets the cap'n'collar (CNC) basic basic-region leucine zipper (bZIP) factor to the endoplasmic reticulum (ER), but it is unknown how its activity is controlled topologically within membranes. Herein, we report a hitherto unknown mechanism by which the transactivation activity of Nrf1 is controlled through its membrane-topology. Thus after Nrf1 is anchored within ER membranes, its acidic transactivation domains (TADs), including the Asn/Ser/Thr-rich (NST) glycodomain situated between acidic domain 1 (AD1) and AD2, are transiently translocated into the lumen of the ER, where NST is glycosylated in the presence of glucose to yield an inactive 120-kDa Nrf1 glycoprotein. Subsequently, portions of the TADs partially repartition across membranes into the cyto/nucleoplasmic compartments, whereupon an active 95-kDa form of Nrf1 accumulates, a process that is more obvious in glucose-deprived cells and may involve deglycosylation. The repartitioning of Nrf1 out of membranes is monitored within this protein by its acidic-hydrophobic amphipathic glucose-responsive domains, particularly the Neh5L subdomain within AD1. Therefore, the membrane-topological organization of Nrf1 dictates its post-translational modifications (i.e. glycosylation, the putative deglycosylation and selective proteolysis), which together control its ability to transactivate target genes.

  3. Targeting tumor antigens to secreted membrane vesicles in vivo induces efficient antitumor immune responses.

    Science.gov (United States)

    Zeelenberg, Ingrid S; Ostrowski, Matias; Krumeich, Sophie; Bobrie, Angélique; Jancic, Carolina; Boissonnas, Alexandre; Delcayre, Alain; Le Pecq, Jean-Bernard; Combadière, Béhazine; Amigorena, Sebastian; Théry, Clotilde

    2008-02-15

    Expression of non-self antigens by tumors can induce activation of T cells in vivo, although this activation can lead to either immunity or tolerance. CD8+ T-cell activation can be direct (if the tumor expresses MHC class I molecules) or indirect (after the capture and cross-presentation of tumor antigens by dendritic cells). The modes of tumor antigen capture by dendritic cells in vivo remain unclear. Here we examine the immunogenicity of the same model antigen secreted by live tumors either in association with membrane vesicles (exosomes) or as a soluble protein. We have artificially addressed the antigen to secreted vesicles by coupling it to the factor VIII-like C1C2 domain of milk fat globule epidermal growth factor-factor VIII (MFG-E8)/lactadherin. We show that murine fibrosarcoma tumor cells that secrete vesicle-bound antigen grow slower than tumors that secrete soluble antigen in immunocompetent, but not in immunodeficient, host mice. This growth difference is due to the induction of a more potent antigen-specific antitumor immune response in vivo by the vesicle-bound than by the soluble antigen. Finally, in vivo secretion of the vesicle-bound antigen either by tumors or by vaccination with naked DNA protects against soluble antigen-secreting tumors. We conclude that the mode of secretion can determine the immunogenicity of tumor antigens and that manipulation of the mode of antigen secretion may be used to optimize antitumor vaccination protocols.

  4. Automated recognition of cell phenotypes in histology images based on membrane- and nuclei-targeting biomarkers

    International Nuclear Information System (INIS)

    Karaçalı, Bilge; Vamvakidou, Alexandra P; Tözeren, Aydın

    2007-01-01

    Three-dimensional in vitro culture of cancer cells are used to predict the effects of prospective anti-cancer drugs in vivo. In this study, we present an automated image analysis protocol for detailed morphological protein marker profiling of tumoroid cross section images. Histologic cross sections of breast tumoroids developed in co-culture suspensions of breast cancer cell lines, stained for E-cadherin and progesterone receptor, were digitized and pixels in these images were classified into five categories using k-means clustering. Automated segmentation was used to identify image regions composed of cells expressing a given biomarker. Synthesized images were created to check the accuracy of the image processing system. Accuracy of automated segmentation was over 95% in identifying regions of interest in synthesized images. Image analysis of adjacent histology slides stained, respectively, for Ecad and PR, accurately predicted regions of different cell phenotypes. Image analysis of tumoroid cross sections from different tumoroids obtained under the same co-culture conditions indicated the variation of cellular composition from one tumoroid to another. Variations in the compositions of cross sections obtained from the same tumoroid were established by parallel analysis of Ecad and PR-stained cross section images. Proposed image analysis methods offer standardized high throughput profiling of molecular anatomy of tumoroids based on both membrane and nuclei markers that is suitable to rapid large scale investigations of anti-cancer compounds for drug development

  5. Radioimmunodetection of membrane type-1 matrix metalloproteinase relevant to tumor malignancy with a pre-targeting method

    International Nuclear Information System (INIS)

    Sano, Kohei; Temma, Takashi; Kuge, Yuji; Kudo, Takashi; Kamihashi, Junko; Saji, Hideo; Zhao, Songji

    2010-01-01

    Since membrane type-1 matrix metalloproteinase (MT1-MMP) is exclusively expressed in tumors and is closely associated with metastasis and invasion, MT1-MMP is a potential target of radiotracers for the evaluation of tumor malignancy. In this study, we planned to visualize MT1-MMP in vivo by a two-step pre-targeting strategy using a streptavidin (SAv)-biotin system combined with anti-MT1-MMP monoclonal immunoglobulin (IgG) (anti-MT1-MMP monoclonal antibody (mAb)). Streptavidinylated anti-MT1-MMP mAb was synthesized by reacting biotinylated anti-MT1-MMP mAb with SAv. In the pre-targeting study, FM3A mouse breast carcinoma-implanted mice were injected with anti-MT1-MMP mAb-SAv, followed 72 h later with radioiodinated biotin, (3-[ 123/125 I]iodobenzoyl)norbiotinamide( 123/125 I-IBB). Biodistribution and imaging (single photon emission computed tomography (SPECT)/CT) data were collected at several time points in the 24 h period following introduction of the tracer. The comparison groups were injected with 125 I-IBB alone or with 125 I-IBB pre-targeted with negative control IgG-SAv. In the pre-targeting study for MT1-MMP, within 1 h of tracer injection, rapid tumor uptake and abrupt clearance from the blood of radioactivity (2.22, 0.87% injected dose/g at 1 h) were observed. The tumor to blood (T/B) radioactivity ratios were significantly higher than those from mice dosed with the pre-targeting negative control (p 125 I-IBB alone did not accumulate in tumors. SPECT/CT image analysis of FM3A bearing mice showed high-contrast tumor images after 3 h with minimal blood-pool activity. The present study that uses a pre-targeting method showed high T/B radioactivity ratios and clear tumor images of MT1-MMP. This imaging method may be useful for the clinical diagnosis of malignant tumors. (author)

  6. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

    Directory of Open Access Journals (Sweden)

    Rust Steven

    2013-02-01

    Full Text Available Abstract Background The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. Methods The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. Results All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated

  7. KSHV Entry and Trafficking in Target Cells—Hijacking of Cell Signal Pathways, Actin and Membrane Dynamics

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    Binod Kumar

    2016-11-01

    Full Text Available Kaposi’s sarcoma associated herpesvirus (KSHV is etiologically associated with human endothelial cell hyperplastic Kaposi’s sarcoma and B-cell primary effusion lymphoma. KSHV infection of adherent endothelial and fibroblast cells are used as in vitro models for infection and KSHV enters these cells by host membrane bleb and actin mediated macropinocytosis or clathrin endocytosis pathways, respectively. Infection in endothelial and fibroblast cells is initiated by the interactions between multiple viral envelope glycoproteins and cell surface associated heparan sulfate (HS, integrins (α3β1, αVβ3 and αVβ5, and EphA2 receptor tyrosine kinase (EphA2R. This review summarizes the accumulated studies demonstrating that KSHV manipulates the host signal pathways to enter and traffic in the cytoplasm of the target cells, to deliver the viral genome into the nucleus, and initiate viral gene expression. KSHV interactions with the cell surface receptors is the key platform for the manipulations of host signal pathways which results in the simultaneous induction of FAK, Src, PI3-K, Rho-GTPase, ROS, Dia-2, PKC ζ, c-Cbl, CIB1, Crk, p130Cas and GEF-C3G signal and adaptor molecules that play critical roles in the modulation of membrane and actin dynamics, and in the various steps of the early stages of infection such as entry and trafficking towards the nucleus. The Endosomal Sorting Complexes Required for Transport (ESCRT proteins are also recruited to assist in viral entry and trafficking. In addition, KSHV interactions with the cell surface receptors also induces the host transcription factors NF-κB, ERK1/2, and Nrf2 early during infection to initiate and modulate viral and host gene expression. Nuclear delivery of the viral dsDNA genome is immediately followed by the host innate responses such as the DNA damage response (DDR, inflammasome and interferon responses. Overall, these studies form the initial framework for further studies of

  8. Sensory-Analytical Comparison of the Aroma of Different Horseradish Varieties (Armoracia rusticana

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    Eva-Maria Kroener

    2018-05-01

    Full Text Available Horseradish (Armoracia rusticana is consumed and valued for the characteristic spicy aroma of its roots in many countries all over the world. In our present study we compare six different horseradish varieties that were grown under comparable conditions, with regard to their aroma profiles, using combined sensory-analytical methods. Horseradish extracts were analyzed through gas chromatography-olfactometry (GC-O and their aroma-active compounds ranked according to their smell potency using the concept of aroma extract dilution analysis (AEDA. Identification was carried out through comparison of retention indices, odor qualities and mass spectra with those of reference substances. Besides some differences in relative ratios, we observed some main odorants that were common to all varieties such as 3-isopropyl-2-methoxypyrazine and allyl isothiocyanate, but also characteristics for specific varieties such as higher contents for 3-isopropyl-2-methoxypyrazine in variety Nyehemes. Moreover, three odorous compounds were detected that have not been described in horseradish roots before.

  9. Preferential hydroxylation over epoxidation catalysis by a horseradish peroxidase mutant: a cytochrome P450 mimic.

    Science.gov (United States)

    de Visser, Sam P

    2007-10-25

    Density functional theory calculations are presented on the catalytic properties of a horseradish peroxidase mutant whereby the axial nitrogen atom is replaced by phosphorus. This mutant has never been studied experimentally and only one theoretical report on this system is known (de Visser, S. P. J. Phys. Chem. B 2006, 110, 20759-20761). Thus, a one-atom substitution in horseradish peroxidase changes the properties of the catalytic center of the enzyme to more cytochrome P450-type qualities. In particular, the phosphorus-substituted horseradish peroxidase mutant reacts with substrates via a unique reactivity pattern, whereby alkanes are regioselectively hydroxylated even in the presence of a double bond. Reaction barriers of propene epoxidation and hydroxylation are almost identical to ones observed for a cytochrome P450 catalyst and significantly higher than those obtained for a horseradish peroxidase catalyst. It is shown that the regioselectivity difference is entropy and thermally driven and that the electron-transfer processes that occur during the reaction mechanism follow cytochrome P450-type patterns in the hydroxylation reaction.

  10. Chemical Composition and Rheological Properties of Set Yoghurt Prepared from Skimmed Milk Treated with Horseradish Peroxidase

    Directory of Open Access Journals (Sweden)

    Yan Wen

    2012-01-01

    Full Text Available The aim of this work is to determine the impact of an enzymatic treatment on the fermentation and rheological properties of set yoghurt prepared from skimmed milk. Skimmed bovine milk was treated with horseradish peroxidase added at the level of 645 U per g of proteins in the presence (addition level of 7.8 mmol per L of milk or absence of ferulic acid as a cross-linking agent, and used to prepare set yoghurt with commercial direct vat set starter culture. The evaluation showed that the treatment of skimmed milk with horseradish peroxidase enhanced its apparent viscosity, and storage and loss moduli. The prepared yoghurt contained protein, fat and total solids at 3.49–3.59, 0.46–0.52 and 15.23–15.43 %, respectively, had titratable acidity of 0.83–0.88 %, and no significant difference in the composition was found among the yoghurt samples (p>0.05. Compared to the control yoghurt, the yoghurt prepared from the milk treated with horseradish peroxidase had a higher apparent viscosity, storage and loss moduli and flow behavior indices, especially when ferulic acid was added. Yoghurt samples from the skimmed milk treated either with horseradish peroxidase only or with the additional ferulic acid treatment had better structural reversibility, because their hysteresis loop area during rheological analysis was larger (p<0.05.

  11. Sodium modulates opioid receptors through a membrane component different from G-proteins. Demonstration by target size analysis

    International Nuclear Information System (INIS)

    Ott, S.; Costa, T.; Herz, A.

    1988-01-01

    The target size for opioid receptor binding was studied after manipulations known to affect the interactions between receptor and GTP-binding regulatory proteins (G-proteins). Addition of GTP or its analogs to the binding reaction, exposure of intact cells to pertussis toxin prior to irradiation, or treatment of irradiated membranes with N-ethylmaleimide did not change the target size (approximately equal to 100 kDa) for opioid receptors in NG 108-15 cells and rat brain. These data suggest that the 100-kDa species does not include an active subunit of a G-protein or alternatively that GTP does not promote the dissociation of the receptor-G-protein complex. The presence of Na+ (100 mM) in the radioligand binding assay induced a biphasic decay curve for agonist binding and a flattening of the monoexponential decay curve for a partial agonist. In both cases the effect was explained by an irradiation-induced loss of the low affinity state of the opioid receptor produced by the addition of Na+. This suggests that an allosteric inhibitor that mediates the effect of sodium on the receptor is destroyed at low doses of irradiation, leaving receptors which are no longer regulated by sodium. The effect of Na+ on target size was slightly increased by the simultaneous addition of GTP but was not altered by pertussis toxin treatment. Thus, the sodium unit is distinct from G-proteins and may represent a new component of the opioid receptor complex. Assuming a simple bimolecular model of one Na+ unit/receptor, the size of this inhibitor can be measured as 168 kDa

  12. Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

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    Ruben R Bender

    2016-06-01

    Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

  13. Membrane-Dependent Bystander Effect Contributes to Amplification of the Response to Alpha-Particle Irradiation in Targeted and Nontargeted Cells

    International Nuclear Information System (INIS)

    Hanot, Maite; Hoarau, Jim; Carriere, Marie; Angulo, Jaime F.; Khodja, Hicham

    2009-01-01

    Purpose: Free radicals are believed to play an active role in the bystander response. This study investigated their origin as well as their temporal and spatial impacts in the bystander effect. Methods and Materials: We employed a precise alpha-particle microbeam to target a small fraction of subconfluent osteoblastic cells (MC3T3-E1). γH2AX-53BP1 foci, oxidative metabolism changes, and micronuclei induction in targeted and bystander cells were assessed. Results: Cellular membranes and mitochondria were identified as two distinct reactive oxygen species producers. The global oxidative stress observed after irradiation was significantly attenuated after cells were treated with filipin, evidence for the primal role of membrane in the bystander effect. To determine the membrane's impact at a cellular level, micronuclei yield was measured when various fractions of the cell population were individually targeted while the dose per cell remained constant. Induction of micronuclei increased in bystander cells as well as in targeted cells and was attenuated by filipin treatment, demonstrating a role for bystander signals between irradiated cells in an autocrine/paracrine manner. Conclusions: A complex interaction of direct irradiation and bystander signals leads to a membrane-dependent amplification of cell responses that could influence therapeutic outcomes in tissues exposed to low doses or to environmental exposure.

  14. The Vasopressin Type-2 Receptor and Prostaglandin Receptors EP2 and EP4 can Increase Aquaporin-2 Plasma Membrane Targeting Through a cAMP Independent Pathway

    DEFF Research Database (Denmark)

    Olesen, Emma Tina Bisgaard; Moeller, Hanne Bjerregaard; Assentoft, Mette

    2016-01-01

    Apical membrane targeting of the collecting duct water channel aquaporin-2 (AQP2) is essential for body water balance. As this event is regulated by Gs coupled 7-transmembrane receptors such as the vasopressin type 2 receptor (V2R) and the prostanoid receptors EP2 and EP4, it is believed to be c...

  15. Combined cross-linked enzyme aggregates of horseradish peroxidase and glucose oxidase for catalyzing cascade chemical reactions.

    Science.gov (United States)

    Nguyen, Le Truc; Yang, Kun-Lin

    2017-05-01

    Cascade reactions involved unstable intermediates are often encountered in biological systems. In this study, we developed combined cross-linked enzyme aggregates (combi-CLEA) to catalyze a cascade reaction which involves unstable hydrogen peroxide as an intermediate. The combi-CLEA contains two enzymes̶ glucose oxidase (GOx) and horseradish peroxidase (HRP) which are cross-linked together as solid aggregates. The first enzyme GOx catalyzes the oxidation of glucose and produces hydrogen peroxide, which is used by the second enzyme HRP to oxidize 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS). The apparent reaction rate of the cascade reaction reaches 10.5±0.5μM/min when the enzyme ratio is 150:1 (GOx:HRP). Interestingly, even in the presence of catalase, an enzyme that quickly decomposes hydrogen peroxide, the reaction rate only decreases by 18.7% to 8.3±0.3μM/min. This result suggests that the intermediate hydrogen peroxide is not decomposed by catalase due to a short diffusion distance between GOx and HRP in the combi-CLEA. Scanning electron microscopy images suggest that combi-CLEA particles are hollow spheres and have an average diameter around 250nm. Because of their size, combi-CLEA particles can be entrapped inside a nylon membrane for detecting glucose by using the cascade reaction. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Permeability of skin and oral mucosa to water and horseradish peroxidase as related to the thickness of the permeability barrier

    International Nuclear Information System (INIS)

    Squier, C.A.; Hall, B.K.

    1985-01-01

    The permeability of porcine skin and keratinized and nonkeratinized oral mucosa to tritium-labeled water and horseradish peroxidase (HRPO) was determined using perfusion chambers. Small blocks from each tissue were also incubated with HRPO and the extent of penetration visualized microscopically; this enabled measurements to be made of the thickness of the permeability barrier to this water-soluble tracer. Results obtained after inverting the oral mucosa in the chambers or adding metabolic inhibitors indicated that both compounds diffuse across the tissue. The permeability constants derived directly in the study showed that skin was less permeable than oral mucosa and that the floor of the mouth was significantly more permeable than all other regions. When these constants were normalized in terms of a standard permeability barrier thickness and the different tissues compared, the values obtained for skin were again less than those of the oral regions but, of these, the buccal mucosa was significantly higher. The difference in permeability between epidermis and keratinized oral epithelium may be due to differences in the volume density of membrane-coating granules known to exist between the tissues; differences between the oral mucosal regions may reflect differences in the nature of the intercellular barrier material

  17. Prostate-specific membrane antigen targeted protein contrast agents for molecular imaging of prostate cancer by MRI

    Science.gov (United States)

    Pu, Fan; Salarian, Mani; Xue, Shenghui; Qiao, Jingjuan; Feng, Jie; Tan, Shanshan; Patel, Anvi; Li, Xin; Mamouni, Kenza; Hekmatyar, Khan; Zou, Juan; Wu, Daqing; Yang, Jenny J.

    2016-06-01

    Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high resolution has yet to be achieved due to the lack of contrast agents with significantly improved relaxivity for sensitivity, targeting capabilities and metal selectivity. We have previously reported our creation of a novel class of protein Gd3+ contrast agents, ProCA32, which displayed significantly improved relaxivity while exhibiting strong Gd3+ binding selectivity over physiological metal ions. In this study, we report our effort in further developing biomarker-targeted protein MRI contrast agents for molecular imaging of PSMA. Among three PSMA targeted contrast agents engineered with addition of different molecular recognition sequences, ProCA32.PSMA exhibits a binding affinity of 1.1 +/- 0.1 μM for PSMA while the metal binding affinity is maintained at 0.9 +/- 0.1 × 10-22 M. In addition, ProCA32.PSMA exhibits r1 of 27.6 mM-1 s-1 and r2 of 37.9 mM-1 s-1 per Gd (55.2 and 75.8 mM-1 s-1 per molecule r1 and r2, respectively) at 1.4 T. At 7 T, ProCA32.PSMA also has r2 of 94.0 mM-1 s-1 per Gd (188.0 mM-1 s-1 per molecule) and r1 of 18.6 mM-1 s-1 per Gd (37.2 mM-1 s-1 per molecule). This contrast capability enables the first MRI enhancement dependent on PSMA expression levels in tumor bearing mice using both T1 and T2-weighted MRI at 7 T. Further development of these PSMA-targeted contrast agents are expected to be used for the precision imaging of prostate cancer at an early stage and to monitor disease progression and staging, as well as determine the effect of therapeutic treatment by non-invasive evaluation of the PSMA level using MRI.Prostate-specific membrane antigen (PSMA) is one of the most specific cell surface markers for prostate cancer diagnosis and targeted treatment. However, achieving molecular imaging using non-invasive MRI with high

  18. The role of aspartic acid residues 405 and 416 of the kidney isotype of sodium-bicarbonate cotransporter 1 in its targeting to the plasma membrane

    Science.gov (United States)

    Kucher, Volodymyr; Li, Emily Y.; Conforti, Laura; Zahedi, Kamyar A.

    2012-01-01

    The NH2 terminus of the sodium-bicarbonate cotransporter 1 (NBCe1) plays an important role in its targeting to the plasma membrane. To identify the amino acid residues that contribute to the targeting of NBCe1 to the plasma membrane, polarized MDCK cells were transfected with expression constructs coding for green fluorescent protein (GFP)-tagged NBCe1 NH2-terminal deletion mutants, and the localization of GFP-tagged proteins was analyzed by confocal microscopy. Our results indicate that the amino acids between residues 399 and 424 of NBCe1A contain important sequences that contribute to its localization to the plasma membrane. Site-directed mutagenesis studies showed that GFP-NBCe1A mutants D405A and D416A are retained in the cytoplasm of the polarized MDCK epithelial cells. Examination of functional activities of D405A and D416A reveals that their activities are reduced compared with the wild-type NBCe1A. Similarly, aspartic acid residues 449 and 460 of pancreatic NBCe1 (NBCe1B), which correspond to residues 405 and 416 of NBCe1A, are also required for its full functional activity and accurate targeting to the plasma membrane. In addition, while replacement of D416 with glutamic acid did not affect the targeting or functional activity of NBCe1A, substitution of D405 with glutamic acid led to the retention of the mutated protein in the intracellular compartment and impaired functional activity. These studies demonstrate that aspartic acid residues 405 and 416 in the NH2 terminus of NBCe1A are important in its accurate targeting to the plasma membrane. PMID:22442137

  19. Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

    Energy Technology Data Exchange (ETDEWEB)

    Jensen, J.; Norby, J.G.

    1988-12-05

    Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper.

  20. Membrane-bound Na,K-ATPase: target size and radiation inactivation size of some of its enzymatic reactions

    International Nuclear Information System (INIS)

    Jensen, J.; Norby, J.G.

    1988-01-01

    Frozen samples of membrane-bound pig kidney Na,K-ATPase were subjected to target size analysis by radiation inactivation with 10-MeV electrons at -15 degrees C. The various properties investigated decreased monoexponentially with radiation dose, and the decay constants, gamma, were independent of the presence of other proteins and of sucrose concentrations above 0.25 M. The temperature factor was the same as described by others. Irradiation of four proteins of known molecular mass, m, showed that gamma for protein integrity was proportional to m with a proportionality factor about 20% higher than that conventionally used. By this standard curve, glucose-6-phosphate dehydrogenase activity used as internal standard gave a radiation inactivation size of 110 +/- 5 kDa, very close to m = 104-108 kDa for the dimer, as expected. For Na+/K+-transporting ATPase the following target sizes and radiation inactivation size values were very close to m = 112 kDa for the alpha-peptide: peptide integrity of alpha, 115 kDa; unmodified binding sites for ATP and vanadate, 108 kDa; K+-activated p-nitrophenylphosphatase activity, 106 kDa. There was thus no sign of dimerization of the alpha-peptide or involvement of the beta-peptide. In contrast, optimal Na+/K+-transporting ATPase activity had a radiation inactivation size = 189 +/- 7 kDa, and total nucleotide binding capacity corresponded to 72 +/- 3 kDa. These latter results will be extended and discussed in a forthcoming paper

  1. Peptide-Based Membrane Fusion Inhibitors Targeting HCoV-229E Spike Protein HR1 and HR2 Domains

    Directory of Open Access Journals (Sweden)

    Shuai Xia

    2018-02-01

    Full Text Available Human coronavirus 229E (HCoV-229E infection in infants, elderly people, and immunocompromised patients can cause severe disease, thus calling for the development of effective and safe therapeutics to treat it. Here we reported the design, synthesis and characterization of two peptide-based membrane fusion inhibitors targeting HCoV-229E spike protein heptad repeat 1 (HR1 and heptad repeat 2 (HR2 domains, 229E-HR1P and 229E-HR2P, respectively. We found that 229E-HR1P and 229E-HR2P could interact to form a stable six-helix bundle and inhibit HCoV-229E spike protein-mediated cell-cell fusion with IC50 of 5.7 and 0.3 µM, respectively. 229E-HR2P effectively inhibited pseudotyped and live HCoV-229E infection with IC50 of 0.5 and 1.7 µM, respectively. In a mouse model, 229E-HR2P administered intranasally could widely distribute in the upper and lower respiratory tracts and maintain its fusion-inhibitory activity. Therefore, 229E-HR2P is a promising candidate for further development as an antiviral agent for the treatment and prevention of HCoV-229E infection.

  2. Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells

    Science.gov (United States)

    Hafner, Anne E.; Rieger, Heiko

    2016-12-01

    Intracellular transport is vital for the proper functioning and survival of a cell. Cargo (proteins, vesicles, organelles, etc) is transferred from its place of creation to its target locations via molecular motor assisted transport along cytoskeletal filaments. The transport efficiency is strongly affected by the spatial organization of the cytoskeleton, which constitutes an inhomogeneous, complex network. In cells with a centrosome microtubules grow radially from the central microtubule organizing center towards the cell periphery whereas actin filaments form a dense meshwork, the actin cortex, underneath the cell membrane with a broad range of orientations. The emerging ballistic motion along filaments is frequently interrupted due to constricting intersection nodes or cycles of detachment and reattachment processes in the crowded cytoplasm. In order to investigate the efficiency of search strategies established by the cell’s specific spatial organization of the cytoskeleton we formulate a random velocity model with intermittent arrest states. With extensive computer simulations we analyze the dependence of the mean first passage times for narrow escape problems on the structural characteristics of the cytoskeleton, the motor properties and the fraction of time spent in each state. We find that an inhomogeneous architecture with a small width of the actin cortex constitutes an efficient intracellular search strategy.

  3. Generation and evaluation of mammalian secreted and membrane protein expression libraries for high-throughput target discovery.

    Science.gov (United States)

    Panavas, Tadas; Lu, Jin; Liu, Xuesong; Winkis, Ann-Marie; Powers, Gordon; Naso, Michael F; Amegadzie, Bernard

    2011-09-01

    Expressed protein libraries are becoming a critical tool for new target discovery in the pharmaceutical industry. In order to get the most meaningful and comprehensive results from protein library screens, it is essential to have library proteins in their native conformation with proper post-translation modifications. This goal is achieved by expressing untagged human proteins in a human cell background. We optimized the transfection and cell culture conditions to maximize protein expression in a 96-well format so that the expression levels were comparable with the levels observed in shake flasks. For detection purposes, we engineered a 'tag after stop codon' system. Depending on the expression conditions, it was possible to express either native or tagged proteins from the same expression vector set. We created a human secretion protein library of 1432 candidates and a small plasma membrane protein set of about 500 candidates. Utilizing the optimized expression conditions, we expressed and analyzed both libraries by SDS-PAGE gel electrophoresis and Western blotting. Two thirds of secreted proteins could be detected by Western-blot analyses; almost half of them were visible on Coomassie stained gels. In this paper, we describe protein expression libraries that can be easily produced in mammalian expression systems in a 96-well format, with one protein expressed per well. The libraries and methods described allow for the development of robust, high-throughput functional screens designed to assay for protein specific functions associated with a relevant disease-specific activity. Copyright © 2011 Elsevier Inc. All rights reserved.

  4. Outer Membrane Protein A Conservation among Orientia tsutsugamushi Isolates Suggests Its Potential as a Protective Antigen and Diagnostic Target

    Directory of Open Access Journals (Sweden)

    Sean M. Evans

    2018-06-01

    Full Text Available Scrub typhus threatens one billion people in the Asia-Pacific area and cases have emerged outside this region. It is caused by infection with any of the multitude of strains of the bacterium Orientia tsutsugamushi. A vaccine that affords heterologous protection and a commercially-available molecular diagnostic assay are lacking. Herein, we determined that the nucleotide and translated amino acid sequences of outer membrane protein A (OmpA are highly conserved among 51 O. tsutsugamushi isolates. Molecular modeling revealed the predicted tertiary structure of O. tsutsugamushi OmpA to be very similar to that of the phylogenetically-related pathogen, Anaplasma phagocytophilum, including the location of a helix that contains residues functionally essential for A. phagocytophilum infection. PCR primers were developed that amplified ompA DNA from all O. tsutsugamushi strains, but not from negative control bacteria. Using these primers in quantitative PCR enabled sensitive detection and quantitation of O. tsutsugamushi ompA DNA from organs and blood of mice that had been experimentally infected with the Karp or Gilliam strains. The high degree of OmpA conservation among O. tsutsugamushi strains evidences its potential to serve as a molecular diagnostic target and justifies its consideration as a candidate for developing a broadly-protective scrub typhus vaccine.

  5. Spatial organization of the cytoskeleton enhances cargo delivery to specific target areas on the plasma membrane of spherical cells.

    Science.gov (United States)

    Hafner, Anne E; Rieger, Heiko

    2016-11-15

    Intracellular transport is vital for the proper functioning and survival of a cell. Cargo (proteins, vesicles, organelles, etc) is transferred from its place of creation to its target locations via molecular motor assisted transport along cytoskeletal filaments. The transport efficiency is strongly affected by the spatial organization of the cytoskeleton, which constitutes an inhomogeneous, complex network. In cells with a centrosome microtubules grow radially from the central microtubule organizing center towards the cell periphery whereas actin filaments form a dense meshwork, the actin cortex, underneath the cell membrane with a broad range of orientations. The emerging ballistic motion along filaments is frequently interrupted due to constricting intersection nodes or cycles of detachment and reattachment processes in the crowded cytoplasm. In order to investigate the efficiency of search strategies established by the cell's specific spatial organization of the cytoskeleton we formulate a random velocity model with intermittent arrest states. With extensive computer simulations we analyze the dependence of the mean first passage times for narrow escape problems on the structural characteristics of the cytoskeleton, the motor properties and the fraction of time spent in each state. We find that an inhomogeneous architecture with a small width of the actin cortex constitutes an efficient intracellular search strategy.

  6. Conformational stabilization of the membrane embedded targeting domain of the lysosomal peptide transporter TAPL for solution NMR

    Energy Technology Data Exchange (ETDEWEB)

    Tumulka, Franz [Goethe-University Frankfurt, Institute of Biochemistry, Biocenter (Germany); Roos, Christian; Loehr, Frank [Goethe-University Frankfurt, Institute of Biophysical Chemistry, Biocenter (Germany); Bock, Christoph [Goethe-University Frankfurt, Institute of Biochemistry, Biocenter (Germany); Bernhard, Frank; Doetsch, Volker [Goethe-University Frankfurt, Institute of Biophysical Chemistry, Biocenter (Germany); Abele, Rupert, E-mail: abele@em.uni-frankfurt.de [Goethe-University Frankfurt, Institute of Biochemistry, Biocenter (Germany)

    2013-10-15

    The ATP binding cassette transporter TAPL translocates cytosolic peptides into the lumen of lysosomes driven by the hydrolysis of ATP. Functionally, this transporter can be divided into coreTAPL, comprising the transport function, and an additional N-terminal transmembrane domain called TMD0, which is essential for lysosomal targeting and mediates the interaction with the lysosomal associated membrane proteins LAMP-1 and LAMP-2. To elucidate the structure of this unique domain, we developed protocols for the production of high quantities of cell-free expressed TMD0 by screening different N-terminal expression tags. Independently of the amino acid sequence, high expression was detected for AU-rich sequences in the first seven codons, decreasing the free energy of RNA secondary structure formation at translation initiation. Furthermore, avoiding NGG codons in the region of translation initiation demonstrated a positive effect on expression. For NMR studies, conditions were optimized for high solubilization efficiency, long-term stability, and high quality spectra. A most critical step was the careful exchange of the detergent used for solubilization by the detergent dihexanoylphosphatidylcholine. Several constructs of different size were tested in order to stabilize the fold of TMD0 as well as to reduce the conformation exchange. NMR spectra with sufficient resolution and homogeneity were finally obtained with a TMD0 derivative only modified by a C-terminal His{sub 10}-tag and containing a codon optimized AT-rich sequence.

  7. Evaluation of an Aqueous Extract from Horseradish Root (Armoracia rusticana Radix) against Lipopolysaccharide-Induced Cellular Inflammation Reaction

    OpenAIRE

    Herz, Corinna; Tran, Hoai Thi Thu; M?rton, Melinda-Rita; Maul, Ronald; Baldermann, Susanne; Schreiner, Monika; Lamy, Evelyn

    2017-01-01

    Horseradish (Armoracia rusticana) is a perennial crop and its root is used in condiments. Traditionally, horseradish root is used to treat bacterial infections of the respiratory tract and urinary bladder. The antiphlogistic activity, determined in activated primary human peripheral blood mononuclear cells (PBMC), was evaluated for an aqueous extract and its subfractions, separated by HPLC. Compound analysis was done by UHPLC-QToF/MS and GC-MS. The aqueous extract concentration-dependently in...

  8. Horseradish peroxidase embedded in polyacrylamide nanoparticles enables optical detection of reactive oxygen species

    DEFF Research Database (Denmark)

    Poulsen, A.K.; Scharff-Poulsen, Anne Marie; Olsen, L.F.

    2007-01-01

    We have synthesized and characterized new nanometer-sized polyacrylamide particles containing horseradish peroxidase and fluorescent dyes. Proteins and dyes are encapsulated by radical polymerization in inverse microemulsion. The activity of the encapsulated enzyme has been examined and it mainta......We have synthesized and characterized new nanometer-sized polyacrylamide particles containing horseradish peroxidase and fluorescent dyes. Proteins and dyes are encapsulated by radical polymerization in inverse microemulsion. The activity of the encapsulated enzyme has been examined...... for quantification of hydrogen peroxide and other reactive oxygen species in microenvironments, and we propose that the particles may find use as nanosensors for use in, e.g., living cells. (C) 2007 Elsevier Inc. All rights reserved....

  9. Antibacterial activity of alkyl gallates is a combination of direct targeting of FtsZ and permeabilization of bacterial membranes

    NARCIS (Netherlands)

    Krol, Ewa; de Sousa Borges, Anabela; da Silva, Isabel; Polaquini, Carlos; Regasini, Luis; Ferreira, Henrique; Scheffers, Dirk

    2015-01-01

    Alkyl gallates are compounds with reported antibacterial activity. One of the modes of action is binding of the alkyl gallates to the bacterial membrane and interference with membrane integrity. However, alkyl gallates also cause cell elongation and disruption of cell division in the important plant

  10. The C-terminal hypervariable domain targets Aradopsis ROP9 to the invaginated pollen tube plasma membrane

    Science.gov (United States)

    Rop9 is a small GTPase of the Type II class, whereas the often studied type I Rops play roles during pollen tube growth. In pollen, Rop9 is located at the invaginated plasma membrane that surrounds the sperm cells, whereas type I Rops are located at the apical membrane of the pollen tube. The C-ter...

  11. A mitochondrially targeted compound delays aging in yeast through a mechanism linking mitochondrial membrane lipid metabolism to mitochondrial redox biology

    Directory of Open Access Journals (Sweden)

    Michelle T. Burstein

    2014-01-01

    Full Text Available A recent study revealed a mechanism of delaying aging in yeast by a natural compound which specifically impacts mitochondrial redox processes. In this mechanism, exogenously added lithocholic bile acid enters yeast cells, accumulates mainly in the inner mitochondrial membrane, and elicits an age-related remodeling of phospholipid synthesis and movement within both mitochondrial membranes. Such remodeling of mitochondrial phospholipid dynamics progresses with the chronological age of a yeast cell and ultimately causes significant changes in mitochondrial membrane lipidome. These changes in the composition of membrane phospholipids alter mitochondrial abundance and morphology, thereby triggering changes in the age-related chronology of such longevity-defining redox processes as mitochondrial respiration, the maintenance of mitochondrial membrane potential, the preservation of cellular homeostasis of mitochondrially produced reactive oxygen species, and the coupling of electron transport to ATP synthesis.

  12. Stability and Catalytic Kinetics of Horseradish Peroxidase Confined in Nanoporous SBA-15

    DEFF Research Database (Denmark)

    Ikemoto, Hediki; Chi, Qijin; Ulstrup, Jens

    2010-01-01

    We have synthesized nanoporous silica, SBA-15 in the 1 m size range with the pore diameter of 7.6 nm. The redox enzyme horseradish peroxidase (HRP) was entrapped in the pores to form nanostructured hybrid materials. The catalytic activity of free and immobilized enzyme was first compared at room...... likely due to different hydrogen bonding of water and increased hydration strength of the protein inside the nanopores....

  13. Tumor cell proliferation and cyclooxygenase inhibitory constituents in horseradish (Armoracia rusticana) and Wasabi (Wasabia japonica).

    Science.gov (United States)

    Weil, Marvin J; Zhang, Yanjun; Nair, Muraleedharan G

    2005-03-09

    Cyclooxygenase and human tumor cell growth inhibitory extracts of horseradish (Armoracia rusticana) and wasabi (Wasabia japonica) rhizomes upon purification yielded active compounds 1-3 from horseradish and 4 and 5 from wasabi rhizomes. Spectroscopic analyses confirmed the identities of these active compounds as plastoquinone-9 (1), 6-O-acyl-beta-d-glucosyl-beta-sitosterol (2), 1,2-dilinolenoyl-3-galactosylglycerol (3), linolenoyloleoyl-3-beta-galactosylglycerol (4), and 1,2-dipalmitoyl-3-beta-galactosylglycerol (5). 3-Acyl-sitosterols, sinigrin, gluconasturtiin, and phosphatidylcholines isolated from horseradish and alpha-tocopherol and ubiquinone-10 from wasabi rhizomes isolated were inactive in our assays. At a concentration of 60 microg/mL, compounds 1 and 2 selectively inhibited COX-1 enzyme by 28 and 32%, respectively. Compounds 3, 4, and 5 gave 75, 42, and 47% inhibition of COX-1 enzyme, respectively, at a concentration of 250 microg/mL. In a dose response study, compound 3 inhibited the proliferation of colon cancer cells (HCT-116) by 21.9, 42.9, 51.2, and 68.4% and lung cancer cells (NCI-H460) by 30, 39, 44, and 71% at concentrations of 7.5, 15, 30, and 60 microg/mL, respectively. At a concentration of 60 microg/mL, compound 4 inhibited the growth of colon, lung, and stomach cancer cells by 28, 17, and 44%, respectively. This is the first report of the COX-1 enzyme and cancer cell growth inhibitory monogalactosyl diacylglycerides from wasabi and horseradish rhizomes.

  14. Using minced horseradish roots and peroxides for the deodorization of swine manure: a pilot scale study.

    Science.gov (United States)

    Govere, Ephraim M; Tonegawa, Masami; Bruns, Mary Ann; Wheeler, Eileen F; Kephart, Kenneth B; Voigt, Jean W; Dec, Jerzy

    2007-04-01

    Enzymes that have proven to be capable of removing toxic compounds from water and soil may also be useful in the deodorization of animal manures. Considering that pork production in the US is a $40-billion industry with over half a million workers, odor control to protect air quality in the neighboring communities must be considered an essential part of managing livestock facilities. This pilot scale (20-120 L) study tested the use of minced horseradish (Armoracia rusticana L.) roots (1:10 roots to swine slurry ratio), with calcium peroxide (CaO(2) at 34 mM) or hydrogen peroxide (H(2)O(2) at 68 mM), to deodorize swine slurry taken from a 40,000-gallon storage pit at the Pennsylvania State University's Swine Center. Horseradish is known to contain large amounts of peroxidase, an enzyme that, in the presence of peroxides, can polymerize phenolic odorants and thus reduce the malodor. Twelve compounds commonly associated with malodor (seven volatile fatty acids or VFAs, three phenolic compounds and two indolic compounds) were used as odor indicators. Their concentration in swine slurry before and after treatment was determined by gas chromatography (GC) to assess the deodorization effect. The pilot scale testing demonstrated a complete removal of phenolic odorants (with a detection limit of 0.5 mg L(-1)) from the swine slurry, which was consistent with our previous laboratory experiments using 30-mL swine slurry samples. Horseradish could be recycled (reused) five times while retaining significant reduction in the concentration of phenolic odorants. In view of these findings, inexpensive plant materials, such as horseradish, represent a promising tool for eliminating phenolic odorants from swine slurry.

  15. Sulfate determines the glucosinolate concentration of horseradish in vitro plants (Armoracia rusticana Gaertn., Mey. & Scherb.).

    Science.gov (United States)

    Alnsour, Mohammad; Kleinwächter, Maik; Böhme, Julia; Selmar, Dirk

    2013-03-15

    Horseradish plants (Armoracia rusticana) contain high concentrations of glucosinolates. Former studies have revealed that Armoracia plants cultivated in vitro have markedly lower glucosinolate concentrations than those grown in soils. Yet, these studies neglected that the sulfate concentration in the growth medium may have had a strong impact on glucosinolate metabolism. Accordingly, in this study horseradish in vitro plants were cultivated with differing sulfate concentrations and the glucosinolate concentrations were quantified by ion pair HPLC. Cultivation in 1.7 mmol L(-1) sulfate (as used in the prior studies) resulted in the accumulation of 16.2 µmol g(-1) DW glucosinolates, while the glucosinolate concentration increased to more than 23 µmol g(-1) DW when 23.5 mmol L(-1) sulfate was used in the medium. Correspondingly, the glucosinolate concentration decreased to 1.6 µmol g(-1) DW when sulfate concentration was lowered to 0.2 mmol L(-1). Since the glucosinolate accumulation in relation to the sulfate concentration follows a typical saturation curve, we deduce that the availability of sulfate determines the glucosinolate concentration in horseradish in vitro plants. © 2012 Society of Chemical Industry.

  16. Study on a hydrogen peroxide biosensor based on horseradish peroxidase/GNPs-thionine/chitosan

    International Nuclear Information System (INIS)

    Kang Xiaobin; Pang Guangchang; Liang Xinyi; Wang Meng; Liu Jing; Zhu Weiming

    2012-01-01

    Highlights: ► Glutaraldehyde was used as the bridge linking agent to covalently bonded thionine in chitosan, which is more stable and could effectively prevalent leakage of the electronic mediator. ► The effect of GNPs adsorbed HRP was first accurately characterized by bio-layer interferometry using the ForteBio Octer system. ► The application of self-assembly technology increases the biosensor stability. - Abstract: A novel hydrogen peroxide biosensor based on horseradish peroxidase/GNPs-thionine/chitosan has been developed. Gold nanoparticles fixed with horseradish peroxidase were adsorbed on glassy carbon electrode by the chitosan which cross-linked with the electron mediator of horseradish peroxidase as the bridge linking agent. The assembly procedures were monitored by UV–visible spectral scanning, bio-layer interferometry, cyclic voltammetric and alternating current impedance. The chronoamperometry was used to measure hydrogen peroxide. The hydrogen peroxide biosensor linear range of detection is 1 × 10 −7 –1 × 10 −4 mol/L, detection limit up to 5.0 × 10 −8 mol/L. Moreover the stability, reproducibility and selectivity of the biosensor were also studied and the results confirmed that the biosensor exhibit fast response to hydrogen peroxide and possess high sensitivity, good reproducibility and long-term stability.

  17. New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA

    Directory of Open Access Journals (Sweden)

    Hidetoshi Arakawa

    2012-04-01

    Full Text Available Horseradish peroxidase (HRP is generally used as a label enzyme in enzyme immunoassay (EIA. The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm.The measurable range of HRP was 1.0×10−18 to 1.0×10−15 mol/assay, with a detection limit of 1.0×10−18 mol/assay. The coefficient of variation (CV, n=8 was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH EIA using HRP as the label enzyme. Keywords: Sesamol, Fluorescence, Enzyme immunoassay (EIA, Horseradish peroxidase (HRP, Thyroid stimulating hormone (TSH

  18. Study of Horseradish Peroxidase Fixed on Mesoporous Materials as a Chemical Reaction Catalyst

    Science.gov (United States)

    Gao, Mengdan; Dai, Rongji

    2017-12-01

    Nanostructured mesoporous materials is a new type of porous materials, which has been widely used. It has excellent capability in enzymes immobilization, but modification on the chemical bonds of the enzyme reduce the enzymatic activity and rarely used in chemical reactions. The horseradish peroxidase was immobilized on the mesoporous materials with appropriate aperture and its activity and stability was evaluated when catalyzing the nitration reaction of amines and oxidation reaction of thiourea. The optimum mesoporous material to fix the horseradish peroxidase can be obtained by mixing polyoxyethylene - polyoxypropylene-pol, yoxyethylene(P123), 1,3,5-trimethylbenzene(TMB), and tetramethoxysilane (TMOS) at a ratio of 10:1:1, whose surface area and pore volume and pore diameter calculated by BET and BJH model were 402.903m2/g, 1.084cm2/g, 1.084cm2/g respectively. The horseradish peroxidase, immobilized on the mesoporous materials, was applied for catalyzing the nitration reaction of anilines and oxidation reaction of thiourea, produced a high product yield and can be recycled. Thus, it is a strong candidate as a catalysts for oxidation reactions, to be produced at industral scale, due to its high efficiency and low cost.

  19. Characterization of the immersion properties of the peripheral membrane anchor of the FATC domain of the kinase "target of rapamycin" by NMR, oriented CD spectroscopy, and MD simulations.

    Science.gov (United States)

    Sommer, Lisa A M; Janke, J Joel; Bennett, W F Drew; Bürck, Jochen; Ulrich, Anne S; Tieleman, D Peter; Dames, Sonja A

    2014-05-08

    The multidomain ser/thr kinase "target of rapamycin" (TOR) centrally controls eukaryotic growth and metabolism. The C-terminal FATC domain is important for TOR regulation and was suggested to directly mediate TOR-membrane interactions. Here, we present a detailed characterization of the membrane immersion properties of the oxidized and reduced yeast TOR1 FATC domain (2438-2470 = y1fatc). The immersion depth was characterized by NMR-monitored interaction studies with DPC micelles containing paramagnetically tagged 5- or 16-doxyl stearic acid (5-/16-SASL) and by analyzing the paramagnetic relaxation enhancement (PRE) from Mn(2+) in the solvent. Complementary MD-simulations of micellar systems in the absence and presence of protein showed that 5-/16-SASL can move in the micelle and that 16-SASL can bend such that the doxyl group is close to the headgroup region and not deep in the interior as commonly assumed. Based on oriented CD (OCD) data, the single α-helix of oxidized/reduced y1fatc has an angle to the membrane normal of ∼30-60°/∼35-65° in neutral and ∼5-35°/∼0-30° in negatively charged bilayers. The presented experimentally well-founded models help to better understand how this redox-sensitive peripheral membrane anchor may be part of a network of protein-protein and protein-membrane interactions regulating TOR localization at different cellular membranes. Moreover, the presented work provides a good methodological reference for the structural characterization of other peripherally membrane associating proteins.

  20. Human cytotoxic T-lymphocyte membrane-camouflaged nanoparticles combined with low-dose irradiation: a new approach to enhance drug targeting in gastric cancer

    Directory of Open Access Journals (Sweden)

    Zhang L

    2017-03-01

    Full Text Available Lianru Zhang, Rutian Li, Hong Chen, Jia Wei, Hanqing Qian, Shu Su, Jie Shao, Lifeng Wang, Xiaoping Qian, Baorui Liu The Comprehensive Cancer Centre of Drum Tower Hospital, Medical School of Nanjing University and Clinical Cancer Institute of Nanjing University, Nanjing, People’s Republic of China Abstract: Cell membrane-derived nanoparticles are becoming more attractive because of their ability to mimic many features of their source cells. This study reports on a biomimetic delivery platform based on human cytotoxic T-lymphocyte membranes. In this system, the surface of poly-lactic-co-glycolic acid nanoparticles was camouflaged using T-lymphocyte membranes, and local low-dose irradiation (LDI was used as a chemoattractant for nanoparticle targeting. The T-lymphocyte membrane coating was verified using dynamic light scattering, transmission electron microscopy, and confocal laser scanning microscopy. This new platform reduced nanoparticle phagocytosis by macrophages to 23.99% (P=0.002. Systemic administration of paclitaxel-loaded T-lymphocyte membrane-coated nanoparticles inhibited the growth of human gastric cancer by 56.68% in Balb/c nude mice. Application of LDI at the tumor site significantly increased the tumor growth inhibition rate to 88.50%, and two mice achieved complete remission. Furthermore, LDI could upregulate the expression of adhesion molecules in tumor vessels, which is important in the process of leukocyte adhesion and might contribute to the localization of T-lymphocyte membrane-encapsulated nanoparticles in tumors. Therefore, this new drug-delivery platform retained both the long circulation time and tumor site accumulation ability of human cytotoxic T lymphocytes, while local LDI could significantly enhance tumor localization. Keywords: cell membrane, drug delivery system, gastric cancer, low-dose irradiation, nanoparticles

  1. Overview of online two-dimensional liquid chromatography based on cell membrane chromatography for screening target components from traditional Chinese medicines.

    Science.gov (United States)

    Muhammad, Saqib; Han, Shengli; Xie, Xiaoyu; Wang, Sicen; Aziz, Muhammad Majid

    2017-01-01

    Cell membrane chromatography is a simple, specific, and time-saving technique for studying drug-receptor interactions, screening of active components from complex mixtures, and quality control of traditional Chinese medicines. However, the short column life, low sensitivity, low column efficiency (so cannot resolve satisfactorily mixture of compounds), low peak capacity, and inefficient in structure identification were bottleneck in its application. Combinations of cell membrane chromatography with multidimensional chromatography such as two-dimensional liquid chromatography and high sensitivity detectors like mass have significantly reduced many of the above-mentioned shortcomings. This paper provides an overview of the current advances in online two-dimensional-based cell membrane chromatography for screening target components from traditional Chinese medicines with particular emphasis on the instrumentation, preparation of cell membrane stationary phase, advantages, and disadvantages compared to alternative approaches. The last section of the review summarizes the applications of the online two-dimensional high-performance liquid chromatography based cell membrane chromatography reported since its emergence to date (2010-June 2016). © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Expression and Activation of Horseradish Peroxidase-Protein A/G Fusion Protein in Silkworm Larvae for Diagnostic Purposes.

    Science.gov (United States)

    Xxxx, Patmawati; Minamihata, Kosuke; Tatsuke, Tsuneyuki; Lee, Jae Man; Kusakabe, Takahiro; Kamiya, Noriho

    2018-06-01

    Recombinant protein production can create artificial proteins with desired functions by introducing genetic modifications to the target proteins. Horseradish peroxidase (HRP) has been used extensively as a reporter enzyme in biotechnological applications; however, recombinant production of HRP has not been very successful, hampering the utilization of HRP with genetic modifications. A fusion protein comprising an antibody binding protein and HRP will be an ideal bio-probe for high-quality HRP-based diagnostic systems. A HRP-protein A/G fusion protein (HRP-pAG) is designed and its production in silkworm (Bombyx mori) is evaluated for the first time. HRP-pAG is expressed in a soluble apo form, and is activated successfully by incubating with hemin. The activated HRP-pAG is used directly for ELISA experiments and retains its activity over 20 days at 4 °C. Moreover, HRP-pAG is modified with biotin by the microbial transglutaminase (MTG) reaction. The biotinylated HRP-pAG is conjugated with streptavidin to form a HRP-pAG multimer and the multimeric HRP-pAG produced higher signals in the ELISA system than monomeric HRP-pAG. The successful production of recombinant HRP in silkworm will contribute to creating novel HRP-based bioconjugates as well as further functionalization of HRP by applying enzymatic post-translational modifications. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. GRP1 PH Domain, Like AKT1 PH Domain, Possesses a Sentry Glutamate Residue Essential for Specific Targeting to Plasma Membrane PI(3,4,5)P3

    Science.gov (United States)

    Pilling, Carissa; Landgraf, Kyle E.; Falke, Joseph J.

