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
Oxidative decarboxylation of unsaturated fatty acids
Klis, F. van der; Hoorn, M.H. van den; Blaauw, R. ; Haveren, J. van; Es, D.S. van - \ 2011
European Journal of Lipid Science and Technology 113 (2011)5. - ISSN 1438-7697 - p. 562 - 571.
alkyl radicals - carboxylic-acids - complexes - alkenes - peroxydisulfate - silver(ii) - mechanism - olefins
Long-chain internal olefins were prepared by silver(II)-catalyzed oxidative decarboxylation of unsaturated fatty acids by sodium peroxydisulfate. Similar to saturated carboxylic acids, 1-alkenes were the major decarboxylation product in the additional presence of copper(II), whereas in the absence of copper(II) alkanes were predominantly formed. In both cases, the internal unsaturation of the fatty acids remained largely intact, although the moderate yields indicated that side reactions occurred to a significant extent. The simple procedure makes this multistep one-pot reaction useful for the synthesis of a variety of internally unsaturated hydrocarbons. The purified products, almost all of which are prepared for the first time, may serve as reference compounds for studies on the heterogeneously catalyzed decarboxylation of triglycerides and fatty acids in the absence of hydrogen.
Analysis of the polymerization initiation and activity of Pasteurella multocida heparosan synthase PmHS2, an enzyme with glycosyltransferase and UDP-sugar hydrolase activity
Chavaroche, A.A.E. ; Broek, L.A.M. van den; Springer, J. ; Boeriu, C. ; Eggink, G. - \ 2011
Journal of Biological Chemistry 286 (2011)3. - ISSN 0021-9258 - p. 1777 - 1785.
hyaluronan synthase - chemoenzymatic synthesis - identification - biosynthesis - glycosidases - transferase - mechanism - polymers - distinct - sulfate
Heparosan synthase catalyzes the polymerization of heparosan [-4GlcUAß1-4GlcNAca1-]n by transferring alternatively the monosaccharide units from UDP-GlcUA and UDP-GlcNAc to an acceptor molecule. Details on the heparosan chain initiation by Pasteurella multocida heparosan synthase PmHS2 and its influence on the polymerization process have not been reported yet. By site directed mutagenesis of PmHS2, the single action transferases PmHS2-GlcUA+ and PmHS2-GlcNAc+ were obtained. When incubated together in the standard polymerization conditions, the PmHS2-GlcUA+/PmHS2-GlcNAc+ showed comparable polymerization properties as determined for PmHS2. We investigated the first step occurring in heparosan chain initiation by the use of the single action transferases and by studying the PmHS2 polymerization process in the presence of heparosan templates and various UDP-sugar concentrations. We observed that PmHS2 favored the initiation of the heparosan chains when incubated in the presence of an excess of UDP-GlcNAc. It resulted in a higher number of heparosan chains with a lower average molecular weight or in the synthesis of two distinct groups of heparosan chain length, in the absence or in the presence of heparosan templates, respectively. These data suggest that PmHS2 transfers GlcUA from UDP-GlcUA moiety to a UDP-GlcNAc acceptor molecule to initiate the heparosan polymerization; as a consequence not only the UDP-sugar concentration but also the amount of each UDP-sugar is influencing the PmHS2 polymerization process. In addition, it was shown that PmHS2 hydrolyzes the UDP-sugars; UDP-GlcUA being more degraded than UDP-GlcNAc. However, PmHS2 incubated in the presence of both UDP-sugars favors the synthesis of heparosan polymers over the hydrolysis of UDP-sugars
The Effects of Long-or Medium-Chain Fat Diets on Glucose Toleance and Myocellular Content of Lipid Intermediates in Rats
Vogel-van den Bosch, H.M. de; Hoeks, J. ; Timmers, S. ; Houten, S.M. ; Dijk, P.J. ; Boon, W.P.C. ; Beurden, D. van; Schaart, G. ; Kersten, A.H. ; Voshol, P.J. ; Wanders, R.J.A. ; Hesselink, M.K. ; Schrauwen, P. - \ 2011
Obesity 19 (2011)4. - ISSN 1930-7381 - p. 792 - 799.
muscle insulin-resistance - skeletal-muscle - differential oxidation - energy-expenditure - acids - triglycerides - metabolism - transport - carnitine - mechanism
Accumulation of triacylglycerols (TAGs) and acylcarnitines in skeletal muscle upon high-fat (HF) feeding is the resultant of fatty acid uptake and oxidation and is associated with insulin resistance. As medium-chain fatty acids (MCFAs) are preferentially ß-oxidized over long-chain fatty acids, we examined the effects of medium-chain TAGs (MCTs) and long-chain TAGs (LCTs) on muscle lipid storage and whole-body glucose tolerance. Rats fed a low-fat (LF), HFLCT, or an isocaloric HFMCT diet displayed a similar body weight gain over 8 weeks of treatment. Only HFLCT increased myocellular TAG (42.3 ± 4.9, 71.9 ± 6.7, and 48.5 ± 6.5 µmol/g for LF, HFLCT, and HFMCT, respectively, P <0.05) and long-chain acylcarnitine content (P <0.05). Neither HF diet increased myocellular diacylglycerol (DAG) content. Intraperitoneal (IP) glucose tolerance tests (1.5 g/kg) revealed a significantly decreased glucose tolerance in the HFMCT compared to the HFLCT-fed rats (802 ± 40, 772 ± 18, and 886 ± 18 area under the curve for LF, HFLCT, and HFMCT, respectively, P <0.05). Finally, no differences in myocellular insulin signaling after bolus insulin injection (10 U/kg) were observed between LF, HFLCT, or HFMCT-fed rats. These results show that accumulation of TAGs and acylcarnitines in skeletal muscle in the absence of body weight gain do not impede myocellular insulin signaling or whole-body glucose intolerance.
Carbon chain length and the stimulus problem in oldfaction
Boesveldt, S. ; Olsson, M. ; Lundstrom, J.N. - \ 2010
Behavioural Brain Research 215 (2010)1. - ISSN 0166-4328 - p. 110 - 113.
physicochemical dimensions - discrimination ability - homologous series - odor mixtures - quality - perception - mechanism - exposure - receptor
Understanding how odour quality perception is encoded in its molecular properties arguably poses one of the most significant problems in olfaction. Determining the odour structure–quality relationships of structurally similar odorants could provide a key tool to this problem. We tentatively explored whether a mixture of two molecules, differing only in carbon chain length (C), would yield the same percept as a single odorant with an intermediate carbon chain length, akin to colour vision, or be perceived as a different quality. Ability to discriminate between pairs of iso-intense solutions of n-butanol (4C), n-propanol (3C), n-pentanol (5C), and an intermediate 50/50 molecular weight mixture of n-propanol and n-pentanol (3C/5C) was assessed in 20 healthy young adults. We found that participants were able to discriminate 4C from the 50/50 molecular weight mixture of n-propanol and n-pentanol (3C/5C), and also from the other alcohols. In conclusion, we successfully replicated previous data demonstrating that participants are able to discriminate between structurally similar alcohols, and, more importantly, the present study shows that an odour mixture of two molecules differing only in carbon chain length is clearly distinguishable from a single odorant with an intermediate carbon chain length. These findings suggest that although carbon chain length matters to odour quality, carbon chain length is not a physical continuum within homologous series of substances that corresponds to a single qualitative dimension akin to the wavelength–hue relation for monochromatic light
StyA1 and StyA2B from Rhodococcus opacus 1CP: a Multifunctional Styrene Monooxygenase System
Tischler, D. ; Kermer, R. ; Groning, J.A.D. ; Kaschabek, S.R. ; Berkel, W.J.H. van; Schlomann, M. - \ 2010
Journal of Bacteriology 192 (2010)19. - ISSN 0021-9193 - p. 5220 - 5227.
whole-cell biocatalyst - (s)-styrene oxide - escherichia-coli - functional-analysis - strain vlb120 - flavin - degradation - regeneration - mechanism - cytochrome-p450
Two-component flavoprotein monooxygenases are emerging biocatalysts that generally consist of a monooxygenase and a reductase component. Here we show that Rhodococcus opacus 1CP encodes a multifunctional enantioselective flavoprotein monooxygenase system composed of a single styrene monooxygenase (SMO) (StyA1) and another styrene monooxygenase fused to an NADH-flavin oxidoreductase (StyA2B). StyA1 and StyA2B convert styrene and chemical analogues to the corresponding epoxides at the expense of FADH(2) provided from StyA2B. The StyA1/StyA2B system presents the highest monooxygenase activity in an equimolar ratio of StyA1 and StyA2B, indicating (transient) protein complex formation. StyA1 is also active when FADH(2) is supplied by StyB from Pseudomonas sp. VLB120 or PheA2 from Rhodococcus opacus 1CP. However, in both cases the reductase produces an excess of FADH(2), resulting in a high waste of NADH. The epoxidation rate of StyA1 heavily depends on the type of reductase. This supports that the FADH(2)-induced activation of StyA1 requires interprotein communication. We conclude that the StyA1/StyA2B system represents a novel type of multifunctional flavoprotein monooxygenase. Its unique mechanism of cofactor utilization provides new opportunities for biotechnological applications and is highly relevant from a structural and evolutionary point of view.
