Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Catalytic and hydrodynamic properties of styrene monooxygenases from Rhocodoccus opacus 1CP are modulated by cofactor binding.
    Riedel, A. ; Heine, T. ; Westphal, A.H. ; Conrad, C. ; Rathsack, P. ; Berkel, W.J.H. van; Tischler, D. - \ 2015
    AMB Express 5 (2015). - ISSN 2191-0855 - 11 p.
    recombinant escherichia-coli - pseudomonas-fluorescens st - functional-analysis - crystal-structure - catabolism genes - strain vlb120 - putida ca-3 - degradation - mechanism - oxide
    Styrene monooxygenases (SMOs) are flavoenzymes catalyzing the epoxidation of styrene into styrene oxide. SMOs are composed of a monooxygenase (StyA) and a reductase (StyB). The latter delivers reduced FAD to StyA on the expense of NADH. We identified Rhodococcus opacus 1CP as the first microorganism to possess three different StyA isoforms occurring in two systems StyA1/StyA2B and StyA/StyB, respectively. The hydrodynamic properties of StyA isozymes were found to be modulated by the binding of the (reduced) FAD cofactor. StyA1 and SyA2B mainly occur as dimers in their active forms while StyA is a monomer. StyA1 showed the highest epoxidation activity and excellent enantioselectivity in aromatic sulfoxidation. The hydrodynamic and biocatalytic properties of SMOs from strain 1CP are of relevance for investigation of possible industrial applications.
    A generic microfluidic biosensor of G protein-coupled receptor activation - impedance measurements of reversible morphological changes of reverse transfected HEK293 cells on microelectrodes
    Srivastava, S.K. ; Ramaneti, R. ; Roelse, M. ; Duy Tong, H. ; Vrouwe, E.X. ; Brinkman, A.G.M. ; Smet, L.C.P.M. de; Rijn, C.J.M. van; Jongsma, M.A. - \ 2015
    RSC Advances : An international journal to further the chemical sciences 5 (2015). - ISSN 2046-2069 - p. 52563 - 52570.
    drug discovery - assays - technology - mechanism - responses - targets - design
    Impedance spectroscopy of cell lines on interdigitated electrodes (IDEs) is an established method of monitoring receptor-specific cell shape changes in response to certain analytes. Normally, assays are done in multiwells making it a bulky, static and single use procedure. Here, we present a biosensor allowing sequential application of biological test samples with an automated microfluidic system. It is capable of monitoring relative changes in impedance using castellated IDEs of 250–500 mm diameter, covered with stable or reverse transfected HEK293 cells. Reversible activation of the Neurokinin 1 (NK1) receptor in stable cell lines was observed in response to a series of 5 minute exposures from 1 pM–10 nM of the specific ligand Substance P (SP) using impedance measurements at 10 mV and 15 kHz. An optimal flow speed of 10 ml min 1 was chosen for the 10 ml flow cell. The EC50 of 10 pM was about 10 times lower than the EC50 based on measuring changes in the calcium ion concentration. The method was also shown to work with reverse transfected cells. Plasmid DNA encoding the NK1 gene was spotted onto the electrodes and pre-incubated with a transfection agent. The overlaid HEK293 cells were subsequently transfected by the underlying DNA. After challenge with SP, the cells induced an activation response similar to the stable cell line. The microfluidic micro-electrode reverse transfection system opens up possibilities to perform parallel measurements on IDE arrays with distinct receptors per IDE in a single flow channel .
    Controlling the structure and length of self-synthesizing supramolecular polymers through nucleated growth and disassembly
    Pal, A. ; Malakoutikhah, M. ; Leonetti, G. ; Tezcan, M. ; Colomb-Delsuc, M. ; Nguyen, V.D. ; Gucht, J. van der; Otto, S. - \ 2015
    Angewandte Chemie-International Edition 54 (2015)27. - ISSN 1433-7851 - p. 7852 - 7856.
    dynamic combinatorial libraries - systems chemistry - co-micelles - polymerization - replication - driven - complexity - mechanism - selection
    Directing self-assembly processes out-of-equilibrium to yield kinetically trapped materials with well-defined dimensions remains a considerable challenge. Kinetically controlled assembly of self-synthesizing peptide-functionalized macrocycles through a nucleation–growth mechanism is reported. Spontaneous fiber formation in this system is effectively shut down as most of the material is diverted into metastable non-assembling trimeric and tetrameric macrocycles. However, upon adding seeds to this mixture, well-defined fibers with controllable lengths and narrow polydispersities are obtained. This seeded growth strategy also allows access to supramolecular triblock copolymers. The resulting noncovalent assemblies can be further stabilized through covalent capture. Taken together, these results show that self-synthesizing materials, through their interplay between dynamic covalent bonds and noncovalent interactions, are uniquely suited for out-of-equilibrium self-assembly.
    2-Amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazin-3-one as an unexpected product from reduction of 5-methyl-2-nitrophenol
    Jansze, S.M. ; Saggiomo, V. ; Marcelis, A.T.M. ; Lutz, M. ; Velders, A.H. - \ 2015
    Tetrahedron Letters 56 (2015)9. - ISSN 0040-4039 - p. 1060 - 1062.
    aromatic nitro-compounds - phenoxazinone synthase - azo-compounds - aminophenol - mechanism - cells
    When attempting to synthesize symmetric 2,2'-dihydroxy-4,4'-dimethyl-azobenzene from 5-methyl-2-nitrophenol by reductive methods based on two literature procedures, an unexpected product was isolated in 30% yield. Full analysis by mass spectrometry, NMR spectroscopy, and single-crystal X-ray structure analysis, proved this product to be tricyclic 2-amino-4,4a-dihydro-4a,7-dimethyl-3H-phenoxazin-3-one. This Letter conveys a warning regarding reductive synthetic routes toward azobenzenes. We also present a novel reductive synthetic route for phenoxazines, an important class of tricyclic compounds.
    Effects of nocturnal illumination on life-history decisions and fitness in two wild songbird species
    Jong, M.J. de; Ouyang, J. ; Silva, A. Da; Grunsven, R.H.A. van; Kempenaers, B. ; Visser, M.E. ; Spoelstra, K. - \ 2015
    Philosophical Transactions of the Royal Society B. Biological sciences 370 (2015). - ISSN 0962-8436 - 8 p.
    chemical magnetoreception - photoperiodic control - birds - light - mechanism - success - vision - dawn - date
    The effects of artificial night lighting on animal behaviour and fitness are largely unknown. Most studies report short-term consequences in locations that are also exposed to other anthropogenic disturbance. We know little about how the effects of nocturnal illumination vary with different light colour compositions. This is increasingly relevant as the use of LED lights becomes more common, and LED light colour composition can be easily adjusted. We experimentally illuminated previously dark natural habitat with white, green and red light, and measured the effects on life-history decisions and fitness in two free-living songbird species, the great tit (Parus major) and pied flycatcher (Ficedula hypoleuca) in two consecutive years. In 2013, but not in 2014, we found an effect of light treatment on lay date, and of the interaction of treatment and distance to the nearest lamp post on chick mass in great tits but not in pied flycatchers. We did not find an effect in either species of light treatment on breeding densities, clutch size, probability of brood failure, number of fledglings and adult survival. The finding that light colour may have differential effects opens up the possibility to mitigate negative ecological effects of nocturnal illumination by using different light spectra.
    Authentication of Geographical Origin and Crop System of Grape Juices by Phenolic Compounds and Antioxidant Activity Using Chemometrics
    Granato, D. ; Koot, A.H. ; Schnitzler, E. ; Ruth, S.M. van - \ 2015
    Journal of Food Science 80 (2015)3. - ISSN 0022-1147 - p. C584 - C593.
    in-vitro - red wines - oxidative stress - fruit juices - rats - capacity - vivo - mechanism - profile
    The main goal of this work was to propose an authentication model based on the phenolic composition and antioxidant and metal chelating capacities of purple grape juices produced in Brazil and Europe in order to assess their typicality. For this purpose, organic, conventional, and biodynamic grape juices produced in Brazil (n = 65) and in Europe (n = 31) were analyzed and different multivariate class-modeling and classification statistical techniques were employed to differentiate juices based on the geographical origin and crop system. Overall, Brazilian juices, regardless of the crop system adopted, presented higher contents of total phenolic compounds and flavonoids, total monomeric anthocyanins, proanthocyanidins, flavonols, flavanols, cyanidin-3-glucoside, delphinidin-3-glucoside, and malvidin-3,5-glucoside. No differences were observed for trans-resveratrol, malvidin-3-glucoside, and pelargonidin-3-glucoside between countries and among crop systems. A total of 91% of Brazilian and 97% of European juices were adroitly classified using partial least squares discriminant analysis when the producing region was considered (92% efficiency), in which the free-radical scavenging activity toward 2,2-diphenyl-1-picrylhydrazyl, content of total phenolic compounds, gallic acid, and malvidin-3-glucoside were the variables responsible for the classification. Intraregional models based on soft independent modeling of class analogy were able to differentiate organic from conventional Brazilian juices as well as conventional and organic/biodynamic European juices.