    2011-01-01

    During the appearance of the signaling lipid PI(3,4,5)P3, an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P3-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P2 and bind the rare PI(3,4,5)P3 target lipid with sufficiently high affinity. Our previous study of the E17K mutant of protein kinase B (AKT1) PH domain, together with evidence from Carpten et al (1), revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P2, thereby playing an essential role in specific PI(3,4,5)P3 targeting (2). The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P3-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P2 affinity and constitutive plasma membrane targeting. To test this hypothesis the present study investigates the E345 residue, a putative sentry glutamate, of General Receptor for Phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into GRP1 PH domain enhances PI(4,5)P2 affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P2 releases E345K GRP1 PH domain into the cytoplasm and the efficiency of this release increases when target Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K (1, 3). Analysis of available PH domain structures suggests that a lone glutamate residue (or, in some cases an aspartate) is a common, perhaps ubiquitous, feature of PI(3,4,5)P3-specific binding

  4. An N-terminal peptide extension results in efficient expression, but not secretion, of a synthetic horseradish peroxidase gene in transgenic tobacco.

    Science.gov (United States)

    Kis, Mihaly; Burbridge, Emma; Brock, Ian W; Heggie, Laura; Dix, Philip J; Kavanagh, Tony A

    2004-03-01

    Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N-terminal and C-terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N-terminal or the C-terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV-35S) or the tobacco RUBISCO-SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium-mediated transformation. To study the effects of the N- and C-terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. Transgenic tobacco plants can exhibit a ten-fold increase in peroxidase activity compared with wild-type tobacco levels, and the majority of this activity is located in the symplast. The N-terminal extension is essential for the production of high levels of recombinant protein, while the C-terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N-terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been generated with greatly elevated cytosolic peroxidase activity, and smaller increases in apoplastic

  5. Plasma membrane associated, virus-specific polypeptides required for the formation of target antigen complexes recognized by virus-specific cytotoxic T lymphocytes

    International Nuclear Information System (INIS)

    Domber, E.A.

    1986-01-01

    These studies were undertaken to define some of the poxvirus-specific target antigens which are synthesized in infected cells and recognized by vaccinia virus-specific CTLs (VV-CTLs). Since vaccinia virus infected, unmanipulated target cells express numerous virus-specific antigens on the plasma membrane, attempts were made to manipulate expression of the poxvirus genome after infection so that one or a few defined virus-specified antigens were expressed on the surface of infected cells. In vitro [ 51 Cr]-release assays determined that viral DNA synthesis and expression of late viral proteins were not necessary to form a target cell which was fully competent for lysis by VV-CTLs. Under the conditions employed in these experiments, 90-120 minutes of viral protein synthesis were necessary to produce a competent cell for lysis by VV-CTLs. In order to further inhibit the expression of early viral proteins in infected cells, partially UV-inactivated vaccinia virus was employed to infect target cells. It was determined that L-cells infected with virus preparations which had been UV-irradiated for 90 seconds were fully competent for lysis by VV-CTLs. Cells infected with 90 second UV-irr virus expressed 3 predominant, plasma membrane associated antigens of 36-37K, 27-28K, and 19-17K. These 3 viral antigens represent the predominant membrane-associated viral antigens available for interaction with class I, major histocompatibility antigens and hence are potential target antigens for VV-CTLs

  6. The C-terminal HRET sequence of Kv1.3 regulates gating rather than targeting of Kv1.3 to the plasma membrane.

    Science.gov (United States)

    Voros, Orsolya; Szilagyi, Orsolya; Balajthy, András; Somodi, Sándor; Panyi, Gyorgy; Hajdu, Péter

    2018-04-12

    Kv1.3 channels are expressed in several cell types including immune cells, such as T lymphocytes. The targeting of Kv1.3 to the plasma membrane is essential for T cell clonal expansion and assumed to be guided by the C-terminus of the channel. Using two point mutants of Kv1.3 with remarkably different features compared to the wild-type Kv1.3 (A413V and H399K having fast inactivation kinetics and tetraethylammonium-insensitivity, respectively) we showed that both Kv1.3 channel variants target to the membrane when the C-terminus was truncated right after the conserved HRET sequence and produce currents identical to those with a full-length C-terminus. The truncation before the HRET sequence (NOHRET channels) resulted in reduced membrane-targeting but non-functional phenotypes. NOHRET channels did not display gating currents, and coexpression with wild-type Kv1.3 did not rescue the NOHRET-A413V phenotype, no heteromeric current was observed. Interestingly, mutants of wild-type Kv1.3 lacking HRET(E) (deletion) or substituted with five alanines for the HRET(E) motif expressed current indistinguishable from the wild-type. These results demonstrate that the C-terminal region of Kv1.3 immediately proximal to the S6 helix is required for the activation gating and conduction, whereas the presence of the distal region of the C-terminus is not exclusively required for trafficking of Kv1.3 to the plasma membrane.

  7. Nanobody Based Immunoassay for Human Soluble Epoxide Hydrolase Detection Using Polymeric Horseradish Peroxidase (PolyHRP) for Signal Enhancement: The Rediscovery of PolyHRP?

    Science.gov (United States)

    Li, Dongyang; Cui, Yongliang; Morisseau, Christophe; Gee, Shirley J; Bever, Candace S; Liu, Xiangjiang; Wu, Jian; Hammock, Bruce D; Ying, Yibin

    2017-06-06

    Soluble epoxide hydrolase (sEH) is a potential pharmacological target for treating hypertension, vascular inflammation, cancer, pain, and multiple cardiovascular related diseases. A variable domain of the heavy chain antibody (termed single domain antibody (sdAb), nanobody, or VHH) possesses the advantages of small size, high stability, ease of genetic manipulation, and ability for continuous manufacture, making such nanobody a superior choice as an immunoreagent. In this work, we developed an ultrasensitive nanobody based immunoassay for human sEH detection using polymeric horseradish peroxidase (PolyHRP) for signal enhancement. Llama nanobodies against human sEH were used as the detection antibody in sandwich enzyme linked immunosorbent assays (ELISA) with polyclonal anti-sEH as the capture antibody. A conventional sandwich ELISA using a horseradish peroxidase (HRP) labeled anti-hemeagglutinin (HA) tag as the tracer showed a marginal sensitivity (0.0015 optical density (OD)·mL/ng) and limit of detection (LOD) of 3.02 ng/mL. However, the introduction of the PolyHRP as the tracer demonstrated a 141-fold increase in the sensitivity (0.21 OD·mL/ng) and 57-fold decrease in LOD (0.05 ng/mL). Systematic comparison of three different tracers in four ELISA formats demonstrated the overwhelming advantage of PolyHRP as a label for nanobody based immunoassay. This enhanced sEH immunoassay was further evaluated in terms of selectivity against other epoxide hydrolases and detection of the target protein in human tissue homogenate samples. Comparison with an enzyme activity based assay and a Western blot for sEH detection reveals good correlation with the immunoassay. This work demonstrates increased competiveness of nanobodies for practical sEH protein detection utilizing PolyHRP. It is worthwhile to rediscover the promising potential of PolyHRP in nanobody and other affinity based methods after its low-profile existence for decades.

  8. Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

    Science.gov (United States)

    Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

    2000-01-01

    BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confocal laser microscopy of isolated rat hepatocytes probed with 10 LKM1 positive sera (five from patients with AIH and five from patients with chronic HCV infection) and a rabbit polyclonal anti-CYP2D6 serum.
RESULTS—Serum from both types of patient stained the plasma membrane of non-permeabilised cells, where the fluorescent signal could be visualised as discrete clumps. Conversely, permeabilised hepatocytes showed diffuse submembranous/cytoplasmic staining. Adsorption with recombinant CYP2D6 substantially reduced plasma membrane staining and LKM1 immunoblot reactivity. Plasma membrane staining of LKM1 colocalised with that of anti-CYP2D6. Immunoprecipitation experiments showed that a single 50 kDa protein recognised by anti-CYP2D6 can be isolated from the plasma membrane of intact hepatocytes.
CONCLUSIONS—AIH and HCV related LKM1 recognise CYP2D6 exposed on the plasma membrane of isolated hepatocytes. This observation supports the notion that anti-CYP2D6 autoreactivity may be involved in the pathogenesis of liver damage.


Keywords: liver/kidney microsomal antibody type 1; autoimmunity; autoimmune hepatitis; hepatitis C virus infection; confocal microscopy PMID:10716687

  9. Kinase Associated-1 Domains Drive MARK/PAR1 Kinases to Membrane Targets by Binding Acidic Phospholipids

    Energy Technology Data Exchange (ETDEWEB)

    Moravcevic, Katarina; Mendrola, Jeannine M.; Schmitz, Karl R.; Wang, Yu-Hsiu; Slochower, David; Janmey, Paul A.; Lemmon, Mark A. (UPENN-MED)

    2011-09-28

    Phospholipid-binding modules such as PH, C1, and C2 domains play crucial roles in location-dependent regulation of many protein kinases. Here, we identify the KA1 domain (kinase associated-1 domain), found at the C terminus of yeast septin-associated kinases (Kcc4p, Gin4p, and Hsl1p) and human MARK/PAR1 kinases, as a membrane association domain that binds acidic phospholipids. Membrane localization of isolated KA1 domains depends on phosphatidylserine. Using X-ray crystallography, we identified a structurally conserved binding site for anionic phospholipids in KA1 domains from Kcc4p and MARK1. Mutating this site impairs membrane association of both KA1 domains and intact proteins and reveals the importance of phosphatidylserine for bud neck localization of yeast Kcc4p. Our data suggest that KA1 domains contribute to coincidence detection, allowing kinases to bind other regulators (such as septins) only at the membrane surface. These findings have important implications for understanding MARK/PAR1 kinases, which are implicated in Alzheimer's disease, cancer, and autism.

  10. A novel and efficient oxidative functionalization of lignin by layer-by-layer immobilised Horseradish peroxidase.

    Science.gov (United States)

    Perazzini, Raffaella; Saladino, Raffaele; Guazzaroni, Melissa; Crestini, Claudia

    2011-01-01

    Horseradish peroxidase (HRP) was chemically immobilised onto alumina particles and coated by polyelectrolytes layers, using the layer-by-layer technique. The reactivity of the immobilised enzyme was studied in the oxidative functionalisation of softwood milled wood and residual kraft lignins and found higher than the free enzyme. In order to investigate the chemical modifications in the lignin structure, quantitative (31)P NMR was used. The immobilised HRP showed a higher reactivity with respect to the native enzyme yielding extensive depolymerisation of lignin. Copyright © 2010 Elsevier Ltd. All rights reserved.

  11. Engineered CAR T Cells Targeting the Cancer-Associated Tn-Glycoform of the Membrane Mucin MUC1 Control Adenocarcinoma

    DEFF Research Database (Denmark)

    Posey, Avery D; Schwab, Robert D; Boesteanu, Alina C

    2016-01-01

    with sufficient cancer-specific expression. The majority of CAR targets have been normal self-antigens on dispensable hematopoietic tissues or overexpressed shared antigens. Here, we established that abnormal self-antigens can serve as targets for tumor rejection. We developed a CAR that recognized cancer...

  12. Electrospun Chitosan-Gelatin Biopolymer Composite Nanofibers for Horseradish Peroxidase Immobilization in a Hydrogen Peroxide Biosensor

    Directory of Open Access Journals (Sweden)

    Siriwan Teepoo

    2017-10-01

    Full Text Available A biosensor based on chitosan-gelatin composite biopolymers nanofibers is found to be effective for the immobilization of horseradish peroxidase to detect hydrogen peroxide. The biopolymer nanofibers were fabricated by an electrospining technique. Upon optimization of synthesis parameters, biopolymers nanofibers, an average of 80 nm in diameter, were obtained and were then modified on the working electrode surface. The effects of the concentration of enzyme, pH, and concentration of the buffer and the working potential on the current response of the nanofibers-modified electrode toward hydrogen peroxide were optimized to obtain the maximal current response. The results found that horseradish peroxidase immobilization on chitosan-gelatin composite biopolymer nanofibers had advantages of fast response, excellent reproducibility, high stability, and showed a linear response to hydrogen peroxide in the concentration range from 0.1 to 1.7 mM with a detection limit of 0.05 mM and exhibited high sensitivity of 44 µA∙mM−1∙cm−2. The developed system was evaluated for analysis of disinfectant samples and showed good agreement between the results obtained by the titration method without significant differences at the 0.05 significance level. The proposed strategy based on chitosan-gelatin composite biopolymer nanofibers for the immobilization of enzymes can be extended for the development of other enzyme-based biosensors.

  13. A novel life cycle arising from leaf segments in plants regenerated from horseradish hairy roots.

    Science.gov (United States)

    Mano, Y; Matsuhashi, M

    1995-03-01

    Horseradish (Armoracia rusticana) hairy root clones were established from hairy roots which were transformed with the Ri plasmid in Agrobacterium rhizogenes 15834. The transformed plants, which were regenerated from hairy root clones, had thicker roots with extensive lateral branches and thicker stems, and grew faster compared with non-transformed horseradish plants. Small sections of leaves of the transformed plants generated adventitious roots in phytohormone-free G (modified Gamborg's) medium. Root proliferation was followed by adventitious shoot formation and plant regeneration. Approximately twenty plants were regenerated per square centimeter of leaf. The transformed plants were easily transferable from sterile conditions to soil. When leaf segments of the transformed plants were cultured in a liquid fertilizer under non-sterile conditions, adventitious roots were generated at the cut ends of the leaves. Adventitious shoots were generated at the boundary between the leaf and the adventitious roots and developed into complete plants. This novel life cycle arising from leaf segments is a unique property of the transformed plants derived from hairy root clones.

  14. Transient Expression and Purification of Horseradish Peroxidase C in Nicotiana benthamiana.

    Science.gov (United States)

    Huddy, Suzanne M; Hitzeroth, Inga I; Meyers, Ann E; Weber, Brandon; Rybicki, Edward P

    2018-01-01

    Horseradish peroxidase (HRP) is a commercially important reagent enzyme used in molecular biology and in the diagnostic product industry. It is typically purified from the roots of the horseradish ( Armoracia rusticana ); however, this crop is only available seasonally, yields are variable and often low, and the product is a mixture of isoenzymes. Engineering high-level expression in transiently transformed tobacco may offer a solution to these problems. In this study, a synthetic Nicotiana benthamiana codon-adapted full-length HRP isoenzyme gene as well as C-terminally truncated and both N- and C-terminally truncated versions of the HRP C gene were synthesized, and their expression in N. benthamiana was evaluated using an Agrobacterium tumefaciens -mediated transient expression system. The influence on HRP C expression levels of co-infiltration with a silencing suppressor (NSs) construct was also evaluated. Highest HRP C levels were consistently obtained using either the full length or C-terminally truncated HRP C constructs. HRP C purification by ion exchange chromatography gave an overall yield of 54% with a Reinheitszahl value of >3 and a specific activity of 458 U/mg. The high level of HRP C production in N. benthamiana in just five days offers an alternative, viable, and scalable system for production of this commercially significant enzyme.

  15. Nematicidal activity of allylisothiocyanate from horseradish (Armoracia rusticana) roots against Meloidogyne incognita.

    Science.gov (United States)

    Aissani, Nadhem; Tedeschi, Paola; Maietti, Annalisa; Brandolini, Vincenzo; Garau, Vincenzo Luigi; Caboni, Pierluigi

    2013-05-22

    In recent years, there has been a great development in the search for new natural pesticides for crop protection aiming a partial or total replacement of currently used chemical nematicides. Glucosinolate breakdown products are volatile and are therefore good candidates for nematodes fumigants. In this article, the methanol-aqueous extract (1:1, w/v) of horseradish (Armoracia rusticana) fresh roots (MAH) was in vitro tested for nematicidal activity against second stage (J2) Meloidogyne incognita. The EC50 of MAH after 3 days of J2 immersion in test solutions was 251 ± 46 mg/L. The chemical composition analysis of the extract carried out by the GC-MS technique showed that allylisothicyanate was the most abundant compound. This pure compound induced J2 paralysis with an EC50 of 52.6 ± 45.6 and 6.6 ± 3.4 mg/L after 1 h and 3 days of incubation. The use of LC-MS/MS showed for the first time that horseradish root is rich in polyphenols. The study of isothiocyanate degradation in soil showed that allylisothiocyanate was the most quickly degradable compound (half-life <10 min), whereas no significant differences in half-life time were noted between degradation in regular and autoclaved soil.

  16. Construction of a horseradish peroxidase resistant toward hydrogen peroxide by saturation mutagenesis.

    Science.gov (United States)

    Asad, Sedigheh; Dastgheib, Seyed Mohammad Mehdi; Khajeh, Khosro

    2016-11-01

    Horseradish peroxidase (HRP) with a variety of potential biotechnological applications is still isolated from the horseradish root as a mixture of different isoenzymes with different biochemical properties. There is an increasing demand for preparations of high amounts of pure enzyme but its recombinant production is limited because of the lack of glycosylation in Escherichia coli and different glycosylation patterns in yeasts which affects its stability parameters. The goal of this study was to increase the stability of non-glycosylated enzyme, which is produced in E. coli, toward hydrogen peroxide via mutagenesis. Asparagine 268, one of the N-glycosylation sites of the enzyme, has been mutated via saturation mutagenesis using the megaprimer method. Modification and miniaturization of previously described protocols enabled screening of a library propagated in E. coli XJb (DE3). The library of mutants was screened for stability toward hydrogen peroxide with azinobis (ethylbenzthiazoline sulfonate) as a reducing substrate. Asn268Gly mutant, the top variant from the screening, exhibited 18-fold increased stability toward hydrogen peroxide and twice improved thermal stability compared with the recombinant HRP. Moreover, the substitution led to 2.5-fold improvement in the catalytic efficiency with phenol/4-aminoantipyrine. Constructed mutant represents a stable biocatalyst, which may find use in medical diagnostics, biosensing, and bioprocesses. © 2015 International Union of Biochemistry and Molecular Biology, Inc.

  17. New fluorimetric assay of horseradish peroxidase using sesamol as substrate and its application to EIA.

    Science.gov (United States)

    Arakawa, Hidetoshi; Nakabayashi, Shigeo; Ohno, Ken-Ichi; Maeda, Masako

    2012-04-01

    Horseradish peroxidase (HRP) is generally used as a label enzyme in enzyme immunoassay (EIA). The procedure used for HRP detection in EIA is critical for sensitivity and precision. This paper describes a novel fluorimetric assay for horseradish peroxidase (HRP) using sesamol as substrate. The principle of the assay is as follow: sesamol (3,4-methylenedioxy phenol) is reacted enzymatically in the presence of hydrogen peroxide to produce dimeric sesamol. The dimer is fluorescent and can be detected sensitively at ex. 347 nm, em. 427 nm. The measurable range of HRP was 1.0×10 -18 to 1.0×10 -15  mol/assay, with a detection limit of 1.0×10 -18  mol/assay. The coefficient of variation (CV, n =8) was examined at each point on the standard curve, with a mean CV percentage of 3.8%. This assay system was applied to thyroid stimulating hormone (TSH) EIA using HRP as the label enzyme.

  18. Prostate-specific membrane antigen targeted imaging and therapy of prostate cancer using a PSMA inhibitor as a homing ligand.

    Science.gov (United States)

    Kularatne, Sumith A; Wang, Kevin; Santhapuram, Hari-Krishna R; Low, Philip S

    2009-01-01

    Prostate cancer (PCa) is a major cause of mortality and morbidity in Western society today. Current methods for detecting PCa are limited, leaving most early malignancies undiagnosed and sites of metastasis in advanced disease undetected. Major deficiencies also exist in the treatment of PCa, especially metastatic disease. In an effort to improve both detection and therapy of PCa, we have developed a PSMA-targeted ligand that delivers attached imaging and therapeutic agents selectively to PCa cells without targeting normal cells. The PSMA-targeted radioimaging agent (DUPA-(99m)Tc) was found to bind PSMA-positive human PCa cells (LNCaP cell line) with nanomolar affinity (K(D) = 14 nM). Imaging and biodistribution studies revealed that DUPA-(99m)Tc localizes primarily to LNCaP cell tumor xenografts in nu/nu mice (% injected dose/gram = 11.3 at 4 h postinjection; tumor-to-muscle ratio = 75:1). Two PSMA-targeted optical imaging agents (DUPA-FITC and DUPA-rhodamine B) were also shown to efficiently label PCa cells and to internalize and traffic to intracellular endosomes. A PSMA-targeted chemotherapeutic agent (DUPA-TubH) was demonstrated to kill PSMA-positive LNCaP cells in culture (IC(50) = 3 nM) and to eliminate established tumor xenografts in nu/nu mice with no detectable weight loss. Blockade of tumor targeting upon administration of excess PSMA inhibitor (PMPA) and the absence of targeting to PSMA-negative tumors confirmed the specificity of each of the above targeted reagents for PSMA. Tandem use of the imaging and therapeutic agents targeted to the same receptor could allow detection, staging, monitoring, and treatment of PCa with improved accuracy and efficacy.

  19. High-expression β(1) adrenergic receptor/cell membrane chromatography method based on a target receptor to screen active ingredients from traditional Chinese medicines.

    Science.gov (United States)

    Yue, Yuan; Xue, Hui; Wang, Xin; Yang, Qian; Song, Yanhong; Li, Xiaoni

    2014-02-01

    β-Adrenergic receptors are important targets for drug discovery. We have developed a new β1 -adrenergic receptor cell membrane chromatography (β1 AR-CMC) with offline ultra-performance LC (UPLC) and MS method for screening active ingredients from traditional Chinese medicines. In this study, Chinese hamster ovary-S cells with high β1 AR expression levels were established and used to prepare a cell membrane stationary phase in a β1 AR-CMC model. The retention fractions were separated and identified by the UPLC-MS system. The screening results found that isoimperatorin from Rhizoma et Radix Notopterygii was the targeted component that could act on β1 AR in similar manner of metoprolol as a control drug. In addition, the biological effects of active component were also investigated in order to search for a new type of β1 AR antagonist. It will be a useful method for drug discovery as a leading compound resource. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Liver/kidney microsomal antibody type 1 targets CYP2D6 on hepatocyte plasma membrane

    OpenAIRE

    Muratori, L; Parola, M; Ripalti, A; Robino, G; Muratori, P; Bellomo, G; Carini, R; Lenzi, M; Landini, M; Albano, E; Bianchi, F

    2000-01-01

    BACKGROUND—Liver/kidney microsomal antibody type 1 (LKM1) is the marker of type 2 autoimmune hepatitis (AIH) and is detected in up to 6% of patients with hepatitis C virus (HCV) infection. It recognises linear and conformational epitopes of cytochrome P450IID6 (CYP2D6) and may have liver damaging activity, provided that CYP2D6 is accessible to effector mechanisms of autoimmune attack.
METHODS—The presence of LKM1 in the plasma membrane was investigated by indirect immunofluorescence and confo...

  1. A New Crucial Protein Interaction Element That Targets the Adenovirus E4-ORF1 Oncoprotein to Membrane Vesicles▿

    OpenAIRE

    Chung, Sang-Hyuk; Frese, Kristopher K.; Weiss, Robert S.; Prasad, B. V. Venkataram; Javier, Ronald T.

    2007-01-01

    Human adenovirus type 9 exclusively elicits mammary tumors in experimental animals, and the primary oncogenic determinant of this virus is the E4-ORF1 oncogene, as opposed to the well-known E1A and E1B oncogenes. The tumorigenic potential of E4-ORF1, as well as its ability to oncogenically stimulate phosphatidylinositol 3-kinase (PI3K), depends on a carboxyl-terminal PDZ domain-binding motif (PBM) that mediates interactions with several different membrane-associated cellular PDZ proteins, inc...

  2. Horseradish peroxidase-catalyzed oligomerization of ferulic acid on a template of a tyrosine-containing tripeptide

    NARCIS (Netherlands)

    Oudgenoeg, G.; Dirksen, E.; Ingemann, S.; Hilhorst, R.; Gruppen, H.; Boeriu, C.G.; Piersma, S.R.; Berkel, W.J.H. van; Laane, C.; Voragen, A.G.J.

    2002-01-01

    Ferulic acid (FA) is an abundantly present phenolic constituent of plant cell walls. Kinetically controlled incubation of FA and the tripeptide Gly-Tyr-Gly (GYG) with horseradish peroxidase and H2O2 yielded a range of new cross-linked products. Two predominant series of hetero-oligomers of FA linked

  3. Use of additives to enhance the removal of phenols from water treated with horseradish and hydrogen peroxide.

    Science.gov (United States)

    Tonegawa, Masami; Dec, Jerzy; Bollag, Jean-Marc

    2003-01-01

    Use of additives, such as polyethylene glycol (PEG), selected surfactants, chitosan gel, or activated carbon, has been shown to enhance enzymatic treatment of water polluted with organic compounds. In this study, additives were used to facilitate the removal of 2,4-dichlorophenol (2,4-DCP) from water using minced horseradish (Armoracia rusticana P. Gaertn. et al.) as a carrier of peroxidase activity. The specific objectives of the study were to (i) enhance the pollutant removal activity of minced horseradish by the addition of PEG and other additives (e.g., Tween 20, Triton X-100, and rhamnolipid); (ii) eliminate colored reaction products by the addition of chitosan; and (iii) eliminate color by amending treated water with activated carbon. The disappearance of 2,4-DCP in horseradish-treated water samples amended with PEG or various surfactants (75-90%) was greatly increased over that observed in nonamended samples (29%). The effect of PEG depended on its average molecular weight. As indicated by visible spectrophotometry, enclosing horseradish pieces between two sealed chitosan films completely eliminated colored reaction products; however, the decolorization was accompanied by a reduction in 2,4-DCP removal (from 95 to 60%). On the other hand, commercially available activated carbon completely removed colored reaction products from the treated water without reducing the removal efficiency. Based on the results obtained, it can be concluded that the use of additives may considerably improve the quality of wastewater treated by plant materials.

  4. Redirecting the Cyanobacterial Bicarbonate Transporters BicA and SbtA to the Chloroplast Envelope: Soluble and Membrane Cargos Need Different Chloroplast Targeting Signals in Plants.

    Directory of Open Access Journals (Sweden)

    Vivien eRolland

    2016-02-01

    Full Text Available Most major crops used for human consumption are C3 plants, which yields are limited by photosynthetic inefficiency. To circumvent this, it has been proposed to implement the cyanobacterial CO2-concentrating mechanism (CCM, principally consisting of bicarbonate transporters and carboxysomes, into plant chloroplasts. As it is currently not possible to recover homoplasmic transplastomic monocots, foreign genes must be introduced in these plants via nuclear transformation. Consequently, it is paramount to ensure that resulting proteins reach the appropriate sub-cellular compartment, which for cyanobacterial transporters BicA and SbtA, is the chloroplast inner-envelope membrane (IEM. At present, targeting signals to redirect large transmembrane proteins from non-chloroplastic organisms to plant chloroplast envelopes are unknown. The goal of this study was to identify such signals, using agrobacteria-mediated transient expression and confocal microscopy to determine the sub-cellular localization of ~37 GFP-tagged chimeras. Initially, fragments of chloroplast proteins known to target soluble cargos to the stroma were tested for their ability to redirect BicA, but they proved ineffective. Next, different N-terminal regions from Arabidopsis IEM transporters were tested. We demonstrated that the N-terminus of AtHP59, AtPLGG1 or AtNTT1 (92-115 amino acids, containing a cleavable chloroplast transit peptide (cTP and a membrane protein leader (MPL, was sufficient to redirect BicA or SbtA to the chloroplast envelope. This constitutes the first evidence that nuclear-encoded transmembrane proteins from non-chloroplastic organisms can be targeted to the envelope of plant chloroplasts; a finding which represents an important advance in chloroplast engineering by opening up the door to further manipulation of the chloroplastic envelope.

  5. Study on the Matching Relationship between Polymer Hydrodynamic Characteristic Size and Pore Throat Radius of Target Block S Based on the Microporous Membrane Filtration Method

    Directory of Open Access Journals (Sweden)

    Li Yiqiang

    2014-01-01

    Full Text Available The concept of the hydrodynamic characteristic size of polymer was proposed in this study, to characterize the size of aggregates of many polymer molecules in the polymer percolation process. The hydrodynamic characteristic sizes of polymers used in the target block S were examined by employing microporous membrane filtration method, and the factors were studied. Natural core flow experiments were conducted in order to set up the flow matching relationship plate. According to the flow matching plate, the relationship between the hydrodynamic characteristic size of polymer and pore throat radius obtained from core mercury injection data was found. And several suitable polymers for different reservoirs permeability were given. The experimental results of microporous membrane filtration indicated that the hydrodynamic characteristic size of polymer maintained a good nonlinear relationship with polymer viscosity; the value increased as the molecular weight and concentration of the polymer increased and increased as the salinity of dilution water decreased. Additionally, the hydrodynamic characteristic size decreased as the pressure increased, so the hydrodynamic characteristic size ought to be determined based on the pressure of the target block. In the core flow studies, good matching of polymer and formation was identified as polymer flow pressure gradient lower than the fracture pressure gradient of formation. In this case, good matching that was the pore throat radius should be larger than 10 times the hydrodynamic characteristic size of polymer in this study. Using relationship, more matching relationship between the hydrodynamic characteristic sizes of polymer solutions and the pore throat radius of target block was determined.

  6. Ethyl p-coumarate exerts antifungal activity in vitro and in vivo against fruit Alternaria alternata via membrane-targeted mechanism.

    Science.gov (United States)

    Li, Wusun; Yuan, Shuzhi; Sun, Jing; Li, Qianqian; Jiang, Weibo; Cao, Jiankang

    2018-08-02

    by the severe occurrence of lipid peroxidation of the pathogen treated with EpCA. Taken together, these results indicated that EpCA exerts antifungal activity via membrane-targeted mechanism and it would be a promising candidate to control postharvest diseases of fruits. Copyright © 2018 Elsevier B.V. All rights reserved.

  7. Insecticidal activity and fungitoxicity of plant extracts and components of horseradish (Armoracia rusticana) and garlic (Allium sativum).

    Science.gov (United States)

    Tedeschi, Paola; Leis, Marilena; Pezzi, Marco; Civolani, Stefano; Maietti, Annalisa; Brandolini, Vincenzo

    2011-01-01

    To avoid environmental pollution and health problems caused by the use of traditional synthetic pesticides, there is a trend to search for naturally occurring toxicants from plants. Among the compounds discussed for anti-fungal and insecticidal activity, the natural extracts from garlic and horseradish have attracted considerable attention. The objective of this study is to determine the insecticidal and anti-fungal activity of Armoracia rusticana and Allium sativum L. extracts against larvae of Aedes albopictus (Skuse) and some pathogenic fungi. For the insecticidal test, horseradish and garlic extracts were prepared from fresh plants (cultivated in Emilia Romagna region) in a solution of ethanol 80 % and the two different solutions were used at different concentrations (for the determination of the lethal dose) against the fourth instar mosquito's larvae. The fungicidal test was carried out by the agar plates technique using garlic and horseradish extracts in a 10 % ethanol solution against the following organisms: Sclerotium rolfsii Sacc., Trichoderma longibrachiatum, Botrytis cinerea Pers., Fusarium oxysporum Schlecht. and Fusarium culmorum (Wm. G. Sm.) Sacc. The first results demonstrated that the horseradish ethanol extracts present only a fungistatic activity against Sclerotium rolfsii Sacc., Fusarium oxysporum Schlecht. and F. culmorum (Wm.G. Sm) Sacc. while garlic extracts at the same concentration provided a good fungicidal activity above all against Botrytis cinerea Pers. and S. rolfsii. A. rusticana and A. sativum preparations showed also an interesting and significant insecticidal activity against larvae of A. albopictus, even if horseradish presented a higher efficacy (LC₅₀ value of 2.34 g/L), approximately two times higher than garlic one (LC₅₀ value of 4.48 g/L).

  8. Alterations in blood-brain barrier function following acute hypertension: comparison of the blood-to-brain transfer of horseradish peroxidase with that of alpha-aminisobutyric acid

    International Nuclear Information System (INIS)

    Ellison, M.D.B.

    1985-01-01

    The blood-brain barrier (BBB) selectively restricts the blood-to-brain passage of many solutes owing to unique properties of cerebrovascular endothelial cell membranes. To date, experimental study of the BBB has been accomplished primarily through the use of two different methodological approaches. Morphological studies have mostly employed large molecular weight (MW) tracers to detect morphological alterations underlying increased permeability. Physiological studies, employing smaller, more physiologic tracers have successfully described, quantitatively, certain functional aspects of blood-to-brain transfer. The current work attempts to merge these two approaches and to consider barrier function/dysfunction from both a morphological and a functional perspective. Specifically, the study compares in rats, following acute hypertension, the cerebrovascular passage of 14 C-alpha-aminoisobutyric acid (AIB) and that of horseradish peroxidase (HRP). The blood-to-brain passage of AIB and HRP were compared following acute hypertension, with regard to both the distributions of the tracer extravasation patterns and the magnitude of tracer extravasation. The results of this study suggest that traditional morphological barrier studies alone do not reveal all aspects of altered barrier status and that multiple mechanisms underlying increased BBB permeability may operate simultaneously during BBB dysfunction

  9. Proximity does not contribute to activity enhancement in the glucose oxidase-horseradish peroxidase cascade

    Science.gov (United States)

    Zhang, Yifei; Tsitkov, Stanislav; Hess, Henry

    2016-12-01

    A proximity effect has been invoked to explain the enhanced activity of enzyme cascades on DNA scaffolds. Using the cascade reaction carried out by glucose oxidase and horseradish peroxidase as a model system, here we study the kinetics of the cascade reaction when the enzymes are free in solution, when they are conjugated to each other and when a competing enzyme is present. No proximity effect is found, which is in agreement with models predicting that the rapidly diffusing hydrogen peroxide intermediate is well mixed. We suggest that the reason for the activity enhancement of enzymes localized by DNA scaffolds is that the pH near the surface of the negatively charged DNA nanostructures is lower than that in the bulk solution, creating a more optimal pH environment for the anchored enzymes. Our findings challenge the notion of a proximity effect and provide new insights into the role of DNA scaffolds.

  10. Direct electrochemistry and electrocatalysis of horseradish peroxidase based on halloysite nanotubes/chitosan nanocomposite film

    Energy Technology Data Exchange (ETDEWEB)

    Sun Xiumei; Zhang Yao; Shen Hebai [Department of Chemistry, College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Road, Shanghai 200234 (China); Jia Nengqin, E-mail: nqjia@shnu.edu.c [Department of Chemistry, College of Life and Environmental Sciences, Shanghai Normal University, 100 Guilin Road, Shanghai 200234 (China)

    2010-12-30

    The novel halloysite nanotubes/chitosan (HNTs/Chi) composite films were firstly explored to utilize for the immobilization of horseradish peroxidase (HRP) and their bioelectrochemical properties were studied, in which the biopolymer chitosan was used as a binder to increase film adherence on glassy carbon (GC) electrode. UV-vis and FTIR spectroscopy demonstrated that HRP in the composite film could retain its native secondary structure. A pair of well-defined redox peaks of HRP was obtained at the HRP/HNTs/Chi composite film-modified electrode, exhibiting its fast direct electron transfer (DET). Furthermore, the immobilized HRP displayed its good electrocatalytic activity for the reduction of hydrogen peroxide (H{sub 2}O{sub 2}). The results demonstrate that the HNTs/Chi composite film may improve the enzyme loading with the retention of bioactivity and greatly promote the direct electron transfer, which can be attributed to its unique tubular structure, high specific surface area, and good biocompatibility.

  11. Well-Defined Macromolecules Using Horseradish Peroxidase as a RAFT Initiase.

    Science.gov (United States)

    Danielson, Alex P; Bailey-Van Kuren, Dylan; Lucius, Melissa E; Makaroff, Katherine; Williams, Cameron; Page, Richard C; Berberich, Jason A; Konkolewicz, Dominik

    2016-02-01

    Enzymatic catalysis and control over macromolecular architectures from reversible addition-fragmentation chain transfer polymerization (RAFT) are combined to give a new method of making polymers. Horseradish peroxidase (HRP) is used to catalytically generate radicals using hydrogen peroxide and acetylacetone as a mediator. RAFT is used to control the polymer structure. HRP catalyzed RAFT polymerization gives acrylate and acrylamide polymers with relatively narrow molecular weight distributions. The polymerization is rapid, typically exceeding 90% monomer conversion in 30 min. Complex macromolecular architectures including a block copolymer and a protein-polymer conjugate are synthesized using HRP to catalytically initiate RAFT polymerization. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Electrochemical properties of seamless three-dimensional carbon nanotubes-grown graphene modified with horseradish peroxidase.

    Science.gov (United States)

    Komori, Kikuo; Terse-Thakoor, Trupti; Mulchandani, Ashok

    2016-10-01

    Horseradish peroxidase (HRP) was immobilized through sodium dodecyl sulfate (SDS) on the surface of a seamless three-dimensional hybrid of carbon nanotubes grown at the graphene surface (HRP-SDS/CNTs/G) and its electrochemical properties were investigated. Compared with graphene alone electrode modified with HRP via SDS (HRP-SDS/G electrode), the surface coverage of electroactive HRP at the CNTs/G electrode surface was approximately 2-fold greater because of CNTs grown at the graphene surface. Based on the increase in the surface coverage of electroactive HRP, the sensitivity to H2O2 at the HRP-SDS/CNTs/G electrode was higher than that at the HRP-SDS/G electrode. The kinetics of the direct electron transfer from the CNTs/G electrode to compound I and II of modified HRP was also analyzed. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. Modifying sulfomethylated alkali lignin by horseradish peroxidase to improve the dispersibility and conductivity of polyaniline

    Science.gov (United States)

    Yang, Dongjie; Huang, Wenjing; Qiu, Xueqing; Lou, Hongming; Qian, Yong

    2017-12-01

    Pine and wheat straw alkali lignin (PAL and WAL) were sulfomethylated to improve water solubility, polymerized with horseradish peroxidase (HRP) to improve the molecular weight (Mw) and applied to dope and disperse polyaniline (PANI). The structural effect of lignin from different origins on the reactivities of sulfomethylation and HRP polymerization was investigated. The results show that WAL with less methoxyl groups and lower Mw have higher reactivity in sulfomethylation (SWAL). More phenolic hydroxyl groups and lower Mw benefit the HRP polymerization of sulfomethylated PAL (SPAL). Due to the natural three-dimensional aromatic structure and introduced sulfonic groups, SPAL and SWAL could effectively dope and disperse PANI in water by π-π stacking and electrostatic interaction. HRP modified SPAL (HRP-SPAL) with much higher sulfonation degree and larger Mw significantly increased the conductivity and dispersibility of lignin/PANI composites.

  14. An N‐terminal Peptide Extension Results in Efficient Expression, but not Secretion, of a Synthetic Horseradish Peroxidase Gene in Transgenic Tobacco

    Science.gov (United States)

    KIS, MIHALY; BURBRIDGE, EMMA; BROCK, IAN W.; HEGGIE, LAURA; DIX, PHILIP J.; KAVANAGH, TONY A.

    2004-01-01

    • Background and Aims Native horseradish (Armoracia rusticana) peroxidase, HRP (EC 1.11.1.7), isoenzyme C is synthesized with N‐terminal and C‐terminal peptide extensions, believed to be associated with protein targeting. This study aimed to explore the specific functions of these extensions, and to generate transgenic plants with expression patterns suitable for exploring the role of peroxidase in plant development and defence. • Methods Transgenic Nicotiana tabacum (tobacco) plants expressing different versions of a synthetic horseradish peroxidase, HRP, isoenzyme C gene were constructed. The gene was engineered to include additional sequences coding for either the natural N‐terminal or the C‐terminal extension or both. These constructs were placed under the control of a constitutive promoter (CaMV‐35S) or the tobacco RUBISCO‐SSU light inducible promoter (SSU) and introduced into tobacco using Agrobacterium‐mediated transformation. To study the effects of the N‐ and C‐terminal extensions, the localization of recombinant peroxidase was determined using biochemical and molecular techniques. • Key Results Transgenic tobacco plants can exhibit a ten‐fold increase in peroxidase activity compared with wild‐type tobacco levels, and the majority of this activity is located in the symplast. The N‐terminal extension is essential for the production of high levels of recombinant protein, while the C‐terminal extension has little effect. Differences in levels of enzyme activity and recombinant protein are reflected in transcript levels. • Conclusions There is no evidence to support either preferential secretion or vacuolar targeting of recombinant peroxidase in this heterologous expression system. This leads us to question the postulated targeting roles of these peptide extensions. The N‐terminal extension is essential for high level expression and appears to influence transcript stability or translational efficiency. Plants have been

  15. A proteomic screen reveals the mitochondrial outer membrane protein Mdm34p as an essential target of the F-box protein Mdm30p.

    Science.gov (United States)

    Ota, Kazuhisa; Kito, Keiji; Okada, Satoshi; Ito, Takashi

    2008-10-01

    Ubiquitination plays various critical roles in eukaryotic cellular regulation and is mediated by a cascade of enzymes including ubiquitin protein ligase (E3). The Skp1-Cullin-F-box protein complex comprises the largest E3 family, in each member of which a unique F-box protein binds its targets to define substrate specificity. Although genome sequencing uncovers a growing number of F-box proteins, most of them have remained as "orphans" because of the difficulties in identification of their substrates. To address this issue, we tested a quantitative proteomic approach by combining the stable isotope labeling by amino acids in cell culture (SILAC), parallel affinity purification (PAP) that we had developed for efficient enrichment of ubiquitinated proteins, and mass spectrometry (MS). We applied this SILAC-PAP-MS approach to compare ubiquitinated proteins between yeast cells with and without over-expressed Mdm30p, an F-box protein implicated in mitochondrial morphology. Consequently, we identified the mitochondrial outer membrane protein Mdm34p as a target of Mdm30p. Furthermore, we found that mitochondrial defects induced by deletion of MDM30 are not only recapitulated by a mutant Mdm34p defective in interaction with Mdm30p but alleviated by ubiquitination-mimicking forms of Mdm34p. These results indicate that Mdm34p is a physiologically important target of Mdm30p.

  16. Decolorization of Anthraquinonic Dyes from Textile Effluent Using Horseradish Peroxidase: Optimization and Kinetic Study

    Directory of Open Access Journals (Sweden)

    Nataša Ž. Šekuljica

    2015-01-01

    Full Text Available Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C. The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes.

  17. Decolorization of Anthraquinonic Dyes from Textile Effluent Using Horseradish Peroxidase: Optimization and Kinetic Study

    Science.gov (United States)

    Šekuljica, Nataša Ž.; Prlainović, Nevena Ž.; Stefanović, Andrea B.; Žuža, Milena G.; Čičkarić, Dragana Z.; Mijin, Dušan Ž.; Knežević-Jugović, Zorica D.

    2015-01-01

    Two anthraquinonic dyes, C.I. Acid Blue 225 and C.I. Acid Violet 109, were used as models to explore the feasibility of using the horseradish peroxidase enzyme (HRP) in the practical decolorization of anthraquinonic dyes in wastewater. The influence of process parameters such as enzyme concentration, hydrogen peroxide concentration, temperature, dye concentration, and pH was examined. The pH and temperature activity profiles were similar for decolorization of both dyes. Under the optimal conditions, 94.7% of C.I. Acid Violet 109 from aqueous solution was decolorized (treatment time 15 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.4 mM, dye concentration 30 mg/L, pH 4, and temperature 24°C) and 89.36% of C.I. Acid Blue 225 (32 min, enzyme concentration 0.15 IU/mL, hydrogen peroxide concentration 0.04 mM, dye concentration 30 mg/L, pH 5, and temperature 24°C). The mechanism of both reactions has been proven to follow the two substrate ping-pong mechanism with substrate inhibition, revealing the formation of a nonproductive or dead-end complex between dye and HRP or between H2O2 and the oxidized form of the enzyme. Both chemical oxygen demand and total organic carbon values showed that there was a reduction in toxicity after the enzymatic treatment. This study verifies the viability of use of horseradish peroxidase for the wastewaters treatment of similar anthraquinonic dyes. PMID:25685837

  18. An Enzyme Switch Employing Direct Electrochemical Communication between Horseradish Peroxidase and a Poly(aniline) Film.

    Science.gov (United States)

    Bartlett, P N; Birkin, P R; Wang, J H; Palmisano, F; De Benedetto, G

    1998-09-01

    An enzyme switch, or microelectrochemical enzyme transistor, responsive to hydrogen peroxide was made by connecting two carbon band electrodes (∼10 μm wide, 4.5 mm long separated by a 20-μm gap) with an anodically grown film of poly(aniline). Horseradish peroxidase (EC 1.11.1.7) was either adsorbed onto the poly(aniline) film or immobilized in an insulating poly(1,2-diaminobenzene) polymer grown electrochemically on top of the poly(aniline) film to complete the device. In the completed device, the conductivity of the poly(aniline) film changes from conducting (between - 0.05 and + 0.3 V vs SCE at pH 5) to insulating (>+0.3 V vs SCE at pH 5) on addition of hydrogen peroxide. The change in conductivity is brought about by oxidation of the poly(aniline) film by direct electrochemical communication between the enzyme and the conducting polymer. This was confirmed by measuring the potential of the poly(aniline) film during switching of the conductivity in the presence of hydrogen peroxide. The devices can be reused by rereducing the poly(aniline) electrochemically to a potential below +0.3 V vs SCE. A blind test showed that the device can be used to determine unknown concentrations of H(2)O(2) in solution and that, when used with hydrogen peroxide concentrations below 0.5 mmol dm(-)(3), the same device maybe reused several times. The possible development of devices of this type for use in applications requiring the measurement of low levels of hydrogen peroxide or horseradish peroxidase is discussed.

  19. Oxidative Stress in Horseradish (Armoracia lapathifolia Gilib. Tissues Grown in vitro

    Directory of Open Access Journals (Sweden)

    Petra Peharec

    2011-01-01

    Full Text Available In a previous study it was reported that transformed tissue of horseradish (Armoracia lapathifolia Gilib., obtained by infection of leaf explants with A. tumefaciens, developed two tumour lines with different morphology. One line grew as a completely unorganized tissue (TN – tumour tissue, while the other line grew as a partially organized teratogenous tumour with malformed hyperhydric shoots (TM – teratoma tissue, but did not regenerate the whole plant of normal morphology. The factor responsible for this problem could be the increased production of reactive oxygen species (ROS. Therefore, in this study a possible involvement of activated oxygen metabolism in dedifferentiation and hyperhydricity in TM and TN tissues is investigated. Elevated values of malondialdehyde and protein carbonyl contents found in TM and TN, in comparison with plantlet leaf, confirm the presence of oxidative stress. However, lower H2O2 content was measured in TM and TN. Lipoxygenase (LOX activity was more pronounced in TM and especially in TN compared to leaf, which suggests that the LOX-dependent peroxidation of fatty acids might be one of the causes of oxidative damage. Moreover, significantly higher peroxidase (PRX and ascorbate peroxidase (APX activity as well as the increased number of their isoforms was found in transformed TM and TN in comparison with leaf. On the other hand, significantly lower superoxide dismutase (SOD activity was found in TM and TN, which correlates with lower H2O2 content. High catalase (CAT activity measured in leaf and partially organized TM is consistent with the role of CAT in growth and differentiation. In conclusion, in horseradish transformed tissues that underwent dedifferentiation and hyperhydricity, prominent oxidative damage was found. This result suggests that oxidative stress could be associated with the inability of partially organized teratogenous TM to regenerate plantlets with normal morphology.

  20. A cell-penetrating peptide analogue, P7, exerts antimicrobial activity against Escherichia coli ATCC25922 via penetrating cell membrane and targeting intracellular DNA.

    Science.gov (United States)

    Li, Lirong; Shi, Yonghui; Cheng, Xiangrong; Xia, Shufang; Cheserek, Maureen Jepkorir; Le, Guowei

    2015-01-01

    The antibacterial activities and mechanism of a new P7 were investigated in this study. P7 showed antimicrobial activities against five harmful microorganisms which contaminate and spoil food (MIC=4-32 μM). Flow cytometry and scanning electron microscopy analyses demonstrated that P7 induced pore-formation on the cell surface and led to morphological changes but did not lyse cell. Confocal fluorescence microscopic observations and flow cytometry analysis expressed that P7 could penetrate the Escherichia coli cell membrane and accumulate in the cytoplasm. Moreover, P7 possessed a strong DNA binding affinity. Further cell cycle analysis and change in gene expression analysis suggested that P7 induced a decreased expression in the genes involved in DNA replication. Up-regulated expression genes encoding DNA damage repair. This study suggests that P7 could be applied as a candidate for the development of new food preservatives as it exerts its antibacterial activities by penetrating cell membranes and targets intracellular DNA. Copyright © 2014 Elsevier Ltd. All rights reserved.

  1. Flunarizine Prevents Hepatitis C Virus Membrane Fusion in a Genotype-dependent Manner by Targeting the Potential Fusion Peptide within E1

    Science.gov (United States)

    Perin, Paula M.; Haid, Sibylle; Brown, Richard J. P.; Doerrbecker, Juliane; Schulze, Kai; Zeilinger, Carsten; von Schaewen, Markus; Heller, Brigitte; Vercauteren, Koen; Luxenburger, Eva; Baktash, Yasmine M.; Vondran, Florian W. R.; Speerstra, Sietkse; Awadh, Abdullah; Mukhtarov, Furkat; Schang, Luis M; Kirschning, Andreas; Müller, Rolf; Guzman, Carlos A.; Kaderali, Lars; Randall, Glenn; Meuleman, Philip; Ploss, Alexander; Pietschmann, Thomas

    2015-01-01

    To explore mechanisms of hepatitis C virus (HCV) replication we screened a compound library including licensed drugs. Flunarizine, a diphenylmethylpiperazine used to treat migraine, inhibited HCV cell entry in vitro and in vivo in a genotype-dependent fashion. Analysis of mosaic viruses between susceptible and resistant strains revealed that E1 and E2 glycoproteins confer susceptibility to flunarizine. Time of addition experiments and single particle tracking of HCV demonstrated that flunarizine specifically prevents membrane fusion. Related phenothiazines and pimozide also inhibited HCV infection and preferentially targeted HCV genotype 2 viruses. However, phenothiazines and pimozide exhibited improved genotype coverage including the difficult to treat genotype 3. Flunarizine-resistant HCV carried mutations within the alleged fusion peptide and displayed cross-resistance to these compounds, indicating that these drugs have a common mode of action. Conclusion: These observations reveal novel details about HCV membrane fusion. Moreover, flunarizine and related compounds represent first-in-class HCV fusion inhibitors that merit consideration for repurposing as cost-effective component of HCV combination therapies. PMID:26248546

  2. Intramolecular dynamics within the N-Cap-SH3-SH2 regulatory unit of the c-Abl tyrosine kinase reveal targeting to the cellular membrane.

    Science.gov (United States)

    de Oliveira, Guilherme A P; Pereira, Elen G; Ferretti, Giulia D S; Valente, Ana Paula; Cordeiro, Yraima; Silva, Jerson L

    2013-09-27

    c-Abl is a key regulator of cell signaling and is under strict control via intramolecular interactions. In this study, we address changes in the intramolecular dynamics coupling within the c-Abl regulatory unit by presenting its N-terminal segment (N-Cap) with an alternative function in the cell as c-Abl becomes activated. Using small angle x-ray scattering, nuclear magnetic resonance, and confocal microscopy, we demonstrate that the N-Cap and the Src homology (SH) 3 domain acquire μs-ms motions upon N-Cap association with the SH2-L domain, revealing a stabilizing synergy between these segments. The N-Cap-myristoyl tether likely triggers the protein to anchor to the membrane because of these flip-flop dynamics, which occur in the μs-ms time range. This segment not only presents the myristate during c-Abl inhibition but may also trigger protein localization inside the cell in a functional and stability-dependent mechanism that is lost in Bcr-Abl(+) cells, which underlie chronic myeloid leukemia. This loss of intramolecular dynamics and binding to the cellular membrane is a potential therapeutic target.