Overall Charge and Local Charge Density of Pectin Determines the Enthalpic and Entropic Contributions to Complexation with ß-Lactoglobulin
Sperber, B.L.H.M. ; Cohen Stuart, M.A. ; Schols, H.A. ; Voragen, A.G.J. ; Norde, W. - \ 2010
Biomacromolecules 11 (2010)12. - ISSN 1525-7797 - p. 3578 - 3583.
isothermal titration calorimetry - polyelectrolyte complexation - milk-proteins - acid - isolate - gum - chromatography - hydrocolloids - association - mechanism
The complex formation between ß-lactoglobulin and pectins of varying overall charge and local charge density were investigated. Isothermal titration calorimetry experiments were carried out to determine the enthalpic contribution to the complex formation at pH 4.25 and various ionic strengths. Complex formation was found to be an exothermic process for all conditions. Combination with previously published binding constants by Sperber et al. (Sperber, B. L. H. M.; Cohen Stuart, M. A.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246-3252) allows for the determination of the changes in the Gibbs energy and the change in entropy of the system upon complex formation between ß-lactoglobulin and pectin. The local charge density of pectin is found to determine the balance between enthalpic and entropic contributions. For a high local charge density pectin, the main contribution to the Gibbs energy is of an enthalpic nature, supported by a favorable entropy effect due to the release of small counterions. A pectin with a low local charge density has a more even distribution of the enthalpic and entropic part to the change of the Gibbs energy. The enthalpic part is reduced due to the lower charge density, while the relative increase of the entropic contribution is thought to be caused by a change in the location of the binding place for pectin on the ß-lactoglobulin molecule. The association of the hydrophobic methyl esters on pectin with an exposed hydrophobic region on ß-lg results in the release of water molecules from the hydrophobic region and surrounding the methyl esters of the pectin molecule. An increase in the ionic strength decreases the enthalpic contribution due to the shielding of electrostatic attraction in favor of the entropic contribution, supporting the idea that the release of water molecules from hydrophobic areas plays a part in the complex formation.
Launched at 36,000g
Leeuwen, J.L. van - \ 2010
Science 329 (2010)5990. - ISSN 0036-8075 - p. 395 - 396.
sphagnum - mechanism - size
Most mosses, including peat mosses of the genus Sphagnum (about 285 species), disperse their spores by turbulent wind (1). In still air, spores (22 to 45 µm in size) sink at only 0.5 to 2 cm/s, ideal for wind dispersal (2). However, spore capsules, positioned on a short stalk, grow to heights of about 1 cm and do not extend into the atmospheric turbulent boundary layer (more than 10 cm above ground). Peat mosses solve this problem with an “air gun” mechanism that explosively discharges spores from a pressurized cylindrical capsule µ2 mm in length (see the figure), projecting spores over 10 to 20 cm (1–3). On page 406 of this issue, Whitaker and Edwards (4) report that an upward-traveling turbulent vortex ring of spores and air is formed by this explosion within less than 0.2 ms, carrying sufficient momentum to reach the turbulent boundary layer
Size-exclusion chromatographic protein refolding: Fundamentals, modelling and operation
Freydell, E.J. ; Wielen, L. van der; Eppink, M.H.M. ; Ottens, M. - \ 2010
Journal of Chromatography. A, Including electrophoresis and other separation methods 1217 (2010)49. - ISSN 0021-9673 - p. 7723 - 7737.
refractive-index detectors - nonlinear chromatography - diffusion-coefficients - liquid-chromatography - light-scattering - human proinsulin - aggregation - lysozyme - mechanism - media
Size-exclusion chromatography (SEC) has proven its capability to refold a variety of proteins using a range of gel filtration column materials, demonstrated in the growing body of experimental evidence. However, little effort has been allocated to the development of mechanistic models describing size-exclusion chromatographic refolding reactors (SECRR). Mechanistic models are important since they provide a link between process variables like denatured and reduced protein feed concentration (C(f,D&R)), flow rate, column length, etc., and performance indicators like refolding yield (Y(N)), thereby opening the possibility for in silico design of SECRRs. A critical step, in the formulation of such models, is the selection of an adequate reaction mechanism, which provides the direct link between the separation and the refolding yield. Therefore, in this work we present a methodology using a SEC refolding reactor model, supported by a library of reaction mechanisms, to estimate a suitable reaction scheme using experimental SEC refolding data. SEC refolding data is used since it provides information about the mass distribution of monomers and aggregates after refolding, information not readily available from batch dilution refolding data alone. Additionally, this work presents (1) a systematic analysis of the reaction mechanisms considered using characteristic time analysis and Damköhler maps, revealing (a) the direct effect of a given reaction mechanism on the shape of the SEC refolding chromatogram (number of peaks and resolution) and (b) the effect that the competition between convection, refolding and aggregation is likely to have on the SEC refolding yield; (2) a comparison between the SECR reactor and the batch dilution refolding reactor based on mechanistic modeling, quantitatively showing the advantages of the former over the latter; and (3) the successful application of the modeling based strategy to study the SEC refolding data of an industrially relevant protein. In principle, the presented modeling strategy can be applied to any protein refolded using any gel filtration material, providing the proper mass balances and activity measurements are available.
How the projection domains of NF-L and alpha-internexin determine the conformations of NF-M and NF-H in neurofilaments
Leermakers, F.A.M. ; Zhulina, E.B. - \ 2010
European Biophysics Journal 39 (2010)9. - ISSN 0175-7571 - p. 1323 - 1334.
consistent-field theory - intermediate-filaments - axonal-transport - brush - sidearms - polypeptides - architecture - simulations - transition - mechanism
Making use of a numerical self-consistent field method and polymer brush concepts, we model the solvated corona of neurofilaments (NF) composed of projection domains (unstructured tails) of constituent proteins. Projections are modeled with amino acid resolution. We focus on the importance of the two shortest ones (alpha-internexin and NF-L) in regulating the conformations of the two longer ones (NF-M and NF-H) in an isolated NF. We take the wild-type NF with no alpha-internexin as the reference, for which the phosphorylation-induced translocation of M- and H-tails has been examined previously. We demonstrate that a subbrush of L-tails creates an electrostatic potential profile with an approximately parabolic shape. An experimentally relevant (2:1) ratio of L- to alpha-projections reduces the charge density of the L subbrush and shifts the translocation transition of the H-tails to slightly higher degrees of phosphorylation. Replacing all L-tails by alpha-projections destroys the substructure of the NF corona and this alters the NF response to the phosphorylation of long tails
Lysozyme uptake by oxidized starch polymer microgels
Li, Y. ; Vries, R.J. de; Kleijn, J.M. ; Slaghek, T.M. ; Timmermans, J. ; Cohen Stuart, M.A. ; Norde, W. - \ 2010
Biomacromolecules 11 (2010)7. - ISSN 1525-7797 - p. 1754 - 1762.
linked high amylose - protein-polysaccharide complexes - poly(acrylic acid) microgels - controlled-release - polyelectrolyte brushes - beta-lactoglobulin - globular-proteins - hydrogels - adsorption - mechanism
With the aim of determining suitable conditions for uptake and release of globular proteins on microgels, we studied the interaction between phosphated, highly cross-linked, negatively charged oxidized potato starch polymer (OPSP) microgel particles and lysozyme from hen eggs. Our microgel shows a typical protein-induced deswelling behavior for charged microgels. The protein distributes rather homogenously through the microgel. We found that at low salt concentration the saturation protein uptake Gsat increases with increasing pH. This is because the binding capacity is mainly determined by charge compensation: with increasing pH, the (positive) charge on the lysozyme molecules decreases, while the (negative) charge of the microgel particles increases. Therefore, more protein molecules are needed to compensate for the charge on the gel and the binding capacity increases. The protein binding affinity, however, decreases sharply with increasing pH, presumably because this affinity is mainly sensitive to the lysozyme charge density. At high pH the binding affinity is relatively low, and by adding salt, the protein can easily be released from the gel. This leads to a maximum in the curves of Gsat versus pH, and this maximum shifts to lower pH values with increasing ionic strength. We conclude that, for protein uptake and release applications, the present system works best around pH 5 due to a sufficiently high binding affinity and a sufficiently high binding capacity.
The polymer brush model of neurofilament projections: Effect of protein composition
Zhulina, E.B. ; Leermakers, F.A.M. - \ 2010
Biophysical Journal 98 (2010)3. - ISSN 0006-3495 - p. 462 - 469.
terminal tail domain - intermediate-filaments - axonal-transport - triplet proteins - nf-m - subunit - mechanism - calibers
Applying self-consistent field theory, we consider a coarse-grained model for the polymerlike projections of neurofilament (NF) proteins that form a brush structure around neurofilaments. We focus on effects of molecular composition, which is the relative occurrence of NF-H, NF-M, and NF-L proteins, on the organization of NF projection domains. We consider NF brushes with selectively truncated projections, and with a varied ratio L:H:M of constituent tails. Our conclusion is that the NF brush structure is remarkably tolerant with respect to the variation in M and H chains. Results compare favorably with experimental data on model animals, provided that due attention is paid on the level of phosphorylation of the KSP repeats
Genome-Wide Gene Expression Analysis in Response to Organophosphorus Pesticide Chlorpyrifos and Diazion in C.Elegans
Viñuela Rodriguez, A. ; Snoek, L.B. ; Riksen, J.A.G. ; Kammenga, J.E. - \ 2010
PLoS ONE 5 (2010)8. - ISSN 1932-6203 - 8 p.