    Natural loss-of-function mutation of EDR1 conferring resistance to tomato powdery mildew in Arabidopsis thaliana accession C24
    Gao, D. ; Appiano, M. ; Huibers, R.P. ; Loonen, A.E.H.M. ; Visser, R.G.F. ; Wolters, A.M.A. ; Bai, Y. - \ 2015
    Molecular Plant Pathology 16 (2015)1. - ISSN 1464-6722 - p. 71 - 82.
    salicylic-acid - downy mildew - gene - defense - plants - microsatellites - mechanism - evolution - cloning - kinase
    To screen for potentially novel types of resistance to tomato powdery mildew Oidium neolycopersici, a disease assay was performed on 123 Arabidopsis thaliana accessions. Forty accessions were fully resistant, and one, C24, was analysed in detail. By quantitative trait locus (QTL) analysis of an F2 population derived from C24 × Sha (susceptible accession), two QTLs associated with resistance were identified in C24. Fine mapping of QTL-1 on chromosome 1 delimited the region to an interval of 58¿kb encompassing 15 candidate genes. One of these was Enhanced Disease Resistance 1 (EDR1). Evaluation of the previously obtained edr1 mutant of Arabidopsis accession Col-0, which was identified because of its resistance to powdery mildew Golovinomyces cichoracearum, showed that it also displayed resistance to O.¿neolycopersici. Sequencing of EDR1 in our C24 germplasm (referred to as C24-W) revealed two missing nucleotides in the second exon of EDR1 resulting in a premature stop codon. Remarkably, C24 obtained from other laboratories does not contain the EDR1 mutation. To verify the identity of C24-W, a DNA region containing a single nucleotide polymorphism (SNP) unique to C24 was sequenced showing that C24-W contains the C24-specific nucleotide. C24-W showed enhanced resistance to O.¿neolycopersici compared with C24 not containing the edr1 mutation. Furthermore, C24-W displayed a dwarf phenotype, which was not associated with the mutation in EDR1 and was not caused by the differential accumulation of pathogenesis-related genes. In conclusion, we identified a natural edr1 mutant in the background of C24.
    The impact of elevated water nitrite concentration on physiology, growth and feed intake of African catfish Clarias gariepinus (Burchell 1822)
    Roques, J.A.C. ; Schram, E. ; Spanings, T. ; Schaik, T. van; Abbink, W. ; Boerrigter, J. ; Vries, P. de; Vis, J.W. van de; Flik, G. - \ 2015
    Aquaculture Research 46 (2015)6. - ISSN 1355-557X - p. 1384 - 1395.
    channel catfish - rainbow-trout - fresh-water - ictalurus-punctatus - oncorhynchus-mykiss - na+/k+-atpase - carpio l. - toxicity - chloride - mechanism
    The nitrite threshold concentration in rearing water of African catfish (Clarias gariepinus) was assessed. African catfish with an initial mean (SD) weight of 219.7 (57.8) g were exposed to an increasing range of water nitrite from 6 (Control) to 928 µM nitrite for 28 days. Mean (SD) plasma nitrite concentrations increased from 5.0 (3.6) to 32.5 (12.6) µM at 928 µM ambient nitrite. The increase in nitrite was accompanied by gradual increase in plasma nitrate from 41.6 (28.4) µM to 420.2 (106.4) µM. Haematocrit, haemoglobin, methemoglobin, plasma concentrations of cortisol, glucose, lactate, osmolality, gill morphology and branchial Na+/K+-ATPase activity were not affected. Feed intake, final weight, SGR, FCR and mortality were not affected. We advise not to exceed a water nitrite concentration of 43 µM (0.6 mg L-1 NO2--N) to prevent the risk of reduced growth and feed intake in African catfish aquaculture.
    One-step synthesis of delta-MnO2 nanoparticles using ascorbic acid and their scavenging properties to Pb(II), Zn(II) and methylene blue
    Wang, M.X. ; Pang, P. ; Koopal, L.K. ; Qiu, G.H. ; Wang, Y. ; Liu, F. - \ 2014
    Materials Chemistry and Physics 148 (2014)3. - ISSN 0254-0584 - p. 1149 - 1156.
    high-temperature decomposition - manganese oxide - structural evolution - oxidation-state - layered mno2 - birnessite - adsorption - mechanism - nanobelts - dissolution
    To obtain delta-MnO2 particles with a large specific surface area, MnO2 was synthesized in an ice-water bath using ascorbic acid (AA) to reduce KMnO4. At pH 3 and 5 and KMnO4/AA molar ratios of 8/1 and 10/1, nanoparticles of delta-MnO2 were produced. The specific surface areas (SSAs) of the samples ranged from 163 to 207 m(2)/g. The Mn average oxidation state of the samples ranged from 3.88 to 3.98 and increased with the KMnO4/AA ratio and pH. The adsorption of the samples with respect to metal ion revealed pseudo adsorption capacities of 3425 mmol Pb2+/kg and 1789 mmol Zn2+/kg. The decolorization behaviors of sample S10-5 (produced at pH 5 and KMnO4/AA molar ratios of 10/1) to methylene blue (MB) were compared at different pH values and temperatures. After 120 min at room temperature, 97% of the MB was adsorbed, and approximately 68% was oxidized. The adsorbed amount and the level of oxidation increased with increasing temperature and decreased with increasing pH. (C) 2014 Elsevier B.V. All rights reserved.
    Effect of Selected Plant Extracts and D- and L-Lysine on the Cyanobacterium Microcystis aeruginosa
    Lurling, M.F.L.L.W. ; Oosterhout, J.F.X. - \ 2014
    Water 6 (2014)6. - ISSN 2073-4441 - p. 1807 - 1825.
    moringa-oleifera seeds - controlling eutrophication - fresh-water - amino-acid - blooms - coagulation - inhibition - mechanism - substances - phosphorus
    We tested extracts from Fructus mume, Salvia miltiorrhiza and Moringa oleifera as well as L-lysine and D-Lysine as curative measures to rapidly suppress the cyanobacterium Microcystis aeruginosa NIVA-CYA 43. We tested these compounds under similar conditions to facilitate comparisons. We hypothesized that for each compound, relatively low concentrations—i.e., 5–50 mg L-1, would reduce M. aeruginosa biomass. At these low concentrations, only L-lysine caused a decline in M. aeruginosa biomass at =4.3 mg L-1. F. mume extract was effective to do so at high concentrations, i.e., at =240 mg L-1, but the others were virtually non-effective. Low pH caused by organic acids is a probable explanation for the effect of F. mume extract. No complete wipe-outs of the experimental population were achieved as Photosystem II efficiency showed a recovery after six days. L-lysine may be effective at low concentrations—meaning low material costs. However, the effect of L-lysine seems relatively short-lived. Overall, the results of our study did not support the use of the tested plant extracts and amino-acid as promising candidates for curative application in M. aeruginosa bloom control.
    Probing the relation between protein–protein interactions and DNA binding for a linker mutant of the bacterial nucleoid protein H-NS
    Giangrossi, M. ; Wintraecken, K. ; Spurio, R. ; Vries, R.J. de - \ 2014
    Biochimica et Biophysica Acta. Proteins & Proteomics 1844 (2014)2. - ISSN 1570-9639 - p. 339 - 345.
    in-vivo oligomerization - virulence gene icsa - escherichia-coli - curved dna - structuring protein - shigella-flexneri - organization - domain - mechanism - transcription
    We have investigated the relationship between oligomerization in solution and DNA binding for the bacterial nucleoid protein H-NS. This was done by comparing oligomerization and DNA binding of H-NS with that of a H-NS D68V-D71V linker mutant. The double linker mutation D68V-D71V, that makes the linker significantly more hydrophobic, leads to a dramatically enhanced and strongly temperature-dependent H-NS oligomerization in solution, as detected by dynamic light scattering. The DNA binding affinity of H-NS D68V-D71V for the hns promoter region is lower and has stronger temperature dependence than that of H-NS. DNase I footprinting experiments show that at high concentrations, regions protected by H-NS D68V-D71V are larger and less defined than for H-NS. In vitro transcription assays show that the enhanced protection also leads to enhanced transcriptional repression. Whereas the lower affinity of the H-NS D68V-D71V for DNA could be caused by competition between oligomerization in solution and oligomerization on DNA, the larger size of protected regions clearly confirms the notion that cooperative binding of H-NS to DNA is related to protein–protein interactions. These results emphasize the relative contributions of protein–protein interactions and substrate-dependent oligomerization in the control of gene repression operated by H-NS.
    Influence of water availability on the enzymatic hydrolysis of proteins
    Butré, C.I. ; Wierenga, P.A. ; Gruppen, H. - \ 2014
    Process Biochemistry 49 (2014)11. - ISSN 1359-5113 - p. 1903 - 1912.
    substrate-inhibition - functional-properties - ionic-strength - amino-acid - hydration - nmr - macromolecules - mechanism - kinetics - protease
    The overall rate of enzymatic protein hydrolysis decreases with increasing protein concentration (0.1–30% (w/v)) at constant enzyme/substrate ratio. To understand the role of water, the amount of available water was expressed as the ratio between free and bound water and experimentally determined from water activity and T2 relaxation time (NMR) measurements. At low protein concentrations a large excess of water is present (1.5 × 106 water molecules per protein molecule at 0.1% (w/v) whey protein isolate (WPI), but only 3984 at 30% (w/v) WPI. Assuming that 357 molecules of water are needed for full hydration of the protein, these values correspond to a 4280 and 11 times excess of water, showing that at 30% (w/v) WPI the amount of water becomes limited. At the same time, only a small decrease was observed in water activity (1.00–0.997 for 0.1–30% (w/v) WPI), and an increase of bound water measured by NMR (
    Quantification of variability in trichome patterns
    Greese, B. ; Huelskamp, M. ; Fleck, C. - \ 2014
    Frontiers in Plant Science 5 (2014). - ISSN 1664-462X
    reaction-diffusion systems - to-cell variability - gene-expression - spatial-pattern - biological-systems - lateral inhibition - voronoi diagrams - differentiation - arabidopsis - mechanism
    While pattern formation is studied in various areas of biology, little is known about the intrinsic noise leading to variations between individual realizations of the pattern. One prominent example for de novo pattern formation in plants is the patterning of trichomes on Arabidopsis leaves, which involves genetic regulation and cell-to-cell communication. These processes are potentially variable due to , e.g., the abundance of cell components or environmental conditions. To elevate the understanding of the regulatory processes underlying the pattern formation it is crucial to quantitatively analyze the variability in naturally occurring patterns. Here, we review recent approaches towards characterization of noise on trichome initiation. We present methods for the quantification of spatial patterns, which are the basis for data-driven mathematical modeling and enable the analysis of noise from different sources. Besides the insight gained on trichome formation, the examination of observed trichome patterns also shows that highly regulated biological processes can be substantially affected by variability.