  3. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Rama Garimella

    2017-03-01

    Full Text Available Osteosarcoma (OS is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a inflammation and immunity; (b formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium, quantity of gap junctions and skeletogenesis; (c bone mineral density; and (d cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12, bone morphogenetic factor-1 (BMP1, SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4, Matrix extracellular phosphoglycoprotein (MEPE, Integrin, β4 (ITGBP4, Matrix Metalloproteinase -1, -28 (MMP1 and MMP28, and signal transducer and activator of transcription-4 (STAT4 in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7, but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

  4. Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

    Science.gov (United States)

    Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

    2017-01-01

    Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology. PMID:28300755

  5. Limited and selective transfer of plasma membrane glycoproteins to membrane of secondary lysosomes

    International Nuclear Information System (INIS)

    Haylett, T.; Thilo, L.

    1986-01-01

    Radioactive galactose, covalently bound to cell surface glycoconjugates on mouse macrophage cells, P388D 1 , was used as a membrane marker to study the composition, and the kinetics of exchange, of plasma membrane-derived constituents in the membrane of secondary lysosomes. Secondary lysosomes were separated from endosomes and plasma membrane by self-forming Percoll density gradients. Horseradish peroxidase, taken up by fluid-phase pinocytosis, served as a vesicle contents marker to monitor transfer of endosomal contents into secondary lysosomes. Concurrently, the fraction of plasma membrane-derived label of secondary lysosomes increased by first order kinetics from 4 PAGE, labeled molecules of M/sub r/ 160-190 kD were depleted and of the M/sub r/ 100-120 kD were enriched in lysosome membrane compared with the relative composition of label on the cell surface. No corresponding selectivity was observed for the degradation of label, with all M/sub r/ classes being affected to the same relative extent. The results indicate that endocytosis-derived transfer of plasma membrane constitutents to secondary lysosomes is a limited and selective process, and that only ∼1% of internalized membrane is recycled via a membrane pool of secondary lysosomes

  6. Plasma membrane profiling defines an expanded class of cell surface proteins selectively targeted for degradation by HCMV US2 in cooperation with UL141.

    Directory of Open Access Journals (Sweden)

    Jye-Lin Hsu

    2015-04-01

    Full Text Available Human cytomegalovirus (HCMV US2, US3, US6 and US11 act in concert to prevent immune recognition of virally infected cells by CD8+ T-lymphocytes through downregulation of MHC class I molecules (MHC-I. Here we show that US2 function goes far beyond MHC-I degradation. A systematic proteomic study using Plasma Membrane Profiling revealed US2 was unique in downregulating additional cellular targets, including: five distinct integrin α-chains, CD112, the interleukin-12 receptor, PTPRJ and thrombomodulin. US2 recruited the cellular E3 ligase TRC8 to direct the proteasomal degradation of all its targets, reminiscent of its degradation of MHC-I. Whereas integrin α-chains were selectively degraded, their integrin β1 binding partner accumulated in the ER. Consequently integrin signaling, cell adhesion and migration were strongly suppressed. US2 was necessary and sufficient for degradation of the majority of its substrates, but remarkably, the HCMV NK cell evasion function UL141 requisitioned US2 to enhance downregulation of the NK cell ligand CD112. UL141 retained CD112 in the ER from where US2 promoted its TRC8-dependent retrotranslocation and degradation. These findings redefine US2 as a multifunctional degradation hub which, through recruitment of the cellular E3 ligase TRC8, modulates diverse immune pathways involved in antigen presentation, NK cell activation, migration and coagulation; and highlight US2's impact on HCMV pathogenesis.

  7. One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III).

    Science.gov (United States)

    Guo, Shaofen; Cao, Rui; Lu, Aihua; Zhou, Qing; Lu, Tianhong; Ding, Xiaolan; Li, Chaojun; Huang, Xiaohua

    2008-05-01

    One of the possible mechanisms for the inhibition effect of Tb(III) on peroxidase activity in horseradish (Armoracia rusticana) treated with Tb(III) was investigated using some biophysical and biochemical methods. Firstly, it was found that a large amount of Tb(III) can be distributed on the cell wall, that some Tb(III) can enter into the horseradish cell, indicating that peroxidase was mainly distributed on cell wall, and thus that Tb(III) would interact with horseradish peroxidase (HRP) in the plant. In addition, peroxidase bioactivity was decreased in the presence of Tb(III). Secondly, a new peroxidase-containing Tb(III) complex (Tb-HRP) was obtained from horseradish after treatment with Tb(III); the molecular mass of Tb-HRP is near 44 kDa and the pI is about 8.80. Thirdly, the electrocatalytic activity of Tb-HRP is much lower than that of HRP obtained from horseradish without treatment with Tb(III). The decrease in the activity of Tb-HRP is due to the destruction (unfolding) of the conformation in Tb-HRP. The planarity of the heme active center in the Tb-HRP molecule was increased and the extent of exposure of Fe(III) in heme was decreased, leading to inhibition of the electron transfer. The microstructure change in Tb-HRP might be the result of the inhibition effect of Tb(III) on peroxidase activity in horseradish.

  8. A membrane-based ELISA assay for the herbicide Isoproturon in soil samples

    OpenAIRE

    Baskeyfield, Damian E. H.; Davis, Frank; Magan, Naresh; Tothill, Ibtisam E.

    2012-01-01

    A membrane based enzyme linked immunosorbent assay (MELISA) for the detection of a common herbicide, isoproturon is described. A heterogeneous competitive ELISA was the format chosen for isoproturon detection. An immunoassay system with a horseradish peroxidase (HRP) labeled polyclonal antibody preparation was developed and characterized before suitable sensitivity and selectivity for isoproturon were attained. After development as a microtiter plate immunoassay, the system was transferred to...

  9. The metabolically active subpopulation in Pseudomonas aeruginosa biofilms survives exposure to membrane-targeting antimicrobials via distinct molecular mechanisms

    DEFF Research Database (Denmark)

    Chiang, Wen-Chi; Pamp, Sünje Johanna; Nilsson, Martin

    2012-01-01

    encoding lipopolysaccharide modification enzymes, as well as on the mexAB-oprM, mexCD-oprJ, and muxABC-opmB genes encoding antimicrobial efflux pumps, but does not depend on the mexPQ-opmE efflux pump genes. Development of chlorhexidine-tolerant subpopulations was found to depend on the mexCD-oprJ genes......, but does not depend on the pmr, mexAB-oprM, mexPQ-opmE, or muxABC-opmB genes. Tolerance to SDS and EDTA in P. aeruginosa biofilms is linked to metabolically active cells, but does not depend on the pmr, mexAB, mexCD, mexPQ, or muxABC genes. Our data suggest that the active subpopulation in P. aeruginosa......-targeting compounds colistin, EDTA, SDS, and chlorhexidine resulted in the same spatial distribution of live and dead bacteria, we investigated whether tolerance to these compounds originated from the same molecular mechanisms. Development of colistin-tolerant subpopulations was found to depend on the pmr genes...

  10. Evaluating strategies to improve glucosinolate concentration and root yield of field-grown horseradish in a Mediterranean environment: preliminary results

    Directory of Open Access Journals (Sweden)

    Anna Rita Rivelli

    2016-03-01

    Full Text Available Horseradish is grown for its enlarged taproot that is widely used as a dish condiment and as a source of horseradish peroxidase. Nowadays, the species is gaining great interest due to the richness in bioactive compounds that besides providing a high nutritional value are tested for innovative applications in different fields. Nevertheless, the effect of crop management on root yield and glucosinolates (GLS biosynthesis is poorly documented. Aim of this study was to evaluate the root yield and GLS concentration of two field-grown horseradish accessions (Cor and Mon grown with nitrogen (N alone and both N and sulphur (S (-N-S, +N-S and +N+S treatments and harvested at different times [late autumn (LA, 2011 and 2012, early spring (ES, 2012]. Yield increased throughout the harvests up to 48% on average of the fertilised treatments and 25% in the unfertilised control. Conversely, root GLS concentration significantly declined in the unfertilised control throughout the harvests [from 7.6 in LA_2011 to 1.43 μmol/g dry matter (DM in LA_2012] while it highly increased in plants grown with N alone and with both N and S by 46 and 98%, respectively, from LA_2011 to ES_2012 (up to 11.9 and 21.1 μmol/g DM, respectively; then it drastically decreased by 80% on average, in the next harvest. Among individual GLS, the concentration of sinigrin and nasturtin similarly varied as effect of the analysed factors, showing the highest values in Cor accession. The data show that although the level of GLS is highly dependent on genotype, fertilisation and harvesting date may play a primary role in determining the yield and GLS concentration in horseradish root.

  11. Glucosinolate profile and distribution among plant tissues and phenological stages of field-grown horseradish.

    Science.gov (United States)

    Agneta, Rosa; Lelario, Filomena; De Maria, Susanna; Möllers, Christian; Bufo, Sabino Aurelio; Rivelli, Anna Rita

    2014-10-01

    Profile and distribution of glucosinolates (GLS) were detected in plant tissues of horseradish at different developmental stages: beginning of vegetative re-growth, flowering and silique formation. The GLS profile varied widely in the different tissues: we identified 17 GLS in roots and sprouts, one of which was not previously characterized in horseradish, i.e. the 2(S)-hydroxy-2-phenylethyl-GLS (glucobarbarin) and/or 2(R)-hydroxy-2-phenylethyl-GLS (epiglucobarbarin), 11 already found in the roots, including the putative 2-methylsulfonyl-oxo-ethyl-GLS, and 5 previously recognized only in the sprouts. Fifteen of those GLS were also identified in young and cauline leaves, 12 in the mature leaves and 13 in the inflorescences. No difference in GLS profile was observed in plant among the phenological stages. Differences in concentrations of GLS, quantified as desulfated, were found in plant. At the beginning of vegetative re-growth, sprouts while showing the same profile of the roots were much richer in GLS having the highest total GLS concentrations (117.5 and 7.7μmolg(-1) dry weight in sprouts and roots, respectively). During flowering and silique forming stages, the roots still maintained lower amount of total GLS (7.4μmolg(-1) of dry weight, on average) with respect to the epigeous tissues, in which mature and young leaves showed the highest total concentrations (70.5 and 73.8μmolg(-1) of dry weight on average, respectively). Regardless of the phenological stages, the aliphatic GLS were always predominant in all tissues (95%) followed by indolic (2.6%) and benzenic (2.4%) GLS. Sinigrin contributed more than 90% of the total GLS concentration. Aliphatic GLS concentrations were much higher in the epigeous tissues, particularly in the mature and young leaves, while benzenic and indolic GLS concentrations were higher in the roots. Through the phenological stages, GLS concentration increased in young and mature leaves and decreased in cauline leaves and inflorescences

  12. Correlation of quinone reductase activity and allyl isothiocyanate formation among different genotypes and grades of horseradish roots.

    Science.gov (United States)

    Ku, Kang-Mo; Jeffery, Elizabeth H; Juvik, John A; Kushad, Mosbah M

    2015-03-25

    Horseradish (Armoracia rusticana) is a perennial crop and its ground root tissue is used in condiments because of the pungency of the glucosinolate (GS)-hydrolysis products allyl isothiocyanate (AITC) and phenethyl isothiocyanate (PEITC) derived from sinigrin and gluconasturtiin, respectively. Horseradish roots are sold in three grades: U.S. Fancy, U.S. No. 1, and U.S. No. 2 according to the USDA standards. These grading standards are primarily based on root diameter and length. There is little information on whether root grades vary in their phytochemical content or potential health promoting properties. This study measured GS, GS-hydrolysis products, potential anticancer activity (as quinone reductase inducing activity), total phenolic content, and antioxidant activities from different grades of horseradish accessions. U.S. Fancy showed significantly higher sinigrin and AITC concentrations than U.S. No. 1 ,whereas U.S. No. 1 showed significantly higher concentrations of 1-cyano 2,3-epithiopropane, the epithionitrile hydrolysis product of sinigrin, and significantly higher total phenolic concentrations than U.S. Fancy.

  13. A Novel ¹¹¹In-Labeled Anti-Prostate-Specific Membrane Antigen Nanobody for Targeted SPECT/CT Imaging of Prostate Cancer.

    Science.gov (United States)

    Chatalic, Kristell L S; Veldhoven-Zweistra, Joke; Bolkestein, Michiel; Hoeben, Sander; Koning, Gerben A; Boerman, Otto C; de Jong, Marion; van Weerden, Wytske M

    2015-07-01

    Prostate-specific membrane antigen (PSMA) is overexpressed in prostate cancer (PCa) and a promising target for molecular imaging and therapy. Nanobodies (single-domain antibodies, VHH) are the smallest antibody-based fragments possessing ideal molecular imaging properties, such as high target specificity and rapid background clearance. We developed a novel anti-PSMA Nanobody (JVZ-007) for targeted imaging and therapy of PCa. Here, we report on the application of the (111)In-radiolabeled Nanobody for SPECT/CT imaging of PCa. A Nanobody library was generated by immunization of a llama with 4 human PCa cell lines. Anti-PSMA Nanobodies were captured by biopanning on PSMA-overexpressing cells. JVZ-007 was selected for evaluation as an imaging probe. JVZ-007 was initially produced with a c-myc-hexahistidine (his) tag allowing purification and detection. The c-myc-his tag was subsequently replaced by a single cysteine at the C terminus, allowing site-specific conjugation of chelates for radiolabeling. JVZ-007-c-myc-his was conjugated to 2-(4-isothiocyanatobenzyl)-diethylenetriaminepentaacetic acid (p-SCN-DTPA) via the lysines, whereas JVZ-007-cys was conjugated to maleimide-DTPA via the C-terminal cysteine. PSMA targeting was analyzed in vitro by cell-binding experiments using flow cytometry, autoradiography, and internalization assays with various PCa cell lines and patient-derived xenografts (PDXs). The targeting properties of radiolabeled Nanobodies were evaluated in vivo in biodistribution and SPECT/CT imaging experiments, using nude mice bearing PSMA-positive PC-310 and PSMA-negative PC-3 tumors. JVZ-007 was successfully conjugated to DTPA for radiolabeling with (111)In at room temperature. (111)In-JVZ007-c-myc-his and (111)In-JVZ007-cys internalized in LNCaP cells and bound to PSMA-expressing PDXs and, importantly, not to PSMA-negative PDXs and human kidneys. Good tumor targeting and fast blood clearance were observed for (111)In-JVZ-007-c-myc-his and (111)In

  14. Nanodisc-solubilized membrane protein library reflects the membrane proteome

    OpenAIRE

    Marty, Michael T.; Wilcox, Kyle C.; Klein, William L.; Sligar, Stephen G.

    2013-01-01

    The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membr...

  15. Inhibition mechanism of lanthanum ion on the activity of horseradish peroxidase in vitro

    Science.gov (United States)

    Guo, Shaofen; Wang, Lihong; Lu, Aihua; Lu, Tianhong; Ding, Xiaolan; Huang, Xiaohua

    2010-02-01

    In order to understand the inhibition mechanism of lanthanum ion (La 3+) on the activity of horseradish peroxidase (HRP), the effects of La 3+ on the activity, electron transfer and conformation of HRP in vitro were investigated by using cyclic voltammetry (CV), atomic force microscopy (AFM), circular dichroism (CD), high performance liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectroscopy (MALDI-TOF/MS) and inductively coupled plasma mass spectrometry (ICP-MS). It was found that La 3+ can combine with the amide groups of the polypeptide chain in HRP molecule, forming the complex of La 3+ and HRP (La-HRP). The formation of the La-HRP complex causes the destruction of the native structure of HRP molecule, leading to the decrease in the non-planarity of the porphyrin ring in the heme group of HRP molecule, and then in the exposure extent of active center, Fe(III) of the porphyrin ring of HRP molecule. Thus, the direct electrochemical and catalytic activities of HRP are decreased. It is a possible inhibition mechanism of La 3+ on the activity of peroxidase.

  16. Luminol, horseradish peroxidase, and glucose oxidase ternary functionalized graphene oxide for ultrasensitive glucose sensing.

    Science.gov (United States)

    Li, Fang; Ma, Wenjing; Liu, Jiachang; Wu, Xiang; Wang, Yan; He, Jianbo

    2018-01-01

    Luminol, horseradish peroxidase (HRP), and glucose oxidase (GOx) ternary functionalized graphene oxide (HRP/GOx-luminol-GO) with excellent chemiluminescence (CL) activity and specific enzymatic property was prepared via a simple and general strategy for the first time. In this approach, luminol functionalized GO (luminol-GO) was prepared by gently stirring GO with luminol. Then HRP and GOx were further co-immobilized onto the surface of luminol-GO by storing HRP and GOx with luminol-GO at 4 °C overnight, to form HRP/GOx-luminol-GO bionanocomposites. The synthesized HRP/GOx-luminol-GO could react with H 2 O 2 generated from GOx catalyzed glucose oxidization reaction, to produce strong CL emission in the presence of co-immobilized HRP. Thus, we developed an ultrasensitive, homogeneous, reagentless, selective, and simple CL sensing system for glucose detection. The resulting biosensors exhibited ultra-wide linear range from 5.0 nM to 5.0 mM, and an ultra-low detection limit of 1.2 nM, which was more than 3 orders of magnitude lower than previously reported methods. Furthermore, the sensing system was successfully applied for the detection of glucose in human blood samples.

  17. Mechanisms for Covalent Immobilization of Horseradish Peroxidase on Ion-Beam-Treated Polyethylene

    Directory of Open Access Journals (Sweden)

    Alexey V. Kondyurin

    2012-01-01

    Full Text Available The surface of polyethylene was modified by plasma immersion ion implantation. Structure changes including carbonization and oxidation were observed. High surface energy of the modified polyethylene was attributed to the presence of free radicals on the surface. The surface energy decay with storage time after treatment was explained by a decay of the free radical concentration while the concentration of oxygen-containing groups increased with storage time. Horseradish peroxidase was covalently attached onto the modified surface by the reaction with free radicals. Appropriate blocking agents can block this reaction. All aminoacid residues can take part in the covalent attachment process, providing a universal mechanism of attachment for all proteins. The native conformation of attached protein is retained due to hydrophilic interactions in the interface region. The enzymatic activity of covalently attached protein remained high. The long-term activity of the modified layer to attach protein is explained by stabilisation of unpaired electrons in sp2 carbon structures. A high concentration of free radicals can give multiple covalent bonds to the protein molecule and destroy the native conformation and with it the catalytic activity. The universal mechanism of protein attachment to free radicals could be extended to various methods of radiation damage of polymers.

  18. Self-Assembled Complexes of Horseradish Peroxidase with Magnetic Nanoparticles Showing Enhanced Peroxidase Activity

    KAUST Repository

    Corgié, Stéphane C.

    2012-02-15

    Bio-nanocatalysts (BNCs) consisting of horseradish peroxidase (HRP) self-assembled with magnetic nanoparticles (MNPs) enhance enzymatic activity due to the faster turnover and lower inhibition of the enzyme. The size and magnetization of the MNPs affect the formation of the BNCs, and ultimately control the activity of the bound enzymes. Smaller MNPs form small clusters with a low affinity for the HRP. While the turnover for the bound fraction is drastically increased, there is no difference in the H 2O 2 inhibitory concentration. Larger MNPs with a higher magnetization aggregate in larger clusters and have a higher affinity for the enzyme and a lower substrate inhibition. All of the BNCs are more active than the free enzyme or the MNPs (BNCs > HRP ≤laquo; MNPs). Since the BNCs show surprising resilience in various reaction conditions, they may pave the way towards new hybrid biocatalysts with increased activities and unique catalytic properties for magnetosensitive enzymatic reactions. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  19. Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

    International Nuclear Information System (INIS)

    Ran Qin; Peng Ru; Liang Cong; Ye Siqiu; Xian Yuezhong; Zhang Wenjing; Jin Litong

    2011-01-01

    Graphical abstract: Hydrogen peroxide biosensor was developed based on electrochemically assisted aryldiazonium salt chemistry and click chemistry. Highlights: → A simple, versatile two-step approach, which is based on electrochemically assisted aryldiazonium salt chemistry and Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction has been developed for covalent redox proteins immobilization and biosensing for the first time. In this work, azido group modified HRP was covalently grafted on 4-ethylnylphenyl diazonium compound via CuAAC reaction and a novel electrochemical hydrogen peroxide biosensor was successfully fabricated. - Abstract: In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H 2 O 2 with a linear range of 5.0 x 10 -6 to 9.3 x 10 -4 mol L -1 .

  20. Direct electrochemistry of horseradish peroxidase immobilized on electrografted 4-ethynylphenyl film via click chemistry

    Energy Technology Data Exchange (ETDEWEB)

    Ran Qin; Peng Ru; Liang Cong; Ye Siqiu [Department of Chemistry, East China Normal University, Shanghai 200062 (China); Xian Yuezhong, E-mail: yzxian@chem.ecnu.edu.cn [Department of Chemistry, East China Normal University, Shanghai 200062 (China); Zhang Wenjing; Jin Litong [Department of Chemistry, East China Normal University, Shanghai 200062 (China)

    2011-07-04

    Graphical abstract: Hydrogen peroxide biosensor was developed based on electrochemically assisted aryldiazonium salt chemistry and click chemistry. Highlights: > A simple, versatile two-step approach, which is based on electrochemically assisted aryldiazonium salt chemistry and Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction has been developed for covalent redox proteins immobilization and biosensing for the first time. In this work, azido group modified HRP was covalently grafted on 4-ethylnylphenyl diazonium compound via CuAAC reaction and a novel electrochemical hydrogen peroxide biosensor was successfully fabricated. - Abstract: In this paper, a simple two-step approach for redox protein immobilization was introduced. Firstly, alkynyl-terminated film was formed on electrode surface by electrochemical reduction of 4-ethylnylphenyl (4-EP) diazonium compound. Then, horseradish peroxidase (HRP) modified with azido group was covalently immobilized onto the electrografted film via click reaction. Reflection absorption infrared (RAIR) spectroscopy and electrochemical methods were used to characterize the modification process. The results indicate that HRP retains its native structure and shows fast direct electron transfer. Moreover, the immobilized HRP shows excellent electrocatalytic reduction activity toward H{sub 2}O{sub 2} with a linear range of 5.0 x 10{sup -6} to 9.3 x 10{sup -4} mol L{sup -1}.

  1. Direct electrochemistry and electrocatalysis of horseradish peroxidase immobilized in graphene oxide–Nafion nanocomposite film

    International Nuclear Information System (INIS)

    Zhang Lili; Cheng Huhu; Zhang Huimin; Qu Liangti

    2012-01-01

    Direct electron transfer of horseradish peroxidase (HRP) immobilized in graphene oxide (GO)–Nafion nanocomposite film and its application as a new biosensor was investigated with electrochemical methods. Immobilized HRP shows a pair of well-defined redox waves and retains its bioelectrocatalytic activity for the reduction of H 2 O 2 and O 2 . As a new sensor with excellent electrocatalytic response to the reduction of H 2 O 2 and O 2 , calibrations with good linear relationships were obtained from 1.0 μmol L −1 to 1.0 mmol L −1 for H 2 O 2 and from 0.5 μmol L −1 to 18.6 μmol L −1 for O 2 with the detection limits of 4.0 × 10 −7 mol L −1 for H 2 O 2 and 1.0 × 10 −7 mol L −1 for O 2 at a signal-to-noise ratio of 3. Additionally, the responses showed Michaelis–Menten behavior with K m app values of 0.684 mmol L −1 for H 2 O 2 and 0.0160 mmol L −1 for O 2 . Moreover, the cathodic peak current of an HRP/GO/Nafion/GCE biosensor decreases by less than 5% after 4 weeks. These results reveal that GO can be conveniently incorporated into a polymer nanocomposite for fabrication of new GO-based biosensors.

  2. Structure-activity relationships and molecular docking of thirteen synthesized flavonoids as horseradish peroxidase inhibitors.

    Science.gov (United States)

    Mahfoudi, Reguia; Djeridane, Amar; Benarous, Khedidja; Gaydou, Emile M; Yousfi, Mohamed

    2017-10-01

    For the first time, the structure-activity relationships of thirteen synthesized flavonoids have been investigated by evaluating their ability to modulate horseradish peroxidase (HRP) catalytic activity. Indeed, a modified spectrophotometrically method was carried out and optimized using 4-methylcatechol (4-MC) as peroxidase co-substrate. The results show that these flavonoids exhibit a great capacity to inhibit peroxidase with Ki values ranged from 0.14±0.01 to 65±0.04mM. Molecular docking has been achieved using Auto Dock Vina program to discuss the nature of interactions and the mechanism of inhibition. According to the docking results, all the flavonoids have shown great binding affinity to peroxidase. These molecular modeling studies suggested that pyran-4-one cycle acts as an inhibition key for peroxidase. Therefore, potent peroxidase inhibitors are flavonoids with these structural requirements: the presence of the hydroxyl (OH) group in 7, 5 and 4' positions and the absence of the methoxy (O-CH 3 ) group. Apigenin contributed better in HRP inhibitory activity. The present study has shown that the studied flavonoids could be promising HRP inhibitors, which can help in developing new molecules to control thyroid diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Direct Electrochemistry of Horseradish Peroxidase on NiO Nanoflower Modified Electrode and Its Electrocatalytic Activity

    Directory of Open Access Journals (Sweden)

    Lijun Yan

    2016-09-01

    Full Text Available In this paper nickel oxide (NiO nanoflower was synthesized and used for the realization of direct electrochemistry of horseradish peroxidase (HRP. By using carbon ionic liquid electrode (CILE as the substrate electrode, NiO-HRP composite was casted on the surface of CILE with chitosan (CTS as the film forming material and the modified electrode was denoted as CTS/NiO-HRP/CILE. UV-Vis absorption and FT-IR spectra confirmed that HRP retained its native structure after mixed with NiO nanoflower. Direct electron transfer of HRP on the modified electrode was investigated by cyclic voltammetry with a pair of quasi-reversible redox waves appeared, indicating that the presence of NiO nanoflower on the electrode surface could accelerate the electron transfer rate between the electroactive center of HRP and the substrate electrode. Electrochemical behaviors of HRP on the modified electrode were carefully investigated. The HRP modified electrode showed excellent electrocatalytic activity to the reduction of trichloroacetic acid with wider linear range and lower detection limit. Therefore the presence of NiO nanoflower could provide a friendly biocompatible interface for immobilizing biomolecules and keeping their native structure. The fabricated electrochemical biosensor displayed the advantages such as high sensitivity, good reproducibility and long-term stability. This work is licensed under a Creative Commons Attribution 4.0 International License.

  4. Effect of ozone and histamine on airway permeability to horseradish peroxidase in guinea pigs

    International Nuclear Information System (INIS)

    Miller, P.D.; Gordon, T.; Warnick, M.; Amdur, M.O.

    1986-01-01

    Airway permeability was studied in groups of male guinea pigs at 2, 8, and 24 h after a 1-h exposure to 1 ppm ozone or at 2 h after a 1-h exposure to filtered air (control). Intratracheal administration of 2 mg horseradish peroxidase (HRP) was followed by blood sampling at 5-min intervals up to 30 min. The rate of appearance of HRP in plasma was significantly higher at 2 and 8 h after ozone exposure than that found in animals examined 2 h after air exposure or 24 h after ozone exposure. A dose of 0.12 mg/kg of subcutaneous histamine given after the 15 min blood sample significantly increased the already elevated permeability seen at 2 h post ozone, but had no effect on animals exposed to filtered air 2 h earlier or to ozone 24 h earlier. No difference was seen in the amount of subcutaneous radiolabeled histamine in the lungs of animals exposed 2 h earlier either to air or to ozone. These data indicate that a short-term exposure to ozone produced a reversible increase in respiratory epithelial permeability to HRP in guinea pigs. The potentiation of this increased permeability by histamine may be another manifestation of ozone-induced hyperreactivity

  5. Horseradish peroxidase-screen printed biosensors for determination of Ochratoxin A.

    Science.gov (United States)

    Alonso-Lomillo, M Asunción; Domínguez-Renedo, Olga; Román, Lorena Del Torno-de; Arcos-Martínez, M Julia

    2011-02-28

    This work summarizes the manufacturing procedure of Horseradish peroxidase (HRP) based biosensors for the determination of the mycotoxin Ochratoxin A (OTA). The biosensors have been fabricated using the single technology of screen-printing. That is to say, an HRP containing ink has been directly screen-printed onto carbon electrodes, which offers a higher rapidity and simplicity in the manufacturing process of biosensors for OTA determination. The formal redox potential of the Fe(III/II) moiety of HRP has been used to demonstrate the effective loading of enzyme into the ink. The chronoamperometric oxidation current registered has been successfully related to the concentration of OTA in solution from different samples, including beer ones. Under the optimum conditions of the experimental variables, precision in terms of reproducibility and repeatability has been calculated in the concentration range from 23.85 to 203.28 nM. A relative standard deviation for the slopes of 10% (n = 4) was obtained for reproducibility. In the case of repeatability, the biosensor retained a 30% of the initial sensitivity after the third calibration. The average capability of detection for 0.05% probabilities of false positive and negative was 26.77 ± 3.61 nM (α = 0.05 and β=0.05, n = 3). Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Comparison of monomeric and polymeric horseradish peroxidase as labels in competitive ELISA for small molecule detection

    International Nuclear Information System (INIS)

    Li, Dongyang; Ying, Yibin; Wu, Jian; Niessner, Reinhard; Knopp, Dietmar

    2013-01-01

    We have developed a simple and sensitive competitive enzyme-linked immunosorbent assay (ELISA) to determine aflatoxin B1 (as a model small analyte) and using streptavidin-polymeric horseradish peroxidase complex (SApolyHRP) as a label for signal amplification. The performance of the assay was evaluated by comparing it with the classical indirect competitive ELISA using HRP labeled anti-mouse IgG as the tracer antibody. The results indicate that the SApolyHRP-based competitive ELISA exhibits a typically 2.4-fold steeper slope of the linear working range of the calibration curve compared to the monomeric HRP based classical ELISA, i.e., the sensitivity was increased. The SApolyHRP conjugate causes a typically 19-fold stronger signal generation in comparison to the traditional HRP labeled anti-mouse IgG at the same concentration (25 ng mL −1 ). Moreover, the SApolyHRP-based assay has a much wider linear range and a 3.8-fold better signal-to-noise ratio. Considering its simplicity, sensitivity and ease of operation, this competitive ELISA is considered to be a promising tool for small molecule immuno detection. (author)

  7. Competitive horseradish peroxidase-linked aptamer assay for sensitive detection of Aflatoxin B1.

    Science.gov (United States)

    Sun, Linlin; Zhao, Qiang

    2018-03-01

    Aflatoxin B1 (AFB1) is one of highly toxic mycotoxins and a known human carcinogen. The frequent contamination of AFB1 in food products and large health risk of AFB1 have raised global concerns. Sensitive detection of AFB1 is of vital importance and highly demanded. Herein, we reported a competitive horseradish peroxidase (HRP)-linked aptamer assay for AFB1, combining the advantages of aptamer for affinity binding and enzyme label for signal amplification. In this assay, free AFB1 in solution competed with a covalent conjugate of bovine serum albumin-AFB1 (BSA-AFB1) coated on the wells of microplate in binding to the HRP-labeled aptamer probe. HRP attached on BSA-AFB1 in the wells catalyzed the conversion of substrates into products, allowing the final detection of AFB1 through measurement of the generated products. When TMB (3,3',5,5'-tetramethylbenzidine dihydrochloride) was used as substrate, absorbance analysis of the product of enzyme reaction enabled the detection of AFB1 at 0.2nM. We further lowered the detection limit of AFB1 to 0.01nM through chemiluminescence analysis by using chemiluminescence substrate of HRP. This assay enabled the detection of AFB1 in complex sample matrix, such as diluted white wine and maize flour. This assay provides a simple, sensitive and rapid method for AFB1 determination. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Thermal stability and degradation kinetics of polyphenols and polyphenylenediamines enzymatically synthesized by horseradish peroxidase

    International Nuclear Information System (INIS)

    Park, Hansol; Ryu, Keungarp; Kwon, Oyul

    2015-01-01

    Various substituted phenols and phenylenediamines were enzymatically polymerized by horseradish peroxidase in 80% (v/v) organic solvents-aqueous buffer (100 mM sodium acetate, pH 5) mixtures with H 2 O 2 as the oxidant. The thermal stability of the polymers was investigated by thermogravimetric analysis (TGA) and represented by the char yield (wt% of the initial polymer mass) after being heated at 800 .deg. C. Poly(p-phenylphenol) had the highest thermal stability among the synthesized polymers with a char yield of 47 wt%. The polymers containing amino groups such as poly(p-aminophenol) and polyphenylenediamines were also shown to possess high thermal stabilities. The activation energies for the thermal degradation of the polymers determined by derivative thermogravimetric analysis (DTG) using Horowitz-Metzger's pseudo-first-order kinetics were in the range between 23-65 kJ/mol and comparable to those of the chemically synthesized polymers. Dynamic structural changes of the enzymatically synthesized polymers upon heating were studied by differential scanning calorimetry (DSC). The DSC curves of poly(p-phenylphenol) showed a broad exothermic peaks between 150-250 .deg. C, indicating that the polymer undergoes complex structural transitions in the temperature range. On the other hand, the DSC curves of the poly(p-aminophenol) and the poly(p-phenylenediamine) which contain amino groups showed strong sharp endothermic peaks near 150 .deg. C, implying that these polymers possess homogeneous oriented structures which undergo a concerted structural disintegration upon heating.

  9. Quercetin oxidation by horseradish peroxidase: The effect of UV-B irradiation

    Directory of Open Access Journals (Sweden)

    Savić Saša R.

    2013-01-01

    Full Text Available Horseradish peroxidase (HRP, a highly-investigated member of the peroxidase family has been known, among many other biological activities, to catalyze the oxidation of flavonoids and phenolic substrates overall, including quercetin. On the other hand, quercetin is very well known for its antioxidant activities, which in the case of UV external radiation is exibited partly in a preventive manner since it is an excellent UV-absorber. Therefore the aim of this investigation is to study quercetin oxidation by HRP in phosphate buffer under the conditions of UV-stress, i.e. continuous, prolonged UV-B irradiation. The results show that while UV-B irradiation affects the activity of HRP, and the overal rate of quercetin oxidation by HRP, it probably has very little effect on it for longer UV-B-irradiation periods (>30 min. [Acknowledgements. This work was supported by the Ministry of Education and Science of the Republic of Serbia under Project No.TR-34012 and OI-172044

  10. Horseradish peroxidase immobilized on copper surfaces and applications in selective electrocatalysis of p-dihydroxybenzene

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Chuntao, E-mail: tsyj1992@126.com [Department of Chemistry, Taiyuan Normal University, Taiyuan 030031 (China); Institute of Energy and Environmental Electrochemistry, Taiyuan Normal University, Taiyuan 030031 (China); Luo, Xiaoxiao [Department of Natural Science, Michigan State University, MI 48823,USA (United States); Jia, Zehui [Department of Chemistry, Taiyuan Normal University, Taiyuan 030031 (China); Institute of Energy and Environmental Electrochemistry, Taiyuan Normal University, Taiyuan 030031 (China); Shi, Qinghua; Zhu, Ritao [Department of Chemistry, Taiyuan Normal University, Taiyuan 030031 (China)

    2017-06-01

    Abstract: Horseradish Peroxidase (HRP) was immobilized on copper surfaces with the linker of L-Cysteine (L-Cys) self-assembled films to form Cu/L-Cys/HRP electrodes. The activity of HRP can be preserved by the Cu/L-Cys self-assembled films. The Cu/L-Cys/HRP electrodes can be used for the selective electrocatalytic oxidase of p-dihydroxybenzen in absent of H{sub 2}O{sub 2}. The optimum pH for electrocatalyzing p-dihydroxybenzen was 5.5 or 7.0, which corresponds to the isoelectric points of L-Cys and HRP, respectively. X-ray photoelectron spectroscopy (XPS) provided the evidence that L-Cys linked with Cu surface by the Cu− S bond. Fourier transform infrared spectroscopy (FTIR) analyses indicated that aromatic plane of p-dihydroxybenzen was connected parallel to porphyrin ring of heme in HRP. Quantum chemical calculation of density functional theory (DFT) revealed that symmetry of molecular structure and minimum space steric hindrance for p-dihydroxybenzen were benefit to combination with HRP. Moreover, the lowest energy of LUMO and most negative charges of oxygen atom on hydroxyl group of p-dihydroxybenzen were advantage to lose the hydrogen atom of hydroxyl group to be oxided.

  11. Improved activity of immobilized horseradish peroxidase on gold nanoparticles in the presence of bovine serum albumin

    International Nuclear Information System (INIS)

    Ni, Yuyang; Li, Jun; Huang, Zhenzhen; He, Ke; Zhuang, Jiaqi; Yang, Wensheng

    2013-01-01

    The using of macromolecular additives is known to be a simple and effective way to improve the activity of immobilized enzymes on solid support, yet the mechanism has not been well understood. Taking horseradish peroxidase (HRP) as an example, only 30 % of its catalytic activity was kept after being immobilized on the surface of 25-nm Au nanoparticles, mainly attributed to the conformational change of the heme-containing active site. The catalytic activity of HRP was significantly improved to 80 % when a certain amount of bovine serum albumin (BSA) was added at the initial stage of the immobilization. Systematic spectral investigation indicated that the addition of BSA inhibited the tertiary structure change around the active site, which was a prerequisite for improved activity of the immobilized HRP. Steady-state kinetic analyses revealed that the introduction of BSA could effectively improve the turnover rate of substrate to product in spite of slight reduced affinity to substrates, which also contributed to the improved catalytic activity

  12. Cooxidation of styrene by horseradish peroxidase and phenols. A biochemical model for protein-mediated cooxidation

    International Nuclear Information System (INIS)

    Ortiz de Montellano, P.R.; Grab, L.A.

    1987-01-01

    Styrene is oxidized to styrene oxide and benzaldehyde when incubated with horseradish peroxidase, H 2 O 2 , and 4-methylphenol. Styrene oxide is not formed in the absence of any of these reaction components or of molecular oxygen. The coupling products 2-(4-methylphenoxy)-1-phenylethane, 2-(4-methylphenoxy)-1-phenylethan-1-ol, and 2-(4-methylphenoxy)-2-phenylethan-1-ol are not formed, but the ortho-linked dimer of 4-methylphenol is a major product. The epoxide oxygen is labeled in the presence of 18 O 2 but not H 2 18 O 2 . Styrene oxide formation is not inhibited by mannitol or superoxide dismutase. The stereochemistry of trans-[1- 2 H]styrene is partially scrambled in the epoxide product. EPR signals attributable to the 2,4-dihydroxyl-5-methylphenoxy radical, a product of the oxidation of 4-methylcatechol, are observed if Zn 2+ is added to stabilize the radical. This radical is only detected in the presence of styrene. The results imply that styrene is epoxidized by the hydroperoxy radical generated by addition of molecular oxygen to the 4-methylphenoxy radical. The epoxidation mimics the chemistry proposed to occur in the protein-mediated cooxidation of styrene by hemoglobin and myoglobin

  13. Development of (99m)Tc-labeled asymmetric urea derivatives that target prostate-specific membrane antigen for single-photon emission computed tomography imaging.

    Science.gov (United States)

    Kimura, Hiroyuki; Sampei, Sotaro; Matsuoka, Daiko; Harada, Naoya; Watanabe, Hiroyuki; Arimitsu, Kenji; Ono, Masahiro; Saji, Hideo

    2016-05-15

    Prostate-specific membrane antigen (PSMA) is expressed strongly in prostate cancers and is, therefore, an attractive diagnostic and radioimmunotherapeutic target. In contrast to previous reports of PMSA-targeting (99m)Tc-tricarbonyl complexes that are cationic or lack a charge, no anionic (99m)Tc-tricarbonyl complexes have been reported. Notably, the hydrophilicity conferred by both cationic and anionic charges leads to rapid hepatobiliary clearance, whereas an anionic charge might better enhance renal clearance relative to a cationic charge. Therefore, an improvement in rapid clearance would be expected with either cationic or anionic charges, particularly anionic charges. In this study, we designed and synthesized a novel anionic (99m)Tc-tricarbonyl complex ([(99m)Tc]TMCE) and evaluated its use as a single-photon emission computed tomography (SPECT) imaging probe for PSMA detection. Direct synthesis of [(99m)Tc]TMCE from dimethyl iminodiacetate, which contains both the asymmetric urea and succinimidyl moiety important for PSMA binding, was performed using our microwave-assisted one-pot procedure. The chelate formation was successfully achieved even though the precursor included a complicated bioactive moiety. The radiochemical yield of [(99m)Tc]TMCE was 12-17%, with a radiochemical purity greater than 98% after HPLC purification. [(99m)Tc]TMCE showed high affinity in vitro, with high accumulation in LNCaP tumors and low hepatic retention in biodistribution and SPECT/CT studies. These findings warrant further evaluation of [(99m)Tc]TMCE as an imaging agent and support the benefit of this strategy for the design of other PSMA imaging probes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Immunohistochemical expression of mitochondrial membrane complexes (MMCs) I, III, IV and V in malignant and benign periampullary epithelium: a potential target for drug therapy of periampullary cancer?

    International Nuclear Information System (INIS)

    Aloysius, Mark M; Zaitoun, Abed M; Bates, Timothy E; Ilyas, Mohammad; Constantin-Teodosiu, Dumitru; Rowlands, Brian J; Lobo, Dileep N

    2010-01-01

    Mitochondrial membrane complexes (MMCs) are key mediators of cellular oxidative phosphorylation, and inhibiting them could lead to cell death. No published data are available on the relative abundance of MMCs in different periampullary cancers. Therefore, we studied the expression profile of MMCs I, III, IV and V in periampullary cancers, reactive pancreatitis, normal pancreas and chronic pancreatitis. This was a retrospective study on tissue microarrays constructed from formalin-fixed paraffin-embedded tissue from 126 consecutive patients (cancer = 104, chronic pancreatitis = 22) undergoing pancreatic resections between June 2001 and June 2006. 78 specimens of chronic pancreatitis tissue were obtained adjacent to areas of cancer. Normal pancreatic tissue was obtained from the resection specimens in a total of 30 patients. Metastatic tumours in 61 regional lymph nodes from 61 patients were also studied. MMCs I, III, IV and V were highly expressed (p < 0.05) in all primary periampullary cancers compared with metastatic lymph nodes and adjacent benign pancreas. MMCs III, IV and V were highly expressed in all cancers regardless of type compared with chronic pancreatitis (p < 0.05). Higher expression of MMCs I and V was associated with better survival and may, in part, relate to lower expression of these MMCs in poorly differentiated tumours compared with well and moderately differentiated tumours. Differential expression of MMCs III, IV and V in primary periampullary cancers compared with adjacent benign periampullary tissue and chronic pancreatitis is a novel finding, which may render them attractive anticancer targets

  15. Matriptase and prostasin are expressed in human skin in an inverse trend over the course of differentiation and are targeted to different regions of the plasma membrane

    Directory of Open Access Journals (Sweden)

    Chih-Hsin Lai

    2016-10-01

    Full Text Available Matriptase and prostasin, acting as a tightly coupled proteolytic cascade, were reported to be required for epidermal barrier formation in mouse skin. Here we show that, in human skin, matriptase and prostasin are expressed with an inverse pattern over the course of differentiation. Matriptase was detected primarily in epidermal basal keratinocytes and the basaloid cells in the outer root sheath of hair follicles and the sebaceous gland, where prostasin was not detected. In contrast, prostasin was detected primarily in differentiated cells in the epidermal granular layer, the inner root sheath of hair follicles, and the sebaceous gland, where matriptase expression is negligible. While co-expressed in the middle stage of differentiation, prostasin was detected as polarized patches, and matriptase at intercellular junctions. Targeting to different subcellular localizations is also observed in HaCaT human keratinocytes, in which matriptase was detected primarily at intercellular junctions, and prostasin primarily on membrane protrusion. Furthermore, upon induction of zymogen activation, free active prostasin remains cell-associated and free active matriptase is rapidly shed into the extracellular milieu. Our data suggest that matriptase and prostasin likely function as independent entities in human skin rather than as a tightly coupled proteolytic cascade as observed in mouse skin.

  16. Rice calcium-dependent protein kinase OsCPK17 targets plasma membrane intrinsic protein and sucrose phosphate synthase and is required for a proper cold stress response

    KAUST Repository

    Almadanim, M. Cecília

    2017-01-19

    Calcium-dependent protein kinases (CDPKs) are involved in plant tolerance mechanisms to abiotic stresses. Although CDPKs are recognized as key messengers in signal transduction, the specific role of most members of this family remains unknown. Here we test the hypothesis that OsCPK17 plays a role in rice cold stress response by analyzing OsCPK17 knockout, silencing, and overexpressing rice lines under low temperature. Altered OsCPK17 gene expression compromises cold tolerance performance, without affecting the expression of key cold stress-inducible genes. A comparative phosphoproteomic approach led to the identification of six potential in vivo OsCPK17 targets, which are associated with sugar and nitrogen metabolism, and with osmotic regulation. To test direct interaction, in vitro kinase assays were performed, showing that the sucrose phosphate synthase OsSPS4, and the aquaporin OsPIP2;1/OsPIP2;6 are phosphorylated by OsCPK17 in a calcium-dependent manner. Altogether, our data indicates that OsCPK17 is required for a proper cold stress response in rice, likely affecting the activity of membrane channels and sugar metabolism.

  17. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  18. 18F-DCFBC Prostate-Specific Membrane Antigen-Targeted PET/CT Imaging in Localized Prostate Cancer: Correlation With Multiparametric MRI and Histopathology.

    Science.gov (United States)

    Turkbey, Baris; Mena, Esther; Lindenberg, Liza; Adler, Stephen; Bednarova, Sandra; Berman, Rose; Ton, Anita T; McKinney, Yolanda; Eclarinal, Philip; Hill, Craig; Afari, George; Bhattacharyya, Sibaprasad; Mease, Ronnie C; Merino, Maria J; Jacobs, Paula M; Wood, Bradford J; Pinto, Peter A; Pomper, Martin G; Choyke, Peter L

    2017-10-01

    To assess the ability of (N-[N-[(S)-1,3-dicarboxypropyl]carbamoyl]-4-F-fluorobenzyl-L-cysteine) (F-DCFBC), a prostate-specific membrane antigen-targeted PET agent, to detect localized prostate cancer lesions in correlation with multiparametric MRI (mpMRI) and histopathology. This Health Insurance Portability and Accountability Act of 1996-compliant, prospective, institutional review board-approved study included 13 evaluable patients with localized prostate cancer (median age, 62.8 years [range, 51-74 years]; median prostate-specific antigen, 37.5 ng/dL [range, 3.26-216 ng/dL]). Patients underwent mpMRI and F-DCFBC PET/CT within a 3 months' window. Lesions seen on mpMRI were biopsied under transrectal ultrasound/MRI fusion-guided biopsy, or a radical prostatectomy was performed. F-DCFBC PET/CT and mpMRI were evaluated blinded and separately for tumor detection on a lesion basis. For PET image analysis, MRI and F-DCFBC PET images were fused by using software registration; imaging findings were correlated with histology, and uptake of F-DCFBC in tumors was compared with uptake in benign prostatic hyperplasia nodules and normal peripheral zone tissue using the 80% threshold SUVmax. A total of 25 tumor foci (mean size, 1.8 cm; median size, 1.5 cm; range, 0.6-4.7 cm) were histopathologically identified in 13 patients. Sensitivity rates of F-DCFBC PET/CT and mpMRI were 36% and 96%, respectively, for all tumors. For index lesions, the largest tumor with highest Gleason score, sensitivity rates of F-DCFBC PET/CT and mpMRI were 61.5% and 92%, respectively. The average SUVmax for primary prostate cancer was higher (5.8 ± 4.4) than that of benign prostatic hyperplasia nodules (2.1 ± 0.3) or that of normal prostate tissue (2.1 ± 0.4) at 1 hour postinjection (P = 0.0033). The majority of index prostate cancers are detected with F-DCFBC PET/CT, and this may be a prognostic indicator based on uptake and staging. However, for detecting prostate cancer with high sensitivity, it

  19. Membrane dynamics

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    Current topics include membrane-protein interactions with regard to membrane deformation or curvature sensing by BAR domains. Also, we study the dynamics of membrane tubes of both cells and simple model membrane tubes. Finally, we study membrane phase behavior which has important implications...... for the lateral organization of membranes as wells as for physical properties like bending, permeability and elasticity...

  20. Size-dependent tuning of horseradish peroxidase bioreactivity by gold nanoparticles

    Science.gov (United States)

    Wu, Haohao; Liu, Yi; Li, Meng; Chong, Yu; Zeng, Mingyong; Lo, Y. Martin; Yin, Jun-Jie

    2015-02-01

    Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the α-helicity of the enzyme to extents inversely related to their size. Au-5 nm inhibited both HRP peroxidase activity toward 3,3',5,5'-tetramethylbenzidine and HRP compound I/II reactivity toward 5,5-dimethyl-1-pyrroline N-oxide. Au-5 nm enhanced the HRP peroxidase activity toward ascorbic acid and the HRP compound I/II reactivity toward redox-active residues in the HRP protein moiety. Further, Au-5 nm also decreased the catalase- and oxidase-like activities of HRP. Au-10 nm showed similar, but weaker effects, while Au-15 nm, Au-30 nm and Au-60 nm had no effect. Results suggest that AuNPs can size-dependently enhance or inhibit HRP bioreactivity toward substrates with different redox potentials via a mechanism involving extension of the HRP substrate access channel and decline in the redox potentials of HRP catalytic intermediates.Molecules with diverse biological functions, such as heme peroxidases, can be useful tools for identifying potential biological effects of gold nanoparticles (AuNPs) at the molecular level. Here, using UV-Vis, circular dichroism, dynamic light scattering, and electron spin resonance spectroscopy, we report tuning of horseradish peroxidase (HRP) bioactivity by reactant-free AuNPs with diameters of 5, 10, 15, 30 and 60 nm (Au-5 nm, Au-10 nm, Au-15 nm, Au-30 nm and Au-60 nm). HRP conjugation to AuNPs was observed with only Au-5 nm and Au-10 nm prominently increasing the

  1. Thermal stability and degradation kinetics of polyphenols and polyphenylenediamines enzymatically synthesized by horseradish peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Park, Hansol; Ryu, Keungarp [University of Ulsan, Ulsan (Korea, Republic of); Kwon, Oyul [Seoul National University of Science and Technology, Seoul (Korea, Republic of)

    2015-09-15

    Various substituted phenols and phenylenediamines were enzymatically polymerized by horseradish peroxidase in 80% (v/v) organic solvents-aqueous buffer (100 mM sodium acetate, pH 5) mixtures with H{sub 2}O{sub 2} as the oxidant. The thermal stability of the polymers was investigated by thermogravimetric analysis (TGA) and represented by the char yield (wt% of the initial polymer mass) after being heated at 800 .deg. C. Poly(p-phenylphenol) had the highest thermal stability among the synthesized polymers with a char yield of 47 wt%. The polymers containing amino groups such as poly(p-aminophenol) and polyphenylenediamines were also shown to possess high thermal stabilities. The activation energies for the thermal degradation of the polymers determined by derivative thermogravimetric analysis (DTG) using Horowitz-Metzger's pseudo-first-order kinetics were in the range between 23-65 kJ/mol and comparable to those of the chemically synthesized polymers. Dynamic structural changes of the enzymatically synthesized polymers upon heating were studied by differential scanning calorimetry (DSC). The DSC curves of poly(p-phenylphenol) showed a broad exothermic peaks between 150-250 .deg. C, indicating that the polymer undergoes complex structural transitions in the temperature range. On the other hand, the DSC curves of the poly(p-aminophenol) and the poly(p-phenylenediamine) which contain amino groups showed strong sharp endothermic peaks near 150 .deg. C, implying that these polymers possess homogeneous oriented structures which undergo a concerted structural disintegration upon heating.