nematode caenorhabditis-elegans - binary-mixture - microarray data - in-vitro - toxicity - model - mechanism - rat - neurotoxicity - bioconductor
pesticides (OPs) were originally designed to affect the nervous system by inhibiting the enzyme acetylcholinesterase, an important regulator of the neurotransmitter acetylcholine. Over the past years evidence is mounting that these compounds affect many other processes. Little is known, however, about gene expression responses against OPs in the nematode Caenorhabditis elegans. This is surprising because C. elegans is extensively used as a model species in toxicity studies. To address this question we performed a microarray study in C. elegans which was exposed for 72 hrs to two widely used Ops, chlorpyrifos and diazinon, and a low dose mixture of these two compounds. Our analysis revealed transcriptional responses related to detoxification, stress, innate immunity, and transport and metabolism of lipids in all treatments. We found that for both compounds as well as in the mixture, these processes were regulated by different gene transcripts. Our results illustrate intense, and unexpected crosstalk between gene pathways in response to chlorpyrifos and diazinon in C. elegans
Identifying hybridizing taxa within the Daphnia longispina species complex: a comparison of genetic methods and phenotypic approaches
Dlouha, S. ; Thielsch, A. ; Kraus, R.H.S. ; Seda, J. ; Schwenk, K. ; Petrusek, S. - \ 2010
Hydrobiologia 643 (2010)1. - ISSN 0018-8158 - p. 107 - 122.
interspecific hybridization - cyclomorphic daphnia - sexual reproduction - concerted evolution - european daphnia - galeata complex - cladocera - crustacea - differentiation - mechanism
Daphnia galeata Sars, D. longispina O. F. Muller and D. cucullata Sars (Crustacea: Cladocera) are closely related species which often produce interspecific hybrids in natural populations. Several marker systems are available for taxon determination in this hybridizing complex, but their performance and reliability has not been systematically assessed. We compared results from identifications by three molecular methods. More than 1,200 individuals from 10 localities in the Czech Republic were identified as parental species or hybrids by allozyme electrophoresis and the analysis of the restriction fragment length polymorphism of the internal transcribed spacer (ITS-RFLP); over 440 of them were additionally analyzed and identified by 12 microsatellite loci. Identification by microsatellite markers corresponded well with allozyme analyses. However, consistent discrepancies between ITS-RFLP and other markers were observed in two out of 10 studied localities. Although some marker discrepancies may have been caused by occasional recent introgression, consistent deviations between ITS-RFLP and other markers suggest a long-term maintenance of introgressed alleles. These results warn against its use as a sole identification method in field studies. Additionally, we quantitatively evaluated the discriminatory power of geometric morphometric (elliptic Fourier) analysis of body shapes based on photos of over 1,300 individuals pre-classified by allozyme markers. Furthermore, a randomly selected subset of 240 individuals was independently determined from photos by several experts. Despite a tendency for morphological divergence among parental Daphnia species, some taxa (especially D. galeata, D. longispina, and their hybrids) substantially overlapped in their body shapes. This was reflected in different determination success for particular species and hybrids in discriminant analysis based on shape data as well as from photographs
Sieve tube geometry in relation to phloem flow
Mullendore, D.L. ; Windt, C.W. ; As, H. van; Knoblauch, M. - \ 2010
The Plant Cell 22 (2010)3. - ISSN 1040-4651 - p. 579 - 593.
distance water transport - ricinus-communis - mass-flow - callose substance - xylem flow - p-protein - translocation - long - mechanism - element
Sieve elements are one of the least understood cell types in plants. Translocation velocities and volume flow to supply sinks with photoassimilates greatly depend on the geometry of the microfluidic sieve tube system and especially on the anatomy of sieve plates and sieve plate pores. Several models for phloem translocation have been developed, but appropriate data on the geometry of pores, plates, sieve elements, and flow parameters are lacking. We developed a method to clear cells from cytoplasmic constituents to image cell walls by scanning electron microscopy. This method allows high-resolution measurements of sieve element and sieve plate geometries. Sieve tube–specific conductivity and its reduction by callose deposition after injury was calculated for green bean (Phaseolus vulgaris), bamboo (Phyllostachys nuda), squash (Cucurbita maxima), castor bean (Ricinus communis), and tomato (Solanum lycopersicum). Phloem sap velocity measurements by magnetic resonance imaging velocimetry indicate that higher conductivity is not accompanied by a higher velocity. Studies on the temporal development of callose show that small sieve plate pores might be occluded by callose within minutes, but plants containing sieve tubes with large pores need additional mechanisms
Migration of gluten under shear flow: influence of process parameters on separation behaviour
Peighambardoust, S.H. ; Goot, A.J. van der - \ 2010
Food Chemistry 118 (2010)3. - ISSN 0308-8146 - p. 712 - 718.
wheat-flour - radial migration - dilute-solutions - dough - starch - microstructure - fractionation - suspensions - molecules - mechanism
The effect of processing conditions on the shear-induced migration of starch and gluten was described. A shearing device was used to induce a separation of wheat dough into a gluten rich fraction and a starch phase. A two-stage mechanism for separation was observed: first local aggregation of gluten, followed by migration of the gluten domains to the apex of the cone. The process was not strongly influenced by variations in the process conditions, possibly because a change in process parameter could have opposing effects on stages 1 and 2. Severe process conditions can lead to gluten damage and thereby a reduction in the driving force for separation. A relatively low temperature (10–15 °C) and low rotational speed (10 rpm) had a positive effect on the gluten migration inside the shearing device. A simple model was proposed to explain the effect of process parameters on the second stage of the gluten migration process
Unravelling the kinetics of the formation of acrylamide in the Maillard reaction of fructose and asparagine by multiresponse modelling
Knol, J.J. ; Linssen, J.P.H. ; Boekel, M.A.J.S. van - \ 2010
Food Chemistry 120 (2010)4. - ISSN 0308-8146 - p. 1047 - 1057.
n-(1-deoxy-d-fructos-1-yl)-glycine degradation pathways - sugar-casein systems - potato crisps - formation/elimination reactions - phosphate buffer - reaction cascade - food-science - mechanism - products - glucose
A kinetic model for the formation of acrylamide in a fructose–asparagine reaction system at initial pH 5.5 is proposed, based on an approach called multiresponse kinetic modelling. The formation of acetic acid and formic acid from the degradation of fructose and its isomer glucose was included in the proposed kinetic model. The kinetic model suggests that the effect of temperature on acrylamide formation with fructose is more due to the preceding steps with the formation of the Schiff base. The use of fructose and lower pH resulted in a higher yield of acrylamide (3%), suggesting that both can play an important role in acrylamide mitigation. Furthermore, these models have shown that, at high temperatures (120–200 °C), the Maillard reaction rapidly goes into the advanced stages, forming high amounts of organic acids and high molecular weight melanoidins. Overall, these mechanistic models provide more insight of the formation of acrylamide in a quantitative way.
Nucleases Encoded by Integraded Elements CJIE2 and CJIE4 Inhibit Natural Transformation of Campylobacter Jejuni
Gaasbeek, E.J. ; Wagenaar, J.A. ; Guilhabert, M.R. ; Putten, J.P. van; Parker, C.T. ; Wal, F.J. van der - \ 2010
Journal of Bacteriology 192 (2010)4. - ISSN 0021-9193 - p. 936 - 941.
serratia-marcescens endonuclease - horizontal gene-transfer - diversity - bacteria - sequence - dnase - identification - mutagenesis - mechanism - strains
The species Campylobacter jejuni is naturally competent for DNA uptake; nevertheless, nonnaturally transformable strains do exist. For a subset of strains we previously showed that a periplasmic DNase, encoded by dns, inhibits natural transformation in C. jejuni. In the present study, genetic factors coding for DNase activity in absence of dns were identified. DNA arrays indicated that nonnaturally transformable dns-negative strains contain putative DNA/RNA non-specific endonucleases encoded by CJE0566 and CJE1441 of strain RM1221. These genes are located on C. jejuni integrated element 2 and 4. Expression of CJE0566 and CJE1441 from strain RM1221 and a homologous gene from strain 07479 in DNase-negative Escherichia coli and C. jejuni strains indicated that these genes code for DNases. Genetic transfer of the genes to a naturally transformable C. jejuni strain resulted in a decreased efficiency of natural transformation. Modelling suggests that the C. jejuni DNases belong to the Serratia nuclease family. Overall, the data indicate that the acquisition of prophage encoded DNA/RNA non-specific endonucleases inhibits the natural transformability of C. jejuni through hydrolysis of DNA
Potency of isothiocyanates to induce luciferase reporter gene expression via the electrophile-responsive element from murine glutathione S-transferase Ya
Vermeulen, M. ; Boerboom, A.M.J.F. ; Blankvoort, B.M.G. ; Aarts, J.M.M.J.G. ; Rietjens, I. ; Bladeren, P.J. van; Vaes, W.H.J. - \ 2009
Toxicology in Vitro 23 (2009)4. - ISSN 0887-2333 - p. 617 - 621.