    Shear structuring as a new method to make anisotropic structures from soy-gluten blends
    Grabowska, K.J. ; Tekidou, S. ; Boom, R.M. ; Goot, A.J. van der - \ 2014
    Food Research International 64 (2014). - ISSN 0963-9969 - p. 743 - 751.
    starch-zein blends - wheat-flour - electron-microscopy - globular-proteins - extrusion-cooking - behavior - microstructure - dispersions - consumption - mechanism
    The concept of shear-induced structuring was applied to concentrated blends of soy protein isolate (SPI) and wheat gluten (WG) to create novel semi-solid food textures. Concurrent simple shear deformation and heating (95 °C) of the protein blends generated original structures consisting of fibers or layers. The ratio of SPI to vital WG and the final concentration determined the morphology of the structure. It is hypothesized that the spatial distribution of the SPI-rich phase and the WG-rich phase in a blend was altered by the shear flow. When both phases became aligned horizontally in the shear cell, a fibrous structure was formed; when they became aligned vertically in the shear cell, a layered structure was formed. The structures obtained were analyzed visually and using texture analysis and scanning electron microscopy.
    Plasma Micro-Nanotextured, Scratch, Water and Hexadecane Resistant, Superhydrophobic, and Superamphiphobic Polymeric Surfaces with Perfluorinated Monolayers
    Ellinas, K. ; Pujari, S.P. ; Dragatogiannis, D.A. ; Charitidis, C.A. ; Tserepi, A. ; Zuilhof, H. ; Gogolides, E. - \ 2014
    ACS Applied Materials and Interfaces 6 (2014)9. - ISSN 1944-8244 - p. 6510 - 6524.
    self-assembled monolayers - contact-angle hysteresis - superoleophobic surfaces - superomniphobic surfaces - fabrication - friction - design - wettability - mechanism - adhesive
    Superhydrophobic and superamphiphobic toward superoleophobic polymeric surfaces of polymethyl methacrylate (PMMA), polyether ether ketone (PEEK), and polydimethyl siloxane (PDMS) are fabricated in a two-step process: (1) plasma texturing (i.e., ion-enhanced plasma etching with simultaneous roughening), with varying plasma chemistry depending on the polymer, and subsequently (2) grafting of self-assembled perfluorododecyltrichlorosilane monolayers (SAMs). Depending on the absence or not of an etch mask (i.e., colloidal microparticle self-assembly on it), random or ordered hierarchical micro-nanotexturing can be obtained. We demonstrate that stable organic monolayers can be grafted onto all these textured polymeric surfaces. After the monolayer deposition, the initially hydrophilic polymeric surfaces become superamphiphobic with static contact angles for water and oils >153°, for hexadecane >142°, and hysteresis
    Potato and Mushroom Polyphenol Oxidase Activities Are Differently Modulated by Natural Plant Extracts
    Kuijpers, T.F.M. ; Herk, T. van; Vincken, J.P. ; Janssen, R.H. ; Narh, D.L. ; Berkel, W.J.H. van; Gruppen, H. - \ 2014
    Journal of Agricultural and Food Chemistry 62 (2014)1. - ISSN 0021-8561 - p. 214 - 221.
    tyrosinase inhibitors - chlorogenic acid - ilex-paraguariensis - constituents - activation - licorice - identification - mechanism - agents - sds
    Enzymatic browning is a major quality issue in fruit and vegetable processing and can be counteracted by different natural inhibitors. Often, model systems containing a single polyphenol oxidase (PPO) are used to screen for new inhibitors. To investigate the impact of the source of PPO on the outcome of such screening, this study compared the effect of 60 plant extracts on the activity of PPO from mushroom (Agaricus bisporus, AbPPO) and PPO from potato (Solanum tuberosum, StPPO). Some plant extracts had different effects on the two PPOs: an extract that inhibited one PPO could be an activator for the other. As an example of this, the mate (Ilex paraguariensis) extract was investigated in more detail. In the presence of mate extract, oxygen consumption by AbPPO was found to be reduced >5-fold compared to a control reaction, whereas that of StPPO was increased >9-fold. RP-UHPLC-MS analysis showed that the mate extract contained a mixture of phenolic compounds and saponins. Upon incubation of mate extract with StPPO, phenolic compounds disappeared completely and saponins remained. Flash chromatography was used to separate saponins and phenolic compounds. It was found that the phenolic fraction was mainly responsible for inhibition of AbPPO and activation of StPPO. Activation of StPPO was probably caused by activation of latent StPPO by chlorogenic acid quinones.
    Kinetic and structural analysis of two transferase domains inPasteurella multocida hyaluronan synthase
    Kooy, F.K. ; Beeftink, H.H. ; Eppink, M.H.M. ; Tramper, J. ; Eggink, G. ; Boeriu, C.G. - \ 2014
    Journal of Molecular Catalysis. B, Enzymatic 102 (2014). - ISSN 1381-1177 - p. 138 - 145.
    blood-group-b - enzymological characterization - conformational-changes - n-acetylglucosamine - crystal-structure - group-a - glycosyltransferase - polypeptide - mechanism - substrate
    Pasteurella multocida hyaluronan synthase (PmHAS) encompasses two transferase domains that elongatea growing hyaluronan (HA) oligosaccharide chain by addition of either GlcNAc or GlcUA residues froma corresponding UDP-sugar. Initial velocity studies of single-step elongations were conducted for bothdomains by independently varying the concentrations of the HA oligosaccharide and the UDP-sugar.Two-substrate models were discriminated by their goodness-of-fit parameters and by dead-end inhi-bition studies. A mechanistic shift from a steady-state ordered bi-bi to rapid equilibrium ordered bi-bimechanism was observed at the NAc-site between the HA6and HA8elongation. This shift was invokedby a minor reduction in turnover number kcat. Both NAc- and UA-transferase domains follow a sequentialkinetic mechanism, most likely an ordered one in which the UDP-sugar donor binds first, followed bythe HA oligosaccharide. After transfer of the sugar moiety, both products are released, first the elongatedHA oligosaccharide and then the UDP sugar. This mechanism was visualized with a structural model ofPmHAS that presented two flexible loops, one in each transferase domain; these loops form a bridgeabove the active site.
    Use of dynamic membranes for the preparation of vitamin E-loaded lipid particles: An alternative to prevent fouling observed in classical cross-flow emulsification
    Lauoini, A. ; Charcosset, C. ; Fessi, H. ; Schroën, C.G.P.H. - \ 2014
    Chemical Engineering Journal 236 (2014). - ISSN 1385-8947 - p. 498 - 505.
    droplet break-up - multiple emulsions - process parameters - drug-delivery - encapsulation - homogenization - clearance - contactor - mechanism - beds
    Solid lipid particles (SLP) were introduced at the beginning of the 1990s as an alternative to encapsulation systems such as emulsions and liposomes used in cosmetic and pharmaceutical preparations. The present paper investigated for the first time the preparation of SLP based on premix emulsification with packed beds of micron-sized glass beads. A coarse pre-emulsion was prepared by mixing the aqueous phase (water and Tween 80) and the lipid phase (Precirol and vitamin E) under magnetic stirring at 1200 rpm during 15 min, followed by passing the premix through the glass beads layer. SLP were formed by cooling to room temperature of the final emulsion. SLP were successfully produced under various conditions, but was most optimally carried out by extruding a coarse O/W emulsion 6 times under a pressure of 2 bar through a dynamic membrane. For example, when a 2 mm layer of glass beads sized 63 lm was used, the premix size of 5 lm was reduced to 1.5 lm. It was found that particle size tended to decrease with increasing feed pressure, increasing number of passes, decreasing glass bead size and decreasing bed height. Even more importantly, the dynamic membrane was hardly prone to fouling compared to the membranes used in traditional cross-flow emulsification which typically need small pore size for the production of particles of similar size. In addition, the small beads could be easily cleaned by disintegrating the bed. The preparation process developed was easy to use, easy to scale-up, and the particle size could be controlled by appropriate choice of process parameters.
    Rooting plant development
    Scheres, B. - \ 2013
    Development 140 (2013)5. - ISSN 0950-1991 - p. 939 - 941.
    arabidopsis root - cell fate - meristem - differentiation - mechanism - shoot - framework - epidermis - division - pattern
    In 1993, we published a paper in Development detailing the anatomical structure of the Arabidopsis root. The paper described how root growth was maintained by the precisely tuned activity of a small set of 'initials', which acted as the source of dividing and differentiating cells, and how these stem cell-like cells surrounded a few infrequently dividing cells. This work underpinned subsequent research on root developmental biology and sparked a detailed molecular analysis of how stem cell groups are positioned and maintained in plants.
    Cell death of gamma interferon-stimulated human fibroblasts upon toxoplasma gondii infection induces early parasite egress and limits parasite replication
    Niedelman, W. ; Sprokholt, J.K. ; Clough, B. ; Frickel, E. ; Saeij, J.P.J. - \ 2013
    Infection and Immunity 81 (2013)12. - ISSN 0019-9567 - p. 4341 - 4349.
    ifn-gamma - indoleamine 2,3-dioxygenase - intracellular pathogen - autophagy - iron - macrophages - tryptophan - resistance - mechanism - triggers
    The intracellular protozoan parasite Toxoplasma gondii is a major food-borne illness and opportunistic infection for the immunosuppressed. Resistance to Toxoplasma is dependent on gamma interferon (IFN-¿) activation of both hematopoietic and nonhematopoietic cells. Although IFN-¿-induced innate immunity in nonhematopoietic cells has been extensively studied in mice, it remains unclear what resistance mechanisms are relied on in nonhematopoietic human cells. Here, we report an IFN-¿-induced mechanism of resistance to Toxoplasma in primary human foreskin fibroblasts (HFFs) that does not depend on the deprivation of tryptophan or iron. In addition, infection is still controlled in HFFs deficient in the p65 guanylate binding proteins GBP1 or GBP2 and the autophagic protein ATG5. Resistance is coincident with host cell death that is not dependent on the necroptosis mediator RIPK3 or caspases and is correlated with early egress of the parasite before replication. This IFN-¿-induced cell death and early egress limits replication in HFFs and could promote clearance of the parasite by immune cells.