  2. Engineering a horseradish peroxidase C stable to radical attacks by mutating multiple radical coupling sites.

    Science.gov (United States)

    Kim, Su Jin; Joo, Jeong Chan; Song, Bong Keun; Yoo, Young Je; Kim, Yong Hwan

    2015-04-01

    Peroxidases have great potential as industrial biocatalysts. In particular, the oxidative polymerization of phenolic compounds catalyzed by peroxidases has been extensively examined because of the advantage of this method over other conventional chemical methods. However, the industrial application of peroxidases is often limited because of their rapid inactivation by phenoxyl radicals during oxidative polymerization. In this work, we report a novel protein engineering approach to improve the radical stability of horseradish peroxidase isozyme C (HRPC). Phenylalanine residues that are vulnerable to modification by the phenoxyl radicals were identified using mass spectrometry analysis. UV-Vis and CD spectra showed that radical coupling did not change the secondary structure or the active site of HRPC. Four phenylalanine (Phe) residues (F68, F142, F143, and F179) were each mutated to alanine residues to generate single mutants to examine the role of these sites in radical coupling. Despite marginal improvement of radical stability, each single mutant still exhibited rapid radical inactivation. To further reduce inactivation by radical coupling, the four substitution mutations were combined in F68A/F142A/F143A/F179A. This mutant demonstrated dramatic enhancement of radical stability by retaining 41% of its initial activity compared to the wild-type, which was completely inactivated. Structure and sequence alignment revealed that radical-vulnerable Phe residues of HPRC are conserved in homologous peroxidases, which showed the same rapid inactivation tendency as HRPC. Based on our site-directed mutagenesis and biochemical characterization, we have shown that engineering radical-vulnerable residues to eliminate multiple radical coupling can be a good strategy to improve the stability of peroxidases against radical attack. © 2014 Wiley Periodicals, Inc.

  3. Horseradish extract promotes urinary bladder carcinogenesis when administered to F344 rats in drinking water.

    Science.gov (United States)

    Cho, Young-Man; Hasumura, Mai; Imai, Toshio; Takami, Shigeaki; Nishikawa, Akiyoshi; Ogawa, Kumiko

    2017-07-01

    Horseradish extract (HRE), consisting mainly of a mixture of allyl isothiocyanate and other isothiocyanates, has been used as a food additive. To evaluate the potential hazards of HRE, a 104-week chronic study, a 2-week analysis of cell proliferation in the urinary bladder and a medium-term promotion bioassay of HRE were conducted with administration at concentrations of up to 0.04% HRE in the drinking water to male F344 rats. In the 104-week chronic study with 32 male rats per group, no treatment-related increases in the incidences of neoplastic lesions in any organ, including urinary bladder, were observed, except for simple hyperplasia in the urinary bladder in rats treated with HRE at concentrations of more than 0.01% (5.0 mg kg -1 body weight day -1 ). In the promotion study, HRE treatment after N-butyl-N-(4-hydroxybutyl)nitrosamine initiation caused a clear increase in papillary or nodular hyperplasia, papilloma, and urothelial carcinoma of the urinary bladder in the groups given HRE for 13 weeks at doses higher than 0.005%, 0.01%, and 0.04% (2.7, 5.4 and 20.5 mg kg -1 body weight day -1 ), respectively. In the 2-week cell proliferation analysis, treatment with HRE at concentrations greater than 0.005% (3.9 mg kg -1 body weight day -1 ) caused transient increases in 5-bromo-2'-deoxyuridine labeling indices in the urothelium. Although clear tumor induction was not observed, administration of relatively low-dose HRE increased cell proliferation in the urothelium and exerted obvious promoting effects on rat urinary bladder carcinogenesis. Further studies are needed to elucidate the mode of action of HRE in the rat urinary bladder to facilitate data extrapolation from the present study and provide insights into risk assessment. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

  4. Conformation and activity alteration of horseradish peroxidase induced by the interaction with gene carrier polyethyleneimines

    Science.gov (United States)

    Huang, Aimin; Wei, Bangzhi; Mo, Junyong; Wang, Yajing; Ma, Lin

    2018-01-01

    Polyethyleneimine (PEI) has long been considered as "golden standard" for polymeric gene delivery carriers. However the molecular basis of the cytotoxicity of PEI is poorly understood. Little is known about the effects of PEI on the structure and functions of biomacromolecules. In this work, fluorescence, UV-vis absorption, circular dichroism spectroscopy were conducted to investigate the influence of PEI of average molecular weight 25, 10 and 1.8 kDa (denoted as PEI25k, PEI10k and PEI1.8k) on the conformation of horseradish peroxidase (HRP) and its catalytic efficiency. Zeta-potential measurement and isothermal titration calorimetry were used to reveal the mechanism of the interaction between PEIs and HRP. PEIs were found to bind onto the surface of HRP predominantly via hydrophobic interaction and hydrogen bond or van der Waals interaction. The complex formation between HRP and PEI induced a more compact conformation of the enzyme and an increased hydrophobicity of the microenvironment surrounding heme pocket. The conformational change of HRP had little impact on the affinity towards H2O2 and phenol. However, the increase in the non-planarity of porphyrin ring in the heme group led to an increase in the exposure degree of the active center and thus an enhancement of catalytic efficiency of HRP in the presence of high molecular weight PEIs (PEI25k and PEI10k). The polymer size played an important role in PEI-HRP interaction. PEI of low molecular weight (PEI1.8k) was less efficient to alter the conformation and catalytic activity of HRP in aqueous solutions.

  5. Mode of bindings of zinc oxide nanoparticles to myoglobin and horseradish peroxidase: A spectroscopic investigations

    Science.gov (United States)

    Mandal, Gopa; Bhattacharya, Sudeshna; Ganguly, Tapan

    2011-07-01

    The interactions between two heme proteins myoglobin (HMb) and horseradish peroxidase (HRP) with zinc oxide (ZnO) nanoparticles are investigated by using UV-vis absorption, steady state fluorescence, synchronous fluorescence, time-resolved fluorescence, FT-IR, atomic force microscopy (AFM) and circular dichroism (CD) techniques under physiological condition of pH˜7.4. The presence of mainly static mode in fluorescence quenching mechanism of HMb and HRP by ZnO nanoparticle indicates the possibility of formation of ground state complex. The processes of bindings of ZnO nanoparticles with the two proteins are spontaneous molecular interaction procedures. In both cases hydrogen bonding plays a major role. The circular dichroism (CD) spectra reveal that a helicity of the proteins is reduced by increasing ZnO nanoparticle concentration although the α-helical structures of HMb and HRP retain their identity. On binding to the ZnO nanoparticles the secondary structure of HRP molecules (or HMb molecules) remains unchanged while there is a substantial change in the environment of the tyrosin active site in case of HRP molecules and tryptophan active site in case of HMb molecules. Tapping mode atomic force microscopy (AFM) was applied for the investigation the structure of HRP adsorbed in the environment of nanoparticles on the silicon and on the bare silicon. HRP molecules adsorb and aggregate on the mica with ZnO nanoparticle. The aggregation indicates an attractive interaction among the adsorbed molecules. The molecules are randomly distributed on the bare silicon wafer. The adsorption of HRP in the environment of ZnO nanoparticle changes drastically the domains due to a strong interaction between HRP and ZnO nanoparticles. Similar situation is observed in case of HMb molecules. These findings demonstrate the efficacy of biomedical applications of ZnO nanoparticles as well as in elucidating their mechanisms of action as drugs in both human and plant systems.

  6. The Molecular Mechanism of the Catalase-like Activity in Horseradish Peroxidase.

    Science.gov (United States)

    Campomanes, Pablo; Rothlisberger, Ursula; Alfonso-Prieto, Mercedes; Rovira, Carme

    2015-09-02

    Horseradish peroxidase (HRP) is one of the most relevant peroxidase enzymes, used extensively in immunochemistry and biocatalysis applications. Unlike the closely related catalase enzymes, it exhibits a low activity to disproportionate hydrogen peroxide (H2O2). The origin of this disparity remains unknown due to the lack of atomistic information on the catalase-like reaction in HRP. Using QM(DFT)/MM metadynamics simulations, we uncover the mechanism for reduction of the HRP Compound I intermediate by H2O2 at atomic detail. The reaction begins with a hydrogen atom transfer, forming a peroxyl radical and a Compound II-like species. Reorientation of the peroxyl radical in the active site, concomitant with the transfer of the second hydrogen atom, is the rate-limiting step, with a computed free energy barrier (18.7 kcal/mol, ∼ 6 kcal/mol higher than the one obtained for catalase) in good agreement with experiments. Our simulations reveal the crucial role played by the distal pocket residues in accommodating H2O2, enabling formation of a Compound II-like intermediate, similar to catalases. However, out of the two pathways for Compound II reduction found in catalases, only one is operative in HRP. Moreover, the hydrogen bond network in the distal side of HRP compensates less efficiently than in catalases for the energetic cost required to reorient the peroxyl radical at the rate-determining step. The distal Arg and a water molecule in the "wet" active site of HRP have a substantial impact on the reaction barrier, compared to the "dry" active site in catalase. Therefore, the lower catalase-like efficiency of heme peroxidases compared to catalases can be directly attributed to the different distal pocket architecture, providing hints to engineer peroxidases with a higher rate of H2O2 disproportionation.

  7. Effects of molecular confinement and crowding on horseradish peroxidase kinetics using a nanofluidic gradient mixer.

    Science.gov (United States)

    Wichert, William R A; Han, Donghoon; Bohn, Paul W

    2016-03-07

    The effects of molecular confinement and crowding on enzyme kinetics were studied at length scales and under conditions similar to those found in biological cells. These experiments were carried out using a nanofluidic network of channels constituting a nanofluidic gradient mixer, providing the basis for measuring multiple experimental conditions simultaneously. The 100 nm × 40 μm nanochannels were wet etched directly into borosilicate glass, then annealed and characterized with fluorescein emission prior to kinetic measurements. The nanofluidic gradient mixer was then used to measure the kinetics of the conversion of the horseradish peroxidase (HRP)-catalyzed conversion of non-fluorescent Amplex Red (AR) to the fluorescent product resorufin in the presence of hydrogen peroxide (H2O2). The design of the gradient mixer allows reaction kinetics to be studied under multiple (five) unique solution compositions in a single experiment. To characterize the efficiency of the device the effects of confinement on HRP-catalyzed AR conversion kinetics were studied by varying the starting ratio of AR : H2O2. Equimolar concentrations of Amplex Red and H2O2 yielded the highest reaction rates followed by 2 : 1, 1 : 2, 5 : 1, and finally 1 : 5 [AR] : [H2O2]. Under all conditions, initial reaction velocities were decreased by excess H2O2. Crowding effects on kinetics were studied by increasing solution viscosity in the nanochannels in the range 1.0-1.6 cP with sucrose. Increasing the solution viscosities in these confined geometries decreases the initial reaction velocity at the highest concentration from 3.79 μM min(-1) at 1.00 cP to 0.192 μM min(-1) at 1.59 cP. Variations in reaction velocity are interpreted in the context of models for HRP catalysis and for molecular crowding.

  8. Humanised IgG1 antibody variants targeting membrane-bound carcinoembryonic antigen by antibody-dependent cellular cytotoxicity and phagocytosis.

    Science.gov (United States)

    Ashraf, S Q; Umana, P; Mössner, E; Ntouroupi, T; Brünker, P; Schmidt, C; Wilding, J L; Mortensen, N J; Bodmer, W F

    2009-11-17

    The effect of glycoengineering a membrane specific anti-carcinoembryonic antigen (CEA) (this paper uses the original term CEA for the formally designated CEACAM5) antibody (PR1A3) on its ability to enhance killing of colorectal cancer (CRC) cell lines by human immune effector cells was assessed. In vivo efficacy of the antibody was also tested. The antibody was modified using EBNA cells cotransfected with beta-1,4-N-acetylglucosaminyltransferase III and the humanised hPR1A3 antibody genes. The resulting alteration of the Fc segment glycosylation pattern enhances the antibody's binding affinity to the FcgammaRIIIa receptor on human immune effector cells but does not alter the antibody's binding capacity. Antibody-dependent cellular cytotoxicity (ADCC) is inhibited in the presence of anti-FcgammaRIII blocking antibodies. This glycovariant of hPR1A3 enhances ADCC 10-fold relative to the parent unmodified antibody using either unfractionated peripheral blood mononuclear or natural killer (NK) cells and CEA-positive CRC cells as targets. NK cells are far more potent in eliciting ADCC than either freshly isolated monocytes or granulocytes. Flow cytometry and automated fluorescent microscopy have been used to show that both versions of hPR1A3 can induce antibody-dependent cellular phagocytosis (ADCP) by monocyte-derived macrophages. However, the glycovariant antibody did not mediate enhanced ADCP. This may be explained by the relatively low expression of FcgammaRIIIa on cultured macrophages. In vivo studies show the efficacy of glycoengineered humanised IgG1 PR1A3 in significantly improving survival in a CRC metastatic murine model. The greatly enhanced in vitro ADCC activity of the glycoengineered version of hPR1A3 is likely to be clinically beneficial.

  9. A young root-specific gene (ArMY2) from horseradish encoding a MYR II myrosinase with kinetic preference for the root-specific glucosinolate gluconasturtiin.

    Science.gov (United States)

    Loebers, Andreas; Müller-Uri, Frieder; Kreis, Wolfgang

    2014-03-01

    The pungent taste of horseradish is caused by isothiocyanates which are released from glucosinolates by myrosinases. These enzymes are encoded by genes belonging to one of two subfamilies, termed MYR I and MYR II, respectively. A MYR II-type myrosinase gene was identified for the first time in horseradish. The gene termed ArMY2 was only expressed in young roots. A full-length cDNA encoding a myrosinase termed ArMy2 was isolated and heterologously expressed in Pichia pastoris. The recombinant His-tagged enzyme was characterized biochemically. Substrate affinity was 5 times higher towards gluconasturtiin than towards sinigrin. Gluconasturtiin was found to be the most abundant glucosinolate in young horseradish roots while sinigrin dominated in storage roots and leaves. This indicates that a specialized glucosinolate-myrosinase defense system might be active in young roots. Copyright © 2013 Elsevier Ltd. All rights reserved.

  10. Embryo yolk sac membrane kynurenine formamidase of l-tryptophan to NAD+ pathway as a primary target for organophosphorus insecticides (OPI) in OPI-induced NAD-associated avian teratogenesis.

    Science.gov (United States)

    Seifert, Josef

    2017-10-01

    The objective of this study was to provide in ovo evidence for the proposed role of kynurenine formamidase of l-tryptophan to NAD + pathway in embryo yolk sac membranes as a primary target for organophosphorus insecticide (OPI) teratogens in OPI-induced NAD-associated avian teratogenesis. Slices prepared from yolk sac membranes or embryo livers of chicken eggs treated with the OPI dicrotophos and/or methyl parathion were incubated with l-tryptophan. Yolk sac membrane slices metabolized l-tryptophan in the pathway to NAD + before that function was established in livers. OPI interfered in ovo with the second step of l-tryptophan to NAD + biosynthesis by inhibiting kynurenine formamidase. Its inhibition due to the teratogen dicrotophos occurred in yolk sac membranes during the period of embryo highest susceptibility to OPI teratogens in contrast to delayed and lower inhibition caused by the nonteratogen methyl parathion. Both OPI affected liver kynurenine formamidase in a similar manner. The onsets of liver enzyme inhibition, however, were delayed by about two days and occurred at the time of the reduced embryo susceptibility to teratogens. The early disruption of l-tryptophan metabolism and higher inhibition of kynurenine formamidase in yolk sac membranes may be the factors that determine action of OPI as teratogens in chicken embryos. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. The interaction of antimicrobial peptides with the membrane and intracellular targets of Staphylococcus aureus investigated by ATP leakage, DNA-binding analysis, and the expression of a LexA-controlled gene, recA

    DEFF Research Database (Denmark)

    Gottschalk, Sanne; Thomsen, Line Elnif

    2017-01-01

    The analysis of how antimicrobial peptides (AMPs) interact with bacterial membranes and intracellular targets is important for our understanding of how these molecules affect bacteria. Increased knowledge may aid the design of AMPs that work on their target bacterium without inducing bacterial...... resistance. Here, we describe different methods to investigate the mode of action of peptides against the Gram-positive bacterium Staphylococcus aureus. ATP leakage analysis can be used to evaluate the ability of AMPs to perturb bacteria. DNA-binding and SOS response induction can be analyzed to investigate...

  12. Liver plasma membranes: an effective method to analyze membrane proteome.

    Science.gov (United States)

    Cao, Rui; Liang, Songping

    2012-01-01

    Plasma membrane proteins are critical for the maintenance of biological systems and represent important targets for the treatment of disease. The hydrophobicity and low abundance of plasma membrane proteins make them difficult to analyze. The protocols given here are the efficient isolation/digestion procedures for liver plasma membrane proteomic analysis. Both protocol for the isolation of plasma membranes and protocol for the in-gel digestion of gel-embedded plasma membrane proteins are presented. The later method allows the use of a high detergent concentration to achieve efficient solubilization of hydrophobic plasma membrane proteins while avoiding interference with the subsequent LC-MS/MS analysis.

  13. Correlation of glucosinolate content to myrosinase activity in horseradish (Armoracia rusticana).

    Science.gov (United States)

    Li, Xian; Kushad, Mosbah M

    2004-11-17

    Fully developed horseradish (Armoracia rusticana Gaertn., Mey., & Scherb.) roots from 27 accessions and leaves from a subset of 9 accessions were evaluated for glucosinolates and myrosinase enzyme activity. Eight different glucosinolates were detected (based on HPLC retention times as desulfoglucosinolates) in both root and leaf tissues. The sum of these glucosinolates, referred to as total, ranged from 2 to 296 micromol g(-1) of dry weight (DW) in both tissues. Four glucosinolates (sinigrin, glucobrassicin, neoglucobrassicin, and gluconasturtiin) were detected in major quantities. In fully developed roots, sinigrin concentration represented approximately 83%, gluconasturtiin approximately 11%, and glucobrassicin approximately 1% of the total glucosinolates. Approximately the same proportions of individual glucosinolates appeared in fully developed leaves, except that glucobrassicin was substituted by neoglucobrassicin and gluconasturtiin concentration was significantly lower (<1%). At least four other glucosinolates were detected in very small quantities (<1%) in both roots and leaves. Myrosinase (beta-thioglucoside glucohydrolase, EC 3.2.3.1) is the enzyme responsible for the hydrolysis of the parent glucosinolates into biologically active products. Very little is known about myrosinase activity and the correlation of its activity to total and individual glucosinolates in plant tissues. Significant differences in myrosinase activity were detected between the roots and leaves, ranging from 1.2 to 57.1 units g(-1) of DW. Data showed no correlation between myrosinase activity and total and/or individual glucosinolates in the roots. However, in the leaves, significant correlations were found between myrosinase activity and total glucosinolates (0.78 at P = 0.01) and between myrosinase activity and sinigrin (0.80 at P = 0.01). Glucosinolates content and myrosinase activity were also correlated in young and fully developed roots and leaves and during tissue crushing

  14. Preparation of goat and rabbit anti-camel immunoglobulin G whole molecule labeled with horseradish peroxidase

    Directory of Open Access Journals (Sweden)

    Eman Hussein Abdel-Rahman

    2017-01-01

    Full Text Available Aim: As the labeled anti-camel immunoglobulins (Igs with enzymes for enzyme-linked immunosorbent assay (ELISA are unavailable in the Egyptian market, the present investigation was directed for developing local labeled anti-camel IgG with horseradish peroxidase (HRP to save hard curacy. Materials and Methods: For purification of camel IgG whole molecule, camel sera was preliminary precipitated with 50% saturated ammonium sulfate and dialyzed against 15 mM phosphate-buffered saline pH 7.2 then concentrated. This preparation was further purified by protein A sepharose affinity column chromatography. The purity of the eluted camel IgG was tested by sodium dodecyl sulfate polyacrylamide gel electrophoresi. Anti-camel IgG was prepared by immunization of goats and rabbits separately, with purified camel IgG. The anti-camel IgG was purified by protein A sepharose affinity column chromatography. Whole molecule anti-camel IgG was conjugated with HRP using glutraldehyde based assay. Sensitivity and specificity of prepared conjugated secondary antibodies were detected using positive and negative camel serum samples reacted with different antigens in ELISA, respectively. The potency of prepared conjugated antibodies was evaluated compared with protein A HRP. The stability of the conjugate at −20°C during 1 year was assessed by ELISA. Results: The electrophoretic profile of camel IgG showed four bands of molecular weight 63, 52, 40 and 33 kDa. The recorded sensitivity and specificity of the product are 100%. Its potency is also 100% compared to 58-75% of commercial protein A HRP. The conjugates are stable for 1 year at −20°C as proved by ELISA. Conclusion: Collectively, this study introduces goat and rabbit anti-camel IgG whole molecules with simple, inexpensive method, with 100% sensitivity, 100% specificity and stability up to 1 year at −20°C. The important facet of the current study is saving hard curacy. Future investigations are necessary for

  15. Immobilization of horseradish peroxidase onto kaolin by glutaraldehyde method and its application in decolorization of anthraquinone dye

    Directory of Open Access Journals (Sweden)

    Šekuljica Nataša Ž.

    2016-01-01

    Full Text Available The problem of environmental pollution day by day becomes more worrisome, primarily due to the large amounts of wastewater contaminated with various harmful organic compounds, discharged into the environment untreated or partially clean. Feasibility of use of horseradish peroxidase (Amoracia rusticana in the synthetic dyes decolorization was approved by many researchers. Among a number of supports used for the immobilization, it was found that natural clay, kaolin has excellent features which are a precondition for obtaining biocatalysts with the excellent performances. For this reason, a horseradish peroxidase was immobilized onto kaolin using glutaraldehyde as a cross-linking agent. Obtained biocatalyst was applied in the decolorization of anthraquinone dye C. I. Acid Violet 109. Under determined optimal conditions (pH 4.0, hydrogen peroxide concentration 0.6 mM, dye concentration 30 mg L-1, temperature 24ºC around 76 % of dye decolorization was achieved. Reusability study showed that resulting biocatalyst was possible to apply four times in the desired reaction with relatively high decolorization percentage. [Projekat Ministarstva nauke Republike Srbije, br. III-46010 i br. 172013

  16. Chaperone-like activity of β-casein and its effect on residual in vitro activity of horseradish peroxidase

    DEFF Research Database (Denmark)

    Sulewska, Anna Maria; Olsen, Karsten; Sørensen, Jens Christian

    2014-01-01

    , as similar experiment with bovine serum albumin resulted in residual activity of horseradish peroxidase that was significantly lower than without any addition. The effect of β-casein on HRP disappears when pH is below the isoelectric point of β-casein. It was also proven by light scattering studies that β...... proteins. Incubating HRP (0.1 mg mL-1) for 10 min at 72 °C resulted in residual activity of 59 ± 5%, while addition of 1 mg mL-1 β-casein resulted in increase in residual activity up to 85 ± 1%. Increased residual activity is not merely attributed to an effect of higher total protein concentration......-casein interacts with horseradish peroxidase when the temperature was increased from 25 to 70 °C whereas interactions seem to cease when temperature was lowered back to 25 °C. This study highlights how specific proteins can influence enzyme activity, which is of potential importance for various industries...

  17. Enzymatic removal of phenol and p-chlorophenol in enzyme reactor: Horseradish peroxidase immobilized on magnetic beads

    International Nuclear Information System (INIS)

    Bayramoglu, Guelay; Arica, M. Yakup

    2008-01-01

    Horseradish peroxidase was immobilized on the magnetic poly(glycidylmethacrylate-co-methylmethacrylate) (poly(GMA-MMA)), via covalent bonding and used for the treatment of phenolic wastewater in continuous systems. For this purposes, horseradish peroxidase (HRP) was covalently immobilized onto magnetic poly(GMA-MMA) beds using glutaraldehyde (GA) as a coupling agent. The maximum HRP immobilization capacity of the magnetic poly(GMA-MMA)-GA beads was 3.35 mg g -1 . The immobilized HRP retained 79% of the activity of the free HRP used for immobilization. The immobilized HRP was used for the removal of phenol and p-chlorophenol via polymerization of dissolved phenols in the presence of hydrogen peroxide (H 2 O 2 ). The effect of pH and temperature on the phenol oxidation rate was investigated. The results were compared with the free HRP, which showed that the optimum pH value for the immobilized HRP is similar to that for the free HRP. The optimum pH value for free and immobilized HRP was observed at pH 7.0. The optimum temperature for phenols oxidation with immobilized HRP was between 25 and 35 deg. C and the immobilized HRP has more resistance to temperature inactivation than that of the free form. Finally, the immobilized HRP was operated in a magnetically stabilized fluidized bed reactor, and phenols were successfully removed in the enzyme reactor

  18. Improved n-butanol production via co-expression of membrane-targeted tilapia metallothionein and the clostridial metabolic pathway in Escherichia coli.

    Science.gov (United States)

    Chin, Wei-Chih; Lin, Kuo-Hsing; Liu, Chun-Chi; Tsuge, Kenji; Huang, Chieh-Chen

    2017-04-11

    N-Butanol has favorable characteristics for use as either an alternative fuel or platform chemical. Bio-based n-butanol production using microbes is an emerging technology that requires further development. Although bio-industrial microbes such as Escherichia coli have been engineered to produce n-butanol, reactive oxygen species (ROS)-mediated toxicity may limit productivity. Previously, we show that outer-membrane-targeted tilapia metallothionein (OmpC-TMT) is more effective as an ROS scavenger than human and mouse metallothioneins to reduce oxidative stress in the host cell. The host strain (BUT1-DE) containing the clostridial n-butanol pathway displayed a decreased growth rate and limited n-butanol productivity, likely due to ROS accumulation. The clostridial n-butanol pathway was co-engineered with inducible OmpC-TMT in E. coli (BUT3-DE) for simultaneous ROS removal, and its effect on n-butanol productivity was examined. The ROS scavenging ability of cells overexpressing OmpC-TMT was examined and showed an approximately twofold increase in capacity. The modified strain improved n-butanol productivity to 320 mg/L, whereas the control strain produced only 95.1 mg/L. Transcriptomic analysis revealed three major KEGG pathways that were significantly differentially expressed in the BUT3-DE strain compared with their expression in the BUT1-DE strain, including genes involved in oxidative phosphorylation, fructose and mannose metabolism and glycolysis/gluconeogenesis. These results indicate that OmpC-TMT can increase n-butanol production by scavenging ROS. The transcriptomic analysis suggested that n-butanol causes quinone malfunction, resulting in oxidative-phosphorylation-related nuo operon downregulation, which would diminish the ability to convert NADH to NAD + and generate proton motive force. However, fructose and mannose metabolism-related genes (fucA, srlE and srlA) were upregulated, and glycolysis/gluconeogenesis-related genes (pfkB, pgm) were

  19. Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

    Directory of Open Access Journals (Sweden)

    T. Jiříček

    2017-01-01

    Full Text Available Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest flux was achieved with the thinnest membranes and the best energy efficiency was achieved with the thickest membranes. All membranes had salt retention above 99%. Nanotechnology offers the potential to find modern solutions for desalination of waste waters, by introducing new materials with revolutionary properties, but new membranes must be developed according to the target application.

  20. The GRP1 PH domain, like the AKT1 PH domain, possesses a sentry glutamate residue essential for specific targeting to plasma membrane PI(3,4,5)P(3).

    Science.gov (United States)

    Pilling, Carissa; Landgraf, Kyle E; Falke, Joseph J

    2011-11-15

    During the appearance of the signaling lipid PI(3,4,5)P(3), an important subset of pleckstrin homology (PH) domains target signaling proteins to the plasma membrane. To ensure proper pathway regulation, such PI(3,4,5)P(3)-specific PH domains must exclude the more prevalant, constitutive plasma membrane lipid PI(4,5)P(2) and bind the rare PI(3,4,5)P(3) target lipid with sufficiently high affinity. Our previous study of the E17K mutant of the protein kinase B (AKT1) PH domain, together with evidence from Carpten et al. [Carpten, J. D., et al. (2007) Nature 448, 439-444], revealed that the native AKT1 E17 residue serves as a sentry glutamate that excludes PI(4,5)P(2), thereby playing an essential role in specific PI(3,4,5)P(3) targeting [Landgraf, K. E., et al. (2008) Biochemistry 47, 12260-12269]. The sentry glutamate hypothesis proposes that an analogous sentry glutamate residue is a widespread feature of PI(3,4,5)P(3)-specific PH domains, and that charge reversal mutation at the sentry glutamate position will yield both increased PI(4,5)P(2) affinity and constitutive plasma membrane targeting. To test this hypothesis, we investigated the E345 residue, a putative sentry glutamate, of the general receptor for phosphoinositides 1 (GRP1) PH domain. The results show that incorporation of the E345K charge reversal mutation into the GRP1 PH domain enhances PI(4,5)P(2) affinity 8-fold and yields constitutive plasma membrane targeting in cells, reminiscent of the effects of the E17K mutation in the AKT1 PH domain. Hydrolysis of plasma membrane PI(4,5)P(2) releases the E345K GRP1 PH domain into the cytoplasm, and the efficiency of this release increases when Arf6 binding is disrupted. Overall, the findings provide strong support for the sentry glutamate hypothesis and suggest that the GRP1 E345K mutation will be linked to changes in cell physiology and human pathologies, as demonstrated for AKT1 E17K [Carpten, J. D., et al. (2007) Nature 448, 439-444; Lindhurst, M. J., et al

  1. Red Bell Pepper Chromoplasts Exhibit in Vitro Import Competency and Membrane Targeting of Passenger Proteins from the Thylakoidal Sec and ΔpH Pathways but Not the Chloroplast Signal Recognition Particle Pathway1

    Science.gov (United States)

    Summer, Elizabeth J.; Cline, Kenneth

    1999-01-01

    Chloroplast to chromoplast development involves new synthesis and plastid localization of nuclear-encoded proteins, as well as changes in the organization of internal plastid membrane compartments. We have demonstrated that isolated red bell pepper (Capsicum annuum) chromoplasts contain the 75-kD component of the chloroplast outer envelope translocon (Toc75) and are capable of importing chloroplast precursors in an ATP-dependent fashion, indicating a functional general import apparatus. The isolated chromoplasts were able to further localize the 33- and 17-kD subunits of the photosystem II O2-evolution complex (OE33 and OE17, respectively), lumen-targeted precursors that utilize the thylakoidal Sec and ΔpH pathways, respectively, to the lumen of an internal membrane compartment. Chromoplasts contained the thylakoid Sec component protein, cpSecA, at levels comparable to chloroplasts. Routing of OE17 to the lumen was abolished by ionophores, suggesting that routing is dependent on a transmembrane ΔpH. The chloroplast signal recognition particle pathway precursor major photosystem II light-harvesting chlorophyll a/b protein failed to associate with chromoplast membranes and instead accumulated in the stroma following import. The Pftf (plastid fusion/translocation factor), a chromoplast protein, integrated into the internal membranes of chromoplasts during in vitro assays, and immunoblot analysis indicated that endogenous plastid fusion/translocation factor was also an integral membrane protein of chromoplasts. These data demonstrate that the internal membranes of chromoplasts are functional with respect to protein translocation on the thylakoid Sec and ΔpH pathways. PMID:9952453

  2. Fumigant activity of plant essential oils and components from horseradish (Armoracia rusticana), anise (Pimpinella anisum) and garlic (Allium sativum) oils against Lycoriella ingenua (Diptera: Sciaridae).

    Science.gov (United States)

    Park, Ii-Kwon; Choi, Kwang-Sik; Kim, Do-Hyung; Choi, In-Ho; Kim, Lee-Sun; Bak, Won-Chull; Choi, Joon-Weon; Shin, Sang-Chul

    2006-08-01

    Plant essential oils from 40 plant species were tested for their insecticidal activities against larvae of Lycoriella ingénue (Dufour) using a fumigation bioassay. Good insecticidal activity against larvae of L. ingenua was achieved with essential oils of Chenopodium ambrosioides L., Eucalyptus globulus Labill, Eucalyptus smithii RT Baker, horseradish, anise and garlic at 10 and 5 microL L(-1) air. Horseradish, anise and garlic oils showed the most potent insecticidal activities among the plant essential oils. At 1.25 microL L(-1), horseradish, anise and garlic oils caused 100, 93.3 and 13.3% mortality, but at 0.625 microL L(-1) air this decreased to 3.3, 0 and 0% respectively. Analysis by gas chromatography-mass spectrometry led to the identification of one major compound from horseradish, and three each from anise and garlic oils. These seven compounds and m-anisaldehyde and o-anisaldehyde, two positional isomers of p-anisaldehyde, were tested individually for their insecticidal activities against larvae of L. ingenua. Allyl isothiocyanate was the most toxic, followed by trans-anethole, diallyl disulfide and p-anisaldehyde with LC(50) values of 0.15, 0.20, 0.87 and 1.47 microL L(-1) respectively.

  3. Reversible formation of high-valent-iron-oxo-porphyrin intermediate in heme-based catalysis: revisiting the kinetic model for horseradish peroxidase.

    NARCIS (Netherlands)

    Haandel, van M.J.H.; Primus, J.L.; Teunis, C.; Boersma, M.G.; Osman, A.M.; Veeger, C.; Rietjens, I.M.C.M.

    1998-01-01

    Many heme-containing biocatalysts exert their catalytic action through the initial formation of so-called high-valent-iron-oxo porphyrin intermediates. For horseradish peroxidase the initial intermediate formed has been identified as a high-valent-iron-oxo porphyrin π-radical cation, called compound

  4. Membrane fusion

    DEFF Research Database (Denmark)

    Bendix, Pól Martin

    2015-01-01

    At Stanford University, Boxer lab, I worked on membrane fusion of small unilamellar lipid vesicles to flat membranes tethered to glass surfaces. This geometry closely resembles biological systems in which liposomes fuse to plasma membranes. The fusion mechanism was studied using DNA zippering...... between complementary strands linked to the two apposing membranes closely mimicking the zippering mechanism of SNARE fusion complexes....

  5. Pastry Filled With Horseradish. discomfort city – how to create it

    Directory of Open Access Journals (Sweden)

    Konstantin Lidin

    2007-07-01

    buildings do not make the general impression better: «Try to find a point for panoramic picture for everybody to say: what a beautiful city! Even the perfection of Japanese photographic apparatuses cannot be helpful. You need not a tripod but at least a helicopter… Hundreds of monumental buildings are lost like a mosaic fallen to pieces, which could compose a wonderful picture if architects worked in collaboration with city planners».It can hardly be said that some definite people deliberately and purposefully came to a decision to make the city uncomfortable for living. The situation looks like an unintentional reflective reaction of a primitive living organism – it is a rejection of irritant factor. The city, like a living but unreasonable being, tries to push out its «excess» people. If authorities do not take upon themselves the functions of reason, that is planning and management, the urban environment degrades very quickly. Instead of expected welfare citizens get a pastry filled with horseradish – life dressed with many problems and oppressive discomfort.

  6. Membrane cholesterol effect on the 5-HT2A receptor: Insights into the lipid-induced modulation of an antipsychotic drug target.

    Science.gov (United States)

    Ramírez-Anguita, Juan Manuel; Rodríguez-Espigares, Ismael; Guixà-González, Ramon; Bruno, Agostino; Torrens-Fontanals, Mariona; Varela-Rial, Alejandro; Selent, Jana

    2018-01-01

    The serotonin 5-hydroxytryptamine 2A (5-HT 2A ) receptor is a G-protein-coupled receptor (GPCR) relevant for the treatment of CNS disorders. In this regard, neuronal membrane composition in the brain plays a crucial role in the modulation of the receptor functioning. Since cholesterol is an essential component of neuronal membranes, we have studied its effect on the 5-HT 2A receptor dynamics through all-atom MD simulations. We find that the presence of cholesterol in the membrane increases receptor conformational variability in most receptor segments. Importantly, detailed structural analysis indicates that conformational variability goes along with the destabilization of hydrogen bonding networks not only within the receptor but also between receptor and lipids. In addition to increased conformational variability, we also find receptor segments with reduced variability. Our analysis suggests that this increased stabilization is the result of stabilizing effects of tightly bound cholesterol molecules to the receptor surface. Our finding contributes to a better understanding of membrane-induced alterations of receptor dynamics and points to cholesterol-induced stabilizing and destabilizing effects on the conformational variability of GPCRs. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  7. Protein-induced fusion can be modulated by target membrane lipids through a structural switch at the level of the fusion peptide

    NARCIS (Netherlands)

    Pecheur, EI; Martin, [No Value; Bienvenue, A; Ruysschaert, JM; Hoekstra, D

    2000-01-01

    Regulatory features of protein-induced membrane fusion are largely unclear, particularly at the level of the fusion peptide. Fusion peptides being part of larger protein complexes, such investigations are met with technical limitations. Here, we show that the fusion activity of influenza virus or

  8. Nanodisc-solubilized membrane protein library reflects the membrane proteome.

    Science.gov (United States)

    Marty, Michael T; Wilcox, Kyle C; Klein, William L; Sligar, Stephen G

    2013-05-01

    The isolation and identification of unknown membrane proteins offers the prospect of discovering new pharmaceutical targets and identifying key biochemical receptors. However, interactions between membrane protein targets and soluble ligands are difficult to study in vitro due to the insolubility of membrane proteins in non-detergent systems. Nanodiscs, nanoscale discoidal lipid bilayers encircled by a membrane scaffold protein belt, have proven to be an effective platform to solubilize membrane proteins and have been used to study a wide variety of purified membrane proteins. This report details the incorporation of an unbiased population of membrane proteins from Escherichia coli membranes into Nanodiscs. This solubilized membrane protein library (SMPL) forms a soluble in vitro model of the membrane proteome. Since Nanodiscs contain isolated proteins or small complexes, the SMPL is an ideal platform for interactomics studies and pull-down assays of membrane proteins. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the protein population before and after formation of the Nanodisc library indicates that a large percentage of the proteins are incorporated into the library. Proteomic identification of several prominent bands demonstrates the successful incorporation of outer and inner membrane proteins into the Nanodisc library.

  9. Simultaneous targeting of prostate stem cell antigen and prostate-specific membrane antigen improves the killing of prostate cancer cells using a novel modular T cell-retargeting system.

    Science.gov (United States)

    Arndt, Claudia; Feldmann, Anja; Koristka, Stefanie; Cartellieri, Marc; Dimmel, Maria; Ehninger, Armin; Ehninger, Gerhard; Bachmann, Michael

    2014-09-01

    Recently, we described a novel modular platform technology in which T cell-recruitment and tumor-targeting domains of conventional bispecific antibodies are split to independent components, a universal effector module (EM) and replaceable monospecific/monovalent target modules (TMs) that form highly efficient T cell-retargeting complexes. Theoretically, our unique strategy should allow us to simultaneously retarget T cells to different tumor antigens by combining the EM with two or more different monovalent/monospecific TMs or even with bivalent/bispecific TMs, thereby overcoming limitations of a monospecific treatment such as the selection of target-negative tumor escape variants. In order to advance our recently introduced prostate stem cell antigen (PSCA)-specific modular system for a dual-targeting of prostate cancer cells, two additional TMs were constructed: a monovalent/monospecific TM directed against the prostate-specific membrane antigen (PSMA) and a bivalent/bispecific TM (bsTM) with specificity for PSMA and PSCA. The functionality of the novel dual-targeting strategies was analyzed by performing T cell activation and chromium release assays. Similar to the PSCA-specific modular system, the novel PSMA-specific modular system mediates an efficient target-dependent and -specific tumor cell lysis at low E:T ratios and picomolar Ab concentrations. Moreover, by combination of the EM with either the bispecific TM directed to PSMA and PSCA or both monospecifc TMs directed to either PSCA or PSMA, dual-specific targeting complexes were formed which allowed us to kill potential escape variants expressing only one or the other target antigen. Overall, the novel modular system represents a promising tool for multiple tumor targeting. © 2014 Wiley Periodicals, Inc.

  10. Retrograde and transganglionic transport of horseradish peroxidase-conjugated cholera toxin B subunit, wheatgerm agglutinin and isolectin B4 from Griffonia simplicifolia I in primary afferent neurons innervating the rat urinary bladder.

    Science.gov (United States)

    Wang, H F; Shortland, P; Park, M J; Grant, G

    1998-11-01

    In the present study, we investigated and compared the ability of the cholera toxin B subunit, wheat germ agglutinin and isolectin B4 from Griffonia simplicifolia I conjugated to horseradish peroxidase, to retrogradely and transganglionically label visceral primary afferents after unilateral injections into the rat urinary bladder wall. Horseradish peroxidase histochemical or lectin-immunofluorescence histochemical labelling of bladder afferents was seen in the L6-S1 spinal cord segments and in the T13-L2 and L6-S1 dorsal root ganglia. In the lumbosacral spinal cord, the most intense and extensive labelling of bladder afferents was seen when cholera toxin B subunit-horseradish peroxidase was injected. Cholera toxin B subunit-horseradish peroxidase-labelled fibres were found in Lissauer's tract, its lateral and medial collateral projections, and laminae I and IV-VI of the spinal gray matter. Labelled fibres were numerous in the lateral collateral projection and extended into the spinal parasympathetic nucleus. Labelling from both the lateral and medial projections extended into the dorsal grey commissural region. Wheat germ agglutinin-horseradish peroxidase labelling produced a similar pattern but was not as dense and extensive as that of cholera toxin B subunit-horseradish peroxidase. The isolectin B4 from Griffonia simplicifolia I-horseradish peroxidase-labelled fibres, on the other hand, were fewer and only observed in the lateral collateral projection and occasionally in lamina I. Cell profile counts showed that a larger number of dorsal root ganglion cells were labelled with cholera toxin B subunit-horseradish peroxidase than with wheat germ agglutinin- or isolectin B4-horseradish peroxidase. In the L6-S1 dorsal root ganglia, the majority (81%) of the cholera toxin B subunit-, and almost all of the wheat germ agglutinin- and isolectin B4-immunoreactive cells were RT97-negative (an anti-neurofilament antibody that labels dorsal root ganglion neurons with

  11. Fundamentals of membrane bioreactors materials, systems and membrane fouling

    CERN Document Server

    Ladewig, Bradley

    2017-01-01

    This book provides a critical, carefully researched, up-to-date summary of membranes for membrane bioreactors. It presents a comprehensive and self-contained outline of the fundamentals of membrane bioreactors, especially their relevance as an advanced water treatment technology. This outline helps to bring the technology to the readers’ attention, and positions the critical topic of membrane fouling as one of the key impediments to its more widescale adoption. The target readership includes researchers and industrial practitioners with an interest in membrane bioreactors.

  12. Competitive photometric enzyme immunoassay for fullerene C60 and its derivatives using a fullerene conjugated to horseradish peroxidase

    International Nuclear Information System (INIS)

    Hendrickson, Olga D.; Smirnova, Natalya I.; Zherdev, Anatoly V.; Dzantiev, Boris B.; Sveshnikov, Peter G.

    2016-01-01

    The article describes a highly sensitive single-step microplate enzyme immunoassay of the ELISA type for fullerene C 60 and its derivatives. Monoclonal anti-fullerene antibodies and a conjugate between fullerene and horseradish peroxidase were used as specific reagents. A direct competitive ELISA was carried out that was based on antibodies immobilized in the well of a microtiter plate, a peroxidase-labeled antigen, and detection via the dye formed from 3,3′,5,5′-tetramethylbenzidine and hydrogen peroxide. Both pristine fullerene C 60 and its water-soluble forms can be determined. The detection limits are 1.5 ng∙mL −1 for fullerene C 60 , and between 0.1 and 1.3 ng∙mL −1 for its derivatives. This ELISA format allows for almost two-fold reduction of the time needed for the assay in comparison to indirect scheme with labeled antibodies. (author)

  13. A H2O2 Biosensor Based on Immobilization of HorseradishPeroxidase in a Gelatine Network Matrix

    Directory of Open Access Journals (Sweden)

    Jun-Jie Zhu

    2005-05-01

    Full Text Available A simple and promising H2O2 biosensor has been developed by successfulentrapment of horseradish peroxidase (HRP in a gelatine matrix which was cross-linkedwith formaldehyde. The large microscopic surface area and porous morphology of thegelatine matrix lead to high enzyme loading and the enzyme entrapped in this matrix canretain its bioactivity. This biosensor exhibited a fast amperometric response to hydrogenperoxide (H2O2. The linear range for H2O2 determination was from 2.5×10-5 to2.5×10-3 M, with a detection limit of 2.0×10-6 M based on S / N = 3. This biosensorpossessed very good reproducibility.

  14. Radiation effects on cell membranes

    International Nuclear Information System (INIS)

    Koeteles, G.J.

    1982-01-01

    The recent developments in the field of membrane biology of eukaryotic cells result in revival of relevant radiobiological studies. The spatial relations and chemical nature of membrane components provide rather sensitive targets. Experimental data are presented concerning the effects of relatively low doses of X-irradiation and low concentration of tritiated water (HTO) on various receptor functions - concanavalin A, cationized ferritin, poliovirus - of plasma membranes of animal and human cells which point to early and temporary disturbances of the composite structures and functions of membranes. References are given to the multifold roles of radiationinduced membrane phenomena on the development and regeneration of radiation injuries. (orig.)

  15. Radiation effects on cell membranes

    Energy Technology Data Exchange (ETDEWEB)

    Koeteles, G.J.

    1982-11-01

    The recent developments in the field of membrane biology of eukaryotic cells result in revival of relevant radiobiological studies. The spatial relations and chemical nature of membrane components provide rather sensitive targets. Experimental data are presented concerning the effects of relatively low doses of X-irradiation and low concentration of tritiated water (HTO) on various receptor functions - concanavalin A, cationized ferritin, poliovirus - of plasma membranes of animal and human cells which point to early and temporary disturbances of the composite structures and functions of membranes. References are given to the multifold roles of radiationinduced membrane phenomena on the development and regeneration of radiation injuries.

  16. Liquid-phase membrane extraction of targeted pesticides from manufacturing wastewaters in a hollow fibre contactor with feed-stream recycle.

    Science.gov (United States)

    Đorđević, Jelena; Vladisavljević, Goran T; Trtić-Petrović, Tatjana

    2017-01-01

    A two-phase membrane extraction in a hollow fibre contactor with feed-stream recycle was applied to remove selected pesticides (tebufenozide, linuron, imidacloprid, acetamiprid and dimethoate) from their mixed aqueous solutions. The contactor consisted of 50 polypropylene hollow fibres impregnated with 5% tri-n-octylphosphine oxide in di-n-hexyl ether. For low-polar pesticides with log P ≥ 2 (tebufenozide and linuron), the maximum removal efficiency increased linearly from 85% to 96% with increasing the feed flow rate. The maximum removal efficiencies of more polar pesticides were significantly higher under feed recirculation (86%) than in a continuous single-pass operation (30%). It was found from the Wilson's plot that the mass transfer resistance of the liquid membrane can be neglected for low-polar pesticides. The pesticide removals from commercial formulations were similar to those from pure pesticide solutions, indicating that built-in adjuvants did not affect the extraction process.

  17. Bovine lactoferricin causes apoptosis in Jurkat T-leukemia cells by sequential permeabilization of the cell membrane and targeting of mitochondria

    International Nuclear Information System (INIS)

    Mader, Jamie S.; Richardson, Angela; Salsman, Jayme; Top, Deniz; Antueno, Roberto de; Duncan, Roy; Hoskin, David W.

    2007-01-01

    Bovine lactoferricin (LfcinB) is a cationic antimicrobial peptide that kills Jurkat T-leukemia cells by the mitochondrial pathway of apoptosis. However, the process by which LfcinB triggers mitochondria-dependent apoptosis is not well understood. Here, we show that LfcinB-induced apoptosis in Jurkat T-leukemia cells was preceded by LfcinB binding to, and progressive permeabilization of the cell membrane. Colloidal gold electron microscopy revealed that LfcinB entered the cytoplasm of Jurkat T-leukemia cells prior to the onset of mitochondrial depolarization. LfcinB was not internalized by endocytosis because endocytosis inhibitors did not prevent LfcinB-induced cytotoxicity. Furthermore, intracellular delivery of LfcinB via fusogenic liposomes caused the death of Jurkat T-leukemia cells, as well as normal human fibroblasts. Collectively, these findings suggest that LfcinB caused damage to the cell membrane that allowed LfcinB to enter the cytoplasm of Jurkat T-leukemia cells and mediate cytotoxicity. In addition, confocal microscopy showed that intracellular LfcinB co-localized with mitochondria in Jurkat T-leukemia cells, while flow cytometry and colloidal gold electron microscopy showed that LfcinB rapidly associated with purified mitochondria. Furthermore, purified mitochondria treated with LfcinB rapidly lost transmembrane potential and released cytochrome c. We conclude that LfcinB-induced apoptosis in Jurkat T-leukemia cells resulted from cell membrane damage and the subsequent disruption of mitochondrial membranes by internalized LfcinB

  18. Membrane fusion and exocytosis.

    Science.gov (United States)

    Jahn, R; Südhof, T C

    1999-01-01

    Membrane fusion involves the merger of two phospholipid bilayers in an aqueous environment. In artificial lipid bilayers, fusion proceeds by means of defined transition states, including hourglass-shaped intermediates in which the proximal leaflets of the fusing membranes are merged whereas the distal leaflets are separate (fusion stalk), followed by the reversible opening of small aqueous fusion pores. Fusion of biological membranes requires the action of specific fusion proteins. Best understood are the viral fusion proteins that are responsible for merging the viral with the host cell membrane during infection. These proteins undergo spontaneous and dramatic conformational changes upon activation. In the case of the paradigmatic fusion proteins of the influenza virus and of the human immunodeficiency virus, an amphiphilic fusion peptide is inserted into the target membrane. The protein then reorients itself, thus forcing the fusing membranes together and inducing lipid mixing. Fusion of intracellular membranes in eukaryotic cells involves several protein families including SNAREs, Rab proteins, and Sec1/Munc-18 related proteins (SM-proteins). SNAREs form a novel superfamily of small and mostly membrane-anchored proteins that share a common motif of about 60 amino acids (SNARE motif). SNAREs reversibly assemble into tightly packed helical bundles, the core complexes. Assembly is thought to pull the fusing membranes closely together, thus inducing fusion. SM-proteins comprise a family of soluble proteins that bind to certain types of SNAREs and prevent the formation of core complexes. Rab proteins are GTPases that undergo highly regulated GTP-GDP cycles. In their GTP form, they interact with specific proteins, the effector proteins. Recent evidence suggests that Rab proteins function in the initial membrane contact connecting the fusing membranes but are not involved in the fusion reaction itself.

  19. Novicidin interactions with phospholipid membranes

    DEFF Research Database (Denmark)

    Balakrishnan, Vijay Shankar

    Antimicrobial peptides target bacterial cell membranes and are considered as potential antibiotics. Their interactions with cell membranes are studied using different approaches. This thesis comprises of the biophysical investigations on the antimicrobial peptide Novicidin, interacting with lipos......Antimicrobial peptides target bacterial cell membranes and are considered as potential antibiotics. Their interactions with cell membranes are studied using different approaches. This thesis comprises of the biophysical investigations on the antimicrobial peptide Novicidin, interacting...... with liposomes. The lipid-induced changes in the peptide due to membrane binding, and the peptide-induced changes in the membrane properties were investigated using various spectroscopic and calorimetric methods, and the structural and thermodynamic aspects of peptide-lipid interactions are discussed. This helps...

  20. Membrane Biophysics

    CERN Document Server

    Ashrafuzzaman, Mohammad

    2013-01-01

    Physics, mathematics and chemistry all play a vital role in understanding the true nature and functioning of biological membranes, key elements of living processes. Besides simple spectroscopic observations and electrical measurements of membranes we address in this book the phenomena of coexistence and independent existence of different membrane components using various theoretical approaches. This treatment will be helpful for readers who want to understand biological processes by applying both simple observations and fundamental scientific analysis. It provides a deep understanding of the causes and effects of processes inside membranes, and will thus eventually open new doors for high-level pharmaceutical approaches towards fighting membrane- and cell-related diseases.