consensus sequence - mercapturic acids - identification - vegetables - sulforaphane - activation - mechanism - enzymes - fruit - risk
Isothiocyanates are electrophiles that are able to induce phase II biotransformation enzyme gene expression via an electrophile-responsive element (EpRE) in the gene regulatory region. To study the potency of different isothiocyanates to induce the expression of EpRE-regulated genes, a Hepa-1c1c7 luciferase reporter cell line was exposed to structurally different isothiocyanates. The reporter cell line, EpRE(mGST-Ya)–LUX, contains the EpRE from the regulatory region of the mouse glutathione S-transferase Ya gene. Isothiocyanates containing a methyl-sulfur side chain, e.g. sulforaphane, showed a lower EC50 (0.8–3.2 µM) and a comparable induction factor (17–22.4) compared to the structurally different isothiocyanates containing an alkyl or aromatic side chain, e.g. allyl and phenylethyl isothiocyanate (EC50 3.9–6.5 µM, induction factor 17.5–23). After 24 h of exposure, on average (±SD) 23 ± 5% of the isothiocyanate was found in the cells and 77% in the cell medium. Isothiocyanates prove to be strong inducers of electrophile-responsive element-mediated gene expression at physiological concentrations. The here described luciferase reporter cell line is a suitable assay to measure the potency of compounds to induce EpRE-regulated gene expression
Influence of process conditions on the separation behaviour of starch-gluten systems
Zalm, E.E.J. van der; Goot, A.J. van der; Boom, R.M. - \ 2009
Journal of Food Engineering 95 (2009)4. - ISSN 0260-8774 - p. 572 - 578.
wheat-flour - mixing time - migration - shear - ultracentrifugation - microstructure - mechanism
Separation of wheat flour into its constituents starch and gluten was studied using a cone-cone shearing device, with emphasis on the effect of rotation rate, processing time, temperature and water content. This study confirms the two step mechanism previously proposed for the gluten migration: aggregation of gluten protein into gluten domains that subsequently migrate to the apex of the cone. The results show that optimal process conditions for gluten migration are different from the process conditions for gluten aggregation. While gluten agglomeration (step 1) benefits from high temperature, low rotation rate and high water content, gluten migration (step 2) is positively influenced by a high dough viscosity and higher rotation rate
Salt-Induced Disintegration of Lysozyme-Containing Polyelectrolyte Complex Micelles
Lindhoud, S. ; Cohen Stuart, M.A. ; Norde, W. ; Vries, R.J. de; Schweins, R. ; Voorhaar, L. - \ 2009
Langmuir 25 (2009)19. - ISSN 0743-7463 - p. 11425 - 11430.
entrapping enzyme molecules - coacervate core micelles - electrostatic complexes - neutron-scattering - protein complexes - block-copolymers - acrylamide - stability - nanoparticles - mechanism
The salt-induced disintegration of lysozyme-filled polyelectrolyte complex micelles, consisting of positively charged homopolymers (PDMAEMA150), negatively charged diblock copolymers (PAA42-PAAm417), and lysozyme, has been studied with dynamic light scattering (DLS) and small-angle neutron scattering (SANS). These measurements show that, from 0 to 0.2 M NaCl, both the hydrodynamic radius (Rh) and the core radius (Rcore) decrease with increasing salt concentration. This suggests that the micellar structures rearrange. Moreover, from 0.2 to 0.4 M NaCl the light-scattering intensity is constant. In this salt interval, the hydrodynamic radius increases, has a maximum at 0.3 M NaCl, and subsequently decreases. This behavior is observed in both a lysozyme-containing system and a system without lysozyme. The SANS measurements on the lysozyme-filled micelles do not show increased intensity or a larger core radius at 0.3 M NaCl. This indicates that from 0.2 to 0.4 M NaCl another structure is formed, consisting of just the diblock copolymer and the homopolymer, because at 0.12 M NaCl the lysozyme-PAA42-PAAm417 complex has disintegrated. One may expect that the driving force for the formation of the complex in this salt range is other than electrostatic
Absence of Connexin 40 gene polymorphism, as a marker of undetected atrial fibrillation in patients with unexplained cerebral ischemic events
Chaldoupi, S.M. ; Soedamah-Muthu, S.S. ; Regieli, J. ; Werf, C. van de; Nelen, M. ; Smagt, J.J. van der; Algra, A. ; Hauer, R.N.W. ; Doevendans, P.A. ; Loh, P.C. - \ 2009
European Journal of Cardiovascular Prevention and Rehabilitation 16 (2009)5. - ISSN 1741-8267 - p. 616 - 620.
independent risk factor - blood-flow - follow-up - stroke - manifestations - disease - association - coagulation - tachycardia - mechanism
Background - Atrial fibrillation (AF) is a major cause of cerebral infarction. Idiopathic AF is strongly associated with the human minor Connexin 40 (Cx40) promotor polymorphism. We examined the prevalence of the minor Cx40 allele in patients with cerebral ischemia and no other cardiovascular disease (CVD), as an indication of underlying idiopathic AF. Methods - In patients with cerebral ischemia without prior CVD (n=225), DNA analysis of the Cx40 minor allele (-44 G¿A) was performed. Patients were divided into those with a normal electrocardiographic (ECG) findings (group A, n=164), with ECG abnormalities (group B, n=51) and those with normal ECG and documented episodes of AF (group C, n=10). On the basis of echocardiography (ECHO) data availability, further subgroups were defined: normal ECG and ECHO (group D, n=45); ECG or ECHO abnormalities (group E, n=22); and normal ECG and ECHO and documented AF episodes (group F, n=8). The prevalence of Cx40 promotor polymorphism was compared among all the subgroups. Results - The average age was 58.7 years (±11.5) and 64.4% were men. Patients with episodes of AF and those with abnormal ECG or ECHO results (B+C or E+F) did not show a higher prevalence of the minor allele genotype (AA vs. GG) compared with the normal ECG/ECHO groups (A or D) (group A, odds ratio=1.04, 95% confidence interval: 0.26–4.11 and group D, odds ratio=0.38, 95% confidence interval: 0.04–3.63). Conclusion - In patients with cerebral ischemic events, without prior CVD, a higher prevalence of the Cx40 gene polymorphism, as a marker of underlying idiopathic AF appeared to be absent
ESR ST study of hydroxyl radical generation in wet peroxide system catalyzed by heterogeneous ruthenium
Rokhina, E.V. ; Golovina, E.A. ; As, H. van; Virkutyte, J. - \ 2009
Chemosphere 77 (2009)1. - ISSN 0045-6535 - p. 148 - 150.
oxidation - decomposition - pollutants - mechanism - kinetics - surface - phenol - water - h2o2
Ru-based catalysts gained popularity because of their applicability for a variety of processes, including carbon monoxide oxidation, wet air catalytic oxidation and wastewater treatment. The focus of a current study was generation of hydroxyl radicals in the wet peroxide system catalyzed by heterogeneous ruthenium, spin-trapped by DEPMPO and DIPPMPO by means of electron spin resonance spin-trapping technique (ESR ST). The mechanism of free radicals formation was proposed via direct cleavage of hydrogen peroxide over ruthenium active sites. The chemical reactions occurring in the system were introduced according to the experimental results. Also, radical production rate was assessed based on concentration changes of species involved in the bulk liquid phase oxidation
Picosecond Fluorescence Relaxation Spectroscopy of the Calcium-Discharged Photoproteins Aequorin and Obelin
Oort, B.F. van; Eremeeva, E. ; Koehorst, R.B.M. ; Laptenok, S. ; Amerongen, H. van; Berkel, W.J.H. van; Malikova, N.P. ; Markova, S.V. ; Vysotski, E.S. ; Visser, A.J.W.G. ; Lee, J. - \ 2009
Biochemistry 48 (2009)44. - ISSN 0006-2960 - p. 10486 - 10491.
ca2+-regulated photoproteins - violet bioluminescence - angstrom resolution - recombinant obelin - crystal-structure - w92f obelin - coelenterazine - mechanism - expression - proteins
Addition of calcium ions to the Ca2+-regulated photoproteins, such as aequorin and obelin, produces a blue bioluminescence originating from a fluorescence transition of the protein-bound product, coelenteramide. The kinetics of several transient fluorescent species of the bound coelenteramide is resolved after picosecond-laser excitation and streak camera detection. The initially formed spectral distributions at picosecond-times are broad, evidently comprised of two contributions, one at higher energy (25000 cm-1) assigned as from the Ca2+-discharged photoprotein-bound coelenteramide in its neutral state. This component decays much more rapidly (t1/2 2 ps) in the case of the Ca2+-discharged obelin than aequorin (t1/2 30 ps). The second component at lower energy shows several intermediates in the 150-500 ps times, with a final species having spectral maxima 19400 cm-1, bound to Ca2+-discharged obelin, and 21300 cm-1, bound to Ca2+-discharged aequorin, and both have a fluorescence decay lifetime of 4 ns. It is proposed that the rapid kinetics of these fluorescence transients on the picosecond time scale, correspond to times for relaxation of the protein structural environment of the binding cavity
Biofluiddynamic scaling of flapping, spinning and translating fins and wings
Lentink, D. ; Dickinson, M.H. - \ 2009
Journal of Experimental Biology 212 (2009)16. - ISSN 0022-0949 - p. 2691 - 2704.