    The Reaction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1 Provide an Understanding of the para-Hydroxylation Enzyme Catalytic Cycle
    Sucharitakul, J. ; Tongsook, C. ; Pakotiprapha, D. ; Berkel, W.J.H. van; Chaiyen, P. - \ 2013
    Journal of Biological Chemistry 288 (2013)49. - ISSN 0021-9258 - p. 35210 - 35221.
    para-hydroxybenzoate hydroxylase - p-hydroxyphenylacetate 3-hydroxylase - 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase - steady-state - ornithine hydroxylase - vibrio-campbellii - crystal-structure - gentisic acid - in-vitro - mechanism
    3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is an NADH-specific flavoprotein monooxygenase that catalyzes the para-hydroxylation of 3-hydroxybenzoate (3HB) to form 2,5-dihydroxybenzoate (2,5-DHB). Based on results from stopped-flow spectrophotometry, the reduced enzyme-3HB complex reacts with oxygen to form a C4a-peroxy flavin with a rate constant of 1.13 ± 0.01 × 10(6) m(-1) s(-1) (pH 8.0, 4 °C). This intermediate is subsequently protonated to form a C4a-hydroperoxyflavin with a rate constant of 96 ± 3 s(-1). This step shows a solvent kinetic isotope effect of 1.7. Based on rapid-quench measurements, the hydroxylation occurs with a rate constant of 36 ± 2 s(-1). 3HB6H does not exhibit substrate inhibition on the flavin oxidation step, a common characteristic found in most ortho-hydroxylation enzymes. The apparent kcat at saturating concentrations of 3HB, NADH, and oxygen is 6.49 ± 0.02 s(-1). Pre-steady state and steady-state kinetic data were used to construct the catalytic cycle of the reaction. The data indicate that the steps of product release (11.7 s(-1)) and hydroxylation (36 ± 2 s(-1)) partially control the overall turnover
    FAD C(4a)-hydroxide stabilized in a naturally fused styrene monooxygenase
    Tischler, D. ; Schlömann, M. ; Berkel, W.J.H. van; Gassner, G.T. - \ 2013
    FEBS Letters 587 (2013)23. - ISSN 0014-5793 - p. 3848 - 3852.
    rhodococcus-opacus 1cp - phenol hydroxylase - wild-type - mechanism
    StyA2B represents a new class of styrene monooxygenases that integrates flavin-reductase and styrene-epoxidase activities into a single polypeptide. This naturally-occurring fusion protein offers new avenues for studying and engineering biotechnologically relevant enantioselective biochemical epoxidation reactions. Stopped-flow kinetic studies of StyA2B reported here identify reaction intermediates similar to those reported for the separate reductase and epoxidase components of related two-component systems. Our studies identify substrate epoxidation and elimination of water from the FAD C(4a)-hydroxide as rate-limiting steps in the styrene epoxidation reaction. Efforts directed at accelerating these reaction steps are expected to greatly increase catalytic efficiency and the value of StyA2B as biocatalyst.
    Increased plasma citrulline in mice marks diet-induced obesity and may predict the development of the metabolic syndrome
    Sailer, M. ; Dahlhoff, C. ; Giesbertz, P. ; Eidens, M.K. ; Wit, N.J.W. de; Rubio-Aliaga, I. ; Boekschoten, M.V. ; Müller, M.R. ; Daniel, H. - \ 2013
    PLoS ONE 8 (2013)5. - ISSN 1932-6203
    amino-acid transporter - skeletal-muscle cells - arginine bioavailability ratios - high-fat diet - insulin-resistance - l-alanine - protein - liver - secretion - mechanism
    Article About the Authors Metrics Comments Related Content Abstract Introduction Results Discussion Materials and Methods Supporting Information Acknowledgments Author Contributions References Reader Comments (0) Figures Abstract In humans, plasma amino acid concentrations of branched-chain amino acids (BCAA) and aromatic amino acids (AAA) increase in states of obesity, insulin resistance and diabetes. We here assessed whether these putative biomarkers can also be identified in two different obesity and diabetic mouse models. C57BL/6 mice with diet-induced obesity (DIO) mimic the metabolic impairments of obesity in humans characterized by hyperglycemia, hyperinsulinemia and hepatic triglyceride accumulation. Mice treated with streptozotocin (STZ) to induce insulin deficiency were used as a type 1 diabetes model. Plasma amino acid profiling of two high fat (HF) feeding trials revealed that citrulline and ornithine concentrations are elevated in obese mice, while systemic arginine bioavailability (ratio of plasma arginine to ornithine + citrulline) is reduced. In skeletal muscle, HF feeding induced a reduction of arginine levels while citrulline levels were elevated. However, arginine or citrulline remained unchanged in their key metabolic organs, intestine and kidney. Moreover, the intestinal conversion of labeled arginine to ornithine and citrulline in vitro remained unaffected by HF feeding excluding the intestine as prime site of these alterations. In liver, citrulline is mainly derived from ornithine in the urea cycle and DIO mice displayed reduced hepatic ornithine levels. Since both amino acids share an antiport mechanism for mitochondrial import and export, elevated plasma citrulline may indicate impaired hepatic amino acid handling in DIO mice. In the insulin deficient mice, plasma citrulline and ornithine levels also increased and additionally these animals displayed elevated BCAA and AAA levels like insulin resistant and diabetic patients. Therefore, type 1 diabetic mice but not DIO mice show the “
    Analysis of steady-state Förster resonance energy transfer data by avoiding pitfalls: Interaction of JAK2 tyrosine kinase with N-methylanthraniloyl nucleotides.
    Niranjan, Y. ; Ungureanu, D. ; Hammarén, H. ; Sanz-Sanz, A. ; Westphal, A.H. ; Borst, J.W. ; Silvennoinen, O. ; Hilhorst, M.H. - \ 2013
    Analytical Biochemistry 442 (2013)2. - ISSN 0003-2697 - p. 213 - 222.
    pseudokinase domain - protein-kinase - fluorescence - atp - binding - receptor - analogs - site - autophosphorylation - mechanism
    Förster resonance energy transfer (FRET) between the fluorescent ATP analogue 2'/3'-(N-methyl-anthraniloyl)-adenosine-5'-triphosphate (MANT–ATP) and enzymes is widely used to determine affinities for ATP–protein binding. However, in analysis of FRET fluorescence data, several important parameters are often ignored, resulting in poor accuracy of the calculated dissociation constant (Kd). In this study, we systematically analyze factors that interfere with Kd determination and describe methods for correction of primary and secondary inner filter effects that extend the use of the FRET method to higher MANT nucleotide concentrations. The interactions of the fluorescent nucleotide analogues MANT–ATP, MANT–ADP [2'/3'-O-(N-methylanthraniloyl) adenosine diphosphate], and MANT–AMP [2'/3'-O-(N-methylanthraniloyl) adenosine monophosphate] with the JAK2 tyrosine kinase domain are characterized. Taking all interfering factors into consideration, we found that JAK2 binds MANT–ATP tightly with a Kd of 15 to 25 nM and excluded the presence of a second binding site. The affinity for MANT–ADP is also tight with a Kd of 50 to 80 nM, whereas MANT–AMP does not bind. Titrations of JAK2 JH1 with nonhydrolyzable ATP analogue MANT–ATP-¿-S [2'/3'-O-(N-methylanthraniloyl) adenosine-5'-(thio)- triphosphate] yielded a Kd of 30 to 50 nM. The methods demonstrated here are applicable to other enzyme–fluorophore combinations and are expected to help improve the analysis of steady-state FRET data in MANT nucleotide binding studies and to obtain more accurate results for the affinities of nucleotide binding proteins.
    Conformational landscapes of DNA polymerase I and mutator derivates establish fidelity checkpoints for nucleotide insertion
    Hohlbein, J.C. ; Aigrain, L. ; Craggs, T.D. ; Bermek, O. ; Potapova, O. ; Shoolizadeh, P. ; Grindley, N.D.F. ; Joyce, C.M. ; Kapanidis, A.N. - \ 2013
    Nature Communications 4 (2013). - ISSN 2041-1723 - 11 p.
    single-molecule fret - resonance energy-transfer - probability-distribution analysis - alternating-laser excitation - klenow fragment - escherichia-coli - active-site - photon distribution - mechanism - dynamics
    The fidelity of DNA polymerases depends on conformational changes that promote the rejection of incorrect nucleotides before phosphoryl transfer. Here, we combine single-molecule FRET with the use of DNA polymerase I and various fidelity mutants to highlight mechanisms by which active-site side chains influence the conformational transitions and free-energy landscape that underlie fidelity decisions in DNA synthesis. Ternary complexes of high fidelity derivatives with complementary dNTPs adopt mainly a fully closed conformation, whereas a conformation with a FRET value between those of open and closed is sparsely populated. This intermediate-FRET state, which we attribute to a partially closed conformation, is also predominant in ternary complexes with incorrect nucleotides and, strikingly, in most ternary complexes of low-fidelity derivatives for both correct and incorrect nucleotides. The mutator phenotype of the low-fidelity derivatives correlates well with reduced affinity for complementary dNTPs and highlights the partially closed conformation as a primary checkpoint for nucleotide selection.