  1. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells

    Science.gov (United States)

    Bach, Thomas J

    2013-01-01

    We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL). By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP) pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1), shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA) pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL. PMID:24555083

  2. Specificity of Bacillus thuringiensis endotoxins is correlated with the presence of high-affinity binding sites in the brush border membrane of target insect midguts

    International Nuclear Information System (INIS)

    Hofmann, C.; Vanderbruggen, H.; Hoefte, H.; Van Rie, J.; Jansens, S.; Van Mellaert, H.

    1988-01-01

    Binding studies were performed with two 125 I-labeled Bacillus thuringiensis δ-endotoxins on brush border membrane vesicles prepared from the larval midgut of the tobacco hornworm Manduca sexta or the cabbage butterfly Pieris brassicae. One δ-endotoxin, Bt2-protoxin, is a 130-kDa recombinant crystalline protein from B. thuringiensis subsp. berliner. It kills larvae of both insect species. The active Bt2-toxin is a 60-kDa proteolytic fragment of the Bt2-protoxin. It binds saturably and with high affinity to brush border membrane vesicles from the midgut of both species. The other δ-endotoxin, Bt4412-protoxin, is a 136-kDa crystalline protein from B. thuringiensis subsp. thuringiensis, which is highly toxic for P. brassicae, but not for M. sexta larvae. Bt4412-toxin, obtained after proteolytic activation of Bt4412-protoxin, shows high-affinity saturable binding to P. brassicae vesicles but not to M. sexta vesicles. The correlation between toxicity and specific binding is further strengthened by competition studies. Other B. thuringiensis δ-endotoxins active against M. sexta compete for binding of 125 I-labeled Bt2-toxin to M. sexta vesicles, whereas toxins active against dipteran or coleopteran larvae do not compete. Bt2-toxin and Bt4412-toxin bind to different sites on P. brassicae vesicles

  3. Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

    International Nuclear Information System (INIS)

    Bonekamp, Nina A.; Vormund, Kerstin; Jacob, Ralf; Schrader, Michael

    2010-01-01

    The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.

  4. Human serum albumin supported lipid patterns for the targeted recognition of microspheres coated by membrane based on ss-DNA hybridization

    International Nuclear Information System (INIS)

    Zhang Xiaoming; He Qiang; Cui Yue; Duan Li; Li Junbai

    2006-01-01

    Human serum albumin (HSA) patterns have been successfully fabricated for the deposition of lipid bilayer, 1,2-dimyristoyl-sglycerophosphate (DMPA), by making use of the micro-contact printing (μCP) technique and liposome fusion. Confocal laser scanning microscopy (CLSM) results indicate that lipid bilayer has been assembled in HSA patterns with a good stability. Such well-defined lipid patterns formed on HSA surface create possibility to incorporate specific components like channels or receptors for specific recognition. In view of this, microspheres coated with lipid membranes were immobilized in HSA-supported lipid patterns via the hybridization of complementary ss-DNAs. This procedure enables to transfer solid materials to a soft surface through a specific recognition

  5. Spasmolytic, antimicrobial and cytotoxic activities of 5-phenylpentyl isothiocyanate, a new glucosinolate autolysis product from horseradish (Armoracia rusticana P. Gaertn., B. Mey. & Scherb., Brassicaceae).

    Science.gov (United States)

    Dekić, Milan S; Radulović, Niko S; Stojanović, Nikola M; Randjelović, Pavle J; Stojanović-Radić, Zorica Z; Najman, Stevo; Stojanović, Sanja

    2017-10-01

    Detailed analyses of horseradish autolysates led to the identification of a new natural product, 5-phenylpentyl isothiocyanate (PhPeITC). The structural assignment was corroborated by synthesis, and the identity unequivocally established by spectral means. The occurrence of PhPeITC is the first direct proof of the existence of a 5-phenylpentyl glucosinolate in the aerial parts of this species as one of the possible "mustard oil" precursors. To verify its possible contribution to the horseradish functional food status, horseradish above- and underground autolysates, together with five ω-phenylalkyl isothiocyanates were tested for their spasmolytic, cytotoxic and antimicrobial activities. Specifically, the cytotoxic effect on Caco-2, HeLa (cancer) and MDCK (non-cancer) cell lines was established. Additionally, the five tested ITCs exerted significant spasmolytic activity (on rat distal colon), with PhPeITC being almost 100 times more potent than papaverine. A non-selective antimicrobial activity of all ITCs was revealed in the case of 6 bacterial and 2 fungal strains. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Vesicular transport route of horseradish C1a peroxidase is regulated by N- and C-terminal propeptides in tobacco cells.

    Science.gov (United States)

    Matsui, T; Nakayama, H; Yoshida, K; Shinmyo, A

    2003-10-01

    Peroxidases (PRX, EC 1.11.1.7) are widely distributed across microorganisms, plants, and animals; and, in plants, they have been implicated in a variety of secondary metabolic reactions. In particular, horseradish (Armoracia rusticana) root represents the main source of commercial PRX production. The prxC1a gene, which encodes horseradish PRX (HRP) C, is expressed mainly in the roots and stems of the horseradish plant. HRP C1a protein is shown to be synthesized as a preprotein with both a N-terminal (NTPP) and a C-terminal propeptide (CTPP). These propeptides, which might be responsible for intracellular localization or secretion, are removed before or concomitant with production of the mature protein. We investigated the functional role of HRP C1a NTPP and CTPP in the determination of the vesicular transport route, using an analytical system of transgenically cultured tobacco cells (Nicotiana tabacum, BY2). Here, we report that NTPP and CTPP are necessary and sufficient for accurate localization of mature HRP C1a protein to vacuoles of the vesicular transport system. We also demonstrate that HRP C1a derived from a preprotein lacking CTPP is shunted into the secretory pathway.

  7. Mediatorless amperometric bienzyme glucose biosensor based on horseradish peroxidase and glucose oxidase cross-linked to multiwall carbon nanotubes

    International Nuclear Information System (INIS)

    Xu, Shuxia; Zhou, Shiyi; Zhang, Xinfeng; Qi, Honglan; Zhang, Chengxiao

    2014-01-01

    We report on a bienzyme-channeling sensor for sensing glucose without the aid of mediator. It was fabricated by cross-linking horseradish peroxidase (HRP) and glucose oxidase (GOx) on a glassy carbon electrode modified with multiwalled carbon nanotubes (MWNTs). The bienzyme was cross-linked with the MWNTs by glutaraldehyde and bovine serum albumin. The MWNTs were employed to accelerate the electron transfer between immobilized HRP and electrode. Glucose was sensed by amperometric reduction of enzymatically generated H 2 O 2 at an applied voltage of −50 mV (vs. Ag/AgCl). Factors influencing the preparation and performance of the bienzyme electrode were investigated in detail. The biosensor exhibited a fast and linear response to glucose in the concentration range from 0.4 to 15 mM, with a detection limit of 0.4 mM. The sensor exhibited good selectivity and durability, with a long-term relative standard deviation of <5 %. Analysis of glucose-spiked human serum samples yielded recoveries between 96 and 101 %. (author)

  8. Horseradish peroxidase functionalized gold nanorods as a label for sensitive electrochemical detection of alpha-fetoprotein antigen.

    Science.gov (United States)

    Guo, Jinjin; Han, Xiaowei; Wang, Junchun; Zhao, Junqing; Guo, Zilin; Zhang, Yuzhong

    2015-12-15

    In this study, a novel tracer, horseradish peroxidase (HRP) functionalized gold nanorods (Au NRs) nanocomposites (HRP-Au NRs), was designed to label the signal antibodies for sensitive electrochemical measurement of alpha-fetoprotein (AFP). The preparation of HRP-Au NRs nanocomposites and the labeling of secondary antibody (Ab2) were performed by one-pot assembly of HRP and Ab2 on the surface of Au NRs. The immunosensor was fabricated by assembling carbon nanotubes (CNTs), Au NRs, and capture antibodies (Ab1) on the glassy carbon electrode. In the presence of AFP antigen, the labels were captured on the surface of the Au NRs/CNTs via specific recognition of antigen-antibody, resulting in the signal intensity being clearly increased. Differential pulse voltammetry (DPV) was employed to record the response signal of the immunosensor in phosphate-buffered saline (PBS) containing hydrogen peroxide (H2O2) and 3,3',5,5'-tetramethylbenzidine (TMB). Under optimal conditions, the signal intensity was linearly related to the concentration of AFP in the range of 0.1-100 ng ml(-1), and the limit of detection was 30 pg ml(-1) (at signal/noise [S/N] = 3). Furthermore, the immunoassay method was evaluated using human serum samples, and the recovery obtained was within 99.0 and 102.7%, indicating that the immunosensor has potential clinical applications. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. Bioelectrocatalytic and biosensing properties of horseradish peroxidase covalently immobilized on (3-aminopropyl)trimethoxysilane-modified titanate nanotubes

    International Nuclear Information System (INIS)

    Sovic, David; Gajovic, Andreja; Ivekovic, Damir

    2011-01-01

    Titanate nanotubes (TiNT) surface modified with (3-aminopropyl)trimethoxysilane were employed as a support for covalent immobilization of horseradish peroxidase (HRP) by using 1,4-benzoquinone as a coupling agent. Composite film-electrodes consisting of HRP-modified TiNT embedded into the porous carbon powder/Nafion matrix were fabricated and their applicability in direct bioelectrocatalytic reduction of H 2 O 2 and H 2 O 2 biosensing were investigated. An efficient direct electron transfer between the immobilized HRP molecules and the electrode was observed in the presence of H 2 O 2 at potentials lower than 600 mV (vs. Hg/Hg 2 Cl 2 /3.5 M KCl). For the HRP-TiNT-modified electrodes polarized at 0 mV, a linear dependence of the bioelectrocatalytic current on the concentration of H 2 O 2 was observed up to the concentration of H 2 O 2 equal to 10 μM, with the sensitivity of (1.10 ± 0.01) AM -1 cm -2 and the detection limit of 35 nM.

  10. Ectopic expression of a horseradish peroxidase enhances growth rate and increases oxidative stress resistance in hybrid aspen.

    Science.gov (United States)

    Kawaoka, Akiyoshi; Matsunaga, Etsuko; Endo, Saori; Kondo, Shinkichi; Yoshida, Kazuya; Shinmyo, Atsuhiko; Ebinuma, Hiroyasu

    2003-07-01

    We previously demonstrated that overexpression of the horseradish (Armoracia rusticana) peroxidase prxC1a gene stimulated the growth rate of tobacco (Nicotiana tabacum) plants. Here, the cauliflower mosaic virus 35S::prxC1a construct was introduced into hybrid aspen (Populus sieboldii x Populus grandidentata). The growth rate of these transformed hybrid aspen plants was substantially increased under greenhouse conditions. The average stem length of transformed plants was 25% greater than that of control plants. There was no other obvious phenotypic difference between the transformed and control plants. Fast-growing transformed hybrid aspen showed high levels of expression of prxC1a and had elevated peroxidase activities toward guaiacol and ascorbate. However, there was no increase of the endogenous class I ascorbate peroxidase activities in the transformed plants by separate assay and activity staining of native polyacrylamide gel electrophoresis. Furthermore, calli derived from the transformed hybrid aspen grew faster than those from control plants and were resistant to the oxidative stress imposed by hydrogen peroxide. Therefore, enhanced peroxidase activity affects plant growth rate and oxidative stress resistance.

  11. Two New Preyssler-Type Polyoxometalate-Based Coordination Polymers and Their Application in Horseradish Peroxidase Immobilization.

    Science.gov (United States)

    Du, Jing; Cao, Mei-Da; Feng, Shu-Li; Su, Fang; Sang, Xiao-Jing; Zhang, Lan-Cui; You, Wan-Sheng; Yang, Mei; Zhu, Zai-Ming

    2017-10-17

    Enzyme immobilization is of increasing importance for biocatalysis, for which good supports are critical. Herein, two new Preyssler-type polyoxometalate (POM)-based coordination polymers, namely, {[Cu(H 2 biim) 2 ][{Cu(H 2 biim) 2 (μ-H 2 O)} 2 Cu(H 2 biim)(H 2 O) 2 ]H[({Cu(H 2 biim)(H 2 O) 2 } 0.5 ) 2 ((μ-C 3 HN 2 Cl 2 ){Cu(H 2 biim)} 2 ){Z(H 2 O)P 5 W 30 O 110 }]⋅x H 2 O} n (1: Z=Na, x=9; 2: Z=Ag, x=10; H 2 biim=2,2'-biimidazole) were designed and synthesized. Compounds 1 and 2 exhibit the same skeletons, which contain multiple Cu II complex fragments and penta-supported {ZP 5 W 30 } (Z=Na, Ag) clusters. They were first employed to immobilize horseradish peroxidase (HRP). Results show that compounds 1 and 2 are good supports for HRP immobilization, and exhibit higher enzyme loading, lower loading times, and excellent reusability. The immobilized HRP (HRP/1 or HRP/2) was further applied to detect H 2 O 2 , and good sensitivity, wide linear range, low detection limit, and fast response were achieved. This work shows that POM-based hybrid materials are a new kind of promising support for enzyme immobilization. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Alterations in cytosol free calcium in horseradish roots simultaneously exposed to lanthanum(III) and acid rain.

    Science.gov (United States)

    Zhang, Xuanbo; Wang, Lihong; Zhou, Anhua; Zhou, Qing; Huang, Xiaohua

    2016-04-01

    The extensive use of rare earth elements (REEs) has increased their environmental levels. REE pollution concomitant with acid rain in many agricultural regions can affect crop growth. Cytosol free calcium ions (Ca(2+)) play an important role in almost all cellular activities. However, no data have been reported regarding the role of cytosol free Ca(2+) in plant roots simultaneously exposed to REE and acid rain. In this study, the effects of exposures to lanthanum(III) and acid rain, independently and in combination, on cytosol free Ca(2+) levels, root activity, metal contents, biomass, cytosol pH and La contents in horseradish roots were investigated. The simultaneous exposures to La(III) and acid rain increased or decreased the cytosol free Ca(2+) levels, depending on the concentration of La(III), and these effects were more evident than independent exposure to La(III) or acid rain. In combined exposures, cytosol free Ca(2+) played an important role in the regulation of root activity, metal contents and biomass. These roles were closely related to La(III) dose, acid rain strength and treatment mode (independent exposure or simultaneous exposure). A low concentration of La(III) (20 mg L(-1)) could alleviate the adverse effects on the roots caused by acid rain, and the combined exposures at higher concentrations of La(III) and acid rain had synergic effects on the roots. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. Amperometric inhibition biosensors based on horseradish peroxidase and gold sononanoparticles immobilized onto different electrodes for cyanide measurements.

    Science.gov (United States)

    Attar, Aisha; Cubillana-Aguilera, Laura; Naranjo-Rodríguez, Ignacio; de Cisneros, José Luis Hidalgo-Hidalgo; Palacios-Santander, José María; Amine, Aziz

    2015-02-01

    New biosensors based on inhibition for the detection of cyanide and the comparison of the analytical performances of nine enzyme biosensor designs by using three different electrodes: Sonogel-Carbon, glassy carbon and gold electrodes were discussed. Three different horseradish peroxidase immobilization procedures with and without gold sononanoparticles were studied. The amperometric measurements were performed at an applied potential of -0.15V vs. Ag/AgCl in 50mM sodium acetate buffer solution pH=5.0. The apparent kinetic parameters (Kmapp, Vmaxapp) of immobilized HRP were calculated in the absence of inhibitor (cyanide) by using caffeic acid, hydroquinone, and catechol as substrates. The presence of gold sononanoparticles enhanced the electron transfer reaction and improved the analytical performance of the biosensors. The HRP kinetic interactions reveal non-competitive binding of cyanide with an apparent inhibition constant (Ki) of 2.7μM and I50 of 1.3μM. The determination of cyanide can be achieved in a dynamic range of 0.1-58.6μM with a detection limit of 0.03μM which is lower than those reported by previous studies. Hence this biosensing methodology can be used as a new promising approach for detecting cyanide. Copyright © 2014. Published by Elsevier B.V.

  14. Horseradish peroxidase and toluidine blue covalently immobilized leak-free sol-gel composite biosensor for hydrogen peroxide.

    Science.gov (United States)

    Thenmozhi, K; Narayanan, S Sriman

    2017-01-01

    The enzyme horseradish peroxidase and the water-soluble mediator toluidine blue were covalently immobilized to 3-aminopropyl trimethoxy silane precursor through glutaraldehyde crosslinker. A rigid ceramic composite electrode was fabricated from this modified silane along with graphite powder, which resulted in an amperometric biosensor for H 2 O 2 . The electrochemical behaviour of the modified biosensor was monitored using cyclic voltammetry in the potential range of 0.2V to -0.4V vs SCE. The biosensor exhibited a stable voltammogram with cathodic peak at -0.234V and anodic peak at -0.172V, with a formal potential of -0.203V. Various factors influencing the performance of the biosensor such as buffer solution, pH, temperature and potential were examined for optimizing the working conditions. The modified biosensor exhibited a good catalytic behaviour for the reduction of H 2 O 2 at a lower potential of -0.25V without any barrier from possible interferents. The analytical working range was found to be 0.429μM to 0.455mM of H 2 O 2 with a detection limit of 0.171μM. The fabricated biosensor is robust for long-term usage in addition to the high sensitivity, rapid response and having an advantage of surface renewability by simple mechanical polishing. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Simona Capone

    2015-09-01

    Full Text Available Horseradish peroxidase (HRP, conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris, the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1 was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.

  16. Step-Up Synthesis of Periodic Mesoporous Organosilicas with a Tyrosine Framework and Performance in Horseradish Peroxidase Immobilization.

    Science.gov (United States)

    Wang, Jianqiang; Zhang, Wenqi; Gu, Changqing; Zhang, Wenpei; Zhou, Man; Wang, Zhiwei; Guo, Cheng; Sun, Linbing

    2017-12-14

    New amino-acid-bridged periodic mesoporous organosilicas (PMOs) were constructed by hydrolysis and condensation reactions under acid conditions in the presence of a template. The tyrosine bissilylated organic precursor (TBOS) was first prepared through a multistep reaction by using tyrosine (a natural amino acid) as the starting material. PMOs with the tyrosine framework (Tyr-PMOs) were constructed by simultaneously using TBOS and tetraethoxysilane as complex silicon sources in the condensation process. All the Tyr-PMOs materials were characterized by XRD, FTIR spectroscopy, N 2 adsorption-desorption, TEM, SEM, and solid-state 29 Si NMR spectroscopy to confirm the structure. The horseradish peroxidase (HRP) enzyme was first immobilized on these new Tyr-PMOs materials. Optimal conditions for enzyme adsorption included a temperature of 40 °C, a time of 8 h, and a pH value of 7. Furthermore, the novel Tyr-PMOs materials could store HRP for approximately 40 days and maintained the enzymatic activity, and the Tyr-PMOs-10 % HRP with the best immobilization effect could be reused at least eight times. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Improved biodegradation of synthetic azo dye by horseradish peroxidase cross-linked on nano-composite support.

    Science.gov (United States)

    Sun, Huaiyan; Jin, Xinyu; Long, Nengbing; Zhang, Ruifeng

    2017-02-01

    A ZnO nanowires/macroporous SiO 2 composite was used as support to immobilize horseradish peroxidase (HRP) by in-situ cross-linking method. Using diethylene glycol diglycidyl ether (DDE) as a long-chained cross-linker, it was adsorbed on the surface of ZnO nanowires before reaction with HRPs, the resulted composite was quite different from the traditional cross-linking enzyme aggregates (CLEAs) on both structure and catalytic performance. The immobilized HRP showed high activity in the decolorization of azo dyes. The effect of various conditions such as the loading amount of HRP, solution pH, temperature, contact time and concentration of dye were optimized on the decolorization. The decolorization percentage of Acid Blue 113 and Acid black 10 BX reached as high as 95.4% and 90.3%, respectively. The immobilized HRP gave the highest decolorization rate under dye concentration as 50mg/L and reaction time of 35min. The immobilized HRP exhibited much better resistance to temperature and pH inactivation than free HRP. The storage stability and reusability were greatly improved through the immobilization, from the decolorization of Acid blue 113 it was found that 80.4% of initial efficiency retained after incubation at 4°C for 60 days, and that 79.4% of decolorization efficiency retained after 12 cycles reuse. Copyright © 2016 Elsevier B.V. All rights reserved.

  18. Immobilization of horseradish peroxidase on self-assembled (3-mercaptopropyl)trimethoxysilane film: Characterization, direct electrochemistry, redox thermodynamics and biosensing

    International Nuclear Information System (INIS)

    Wu Fanghua; Hu Zhichao; Xu Jingjing; Tian Yuan; Wang Liwei; Xian Yuezhong; Jin Litong

    2008-01-01

    Highly organized (3-mercaptopropyl)trimethoxysilane (3-MPT) films have been prepared via self-assembled coupled with sol-gel linking technology. Horseradish peroxidase (HRP) is successfully immobilized onto the densely packed three-dimensional (3D) 3-MPT network and the direct electrochemistry of HRP is achieved without any electron mediators or promoters. Redox thermodynamics of HRP on the 3-MPT films, which is obtained from the temperature dependence of the reduction potential, suggests that the positive shift of redox potentials of HRP at the interface of 3-MPT originates from the solvent reorganization effects and conformational change of the polypeptide chain of HRP. Based on the direct electrochemistry and electrocatalytic ability of HRP, a sensitive third-generation amperometric H 2 O 2 biosensor is developed with two linear dependence ranges of 5.0 x 10 -7 to 1.0 x 10 -4 and 1.0 x 10 -4 to 2.0 x 10 -2 mol L -1

  19. The integral membrane protein ITM2A, a transcriptional target of PKA-CREB, regulates autophagic flux via interaction with the vacuolar ATPase.

    Science.gov (United States)

    Namkoong, Sim; Lee, Kang Il; Lee, Jin I; Park, Rackhyun; Lee, Eun-Ju; Jang, Ik-Soon; Park, Junsoo

    2015-01-01

    The PKA-CREB signaling pathway is involved in many cellular processes including autophagy. Recent studies demonstrated that PKA-CREB inhibits autophagy in yeast; however, the role of PKA-CREB signaling in mammalian cell autophagy has not been fully characterized. Here, we report that the integral membrane protein ITM2A expression is positively regulated by PKA-CREB signaling and ITM2A expression interferes with autophagic flux by interacting with vacuolar ATPase (v-ATPase). The ITM2A promoter contains a CRE element, and mutation at the CRE consensus site decreases the promoter activity. Forskolin treatment and PKA expression activate the ITM2A promoter confirming that ITM2A expression is dependent on the PKA-CREB pathway. ITM2A expression results in the accumulation of autophagosomes and interferes with autolysosome formation by blocking autophagic flux. We demonstrated that ITM2A physically interacts with v-ATPase and inhibits lysosomal function. These results support the notion that PKA-CREB signaling pathway regulates ITM2A expression, which negatively regulates autophagic flux by interfering with the function of v-ATPase.

  20. Selective pH-Responsive Core-Sheath Nanofiber Membranes for Chem/Bio/Med Applications: Targeted Delivery of Functional Molecules.

    Science.gov (United States)

    Han, Daewoo; Steckl, Andrew J

    2017-12-13

    Core-sheath fibers using different Eudragit materials were successfully produced, and their controlled multi-pH responses have been demonstrated. Core-sheath fibers made of Eudragit L 100 (EL100) core and Eudragit S 100 (ES100) sheath provide protection and/or controlled release of core material at pH 6 by adjusting the sheath thickness (controlled by the flow rate of source polymer solution). The thickest sheath (∼250 nm) provides the least core release ∼1.25%/h, while the thinnest sheath (∼140 nm) provides much quicker release ∼16.75%/h. Furthermore, switching core and sheath material dramatically altered the pH response. Core-sheath fibers made of ES100 core and EL100 sheath can provide a consistent core release rate, while the sheath release rate becomes higher as the sheath layer becomes thinner. For example, the thinnest sheath (∼120 nm) provides a core and sheath release ratio of 1:2.5, while the thickest sheath (∼200 nm) shows only a ratio of 1:1.7. All core-sheath Eudragit fibers show no noticeable release at pH 5, while they are completely dissolved at pH 7. Extremely high surface area in the porous network of the fiber membranes provides much faster (>30 times) response to external pH changes as compared to that of equivalent cast films.

  1. Application and bioactive properties of CaTI, a trypsin inhibitor from Capsicum annuum seeds: membrane permeabilization, oxidative stress and intracellular target in phytopathogenic fungi cells.

    Science.gov (United States)

    Silva, Marciele S; Ribeiro, Suzanna Ff; Taveira, Gabriel B; Rodrigues, Rosana; Fernandes, Katia Vs; Carvalho, André O; Vasconcelos, Ilka Maria; Mello, Erica Oliveira; Gomes, Valdirene M

    2017-08-01

    During the last few years, a growing number of antimicrobial peptides have been isolated from plants and particularly from seeds. Recent results from our laboratory have shown the purification of a new trypsin inhibitor, named CaTI, from chilli pepper (Capsicum annuum L.) seeds. This study aims to evaluate the antifungal activity and mechanism of action of CaTI on phytopathogenic fungi and detect the presence of protease inhibitors in other species of this genus. Our results show that CaTI can inhibit the growth of the phytopathogenic fungi Colletotrichum gloeosporioides and C. lindemuthianum. CaTI can also permeabilize the membrane of all tested fungi. When testing the inhibitor on its ability to induce reactive oxygen species, an induction of reactive oxygen species (ROS) and nitric oxide (NO) particularly in Fusarium species was observed. Using CaTI coupled to fluorescein isothiocyanate (FITC), it was possible to determine the presence of the inhibitor inside the hyphae of the Fusarium oxysporum fungus. The search for protease inhibitors in other Capsicum species revealed their presence in all tested species. This paper shows the antifungal activity of protease inhibitors such as CaTI against phytopathogenic fungi. Antimicrobial peptides, among which the trypsin protease inhibitor family stands out, are present in different species of the genus Capsicum and are part of the chemical arsenal that plants use to defend themselves against pathogens. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  2. Far Western: probing membranes.

    Science.gov (United States)

    Einarson, Margret B; Pugacheva, Elena N; Orlinick, Jason R

    2007-08-01

    INTRODUCTIONThe far-Western technique described in this protocol is fundamentally similar to Western blotting. In Western blots, an antibody is used to detect a query protein on a membrane. In contrast, in a far-Western blot (also known as an overlay assay) the antibody is replaced by a recombinant GST fusion protein (produced and purified from bacteria), and the assay detects the interaction of this protein with target proteins on a membrane. The membranes are washed and blocked, incubated with probe protein, washed again, and subjected to autoradiography. The GST fusion (probe) proteins are often labeled with (32)P; alternatively, the membrane can be probed with unlabeled GST fusion protein, followed by detection using commercially available GST antibodies. The nonradioactive approach is substantially more expensive (due to the purchase of antibody and detection reagents) than using radioactively labeled proteins. In addition, care must be taken to control for nonspecific interactions with GST alone and a signal resulting from antibody cross-reactivity. In some instances, proteins on the membrane are not able to interact after transfer. This may be due to improper folding, particularly in the case of proteins expressed from a phage expression library. This protocol describes a way to overcome this by washing the membrane in denaturation buffer, which is then serially diluted to permit slow renaturation of the proteins.

  3. Cell-free system for synthesizing membrane proteins cell free method for synthesizing membrane proteins

    Science.gov (United States)

    Laible, Philip D; Hanson, Deborah K

    2013-06-04

    The invention provides an in vitro method for producing proteins, membrane proteins, membrane-associated proteins, and soluble proteins that interact with membrane-associated proteins for assembly into an oligomeric complex or that require association with a membrane for proper folding. The method comprises, supplying intracytoplasmic membranes from organisms; modifying protein composition of intracytoplasmic membranes from organism by modifying DNA to delete genes encoding functions of the organism not associated with the formation of the intracytoplasmic membranes; generating appropriate DNA or RNA templates that encode the target protein; and mixing the intracytoplasmic membranes with the template and a transcription/translation-competent cellular extract to cause simultaneous production of the membrane proteins and encapsulation of the membrane proteins within the intracytoplasmic membranes.

  4. Flux Enhancement in Membrane Distillation Using Nanofiber Membranes

    Directory of Open Access Journals (Sweden)

    T. Jiříček

    2016-01-01

    Full Text Available Membrane distillation (MD is an emerging separation technology, whose largest application potential lies in the desalination of highly concentrated solutions, which are out of the scope of reverse osmosis. Despite many attractive features, this technology is still awaiting large industrial application. The main reason is the lack of commercially available membranes with fluxes comparable to reverse osmosis. MD is a thermal separation process driven by a partial vapour pressure difference. Flux, distillate purity, and thermal efficiency are always in conflict, all three being strictly connected with pore size, membrane hydrophobicity, and thickness. The world has not seen the ideal membrane yet, but nanofibers may offer a solution to these contradictory requirements. Membranes of electrospun PVDF were tested under various conditions on a direct contact (DCMD unit, in order to determine the optimum conditions for maximum flux. In addition, their performance was compared to commonly available PTFE, PE, and PES membranes. It was confirmed that thinner membranes have higher fluxes and a lower distillate purity and also higher energy losses via conduction across the membrane. As both mass and heat transfer are connected, it is best to develop new membranes with a target application in mind, for the specific membrane module and operational conditions.

  5. Human CD34+ cells engineered to express membrane-bound tumor necrosis factor-related apoptosis-inducing ligand target both tumor cells and tumor vasculature.

    Science.gov (United States)

    Lavazza, Cristiana; Carlo-Stella, Carmelo; Giacomini, Arianna; Cleris, Loredana; Righi, Marco; Sia, Daniela; Di Nicola, Massimo; Magni, Michele; Longoni, Paolo; Milanesi, Marco; Francolini, Maura; Gloghini, Annunziata; Carbone, Antonino; Formelli, Franca; Gianni, Alessandro M

    2010-03-18

    Adenovirus-transduced CD34+ cells expressing membrane-bound tumor necrosis factor-related apoptosis-inducing ligand (CD34-TRAIL+ cells) exert potent antitumor activity. To further investigate the mechanism(s) of action of CD34-TRAIL+ cells, we analyzed their homing properties as well as antitumor and antivascular effects using a subcutaneous myeloma model in immunodeficient mice. After intravenous injection, transduced cells homed in the tumor peaking at 48 hours when 188 plus or minus 25 CD45+ cells per 10(5) tumor cells were detected. Inhibition experiments showed that tumor homing of CD34-TRAIL+ cells was largely mediated by vascular cell adhesion molecule-1 and stromal cell-derived factor-1. Both CD34-TRAIL+ cells and soluble (s)TRAIL significantly reduced tumor volume by 40% and 29%, respectively. Computer-aided analysis of TdT-mediated dUTP nick end-labeling-stained tumor sections demonstrated significantly greater effectiveness for CD34-TRAIL+ cells in increasing tumor cell apoptosis and necrosis over sTRAIL. Proteome array analysis indicated that CD34-TRAIL+ cells and sTRAIL activate similar apoptotic machinery. In vivo staining of tumor vasculature with sulfosuccinimidyl-6-(biotinamido) hexanoate-biotin revealed that CD34-TRAIL+ cells but not sTRAIL significantly damaged tumor vasculature, as shown by TdT-mediated dUTP nick end-labeling+ endothelial cells, appearance of hemorrhagic areas, and marked reduction of endothelial area. These results demonstrate that tumor homing of CD34-TRAIL+ cells induces early vascular disruption, resulting in hemorrhagic necrosis and tumor destruction.

  6. Membrane paradigm

    International Nuclear Information System (INIS)

    Price, R.H.; Thorne, K.S.

    1986-01-01

    The membrane paradigm is a modified frozen star approach to modeling black holes, with particles and fields assuming a complex, static, boundary-layer type structure (membrane) near the event horizon. The membrane has no effects on the present or future evolution of particles and fields above itself. The mathematical representation is a combination of a formalism containing terms for the shear and bulk viscosity, surface pressure, momentum, temperature, entropy, etc., of the horizon and the 3+1 formalism. The latter model considers a family of three-dimensional spacelike hypersurfaces in one-dimensional time. The membrane model considers a magnetic field threading the hole and undergoing torque from the hole rotation. The field is cleaned by the horizon and distributed over the horizon so that ohmic dissipation is minimized. The membrane paradigm is invalid inside the horizon, but is useful for theoretically probing the properties of slowly evolving black holes

  7. Membrane processes

    Science.gov (United States)

    Staszak, Katarzyna

    2017-11-01

    The membrane processes have played important role in the industrial separation process. These technologies can be found in all industrial areas such as food, beverages, metallurgy, pulp and paper, textile, pharmaceutical, automotive, biotechnology and chemical industry, as well as in water treatment for domestic and industrial application. Although these processes are known since twentieth century, there are still many studies that focus on the testing of new membranes' materials and determining of conditions for optimal selectivity, i. e. the optimum transmembrane pressure (TMP) or permeate flux to minimize fouling. Moreover the researchers proposed some calculation methods to predict the membrane processes properties. In this article, the laboratory scale experiments of membrane separation techniques, as well their validation by calculation methods are presented. Because membrane is the "heart" of the process, experimental and computational methods for its characterization are also described.

  8. Solubilization of lipids and membrane proteins into nanodiscs : Mode of action and applications of SMA copolymers

    NARCIS (Netherlands)

    Scheidelaar, S.

    2016-01-01

    Cell membranes separate the inside and outside of cells. Membrane proteins in the cell membrane control the traffic of molecules across the membrane and are therefore targets for a lot of drugs: about 50 % of all approved drugs target a membrane protein! Unfortunately, scientists only know little

  9. Primordial membranes

    DEFF Research Database (Denmark)

    Hanczyc, Martin M; Monnard, Pierre-Alain

    2017-01-01

    Cellular membranes, which are self-assembled bilayer structures mainly composed of lipids, proteins and conjugated polysaccharides, are the defining feature of cell physiology. It is likely that the complexity of contemporary cells was preceded by simpler chemical systems or protocells during...... the various evolutionary stages that led from inanimate to living matter. It is also likely that primitive membranes played a similar role in protocell 'physiology'. The composition of such ancestral membranes has been proposed as mixtures of single hydrocarbon chain amphiphiles, which are simpler versions...

  10. Nitroxides protect horseradish peroxidase from H2O2-induced inactivation and modulate its catalase-like activity.

    Science.gov (United States)

    Samuni, Amram; Maimon, Eric; Goldstein, Sara

    2017-08-01

    Horseradish peroxidase (HRP) catalyzes H 2 O 2 dismutation while undergoing heme inactivation. The mechanism underlying this process has not been fully elucidated. The effects of nitroxides, which protect metmyoglobin and methemoglobin against H 2 O 2 -induced inactivation, have been investigated. HRP reaction with H 2 O 2 was studied by following H 2 O 2 depletion, O 2 evolution and heme spectral changes. Nitroxide concentration was followed by EPR spectroscopy, and its reactions with the oxidized heme species were studied using stopped-flow. Nitroxide protects HRP against H 2 O 2 -induced inactivation. The rate of H 2 O 2 dismutation in the presence of nitroxide obeys zero-order kinetics and increases as [nitroxide] increases. Nitroxide acts catalytically since its oxidized form is readily reduced to the nitroxide mainly by H 2 O 2 . The nitroxide efficacy follows the order 2,2,6,6-tetramethyl-piperidine-N-oxyl (TPO)>4-OH-TPO>3-carbamoyl proxyl>4-oxo-TPO, which correlates with the order of the rate constants of nitroxide reactions with compounds I, II, and III. Nitroxide catalytically protects HRP against inactivation induced by H 2 O 2 while modulating its catalase-like activity. The protective role of nitroxide at μM concentrations is attributed to its efficient oxidation by P940, which is the precursor of the inactivated form P670. Modeling the dismutation kinetics in the presence of nitroxide adequately fits the experimental data. In the absence of nitroxide the simulation fits the observed kinetics only if it does not include the formation of a Michaelis-Menten complex. Nitroxides catalytically protect heme proteins against inactivation induced by H 2 O 2 revealing an additional role played by nitroxide antioxidants in vivo. Copyright © 2017 Elsevier B.V. All rights reserved.

  11. How modification of accessible lysines to phenylalanine modulates the structural and functional properties of horseradish peroxidase: a simulation study.

    Directory of Open Access Journals (Sweden)

    Leila Navapour

    Full Text Available Horseradish Peroxidase (HRP is one of the most studied peroxidases and a great number of chemical modifications and genetic manipulations have been carried out on its surface accessible residues to improve its stability and catalytic efficiency necessary for biotechnological applications. Most of the stabilized derivatives of HRP reported to date have involved chemical or genetic modifications of three surface-exposed lysines (K174, K232 and K241. In this computational study, we altered these lysines to phenylalanine residues to model those chemical modifications or genetic manipulations in which these positively charged lysines are converted to aromatic hydrophobic residues. Simulation results implied that upon these substitutions, the protein structure becomes less flexible. Stability gains are likely to be achieved due to the increased number of stable hydrogen bonds, improved heme-protein interactions and more integrated proximal Ca2+ binding pocket. We also found a new persistent hydrogen bond between the protein moiety (F174 and the heme prosthetic group as well as two stitching hydrogen bonds between the connecting loops GH and F'F″ in mutated HRP. However, detailed analysis of functionally related structural properties and dynamical features suggests reduced reactivity of the enzyme toward its substrates. Molecular dynamics simulations showed that substitutions narrow the bottle neck entry of peroxide substrate access channel and reduce the surface accessibility of the distal histidine (H42 and heme prosthetic group to the peroxide and aromatic substrates, respectively. Results also demonstrated that the area and volume of the aromatic-substrate binding pocket are significantly decreased upon modifications. Moreover, the hydrophobic patch functioning as a binding site or trap for reducing aromatic substrates is shrunk in mutated enzyme. Together, the results of this simulation study could provide possible structural clues to explain

  12. Development of horseradish peroxidase-based cross-linked enzyme aggregates and their environmental exploitation for bioremediation purposes.

    Science.gov (United States)

    Bilal, Muhammad; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

    2017-03-01

    In the present study, horseradish peroxidase (HRP), in-house isolated crude cocktail enzyme, from Armoracia rusticana was cross-linked using a new type of cross-linking agent, i.e., ethylene glycol-bis [succinic acid N-hydroxysuccinimide, (EG-NHS)], which is mild in nature as compared to the glutaraldehyde (GA). The HRP-immobilized cross-linked enzyme aggregates (HRP-CLEAs) were developed using a wider range of EG-NHS and notably no adverse effect was observed. In a comparative evaluation, in the case of EG-NHS, a high-level stability in the residual activity was recorded, whereas a sharp decrease was observed in the case of glutaraldehyde. Following initial cross-linker evaluation, the HRP-CLEAs were tested to investigate their bio-catalytic efficacy for bioremediation purposes using a newly developed packed bed reactor system (PBRS). A maximal of 94.26% degradation of textile-based methyl orange dye was recorded within the shortest time frame, following 91.73% degradation of basic red 9, 84.35% degradation of indigo, 81.47% degradation of Rhodamin B, and 73.6% degradation of Rhodamine 6G, respectively, under the same working environment. Notably, the HRP-CLEAs retained almost 60% of its original activity after methyl orange dye degradation in seven consecutive cycles using PBRS. Furthermore, after HRP-CLEAs-mediated treatment in the PBRS, a significant toxicity reduction in the dye samples was recorded as compared to their pristine counterparts. In conclusion, the results suggest that the newly developed HRP-CLEAs have a great potential for industrial exploitation, to tackle numerous industrial dye-based emergent pollutants. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Novel characteristics of horseradish peroxidase immobilized onto the polyvinyl alcohol-alginate beads and its methyl orange degradation potential.

    Science.gov (United States)

    Bilal, Muhammad; Rasheed, Tahir; Iqbal, Hafiz M N; Hu, Hongbo; Wang, Wei; Zhang, Xuehong

    2017-12-01

    Herein, we report the immobilization of in-house isolated horseradish peroxidase (HRP) from Armoracia rusticana with novel characteristics. The HRP was immobilized onto the self-fabricated polyvinyl alcohol-alginate (PVA-alginate) beads using sodium nitrate as a cross-linker. The PVA-alginate beads (2.0mm size) developed using 10% PVA and 1.5% sodium alginate showed maximal immobilization yield. The surface morphologies of the PVA-alginate (control) and immobilized-HRP were characterized by scanning electron microscopy (SEM). The immobilized-HRP retained 64.14% of its initial activity after 10 consecutive substrate-oxidation cycles as compared to the free counterpart. Simultaneously, the thermal stability of the immobilized-HRP was significantly enhanced as compared to the free HRP. The enzyme leakage (E L ) assay was performed by storing the immobilized-HRP in phosphate buffer solution for 30days. Evidently, the leakage of immobilized-HRP was recorded to be 6.98% and 14.82% after 15 and 30days of incubation, respectively. Finally, the immobilized-HRP was used for methyl orange (MO) dye degradation in a batch mode. A noticeable decline in spectral shift accompanied by no appearance of a new peak demonstrated the complete degradation of MO. The degraded fragments of MO were scrutinized by ultra-performance liquid chromatography coupled with mass spectrometry (UPLC-MS). A plausible degradation pathway for MO was proposed based on the identified intermediates. In conclusion, the study portrays the PVA-alginate-immobilized-HRP as a cost-effective and industrially desirable green catalyst, for biotechnological at large and industrial in particular, especially for the treatment of textile dyes or dye-containing industrial waste effluents. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Quantitative determination of trace hydrogen peroxide in the presence of sulfide using the Amplex Red/horseradish peroxidase assay.

    Science.gov (United States)

    Wang, Nannan; Miller, Christopher J; Wang, Peng; Waite, T David

    2017-04-22

    The Amplex Red/horseradish peroxidase (AR/HRP) assay for H 2 O 2 is one of the most sensitive and simple approaches for H 2 O 2 quantification, which is effected by measuring the highly-fluorescent resorufin formed from oxidation of AR by the oxidizing intermediates generated by reaction of HRP and H 2 O 2 . The direct reactions of S(-II) with both H 2 O 2 and resorufin are too slow to be of relevance on analytical timescales, however, the reaction between S(-II) and the HRP/H 2 O 2 oxidizing intermediates is rapid enough to compete with the desired reaction of these oxidizing intermediates with AR, suppressing formation of the resorufin analyte. As this mode of interference can be considered simply a competition between the AR reagent and S(-II) for the intermediate oxidizing species, a simple equation is derived in this work enabling one to correct for this interference and obtain a good estimate of the true H 2 O 2 concentration after measuring the apparent H 2 O 2 concentration and the S(-II) concentration. This mode of interference is general to any compound that can act as a HRP substrate even if not directly reactive with H 2 O 2 . As such, the approach described is widely applicable to many potential reducing interferents and opens up the use of the AR/HRP assay to a much wider range of conditions, as well as demonstrating the utility of explicitly considering the mechanism of the analytical process. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Ultra-high-throughput screening of an in vitro-synthesized horseradish peroxidase displayed on microbeads using cell sorter.

    Directory of Open Access Journals (Sweden)

    Bo Zhu

    Full Text Available The C1a isoenzyme of horseradish peroxidase (HRP is an industrially important heme-containing enzyme that utilizes hydrogen peroxide to oxidize a wide variety of inorganic and organic compounds for practical applications, including synthesis of fine chemicals, medical diagnostics, and bioremediation. To develop a ultra-high-throughput screening system for HRP, we successfully produced active HRP in an Escherichia coli cell-free protein synthesis system, by adding disulfide bond isomerase DsbC and optimizing the concentrations of hemin and calcium ions and the temperature. The biosynthesized HRP was fused with a single-chain Cro (scCro DNA-binding tag at its N-terminal and C-terminal sites. The addition of the scCro-tag at both ends increased the solubility of the protein. Next, HRP and its fusion proteins were successfully synthesized in a water droplet emulsion by using hexadecane as the oil phase and SunSoft No. 818SK as the surfactant. HRP fusion proteins were displayed on microbeads attached with double-stranded DNA (containing the scCro binding sequence via scCro-DNA interactions. The activities of the immobilized HRP fusion proteins were detected with a tyramide-based fluorogenic assay using flow cytometry. Moreover, a model microbead library containing wild type hrp (WT and inactive mutant (MUT genes was screened using fluorescence-activated cell-sorting, thus efficiently enriching the WT gene from the 1:100 (WT:MUT library. The technique described here could serve as a novel platform for the ultra-high-throughput discovery of more useful HRP mutants and other heme-containing peroxidases.

  16. Enzymatic biosensor of horseradish peroxidase immobilized on Au-Pt nanotube/Au-graphene for the simultaneous determination of antioxidants

    Energy Technology Data Exchange (ETDEWEB)

    Wu, Long; Yin, Wenmin; Tang, Kun; Li, Dian; Shao, Kang; Zuo, Yunpeng; Ma, Jing; Liu, Jiawei; Han, Heyou, E-mail: hyhan@mail.hzau.edu.cn

    2016-08-24

    A new electrochemical method has been proposed for the simultaneous determination of butylated hydroxyanisole (BHA) and propyl gallate (PG) in food matrices based on enzymatic biosensors. Spiny Au-Pt nanotubes (SAP NTs) was first synthesized and demonstrated to exhibit intrinsic peroxidase and catalase-like activity. The structure of SAP NTs provides large surface area and favorable medium for electron transfer, on which HRP were immobilized and acted as enzymatic biosensor for the simultaneous detection of BHA and PG. The results revealed that BHA and PG both have well-defined oxidation waves with peak potentials of 624 and 655 mV, respectively. Under the optimal conditions, the method behaved satisfactory analytical performance towards BHA and PG with a wide linear range of 0.3–50 mg L{sup −1} and 0.1–100 mg L{sup −1}, as well as a detection limit of 0.046 mg L{sup −1} and 0.024 mg L{sup −1} (3σ/slope), respectively. Besides, the proposed method exhibits good sensitivity, stability and reproducibility, providing an alternative to fabricate electrode and construct sensitive biosensors. - Highlights: • SAP NTs was synthesized and demonstrated to exhibit intrinsic peroxidase and catalase-like activity. • The structure of SAP NTs provides larger surface area and more favorable medium for electron transfer. • Horseradish peroxidase immobilized on Au-Pt nanotube/Au-graphene acted as enzymatic biosensor. • The simultaneous detection of BHA and PG in food matrices was achieved based on enzymatic biosensors.

  17. Membranous nephropathy

    Science.gov (United States)

    ... skin-lightening creams Systemic lupus erythematosus , rheumatoid arthritis, Graves disease, and other autoimmune disorders The disorder occurs at ... diagnosis. The following tests can help determine the cause of membranous nephropathy: Antinuclear antibodies test Anti-double- ...

  18. Direct electron transfer biosensor for hydrogen peroxide carrying nanocomplex composed of horseradish peroxidase and Au-nanoparticle – Characterization and application to bienzyme systems

    Directory of Open Access Journals (Sweden)

    Yusuke Okawa

    2015-09-01

    Full Text Available A reagentless electrochemical biosensor for hydrogen peroxide was fabricated. The sensor carries a monolayer of nanocomplex composed of horseradish peroxidase and Au-nanoparticle, and responds to hydrogen peroxide through the highly efficient direct electron transfer at a mild electrode potential without any soluble mediator. Formation of the nanocomplex was studied with visible spectroscopy and size exclusion chromatography. The sensor performance was analyzed based on a hydrodynamic electrochemical technique and enzyme kinetics. The sensor was applied to fabrication of sensors for glucose and uric acid through further modification of the nanocomplex-carrying electrode with the corresponding hydrogen peroxide-generating oxidases, glucose oxidase and urate oxidase, respectively.

  19. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

    OpenAIRE

    Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette; Chae, Pil Seok

    2016-01-01

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

  20. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation.

    Science.gov (United States)

    Bae, Hyoung Eun; Mortensen, Jonas S; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K; Loland, Claus J; Byrne, Bernadette; Chae, Pil Seok

    2016-10-04

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research.

  1. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation†

    Science.gov (United States)

    Bae, Hyoung Eun; Mortensen, Jonas S.; Ribeiro, Orquidea; Du, Yang; Ehsan, Muhammad; Kobilka, Brian K.; Loland, Claus J.; Byrne, Bernadette

    2017-01-01

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared to DDM. These agents have potential for use in membrane protein research. PMID:27711401

  2. Regulation of HIV-Gag Expression and Targeting to the Endolysosomal/Secretory Pathway by the Luminal Domain of Lysosomal-Associated Membrane Protein (LAMP-1) Enhance Gag-Specific Immune Response

    Science.gov (United States)

    Lucas, Carolina Gonçalves de Oliveira; Rigato, Paula Ordonhez; Gonçalves, Jorge Luiz Santos; Sato, Maria Notomi; Maciel, Milton; Peçanha, Ligia Maria Torres; August, J. Thomas; de Azevedo Marques, Ernesto Torres; de Arruda, Luciana Barros

    2014-01-01

    We have previously demonstrated that a DNA vaccine encoding HIV-p55gag in association with the lysosomal associated membrane protein-1 (LAMP-1) elicited a greater Gag-specific immune response, in comparison to a DNA encoding the native gag. In vitro studies have also demonstrated that LAMP/Gag was highly expressed and was present in MHCII containing compartments in transfected cells. In this study, the mechanisms involved in these processes and the relative contributions of the increased expression and altered traffic for the enhanced immune response were addressed. Cells transfected with plasmid DNA constructs containing p55gag attached to truncated sequences of LAMP-1 showed that the increased expression of gag mRNA required p55gag in frame with at least 741 bp of the LAMP-1 luminal domain. LAMP luminal domain also showed to be essential for Gag traffic through lysosomes and, in this case, the whole sequence was required. Further analysis of the trafficking pathway of the intact LAMP/Gag chimera demonstrated that it was secreted, at least in part, associated with exosome-like vesicles. Immunization of mice with LAMP/gag chimeric plasmids demonstrated that high expression level alone can induce a substantial transient antibody response, but targeting of the antigen to the endolysosomal/secretory pathways was required for establishment of cellular and memory response. The intact LAMP/gag construct induced polyfunctional CD4+ T cell response, which presence at the time of immunization was required for CD8+ T cell priming. LAMP-mediated targeting to endolysosomal/secretory pathway is an important new mechanistic element in LAMP-mediated enhanced immunity with applications to the development of novel anti-HIV vaccines and to general vaccinology field. PMID:24932692

  3. Axionic membranes

    International Nuclear Information System (INIS)

    Aurilia, A.; Spallucci, E.

    1992-01-01

    A metal ring removed from a soap-water solution encloses a film of soap which can be mathematically described as a minimal surface having the ring as its only boundary. This is known to everybody. In this letter we suggest a relativistic extension of the above fluidodynamic system where the soap film is replaced by a Kalb-Ramand gauge potential B μν (x) and the ring by a closed string. The interaction between the B μν field and the string current excites a new configuration of the system consisting of a relativistic membrane bounded by the string. We call such a classical solution of the equation of motion an axionic membrane. As a dynamical system, the axionic membrane admits a Hamilton-Jacobi formulation which is an extension of the HJ theory of electromagnetic strings. (orig.)