low reynolds-numbers - unsteady aerodynamic performance - insect flight - revolving wings - hovering flight - lift - animals - vortex - drosophila - mechanism
Organisms that swim or fly with fins or wings physically interact with the surrounding water and air. The interactions are governed by the morphology and kinematics of the locomotory system that form boundary conditions to the Navier–Stokes (NS) equations. These equations represent Newton's law of motion for the fluid surrounding the organism. Several dimensionless numbers, such as the Reynolds number and Strouhal number, measure the influence of morphology and kinematics on the fluid dynamics of swimming and flight. There exists, however, no coherent theoretical framework that shows how such dimensionless numbers of organisms are linked to the NS equation. Here we present an integrated approach to scale the biological fluid dynamics of a wing that flaps, spins or translates. Both the morphology and kinematics of the locomotory system are coupled to the NS equation through which we find dimensionless numbers that represent rotational accelerations in the flow due to wing kinematics and morphology. The three corresponding dimensionless numbers are (1) the angular acceleration number, (2) the centripetal acceleration number, and (3) the Rossby number, which measures Coriolis acceleration. These dimensionless numbers consist of length scale ratios, which facilitate their geometric interpretation. This approach gives fundamental insight into the physical mechanisms that explain the differences in performance among flapping, spinning and translating wings. Although we derived this new framework for the special case of a model fly wing, the method is general enough to make it applicable to other organisms that fly or swim using wings or fins
Oxygen exchange between nitrogen oxides and H2O can occur during nitrifier pathways
Kool, D.M. ; Müller, C. ; Wrage, N. ; Oenema, O. ; Groenigen, J.W. van - \ 2009
Soil Biology and Biochemistry 41 (2009)8. - ISSN 0038-0717 - p. 1632 - 1641.
ammonia-oxidizing bacteria - nitrite reductase genes - denitrifying bacteria - aerobic conditions - nitrate reductase - denitrification - n2o - soil - mechanism - water
Interpretation of the oxygen isotopic signature of soil-derived N2O may be flawed when it is based on reaction stoichiometry and fractionation alone. In fact, oxygen (O) exchange between H2O and intermediates of N2O production pathways may largely determine this O isotopic signature. Although in our previous work we conclusively proved the occurrence of O exchange during N2O production by denitrification of NO3¿, its occurrence in N2O production pathways by nitrifiers remains unclear. The aim of this study was to examine the likeliness of O exchange during various stages of N2O production in soil via nitrification, nitrifier denitrification and denitrification. We evaluated a set of scenarios on the presence of such exchange using data from a series of 18O and 15N tracing experiments. The measured actual O incorporation from H2O into N2O (AOI) was compared with the theoretical maximum O incorporation (MOI) from various scenarios that differed in their assumptions on the presence of O exchange. We found that scenarios where O exchange was assumed to occur exclusively during denitrification could not explain the observed AOI, as it exceeded the MOI for 9 out of 10 soils. This demonstrates that additional O exchange must have occurred in N2O production through nitrifier pathways. It remains to be determined in which steps of these pathways O exchange can take place. We conclude that O exchange is likely to be mediated by ammonia oxidizers during NO2¿ reduction (nitrifier denitrification), and that it could possibly occur during NO2¿ oxidation to NO3¿ by nitrite oxidizers as well
ps2, the gene responsible for functional sterility in tomato, due to non-dehiscent anthers, is the result of a mutation in a novel polygalacturonase gene
Gorguet, B.J.M. ; Schipper, E.H. ; Lammeren, A.A.M. van; Visser, R.G.F. ; Heusden, A.W. van - \ 2009
Theoretical and Applied Genetics 118 (2009)6. - ISSN 0040-5752 - p. 1199 - 1209.
arabidopsis-thaliana - endo-polygalacturonase - plant - expression - mechanism - resource - site
The recessive mutation ps-2, which appeared spontaneously in tomato, confers functional male sterility due to non-dehiscent anthers. In this study, we isolated and characterized the PS-2 gene. A single nucleotide mutation in a novel tomato polygalacturonase gene is responsible for the ps-2 phenotype. The mutation in ps-2 is responsible for an alternative splicing during maturation of the pre-mRNA, which leads to an aberrant mRNA. Differentiation between ps-2 and wild type (PS-2) anthers only appears in the final developmental stage in which the stomium remains closed in the mutant. To our knowledge, this is the first functional sterility gene isolated in the Solanaceae family. The specific expression of the Arabidopsis homolog of PS-2 in the anther dehiscence zone suggests a conserved mode of action over the plant kingdom, which means that the repression of PS-2 homologs may be a potential way to introduce functional sterility in other species
Thioflavin T fluorescence assay for ß-lactoglobulin fibrils hindered by DAPH
Kroes-Nijboer, A. ; Lubbersen, Y.S. ; Venema, P. ; Linden, E. van der - \ 2009
Journal of Structural Biology 165 (2009)3. - ISSN 1047-8477 - p. 140 - 145.
amyloid fibrils - ph 2 - aggregation - mechanism - binding
The molecule 4,5-dianilinophthalimide was recently found to be an efficient compound in disaggregating amyloid fibrils involved in the Alzheimer¿s disease. In this study we have investigated whether the compound 4,5-dianilinophthalimide was able to disaggregate fibrils derived from ß-lactoglobulin. In addition to a Thioflavin T fluorescence assay, flow-induced birefringence was used as an independent technique to measure the total length concentration of the fibrils. An additional advantage of the latter technique is that not only the total length concentration, but also the length distribution of the fibrils can be measured. The results from flow-induced birefringence showed that the total amount of fibrils and also the length distribution of the fibrils was not influenced by the addition of 4,5-dianilinophthalimide, even though this was suggested by the results of the Thioflavin T assay. The results of flow-induced birefringence were confirmed by rheological measurements and transmission electron microscopy. Our findings show that the use of a Thioflavin T assay in order to probe the possible disaggregating effect of certain compounds can give misleading results.
Diminished frequency and function of CD4(+) CD25(high) regulatory T cells associated with active uveitis in Vogt-Koyanagi-Harada syndrome
Chen, L. ; Yang, P.Z. ; Zhou, H.Y. ; He, H. ; Ren, X.R. ; Chi, W. ; Wang, L. ; Kijlstra, A. - \ 2008
Investigative ophthalmology and visual science 49 (2008)8. - ISSN 0146-0404 - p. 3475 - 3482.
peripheral-blood - disease - foxp3 - expression - mechanism - sclerosis
PURPOSE. CD4(+)CD25(high) regulatory T (Treg) cells have been shown to be involved in the pathogenesis of autoimmune diseases. Vogt-Koyanagi-Harada (VKH) syndrome is an organ-specific autoimmune disease. This study was designed to phenotypically and functionally characterize peripheral blood CD4(+)CD25(high) Treg cells in VKH patients with active uveitis. METHODS. Blood samples were taken from 30 patients with active VKH, 19 patients with inactive VKH, and 26 healthy controls. Peripheral blood mononuclear cells were subjected to flow cytometry for analysis of phenotypes of the CD4(+)CD25(high) Treg cells. For functional analysis, CD4(+)CD25(high) Treg cells and CD4(+)CD25-T cells were separated by means of magnetic-assisted cell sorting and subsequently cocultured for 6 days. The proliferation of CD4(+)CD25-T cells was measured by [H-3] thymidine incorporation assay. The levels of IFN-gamma, IL-17, and IL-13 in the supernatants were determined by enzyme-linked immunosorbent assay. RESULTS. Significantly decreased frequencies of CD4(+)CD25(high) Treg cells and percentages of FOXP3(+) cells in these Treg cells were shown in patients with active VKH. Treg cells from patients with active VKH showed a significant deficiency in suppressing the proliferation of CD4(+)CD25-T cells and inhibiting the production of IFN-gamma and IL-13 by CD4(+)CD25-T cells. CD4(+)CD25(high) Treg cells from VKH patients or healthy controls did not markedly inhibit or promote IL-17 production. CONCLUSIONS. A significantly decreased frequency and diminished function of CD4(+)CD25(high) Treg cells is associated with active uveitis in patients with VKH syndrome. These results suggest that these dysfunctional CD4(+)CD25(high) Treg cells may play a role in the pathogenesis of uveitis in VKH syndrome.
Identification of strong aggregating regions in soy glycinin upon enzymatic hydrolysis
Kuipers, B.J.H. ; Gruppen, H. - \ 2008
Journal of Agricultural and Food Chemistry 56 (2008)10. - ISSN 0021-8561 - p. 3818 - 3827.
whey-protein isolate - mass-spectrometry - amino-acids - gelation - peptide - coagulation - mechanism - purification - absorption - bromelain
Upon hydrolysis with chymotrypsin, soy glycinin has a strong tendency to aggregate. The regions of glycinin from which the aggregating peptides originate were identified by accumulative-quantitative peptide mapping. To this end, the aggregating peptides were further hydrolyzed with trypsin to obtain peptides of which the sequence can be identified using RP-HPLC-MS/MS. This resulted in a hydrolysate in which 90% of the proteinaceous material was dissolved. The soluble fraction was analyzed using the method of accumulative-quantitative peptide mapping: fractionation using ion exchange chromatography, followed by identification of peptides by RP-HPLC-MS/MS, quantification based on the absorbance at 214 nm, and finally peptide mapping. For the peptide mapping the proportions in which each of the five glycinin subunits are present, as determined by Edman degradation, were taken into account. The results showed that mainly the basic polypeptide and a part of the acidic polypeptide, close to the location of the disulfide bridge between the basic and acidic polypeptides, are present in the aggregating peptide fraction. On the basis of the results obtained, an aggregation mechanism was proposed. The hydrophilic acidic polypeptides shield the hydrophobic basic polypeptides, and the former are preferentially degraded upon hydrolysis. This results in a net increase in hydrophobicity of the remaining material, which mainly consists of the basic polypeptide fragments. This increase in hydrophobicity is proposed to be the driving force in the aggregation of chymotrypsin-derived peptides of glycinin.