    Ultrasound-assisted MnO2 catalyzed homolysis of peracetic acid for phenol degradation: The assessment of process chemistry and kinetics
    Rokhina, E.V. ; Makarova, K. ; Lathinen, M. ; Golovina, E.A. ; As, H. van; Virkutyte, J. - \ 2013
    Chemical Engineering Journal 221 (2013). - ISSN 1385-8947 - p. 476 - 486.
    wet peroxide oxidation - aqueous-solutions - free-radicals - mechanism - systems - water - decomposition - sonochemistry - intermediate - destruction
    The combination of peracetic acid (PAA) and heterogeneous catalyst (MnO2) was used for the degradation of phenol in an aqueous solution in the presence of ultrasound irradiation (US). As a relevant source of free radicals (e.g. OH), peracetic acid was comprehensively studied by means of electron spin resonance (ESR) spin trapping (ST) techniques with the subsequent identification of free radicals by simulation based fitting (SBF) technique. The radical reaction mechanism, where hydroxyl radical was a primary product of OO bond rupture of PAA, was established taking into account radical reactions, occurring during sonolysis. The potential barriers and the reaction heat were determined by basic density function theory (DFT) calculations to estimate whether the proposed radical pathway is possible. The assessment and optimization of the process parameters for MnO2/PAA/US system to eliminate phenol was accomplished with experimental design. Fractional factorial design (FFD) was executed to relate the removal efficiency of phenol with process parameters such as catalyst and PAA concentrations, the presence of ultrasound and the reaction time. The comparative kinetic study of silent and ultrasound-assisted processes revealed the significant difference between these two processes that was mainly attributed to the complex radical system formed during PAA homolysis
    Cortical microtubule arrays are initiated from a nonrandom prepattern driven by atypical microtubule initiation
    Lindeboom, J.J. ; Lioutas, A. ; Deinum, E.E. ; Tindemans, S. ; Ehrhardt, D.W. ; Emons, A.M.C. ; Mulder, B. - \ 2013
    Plant Physiology 161 (2013)3. - ISSN 0032-0889 - p. 1189 - 1201.
    plant-cells - nitella-tasmanica - self-organization - gamma-tubulin - arabidopsis - nucleation - mechanism - orientation - dynamics - reveals
    The ordered arrangement of cortical microtubules in growing plant cells is essential for anisotropic cell expansion and, hence, for plant morphogenesis. These arrays are dismantled when the microtubule cytoskeleton is rearranged during mitosis and reassembled following completion of cytokinesis. The reassembly of the cortical array has often been considered as initiating from a state of randomness, from which order arises at least partly through self-organizing mechanisms. However, some studies have shown evidence for ordering at early stages of array assembly. To investigate how cortical arrays are initiated in higher plant cells, we performed live-cell imaging studies of cortical array assembly in tobacco (Nicotiana tabacum) Bright Yellow-2 cells after cytokinesis and drug-induced disassembly. We found that cortical arrays in both cases did not initiate randomly but with a significant overrepresentation of microtubules at diagonal angles with respect to the cell axis, which coincides with the predominant orientation of the microtubules before their disappearance from the cell cortex in preprophase. In Arabidopsis (Arabidopsis thaliana) root cells, recovery from drug-induced disassembly was also nonrandom and correlated with the organization of the previous array, although no diagonal bias was observed in these cells. Surprisingly, during initiation, only about one-half of the new microtubules were nucleated from locations marked by green fluorescent protein-¿-tubulin complex protein2-tagged ¿-nucleation complexes (¿-tubulin ring complex), therefore indicating that a large proportion of early polymers was initiated by a noncanonical mechanism not involving ¿-tubulin ring complex. Simulation studies indicate that the high rate of noncanonical initiation of new microtubules has the potential to accelerate the rate of array repopulation.
    A physical cross-linking process of cellulose nanofibril gels with shear-controlled fibril orientation
    Fall, A.B. ; Lindstrom, S.B. ; Sprakel, J.H.B. ; Wagberg, L. - \ 2013
    Soft Matter 9 (2013)6. - ISSN 1744-683X - p. 1852 - 1863.
    dynamic light-scattering - microfibrillated cellulose - nanocomposites - polyelectrolyte - mechanism - networks - modulus
    Cellulose nanofibrils constitute the smallest fibrous components of wood, with a width of approximately 4 nm and a length in the micrometer range. They consist of aligned linear cellulose chains with crystallinity exceeding 60%, rendering stiff, high-aspect-ratio rods. These properties are advantageous in the reinforcement components of composites. Cross-linked networks of fibrils can be used as templates into which a polymer enters. In the semi-concentrated regime (i.e. slightly above the overlap concentration), carboxy methylated fibrils dispersed in water have been physically cross-linked to form a volume-spanning network (a gel) by reducing the pH or adding salt, which diminishes the electrostatic repulsion between fibrils. By applying shear during or after this gelation process, we can orient the fibrils in a preferred direction within the gel, for the purpose of fully utilizing the high stiffness and strength of the fibrils as reinforcement components. Using these gels as templates enables precise control of the spatial distribution and orientation of the dispersed phase of the composites, optimizing the potentially very large reinforcement capacity of the nanofibrils.
    The species-specific mode of action of the antimicrobial peptide subtilosin against Listeria monocytogenes Scott A
    Kuijk, S.J.A. van; Noll, K.S. ; Chikindas, M.L. - \ 2012
    Letters in Applied Microbiology 54 (2012)1. - ISSN 0266-8254 - p. 52 - 58.
    bacillus-subtilis - bacteriocins - antibiotics - mechanism - pathogen - growth - acid - ph
    Aims: To elucidate the molecular mechanism of action of the antimicrobial peptide subtilosin against the foodborne pathogen Listeria monocytogenes Scott A. Methods and Results: Subtilosin was purified from a culture of Bacillus amylliquefaciens. The minimal inhibitory concentration of subtilosin against L. moilocytogenes Scott A was determined by broth microdilution method. The effect of subtilosin on the transmembrane electrical potential (Ali) and pH gradient (ApH), and its ability to induce efflux of intracellular ATP, was investigated. Subtilosin fully inhibited L. monocytogencs growth at a concentration of 19 fig Subtilosin caused a partial depletion of the AT and had a similar minor effect on the ApIL There was no significant efflux of intracellular ATP. Conclusion: Subtilosin likely acts upon L. monocytogencs Scott A by perturbing the lipid bilayer of the cellular membrane and causing intracellular damage, leading to eventual cell death. Subtilosin's mode of action against L. monocytogcues Scott A differs from the one previously described for another human path()gen, Cam dnerella vaginalis. Significance and Impact of the Study: This is the first report on the specific mode of action of subtilosin against L. monocytogenes and the first report of a bacteriocin with a species specific mode of action
    Long- and Medium-Chain Fatty Acids Induce Insulin Resistance to a Similar Extent in Humans Despite Marked Differences in Muscle Fat Accumulation
    Hoeks, J. ; Mensink, M.R. ; Hesselink, M.K.C. ; Ekroos, K. ; Schrauwen, P. - \ 2012
    Journal of Clinical Endocrinology and Metabolism 97 (2012)1. - ISSN 0021-972X - p. 208 - 216.
    human skeletal-muscle - intramyocellular lipid-content - prolonged exercise - ceramide content - obese subjects - oxidation - mechanism - men - diacylglycerol - sensitivity
    Context: Animal studies revealed that medium-chain fatty acids (MCFA), due to their metabolic characteristics, are not stored in skeletal muscle and may therefore not give rise to potentially hazardous lipid species impeding insulin signaling. Objective: We here hypothesized that infusion of medium-chain triacylglycerols (MCT) in healthy lean subjects does not lead to ectopic fat accumulation and hence does not result in lipid-induced insulin resistance. Design and Methods: Nine healthy lean male subjects underwent a 6-h hyperinsulinemic-euglycemic clamp with simultaneous infusion of 1) a 100% long-chain triacylglycerols (LCT) emulsion, 2) a 50/50% MCT/LCT emulsion, or 3) glycerol in a randomized crossover design. Muscle biopsies were taken before and after each clamp. Results: MCT/LCT infusion raised plasma free fatty acid levels to a similar level compared with LCT infusion alone. Despite elevated free fatty acid levels, intramyocellular triacylglycerol (IMTG) levels were not affected by the MCT/LCT emulsion, whereas LCT infusion resulted in an approximately 1.6-fold increase in IMTG. These differences in muscle fat accumulation did not result in significant differences in lipid-induced insulin resistance between LCT (- 28%, P = 0.003) andMCT/LCT (-20%, P <0.001). Total skeletal muscle ceramide content as well as lactosyl-and glucosylceramide levels were not affected by any of the interventions. In addition, the distribution pattern of all ceramide species remained unaltered. Conclusions: Although we confirm that MCFA do not lead to ceramide and IMTG accumulation in skeletal muscle tissue in humans, they do induce insulin resistance. These results indicate that, in humans, MCFA may not be beneficial in preventing peripheral insulin resistance. (J Clin Endocrinol Metab 97: 208-216, 2012)
    Campylobacter jejuni is highly susceptible to killing by chicken host defense peptide cathelicidin-2 and suppresses intestinal cathelicidin-2 expression in young broilers
    Dijk, A. van; Herrebout, M. ; Tersteeg-Zijderveld, M.H.G. ; Tjeerdsma-van Bokhoven, J.L.M. ; Bleumink-Pluym, N. ; Jansman, A.J.M. ; Veldhuizen, E.J.A. ; Haagsman, H.P. - \ 2012
    Veterinary Microbiology 160 (2012)3-4. - ISSN 0378-1135 - p. 347 - 354.
    day-of-hatch - enteric infections - resistance - identification - colonization - pathogenesis - heterophils - mechanism - virulence - ll-37
    Little is known about the interactions of chicken host defense peptides (HDPs) with Campylobacter jejuni in young chicks. To examine the role of the chicken HDP, cathelicidin-2 (CATH-2) in host-pathogen interactions we challenged 4-day-old Ross 308 broilers with a chicken-derived C jejuni isolate (WS356) and used the chicken pathogen Salmonella enterica Enteritidis phage type 4 (FGT1) as a reference. Immunohistochemical staining was used to localize CATH-2, C jejuni and Salmonella enteritidis. Intestinal CATH-2 mRNA expression levels were determined by quantitative PCR. Antibacterial activities of CATH-2 peptide against C. jejuni and S. enteritidis isolates were assessed in colony count assays. In contrast to S. enteritidis, C jejuni was not seen to attach to intestinal epithelium and C jejuni challenge did not result in recruitment of CATH-2 containing heterophils to the small intestinal lamina propria. Minimal inhibitory concentrations found for CATH-2 peptide against human- and chicken-derived C. jejuni isolates were similar (0.6-2.5 mu M) and much lower than for S. enteritidis (20 mu M). Compared to wild-type C. jejuni 81116, the lipooligosaccharide (LOS)-deficient 81116 Delta waaF mutant was much more susceptible to CATH-2. Interestingly, CATH-2 mRNA expression levels in the small intestine were significantly lower 48 h p.i. in C jejuni-challenged chicks. These findings indicate that human clinical and chicken-derived C jejuni are equally highly susceptible to chicken CATH-2 peptide and that C jejuni uses LOS to protect itself to some extent against HDPs. Moreover, suppression of intestinal CATH-2 expression levels may be part of the C. jejuni immune evasion strategy.