  4. Radiobiology effects of radiation-induced horseradish peroxidase/indole-3-acetic suicide gene expression in lung cancer cells

    International Nuclear Information System (INIS)

    Xiong Jie; Zhou Yunfeng; Wang Weifeng; Sun Wenjie; Liao Zhengkai; Zhou Fuxiang; Xie Conghua

    2010-01-01

    Objective: To detect specific cell killing effect of radiation combined with horseradish peroxidase (HRP)/indole-3-acetic (IAA) suicide gene therapy controlled by a novel radio-inducible and cancer-specific chimeric gene promoter in lung cancer. Methods: We constructed a plasmid expressing HRP enzyme under the control of chimeric human telomerase reverse transcriptase (hTERT) promoter carrying 6 CArG elements, a plasmid expressing HRP enzyme under the control of hTERT promoter carrying single CArG element, and two control plasmids, which named pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControlluc, respectively. After radiation, the proliferation inhibition and apoptosis induction effect of each type of plasmid in lung cancer cells (A549, SPC-A1) and normal lung cells (hEL) was detected by cell counting and Annexin V-FITC staining. The change of radiosensitivity of lung cancer cells with plasmid system was also detected by clonogenic assays. Results: After a single dose radiation of 6 Gy,the average proliferation inhibition rates of pE6-hTERT-HRP, phTERT-HRP, pControl-HRP, and pControlluc systems were 72.92% ,40.60% , 51.00% and 25.19% (F= 67.31, P< 0.01) in A549 cells, 64.63%, 30.02%, 48.23% and 23.16% (F=64.94, P< 0.01) in SPC-A1 cells, and 20.81%, 18.05%, 44.20% and 18.32% (F=52.19, P<0.01) in normal hEL cells, respectively. The average early apoptosis rates of these four plasmid systems were 36.63%, 22.30%, 24.33% and 12.53% (F =50.99, P <0.01) in A549 cells, 33.73%, 17.37%, 22.43% and 11.20% (F = 20. 76, P < 0.01) in SPC-A1 cells, and 13.53 %, 12.5%, 21.93% and 12.16% (F = 15.08, P < 0.01) in normal hEL cells,respectively. The sensitizing enhancement ratios of the four plasmid systems were 3.45, 2.29, 3.05 and 1.21 in A549 cells, while 2.68, 2.15, 3.05 and 1.21 in SPC-A1 cells, respectively. Conclusions: The new suicide gene system controlled by chimeric promoter may provide a novel therapeutic modality for lung cancer. (authors)

  5. Metamaterial membranes

    International Nuclear Information System (INIS)

    Restrepo-Flórez, Juan Manuel; Maldovan, Martin

    2017-01-01

    We introduce a new class of metamaterial device to achieve separation of compounds by using coordinate transformations and metamaterial theory. By rationally designing the spatial anisotropy for mass diffusion, we simultaneously concentrate different compounds in different spatial locations, leading to separation of mixtures across a metamaterial membrane. The separation of mixtures into their constituent compounds is critically important in biophysics, biomedical, and chemical applications. We present a practical case where a mixture of oxygen and nitrogen diffusing through a polymeric planar matrix is separated. This work opens doors to new paradigms in membrane separations via coordinate transformations and metamaterials by introducing novel properties and unconventional mass diffusion phenomena. (paper)

  6. Activity of the C-terminal-dependent vacuolar sorting signal of horseradish peroxidase C1a is enhanced by its secondary structure.

    Science.gov (United States)

    Matsui, Takeshi; Tabayashi, Ayako; Iwano, Megumi; Shinmyo, Atsuhiko; Kato, Ko; Nakayama, Hideki

    2011-02-01

    Plant class III peroxidase (PRX) catalyzes the oxidation and oxidative polymerization of a variety of phenolic compounds while reducing hydrogen peroxide. PRX proteins are classified into apoplast type and vacuole type based on the absence or the presence of C-terminal propeptides, which probably function as vacuolar sorting signals (VSSs). In this study, in order to improve our understanding of vacuole-type PRX, we analyzed regulatory mechanisms of vacuolar sorting of a model vacuole-type PRX, the C1a isozyme of horseradish (Armoracia rusticana) (HRP C1a). Using cultured transgenic tobacco cells and protoplasts derived from horseradish leaves, we characterized HRP C1a's VSS, which is a 15 amino acid C-terminal propeptide (C15). We found that the C-terminal hexapeptide of C15 (C6), which is well conserved among vacuole-type PRX proteins, forms the core of the C-terminal-dependent VSS. We also found that the function of C6 is enhanced by the remaining N-terminal part of C15 which probably folds into an amphiphilic α-helix.

  7. Biphenyl-metabolizing bacteria in the rhizosphere of horseradish and bulk soil contaminated by polychlorinated biphenyls as revealed by stable isotope probing.

    Science.gov (United States)

    Uhlik, Ondrej; Jecna, Katerina; Mackova, Martina; Vlcek, Cestmir; Hroudova, Miluse; Demnerova, Katerina; Paces, Vaclav; Macek, Tomas

    2009-10-01

    DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [(13)C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase alpha subunits (BphA) from bacteria that incorporated [(13)C]into DNA in 3-day incubations of the soils with [(13)C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl.

  8. Acute effects of mustard, horseradish, black pepper and ginger on energy expenditure, appetite, ad libitum energy intake and energy balance in human subjects

    DEFF Research Database (Denmark)

    Gregersen, Nikolaj Ture; Belza, Anita; Jensen, M.G.

    2013-01-01

    Chilli peppers have been shown to enhance diet-induced thermogenesis (DIT) and reduce energy intake (EI) in some studies, but there are few data on other pungent spices. The primary aim of the present study was to test the acute effects of black pepper (pepper), ginger, horseradish and mustard...... randomly assigned to receive a brunch meal with either pepper (1·3 g), ginger (20 g), horseradish (8·3 g), mustard (21 g) or no spices (placebo). The amounts of spices were chosen from pre-testing to make the meal spicy but palatable. No significant treatment effects were observed on DIT, but mustard...... produced DIT, which tended to be larger than that of placebo (14 %, 59 (se 3) v. 52 (se 2) kJ/h, respectively, P = 0·08). No other spice induced thermogenic effects approaching statistical significance. Subjective measures of appetite (P>0·85), ad libitum EI (P = 0·63) and energy balance (P = 0·67) also...

  9. Chelating polymeric membranes

    KAUST Repository

    Peinemann, Klaus-Viktor; Villalobos Vazquez de la Parra, Luis Francisco; Hilke, Roland

    2015-01-01

    microporous chelating polymeric membrane. Embodiments include, but are not limited to, microporous chelating polymeric membranes, device comprising the membranes, and methods of using and making the same.

  10. Membrane targeting of the yeast exocyst complex

    Czech Academy of Sciences Publication Activity Database

    Pleskot, Roman; Cwiklik, Lukasz; Jungwirth, Pavel; Žárský, Viktor; Potocký, Martin

    2015-01-01

    Roč. 1848, č. 7 (2015), s. 1481-1489 ISSN 0005-2736 R&D Projects: GA ČR GA13-19073S; GA ČR GBP208/12/G016 Grant - others:GA MŠk(CZ) LO1417 Institutional support: RVO:61389030 ; RVO:61388955 ; RVO:61388963 Keywords : The exocyst complex * Exo70p * Sec3p Subject RIV: EB - Genetics ; Molecular Biology; CF - Physical ; Theoretical Chemistry (UFCH-W) Impact factor: 3.687, year: 2015

  11. Horseradish and Watercress

    Directory of Open Access Journals (Sweden)

    Thiel I

    2016-01-01

    Full Text Available iIntroduction:/i Urinary tract infections (UTIs are among the most common bacterial infections affecting women. Due to the growing problem of antibiotic resistances there is an urgent need for alternative herbal medicinal products for the treatment of non-severe lower UTIs. Vaccinium macrocarpon is effective through various mechanisms of action without inducing resistances in microorganisms. Thus, an observational study (n = 48 was conducted with a prepa ration of a standardized cranberry extract (67 mg proanthocyanidines combined with glucosinolates containing extracts of Nasturtium officinale and Armoracia rusticana.br iMethods:/i Women (≥ 18 years with an increase in leukocytes in the urine and clinical symptoms typical for UTIs like dysuria, imperative strangury, augmented or new onset of incontinence and suprapubic pain received one of the tablets containing the combined herbal extracts twice daily during the first week and one tablet per day for the following 4 weeks. After 14 days (t1 and after 35 days (t2 taking the tablets a further survey and control check-up with a urine test have been conducted. Patients with persistent clinical symptoms received an antibiotic, for the others the intake of the tablets with the herbal combined extract preparation was continued.br iResults:/i 34 patients (70,8 % could abstain from taking antibiotics within the first 14 days (t1 and 32 patients (66,7 % within 35 days (t2. 28 patients (87,5 % were without symptoms at t2. No adverse events were observed.br iConclusion:/i This observational study was the first clinical study performed with a combined extract preparation containing Cranberry, Nasturtium and Armoracia, which furthermore supports the concept of using alternative medicine treating non-severe lower UTIs.br bKurzfassung/b:iEinleitung:/i Harnwegsinfektionen (HWIs gehören zu den häufigsten bakteriellen Infektionen, die Frauen betreffen. Durch die wachsende Problematik der Antibiotika-Resistenzen besteht für die Behandlung von unkomplizierten unteren Harnwegsinfektionen (Urethritis, Zystitis, Urethrozystitis ein dringender Bedarf an alternativen pflanzlichen Arzneimitteln. Vaccinium macrocarpon, die amerikanische Cranberry, wirkt durch verschiedene Mechanismen, ohne Resistenzen auszulösen. Aus diesem Grund wurde eine Anwendungsbeobachtung (n = 48 mit einer Zubereitung eines standardisierten Cranberry-Extraktes (67 mg Proanthocyanidine, kombiniert mit senfölglykosidischen Extrakten aus Brunnenkresse (Nasturtium officinale und Meerrettich (Armoracia rusticana, durchgeführt.br iPatienten:/i Frauen (≥ 18 Jahre mit einem Leukozytenanstieg im Harn und klinischen Symptomen typisch für Harnwegsinfektionen wie Dysurie, Harndrang, neu auftretende Inkontinenz und Schmerzen über dem Schambein erhielten in der ersten Woche eine Tablette mit der Extraktkombination zweimal täglich und eine Tablette pro Tag für die folgenden 4 Wochen. Nach 14 Tagen (t1 und nach 35 Tagen (t2 wurde eine weitere Erhebung und Kontrolluntersuchung mit einem Urin- Test durchgeführt. Patientinnen mit anhaltenden klinischen Symptomen bekamen ein Antibiotikum verschrieben, bei den anderen wurde die Einnahme der Extrakte aus Cranberry, Brunnenkresse und Meerrettich fortgesetzt.br iErgebnisse:/i 34 Patientinnen (70,8 % konnten innerhalb der ersten 14 Tage (t1 auf die Einnahme eines Antibiotikums verzichten und 32 Patientinnen (66,7 % benötigten auch nach 35 Tagen (t2 kein Antibiotikum. 28 Patientinnen (87,5 % waren zum Zeitpunkt (t2 ohne Symptome. Es wurden keine Nebenwirkungen beobachtet. Schlussfolgerung: Diese Anwendungsbeobachtung ist die erste Studie mit einem entsprechenden Kombinationsextrakt (Cranberry, Brunnenkresse, Meerrettich, die darüber hinaus das Konzept des Einsatzes alternativer Therapeutika zur Behandlung von unkomplizierten unteren HWIs unterstützt.

  12. Hydrogen Selective Exfoliated Zeolite Membranes

    Energy Technology Data Exchange (ETDEWEB)

    Tsapatsis, Michael [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Daoutidis, Prodromos [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Elyassi, Bahman [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Lima, Fernando [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Iyer, Aparna [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Agrawal, Kumar [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science; Sabnis, Sanket [Univ. of Minnesota, Minneapolis, MN (United States). Department of Chemical Engineering and Materials Science

    2015-04-06

    in terms of performance and economic aspects of the plants. Specifically, simulation and design optimization studies were performed using the developed stand-alone membrane reactor models to identify the membrane selectivity and permeance characteristics necessary to achieve desired targets of CO2 capture and H2 recovery, as well as guide the selection of the optimal reactor design that minimizes the membrane cost as a function of its surface area required. The isothermal membrane reactor model was also integrated into IGCC system models using both the MATLAB and Aspen software platforms and techno-economic analyses of the integrated plants have been carried out to evaluate the feasibility of replacing current technologies for pre-combustion capture by the proposed novel approach in terms of satisfying stream constraints and achieving the DOE target goal of 90% CO2 capture. The results of the performed analyses based on present value of annuity calculations showed break even costs for the membrane reactor within the feasible range for membrane fabrication. However, the predicted membrane performance used in these simulations exceeded the performance achieved experimentally. Therefore, further work is required to improve membrane performance.

  13. Research Advances: Horseradish Peroxide Mixture May End War of the Noses-- Even Low Levels of Benzene Are Hemotoxic--New "Nuclear Battery" Runs 10 Years. 10 Times More Powerful

    Science.gov (United States)

    King, Angela G.

    2005-01-01

    Experts have mixed horseradish root with hydrogen peroxide or calcium peroxide in laboratory studies to get rid of the problem of odors from farm manure. Researchers evaluated how well the system reduced the concentration of volatile fatty acids, indole and skatole, compounds that are also associated with the stink of animal manure.

  14. Robust membrane systems for actinide separations

    International Nuclear Information System (INIS)

    Jarvinen, Gordon D.; McCleskey, T. Mark; Bluhm, Elizabeth A.; Abney, Kent D.; Ehler, Deborah S.; Bauer, Eve; Le, Quyen T.; Young, Jennifer S.; Ford, Doris K.; Pesiri, David R.; Dye, Robert C.; Robison, Thomas W.; Jorgensen, Betty S.; Redondo, Antonio; Pratt, Lawrence R.; Rempe, Susan L.

    2000-01-01

    Our objective in this project is to develop very stable thin membrane structures containing ionic recognition sites that facilitate the selective transport of target metal ions, especially the actinides

  15. Biphenyl-Metabolizing Bacteria in the Rhizosphere of Horseradish and Bulk Soil Contaminated by Polychlorinated Biphenyls as Revealed by Stable Isotope Probing▿ †

    Science.gov (United States)

    Uhlik, Ondrej; Jecna, Katerina; Mackova, Martina; Vlcek, Cestmir; Hroudova, Miluse; Demnerova, Katerina; Paces, Vaclav; Macek, Tomas

    2009-01-01

    DNA-based stable isotope probing in combination with terminal restriction fragment length polymorphism was used in order to identify members of the microbial community that metabolize biphenyl in the rhizosphere of horseradish (Armoracia rusticana) cultivated in soil contaminated with polychlorinated biphenyls (PCBs) compared to members of the microbial community in initial, uncultivated bulk soil. On the basis of early and recurrent detection of their 16S rRNA genes in clone libraries constructed from [13C]DNA, Hydrogenophaga spp. appeared to dominate biphenyl catabolism in the horseradish rhizosphere soil, whereas Paenibacillus spp. were the predominant biphenyl-utilizing bacteria in the initial bulk soil. Other bacteria found to derive carbon from biphenyl in this nutrient-amended microcosm-based study belonged mostly to the class Betaproteobacteria and were identified as Achromobacter spp., Variovorax spp., Methylovorus spp., or Methylophilus spp. Some bacteria that were unclassified at the genus level were also detected, and these bacteria may be members of undescribed genera. The deduced amino acid sequences of the biphenyl dioxygenase α subunits (BphA) from bacteria that incorporated [13C]into DNA in 3-day incubations of the soils with [13C]biphenyl are almost identical to that of Pseudomonas alcaligenes B-357. This suggests that the spectrum of the PCB congeners that can be degraded by these enzymes may be similar to that of strain B-357. These results demonstrate that altering the soil environment can result in the participation of different bacteria in the metabolism of biphenyl. PMID:19700551

  16. Viral membrane fusion

    International Nuclear Information System (INIS)

    Harrison, Stephen C.

    2015-01-01

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism

  17. Viral membrane fusion

    Energy Technology Data Exchange (ETDEWEB)

    Harrison, Stephen C., E-mail: harrison@crystal.harvard.edu

    2015-05-15

    Membrane fusion is an essential step when enveloped viruses enter cells. Lipid bilayer fusion requires catalysis to overcome a high kinetic barrier; viral fusion proteins are the agents that fulfill this catalytic function. Despite a variety of molecular architectures, these proteins facilitate fusion by essentially the same generic mechanism. Stimulated by a signal associated with arrival at the cell to be infected (e.g., receptor or co-receptor binding, proton binding in an endosome), they undergo a series of conformational changes. A hydrophobic segment (a “fusion loop” or “fusion peptide”) engages the target-cell membrane and collapse of the bridging intermediate thus formed draws the two membranes (virus and cell) together. We know of three structural classes for viral fusion proteins. Structures for both pre- and postfusion conformations of illustrate the beginning and end points of a process that can be probed by single-virion measurements of fusion kinetics. - Highlights: • Viral fusion proteins overcome the high energy barrier to lipid bilayer merger. • Different molecular structures but the same catalytic mechanism. • Review describes properties of three known fusion-protein structural classes. • Single-virion fusion experiments elucidate mechanism.

  18. Manipulation of host membranes by bacterial effectors.

    Science.gov (United States)

    Ham, Hyeilin; Sreelatha, Anju; Orth, Kim

    2011-07-18

    Bacterial pathogens interact with host membranes to trigger a wide range of cellular processes during the course of infection. These processes include alterations to the dynamics between the plasma membrane and the actin cytoskeleton, and subversion of the membrane-associated pathways involved in vesicle trafficking. Such changes facilitate the entry and replication of the pathogen, and prevent its phagocytosis and degradation. In this Review, we describe the manipulation of host membranes by numerous bacterial effectors that target phosphoinositide metabolism, GTPase signalling and autophagy.

  19. Membrane shape modulates transmembrane protein distribution.

    Science.gov (United States)

    Aimon, Sophie; Callan-Jones, Andrew; Berthaud, Alice; Pinot, Mathieu; Toombes, Gilman E S; Bassereau, Patricia

    2014-01-27

    Although membrane shape varies greatly throughout the cell, the contribution of membrane curvature to transmembrane protein targeting is unknown because of the numerous sorting mechanisms that take place concurrently in cells. To isolate the effect of membrane shape, we used cell-sized giant unilamellar vesicles (GUVs) containing either the potassium channel KvAP or the water channel AQP0 to form membrane nanotubes with controlled radii. Whereas the AQP0 concentrations in flat and curved membranes were indistinguishable, KvAP was enriched in the tubes, with greater enrichment in more highly curved membranes. Fluorescence recovery after photobleaching measurements showed that both proteins could freely diffuse through the neck between the tube and GUV, and the effect of each protein on membrane shape and stiffness was characterized using a thermodynamic sorting model. This study establishes the importance of membrane shape for targeting transmembrane proteins and provides a method for determining the effective shape and flexibility of membrane proteins. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Robotic membranes

    DEFF Research Database (Denmark)

    Ramsgaard Thomsen, Mette

    2008-01-01

    The relationship between digital and analogue is often constructed as one of opposition. The perception that the world is permeated with underlying patterns of data, describing events and matter alike, suggests that information can be understood apart from the substance to which it is associated......, and that its encoded logic can be constructed and reconfigured as an isolated entity. This disembodiment of information from materiality implies that an event like a thunderstorm, or a material like a body, can be described equally by data, in other words it can be read or written. The following prototypes......, Vivisection and Strange Metabolisms, were developed at the Centre for Information Technology and Architecture (CITA) at the Royal Danish Academy of Fine Arts in Copenhagen as a means of engaging intangible digital data with tactile physical material. As robotic membranes, they are a dual examination...

  1. Bacterial pathogen manipulation of host membrane trafficking.

    Science.gov (United States)

    Asrat, Seblewongel; de Jesús, Dennise A; Hempstead, Andrew D; Ramabhadran, Vinay; Isberg, Ralph R

    2014-01-01

    Pathogens use a vast number of strategies to alter host membrane dynamics. Targeting the host membrane machinery is important for the survival and pathogenesis of several extracellular, vacuolar, and cytosolic bacteria. Membrane manipulation promotes bacterial replication while suppressing host responses, allowing the bacterium to thrive in a hostile environment. This review provides a comprehensive summary of various strategies used by both extracellular and intracellular bacteria to hijack host membrane trafficking machinery. We start with mechanisms used by bacteria to alter the plasma membrane, delve into the hijacking of various vesicle trafficking pathways, and conclude by summarizing bacterial adaptation to host immune responses. Understanding bacterial manipulation of host membrane trafficking provides insights into bacterial pathogenesis and uncovers the molecular mechanisms behind various processes within a eukaryotic cell.

  2. Tandem neopentyl glycol maltosides (TNMs) for membrane protein stabilisation

    DEFF Research Database (Denmark)

    Bae, Hyoung Eun; Mortensen, Jonas S; Ribeiro, Orquidea

    2016-01-01

    A novel class of detergents, designated tandem neopentyl glycol maltosides (TNMs), were evaluated with four target membrane proteins. The best detergent varied depending on the target, but TNM-C12L and TNM-C11S were notable for their ability to confer increased membrane protein stability compared...

  3. Endophytic fungi from the roots of horseradish (Armoracia rusticana) and their interactions with the defensive metabolites of the glucosinolate - myrosinase - isothiocyanate system.

    Science.gov (United States)

    Szűcs, Zsolt; Plaszkó, Tamás; Cziáky, Zoltán; Kiss-Szikszai, Attila; Emri, Tamás; Bertóti, Regina; Sinka, László Tamás; Vasas, Gábor; Gonda, Sándor

    2018-05-09

    The health of plants is heavily influenced by the intensively researched plant microbiome. The microbiome has to cope with the plant's defensive secondary metabolites to survive and develop, but studies that describe this interaction are rare. In the current study, we describe interactions of endophytic fungi with a widely researched chemical defense system, the glucosinolate - myrosinase - isothiocyanate system. The antifungal isothiocyanates are also of special interest because of their beneficial effects on human consumers. Seven endophytic fungi were isolated from horseradish roots (Armoracia rusticana), from the genera Fusarium, Macrophomina, Setophoma, Paraphoma and Oidiodendron. LC-ESI-MS analysis of the horseradish extract incubated with these fungi showed that six of seven strains could decompose different classes of glucosinolates. Aliphatic, aromatic, thiomethylalkyl and indolic glucosinolates were decomposed by different strains at different rates. SPME-GC-MS measurements showed that two strains released significant amounts of allyl isothiocyanate into the surrounding air, but allyl nitrile was not detected. The LC-ESI-MS analysis of many strains' media showed the presence of allyl isothiocyanate - glutathione conjugate during the decomposition of sinigrin. Four endophytic strains also accepted sinigrin as the sole carbon source. Isothiocyanates inhibited the growth of fungi at various concentrations, phenylethyl isothiocyanate was more potent than allyl isothiocyanate (mean IC 50 was 2.30-fold lower). As a control group, ten soil fungi from the same soil were used. They decomposed glucosinolates with lower overall efficiency: six of ten strains had insignificant or weak activities and only three could use sinigrin as a carbon source. The soil fungi also showed lower AITC tolerance in the growth inhibition assay: the median IC 50 values were 0.1925 mM for endophytes and 0.0899 mM for soil fungi. The host's glucosinolates can be used by the tested

  4. ENCAPSULATION OF HORSERADISH PEROXIDASE-GLUCOSE OXIDASE (HRP-GOx IN SILICA AQUAGEL SYNTHESIZED FROM RICE HULL ASH FOR ENZYMATIC REACTION OF GLUCOSE

    Directory of Open Access Journals (Sweden)

    Nuryono Nuryono

    2010-06-01

    Full Text Available In recent years, the sol-gel technique has attracted increasing interest as a unique approach to immobilize biomolecules for bioanalytical applications as well as biochemical and biophysical studies. In this research, encapsulation of Horseradish peroxidase-Glucose oxidase (HRP-GOx enzymes in silica aquagel from rice hull ash by sol-gel process has been carried out. In addition, the effect of several parameters (weight ratio of HRP to GOx, pH, temperature, sodium ion concentration on enzyme activity was studied, as well. Rice hull ash, which was produced by ashing at 700 °C, was extracted it's silika by NaOH solution 1 M at 100 °C for two hours to produce sodium silikate (Na2SiO3 solution. The Na2SiO3 solution with pH of 13 was added with a strong cation exchanger resin, to produce sol solution with the pH of 4. Encapsulation was emphasized by mixing sol solution and phosphate buffer pH 7 containing HRP-GOx solution at volume ratio of buffer to sol solution 1:5. The mixture was transferred into 96-microwell plate and was aged for 24 hours. Enzymatic reaction was carried out by adding chromogenic solution of phenol and 4-aminoantipyrine (4-AAP and b-D-glucose solution (as substrate into the microwell. Enzymatic activity was examined by measuring absorbance of product solution at 490 nm with ELISA reader. Result of enzymatic activity for encapsulated enzymes (SGE was compared to that for free enzymes (EB. Results showed that at the investigated condition, HRP-GOx enzymes gave high activity at weight ratio of HRP to GOx 10:1 and pH 7 for both SGE and EB. Encapsulation caused the enzymes activity decrease to 53.0±0.2 %. However, SGE was observed to be more stable on pH and temperature changes than EB. Study on the effect of sodium concentration showed that the increase of sodium concentration from 0.10 to 0.37 M decreased the enzymatic activity to 56±0.2%. Reusability test showed that the synthesized SGE was reusable with activity decrease of 60

  5. Investigation of glucosinolate profile and qualitative aspects in sprouts and roots of horseradish (Armoracia rusticana) using LC-ESI-hybrid linear ion trap with Fourier transform ion cyclotron resonance mass spectrometry and infrared multiphoton dissociation.

    Science.gov (United States)

    Agneta, Rosa; Rivelli, Anna Rita; Ventrella, Emanuela; Lelario, Filomena; Sarli, Giulio; Bufo, Sabino Aurelio

    2012-08-01

    Within the family of Brassicaceae, an important source of glucosinolates (GLSs) is represented by horseradish ( Armoracia rusticana P. Gaertner, B. Meyer & Scherbius), cultivated for its roots, which are grated fresh or processed into a sauce and used as a condiment. The characteristic pungent flavor of the root depends on the abundance of the bioactive GLS molecules. In crude plant extracts (sprouts and roots) of an accession of horseradish largely diffused in the Basilicata region (southern Italy), which develops many sprouts and produces white, fiery, and sharp-flavored marketable roots, we characterized the GLS profile by LC-ESI-LTQ-FTICR-MS and IRMPD. In sprouts and roots we identified 16 and 11 GLSs, respectively. We confirmed the presence of sinigrin, 4-hydroxyglucobrassicin, glucobrassicin, gluconasturtin, and 4-methoxyglucobrassicin and identified glucoiberin, gluconapin, glucocochlearin, glucoconringianin, glucosativin, glucoibarin, 5-hydroxyglucobrassicin, glucocapparilinearisin or glucobrassicanapin, glucotropaeolin, and glucoarabishirsutain, not previously characterized in horseradish. Of particular note was the presence of the putative 2-methylsulfonyl-oxo-ethyl-GLS.

  6. Efecto de la polietilenimina en la actividad catalítica de la peroxidasa de rábano (horseradish peroxidase inmovilizada en electrodos de oro modificados con monocapas autoensambladas de tioles (SAMs.

    Directory of Open Access Journals (Sweden)

    Pedro R. Matheus

    2009-05-01

    Full Text Available Effect of the Polyethyleneimine in the Activity Catalytic of the horseradish peroxidase Immobilized on Gold Electrodes Modified with a Self-assembled Monolayer of Thiols (SAMs. Studies were conducted bycyclic voltammetry (CV to investigate the effect of the polymer polyethyleneimine (PEI in the electrochemical reversibility of the mediator thionine and thus the catalytic activity of the enzyme horseradish peroxidase of recombinant HRP-NHis (horseradish peroxidase to the has been added to a chain of six histidine in the extreme N-terminal protein. This self produced monolayers of thiols (SAMS on gold electrodes, with chemical modifications obtained through successive stages in the solid phase of the electrode. The gold electrodes were modified with monolayer SAM-TOA-[ANTA/DADOO] -Co2+ [SAM: self-assembled monolayers of thiols, TOA: dithioctic acid, ANTA: nitrilotriacetic acid, DADOO: 1,8-diamino-3,6-dioxa octane]. The results showed that the presence of the polymer improves the electrochemical reversibility of the mediator to endure catalyticcurrents as high as those that are obtained with molar ratios ANTA:DADOO 10:1 in the absence of PEI, and improve the response voltammetric obtained.

  7. Recent advances on polymeric membranes for membrane reactors

    KAUST Repository

    Buonomenna, M. G.; Choi, Seung Hak

    2012-01-01

    . The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane

  8. Thermal experiments in the ADS target model

    International Nuclear Information System (INIS)

    Efanov, A.D.; Orlov, Yu.I.; Sorokin, A.P.; Ivanov, E.F.; Bogoslovskaya, G.P.; Li, N.

    2002-01-01

    Experiments on the development of the target heat model project and method of investigation into heat exchange in target were conducted with the aim of analysis of thermomechanical and strength characteristics of device; experimental data on the temperature distribution in coolant and membrane were obtained. Obtained data demonstrate that the temperature heterogeneity of membrane and coolant are connected with the temperature distribution variability near the membrane. Peculiarities of the experiment are noted: maximal temperature of oscillations at high point of the membrane, and power bearing temperature oscillations in the range 0 - 1 Hz [ru

  9. Magnetically controlled permeability membranes

    KAUST Repository

    Kosel, Jurgen

    2013-10-31

    A bioactive material delivery system can include a thermoresponsive polymer membrane and nanowires distributed within the thermoresponsive polymer membrane. Magnetic activation of a thermoresponsive polymer membrane can take place via altering the magnetization or dimensions of nanowires dispersed or ordered within the membrane matrix.

  10. Magnetically controlled permeability membranes

    KAUST Repository

    Kosel, Jü rgen; Khashab, Niveen M.; Zaher, Amir

    2013-01-01

    A bioactive material delivery system can include a thermoresponsive polymer membrane and nanowires distributed within the thermoresponsive polymer membrane. Magnetic activation of a thermoresponsive polymer membrane can take place via altering the magnetization or dimensions of nanowires dispersed or ordered within the membrane matrix.

  11. CO2 Acquisition Membrane (CAM)

    Science.gov (United States)

    Mason, Larry W.; Way, J. Douglas; Vlasse, Marcus

    2003-01-01

    The objective of CAM is to develop, test, and analyze thin film membrane materials for separation and purification of carbon dioxide (CO2) from mixtures of gases, such as those found in the Martian atmosphere. The membranes are targeted toward In Situ Resource Utilization (ISRU) applications that will operate in extraterrestrial environments and support future unmanned and human space missions. A primary application is the Sabatier Electrolysis process that uses Mars atmosphere CO2 as raw material for producing water, oxygen, and methane for rocket fuel and habitat support. Other applications include use as an inlet filter to collect and concentrate Mars atmospheric argon and nitrogen gases for habitat pressurization, and to remove CO2 from breathing gases in Closed Environment Life Support Systems (CELSS). CAM membrane materials include crystalline faujasite (FAU) zeolite and rubbery polymers such as silicone rubber (PDMS) that have been shown in the literature and via molecular simulation to favor adsorption and permeation of CO2 over nitrogen and argon. Pure gas permeation tests using commercial PDMS membranes have shown that both CO2 permeance and the separation factor relative to other gases increase as the temperature decreases, and low (Delta)P(Sub CO2) favors higher separation factors. The ideal CO2/N2 separation factor increases from 7.5 to 17.5 as temperature decreases from 22 C to -30 C. For gas mixtures containing CO2, N2, and Ar, plasticization decreased the separation factors from 4.5 to 6 over the same temperature range. We currently synthesize and test our own Na(+) FAU zeolite membranes using standard formulations and secondary growth methods on porous alumina. Preliminary tests with a Na(+) FAU membrane at 22 C show a He/SF6 ideal separation factor of 62, exceeding the Knudsen diffusion selectivity by an order of magnitude. This shows that the membrane is relatively free from large defects and associated non-selective (viscous flow) transport

  12. Amperometric hydrogen peroxide biosensor based on the immobilization of horseradish peroxidase on core-shell organosilica-chitosan nanospheres and multiwall carbon nanotubes composite

    International Nuclear Information System (INIS)

    Chen Shihong; Yuan Ruo; Chai Yaqin; Yin Bin; Li Wenjun; Min Ligen

    2009-01-01

    The application of the composites of multiwall carbon nanotubes (MWNTs) and core-shell organosilica-chitosan crosslinked nanospheres as an immobilization matrix for the construction of an amperometric hydrogen peroxide (H 2 O 2 ) biosensor was described. MWNTs and positively charged organosilica-chitosan nanospheres were dispersed in acetic acid solution (0.6 wt%) to achieve organosilica-chitosan/MWNTs composites, which were cast onto a glass carbon electrode (GCE) surface directly. And then, horseradish peroxidase (HRP), as a model enzyme, was immobilized onto it through electrostatic interaction between oppositely charged organosilica-chitosan nanospheres and HRP. The direct electron transfer of HRP was achieved at HRP/organosilica-chitosan/MWNTs/GCE, which exhibited excellent electrocatalytic activity for the reduction of H 2 O 2 . The catalysis currents increased linearly to H 2 O 2 concentration in a wide range of 7.0 x 10 -7 to 2.8 x 10 -3 M, with a sensitivity of 49.8 μA mM -1 cm -2 and with a detection limit of 2.5 x 10 -7 M at 3σ. A Michaelies-Menten constant K M app value was estimated to be 0.32 mM, indicating a high-catalytic activity of HRP. Moreover, the proposed biosensor displayed a rapid response to H 2 O 2 and possessed good stability and reproducibility. When used to detect H 2 O 2 concentration in disinfector samples and sterilized milks, respectively, it showed satisfactory results

  13. Dendritic and axonic fields of Purkinje cells in developing and X-irradiated rat cerebellum. A comparative study using intracellular staining with horseradish peroxidase

    International Nuclear Information System (INIS)

    Crepel, F.; Delhaye-Bouchaud, N.; Dupont, J.L.; Sotelo, C.

    1980-01-01

    Intracellular staining of cerebellar Purkinje cells with horseradish peroxidase was achieved in normal developing rats (8-13 days old), in normal adult rats and in adult rats in which the cerebellum had been degranulated by X-ray treatment. The mono- and multiple innervation of Purkinje cells by climbing fibres was electrophysiologically determined and correlated with their dendritic pattern and axonal field. In immature rats, considerable variations in dendritic arborization were observed between cells at the same age, according to their position in the vermis. In adult X-irradiated animals, a large variety of dendritic shapes was found, confirming previous anatomical data, but no obvious correlation was found between the morphology of the dendrites of Purkinje cells and their synaptic investment by climbing fibres. As regards the axonal field, the adult branching pattern of recurrent axon collaterals was almost established by postnatal day 8, except for some cells which exhibited richer recurrent collaterals. On the other hand, in X-irradiated animals, profuse plexuses were the rule and they originated either from one collateral stem, or from several collaterals, also independently of the number of afferent climbing fibres. The existence of these enlarged recurrent collateral plexuses can be explained by the persistence of an immature stage, and certainly also by the collateral sprouting following the largely impaired innervation of the terminal field during development. These results emphasize the role of the cellular interactions that occur during Purkinje cell growth in the formation of both its axonal and dendritic fields. (author)

  14. A novel biosensor based on the direct electrochemistry of horseradish peroxidase immobilized in the three-dimensional flower-like Bi_2WO_6 microspheres

    International Nuclear Information System (INIS)

    Liu, Hui; Guo, Kai; Duan, Congyue; Chen, Xianjin; Zhu, Zhenfeng

    2016-01-01

    Three-dimensional flower-like Bi_2WO_6 microspheres (3D-Bi_2WO_6 MSs) have been synthesized through a simple hydrothermal method. The morphology and structure of 3D-Bi_2WO_6 MSs were characterized by scanning electron microscopy (SEM) and X-ray diffraction (XRD). The 3D-Bi_2WO_6 MSs subsequently were used to immobilize horseradish peroxidase (HRP) and fabricate a mediator-free biosensor for the detection of H_2O_2. Spectroscopic and electrochemical results reveal that 3D-Bi_2WO_6 MSs constitute an excellent immobilization matrix with biocompatibility for enzymes. Meanwhile, due to unique morphology of the flower-like microspheres, the direct electron transfer of HRP is facilitated and the prepared biosensors display good performances for the detection of H_2O_2 with a wide linear range, including two linear sections: 0.5–100 μM (R"2 = 0.9983) and 100–250 μM (R"2 = 0.9981), as well as an extremely low method detection limit of 0.18 μM. - Highlights: • 3D-Bi_2WO_6 microspheres are used to fabricate a mediator-free biosensor firstly. • The biosensor displays a wide linear range of 0.5–250 μM for H_2O_2. • The biosensor exhibits a low detection limit of 0.18 μM for H_2O_2.

  15. Immobilization of Horseradish Peroxidase on NH2-Modified Magnetic Fe3O4/SiO2 Particles and Its Application in Removal of 2,4-Dichlorophenol

    Directory of Open Access Journals (Sweden)

    Qing Chang

    2014-09-01

    Full Text Available Fe3O4 nanoparticles were prepared by a co-precipitation method with the assistance of ultrasound irradiation, and then coated with silica generated by hydrolysis and condensation of tetraethoxysilane. The silica-coated Fe3O4 nanoparticles were further modified with 3-aminopropyltriethoxysilane, resulting in anchoring of primary amine groups on the surface of the particles. Horseradish peroxidase (HRP was then immobilized on the magnetic core-shell particles by using glutaraldehyde as a crosslinking agent. Immobilization conditions were optimized to obtain the highest relative activity of the immobilized enzyme. It was found the durability of the immobilized enzyme to heating and pH variation were improved in comparison with free HRP. The apparent Michaelis constants of the immobilized HRP and free HRP with substrate were compared, showing that the enzyme activity of the immobilized HRP was close to that of free HRP. The HRP immobilized particles, as an enzyme catalyst, were used to activate H2O2 for degrading 2,4-dichlorophenol. The rapid degradation of 2,4-dichlorophenol indicated that the immobilized enzyme has potential applications for removing organic pollutants.

  16. Horseradish peroxidase and antibody labeled gold nanoparticle probe for amplified immunoassay of ciguatoxin in fish samples based on capillary electrophoresis with electrochemical detection.

    Science.gov (United States)

    Zhang, Zhaoxiang; Liu, Ying; Zhang, Chaoying; Luan, Wenxiu

    2015-03-01

    This paper describes a new amplified immunoassay with horseradish peroxidase (HRP) and antibody (Ab) labeled gold nanoparticles (AuNPs) probe hyphenated to capillary electrophoresis (CE) with electrochemical (EC) detection for ultrasensitive determination of ciguatoxin CTX1B. AuNPs were conjugated with HRP and Ab, and then incubated with limited amount of CTX1B to produce immunocomplex. The immunoreactive sample was injected into capillary for CE separation and EC detection. Enhanced sensitivity was obtained by adopting the AuNPs as carriers of HRP and Ab at high HRP/Ab molar ratio. The calibration curve of CTX1B was in the range of 0.06-90 ng/mL. The detection limit was 0.045 ng/mL, which is 38-fold lower than that of HPLC-MS method for CTX1B analysis. The proposed method was successfully applied for the quantification of CTX1B in contamined fish samples by simultaneously labeling Ab and HRP on AuNPs. The amplified IA with HRP and Ab labeled AuNPs probe hyphenated to CE and EC detection provides a sensitive analytical approach for the determination of trace ciguatoxin in complex samples. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Electrochemical horseradish peroxidase biosensor based on dextran-ionic liquid-V2O5 nanobelt composite material modified carbon ionic liquid electrode

    International Nuclear Information System (INIS)

    Zhu Zhihong; Sun Xiaoying; Wang Yan; Zeng Yan; Sun Wei; Huang Xintang

    2010-01-01

    Direct electrochemistry of horseradish peroxidase (HRP) was realized in a dextran (De), 1-ethyl-3-methylimidazolium ethylsulphate ([EMIM]EtOSO 3 ) and V 2 O 5 nanobelt composite material modified carbon ionic liquid electrode (CILE). Spectroscopic results indicated that HRP retained its native structure in the composite. A pair of well-defined redox peaks of HRP appeared in pH 3.0 phosphate buffer solution with the formal potential of -0.213 V (vs. SCE), which was the characteristic of HRP heme Fe(III)/Fe(II) redox couple. The result was attributed to the specific characteristics of De-IL-V 2 O 5 nanocomposite and CILE, which promoted the direct electron transfer rate of HRP with electrode. The electrochemical parameters of HRP on the composite modified electrode were calculated and the electrocatalysis of HRP to the reduction of trichloroacetic acid (TCA) was examined. Under the optimal conditions the reduction peak current increased with TCA concentration in the range from 0.4 to 16.0 mmol L -1 . The proposed electrode is valuable for the third-generation electrochemical biosensor.

  18. Light microscopic detection of sugar residues in glycoconjugates of salivary glands and the pancreas with lectin-horseradish peroxidase conjugates. I. Mouse.

    Science.gov (United States)

    Schulte, B A; Spicer, S S

    1983-12-01

    Mouse salivary glands and pancreases were stained with a battery of ten horseradish peroxidase-conjugated lectins. Lectin staining revealed striking differences in the structure of oligosaccharides of stored intracellular secretory glycoproteins and glycoconjugates associated with the surface of epithelial cells lining excretory ducts. The percentage of acinar cells containing terminal alpha-N-acetylgalactosamine residues varied greatly in submandibular glands of 30 male mice, but all submandibular acinar cells contained oligosaccharides with terminal sialic acid and penultimate beta-galactose residues. The last named dimer was abundant in secretory glycoprotein of all mucous acinar cells in murine sublingual glands and an additional 20-50% of these cells in all glands contained terminal N-acetylglucosamine residues. In contrast, terminal alpha-N-acetylgalactosamine was abundant in sublingual serous demilune secretions. Serous acinar cells in the exorbital lacrimal gland, posterior lingual gland, parotid gland and pancreas exhibited a staining pattern unique to each organ. In contrast, the apical cytoplasm and surface of striated duct epithelial cells in the submandibular, sublingual, parotid and exorbital lacrimal gland stained similarly. A comparison of staining with conjugated lectins reported biochemically to have very similar carbohydrate binding specificity has revealed some remarkable differences in their reactivity, suggesting different binding specificity for the same terminal sugars having different glycosidic linkages or with different penultimate sugar residues.

  19. Dendritic and axonic fields of Purkinje cells in developing and X-irradiated rat cerebellum. A comparative study using intracellular staining with horseradish peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Crepel, F; Delhaye-Bouchaud, N; Dupont, J L [Paris-5 Univ., 75 (France); Sotelo, C [Hopital Foch, 92 - Suresnes (France). Centre Medico-Chirurgical

    1980-01-01

    Intracellular staining of cerebellar Purkinje cells with horseradish peroxidase was achieved in normal developing rats (8-13 days old), in normal adult rats and in adult rats in which the cerebellum had been degranulated by X-ray treatment. The mono- and multiple innervation of Purkinje cells by climbing fibres was electrophysiologically determined and correlated with their dendritic pattern and axonal field. In immature rats, considerable variations in dendritic arborization were observed between cells at the same age, according to their position in the vermis. In adult X-irradiated animals, a large variety of dendritic shapes was found, confirming previous anatomical data, but no obvious correlation was found between the morphology of the dendrites of Purkinje cells and their synaptic investment by climbing fibres. As regards the axonal field, the adult branching pattern of recurrent axon collaterals was almost established by postnatal day 8, except for some cells which exhibited richer recurrent collaterals. On the other hand, in X-irradiated animals, profuse plexuses were the rule and they originated either from one collateral stem, or from several collaterals, also independently of the number of afferent climbing fibres. The existence of these enlarged recurrent collateral plexuses can be explained by the persistence of an immature stage, and certainly also by the collateral sprouting following the largely impaired innervation of the terminal field during development. These results emphasize the role of the cellular interactions that occur during Purkinje cell growth in the formation of both its axonal and dendritic fields.

  20. Toxic effects of a horseradish extract and allyl isothiocyanate in the urinary bladder after 13-week administration in drinking water to F344 rats.

    Science.gov (United States)

    Hasumura, Mai; Imai, Toshio; Cho, Young-Man; Ueda, Makoto; Hirose, Masao; Nishikawa, Akiyoshi; Ogawa, Kumiko

    2011-01-01

    Subchronic toxicity of a horseradish extract (HRE), consisting mainly of a mixture of allyl isothiocyanate (AITC) and other isothiocyanates, was investigated with administration at concentrations of 0, 0.0125, 0.025 and 0.05% of HRE in drinking water for 13 weeks to male and female F344 rats. For comparison, treatment with 0.0425% of AITC was similarly performed. Body weight gain was reduced in the 0.05% HRE and AITC males as compared to the 0% controls, and the cause was considered at least partly related to decreased water consumption due to the acrid smell of the test substance and decreased food consumption. Serum biochemistry demonstrated increased urea nitrogen in 0.025 and 0.05% HRE and AITC males and 0.0125-0.05% HRE and AITC females, along with decreased total cholesterol in 0.0125-0.05% HRE females. On histopathological assessment, papillary/nodular hyperplasia of bladder mucosa was observed in 0.05% HRE and AITC males and females, in addition to simple mucosal hyperplasia found in all treated groups. Based on the above findings, no-observed-adverse-effect levels (NOAELs) were estimated to be below 0.0125% of HRE for both males and females, corresponding to 9.4 and 8.0 mg/kg body weight/day, respectively, and there appeared to be comparable toxicological properties of HRE to AITC, such as the inductive effect of significant proliferative lesions in the urinary bladder.

  1. Enzymatic oxidation of rutin by horseradish peroxidase: kinetic mechanism and identification of a dimeric product by LC-Orbitrap mass spectrometry.

    Science.gov (United States)

    Savic, Sasa; Vojinovic, Katarina; Milenkovic, Sanja; Smelcerovic, Andrija; Lamshoeft, Marc; Petronijevic, Zivomir

    2013-12-15

    Flavonoid oxidation is important issue in food processing and quality. The kinetic mechanism of enzymatic oxidation of rutin by horseradish peroxidase (HRP) was studied. Rutin oxidation reaction was followed by recording of spectral changes over the time at 360 nm. The studied oxidation is mostly enzymatic and less part non-enzymatic. The reaction with HRP has a higher rate compared with the reaction without of HRP, whereby is part of non-enzymatic reaction about 10% of the total reaction. Kinetic parameters were determined from graphics of linear Michaelis-Menten equation, and it was found that investigated reactions of rutin oxidation by HRP take place in a ping-pong kinetic mechanism. High resolution HPLC-MS analysis of the mixture of oxidized products of rutin revealed the presence of rutin dimer. Because of widely distribution of rutin as well as presence of peroxidases and hydrogen peroxide in fresh foods identification of this enzymatic modification product can be beneficial for foods quality and safety. Copyright © 2013 Elsevier Ltd. All rights reserved.

  2. A silica-dextran nanocomposite as a novel matrix for immobilization of horseradish peroxidase, and its application to sensing hydrogen peroxide

    International Nuclear Information System (INIS)

    Satvekar, Rajshri K.; Rohiwal, S. S.; Raut, A. V.; Karande, V. A.; Tiwale, B. M.; Pawar, S. H.

    2014-01-01

    We report on a novel matrix of sol gel organic–inorganic nanocomposite that was fabricated from silica sol gel and dextran. It was used for the immobilization of horseradish peroxidase (HRP) to give a biosensor for hydrogen peroxide (H 2 O 2 ). The sensor film was characterized by Fourier transform infrared and UV–vis spectroscopy with respect to structural features and the conformation of the enzyme. The topographies of the surface of the electrode were investigated by field emission scanning electron microscopy. The biosensor was used to determine H 2 O 2 quantitatively in the presence of Methylene blue as a mediator with high electron transfer efficiency. A pair of stable and well defined quasi-reversible redox peaks of the HRP [Fe (III)]/HRP [Fe (II)] redox couple was observed at pH 7.0. The biosensor responds to H 2 O 2 in the 0.5 mM to 16.5 mM concentration range, and the limit of detection is 0.5 mM. (author)

  3. Novel Dry-Type Glucose Sensor Based on a Metal-Oxide-Semiconductor Capacitor Structure with Horseradish Peroxidase + Glucose Oxidase Catalyzing Layer

    Science.gov (United States)

    Lin, Jing-Jenn; Wu, You-Lin; Hsu, Po-Yen

    2007-10-01

    In this paper, we present a novel dry-type glucose sensor based on a metal-oxide-semiconductor capacitor (MOSC) structure using SiO2 as a gate dielectric in conjunction with a horseradish peroxidase (HRP) + glucose oxidase (GOD) catalyzing layer. The tested glucose solution was dropped directly onto the window opened on the SiO2 layer, with a coating of HRP + GOD catalyzing layer on top of the gate dielectric. From the capacitance-voltage (C-V) characteristics of the sensor, we found that the glucose solution can induce an inversion layer on the silicon surface causing a gate leakage current flowing along the SiO2 surface. The gate current changes Δ I before and after the drop of glucose solution exhibits a near-linear relationship with increasing glucose concentration. The Δ I sensitivity is about 1.76 nA cm-2 M-1, and the current is quite stable 20 min after the drop of the glucose solution is tested.

  4. Paper Microzone Plates as Analytical Tools for Studying Enzyme Stability: A Case Study on the Stabilization of Horseradish Peroxidase Using Trehalose and SU-8 Epoxy Novolac Resin.

    Science.gov (United States)

    Ganaja, Kirsten A; Chaplan, Cory A; Zhang, Jingyi; Martinez, Nathaniel W; Martinez, Andres W

    2017-05-16

    Paper microzone plates in combination with a noncontact liquid handling robot were demonstrated as tools for studying the stability of enzymes stored on paper. The effect of trehalose and SU-8 epoxy novolac resin (SU-8) on the stability of horseradish peroxidase (HRP) was studied in both a short-term experiment, where the activity of various concentrations of HRP dried on paper were measured after 1 h, and a long-term experiment, where the activity of a single concentration of HRP dried and stored on paper was monitored for 61 days. SU-8 was found to stabilize HRP up to 35 times more than trehalose in the short-term experiment for comparable concentrations of the two reagents, and a 1% SU-8 solution was found to stabilize HRP approximately 2 times more than a 34% trehalose solution in both short- and long-term experiments. The results suggest that SU-8 is a promising candidate for use as an enzyme-stabilizing reagent for paper-based diagnostic devices and that the short-term experiment could be used to quickly evaluate the capacity of various reagents for stabilizing enzymes to identify and characterize new enzyme-stabilizing reagents.

  5. Study of electron transport in the functionalized nanotubes and their impact on the electron transfer in the active site of horseradish peroxidase

    Science.gov (United States)

    Feizabadi, Mina; Ajloo, Davood; Soleymanpour, Ahmad; Faridnouri, Hassan

    2018-05-01

    Electrochemical characterization of functionalized carbon nanotubes (f-CNT) including carboxyl (CNT-COOH), amine (CNT-NH2) and hydroxyl (CNT-OH) functional groups were studied using differential pulse voltammetry (DPV). The current-voltage (I-V) curves were obtained from each system and the effect of f-CNT on redox interaction of horseradish peroxidase (HRP) immobilized on the electrode surface was investigated. The non-equilibrium Green's function (NEGF) combined with density functional theory (DFT) were used to study the transport properties of f-CNT. Additionally, the effect of the number of functional groups on transport properties of CNT, I-V characteristics, electronic transmission coefficients and spatial distribution of f-CNTs have been calculated and analyzed. The results showed that the carboxyl derivative has larger transmission coefficients and current value than other f-CNTs. Then, the effect of functional groups on the electron transport in heme group of HRP is discussed. Finally, the effect of a covalent bond between active site amino acids and amine functional group of CNT was investigated and discussed.

  6. Immobilization of horseradish peroxidase on ZnO nanowires/macroporous SiO2 composites for the complete decolorization of anthraquinone dyes.

    Science.gov (United States)

    Sun, Huaiyan; Jin, Xinyu; Jiang, Feng; Zhang, Ruifeng

    2018-03-01

    A zinc oxide (ZnO) nanowires/macroporous silicon dioxide composite was used as support to immobilize horseradish peroxidase (HRP) simply by in situ cross-linking method. As cross-linker was adsorbed on the surface of ZnO nanowires, the cross-linked HRP was quite different from the traditional cross-linking enzyme aggregates on both structure and catalytic performance. Among three epoxy compounds, diethylene glycol diglycidyl ether (DDE) was the best cross-linker, with which the loading amount of HRP with pI of 5.3 reached as high as 118.1 mg/g and specific activity was up to 14.9 U/mg-support. The mass loss of HRP cross-linked with DDE was negligible during 50-H leaching at 4 °C, and the thermal stability of the immobilized HRP was also quite good. The catalytic performance of immobilized HRP to decolorize anthraquinone dye was explored by using Reactive Blue 19 (RB 19) and Acid Violet 109 (AV 109) as model substrates. The results indicated that the immobilized HRP exhibited high decolorization efficiency and good reusability. The decolorization efficiency reached 94.3% and 95.9% for AV 109 and RB 19 within the first 30 Min, respectively. A complete decolorization of these two dyes has been realized within 2-3 H by using this new biocatalysis system. © 2017 International Union of Biochemistry and Molecular Biology, Inc.