A New Device for Studying Deep-Frying Behavior of Batters and Resulting Crust Properties
Visser, J.E. ; Beukelaer, H.J. de; Hamer, R.J. ; Vliet, T. van - \ 2008
Cereal Chemistry 85 (2008)3. - ISSN 0009-0352 - p. 417 - 424.
oil uptake - fried batters - fat - mechanism - food - crispness - flour
The formation and properties of a crust during and after deep frying are difficult to study. Batter pickup (the amount of batter adhering to a product) and core properties affect crust formation and properties of the crust in such way that it is difficult to compare batters of different viscosity or cores with different properties. Moreover, it is often difficult and laborious to separate the crust/batter from the core. Another problem is the poor reproducibility of many fried products. A deep-fried model (DFM) was designed, making it possible to study crust formation and crust properties without the difficulties stated above. Two different batter types and three cores have been used to test the system. Crusts obtained from the DFM were evaluated on several physiochemical properties and compared with crusts found around commercial deep-fried products. Results show that crusts obtained with the DFM system are comparable to crusts of commercial products. The good reproducibility of the DFM crusts resulted in low variance in analytical results compared with commercial crusts. This high reproducibility, the versatility of the system, and the ease with which the system can be used offer clear benefits for many potential applications.
Combined carbon and hydrogen isotope fractionation investigations for elucidating benzene biodegradation pathways
Fischer, A. ; Herklotz, I. ; Herrmann, S. ; Thullner, M. ; Weelink, S.A.B. ; Stams, A.J.M. ; Richnow, H.H. ; Vogt, C. - \ 2008
Environmental Science and Technology 42 (2008)12. - ISSN 0013-936X - p. 4356 - 4363.
aromatic hydroxylation - anaerobic biodegradation - toluene 4-monooxygenase - aerobic biodegradation - pseudomonas-putida - stable carbon - degradation - transformation - enrichment - mechanism
Recently, combined carbon and hydrogen isotope fractionation investigations have emerged as a powerful tool for the characterization of reaction mechanisms relevant for the removal of organic pollutants. Here, we applied this approach in order to differentiate benzene biodegradation pathways under oxic and anoxic conditions in laboratory experiments. Carbon and hydrogen isotope fractionation of benzene was studied with four different aerobic strains using a monooxygenase or a dioxygenase for the initial benzene attack, a facultative anaerobic chlorate-reducing strain as well as a sulfate-reducing mixed culture. Carbon and hydrogen enrichment factors (epsilon(C), epsilon(H)) varied for the specific pathways and degradation conditions, respectively, so that from the individual enrichment factors only limited information could be obtained for the identification of benzene biodegradation pathways. However, using the slope derived from hydrogen vs carbon isotope discriminations or the ratio of hydrogen to carbon enrichment factors (lambda = deltaH/ deltaC approximately epsilon(H)/epsilon(C)), benzene degradation mechanisms could be distinguished. Although experimentally determined lambda values partially overlapped, ranges could be determined for different benzene biodegradation pathways. Specific lambda values were <2 for dihydroxylation, between 7 and 9 for monohydroxylation, and > 17 for anaerobic degradation. Moreover, variations in lambda values suggest that more than one reaction mechanism exists for monohydroxylation as well as for anaerobic benzene degradation under nitrate-reducing, sulfate-reducing, or methanogenic conditions. Our results show that the combined carbon and hydrogen isotope fractionation approach has potential to elucidate biodegradation pathways of pollutants in field and laboratory microcosm studies
Photoprotection in higher plants: The putative quenching site is conserved in all outer light-harvesting complexes of Photosystem II
Mozzo, M. ; Passarini, F. ; Bassi, R. ; Amerongen, H. van; Croce, R. - \ 2008
Biochimica et Biophysica Acta. B, Bioenergetics 1777 (2008)10. - ISSN 0005-2728 - p. 1263 - 1267.
antenna protein cp26 - energy-dissipation - chlorophyll-a - mutation analysis - green plants - lhcii - mechanism - binding - xanthophylls - identification
In bright sunlight, the amount of energy harvested by plants exceeds the electron transport capacity of Photosystem II in the chloroplasts. The excess energy can lead to severe damage of the photosynthetic apparatus and to avoid this, part of the energy is thermally dissipated via a mechanism called non-photochemical quenching (NPQ). It has been found that LHCII, the major antenna complex of Photosystem II, is involved in this mechanism and it was proposed that its quenching site is formed by the cluster of strongly interacting pigments: chlorophylls 611 and 612 and lutein 620. In the present work we have investigated the interactions between the pigments in this cluster not only for LHCII, but also for the homologous minor antenna complexes CP24, CP26 and CP29. Use was made of wild-type and mutated reconstituted complexes that were analyzed with (low-temperature) absorption and circular-dichroism spectroscopy as well as by biochemical methods. The pigments show strong interactions that lead to highly specific spectroscopic properties that appear to be identical for LHCII, CP26 and CP29. The interactions are similar but not identical for CP24. It is concluded that if the 611/612/620 domain is responsible for the quenching in LHCII, then all these antenna complexes are prepared to act as a quencher. This can explain the finding that none of the Lhcb complexes seems to be strictly required for NPQ while, in the absence of all of them, NPQ is abolished.
Complex coacervate core micro-emulsions
Hofs, P.S. ; Keizer, A. de; Burgh, S. van der; Leermakers, F.A.M. ; Cohen Stuart, M.A. ; Millard, P.E. ; Muller, A.H.E. - \ 2008
Soft Matter 4 (2008)7. - ISSN 1744-683X - p. 1473 - 1482.
block-copolymer micelles - interacting chain molecules - diblock copolymers - statistical-theory - aqueous-media - polyelectrolyte - kinetics - mechanism - polymerization - solubilization
Complex coacervate core micelles form in aqueous solutions from poly(acrylic acid)-block-poly(acrylamide) (PAAxPAAmy, x and y denote degree of polymerization) and poly(N,N-dimethyl aminoethyl methacrylate) (PDMAEMA150) around the stoichiometric charge ratio of the two components. The hydrodynamic radius, Rh, can be increased by adding oppositely charged homopolyelectrolytes, PAA140 and PDMAEMA150, at the stoichiometric charge ratio. Mixing the components in NaNO3 gives particles in highly aggregated metastable states, whose Rh remain unchanged (less than 5% deviation) for at least 1 month. The Rh increases more strongly with increasing addition of oppositely charged homopolyelectrolytes than is predicted by a geometrical packing model, which relates surface and volume of the particles. Preparation in a phosphate buffer ¿ known to weaken the electrostatic interactions between PAA and PDMAEMA ¿ yields swollen particles called complex coacervate core micro-emulsions (C3-Es) whose Rh increase is close to that predicted by the model. These are believed to be in the stable state (lowest free energy). A two-regime increase in Rh is observed, which is attributed to a transition from more star-like to crew-cut-like, as shown by self-consistent field calculations. Varying the length of the neutral and polyelectrolyte block in electrophoretic mobility measurements shows that for long neutral blocks (PAA26PAAm405 and PAA39PAAm381) the -potential is nearly zero. For shorter neutral blocks the -potential is around -10 mV. This shows that the C3-Es have excess charge, which can be almost completely screened by long enough neutral blocks.
Non-Gaussian curvature distribution of actin-propelled biomimetric colloid trajectories
Schmidt, S. ; Gucht, J. van der; Biesheuvel, P.M. ; Weinkamer, R. ; Helfer, E. ; Fery, A. - \ 2008
European Biophysics Journal 37 (2008)8. - ISSN 0175-7571 - p. 1361 - 1366.
comet tails - lipid vesicles - motility - polymerization - listeria - forces - filaments - mechanism - movement - driven
We analyze the motion of colloids propelled by a comet-like tail of polymerizing actin filaments. The curvature of the particle trajectories deviates strongly from a Gaussian distribution, implying that the underlying microscopic processes are fluctuating in a non-independent manner. Trajectories for beads of different size all showed the same non-Gaussian behavior, while the mean curvature decreased weakly with size. A stochastic simulation that includes nucleation, force-dependent dissociation, growth, and capping of filaments, shows that the non-Gaussian curvature distribution can be explained by a positive feedback mechanism in which attached chains under higher tension are more likely to snap
Phase diagram for a mixture of colloids and polymers with equal size
Tuinier, R. ; Smith, P.A. ; Poon, W.C.K. ; Egelhaaf, S.U. ; Aarts, D.G.A.L. ; Lekkerkerker, H.N.W. ; Fleer, G.J. - \ 2008
Europhysics Letters 82 (2008). - ISSN 0295-5075
volume-restriction - behavior - spheres - dispersions - suspensions - cyclohexane - separations - mechanism - chains - latex
We present the phase diagram of a colloid-polymer mixture in which the radius a of the colloidal spheres is approximately the same as the radius R of a polymer coil (q=R/a1). A three-phase coexistence region is experimentally observed, previously only reported for colloid-polymer mixtures with smaller polymer chains (q0.6). A recently developed generalized free-volume theory (GFVT) for mixtures of hard spheres and non-adsorbing excluded-volume polymer chains gives a quantitative description of the phase diagram. Monte Carlo simulations also agree well with experiment
Long-term fish consumption and n-3 fatty acid intake in relation to (sudden) coronary heart disease death: the Zutphen Study
Streppel, M.T. ; Ocke, M.C. ; Boshuizen, H.C. ; Kok, F.J. ; Kromhout, D. - \ 2008
European Heart Journal 29 (2008)16. - ISSN 0195-668X - p. 2024 - 2030.
middle-aged men - cardiac death - risk-factors - cardiovascular-disease - myocardial-infarction - time-scale - mortality - prevention - metaanalysis - mechanism
Aims: To assess the relationship between fish consumption or eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA) intake from fish, and (sudden) coronary death. Methods and results: The impact of recent and long-term fish consumption and EPA+DHA intake on (sudden) coronary death was investigated in the Zutphen Study, a cohort of 1373 men born between 1900 and 1920, and examined repeatedly between 1960 and 2000. Hazard ratios were obtained from time-dependent Cox regression models. The associations between long-term fish consumption, EPA+DHA intake, and (sudden) coronary death were stronger than those of recent consumption. Long-term fish consumption was inversely associated (borderline significant) with coronary heart disease (CHD) death; however, the strength of the association decreased from age 50 [HR: 0.32 (95% CI: 0.13¿0.80)] until age 80 [HR: 1.34 (0.58¿3.12)]. For men with a daily EPA+DHA intake from fish below 250 mg compared with no intake, CHD death risk was reduced to the same extent as for men with a daily intake above 250 mg (P-value for trend: 0.27). Moreover, long-term fatty-fish consumption lowered the risk of sudden coronary death [HR: 0.46 (0.27¿0.78)]. Conclusion: The strength of the association between long-term fish consumption and CHD death decreased with increasing age. Fatty-fish consumption lowered sudden coronary death risk. There was no clear dose¿response relationship between EPA+DHA intake and (sudden) coronary death.