    Influence of cell-to-cell variability on spatial pattern formation
    Greese, B. ; Wester, K. ; Bensch, R. ; Ronneberger, O. ; Timmer, J. ; Huulskamp, M. ; Fleck, C. - \ 2012
    IET Systems Biology 6 (2012)4. - ISSN 1751-8849 - p. 143 - 153.
    stochastic gene-expression - biochemical reactions - lateral inhibition - single-cell - arabidopsis - noise - mechanism - differentiation - stability - tessellations
    Many spatial patterns in biology arise through differentiation of selected cells within a tissue, which is regulated by a genetic network. This is specified by its structure, parameterisation and the noise on its components and reactions. The latter, in particular, is not well examined because it is rather difficult to trace. The authors use suitable local mathematical measures based on the Voronoi diagram of experimentally determined positions of epidermal plant hairs (trichomes) to examine the variability or noise in pattern formation. Although trichome initiation is a highly regulated process, the authors show that the experimentally observed trichome pattern is substantially disturbed by cell-to-cell variations. Using computer simulations, they find that the rates concerning the availability of the protein complex that triggers trichome formation plays a significant role in noise-induced variations of the pattern. The focus on the effects of cell noise yields further insights into pattern formation of trichomes. The authors expect that similar strategies can contribute to the understanding of other differentiation processes by elucidating the role of naturally occurring fluctuations in the concentration of cellular components or their properties
    Cascade-mediated binding and bending of negatively supercoiled DNA
    Westra, E.R. ; Nilges, B. ; Erp, P.B. ; Oost, J. van der; Dame, R.T. ; Brouns, S.J.J. - \ 2012
    RNA Biology 9 (2012)9. - ISSN 1547-6286 - p. 1134 - 1138.
    crispr-cas systems - immune-system - structural basis - rna - bacteria - archaea - defense - interference - recognition - mechanism
    Prokaryotes possess various defense mechanisms against invading DNA. Adaptive defense by CRISPR/Cas relies on incorporation of invader DNA sequences in the host genome. In Escherichia coli, processed transcripts of these incorporated sequences (crRNAs) guide Cascade-mediated invader DNA recognition. ( 1) (-) ( 4) Cascade is a multisubunit ribonucleoprotein complex, consisting of one crRNA and five proteins: Cse1, Cse2, Cas7, Cas5 and Cas6e. ( 1) (, ) ( 2) Cascade-mediated DNA recognition requires a conserved sequence adjacent to the target (protospacer adjacent motif, PAM) and a negatively supercoiled DNA topology. ( 3) (, ) ( 4) While Cse1 carries out PAM recognition, ( 5) the Cascade structure suggests that Cse2 may interact with target DNA in the PAM-distal end of the protospacer. ( 6) Using Electrophoretic Mobility Shift Assays, we here describe the function of the Cse1 and Cse2 subunits in the context of protospacer recognition on negatively supercoiled DNA. While Cse1 is required for nonspecific DNA binding, Cse2 appears to be important for specific binding, presumably by mediating stabilizing interactions with the displaced strand, the R-loop, or both. Furthermore, we performed Scanning Force Microscopy using linearized DNA molecules, which facilitates accurate and reliable measurements of Cascade-mediated bending. This analysis reveals that Cascade binding induces flexibility in the DNA target, most likely due to single stranded DNA regions flanking the R-loop
    Picosecond Kinetics of Light Harvesting and Photoprotective Quenching in Wild-Type and Mutant Phycobilisomes Isolated from the Cyanobacterium Synechocystis PCC 6803
    Tian, L. ; Gwizdala, M. ; Stokkum, I.H.M. van; Koehorst, R.B.M. ; Kirilovsky, D. ; Amerongen, H. van - \ 2012
    Biophysical Journal 102 (2012)7. - ISSN 0006-3495 - p. 1692 - 1700.
    orange carotenoid protein - chlorophyll-binding protein - energy-dissipation - photosystem-ii - molecular architecture - higher-plants - fluorescence - mechanism - organization - photoinhibition
    In high light conditions, cyanobacteria dissipate excess absorbed energy as heat in the light-harvesting phycobilisomes (PBs) to protect the photosynthetic system against photodamage. This process requires the binding of the red active form of the Orange Carotenoid Protein (OCPr), which can effectively quench the excited state of one of the allophycocyanin bilins. Recently, an in vitro reconstitution system was developed using isolated OCP and isolated PBs from Synechocystis PCC 6803. Here we have used spectrally resolved picosecond fluorescence to study wild-type and two mutated PBs. The results demonstrate that the quenching for all types of PBs takes place on an allophycocyanin bilin emitting at 660 nm (APCQ660) with a molecular quenching rate that is faster than (1 ps)-1. Moreover, it is concluded that both the mechanism and the site of quenching are the same in vitro and in vivo. Thus, utilization of the in vitro system should make it possible in the future to elucidate whether the quenching is caused by charge transfer between APCQ660 and OCP or by excitation energy transfer from APCQ660 to the S1 state of the carotenoid—a distinction that is very hard, if not impossible, to make in vivo.
    Interdecadal North-Atlantic meridional overturning circulation variability in EC-EARTH
    Wouters, B. ; Drijfhout, D. ; Hazeleger, W. - \ 2012
    Climate Dynamics 39 (2012)11. - ISSN 0930-7575 - p. 2695 - 2712.
    multidecadal climate variability - sea-surface temperature - ocean-atmosphere gcm - thermohaline circulation - gulf-stream - decadal variability - oscillation - model - mechanism - transport
    The Atlantic meridional overturning circulation (AMOC) in a 600 years pre-industrial run of the newly developed EC-EARTH model features marked interdecadal variability with a dominant time-scale of 50–60 years. An oscillation of approximately 2 Sverdrup (1 Sv = 106 m3 s-1) is identified, which manifests itself as a monopole causing the overturning to simultaneously strengthen (/weaken) and deepen (/shallow) as a whole. Eight years before the AMOC peaks, density in the Labrador-Irminger Sea region reaches a maximum, triggering deep water formation. This density change is caused by a counterclockwise advection of temperature and salinity anomalies at lower latitudes, which we relate to the north-south excursions of the subpolar-subtropical gyre boundary and variations in strength and position of the subpolar gyre and the North Atlantic Current. The AMOC fluctuations are not directly forced by the atmosphere, but occur in a delayed response of the ocean to forcing by the North Atlantic Oscillation, which initiates “intergyre”-gyre fluctuations. Associated with the AMOC is a 60-year sea surface temperature variability in the Atlantic, with a pattern and timescale showing similarities with the real-world Atlantic Multidecadal Variability. This good agreement with observations lends a certain degree of credibility that the mechanism that is described in this article could be seen as representative of the real climate system.
    High prevalence of a fungal prion
    Debets, A.J.M. ; Dalstra, H.J.P. ; Slakhorst, S.M. ; Koopmanschap-Memelink, A.B. ; Hoekstra, R.F. ; Saupe, S.J. - \ 2012
    Proceedings of the National Academy of Sciences of the United States of America 109 (2012)26. - ISSN 0027-8424 - p. 10432 - 10437.
    podospora-anserina - vegetative incompatibility - het-s - heterokaryon incompatibility - neurospora-crassa - meiotic drive - yeast prion - mechanism - diseases - genes
    Prions are infectious proteins that cause fatal diseases in mammals. Prions have also been found in fungi, but studies on their role in nature are scarce. The proposed biological function of fungal prions is debated and varies from detrimental to benign or even beneficial. [Het-s] is a prion of the fungus Podospora anserina. The het-s locus exists as two antagonistic alleles that constitute an allorecognition system: the het-s allele encoding the protein variant capable of prion formation and the het-S allele encoding a protein variant that cannot form a prion. We document here that het-s alleles, capable of prion formation, are nearly twice as frequent as het-S alleles in a natural population of 112 individuals. Then, we report a 92% prevalence of [Het-s] prion infection among the het-s isolates and find evidence of the role of the [Het-s]/het-S allorecognition system on the incidence of infection by a deleterious senescence plasmid. We explain the het-s/het-S allele ratios by the existence of two selective forces operating at different levels. We propose that during the somatic stage, the role of [Het-s]/HET-S in allorecognition leads to frequency-dependent selection for which an equilibrated frequency would be optimal. However, in the sexual cycle, the [Het-s] prion causes meiotic drive favoring the het-s allele. Our findings indicate that [Het-s] is a selected and, therefore, widespread prion whose activity as selfish genetic element is counteracted by balancing selection for allorecognition polymorphism
    Current-Induced Membrane Discharge
    Baeko Andersen, M. ; Soestbergen, M. ; Mani, A. ; Bruus, H. ; Biesheuvel, P.M. ; Bazant, M.Z. - \ 2012
    Physical Review Letters 109 (2012). - ISSN 0031-9007
    ion-exchange membranes - electrochemical thin-films - charge regulation model - concentration polarization - transport phenomena - water dissociation - amphoteric membranes - proton-transfer - electrodialysis - mechanism
    Possible mechanisms for overlimiting current (OLC) through aqueous ion-exchange membranes (exceeding diffusion limitation) have been debated for half a century. Flows consistent with electro-osmotic instability have recently been observed in microfluidic experiments, but the existing theory neglects chemical effects and remains to be quantitatively tested. Here, we show that charge regulation and water self-ionization can lead to OLC by “current-induced membrane discharge” (CIMD), even in the absence of fluid flow, in ion-exchange membranes much thicker than the local Debye screening length. Salt depletion leads to a large electric field resulting in a local pH shift within the membrane with the effect that the membrane discharges and loses its ion selectivity. Since salt co-ions, H+ ions, and OH- ions contribute to OLC, CIMD interferes with electrodialysis (salt counterion removal) but could be exploited for current-assisted ion exchange and pH control. CIMD also suppresses the extended space charge that leads to electro-osmotic instability, so it should be reconsidered in both models and experiments on OLC.