  7. Tetra(p-tolyl)borate-functionalized solvent polymeric membrane: a facile and sensitive sensing platform for peroxidase and peroxidase mimetics.

    Science.gov (United States)

    Wang, Xuewei; Qin, Wei

    2013-07-22

    The determination of peroxidase activities is the basis for enzyme-labeled bioaffinity assays, peroxidase-mimicking DNAzymes- and nanoparticles-based assays, and characterization of the catalytic functions of peroxidase mimetics. Here, a facile, sensitive, and cost-effective solvent polymeric membrane-based peroxidase detection platform is described that utilizes reaction intermediates with different pKa values from those of substrates and final products. Several key but long-debated intermediates in the peroxidative oxidation of o-phenylenediamine (o-PD) have been identified and their charge states have been estimated. By using a solvent polymeric membrane functionalized by an appropriate substituted tetraphenylborate as a receptor, those cationic intermediates could be transferred into the membrane from the aqueous phase to induce a large cationic potential response. Thus, the potentiometric indication of the o-PD oxidation catalyzed by peroxidase or its mimetics can be fulfilled. Horseradish peroxidase has been detected with a detection limit at least two orders of magnitude lower than those obtained by spectrophotometric techniques and traditional membrane-based methods. As an example of peroxidase mimetics, G-quadruplex DNAzymes were probed by the intermediate-sensitive membrane and a label-free thrombin detection protocol was developed based on the catalytic activity of the thrombin-binding G-quadruplex aptamer. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Controlling the shape of membrane protein polyhedra

    Science.gov (United States)

    Li, Di; Kahraman, Osman; Haselwandter, Christoph A.

    2017-03-01

    Membrane proteins and lipids can self-assemble into membrane protein polyhedral nanoparticles (MPPNs). MPPNs have a closed spherical surface and a polyhedral protein arrangement, and may offer a new route for structure determination of membrane proteins and targeted drug delivery. We develop here a general analytic model of how MPPN self-assembly depends on bilayer-protein interactions and lipid bilayer mechanical properties. We find that the bilayer-protein hydrophobic thickness mismatch is a key molecular control parameter for MPPN shape that can be used to bias MPPN self-assembly towards highly symmetric and uniform MPPN shapes. Our results suggest strategies for optimizing MPPN shape for structural studies of membrane proteins and targeted drug delivery.

  9. Targeted Expression of Stromelysin-1 in Mammary Gland Provides Evidence for a Role of Proteinases in Branching Morphogenesis and the Requirement for an Intact Basement Membrane for Tissue-specific Gene Expression

    Energy Technology Data Exchange (ETDEWEB)

    Sympson, Carolyn J; Talhouk, Rabih S; Alexander, Caroline M; Chin, Jennie R; Cliff, Shirley M; Bissell, Mina J; Werb, Zena

    1994-05-01

    The extracellular matrix (ECM) is an important regulator of the differentiated phenotype of mammary epithelial cells in culture. Despite the fact that ECM-degrading enzymes have been implicated in morphogenesis and tissue remodeling, there is little evidence for a direct role for such regulation in vivo. We generated transgenic mice that express autoactivated isoforms of the matrix metalloproteinase stromelysin-1, under the control of the whey acidic protein gene promoter, to examine the effect of inappropriate expression of this enzyme. Stromelysin-1 is implicated as the primary player in the loss of basement membrane and loss of function in the mammary gland during involution. The transgene was expressed at low levels in mammary glands of virgin female mice, leading to an unexpected phenotype: The primary ducts had supernumerary branches and showed precocious development of alveoli that expressed beta-casein at levels similar to that of an early- to mid-pregnant gland. Lactating glands showed high levels of transgene expression, with accumulation at the basement membrane, and a decrease in laminin and collagen IV, resulting in a loss of basement membrane integrity; this was accompanied by a dramatic alteration of alveolar morphology, with decreased size and shrunken lumina containing little beta-casein. During pregnancy, expression of endogenous whey acidic protein and beta-casein was reduced in transgenic glands, confirming the observed dependence of milk protein transcription of ECM in mammary epithelial cells in culture. These data provide direct evidence that stromelysin-1 activity can be morphogenic for mammary epithelial cells, inducing hyperproliferation and differentiation in virgin animals, and that its lytic activity can, indeed, disrupt membrane integrity and reduce mammary-specific function. We conclude that the balance of ECM-degrading enzymes with their inhibitors, and the associated regulation of ECM structure, is crucial for tissue-specific gene

  10. Layer-by-layer cell membrane assembly

    Science.gov (United States)

    Matosevic, Sandro; Paegel, Brian M.

    2013-11-01

    Eukaryotic subcellular membrane systems, such as the nuclear envelope or endoplasmic reticulum, present a rich array of architecturally and compositionally complex supramolecular targets that are as yet inaccessible. Here we describe layer-by-layer phospholipid membrane assembly on microfluidic droplets, a route to structures with defined compositional asymmetry and lamellarity. Starting with phospholipid-stabilized water-in-oil droplets trapped in a static droplet array, lipid monolayer deposition proceeds as oil/water-phase boundaries pass over the droplets. Unilamellar vesicles assembled layer-by-layer support functional insertion both of purified and of in situ expressed membrane proteins. Synthesis and chemical probing of asymmetric unilamellar and double-bilayer vesicles demonstrate the programmability of both membrane lamellarity and lipid-leaflet composition during assembly. The immobilized vesicle arrays are a pragmatic experimental platform for biophysical studies of membranes and their associated proteins, particularly complexes that assemble and function in multilamellar contexts in vivo.

  11. Biophysical EPR Studies Applied to Membrane Proteins

    Science.gov (United States)

    Sahu, Indra D; Lorigan, Gary A

    2015-01-01

    Membrane proteins are very important in controlling bioenergetics, functional activity, and initializing signal pathways in a wide variety of complicated biological systems. They also represent approximately 50% of the potential drug targets. EPR spectroscopy is a very popular and powerful biophysical tool that is used to study the structural and dynamic properties of membrane proteins. In this article, a basic overview of the most commonly used EPR techniques and examples of recent applications to answer pertinent structural and dynamic related questions on membrane protein systems will be presented. PMID:26855825

  12. Enhancing Membrane Protein Identification Using a Simplified Centrifugation and Detergent-Based Membrane Extraction Approach.

    Science.gov (United States)

    Zhou, Yanting; Gao, Jing; Zhu, Hongwen; Xu, Jingjing; He, Han; Gu, Lei; Wang, Hui; Chen, Jie; Ma, Danjun; Zhou, Hu; Zheng, Jing

    2018-02-20

    Membrane proteins may act as transporters, receptors, enzymes, and adhesion-anchors, accounting for nearly 70% of pharmaceutical drug targets. Difficulties in efficient enrichment, extraction, and solubilization still exist because of their relatively low abundance and poor solubility. A simplified membrane protein extraction approach with advantages of user-friendly sample processing procedures, good repeatability and significant effectiveness was developed in the current research for enhancing enrichment and identification of membrane proteins. This approach combining centrifugation and detergent along with LC-MS/MS successfully identified higher proportion of membrane proteins, integral proteins and transmembrane proteins in membrane fraction (76.6%, 48.1%, and 40.6%) than in total cell lysate (41.6%, 16.4%, and 13.5%), respectively. Moreover, our method tended to capture membrane proteins with high degree of hydrophobicity and number of transmembrane domains as 486 out of 2106 (23.0%) had GRAVY > 0 in membrane fraction, 488 out of 2106 (23.1%) had TMs ≥ 2. It also provided for improved identification of membrane proteins as more than 60.6% of the commonly identified membrane proteins in two cell samples were better identified in membrane fraction with higher sequence coverage. Data are available via ProteomeXchange with identifier PXD008456.

  13. Membrane fusion by VAMP3 and plasma membrane t-SNAREs

    International Nuclear Information System (INIS)

    Hu Chuan; Hardee, Deborah; Minnear, Fred

    2007-01-01

    Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of α-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins

  14. Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues.

    Science.gov (United States)

    Yamamoto, Norio; Yamashita, Yoko; Yoshioka, Yasukiyo; Nishiumi, Shin; Ashida, Hitoshi

    2016-08-01

    Membrane proteins account for 70% to 80% of all pharmaceutical targets, indicating their clinical relevance and underscoring the importance of identifying differentially expressed membrane proteins that reflect distinct disease properties. The translocation of proteins from the bulk of the cytosol to the plasma membrane is a critical step in the transfer of information from membrane-embedded receptors or transporters to the cell interior. To understand how membrane proteins work, it is important to separate the membrane fraction of cells. This unit provides a protocol for rapidly obtaining plasma membrane fractions for western blot analysis. © 2016 by John Wiley & Sons, Inc. Copyright © 2016 John Wiley & Sons, Inc.

  15. A membrane-based immunosensor for the analysis of the herbicide isoproturon.

    Science.gov (United States)

    Baskeyfield, Damian E H; Davis, Frank; Magan, Naresh; Tothill, Ibtisam E

    2011-08-12

    A membrane based heterogeneous competitive enzyme-linked immunosorbent assay (ELISA) was used in this work to develop an immunosensor for the detection of a common herbicide, isoproturon. A screen-printed carbon working electrode with carbon counter and silver-silver chloride pseudo-reference electrode was utilized incorporating a membrane fixed into intimate contact with the working electrode to facilitate signal transduction. The membrane containing an immobilized isoproturon-ovalbumin conjugate was laminated onto the carbon working electrode and horseradish peroxidase (HRP) labeled polyclonal antibody was then applied for the competitive assay. Two different amperometric systems, hydroquinone and o-phenylenediamine (OPD) mediation reduction were utilised and the properties of the resultant sensors were compared. A flow injection apparatus was also developed utilising the immunosensor. Limits of detection for isoproturon (LLD(90)) were found to be as low as 0.84 ng mL(-1). The senor was also validated using spiked extracted soil samples and also isoproturon contaminated samples. Copyright © 2011 Elsevier B.V. All rights reserved.

  16. The effect of MEP pathway and other inhibitors on the intracellular localization of a plasma membrane-targeted, isoprenylable GFP reporter protein in tobacco BY-2 cells [v1; ref status: indexed, http://f1000r.es/yx

    Directory of Open Access Journals (Sweden)

    Michael Hartmann

    2013-08-01

    Full Text Available We have established an in vivo visualization system for the geranylgeranylation of proteins in a stably transformed tobacco BY-2 cell line, based on the expression of a dexamethasone-inducible GFP fused to the carboxy-terminal basic domain of the rice calmodulin CaM61, which naturally bears a CaaL geranylgeranylation motif (GFP-BD-CVIL. By using pathway-specific inhibitors it was demonstrated that inhibition of the methylerythritol phosphate (MEP pathway with known inhibitors like oxoclomazone and fosmidomycin, as well as inhibition of the protein geranylgeranyltransferase type 1 (PGGT-1, shifted the localization of the GFP-BD-CVIL protein from the membrane to the nucleus. In contrast, the inhibition of the mevalonate (MVA pathway with mevinolin did not affect the localization. During the present work, this test system has been used to examine the effect of newly designed inhibitors of the MEP pathway and inhibitors of sterol biosynthesis such as squalestatin, terbinafine and Ro48-8071. In addition, we also studied the impact of different post-prenylation inhibitors or those suspected to affect the transport of proteins to the plasma membrane on the localization of the geranylgeranylable fusion protein GFP-BD-CVIL.

  17. Hybrid adsorptive membrane reactor

    Science.gov (United States)

    Tsotsis, Theodore T [Huntington Beach, CA; Sahimi, Muhammad [Altadena, CA; Fayyaz-Najafi, Babak [Richmond, CA; Harale, Aadesh [Los Angeles, CA; Park, Byoung-Gi [Yeosu, KR; Liu, Paul K. T. [Lafayette Hill, PA

    2011-03-01

    A hybrid adsorbent-membrane reactor in which the chemical reaction, membrane separation, and product adsorption are coupled. Also disclosed are a dual-reactor apparatus and a process using the reactor or the apparatus.

  18. Premature rupture of membranes

    Science.gov (United States)

    ... gov/ency/patientinstructions/000512.htm Premature rupture of membranes To use the sharing features on this page, ... water that surrounds your baby in the womb. Membranes or layers of tissue hold in this fluid. ...

  19. Oxygen transport membrane

    DEFF Research Database (Denmark)

    2015-01-01

    The present invention relates to a novel composite oxygen transport membrane as well as its preparation and uses thereof.......The present invention relates to a novel composite oxygen transport membrane as well as its preparation and uses thereof....

  20. Membrane with integrated spacer

    NARCIS (Netherlands)

    Balster, J.H.; Stamatialis, Dimitrios; Wessling, Matthias

    2010-01-01

    Many membrane processes are severely influenced by concentration polarisation. Turbulence promoting spacers placed in between the membranes can reduce the diffusional resistance of concentration polarisation by inducing additional mixing. Electrodialysis (ED) used for desalination suffers from

  1. Ultrasensitive colorimetric immunoassay for hCG detection based on dual catalysis of Au@Pt core-shell nanoparticle functionalized by horseradish peroxidase

    Science.gov (United States)

    Wang, Weiguo; Zou, Yake; Yan, Jinwu; Liu, Jing; Chen, Huixiong; Li, Shan; Zhang, Lei

    2018-03-01

    In this paper, an ultrasensitive colorimetric biosensor for human chorionic gonadotrophin (hCG) detection was designed from bottom-up method based on the dual catalysis of the horseradish peroxidase (HRP) and Au@Pt nanoparticles (NPs) relative to H2O2-TEM system. HRP and monoclonal mouse anti-hCG antibody (β-submit, mAb1) were co-immobilized onto the Au@Pt NP surface to improve catalytic efficiency and specificity, which formed a dual functionalized Au@Pt-HRP probe with the mean size of 42.8 nm (D50). The colorimetric immunoassay was developed for the hCG detection, and the Au@Pt-HRP probe featured a higher sensitivity in the concentration range of 0.4-12.8 IU L- 1 with a low limit of detection (LOD) of 0.1 IU L- 1 compared with the LODs of 0.8 IU L- 1 for BA-ELISA and of 2.0 IU L- 1 for Au@Pt, which indicated that the Au@Pt-HRP probe possessed higher catalytic efficiency with 2.8-fold increase over Au@Pt and 33.8-fold increase over HRP. Also, the Au@Pt-HRP probe exhibited good precision and reproducibility, high specificity and acceptable accuracy with CV being less than 15%. The dual functionalized Au@Pt-HRP probe as a type of signal amplified method was firstly applied in the colorimetric immunoassay for the hCG detection.

  2. Multi-input and -output logic circuits based on bioelectrocatalysis with horseradish peroxidase and glucose oxidase immobilized in multi-responsive copolymer films on electrodes.

    Science.gov (United States)

    Yu, Xue; Lian, Wenjing; Zhang, Jiannan; Liu, Hongyun

    2016-06-15

    Herein, poly(N-isopropylacrylamide-co-N,N'-dimethylaminoethylmethacrylate) copolymer films were polymerized on electrode surface with a simple one-step method, and the enzyme horseradish peroxidase (HRP) was embedded in the films simultaneously, which were designated as P(NiPAAm-co-DMEM)-HRP. The films exhibited a reversible structure change with the external stimuli, such as pH, CO2, temperature and SO4(2-), causing the cyclic voltammetric (CV) response of electroactive K3Fe(CN)6 at the film electrodes to display the corresponding multi-stimuli sensitive ON-OFF behavior. Based on the switchable CV property of the system and the electrochemical reduction of H2O2 catalyzed by HRP in the films and mediated by Fe(CN)6(3-) in solution, a 5-input/3-output logic gate was established. To further increase the complexity of the logic system, another enzyme glucose oxidase (GOD) was added into the films, designated as P(NiPAAm-co-DMEM)-HRP-GOD. In the presence of oxygen, the oxidation of glucose in the solution was catalyzed by GOD in the films, and the produced H2O2 in situ was recognized and electrocatalytically reduced by HRP and mediated by Fe(CN)6(3-). Based on the bienzyme films, a cascaded or concatenated 4-input/3-output logic gate system was proposed. The present work combined the multi-responsive interface with bioelectrocatalysis to construct cascaded logic circuits, which might open a new avenue to develop biocomputing elements with more sophisticated functions and design novel glucose biosensors. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. Enhanced amperometric response of a glucose oxidase and horseradish peroxidase based bienzyme glucose biosensor modified with a film of polymerized toluidine blue containing reduced graphene oxide

    International Nuclear Information System (INIS)

    Wang, Fang; Gong, Wencheng; Wang, Lili; Chen, Zilin

    2015-01-01

    Reduced graphene oxide (RGO) was used to construct a bienzyme biosensor containing horseradish peroxidase (HRP) and glucose oxidase (GOx). A poly(toluidine blue) (pTB) film containing RGO acted as both enzyme immobilization matrix and electron transfer mediator. The bienzyme biosensor was characterized by electrochemical techniques and displays a highly sensitive amperometric response to glucose and hydrogen peroxide (H 2 O 2 ) at a potential as low as −0.1 V (vs. SCE). It is shown that use of RGO causes a strong enhancement on the amperometric responses. H 2 O 2 formed by the action of GOx in the presence of oxygen can be further reduced by HRP in the pTB film contacting the RGO modified electrode. In the absence of oxygen, glucose oxidation proceeds by another mechanism in which electron transfer occurs from GOx to the electrode and with pTB acting as the mediator. Amperometric responses to glucose and H2O2 follow Michaelis-Menten kinetics. The experimental conditions were optimized, and under these conditions glucose can be determined in the 80 μM to 3.0 mM range with a detection limit of 50 μM. H 2 O 2 , in turn, can be quantified in up to 30.0 μM concentration with a detection limit of 0.2 μM. The bienzyme biosensor is reproducible, repeatable and stable. Finally, it has been successfully applied to the determination of glucose in plasma samples. (author)

  4. Production of a conjugate between the rK346 antigen from Leishmania infantum and the horseradish peroxidase C for the detection of rK346 antibodies.

    Science.gov (United States)

    Rengifo-González, Juan; Medina-Mora, Yollyseth; Silva-Barrios, Sasha; Márquez-Contreras, María Elizabeth; Tibisay Ruiz, María; Cáceres, Ana J; Concepción, Juan Luis; Quiñones, Wilfredo

    2016-06-01

    It was designed and characterized a reporter system to be captured by an- tibodies bound to ELISA plates. The system was designed with the rK346 from Leishmania infantum, a highly antigenic and specific protein. The rK346 was coupled to the horseradish peroxidase C (HRPc) from Armoracia rusticana using glutaraldehyde or sulfo-SMCC. Gluta- raldehyde conjugation was performed in two steps. Separation of conjugates was carried out using a Sepharose S-200 in size exclusion chromatography (SEC); fractions were analyzed via HRPc activity and through ELISA plates sensitized with polyclonal anti-rK346 IgG puri- fied from rabbit serum. A heterogeneous population of conjugates rK346-HRPc was obtained with molecular weights ranging between 109.7 ± 16.5 to 67.6 ± 10.1 kDa; with rK346-HRPe stoichiometries of 1:2; 2:1; 3:1; and 2:2. Conjugation using sulfo-SMCC was carried out first by introducing -SH groups onto the HRPc using the SATA reagent and the antigen was modi- fied with sulfo-SMCC during 45 min. Separation and analysis of conjugates was performed similarly as with glutaraldehyde, resulting in a heterogeneous population of conjugates rK346- HRPc with molecular weights between 150.5 ± 22.6 to 80.0 ± 12.0 kDa; with rK346-HRPC stoichiometries of 2:1; 1:2; 2:2; and 1:3, with an increased conjugation efficiency in compari- son with glutaraldehyde. This enables sulfo-SMCC to be used as a potential reagent for cou- pling the antigen to the HRPc, to design an economic, specific and easy method to apply as a reporter system, available to assess individuals at risk and/or at early and late stages of visceral leishmaniasis.

  5. Coupled enzyme reactions performed in heterogeneous reaction media: experiments and modeling for glucose oxidase and horseradish peroxidase in a PEG/citrate aqueous two-phase system.

    Science.gov (United States)

    Aumiller, William M; Davis, Bradley W; Hashemian, Negar; Maranas, Costas; Armaou, Antonios; Keating, Christine D

    2014-03-06

    The intracellular environment in which biological reactions occur is crowded with macromolecules and subdivided into microenvironments that differ in both physical properties and chemical composition. The work described here combines experimental and computational model systems to help understand the consequences of this heterogeneous reaction media on the outcome of coupled enzyme reactions. Our experimental model system for solution heterogeneity is a biphasic polyethylene glycol (PEG)/sodium citrate aqueous mixture that provides coexisting PEG-rich and citrate-rich phases. Reaction kinetics for the coupled enzyme reaction between glucose oxidase (GOX) and horseradish peroxidase (HRP) were measured in the PEG/citrate aqueous two-phase system (ATPS). Enzyme kinetics differed between the two phases, particularly for the HRP. Both enzymes, as well as the substrates glucose and H2O2, partitioned to the citrate-rich phase; however, the Amplex Red substrate necessary to complete the sequential reaction partitioned strongly to the PEG-rich phase. Reactions in ATPS were quantitatively described by a mathematical model that incorporated measured partitioning and kinetic parameters. The model was then extended to new reaction conditions, i.e., higher enzyme concentration. Both experimental and computational results suggest mass transfer across the interface is vital to maintain the observed rate of product formation, which may be a means of metabolic regulation in vivo. Although outcomes for a specific system will depend on the particulars of the enzyme reactions and the microenvironments, this work demonstrates how coupled enzymatic reactions in complex, heterogeneous media can be understood in terms of a mathematical model.

  6. Single turnover studies of oxidative halophenol dehalogenation by horseradish peroxidase reveal a mechanism involving two consecutive one electron steps: toward a functional halophenol bioremediation catalyst.

    Science.gov (United States)

    Sumithran, Suganya; Sono, Masanori; Raner, Gregory M; Dawson, John H

    2012-12-01

    Horseradish peroxidase (HRP) catalyzes the oxidative para-dechlorination of the environmental pollutant/carcinogen 2,4,6-trichlorophenol (2,4,6-TCP). A possible mechanism for this reaction is a direct oxygen atom transfer from HRP compound I (HRP I) to trichlorophenol to generate 2,6-dichloro 1,4-benzoquinone, a two-electron transfer process. An alternative mechanism involves two consecutive one-electron transfer steps in which HRP I is reduced to compound II (HRP II) and then to the ferric enzyme as first proposed by Wiese et al. [F.W. Wiese, H.C. Chang, R.V. Lloyd, J.P. Freeman, V.M. Samokyszyn, Arch. Environ. Contam. Toxicol. 34 (1998) 217-222]. To probe the mechanism of oxidative halophenol dehalogenation, the reactions between 2,4,6-TCP and HRP compounds I or II have been investigated under single turnover conditions (i.e., without excess H(2)O(2)) using rapid scan stopped-flow spectroscopy. Addition of 2,4,6-TCP to HRP I leads rapidly to HRP II and then more slowly to the ferric resting state, consistent with a mechanism involving two consecutive one-electron oxidations of the substrate via a phenoxy radical intermediate. HRP II can also directly dechlorinate 2,4,6-TCP as judged by rapid scan stopped-flow and mass spectrometry. This observation is particularly significant since HRP II can only carry out one-electron oxidations. A more detailed understanding of the mechanism of oxidative halophenol dehalogenation will facilitate the use of HRP as a halophenol bioremediation catalyst. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Effect of the structure of imidazolium cations in [BF4](-)-type ionic liquids on direct electrochemistry and electrocatalysis of horseradish peroxidase in Nafion films.

    Science.gov (United States)

    Lu, Lu; Huang, Xirong; Qu, Yinbo

    2011-10-01

    The direct electrochemistry and bioelectrocatalysis of horseradish peroxidase (HRP) in Nafion films at glassy carbon electrode (GCE) was investigated in three [BF(4)](-)-type room-temperature ionic liquids (ILs) to understand the structural effect of imidazolium cations. The three ILs are 1-ethyl-3-methylimidazolium tetrafluoroborate ([Emim][BF(4)]), 1-butyl-3-methylimidazolium tetrafluoroborate ([Bmim][BF(4)]) and 1-hexyl-3-methylimidazolium tetrafluoroborate ([Hmim][BF(4)]). A small amount of water in the three ILs is indispensable for maintaining the electrochemical activity of HRP in Nafion films, and the optimum water contents decrease with the increase of alkyl chain length on imidazole ring. Analysis shows that the optimum water contents are primarily determined by the hydrophilicity of ILs used. In contrast to aqueous medium, ILs media facilitate the direct electron transfer of HRP, and the electrochemical parameters obtained in different ILs are obviously related to the nature of ILs. The direct electron transfer between HRP and GCE is a surface-confined quasi-reversible single electron transfer process. The apparent heterogeneous electron transfer rate constant decreases gradually with the increase of alkyl chain length on imidazole ring, but the changing extent is relatively small. The electrocatalytic reduction current of H(2)O(2) at the present electrode decreases obviously with the increase of alkyl chain length, and the mass transfer of H(2)O(2) via diffusion in ILs should be responsible for the change. In addition, the modified electrode has good stability and reproducibility; the ability to tolerate high levels of F(-) has been greatly enhanced due to the use of Nafion film. When an appropriate mediator is included in the sensing layer, a sensitive nonaqueous biosensor could be fabricated. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Amperometric carbohydrate antigen 19-9 immunosensor based on three dimensional ordered macroporous magnetic Au film coupling direct electrochemistry of horseradish peroxidase

    Energy Technology Data Exchange (ETDEWEB)

    Zhang, Qi [College of Sciences, Nanjing Tech University, Nanjing 211816 (China); Chen, Xiaojun, E-mail: chenxj_njut@126.com [College of Sciences, Nanjing Tech University, Nanjing 211816 (China); State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing Tech University, Nanjing 210009 (China); Tang, Yin [Zhangjiagang Hospital of Traditional Chinese Medicine, Zhangjiagang 215600 (China); Ge, Lingna; Guo, Buhua [College of Sciences, Nanjing Tech University, Nanjing 211816 (China); Yao, Cheng, E-mail: yaochengnjut@163.com [College of Sciences, Nanjing Tech University, Nanjing 211816 (China)

    2014-03-01

    Highlights: • Three dimensional ordered macroporous magnetic electrode was newly used in electrochemical immunosensor. • The large surface area of macroporous magnetic electrode could improve the immobilized amount of antibody. • Au nanoparticles functionalized SBA-15 was used to immobilize enzyme labeled Ab₂ and enzyme. • Macroporous magnetic electrode and Au nanoparticles composite facilitated the direct electron transfer of enzyme. • The immunoassay avoided adding electron transfer mediator, simplifying the procedure. Abstract: A sandwich-type electrochemical immunosensor for the detection of carbohydrate antigen 19-9 (CA 19-9) antigen based on the immobilization of primary antibody (Ab₁) on three dimensional ordered macroporous magnetic (3DOMM) electrode, and the direct electrochemistry of horseradish peroxidase (HRP) that was used as both the label of secondary antibody (Ab₂) and the blocking reagent. The 3DOMM electrode was fabricated by introducing core–shell Au–SiO₂@Fe₃O₄ nanospheres onto the surface of three dimensional ordered macroporous (3DOM) Au electrode via the application of an external magnet. Au nanoparticles functionalized SBA-15 (Au@SBA-15) was conjugated to the HRP labeled secondary antibody (HRP-Ab₂) through the Au–SH or Au–NH₃⁺ interaction, and HRP was also used as the block reagent. The formation of antigen–antibody complex made the combination of Au@SBA-15 and 3DOMM exhibit remarkable synergistic effects for accelerating direct electron transfer (DET) between HRP and the electrode. Under the optimal conditions, the DET current signal increased proportionally to CA 19-9 concentration in the range of 0.05 to 15.65 U mL⁻¹ with a detection limit of 0.01 U mL⁻¹. Moreover, the immunosensor showed high selectivity, good stability, satisfactory reproducibility and regeneration. Importantly, the developed method was used to assay clinical serum specimens, achieving a good relation with those obtained from

  9. Oxidations of N-(3-indoleethyl) cyclic aliphatic amines by horseradish peroxidase: the indole ring binds to the enzyme and mediates electron-transfer amine oxidation.

    Science.gov (United States)

    Ling, Ke-Qing; Li, Wen-Shan; Sayre, Lawrence M

    2008-01-23

    Although oxidations of aromatic amines by horseradish peroxidase (HRP) are well-known, typical aliphatic amines are not substrates of HRP. In this study, the reactions of N-benzyl and N-methyl cyclic amines with HRP were found to be slow, but reactions of N-(3-indoleethyl) cyclic amines were 2-3 orders of magnitude faster. Analyses of pH-rate profiles revealed a dominant contribution to reaction by the amine-free base forms, the only species found to bind to the enzyme. A metabolic study on a family of congeneric N-(3-indoleethyl) cyclic amines indicated competition between amine and indole oxidation pathways. Amine oxidation dominated for the seven- and eight-membered azacycles, where ring size supports the change in hybridization from sp3 to sp2 that occurs upon one-electron amine nitrogen oxidation, whereas only indole oxidation was observed for the six-membered ring congener. Optical difference spectroscopic binding data and computational docking simulations suggest that all the arylalkylamine substrates bind to the enzyme through their aromatic termini with similar binding modes and binding affinities. Kinetic saturation was observed for a particularly soluble substrate, consistent with an obligatory role of an enzyme-substrate complexation preceding electron transfer. The significant rate enhancements seen for the indoleethylamine substrates suggest the ability of the bound indole ring to mediate what amounts to medium long-range electron-transfer oxidation of the tertiary amine center by the HRP oxidants. This is the first systematic investigation to document aliphatic amine oxidation by HRP at rates consistent with normal metabolic turnover, and the demonstration that this is facilitated by an auxiliary electron-rich aromatic ring.

  10. Structural characterization of a novel full-length transcript promoter from Horseradish Latent Virus (HRLV) and its transcriptional regulation by multiple stress responsive transcription factors.

    Science.gov (United States)

    Khan, Ahamed; Shrestha, Ankita; Bhuyan, Kashyap; Maiti, Indu B; Dey, Nrisingha

    2018-01-01

    The promoter fragment described in this study can be employed for strong transgene expression under both biotic and abiotic stress conditions. Plant-infecting Caulimoviruses have evolved multiple regulatory mechanisms to address various environmental stimuli during the course of evolution. One such mechanism involves the retention of discrete stress responsive cis-elements which are required for their survival and host-specificity. Here we describe the characterization of a novel Caulimoviral promoter isolated from Horseradish Latent Virus (HRLV) and its regulation by multiple stress responsive Transcription factors (TFs) namely DREB1, AREB1 and TGA1a. The activity of full length transcript (Flt-) promoter from HRLV (- 677 to + 283) was investigated in both transient and transgenic assays where we identified H12 (- 427 to + 73) as the highest expressing fragment having ~ 2.5-fold stronger activity than the CaMV35S promoter. The H12 promoter was highly active and near-constitutive in the vegetative and reproductive parts of both Tobacco and Arabidopsis transgenic plants. Interestingly, H12 contains a distinct cluster of cis-elements like dehydration-responsive element (DRE-core; GCCGAC), an ABA-responsive element (ABRE; ACGTGTC) and as-1 element (TGACG) which are known to be induced by cold, drought and pathogen/SA respectively. The specific binding of DREB1, AREB1 and TGA1a to DRE, ABRE and as-1 elements respectively were confirmed by the gel-binding assays using H12 promoter-specific probes. Detailed mutational analysis of the H12 promoter suggested that the presence of DRE-core and as-1 element was indispensable for its activity which was further confirmed by the transactivation assays. Our studies imply that H12 could be a valuable genetic tool for regulated transgene expression under diverse environmental conditions.

  11. Gel layer formation on membranes in Membrane Bioreactors

    NARCIS (Netherlands)

    Van den Brink, P.F.H.

    2014-01-01

    The widespread application of membrane bioreactors (MBRs) for municipal wastewater treatment is hampered by membrane fouling. Fouling increases energy demand, reduces process performance and creates the need for more frequent (chemical) membrane cleaning or replacement. Membrane fouling in MBRs is

  12. Smart membranes for monitoring membrane based desalination processes

    KAUST Repository

    Laleg-Kirati, Taous-Meriem; Karam, Ayman M.

    2017-01-01

    Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and

  13. Model cell membranes

    DEFF Research Database (Denmark)

    Günther-Pomorski, Thomas; Nylander, Tommy; Cardenas Gomez, Marite

    2014-01-01

    The high complexity of biological membranes has motivated the development and application of a wide range of model membrane systems to study biochemical and biophysical aspects of membranes in situ under well defined conditions. The aim is to provide fundamental understanding of processes control...

  14. Idiopathic epiretinal membrane

    NARCIS (Netherlands)

    Bu, Shao-Chong; Kuijer, Roelof; Li, Xiao-Rong; Hooymans, Johanna M M; Los, Leonoor I

    2014-01-01

    Background: Idiopathic epiretinal membrane (iERM) is a fibrocellular membrane that proliferates on the inner surface of the retina at the macular area. Membrane contraction is an important sight-threatening event and is due to fibrotic remodeling. Methods: Analysis of the current literature

  15. Meniscus Membranes For Separation

    Science.gov (United States)

    Dye, Robert C.; Jorgensen, Betty; Pesiri, David R.

    2005-09-20

    Gas separation membranes, especially meniscus-shaped membranes for gas separations are disclosed together with the use of such meniscus-shaped membranes for applications such as thermal gas valves, pre-concentration of a gas stream, and selective pre-screening of a gas stream. In addition, a rapid screening system for simultaneously screening polymer materials for effectiveness in gas separation is provided.

  16. Meniscus membranes for separations

    Science.gov (United States)

    Dye, Robert C [Irvine, CA; Jorgensen, Betty [Jemez Springs, NM; Pesiri, David R [Aliso Viejo, CA

    2004-01-27

    Gas separation membranes, especially meniscus-shaped membranes for gas separations are disclosed together with the use of such meniscus-shaped membranes for applications such as thermal gas valves, pre-concentration of a gas stream, and selective pre-screening of a gas stream. In addition, a rapid screening system for simultaneously screening polymer materials for effectiveness in gas separation is provided.

  17. CERN: Fixed target targets

    Energy Technology Data Exchange (ETDEWEB)

    Anon.

    1993-03-15

    Full text: While the immediate priority of CERN's research programme is to exploit to the full the world's largest accelerator, the LEP electron-positron collider and its concomitant LEP200 energy upgrade (January, page 1), CERN is also mindful of its long tradition of diversified research. Away from LEP and preparations for the LHC proton-proton collider to be built above LEP in the same 27-kilometre tunnel, CERN is also preparing for a new generation of heavy ion experiments using a new source, providing heavier ions (April 1992, page 8), with first physics expected next year. CERN's smallest accelerator, the LEAR Low Energy Antiproton Ring continues to cover a wide range of research topics, and saw a record number of hours of operation in 1992. The new ISOLDE on-line isotope separator was inaugurated last year (July, page 5) and physics is already underway. The remaining effort concentrates around fixed target experiments at the SPS synchrotron, which formed the main thrust of CERN's research during the late 1970s. With the SPS and LEAR now approaching middle age, their research future was extensively studied last year. Broadly, a vigorous SPS programme looks assured until at least the end of 1995. Decisions for the longer term future of the West Experimental Area of the SPS will have to take into account the heavy demand for test beams from work towards experiments at big colliders, both at CERN and elsewhere. The North Experimental Area is the scene of larger experiments with longer lead times. Several more years of LEAR exploitation are already in the pipeline, but for the longer term, the ambitious Superlear project for a superconducting ring (January 1992, page 7) did not catch on. Neutrino physics has a long tradition at CERN, and this continues with the preparations for two major projects, the Chorus and Nomad experiments (November 1991, page 7), to start next year in the West Area. Delicate neutrino oscillation effects could become visible for the first

  18. 3D7-derived Plasmodium falciparum erythrocyte membrane protein 1 is a frequent target of naturally acquired antibodies recognizing protein domains in a particular pattern independent of malaria transmission intensity

    DEFF Research Database (Denmark)

    Joergensen, Louise; Vestergaard, Lasse S; Turner, Louise

    2007-01-01

    Protection against Plasmodium falciparum malaria is largely mediated by IgG against surface Ags such as the erythrocyte membrane protein 1 family (PfEMP1) responsible for antigenic variation and sequestration of infected erythrocytes. PfEMP1 molecules can be divided into groups A, B/A, B, C, and B......, the sequence by which individuals acquired Abs to particular constructs was largely the same in the three villages. This indicates that the pattern of PfEMP1 expression by parasites transmitted at the different sites was similar, suggesting that PfEMP1 expression is nonrandom and shaped by host......-parasite relationship factors operating at all transmission intensities....

  19. Separation membrane development

    Energy Technology Data Exchange (ETDEWEB)

    Lee, M.W. [Savannah River Technology Center, Aiken, SC (United States)

    1998-08-01

    A ceramic membrane has been developed to separate hydrogen from other gases. The method used is a sol-gel process. A thin layer of dense ceramic material is coated on a coarse ceramic filter substrate. The pore size distribution in the thin layer is controlled by a densification of the coating materials by heat treatment. The membrane has been tested by permeation measurement of the hydrogen and other gases. Selectivity of the membrane has been achieved to separate hydrogen from carbon monoxide. The permeation rate of hydrogen through the ceramic membrane was about 20 times larger than Pd-Ag membrane.

  20. CERN: Fixed target targets

    International Nuclear Information System (INIS)

    Anon.

    1993-01-01

    Full text: While the immediate priority of CERN's research programme is to exploit to the full the world's largest accelerator, the LEP electron-positron collider and its concomitant LEP200 energy upgrade (January, page 1), CERN is also mindful of its long tradition of diversified research. Away from LEP and preparations for the LHC proton-proton collider to be built above LEP in the same 27-kilometre tunnel, CERN is also preparing for a new generation of heavy ion experiments using a new source, providing heavier ions (April 1992, page 8), with first physics expected next year. CERN's smallest accelerator, the LEAR Low Energy Antiproton Ring continues to cover a wide range of research topics, and saw a record number of hours of operation in 1992. The new ISOLDE on-line isotope separator was inaugurated last year (July, page 5) and physics is already underway. The remaining effort concentrates around fixed target experiments at the SPS synchrotron, which formed the main thrust of CERN's research during the late 1970s. With the SPS and LEAR now approaching middle age, their research future was extensively studied last year. Broadly, a vigorous SPS programme looks assured until at least the end of 1995. Decisions for the longer term future of the West Experimental Area of the SPS will have to take into account the heavy demand for test beams from work towards experiments at big colliders, both at CERN and elsewhere. The North Experimental Area is the scene of larger experiments with longer lead times. Several more years of LEAR exploitation are already in the pipeline, but for the longer term, the ambitious Superlear project for a superconducting ring (January 1992, page 7) did not catch on. Neutrino physics has a long tradition at CERN, and this continues with the preparations for two major projects, the Chorus and Nomad experiments (November 1991, page 7), to start next year in the West Area. Delicate neutrino oscillation effects could become

  1. Microporous silica membranes

    DEFF Research Database (Denmark)

    Boffa, Vittorio; Yue, Yuanzheng

    2012-01-01

    Hydrothermal stability is a crucial factor for the application of microporous silica-based membranes in industrial processes. Indeed, it is well established that steam exposure may cause densification and defect formation in microporous silica membranes, which are detrimental to both membrane...... permeability and selectivity. Numerous previous studies show that microporous transition metal doped-silica membranes are hydrothermally more stable than pure silica membranes, but less permeable. Here we present a quantitative study on the impact of type and concentration of transition metal ions...... on the microporous structure, stability and permeability of amorphous silica-based membranes, providing information on how to design chemical compositions and synthetic paths for the fabrication of silica-based membranes with a well accessible and highly stabile microporous structure....

  2. Clustering on Membranes

    DEFF Research Database (Denmark)

    Johannes, Ludger; Pezeshkian, Weria; Ipsen, John H

    2018-01-01

    Clustering of extracellular ligands and proteins on the plasma membrane is required to perform specific cellular functions, such as signaling and endocytosis. Attractive forces that originate in perturbations of the membrane's physical properties contribute to this clustering, in addition to direct...... protein-protein interactions. However, these membrane-mediated forces have not all been equally considered, despite their importance. In this review, we describe how line tension, lipid depletion, and membrane curvature contribute to membrane-mediated clustering. Additional attractive forces that arise...... from protein-induced perturbation of a membrane's fluctuations are also described. This review aims to provide a survey of the current understanding of membrane-mediated clustering and how this supports precise biological functions....

  3. Microporous Silica Based Membranes for Desalination

    Directory of Open Access Journals (Sweden)

    João C. Diniz da Costa

    2012-09-01

    Full Text Available This review provides a global overview of microporous silica based membranes for desalination via pervaporation with a focus on membrane synthesis and processing, transport mechanisms and current state of the art membrane performance. Most importantly, the recent development and novel concepts for improving the hydro-stability and separating performance of silica membranes for desalination are critically examined. Research into silica based membranes for desalination has focussed on three primary methods for improving the hydro-stability. These include incorporating carbon templates into the microporous silica both as surfactants and hybrid organic-inorganic structures and incorporation of metal oxide nanoparticles into the silica matrix. The literature examined identified that only metal oxide silica membranes have demonstrated high salt rejections under a variety of feed concentrations, reasonable fluxes and unaltered performance over long-term operation. As this is an embryonic field of research several target areas for researchers were discussed including further improvement of the membrane materials, but also regarding the necessity of integrating waste or solar heat sources into the final process design to ensure cost competitiveness with conventional reverse osmosis processes.

  4. Application of dynamic membranes in anaerobic membranes in anaerobic membrane bioreactor systems

    NARCIS (Netherlands)

    Erşahin, M.E.

    2015-01-01

    Anaerobic membrane bioreactors (AnMBRs) physically ensure biomass retention by the application of a membrane filtration process. With growing application experiences from aerobic membrane bioreactors (MBRs), the combination of membrane and anaerobic processes has received much attention and become

  5. Membrane processes in nuclear technologies

    International Nuclear Information System (INIS)

    Zakrzewska-Trznadel, G.

    2006-01-01

    The treatment of radioactive wastes is necessary taking into account the potential hazard of radioactive substances to human health and surrounding environment. The choice of appropriate technology depends on capital and operational costs, wastes amount and their characteristics, appointed targets of the process, e.g. the values of decontamination factors and volume reduction coefficients. The conventional technologies applied for radioactive waste processing, such as precipitation coupled with sedimentation, ion exchange and evaporation have many drawbacks. These include high energy consumption and formation of secondary wastes, e.g. the sludge from sediment tanks, spent ion exchange adsorbents and regeneration solutions. There are also many limitations of such processes, i.e. foaming and drop entrainment in evaporators, loses of solvents and production of secondary wastes in solvent extraction or bed clogging in ion exchange columns. Membrane processes as the newest achievement of the process engineering can successfully supersede many non-effective, out-of-date methods. But in some instances they can also complement these methods whilst improving the parameters of effluents and purification economy. This monograph presents own research data on the application of recent achievements in the area of membrane processes for solving selected problems in nuclear technology. Relatively big space was devoted to the use of membrane processing of low and intermediate radioactive liquid wastes because of numerous applications of these processes in nuclear centres over the world and also because of the interests of the author that was reflected by her recent research projects and activity. This work presents a review on the membrane methods recently introduced into the nuclear technology against the background of the other, commonly applied separation techniques, with indications of the possibilities and prospects for their further developments. Particular attention was paid

  6. Hollow fibre supported liquid membrane extraction of ...

    African Journals Online (AJOL)

    A simple sample pre-treatment method utilizing hollow fibre supported liquid membrane (HFSLM) was carried out on pharmaceuticals samples comprising of cough syrups (CS1 and CS2) and an anti-inflammatory product (AI). The active ingredients targeted in the extraction process were diphenylhydramine (DPH), ...

  7. Target laboratory

    International Nuclear Information System (INIS)

    Ephraim, D.C.; Pednekar, A.R.

    1993-01-01

    A target laboratory to make stripper foils for the accelerator and various targets for use in the experiments is set up in the pelletron accelerator facility. The facilities available in the laboratory are: (1) D.C. glow discharge setup, (2) carbon arc set up, and (3) vacuum evaporation set up (resistance heating), electron beam source, rolling mill - all for target preparation. They are described. Centrifugal deposition technique is used for target preparation. (author). 3 figs

  8. Probing lipid membrane electrostatics

    Science.gov (United States)

    Yang, Yi

    The electrostatic properties of lipid bilayer membranes play a significant role in many biological processes. Atomic force microscopy (AFM) is highly sensitive to membrane surface potential in electrolyte solutions. With fully characterized probe tips, AFM can perform quantitative electrostatic analysis of lipid membranes. Electrostatic interactions between Silicon nitride probes and supported zwitterionic dioleoylphosphatidylcholine (DOPC) bilayer with a variable fraction of anionic dioleoylphosphatidylserine (DOPS) were measured by AFM. Classical Gouy-Chapman theory was used to model the membrane electrostatics. The nonlinear Poisson-Boltzmann equation was numerically solved with finite element method to provide the potential distribution around the AFM tips. Theoretical tip-sample electrostatic interactions were calculated with the surface integral of both Maxwell and osmotic stress tensors on tip surface. The measured forces were interpreted with theoretical forces and the resulting surface charge densities of the membrane surfaces were in quantitative agreement with the Gouy-Chapman-Stern model of membrane charge regulation. It was demonstrated that the AFM can quantitatively detect membrane surface potential at a separation of several screening lengths, and that the AFM probe only perturbs the membrane surface potential by external field created by the internai membrane dipole moment. The analysis yields a dipole moment of 1.5 Debye per lipid with a dipole potential of +275 mV for supported DOPC membranes. This new ability to quantitatively measure the membrane dipole density in a noninvasive manner will be useful in identifying the biological effects of the dipole potential. Finally, heterogeneous model membranes were studied with fluid electric force microscopy (FEFM). Electrostatic mapping was demonstrated with 50 nm resolution. The capabilities of quantitative electrostatic measurement and lateral charge density mapping make AFM a unique and powerful

  9. Emulsification using microporous membranes

    Directory of Open Access Journals (Sweden)

    Goran T. Vladisavljević

    2011-10-01

    Full Text Available Membrane emulsification is a process of injecting a pure dispersed phase or pre-emulsion through a microporous membrane into the continuous phase. As a result of the immiscibility of the two phases, droplets of the dispersed phase are formed at the outlets of membrane pores. The droplets formed in the process are removed from the membrane surface by applying cross-flow or stirring of the continuous phase or using a dynamic (rotating or vibrating membrane. The most commonly used membrane for emulsification is the Shirasu Porous Glass (SPG membrane, fabricated through spinodal decomposition in a melt consisting of Japanese volcanic ash (Shirasu, boric acid and calcium carbonate. Microsieve membranes are increasingly popular as an alternative to highly tortuous glass and ceramic membranes. Microsieves are usually fabricated from nickel by photolithography and electroplating or they can be manufactured from silicon nitride via Reactive Ion Etching (RIE. An advantage of microsieves compared to the SPG membrane is in much higher transmembrane fluxes and higher tolerance to fouling by the emulsion ingredients due to the existence of short, straight through pores. Unlike conventional emulsification devices such as high-pressure valve homogenisers and rotor-stator devices, membrane emulsification devices permit a precise control over the mean pore size over a wide range and during the process insignificant amount of energy is dissipated as heat. The drop size is primarily determined by the pore size, but it depends also on other parameters, such as membrane wettability, emulsion formulation, shear stress on the membrane surface, transmembrane pressure, etc.

  10. Ice targets

    International Nuclear Information System (INIS)

    Pacheco, C.; Stark, C.; Tanaka, N.; Hodgkins, D.; Barnhart, J.; Kosty, J.

    1979-12-01

    This report presents a description of ice targets that were constructed for research work at the High Resolution Spectrometer (HRS) and at the Energetic Pion Channel and Spectrometer (EPICS). Reasons for using these ice targets and the instructions for their construction are given. Results of research using ice targets will be published at a later date

  11. N-3 fatty acids and membrane microdomains: from model membranes to lymphocyte function.

    Science.gov (United States)

    Shaikh, Saame Raza; Teague, Heather

    2012-12-01

    This article summarizes the author's research on fish oil derived n-3 fatty acids, plasma membrane organization and B cell function. We first cover basic model membrane studies that investigated how docosahexaenoic acid (DHA) targeted the organization of sphingolipid-cholesterol enriched lipid microdomains. A key finding here was that DHA had a relatively poor affinity for cholesterol. This work led to a model that predicted DHA acyl chains in cells would manipulate lipid-protein microdomain organization and thereby function. We then review how the predictions of the model were tested with B cells in vitro followed by experiments using mice fed fish oil. These studies reveal a highly complex picture on how n-3 fatty acids target lipid-protein organization and B cell function. Key findings are as follows: (1) n-3 fatty acids target not just the plasma membrane but also endomembrane organization; (2) DHA, but not eicosapentaenoic acid (EPA), disrupts microdomain spatial distribution (i.e. clustering), (3) DHA alters protein lateral organization and (4) changes in membrane organization are accompanied by functional effects on both innate and adaptive B cell function. Altogether, the research over the past 10 years has led to an evolution of the original model on how DHA reorganizes membrane microdomains. The work raises the intriguing possibility of testing the model at the human level to target health and disease. Copyright © 2012 Elsevier Ltd. All rights reserved.

  12. Horseradish peroxidase-loaded nanospheres attached to hollow gold nanoparticles as signal enhancers in an ultrasensitive immunoassay for alpha-fetoprotein

    International Nuclear Information System (INIS)

    Li, Ya; Yuan, Ruo; Chai, Yaqin; Zhuo, Ying; Su, Huilan; Zhang, Yuxia

    2014-01-01

    We report on a novel electrochemical signal amplification strategy for use in immunoassays. The highly responsive immunoelectrode was constructed in the following way: (1) The surface of a gold electrode was covered with a layer single-walled carbon nanotubes dispersed in chitosane functionalized with L-cysteine; (2) Gold nanoparticles containing protein A and anti-alpha-fetoprotein (anti-AFP) were then covalently attached to the surface via the thiol groups of the chitosane. The electrode is then exposed to the analyte (AFP) which then is bound by the antibody. In the next step, a gold-conjugated secondary antibody is added that was prepared in the following way: (1) Horseradish peroxidase was crosslinked and the resulting spheres were coated with hollow gold nanoparticles (hollow Au-NPs) to give nanospheres of ∼100 nm in diameter. (2) These were the coated with thionine and, in a last step, with secondary antibody. The use of these materials has several attractive features: The HRP-NPs functionalized with hollow Au-NPs possess a large surface area that can load the large amount of secondary antibody. Thionine (Thi) is highly redox active and improves the intensity of the signal. Carbon nanotubes were used because they possess an excellent electron transfer rate and large surface area. Following incubation of the modified electrode (a) with a sample containing AFP, (b) then with the secondary antibody, and (c) with washing buffer, the electrode is placed in a solution containing H 2 O 2 . The HRP in the smart secondary antibody causes the catalytic decomposition of H 2 O 2 and the results in an electrical current that is linearly related to the concentration of AFP in the 0.025 to 5.0 ng mL −1 concentration range. The detection limit for AFP is as low as 8.3 pg mL −1 . We believe that this novel kind of immunoassay represents a promising tool for use in sensitive clinical assays. (author)

  13. Design and development of a highly stable hydrogen peroxide biosensor on screen printed carbon electrode based on horseradish peroxidase bound with gold nanoparticles in the matrix of chitosan.