Pro- and antioxidative properties of medicinal mushroom extracts
Song, W. ; Griensven, L.J.L.D. van - \ 2008
International Journal of Medicinal Mushrooms 10 (2008)4. - ISSN 1521-9437 - p. 315 - 324.
oxidative-stress - hydrogen-peroxide - cancer-cells - apoptosis - mitochondria - polyphenols - tyrosinase - induction - transport - mechanism
Hot water extracts of 2 groups of medicinal mushrooms have been tested from the genera Agaricus, Antrodia, Auricularia, Coprinus, Cordyceps, Hericium, Grifola, Ganoderma, Lentinus, Phellinus, and Trametes for ROS-generating activity in human cells and for DPPH-TEAC antioxidant activity. Group 1 comprised 39 commercial extracts (7 species), and group 2 comprised 12 fruiting body extracts made from 11 different species of culinary-medicinal mushrooms. For both groups, the ROS-generating activity and the antioxidant activity were strongly correlated, as were their respective polysaccharide and polyphenol contents. The extracts differ in their amounts of the latter components but not in the ratio of the two. The slopes of the correlation curves were different for both groups, which is related to the higher polyphenol content of the commercial extracts. It is suggested that possible excess cell defense¿related intracellular ROS generated by mushroom extracts may be downregulated by the antioxidant components present in the same extracts.
Vortex-wake interactions of a flapping foil that models animal swimming and flight
Lentink, D. ; Muijres, F.T. ; Donker-Duyvis, F.J. ; Leeuwen, J.L. van - \ 2008
Journal of Experimental Biology 211 (2008)2. - ISSN 0022-0949 - p. 267 - 273.
unsteady aerodynamic performance - low reynolds-numbers - soap films - insect flight - flow - mechanism - vortices - dynamics - airfoil
The fluid dynamics of many swimming and flying animals involves the generation and shedding of vortices into the wake. Here we studied the dynamics of similar vortices shed by a simple two-dimensional flapping foil in a soap-film tunnel. The flapping foil models an animal wing, fin or tail in forward locomotion. The vortical flow induced by the foil is correlated to (the resulting) thickness variations in the soap film. We visualized these thickness variations through light diffraction and recorded it with a digital high speed camera. This set-up enabled us to study the influence of foil kinematics on vortex-wake interactions. We varied the dimensionless wavelength of the foil (*=4¿24) at a constant dimensionless flapping amplitude (A*=1.5) and geometric angle of attack amplitude (A,geo=15°). The corresponding Reynolds number was of the order of 1000. Such values are relevant for animal swimming and flight. We found that a significant leading edge vortex (LEV) was generated by the foil at low dimensionless wavelengths (*
Cumulative risk assessment of the exposure to organophosphorus and carbamate insecticides in the dutch diet
Boon, P.E. ; Voet, H. van der; Raaij, M.T.M. van; Klaveren, J.D. van - \ 2008
Food and Chemical Toxicology 46 (2008)9. - ISSN 0278-6915 - p. 3090 - 3098.
pesticide-residues - toxicity - mechanism - dioxins - pcbs
We report the acute cumulative exposure to organophosphorus insecticides (OPs) and carbamates in the Dutch population and young children (1-6 years) via the diet. Residue data were derived from Dutch monitoring programmes performed during 2003-2005, and food consumption levels from the Dutch National Food Consumption Survey 1997/1998. The relative potency factor (RPF) approach was used to cumulate the exposure to OPs and carbamates using acephate and oxamyl as index compound respectively. The exposure was estimated using the probabilistic approach, including unit variability and processing effects. We demonstrate that about 3% of the composite samples analysed for OPs and 0.2% for carbamates contain combinations of these pesticides. The P99.9 of exposure to OPs and carbamates in the total Dutch population equals 23 and 0.64mug/kg BW/d respectively. For young children the corresponding exposure levels are 57 and 1.47mug/kg BW/d. When comparing the P99.9 of exposure with the ARfD, 50 and 9mug/kg BW/d for acephate and oxamyl respectively, there is only a possible health risk for young children. Spinach contributed most to the exposure to OPs in both age groups, followed by orange and mandarin. For carbamates apple (sauce) was the main product determining the exposure.
Characterization of a thermostable dihydrodipicolinate synthase from Thermoanaerobacter tengcongensis
Wolterink-van Loo, S. ; Levisson, M. ; Cabrières, M.C. ; Franssen, M.C.R. ; Oost, J. van der - \ 2008
Extremophiles 12 (2008)3. - ISSN 1431-0651 - p. 461 - 469.
escherichia-coli - crystal-structure - l-lysine - beta-semialdehyde - semi-aldehyde - biosynthesis - resolution - mechanism - reductase - acid
Dihydrodipicolinate synthase (DHDPS) catalyses the first reaction of the (S)-lysine biosynthesis pathway in bacteria and plants. The hypothetical gene for dihydrodipicolinate synthase (dapA) of Thermoanaerobacter tengcongensis was found in a cluster containing several genes of the diaminopimelate lysine¿synthesis pathway. The dapA gene was cloned in Escherichia coli, DHDPS was subsequently produced and purified to homogeneity. The T. tengcongensis DHDPS was found to be thermostable (T 0.5 = 3 h at 90°C). The specific condensation of pyruvate and (S)-aspartate-ß -semialdehyde was catalyzed optimally at 80°C at pH 8.0. Enzyme kinetics were determined at 60°C, as close as possible to in vivo conditions. The established kinetic parameters were in the same range as for example E. coli dihydrodipicolinate synthase. The specific activity of the T. tengcongensis DHDPS was relatively high even at 30°C. Like most dihydrodipicolinate synthases known at present, the DHDPS of T. tengcongensis seems to be a tetramer. A structural model reveals that the active site is well conserved. The binding site of the allosteric inhibitor lysine appears not to be conserved, which agrees with the fact that the DHDPS of T. tengcongensis is not inhibited by lysine under physiological conditions.
Effect of gelatinization and hydrolysis conditions on the selectivity of starch hydrolysis with alpha-amylase from Bacillus licheniformis
Baks, T. ; Bruins, M.E. ; Matser, A.M. ; Janssen, A.E.M. ; Boom, R.M. - \ 2008
Journal of Agricultural and Food Chemistry 56 (2008)2. - ISSN 0021-8561 - p. 488 - 495.
high hydrostatic-pressure - wheat-starch - enzymatic-hydrolysis - phase-transformations - temperature - mechanism - enzymes - stability - complexes - products
Enzymatic hydrolysis of starch can be used to obtain various valuable hydrolyzates with different compositions. The effects of starch pretreatment, enzyme addition point, and hydrolysis conditions on the hydrolyzate composition and reaction rate during wheat starch hydrolysis with ¿-amylase from Bacillus licheniformis were compared. Suspensions of native starch or starch gelatinized at different conditions either with or without enzyme were hydrolyzed. During hydrolysis, the oligosaccharide concentration, the dextrose equivalent, and the enzyme activity were determined. We found that the hydrolyzate composition was affected by the type of starch pretreatment and the enzyme addition point but that it was just minimally affected by the pressure applied during hydrolysis, as long as gelatinization was complete. The differences between hydrolysis of thermally gelatinized, high-pressure gelatinized, and native starch were explained by considering the granule structure and the specific surface area of the granules. These results show that the hydrolyzate composition can be influenced by choosing different process sequences and conditions.