    Illuminating the off-pathway nature of the molten globule folding intermediate of an a-ß parallel protein
    Lindhoud, S. ; Westphal, A.H. ; Borst, J.W. ; Mierlo, C.P.M. van - \ 2012
    PLoS ONE 7 (2012)9. - ISSN 1932-6203
    azotobacter-vinelandii apoflavodoxin - refractive-index - fluorescence depolarization - spectroscopic ruler - hydrogen-exchange - energy landscape - state - flavodoxin - aggregation - mechanism
    Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin’s molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an a-ß parallel protein
    Reduction Kinetics of 3-Hydroxybenzoate 6-Hydroxylase from Rhodococcus jostii RHA1
    Sucharitakul, J. ; Wongnate, T. ; Montersino, S. ; Berkel, W.J.H. van; Chaiyen, P. - \ 2012
    Biochemistry 51 (2012)21. - ISSN 0006-2960 - p. 4309 - 4321.
    para-hydroxybenzoate hydroxylase - biochemical-characterization - pseudomonas-fluorescens - acinetobacter-baumannii - p-hydroxyphenylacetate - genus rhodococcus - mechanism - flavoprotein - purification - degradation
    3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M–1 s–1. In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s–1. At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is 51 s–1, whereas without 3-HB, the rate constant is 0.43 s–1 at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme–product (2,5-DHB) complex, with a rate constant of 45 ± 2 s–1. The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em0 for the enzyme–substrate complex is -179 ± 1 mV compared to an Em0 of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme–ligand complex to a fully activated form before flavin reduction takes place
    CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3
    Westra, E.R. ; Erp, P.B.G. ; Künne, T. ; Wong, S.P. ; Staals, R.H.J. ; Seegers, C.L.C. ; Bollen, S. ; Jore, M.M. ; Semenova, E. ; Severinov, K. ; Vos, W.M. de; Dame, R.T. ; Vries, R. de; Brouns, S.J.J. ; Oost, J. van der - \ 2012
    Molecular Cell 46 (2012)5. - ISSN 1097-2765 - p. 595 - 605.
    rna-polymerase - complex - prokaryotes - mechanism - protein - bacteriophage - resistance - sequence - defense - system
    The prokaryotic CRISPR/Cas immune system is based on genomic loci that contain incorporated sequence tags from viruses and plasmids. Using small guide RNA molecules, these sequences act as a memory to reject returning invaders. Both the Cascade ribonucleoprotein complex and the Cas3 nuclease/helicase are required for CRISPR interference in Escherichia coli, but it is unknown how natural target DNA molecules are recognized and neutralized by their combined action. Here we show that Cascade efficiently locates target sequences in negatively supercoiled DNA, but only if these are flanked by a protospacer-adjacent motif (PAM). PAM recognition by Cascade exclusively involves the crRNA-complementary DNA strand. After Cascade-mediated R loop formation, the Cse1 subunit recruits Cas3, which catalyzes nicking of target DNA through its HD-nuclease domain. The target is then progressively unwound and cleaved by the joint ATP-dependent helicase activity and Mg(2+)-dependent HD-nuclease activity of Cas3, leading to complete target DNA degradation and invader neutralization
    Pathways of sulfide oxidation by haloalkaliphilic bacteria in limited-oxygen gas lift bioreactors
    Klok, J.B. ; Bosch, P.L.F. van den; Buisman, C.J.N. ; Stams, A.J.M. ; Keesman, K.J. ; Janssen, A.J.H. - \ 2012
    Environmental Science and Technology 46 (2012)14. - ISSN 0013-936X - p. 7581 - 7586.
    sulfur-oxidizing bacteria - complete genome sequence - alkaline conditions - hydrogen-sulfide - soda lakes - mechanism - removal - reactor
    Physicochemical processes, such as the Lo-cat and Amine-Claus process, are commonly used to remove hydrogen sulfide from hydrocarbon gas streams such as landfill gas, natural gas, and synthesis gas. Biodesulfurization offers environmental advantages, but still requires optimization and more insight in the reaction pathways and kinetics. We carried out experiments with gas lift bioreactors inoculated with haloalkaliphilic sulfide-oxidizing bacteria. At oxygen-limiting levels, that is, below an O(2)/H(2)S mole ratio of 1, sulfide was oxidized to elemental sulfur and sulfate. We propose that the bacteria reduce NAD(+) without direct transfer of electrons to oxygen and that this is most likely the main route for oxidizing sulfide to elemental sulfur which is subsequently oxidized to sulfate in oxygen-limited bioreactors. We call this pathway the limited oxygen route (LOR). Biomass growth under these conditions is significantly lower than at higher oxygen levels. These findings emphasize the importance of accurate process control. This work also identifies a need for studies exploring similar pathways in other sulfide oxidizers such as Thiobacillus bacteria
    Triazole Fungicides Can Induce Cross-Resistance to Medical Triazoles in Aspergillus fumigatus
    Snelders, E. ; Camps, S.M.T. ; Karawajczyk, A. ; Schaftenaar, G. ; Kema, G.H.J. ; Lee, H.A. van der; Klaassen, C.H. ; Melchers, W.J.G. ; Verweij, P.E. - \ 2012
    PLoS ONE 7 (2012)3. - ISSN 1932-6203
    incremental construction algorithm - azole resistance - hematological malignancies - low-prevalence - french cohort - cyp51 - posaconazole - voriconazole - mechanism - evolution
    Background Azoles play an important role in the management of Aspergillus diseases. Azole resistance is an emerging global problem in Aspergillus fumigatus, and may develop through patient therapy. In addition, an environmental route of resistance development has been suggested through exposure to 14a-demethylase inhibitors (DMIs). The main resistance mechanism associated with this putative fungicide-driven route is a combination of alterations in the Cyp51A-gene (TR34/L98H). We investigated if TR34/L98H could have developed through exposure to DMIs. Methods and Findings Thirty-one compounds that have been authorized for use as fungicides, herbicides, herbicide safeners and plant growth regulators in the Netherlands between 1970 and 2005, were investigated for cross-resistance to medical triazoles. Furthermore, CYP51-protein homology modeling and molecule alignment studies were performed to identify similarity in molecule structure and docking modes. Five triazole DMIs, propiconazole, bromuconazole, tebuconazole, epoxiconazole and difenoconazole, showed very similar molecule structures to the medical triazoles and adopted similar poses while docking the protein. These DMIs also showed the greatest cross-resistance and, importantly, were authorized for use between 1990 and 1996, directly preceding the recovery of the first clinical TR34/L98H isolate in 1998. Through microsatellite genotyping of TR34/L98H isolates we were able to calculate that the first isolate would have arisen in 1997, confirming the results of the abovementioned experiments. Finally, we performed induction experiments to investigate if TR34/L98H could be induced under laboratory conditions. One isolate evolved from two copies of the tandem repeat to three, indicating that fungicide pressure can indeed result in these genomic changes. Conclusions Our findings support a fungicide-driven route of TR34/L98H development in A. fumigatus. Similar molecule structure characteristics of five triazole DMIs and the three medical triazoles appear the underlying mechanism of cross resistance development. Our findings have major implications for the assessment of health risks associated with the use of triazole DMIs
    Identifying charge and mass transfer resistances of an oxygen reducing biocathode
    Heijne, A. ter; Schaetzle, O. ; Gimenez, S. ; Fabregat-Santiago, F. ; Bisquert, J. ; Strik, D.P.B.T.B. ; Barrière, F. ; Buisman, C.J.N. ; Hamelers, H.V.M. - \ 2011
    Energy & Environmental Science 4 (2011)12. - ISSN 1754-5692 - p. 5035 - 5043.
    microbial fuel-cells - anode-respiring bacteria - performance - electrodes - biofilm - mechanism - graphite - model
    this study, we identified mass and charge transfer resistances for an oxygen reducing biocathode in a microbial fuel cell (MFC) by electrochemical impedance spectroscopy (EIS). The oxygen reducing biocathode was grown using nitrifying sludge as the inoculum. A standard model for charge transfer at the electrode surface combined with diffusion across a boundary layer was used. EIS measurements were performed under variation of both linear flow velocities and cathode potentials. Fitting the impedance data to the standard model at constant potential and different flow rates confirmed that increasing flow rate had no effect on charge transfer resistance, but led to a decrease in mass transfer resistance. From the variation in cathode potential at constant flow rate, a minimum in charge transfer resistance was found at 0.28 V vs. Ag/AgCl. The minimum in charge transfer resistance could be explained by the combined biochemical and electrochemical kinetics typical for bioelectrochemical systems.
    Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone
    Ismaya, W.T. ; Rozeboom, H.J. ; Weijn, A. ; Mes, J.J. ; Fusetti, F. ; Wichers, H.J. ; Dijkstra, B.W. - \ 2011
    Biochemistry 50 (2011)24. - ISSN 0006-2960 - p. 5477 - 5486.
    polyphenol oxidase - diffraction data - multiple forms - protein - mechanism - sequence - inhibition - refinement - plant - activation
    Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H(2)L(2) tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ~392 residues and two L subunits of ~150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ~100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme
    Characterization of Translocation of Silver Nanoparticles and Effects on Whole-Genome Gene Expression Using an In Vitro Intestinal Epithelium Coculture Model
    Bouwmeester, H. ; Poortman, J.H. ; Peters, R.J.B. ; Wijma, E. ; Kramer, E.H.M. ; Makama, S. ; Puspitaninganindita, K. ; Marvin, H.J.P. ; Peijnenburg, A.A.C.M. ; Hendriksen, P.J.M. - \ 2011
    ACS Nano 5 (2011)5. - ISSN 1936-0851 - p. 4091 - 4103.
    m-cells - caco-2 cells - nano-silver - transport - toxicity - cytotoxicity - mechanism - release - stress - nanotechnologies
    Applications of nanoparticles in the food sector are eminent. Silver nanoparticles are among the most frequently used, making consumer exposure to silver nanoparticles inevitable. Information about uptake through the intestines and possible toxic effects of silver nanoparticles is therefore very important but still lacking. In the present study, we used an in vitro model for the human intestinal epithelium consisting of Caco-2 and M-cells to study the passage of silver nanoparticles and their ionic equivalents and to assess their effects on whole-genome mRNA expression. This in vitro intestine model was exposed to four sizes of silver nanoparticles for 4 h. Exposure to silver ions was included as a control since 6-17% of the silver nanoparticles were found to be dissociated into silver ions. The amount of silver ions that passed the Caco-2 cell barrier was equal for the silver ion and nanoparticle exposures. The nanoparticles induced clear changes in gene expression in a range of stress responses including oxidative stress, endoplasmatic stress response, and apoptosis. The gene expression response to silver nanoparticles, however, was very similar to that of AgNO(3). Therefore, the observed effects of the silver nanoparticles are likely exerted by the silver ions that are released from the nanoparticles.