    Science.gov (United States)

    Tangkuaram, Tanin; Ponchio, Chatchai; Kangkasomboon, Thippayawadee; Katikawong, Panadda; Veerasai, Waret

    2007-04-15

    The design and development of a screen printed carbon electrode (SPCE) on a polyvinyl chloride substrate as a disposable sensor is described. Six configurations were designed on silk screen frames. The SPCEs were printed with four inks: silver ink as the conducting track, carbon ink as the working and counter electrodes, silver/silver chloride ink as the reference electrode and insulating ink as the insulator layer. Selection of the best configuration was done by comparing slopes from the calibration plots generated by the cyclic voltammograms at 10, 20 and 30 mM K(3)Fe(CN)(6) for each configuration. The electrodes with similar configurations gave similar slopes. The 5th configuration was the best electrode that gave the highest slope. Modifying the best SPCE configuration for use as a biosensor, horseradish peroxidase (HRP) was selected as a biomaterial bound with gold nanoparticles (AuNP) in the matrix of chitosan (HRP/AuNP/CHIT). Biosensors of HRP/SPCE, HRP/CHIT/SPCE and HRP/AuNP/CHIT/SPCE were used in the amperometric detection of H(2)O(2) in a solution of 0.1M citrate buffer, pH 6.5, by applying a potential of -0.4V at the working electrode. All the biosensors showed an immediate response to H(2)O(2). The effect of HRP/AuNP incorporated with CHIT (HRP/AuNP/CHIT/SPCE) yielded the highest performance. The amperometric response of HRP/AuNP/CHIT/SPCE retained over 95% of the initial current of the 1st day up to 30 days of storage at 4 degrees C. The biosensor showed a linear range of 0.01-11.3mM H(2)O(2), with a detection limit of 0.65 microM H(2)O(2) (S/N=3). The low detection limit, long storage life and wide linear range of this biosensor make it advantageous in many applications, including bioreactors and biosensors.

  14. G protein-membrane interactions II: Effect of G protein-linked lipids on membrane structure and G protein-membrane interactions.

    Science.gov (United States)

    Casas, Jesús; Ibarguren, Maitane; Álvarez, Rafael; Terés, Silvia; Lladó, Victoria; Piotto, Stefano P; Concilio, Simona; Busquets, Xavier; López, David J; Escribá, Pablo V

    2017-09-01

    G proteins often bear myristoyl, palmitoyl and isoprenyl moieties, which favor their association with the membrane and their accumulation in G Protein Coupled Receptor-rich microdomains. These lipids influence the biophysical properties of membranes and thereby modulate G protein binding to bilayers. In this context, we showed here that geranylgeraniol, but neither myristate nor palmitate, increased the inverted hexagonal (H II ) phase propensity of phosphatidylethanolamine-containing membranes. While myristate and palmitate preferentially associated with phosphatidylcholine membranes, geranylgeraniol favored nonlamellar-prone membranes. In addition, Gαi 1 monomers had a higher affinity for lamellar phases, while Gβγ and Gαβγ showed a marked preference for nonlamellar prone membranes. Moreover, geranylgeraniol enhanced the binding of G protein dimers and trimers to phosphatidylethanolamine-containing membranes, yet it decreased that of monomers. By contrast, both myristate and palmitate increased the Gαi 1 preference for lamellar membranes. Palmitoylation reinforced the binding of the monomer to PC membranes and myristoylation decreased its binding to PE-enriched bilayer. Finally, binding of dimers and trimers to lamellar-prone membranes was decreased by palmitate and myristate, but it was increased in nonlamellar-prone bilayers. These results demonstrate that co/post-translational G protein lipid modifications regulate the membrane lipid structure and that they influence the physico-chemical properties of membranes, which in part explains why G protein subunits sort to different plasma membrane domains. This article is part of a Special Issue entitled: Membrane Lipid Therapy: Drugs Targeting Biomembranes edited by Pablo V. Escribá. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Membrane inlet mass spectrometry reveals that Ceriporiopsis subvermispora bicupin oxalate oxidase is inhibited by nitric oxide.

    Science.gov (United States)

    Moomaw, Ellen W; Uberto, Richard; Tu, Chingkuang

    2014-07-18

    Membrane inlet mass spectrometry (MIMS) uses a semipermeable membrane as an inlet to a mass spectrometer for the measurement of the concentration of small uncharged molecules in solution. We report the use of MIMS to characterize the catalytic properties of oxalate oxidase (E.C. 1.2.3.4) from Ceriporiopsis subvermispora (CsOxOx). Oxalate oxidase is a manganese dependent enzyme that catalyzes the oxygen-dependent oxidation of oxalate to carbon dioxide in a reaction that is coupled with the formation of hydrogen peroxide. CsOxOx is the first bicupin enzyme identified that catalyzes this reaction. The MIMS method of measuring OxOx activity involves continuous, real-time direct detection of oxygen consumption and carbon dioxide production from the ion currents of their respective mass peaks. (13)C2-oxalate was used to allow for accurate detection of (13)CO2 (m/z 45) despite the presence of adventitious (12)CO2. Steady-state kinetic constants determined by MIMS are comparable to those obtained by a continuous spectrophotometric assay in which H2O2 production is coupled to the horseradish peroxidase catalyzed oxidation of 2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonic acid). Furthermore, we used MIMS to determine that NO inhibits the activity of the CsOxOx with a KI of 0.58±0.06 μM. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

  16. Ion-conducting membranes

    Science.gov (United States)

    Masel, Richard I.; Sajjad, Syed Dawar; Gao, Yan; Liu, Zengcai; Chen, Qingmei

    2017-12-26

    An anion-conducting polymeric membrane comprises a terpolymer of styrene, vinylbenzyl-R.sub.s and vinylbenzyl-R.sub.x. R.sub.s is a positively charged cyclic amine group. R.sub.x is at least one constituent selected from the group consisting Cl, OH and a reaction product between an OH or Cl and a species other than a simple amine or a cyclic amine. The total weight of the vinylbenzyl-R.sub.x groups is greater than 0.3% of the total weight of the membrane. In a preferred embodiment, the membrane is a Helper Membrane that increases the faradaic efficiency of an electrochemical cell into which the membrane is incorporated, and also allows product formation at lower voltages than in cells without the Helper Membrane.

  17. Gas separation with membranes

    International Nuclear Information System (INIS)

    Schulz, G.; Michele, H.; Werner, U.

    1982-01-01

    Gas separation with membranes has already been tested in numerous fields of application, e.g. uranium enrichment of H 2 separation. In many of these processes the mass transfer units, so-called permeators, have to be connected in tandem in order to achieve high concentrations. A most economical operating method provides for each case an optimization of the cascades with regard to the membrane materials, construction and design of module. By utilization of the concentration gradient along the membrane a new process development has been accomplished - the continuously operating membrane rectification unit. Investment and operating costs can be reduced considerably for a number of separating processes by combining a membrane rectification unit with a conventional recycling cascade. However, the new procedure requires that the specifications for the module construction, flow design, and membrane properties be reconsidered. (orig.) [de

  18. Arsenic Removal by Liquid Membranes

    Directory of Open Access Journals (Sweden)

    Tiziana Marino

    2015-03-01

    Full Text Available Water contamination with harmful arsenic compounds represents one of the most serious calamities of the last two centuries. Natural occurrence of the toxic metal has been revealed recently for 21 countries worldwide; the risk of arsenic intoxication is particularly high in Bangladesh and India but recently also Europe is facing similar problem. Liquid membranes (LMs look like a promising alternative to the existing removal processes, showing numerous advantages in terms of energy consumption, efficiency, selectivity, and operational costs. The development of different LM configurations has been a matter of investigation by several researching groups, especially for the removal of As(III and As(V from aqueous solutions. Most of these LM systems are based on the use of phosphine oxides as carriers, when the metal removal is from sulfuric acid media. Particularly promising for water treatment is the hollow fiber supported liquid membrane (HFSLM configuration, which offers high selectivity, easy transport of the targeted metal ions, large surface area, and non-stop flow process. The choice of organic extractant(s plays an essential role in the efficiency of the arsenic removal. Emulsion liquid membrane (ELM systems have not been extensively investigated so far, although encouraging results have started to appear in the literature. For such LM configuration, the most relevant step toward efficiency is the choice of the surfactant type and its concentration.

  19. Chelating polymeric membranes

    KAUST Repository

    Peinemann, Klaus-Viktor

    2015-01-22

    The present application offers a solution to the current problems associated with recovery and recycling of precious metals from scrap material, discard articles, and other items comprising one or more precious metals. The solution is premised on a microporous chelating polymeric membrane. Embodiments include, but are not limited to, microporous chelating polymeric membranes, device comprising the membranes, and methods of using and making the same.

  20. Gas separation membranes

    Science.gov (United States)

    Schell, William J.

    1979-01-01

    A dry, fabric supported, polymeric gas separation membrane, such as cellulose acetate, is prepared by casting a solution of the polymer onto a shrinkable fabric preferably formed of synthetic polymers such as polyester or polyamide filaments before washing, stretching or calendering (so called griege goods). The supported membrane is then subjected to gelling, annealing, and drying by solvent exchange. During the processing steps, both the fabric support and the membrane shrink a preselected, controlled amount which prevents curling, wrinkling or cracking of the membrane in flat form or when spirally wound into a gas separation element.

  1. Anion exchange membrane

    Science.gov (United States)

    Verkade, John G; Wadhwa, Kuldeep; Kong, Xueqian; Schmidt-Rohr, Klaus

    2013-05-07

    An anion exchange membrane and fuel cell incorporating the anion exchange membrane are detailed in which proazaphosphatrane and azaphosphatrane cations are covalently bonded to a sulfonated fluoropolymer support along with anionic counterions. A positive charge is dispersed in the aforementioned cations which are buried in the support to reduce the cation-anion interactions and increase the mobility of hydroxide ions, for example, across the membrane. The anion exchange membrane has the ability to operate at high temperatures and in highly alkaline environments with high conductivity and low resistance.

  2. Photoresponsive nanostructured membranes

    KAUST Repository

    Madhavan, Poornima

    2016-07-26

    The perspective of adding stimuli-response to isoporous membranes stimulates the development of separation devices with pores, which would open or close under control of environment chemical composition, temperature or exposure to light. Changes in pH and temperature have been previously investigated. In this work, we demonstrate for the first time the preparation of photoresponsive isoporous membranes, applying self-assembly non-solvent induced phase separation to a new light responsive block copolymer. First, we optimized the membrane formation by using poly(styrene-b-anthracene methyl methacrylate-b-methylmethacrylate) (PS-b-PAnMMA-b-PMMA) copolymer, identifying the most suitable solvent, copolymer block length, and other parameters. The obtained final triblock copolymer membrane morphologies were characterized using atomic force and electron microscopy. The microscopic analysis reveals that the PS-b-PAnMMA-b-PMMA copolymer can form both lamellar and ordered hexagonal nanoporous structures on the membrane top layer in appropriate solvent compositions. The nanostructured membrane emits fluorescence due to the presence of the anthracene mid-block. On irradiation of light the PS-b-PAnMMA-b-PMMA copolymer membranes has an additional stimuli response. The anthracene group undergoes conformational changes by forming [4 + 4] cycloadducts and this alters the membrane\\'s water flux and solute retention. © 2016 The Royal Society of Chemistry.

  3. Biglycan and decorin differentially regulate signaling in the fetal membranes

    Science.gov (United States)

    Wu, Zhiping; Horgan, Casie E.; Carr, Olivia; Owens, Rick T.; Iozzo, Renato V.; Lechner, Beatrice E.

    2014-01-01

    Preterm birth is the leading cause of newborn mortality in the United States and about one third of cases are caused by preterm premature rupture of fetal membranes, a complication that is frequently observed in patients with Ehlers-Danlos Syndrome. Notably, a subtype of Ehlers-Danlos Syndrome is caused by expression of abnormal biglycan and decorin proteoglycans. As compound deficiency of these two small leucine-rich proteoglycans is a model of preterm birth, we investigated the fetal membranes of Bgn−/−;Dcn−/− double-null and single-null mice. Our results showed that biglycan signaling supported fetal membrane remodeling during early gestation in the absence of concomitant changes in TGFβ levels. In late gestation, biglycan signaling acted in a TGFβ–dependent manner to aid in membrane stabilization. In contrast, decorin signaling supported fetal membrane remodeling at early stages of gestation in a TGFβ–dependent manner, and fetal membrane stabilization at later stages of gestation without changes in TGFβ levels. Furthermore, exogenous soluble decorin was capable of rescuing the TGFβ signaling pathway in fetal membrane mesenchymal cells. Collectively, these findings provide novel targets for manipulation of fetal membrane extracellular matrix stability and could represent novel targets for research on preventive strategies for preterm premature rupture of fetal membranes. PMID:24373743

  4. Plasma membrane ATPases

    DEFF Research Database (Denmark)

    Palmgren, Michael Broberg; Bækgaard, Lone; Lopez Marques, Rosa Laura

    2011-01-01

    The plasma membrane separates the cellular contents from the surrounding environment. Nutrients must enter through the plasma membrane in order to reach the cell interior, and toxic metabolites and several ions leave the cell by traveling across the same barrier. Biological pumps in the plasma me...

  5. Polymide gas separation membranes

    Science.gov (United States)

    Ding, Yong; Bikson, Benjamin; Nelson, Joyce Katz

    2004-09-14

    Soluble polyamic acid salt (PAAS) precursors comprised of tertiary and quaternary amines, ammonium cations, sulfonium cations, or phosphonium cations, are prepared and fabricated into membranes that are subsequently imidized and converted into rigid-rod polyimide articles, such as membranes with desirable gas separation properties. A method of enhancing solubility of PAAS polymers in alcohols is also disclosed.

  6. Enantioseparation with liquid membranes

    NARCIS (Netherlands)

    Gössi, Angelo; Riedl, Wolfgang; Schuur, Boelo

    Chiral resolution of racemic products is a challenging and important task in the pharmaceutical, agrochemical, flavor, polymer and fragrances industries. One of the options for these challenging separations is to use liquid membranes. Although liquid membranes have been known for almost four decades

  7. Silicon nitride nanosieve membrane

    NARCIS (Netherlands)

    Tong, D.H.; Jansen, Henricus V.; Gadgil, V.J.; Bostan, C.G.; Berenschot, Johan W.; van Rijn, C.J.M.; Elwenspoek, Michael Curt

    2004-01-01

    An array of very uniform cylindrical nanopores with a pore diameter as small as 25 nm has been fabricated in an ultrathin micromachined silicon nitride membrane using focused ion beam (FIB) etching. The pore size of this nanosieve membrane was further reduced to below 10 nm by coating it with

  8. Membrane capacitive deionization

    NARCIS (Netherlands)

    Biesheuvel, P.M.; Wal, van der A.

    2010-01-01

    Membrane capacitive deionization (MCDI) is an ion-removal process based on applying an electrical potential difference across an aqueous solution which flows in between oppositely placed porous electrodes, in front of which ion-exchange membranes are positioned. Due to the applied potential, ions

  9. Photoresponsive nanostructured membranes

    KAUST Repository

    Madhavan, Poornima; Sutisna, Burhannudin; Sougrat, Rachid; Nunes, Suzana Pereira

    2016-01-01

    The perspective of adding stimuli-response to isoporous membranes stimulates the development of separation devices with pores, which would open or close under control of environment chemical composition, temperature or exposure to light. Changes in pH and temperature have been previously investigated. In this work, we demonstrate for the first time the preparation of photoresponsive isoporous membranes, applying self-assembly non-solvent induced phase separation to a new light responsive block copolymer. First, we optimized the membrane formation by using poly(styrene-b-anthracene methyl methacrylate-b-methylmethacrylate) (PS-b-PAnMMA-b-PMMA) copolymer, identifying the most suitable solvent, copolymer block length, and other parameters. The obtained final triblock copolymer membrane morphologies were characterized using atomic force and electron microscopy. The microscopic analysis reveals that the PS-b-PAnMMA-b-PMMA copolymer can form both lamellar and ordered hexagonal nanoporous structures on the membrane top layer in appropriate solvent compositions. The nanostructured membrane emits fluorescence due to the presence of the anthracene mid-block. On irradiation of light the PS-b-PAnMMA-b-PMMA copolymer membranes has an additional stimuli response. The anthracene group undergoes conformational changes by forming [4 + 4] cycloadducts and this alters the membrane's water flux and solute retention. © 2016 The Royal Society of Chemistry.

  10. Role of plasma membrane surface charges in dictating the feasibility of membrane-nanoparticle interactions

    Science.gov (United States)

    Sinha, Shayandev; Jing, Haoyuan; Sachar, Harnoor Singh; Das, Siddhartha

    2017-12-01

    Receptor-ligand (R-L) binding mediated interactions between the plasma membrane (PM) and a nanoparticle (NP) require the ligand-functionalized NPs to come to a distance of separation (DOS) of at least dRL (length of the R-L complex) from the receptor-bearing membranes. In this letter, we establish that the membrane surface charges and the surrounding ionic environment dictate whether or not the attainment of such a critical DOS is possible. The negatively charged membrane invariably induces a negative electrostatic potential at the NP surface, repelling the NP from the membrane. This is countered by the attractive influences of the thermal fluctuations and van der Waals (vdw) interactions that drive the NP close to the membrane. For a NP approaching the membrane from a distance, the ratio of the repulsive (electrostatic) and attractive (thermal and vdW) effects balances at a critical NP-membrane DOS of dg,c. For a given set of parameters, there can be two possible values of dg,c, namely, dg,c,1 and dg,c,2 with dg,c,1 ≫ dg,c,2. We establish that any R-L mediated NP-membrane interaction is possible only if dRL > dg,c,1. Therefore, our study proposes a design criterion for engineering ligands for a NP that will ensure the appropriate length of the R-L complex in order to ensure the successful membrane-NP interaction in the presence of a given electrostatic environment. Finally, we discuss the manner in which our theory can help designing ligand-grafted NPs for targeted drug delivery, design biomimetics NPs, and also explain various experimental results.

  11. Molecular Interactions at Membranes

    DEFF Research Database (Denmark)

    Jagalski, Vivien

    . Today, we know more than ever before about the properties of biological membranes. Advanced biophysical techniques and sophisticated membrane models allow us to answer specific questions about the structure of the components within membranes and their interactions. However, many detailed structural...... the surface-immobilization of LeuT by exchanging the detergent with natural phosphatidylcholine (PC) lipids. Various surface sensitive techniques, including neutron reflectometry (NR), are employed and finally enabled us to confirm the gross structure of LeuT in a lipid environment as predicted by molecular...... dynamic simulations. In a second study, the co-localization of three toxic plant-derived diterpene resin acids (RAs) within DPPC membranes was investigated. These compounds are reported to disrupt the membrane and increase its fluidity. The RAs used in this study vary in their toxicity while...

  12. Membrane technology and applications

    International Nuclear Information System (INIS)

    Khalil, F.H.

    1997-01-01

    The main purpose of this dissertation is to prepare and characterize some synthetic membranes obtained by radiation-induced graft copolymerization of and A Am unitary and binary system onto nylon-6 films. The optimum conditions at which the grafting process proceeded homogeneously were determined. Some selected properties of the prepared membranes were studied. Differential scanning calorimetry (DSC), thermal gravimetric analysis (TGA), x-ray diffraction (XRD), mechanical properties and U.V./vis, instruments and techniques were used to characterize the prepared membranes. The use of such membranes for the decontamination of radioactive waste and some heavy metal ions as water pollutants were investigated. These grafted membranes showed good cation exchange properties and may be of practical interest in waste water treatment whether this water was radioactive or not. 4 tabs., 68 figs., 146 refs

  13. Membrane Bioreactor (MBR Technology for Wastewater Treatment and Reclamation: Membrane Fouling

    Directory of Open Access Journals (Sweden)

    Oliver Terna Iorhemen

    2016-06-01

    Full Text Available The membrane bioreactor (MBR has emerged as an efficient compact technology for municipal and industrial wastewater treatment. The major drawback impeding wider application of MBRs is membrane fouling, which significantly reduces membrane performance and lifespan, resulting in a significant increase in maintenance and operating costs. Finding sustainable membrane fouling mitigation strategies in MBRs has been one of the main concerns over the last two decades. This paper provides an overview of membrane fouling and studies conducted to identify mitigating strategies for fouling in MBRs. Classes of foulants, including biofoulants, organic foulants and inorganic foulants, as well as factors influencing membrane fouling are outlined. Recent research attempts on fouling control, including addition of coagulants and adsorbents, combination of aerobic granulation with MBRs, introduction of granular materials with air scouring in the MBR tank, and quorum quenching are presented. The addition of coagulants and adsorbents shows a significant membrane fouling reduction, but further research is needed to establish optimum dosages of the various coagulants/adsorbents. Similarly, the integration of aerobic granulation with MBRs, which targets biofoulants and organic foulants, shows outstanding filtration performance and a significant reduction in fouling rate, as well as excellent nutrients removal. However, further research is needed on the enhancement of long-term granule integrity. Quorum quenching also offers a strong potential for fouling control, but pilot-scale testing is required to explore the feasibility of full-scale application.

  14. Membrane Bioreactor (MBR) Technology for Wastewater Treatment and Reclamation: Membrane Fouling.

    Science.gov (United States)

    Iorhemen, Oliver Terna; Hamza, Rania Ahmed; Tay, Joo Hwa

    2016-06-15

    The membrane bioreactor (MBR) has emerged as an efficient compact technology for municipal and industrial wastewater treatment. The major drawback impeding wider application of MBRs is membrane fouling, which significantly reduces membrane performance and lifespan, resulting in a significant increase in maintenance and operating costs. Finding sustainable membrane fouling mitigation strategies in MBRs has been one of the main concerns over the last two decades. This paper provides an overview of membrane fouling and studies conducted to identify mitigating strategies for fouling in MBRs. Classes of foulants, including biofoulants, organic foulants and inorganic foulants, as well as factors influencing membrane fouling are outlined. Recent research attempts on fouling control, including addition of coagulants and adsorbents, combination of aerobic granulation with MBRs, introduction of granular materials with air scouring in the MBR tank, and quorum quenching are presented. The addition of coagulants and adsorbents shows a significant membrane fouling reduction, but further research is needed to establish optimum dosages of the various coagulants/adsorbents. Similarly, the integration of aerobic granulation with MBRs, which targets biofoulants and organic foulants, shows outstanding filtration performance and a significant reduction in fouling rate, as well as excellent nutrients removal. However, further research is needed on the enhancement of long-term granule integrity. Quorum quenching also offers a strong potential for fouling control, but pilot-scale testing is required to explore the feasibility of full-scale application.

  15. GSL-enriched membrane microdomains in innate immune responses.

    Science.gov (United States)

    Nakayama, Hitoshi; Ogawa, Hideoki; Takamori, Kenji; Iwabuchi, Kazuhisa

    2013-06-01

    Many pathogens target glycosphingolipids (GSLs), which, together with cholesterol, GPI-anchored proteins, and various signaling molecules, cluster on host cell membranes to form GSL-enriched membrane microdomains (lipid rafts). These GSL-enriched membrane microdomains may therefore be involved in host-pathogen interactions. Innate immune responses are triggered by the association of pathogens with phagocytes, such as neutrophils, macrophages and dendritic cells. Phagocytes express a diverse array of pattern-recognition receptors (PRRs), which sense invading microorganisms and trigger pathogen-specific signaling. PRRs can recognize highly conserved pathogen-associated molecular patterns expressed on microorganisms. The GSL lactosylceramide (LacCer, CDw17), which binds to various microorganisms, including Candida albicans, is expressed predominantly on the plasma membranes of human mature neutrophils and forms membrane microdomains together with the Src family tyrosine kinase Lyn. These LacCer-enriched membrane microdomains can mediate superoxide generation, migration, and phagocytosis, indicating that LacCer functions as a PRR in innate immunity. Moreover, the interactions of GSL-enriched membrane microdomains with membrane proteins, such as growth factor receptors, are important in mediating the physiological properties of these proteins. Similarly, we recently found that interactions between LacCer-enriched membrane microdomains and CD11b/CD18 (Mac-1, CR3, or αMβ2-integrin) are significant for neutrophil phagocytosis of non-opsonized microorganisms. This review describes the functional role of LacCer-enriched membrane microdomains and their interactions with CD11b/CD18.

  16. Antiproton Target

    CERN Multimedia

    1980-01-01

    Antiproton target used for the AA (antiproton accumulator). The first type of antiproton production target used from 1980 to 1982 comprised a rod of copper 3mm diameter and 120mm long embedded in a graphite cylinder that was itself pressed into a finned aluminium container. This assembly was air-cooled and it was used in conjunction with the Van der Meer magnetic horn. In 1983 Fermilab provided us with lithium lenses to replace the horn with a view to increasing the antiproton yield by about 30%. These lenses needed a much shorter target made of heavy metal - iridium was chosen for this purpose. The 50 mm iridium rod was housed in an extension to the original finned target container so that it could be brought very close to the entrance to the lithium lens. Picture 1 shows this target assembly and Picture 2 shows it mounted together with the lithium lens. These target containers had a short lifetime due to a combination of beam heating and radiation damage. This led to the design of the water-cooled target in...

  17. Microfabricated hydrogen sensitive membranes

    Energy Technology Data Exchange (ETDEWEB)

    Naddaf, A.; Kraetz, L. [Lehrstuhl fuer Thermische Verfahrenstechnik, Technische Universitaet Kaiserslautern (Germany); Detemple, P.; Schmitt, S.; Hessel, V. [Institut fuer Mikrotechnik Mainz GmbH, Mainz (Germany); Faqir, N. [University of Jordan, Amman (Jordan); Bart, H.J.

    2009-01-15

    Thin, defect-free palladium, palladium/copper and palladium/silver hydrogen absorbing membranes were microfabricated. A dual sputtering technique was used to deposit the palladium alloy membranes of only 1 {mu}m thickness on a nonporous silicon substrate. Advanced silicon etching (ASE) was applied on the backside to create a mechanically stable support structure for the thin films. Performance evaluation was carried out for different gases in a temperature range of 20 C to 298 C at a constant differential pressure of 110 kPa at the two sides of the membrane. The composite membranes show an excellent permeation rate of hydrogen, which appears to be 0.05 Pa m{sup 3} s{sup -1} and 0.01.10{sup -3} Pa m{sup 3} s{sup -1} at 20 C for the microfabricated 23 % silver and the 53 % copper composite membranes, respectively. The selectivity to hydrogen over a gas mixture containing, in addition to hydrogen, carbon monoxide, carbon dioxide and nitrogen was measured. The mass spectrometer did not detect any CO{sub 2} or CO, showing that the membrane is completely hydrogen selective. The microfabricated membranes exhibit both high mechanical strength (they easily withstand pressures up to 4 bar) and high thermal stability (up to 650 C). (Abstract Copyright [2009], Wiley Periodicals, Inc.)

  18. Catalytic nanoporous membranes

    Science.gov (United States)

    Pellin, Michael J; Hryn, John N; Elam, Jeffrey W

    2013-08-27

    A nanoporous catalytic membrane which displays several unique features Including pores which can go through the entire thickness of the membrane. The membrane has a higher catalytic and product selectivity than conventional catalysts. Anodic aluminum oxide (AAO) membranes serve as the catalyst substrate. This substrate is then subjected to Atomic Layer Deposition (ALD), which allows the controlled narrowing of the pores from 40 nm to 10 nm in the substrate by deposition of a preparatory material. Subsequent deposition of a catalytic layer on the inner surfaces of the pores reduces pore sizes to less than 10 nm and allows for a higher degree of reaction selectivity. The small pore sizes allow control over which molecules enter the pores, and the flow-through feature can allow for partial oxidation of reactant species as opposed to complete oxidation. A nanoporous separation membrane, produced by ALD is also provided for use in gaseous and liquid separations. The membrane has a high flow rate of material with 100% selectivity. Also provided is a method for producing a catalytic membrane having flow-through pores and discreet catalytic clusters adhering to the inside surfaces of the pores.

  19. Mechanism of Shiga Toxin Clustering on Membranes

    DEFF Research Database (Denmark)

    Pezeshkian, Weria; Gao, Haifei; Arumugam, Senthil

    2017-01-01

    between them. The precise mechanism by which this clustering occurs remains poorly defined. Here, we used vesicle and cell systems and computer simulations to show that line tension due to curvature, height, or compositional mismatch, and lipid or solvent depletion cannot drive the clustering of Shiga...... toxin molecules. By contrast, in coarse-grained computer simulations, a correlation was found between clustering and toxin nanoparticle-driven suppression of membrane fluctuations, and experimentally we observed that clustering required the toxin molecules to be tightly bound to the membrane surface...... molecules (several nanometers), and persist even beyond. This force is predicted to operate between manufactured nanoparticles providing they are sufficiently rigid and tightly bound to the plasma membrane, thereby suggesting a route for the targeting of nanoparticles to cells for biomedical applications....

  20. Rotating bubble membrane radiator

    Science.gov (United States)

    Webb, Brent J.; Coomes, Edmund P.

    1988-12-06

    A heat radiator useful for expelling waste heat from a power generating system aboard a space vehicle is disclosed. Liquid to be cooled is passed to the interior of a rotating bubble membrane radiator, where it is sprayed into the interior of the bubble. Liquid impacting upon the interior surface of the bubble is cooled and the heat radiated from the outer surface of the membrane. Cooled liquid is collected by the action of centrifical force about the equator of the rotating membrane and returned to the power system. Details regarding a complete space power system employing the radiator are given.

  1. Perforin rapidly induces plasma membrane phospholipid flip-flop.

    Directory of Open Access Journals (Sweden)

    Sunil S Metkar

    Full Text Available The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells.

  2. Free and membrane-bound calcium in microgravity and microgravity effects at the membrane level

    Science.gov (United States)

    Belyavskaya, N. A.

    The changes of [Ca^2+]_i controlled is known to play a key regulatory role in numerous cellular processes especially associated with membranes. Previous studies from our laboratory have demonstrated an increase in calcium level in root cells of pea seedlings grown aboard orbital station ``Salyut 6'' /1/. These results: 1) indicate that observed Ca^2+-binding sites of membranes also consist in proteins and phospholipids; 2) suggest that such effects of space flight in membrane Ca-binding might be due to the enhancement of Ca^2+ influx through membranes. In model presented, I propose that Ca^2+-activated channels in plasma membrane in response to microgravity allow the movement of Ca^2+ into the root cells, causing a rise in cytoplasmic free Ca^2+ levels. The latter, in its turn, may induce the inhibition of a Ca^2+ efflux by Ca^2+-activated ATPases and through a Ca^2+/H^+ antiport. It is possible that increased cytosolic levels of Ca^2+ ions have stimulated hydrolysis and turnover of phosphatidylinositols, with a consequent elevation of cytosolic [Ca^2+]_i. Plant cell can response to such a Ca^2+ rise by an enhancement of membranous Ca^2+-binding activities to rescue thus a cell from an abundance of a cytotoxin. A Ca^2+-induced phase separation of membranous lipids assists to appear the structure nonstable zones with high energy level at the boundary of microdomains which are rich by some phospholipid components; there is mixing of molecules of the membranes contacted in these zones, the first stage of membranous fusion, which was found in plants exposed to microgravity. These results support the hypothesis that a target for microgravity effect is the flux mechanism of Ca^2+ to plant cell.

  3. A cellular backline: specialization of host membranes for defence.

    Science.gov (United States)

    Faulkner, Christine

    2015-03-01

    In plant-pathogen interactions, the host plasma membrane serves as a defence front for pathogens that invade from the extracellular environment. As such, the lipid bilayer acts as a scaffold that targets and delivers defence responses to the site of attack. During pathogen infection, numerous changes in plasma membrane composition, organization, and structure occur. There is increasing evidence that this facilitates the execution of a variety of responses, highlighting the regulatory role membranes play in cellular responses. Membrane microdomains such as lipid rafts are hypothesized to create signalling platforms for receptor signalling in response to pathogen perception and for callose synthesis. Further, the genesis of pathogen-associated structures such as papillae and the extra-haustorial membrane necessitates polarization of membranes and membrane trafficking pathways. Unlocking the mechanisms by which this occurs will enable greater understanding of how targeted defences, some of which result in resistance, are executed. This review will survey some of the changes that occur in host membranes during pathogen attack and how these are associated with the generation of defence responses. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  4. Analysis of protein interactions at native chloroplast membranes by ellipsometry.

    Directory of Open Access Journals (Sweden)

    Verena Kriechbaumer

    Full Text Available Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE. We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins.

  5. Membrane Assisted Enzyme Fractionation

    DEFF Research Database (Denmark)

    Yuan, Linfeng

    to the variation in size of the proteins and a reasonable separation factor can be observed only when the size difference is in the order of 10 or more. This is partly caused by concentration polarization and membrane fouling which hinders an effective separation of the proteins. Application of an electric field...... across the porous membrane has been demonstrated to be an effective way to reduce concentration polarization and membrane fouling. In addition, this technique can also be used to separate the proteins based on difference in charge, which to some extent overcome the limitations of size difference...... of proteins on the basis of their charge, degree of hydrophobicity, affinity or size. Adequate purity is often not achieved unless several purification steps are combined thereby increasing cost and reducing product yield. Conventional fractionation of proteins using ultrafiltration membranes is limited...

  6. Fuel cell membrane humidification

    Science.gov (United States)

    Wilson, Mahlon S.

    1999-01-01

    A polymer electrolyte membrane fuel cell assembly has an anode side and a cathode side separated by the membrane and generating electrical current by electrochemical reactions between a fuel gas and an oxidant. The anode side comprises a hydrophobic gas diffusion backing contacting one side of the membrane and having hydrophilic areas therein for providing liquid water directly to the one side of the membrane through the hydrophilic areas of the gas diffusion backing. In a preferred embodiment, the hydrophilic areas of the gas diffusion backing are formed by sewing a hydrophilic thread through the backing. Liquid water is distributed over the gas diffusion backing in distribution channels that are separate from the fuel distribution channels.

  7. Wrinkles in reinforced membranes

    Science.gov (United States)

    Takei, Atsushi; Brau, Fabian; Roman, Benoît; Bico, José.

    2012-02-01

    We study, through model experiments, the buckling under tension of an elastic membrane reinforced with a more rigid strip or a fiber. In these systems, the compression of the rigid layer is induced through Poisson contraction as the membrane is stretched perpendicularly to the strip. Although strips always lead to out-of-plane wrinkles, we observe a transition from out-of-plane to in plane wrinkles beyond a critical strain in the case of fibers embedded into the elastic membranes. The same transition is also found when the membrane is reinforced with a wall of the same material depending on the aspect ratio of the wall. We describe through scaling laws the evolution of the morphology of the wrinkles and the different transitions as a function of material properties and stretching strain.

  8. Novel Catalytic Membrane Reactors

    Energy Technology Data Exchange (ETDEWEB)

    None

    2009-02-01

    This factsheet describes a research project that will focus on the development and application of nonporous high gas flux perfluoro membranes with high temperature rating and excellent chemical resistance.

  9. Temperature responsive track membranes

    International Nuclear Information System (INIS)

    Omichi, H.; Yoshido, M.; Asano, M.; Tamada, H.

    1994-01-01

    A new track membrane was synthesized by introducing polymeric hydrogel to films. Such a monomer as amino acid group containing acryloyl or methacryloyl was either co-polymerized with diethylene glycol-bis-ally carbonate followed by on beam irradiation and chemical etching, or graft co-polymerized onto a particle track membrane of CR-39. The pore size was controlled in water by changing the water temperature. Some films other than CR-39 were also examined. (author). 11 refs, 7 figs

  10. A membrane-bound vertebrate globin.

    Directory of Open Access Journals (Sweden)

    Miriam Blank

    Full Text Available The family of vertebrate globins includes hemoglobin, myoglobin, and other O(2-binding proteins of yet unclear functions. Among these, globin X is restricted to fish and amphibians. Zebrafish (Danio rerio globin X is expressed at low levels in neurons of the central nervous system and appears to be associated with the sensory system. The protein harbors a unique N-terminal extension with putative N-myristoylation and S-palmitoylation sites, suggesting membrane-association. Intracellular localization and transport of globin X was studied in 3T3 cells employing green fluorescence protein fusion constructs. Both myristoylation and palmitoylation sites are required for correct targeting and membrane localization of globin X. To the best of our knowledge, this is the first time that a vertebrate globin has been identified as component of the cell membrane. Globin X has a hexacoordinate binding scheme and displays cooperative O(2 binding with a variable affinity (P(50∼1.3-12.5 torr, depending on buffer conditions. A respiratory function of globin X is unlikely, but analogous to some prokaryotic membrane-globins it may either protect the lipids in cell membrane from oxidation or may act as a redox-sensing or signaling protein.

  11. Inverse colloidal crystal membranes for hydrophobic interaction membrane chromatography.

    Science.gov (United States)

    Vu, Anh T; Wang, Xinying; Wickramasinghe, S Ranil; Yu, Bing; Yuan, Hua; Cong, Hailin; Luo, Yongli; Tang, Jianguo

    2015-08-01

    Hydrophobic interaction membrane chromatography has gained interest due to its excellent performance in the purification of humanized monoclonal antibodies. The membrane material used in hydrophobic interaction membrane chromatography has typically been commercially available polyvinylidene fluoride. In this contribution, newly developed inverse colloidal crystal membranes that have uniform pores, high porosity and, therefore, high surface area for protein binding are used as hydrophobic interaction membrane chromatography membranes for humanized monoclonal antibody immunoglobulin G purification. The capacity of the inverse colloidal crystal membranes developed here is up to ten times greater than commercially available polyvinylidene fluoride membranes with a similar pore size. This work highlights the importance of developing uniform pore size high porosity membranes in order to maximize the capacity of hydrophobic interaction membrane chromatography. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. Fabrication of electrospun nanofibrous membranes for membrane distillation application

    KAUST Repository

    Francis, Lijo

    2013-02-01

    Nanofibrous membranes of Matrimid have been successfully fabricated using an electrospinning technique under optimized conditions. Nanofibrous membranes are found to be highly hydrophobic with a high water contact angle of 130°. Field emission scanning electron microscopy and pore size distribution analysis revealed the big pore size structure of electrospun membranes to be greater than 2 μm and the pore size distribution is found to be narrow. Flat sheet Matrimid membranes were fabricated via casting followed by phase inversion. The morphology, pore size distribution, and water contact angle were measured and compared with the electrospun membranes. Both membranes fabricated by electrospinning and phase inversion techniques were tested in a direct contact membrane distillation process. Electrospun membranes showed high water vapor flux of 56 kg/m2-h, which is very high compared to the casted membrane as well as most of the fabricated and commercially available highly hydrophobic membranes. ©2013 Desalination Publications.

  13. OXYGEN TRANSPORT CERAMIC MEMBRANES

    Energy Technology Data Exchange (ETDEWEB)

    Dr. Sukumar Bandopadhyay; Dr. Nagendra Nagabhushana

    2000-10-01

    This is the third quarterly report on oxygen Transport Ceramic Membranes. In the following, the report describes the progress made by our university partners in Tasks 1 through 6, experimental apparatus that was designed and built for various tasks of this project, thermodynamic calculations, where applicable and work planned for the future. (Task 1) Design, fabricate and evaluate ceramic to metal seals based on graded ceramic powder/metal braze joints. (Task 2) Evaluate the effect of defect configuration on ceramic membrane conductivity and long term chemical and structural stability. (Task 3) Determine materials mechanical properties under conditions of high temperatures and reactive atmospheres. (Task 4) Evaluate phase stability and thermal expansion of candidate perovskite membranes and develop techniques to support these materials on porous metal structures. (Task 5) Assess the microstructure of membrane materials to evaluate the effects of vacancy-impurity association, defect clusters, and vacancy-dopant association on the membrane performance and stability. (Task 6) Measure kinetics of oxygen uptake and transport in ceramic membrane materials under commercially relevant conditions using isotope labeling techniques.

  14. Bacterial membrane proteomics.

    Science.gov (United States)

    Poetsch, Ansgar; Wolters, Dirk

    2008-10-01

    About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.

  15. Biomimetic membranes and methods of making biomimetic membranes

    Science.gov (United States)

    Rempe, Susan; Brinker, Jeffrey C.; Rogers, David Michael; Jiang, Ying-Bing; Yang, Shaorong

    2016-11-08

    The present disclosure is directed to biomimetic membranes and methods of manufacturing such membranes that include structural features that mimic the structures of cellular membrane channels and produce membrane designs capable of high selectivity and high permeability or adsorptivity. The membrane structure, material and chemistry can be selected to perform liquid separations, gas separation and capture, ion transport and adsorption for a variety of applications.

  16. Polyurethane Nanofiber Membranes for Waste Water Treatment by Membrane Distillation

    OpenAIRE

    Jiříček, T.; Komárek, M.; Lederer, T.

    2017-01-01

    Self-sustained electrospun polyurethane nanofiber membranes were manufactured and tested on a direct-contact membrane distillation unit in an effort to find the optimum membrane thickness to maximize flux rate and minimize heat losses across the membrane. Also salt retention and flux at high salinities up to 100 g kg−1 were evaluated. Even though the complex structure of nanofiber layers has extreme specific surface and porosity, membrane performance was surprisingly predictable; the highest ...

  17. Molecular machines open cell membranes.

    Science.gov (United States)

    García-López, Víctor; Chen, Fang; Nilewski, Lizanne G; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B; Robinson, Jacob T; Wang, Gufeng; Pal, Robert; Tour, James M

    2017-08-30

    Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

  18. Molecular machines open cell membranes

    Science.gov (United States)

    García-López, Víctor; Chen, Fang; Nilewski, Lizanne G.; Duret, Guillaume; Aliyan, Amir; Kolomeisky, Anatoly B.; Robinson, Jacob T.; Wang, Gufeng; Pal, Robert; Tour, James M.

    2017-08-01

    Beyond the more common chemical delivery strategies, several physical techniques are used to open the lipid bilayers of cellular membranes. These include using electric and magnetic fields, temperature, ultrasound or light to introduce compounds into cells, to release molecular species from cells or to selectively induce programmed cell death (apoptosis) or uncontrolled cell death (necrosis). More recently, molecular motors and switches that can change their conformation in a controlled manner in response to external stimuli have been used to produce mechanical actions on tissue for biomedical applications. Here we show that molecular machines can drill through cellular bilayers using their molecular-scale actuation, specifically nanomechanical action. Upon physical adsorption of the molecular motors onto lipid bilayers and subsequent activation of the motors using ultraviolet light, holes are drilled in the cell membranes. We designed molecular motors and complementary experimental protocols that use nanomechanical action to induce the diffusion of chemical species out of synthetic vesicles, to enhance the diffusion of traceable molecular machines into and within live cells, to induce necrosis and to introduce chemical species into live cells. We also show that, by using molecular machines that bear short peptide addends, nanomechanical action can selectively target specific cell-surface recognition sites. Beyond the in vitro applications demonstrated here, we expect that molecular machines could also be used in vivo, especially as their design progresses to allow two-photon, near-infrared and radio-frequency activation.

  19. Challenges in the Development of Functional Assays of Membrane Proteins

    Directory of Open Access Journals (Sweden)

    Sophie Demarche

    2012-11-01

    Full Text Available Lipid bilayers are natural barriers of biological cells and cellular compartments. Membrane proteins integrated in biological membranes enable vital cell functions such as signal transduction and the transport of ions or small molecules. In order to determine the activity of a protein of interest at defined conditions, the membrane protein has to be integrated into artificial lipid bilayers immobilized on a surface. For the fabrication of such biosensors expertise is required in material science, surface and analytical chemistry, molecular biology and biotechnology. Specifically, techniques are needed for structuring surfaces in the micro- and nanometer scale, chemical modification and analysis, lipid bilayer formation, protein expression, purification and solubilization, and most importantly, protein integration into engineered lipid bilayers. Electrochemical and optical methods are suitable to detect membrane activity-related signals. The importance of structural knowledge to understand membrane protein function is obvious. Presently only a few structures of membrane proteins are solved at atomic resolution. Functional assays together with known structures of individual membrane proteins will contribute to a better understanding of vital biological processes occurring at biological membranes. Such assays will be utilized in the discovery of drugs, since membrane proteins are major drug targets.

  20. Empirical membrane lifetime model for heavy duty fuel cell systems

    Science.gov (United States)

    Macauley, Natalia; Watson, Mark; Lauritzen, Michael; Knights, Shanna; Wang, G. Gary; Kjeang, Erik

    2016-12-01

    Heavy duty fuel cells used in transportation system applications such as transit buses expose the fuel cell membranes to conditions that can lead to lifetime-limiting membrane failure via combined chemical and mechanical degradation. Highly durable membranes and reliable predictive models are therefore needed in order to achieve the ultimate heavy duty fuel cell lifetime target of 25,000 h. In the present work, an empirical membrane lifetime model was developed based on laboratory data from a suite of accelerated membrane durability tests. The model considers the effects of cell voltage, temperature, oxygen concentration, humidity cycling, humidity level, and platinum in the membrane using inverse power law and exponential relationships within the framework of a general log-linear Weibull life-stress statistical distribution. The obtained model is capable of extrapolating the membrane lifetime from accelerated test conditions to use level conditions during field operation. Based on typical conditions for the Whistler, British Columbia fuel cell transit bus fleet, the model predicts a stack lifetime of 17,500 h and a membrane leak initiation time of 9200 h. Validation performed with the aid of a field operated stack confirmed the initial goal of the model to predict membrane lifetime within 20% of the actual operating time.

  1. Targeted Learning

    CERN Document Server

    van der Laan, Mark J

    2011-01-01

    The statistics profession is at a unique point in history. The need for valid statistical tools is greater than ever; data sets are massive, often measuring hundreds of thousands of measurements for a single subject. The field is ready to move towards clear objective benchmarks under which tools can be evaluated. Targeted learning allows (1) the full generalization and utilization of cross-validation as an estimator selection tool so that the subjective choices made by humans are now made by the machine, and (2) targeting the fitting of the probability distribution of the data toward the targe

  2. Target preparation

    International Nuclear Information System (INIS)

    Hinn, G.M.

    1984-01-01

    A few of the more interesting of the 210 targets prepared in the Laboratory last year are listed. In addition the author continues to use powdered silver mixed with /sup 9,10/BeO to produce sources for accelerator radio dating of Alaskan and South Polar snow. Currently, he is trying to increase production by multiple sample processing. Also the author routinely makes 3 μg/cm 2 cracked slacked carbon stripper foils and is continuing research with some degree of success in making enriched 28 Si targets starting with the oxide

  3. Investigation of Dendrimer-Membrane Interactions

    Science.gov (United States)

    Mecke, Almut; Hessler, Jessica; Lee, Inhan; Banaszak Holl, Mark; Orr, Bradford; Patri, Anil K.; Baker, J. R.

    2003-03-01

    Modified Polyamidoamine (PAMAM) dendrimers show great promise as targeted drug transport agents. Current research efforts point to the possibility of dramatic improvements to conventional chemotherapy by selectively delivering a therapeutic to antigen bearing tumor cells. In order to better understand the uptake mechanism of such devices into cells we are investigating dendrimer-surface adsorption and dendrimer-membrane interactions using atomic force microscopy, light scattering and computer simulations. Model systems consisting of supported DMPC lipid bilayers have shown interesting results suggesting the shape and architecture of nano-devices play an important role for their biologic activity. We are also investigating the effect of targeted drug vehicles on cells in vitro.

  4. Multi-layered nanoparticles for penetrating the endosome and nuclear membrane via a step-wise membrane fusion process.

    Science.gov (United States)

    Akita, Hidetaka; Kudo, Asako; Minoura, Arisa; Yamaguti, Masaya; Khalil, Ikramy A; Moriguchi, Rumiko; Masuda, Tomoya; Danev, Radostin; Nagayama, Kuniaki; Kogure, Kentaro; Harashima, Hideyoshi

    2009-05-01

    Efficient targeting of DNA to the nucleus is a prerequisite for effective gene therapy. The gene-delivery vehicle must penetrate through the plasma membrane, and the DNA-impermeable double-membraned nuclear envelope, and deposit its DNA cargo in a form ready for transcription. Here we introduce a concept for overcoming intracellular membrane barriers that involves step-wise membrane fusion. To achieve this, a nanotechnology was developed that creates a multi-layered nanoparticle, which we refer to as a Tetra-lamellar Multi-functional Envelope-type Nano Device (T-MEND). The critical structural elements of the T-MEND are a DNA-polycation condensed core coated with two nuclear membrane-fusogenic inner envelopes and two endosome-fusogenic outer envelopes, which are shed in stepwise fashion. A double-lamellar membrane structure is required for nuclear delivery via the stepwise fusion of double layered nuclear membrane structure. Intracellular membrane fusions to endosomes and nuclear membranes were verified by spectral imaging of fluorescence resonance energy transfer (FRET) between donor and acceptor fluorophores that had been dually labeled on the liposome surface. Coating the core with the minimum number of nucleus-fusogenic lipid envelopes (i.e., 2) is essential to facilitate transcription. As a result, the T-MEND achieves dramatic levels of transgene expression in non-dividing cells.

  5. Membrane treatment of liquid wastes from radiological decontamination operations.

    Science.gov (United States)

    Svittsov, A A; Khubetsov, S B; Volchek, K

    2011-01-01

    The paper focuses on the evaluation of membrane filtration for the treatment of liquid radioactive streams generated in area decontamination operations. In this work, semi-permeable membranes were demonstrated to be effective reducing the volume of wastewater containing cesium and cobalt by two orders of a magnitude. The efficiency of membrane separation was enhanced by employing additives that enlarged the size of target radionuclide species and improved their rejection by the membranes. This was achieved by chelation with synthetic water-soluble polymers and by adsorption on micro particles of adsorbent coupled with micelle formation. The effect of wastewater composition and that of the radionuclide-binding additives on the volume reduction was investigated. Membrane treatment is expected to help simplify further processing and decrease disposal costs.

  6. Recent advances on polymeric membranes for membrane reactors

    KAUST Repository

    Buonomenna, M. G.

    2012-06-24

    Membrane reactors are generally applied in high temperature reactions (>400 °C). In the field of fine chemical synthesis, however, much milder conditions are generally applicable and polymeric membranes were applied without their damage. The successful use of membranes in membrane reactors is primary the result of two developments concerning: (i) membrane materials and (ii) membrane structures. The selection of a suited material and preparation technique depends on the application the membrane is to be used in. In this chapter a review of up to date literature about polymers and configuration catalyst/ membranes used in some recent polymeric membrane reactors is given. The new emerging concept of polymeric microcapsules as catalytic microreactors has been proposed. © 2012 Bentham Science Publishers. All rights reserved.

  7. Flow and fouling in membrane filters: Effects of membrane morphology

    Science.gov (United States)

    Sanaei, Pejman; Cummings, Linda J.

    2015-11-01

    Membrane filters are widely-used in microfiltration applications. Many types of filter membranes are produced commercially, for different filtration applications, but broadly speaking the requirements are to achieve fine control of separation, with low power consumption. The answer to this problem might seem obvious: select the membrane with the largest pore size and void fraction consistent with the separation requirements. However, membrane fouling (an inevitable consequence of successful filtration) is a complicated process, which depends on many parameters other than membrane pore size and void fraction; and which itself greatly affects the filtration process and membrane functionality. In this work we formulate mathematical models that can (i) account for the membrane internal morphology (internal structure, pore size & shape, etc.); (ii) fouling of membranes with specific morphology; and (iii) make some predictions as to what type of membrane morphology might offer optimum filtration performance.

  8. Smart membranes for monitoring membrane based desalination processes

    KAUST Repository

    Laleg-Kirati, Taous-Meriem

    2017-10-12

    Various examples are related to smart membranes for monitoring membrane based process such as, e.g., membrane distillation processes. In one example, a membrane, includes a porous surface and a plurality of sensors (e.g., temperature, flow and/or impedance sensors) mounted on the porous surface. In another example, a membrane distillation (MD) process includes the membrane. Processing circuitry can be configured to monitor outputs of the plurality of sensors. The monitored outputs can be used to determine membrane degradation, membrane fouling, or to provide an indication of membrane replacement or cleaning. The sensors can also provide temperatures or temperature differentials across the porous surface, which can be used to improve modeling or control the MD process.

  9. Physics of biological membranes

    Science.gov (United States)

    Mouritsen, Ole G.

    The biological membrane is a complex system consisting of an aqueous biomolecular planar aggregate of predominantly lipid and protein molecules. At physiological temperatures, the membrane may be considered a thin (˜50Å) slab of anisotropic fluid characterized by a high lateral mobility of the various molecular components. A substantial fraction of biological activity takes place in association with membranes. As a very lively piece of condensed matter, the biological membrane is a challenging research topic for both the experimental and theoretical physicists who ar