    Responses of gut microbiota and glucose and lipid metabolism to prebiotics in genetic obese and diet-induced leptin-resistant mice
    Everard, A. ; Derrien, M.M.N. ; Possemiers, S. ; Vos, W.M. de; Delzenne, N.M. ; Schrenzel, J. ; Cani, P.D. - \ 2011
    Diabetes 60 (2011)11. - ISSN 0012-1797 - p. 2775 - 2786.
    glucagon-like peptide-1 - inulin-type fructans - phylogenetic microarray - insulin-resistance - inflammation - permeability - endotoxemia - mechanism - rats - adipogenesis
    OBJECTIVE To investigate deep and comprehensive analysis of gut microbial communities and biological parameters after prebiotic administration in obese and diabetic mice. RESEARCH DESIGN AND METHODS Genetic (ob/ob) or diet-induced obese and diabetic mice were chronically fed with prebiotic-enriched diet or with a control diet. Extensive gut microbiota analyses, including quantitative PCR, pyrosequencing of the 16S rRNA, and phylogenetic microarrays, were performed in ob/ob mice. The impact of gut microbiota modulation on leptin sensitivity was investigated in diet-induced leptin-resistant mice. Metabolic parameters, gene expression, glucose homeostasis, and enteroendocrine-related L-cell function were documented in both models. RESULTS In ob/ob mice, prebiotic feeding decreased Firmicutes and increased Bacteroidetes phyla, but also changed 102 distinct taxa, 16 of which displayed a >10-fold change in abundance. In addition, prebiotics improved glucose tolerance, increased L-cell number and associated parameters (intestinal proglucagon mRNA expression and plasma glucagon-like peptide-1 levels), and reduced fat-mass development, oxidative stress, and low-grade inflammation. In high fat-fed mice, prebiotic treatment improved leptin sensitivity as well as metabolic parameters. CONCLUSIONS We conclude that specific gut microbiota modulation improves glucose homeostasis, leptin sensitivity, and target enteroendocrine cell activity in obese and diabetic mice. By profiling the gut microbiota, we identified a catalog of putative bacterial targets that may affect host metabolism in obesity and diabetes
    Reactions between Methanethiol and Biologically Produced Sulfur
    Leerdam, R.C. van; Bosch, P.L.F. van den; Lens, P. ; Janssen, A.J.H. - \ 2011
    Environmental Science and Technology 45 (2011)4. - ISSN 0013-936X - p. 1320 - 1326.
    dissolved sodium sulfide - equilibrium distribution - inorganic polysulfides - desulfurization - mechanism - ions
    Recently, new biotechnological processes have been developed to enable the sustainable removal of organic and inorganic sulfur compounds from liquid and gaseous hydrocarbon streams. In comparison to existing technologies (e.g., caustic scrubbing or iron based redox technologies) far less chemicals are consumed, while reusable elemental sulfur is formed as the main end-product. This research shows that in these processes a number of consecutive reactions occur between methanethiol (MT) from the hydrocarbon stream and the formed biosulfur particles, leading to the formation of (dimethyl) polysulfides. This is an important feature of this family of new bioprocesses as it improves the MT removal efficiency. The reaction kinetics depend on the MT and biosulfur concentration, temperature, and the nature of the biosulfur particles. The first reaction step involves a S(8) ring-opening by nucleophilic attack of MT molecules to form CH(3)S(9)(-). This work shows that CH(3)S(9)(-) reacts to polysulfides (S(3)(2-), S(4)(2-), S(5)(2-)), dimethyl polysulfides [(CH(3))(2)S(2), (CH(3))(2)S(3)], and dissociated H(2)S, while also some longer-chain dimethyl polysulfides [(CH(3))(2)S(4)-(7)] are formed at µM levels. Control experiments using orthorhombic sulfur flower (S(8)) did not reveal these reactions.
    The existence of an insulin-stimulated glucose and non-essential but not essential amino acid substrate interaction in diabetic pigs
    Koopmans, S.J. ; Meulen, J. van der; Wijdenes, J.W. ; Corbijn, H. ; Dekker, R.A. - \ 2011
    BMC Biochemistry 12 (2011). - ISSN 1471-2091 - 11 p.
    protein-metabolism - resistance - mellitus - humans - niddm - gluconeogenesis - sensitivity - mechanism - kinetics - alanine
    Background The generation of energy from glucose is impaired in diabetes and can be compensated by other substrates like fatty acids (Randle cycle). Little information is available on amino acids (AA) as alternative energy-source in diabetes. To study the interaction between insulin-stimulated glucose and AA utilization in normal and diabetic subjects, intraportal hyperinsulinaemic euglycaemic euaminoacidaemic clamp studies were performed in normal (n = 8) and streptozotocin (120 mg/kg) induced diabetic (n = 7) pigs of ~40-45 kg. Results Diabetic vs normal pigs showed basal hyperglycaemia (19.0 ± 2.0 vs 4.7 ± 0.1 mmol/L, P <.001) and at the level of individual AA, basal concentrations of valine and histidine were increased (P <.05) whereas tyrosine, alanine, asparagine, glutamine, glutamate, glycine and serine were decreased (P <.05). During the clamp, diabetic vs normal pigs showed reduced insulin-stimulated glucose clearance (4.4 ± 1.6 vs 16.0 ± 3.0 mL/kg·min, P <.001) but increased AA clearance (166 ± 22 vs 110 ± 13 mL/kg· min, P <.05) at matched arterial euglycaemia (5-7 mmol/L) and euaminoacidaemia (2.8-3.5 mmol/L). The increase in AA clearance was mainly caused by an increase in non-essential AA clearance (93.6 ± 13.8 vs 46.6 ± 5.4 mL/kg·min, P <.01), in particular alanine (14.2 ± 2.4 vs 3.2 ± 0.4 mL/kg·min, P <.001). Essential AA clearance was largely unchanged (72.9 ± 8.5 vs 63.3 ± 8.5 mL/kg· min), however clearances of threonine (P <.05) and tyrosine (P <.01) were increased in diabetic vs normal pigs (8.1 ± 1.3 vs 5.2 ± 0.5, and 14.3 ± 2.5 vs 6.4 ± 0.7 mL/kg· min, respectively). Conclusions The ratio of insulin-stimulated glucose versus AA clearance was decreased 5.4-fold in diabetic pigs, which was caused by a 3.6-fold decrease in glucose clearance and a 2.0-fold increase in non-essential AA clearance. In parallel with the Randle concept (glucose - fatty acid cycle), the present data suggest the existence of a glucose and non-essential AA substrate interaction in diabetic pigs whereby reduced insulin-stimulated glucose clearance seems to be partly compensated by an increase in non-essential AA clearance whereas essential AA are preferentially spared from an increase in clearance.
    Crystallization and preliminary X-ray crystallographic analysis of tyrosinase from the mushroom Agaricus bisporus
    Ismaya, W.T. ; Rozeboom, H.J. ; Schurink, M. ; Boeriu, C.G. ; Wichers, H.J. ; Dijkstra, B.W. - \ 2011
    Acta Crystallographica Section F. Structural Biology and Crystallization Communications 67 (2011)5. - ISSN 1744-3091 - p. 575 - 578.
    diphenolase activities - polyphenol oxidase - monophenolase - expression - mechanism
    Tyrosinase catalyzes the conversion of tyrosine to dihydroxyphenylalanine quinone, which is the main precursor for the biosynthesis of melanin. The enzyme from Agaricus bisporus, the common button mushroom, was purified and crystallized in two different space groups. Crystals belonging to space group P21 (unit-cell parameters a = 104.2, b = 105.0, c = 119.1 Å, ß = 110.6°, four molecules per asymmetric unit) diffracted to 3.0 Å resolution. Crystals belonging to space group P21212 (unit-cell parameters a = 104.0, b = 104.5, c = 108.4 Å, two molecules per asymmetric unit) diffracted to 2.6 Å resolution. It was essential to include 5 mM HoCl3 in all crystallization conditions in order to obtain well diffracting crystals.
    Quality of shear fractionated wheat gluten – comparison to commercial vital wheat gluten
    Zalm, E.E.J. van der; Goot, A.J. van der; Boom, R.M. - \ 2011
    Journal of Cereal Science 53 (2011)2. - ISSN 0733-5210 - p. 154 - 159.
    starch - flour - dough - separation - density - protein - stabilization - mechanism - behavior
    The functional properties of gluten obtained with a shear-induced separation process, recently proposed by Peighambardoust et al. (2008), are compared with a commercially available vital wheat gluten. Two tests were performed. First, a relatively strong wheat flour, Soissons, was enriched with gluten protein. The resulting dough was then evaluated on its kneading performance. Second, a weak flour, Kolibri, was enriched to evaluate the baking properties. The wheat flour enriched with gluten protein obtained via the shear-induced separation process (SCG) showed comparable to improved gluten functionality relative to commercial available vital wheat gluten protein (CVWG). The differences in functionality cannot be directly related to the composition as analyzed with SE-HPLC, because the composition of the gluten materials was rather comparable. The differences in functionality may therefore be related to the different drying techniques used or to the inherent mildness of the shear-induced separation technique
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