Screening of synthetic and plant-derived compounds for (anti)estrogenic and (anti)androgenic activities
Bovee, T.F.H. ; Schoonen, W.G.E.J. ; Hamers, A.R.M. ; Bento, M.J. ; Peijnenburg, A.A.C.M. - \ 2008
Analytical and Bioanalytical Chemistry 390 (2008)4. - ISSN 1618-2642 - p. 1111 - 1119.
human estrogen-receptor - in-vitro - prostate-cancer - breast-cancer - pure antiestrogen - er-beta - alpha - cells - vivo - mechanism
Recently we constructed yeast cells that either express the human estrogen receptor ¿ or the human androgen receptor in combination with a consensus ERE or ARE repeat in the promoter region of a green fluorescent protein (yEGFP) read-out system. These bioassays were proven to be highly specific for their cognate agonistic compounds. In this study the value of these yeast bioassays was assessed for analysis of compounds with antagonistic properties. Several pure antagonists, selective estrogen receptor modulators (SERMs) and plant-derived compounds were tested. The pure antiestrogens ICI 182,780 and RU 58668 were also classified as pure ER antagonists in the yeast estrogen bioassay and the pure antiandrogen flutamide was also a pure AR antagonist in the yeast androgen bioassay. The plant-derived compounds flavone and guggulsterone displayed both antiestrogenic and antiandrogenic activities, while 3,3¿-diindolylmethane (DIM) and equol combined an estrogenic mode of action with an antiandrogenic activity. Indol-3-carbinol (I3C) only showed an antiandrogenic activity. Coumestrol, genistein, naringenin and 8-prenylnaringenin were estrogenic and acted additively, while the plant sterols failed to show any effect. Although hormonally inactive, in vitro and in vivo metabolism of the aforementioned plant sterols may still lead to the formation of active metabolites in other test systems.
Enzyme-induced aggregation and gelation of proteins
Creusot, N.P. ; Gruppen, H. - \ 2007
Biotechnology Advances 25 (2007)6. - ISSN 0734-9750 - p. 597 - 601.
protease-induced aggregation - bacillus-licheniformis - beta-lactoglobulin - alpha-lactalbumin - whey proteins - identification - hydrolysis - mechanism - glu
This paper provides a brief overview of the effects of protein hydrolysis on aggregation and gel forming properties of protein systems. Among the food globular proteins, whey proteins and soy proteins are the most extensively studied for their ability to form different textures upon proteolysis. Recent studies were focused on identifying aggregating peptides and on mechanisms of aggregation and gelation.
Baculovirus envelope fusion proteins F and GP64 exploit distinct receptors to gain entry into cultured insect cells
Westenberg, M. ; Uijtdewilligen, P. ; Vlak, J.M. - \ 2007
Journal of General Virology 88 (2007). - ISSN 0022-1317 - p. 3302 - 3306.
nuclear polyhedrosis-virus - californica multicapsid nucleopolyhedrovirus - membrane-fusion - recombinant baculovirus - lymantria-dispar - mammalian-cells - genome sequence - lines - glycoprotein - mechanism
Group II nucleopolyhedroviruses (NPVs), e.g. Helicoverpa armigera (Hear) NPV and Spodoptera exigua (Se) MNPV (multiple NPV), lack a GP64-like protein that is present in group I NPVs, e.g. Autographa californica (Ac)MNPV, but have an unrelated envelope fusion protein named F. Three AcMNPV viruses were constructed by introducing AcMNPV gp64, HearNPV f or SeMNPV f genes, respectively, into a gp64-negative AcMNPV bacmid. Sf21 cells were incubated with different amounts of inactivated budded virus to occupy receptors and were subsequently infected with a fixed amount of infectious virus to compete for attachment. The results suggest that GP64 and F act on their own and use different receptors, while the two different F proteins exploit the same receptor. Additionally, gp64-null AcMNPV pseudotyped with baculovirus F was, in contrast to GP64, unable to transduce mammalian cells, indicating that mammalian cells do not possess baculovirus F protein receptors despite the structural similarity of baculovirus F to vertebrate viral fusion proteins.
On the stability of (highly aggregated) polyelectrolyte complexes containing a charged block-neutral diblockcopolymer
Hofs, P.S. ; Keizer, A. de; Cohen Stuart, M.A. - \ 2007
The Journal of Physical Chemistry Part B: Condensed Matter, Materials, Surfaces, Interfaces & Biophysical 111 (2007). - ISSN 1520-6106 - p. 5621 - 5627.
protein-polysaccharide interactions - soluble complexes - core micelles - mechanism - kinetics - interpolyelectrolyte - coacervation - exchange - length
Using light scattering and cryogenic transmission electron microscopy, we show that highly aggregated polyelectrolyte complexes (HAPECs) composed of poly([4-(2-aminoethylthio)butylene] hydrochloride)49-block-poly(ethylene oxide)212 and poly(acrylic acid) (PAA) of varying lengths (140, 160, and 2000 monomeric units) are metastable or unstable if the method of preparation is direct mixing of two solutions containing the oppositely charged components. The stability of the resulting HAPECs decreases with decreasing neutral-block content and with increasing deviation from 1:1 mixing (expressed in number of chargeable groups) of the oppositely charged polyelectrolytes, most probably for electrostatic reasons. The difference between the metastable and stable states, obtained with pH titrations, increases with increasing PAA length and increasing pH mismatch between the two solutions with the oppositely charged components
Thioflavin T and Birefringence Assays to Determine the Conversion of Proteins into Fibrils
Bolder, S.G. ; Sagis, L.M.C. ; Venema, P. ; Linden, E. van der - \ 2007
Langmuir 23 (2007). - ISSN 0743-7463 - p. 4144 - 4147.
beta-lactoglobulin gels - amyloid fibrils - whey proteins - low ph - aggregation - fibrillation - fabrication - mechanism - gelation - route
The conversion of protein monomers into fibrils can be determined using the centrifugal filtration method. The results of this method were used to calibrate steady-shear birefringence and Thioflavin T fluorescence measurements. For both measurements, a linear correlation with the fibril concentration was extracted, resulting in two fast assays to determine the fibril concentration quantitatively. From birefringence measurements and the conversion determined using the centrifugal filtration method, we were able to calculate more precise values for the birefringence per unit length of the fibrils (M) and the flexibility of the fibrils (ß)
Novel peptides with tyrosinase inhibitory activity
Schurink, M. ; Berkel, W.J.H. van; Wichers, H.J. ; Boeriu, C.G. - \ 2007
Peptides 28 (2007)3. - ISSN 0196-9781 - p. 485 - 495.
continuous spectrophotometric method - mushroom polyphenol oxidase - diphenolase activities - musca-domestica - spot-synthesis - kojic acid - monophenolase - mechanism - prospects - protein
Tyrosinase inhibition by peptides may find its application in food, cosmetics or medicine. In order to identify novel tyrosinase inhibitory peptides, protein-based peptide libraries made by SPOT synthesis were used to screen for peptides that show direct interaction with tyrosinase. One of the peptide libraries studied consists of overlapping, octameric peptides derived from industrial proteins as ß-casein, ¿-lactalbumin, ß-lactoglobulin, ovalbumin, gliadin, glycinin, and ß-conglycinin. On-membrane activity staining resulted in a set of peptides that are not only able to bind to tyrosinase, but are able to inhibit tyrosinase as well. Peptides containing aspartic or glutamic acid residues usually do not bind very well to tyrosinase. Strong tyrosinase-binding peptides always contain one or more arginine residues, often in combination with phenylalanine, while lysine residues can be found equally among nonbinding peptides as well as moderate tyrosinase-binding peptides. The presence of the hydrophobic, aliphatic residues valine, alanine or leucine appears to be important for tyrosinase inhibition. Therefore, good tyrosinase inhibitory peptides preferably contain arginine and/or phenylalanine in combination with valine, alanine and/or leucine.
Polylactide films formed by immersion precipitation: Effects of additives, nonsolvent, and temperature
Sawalha, H.I.M. ; Schroën, C.G.P.H. ; Boom, R.M. - \ 2007
Journal of Applied Polymer Science 104 (2007)2. - ISSN 0021-8995 - p. 959 - 971.
induced phase-separation - pvdf membrane formation - asymmetric membranes - systems - water - morphology - methanol - transitions - mechanism - behavior
The influence of nonsolvent, crystallinity of the polymer film, and addition of dodecane (a poor solvent for the polymer and for the nonsolvent) on the morphology of polylactides films has been investigated and was related to phase separation behavior. Both amorphous poly-DL-lactide (PDLLA) and crystalline poly-L-lactide (PLLA) were dissolved in dichloromethane, and subsequently films were made by immersion in nonsolvent baths. PDLLA gave dense films without any internal structure, since the structure was not solidified by crystallization or glassification. PLLA films show varying structure depending on the nonsolvent. With methanol, asymmetric morphologies were observed as a result from combined liquid-liquid demixing and crystallization, while with water symmetric spherulitic structures were formed. As a next step, dodecane was added, which is not miscible with the nonsolvent, and we found it to have a strong influence on the morphology of the films. The PDLLA films with dodecane did not collapse: a closed cell structure was obtained. In PLLA films, dodecane speeds up phase separation and induces faster crystallization in the films, and the porosity, size of the pores, and interconnectivity increased. When the PLLA solutions were subjected to a heat pretreatment, crystallization could be postponed, which yielded a cellular structure around dodecane, which did not contain spherulites anymore
In vivo and in vitro effects of tea extracts on enterotoxigenic Escherichia coli-induced intestinal fluid loss in animal models
Bruins, M.J. ; Cermak, R. ; Kiers, J.L. ; Meulen, J. van der; Amelsvoort, J.M.M. van; Klinken, B.J. - \ 2006
Journal of Pediatric Gastroenterology and Nutrition 43 (2006). - ISSN 0277-2116 - p. 459 - 469.
antibacterial activity - net absorption - transport - flavonoids - secretion - mechanism - diarrhea - pigs
OBJECTIVES: Enterotoxigenic Escherichia coli (ETEC) infection is a major cause of dehydrating diarrhoea in infants and early-weaned piglets living under subhygienic conditions. We studied the effect of different tea types and subfractions on the intestinal fluid and electrolyte losses involved in ETEC diarrhoea. MATERIALS AND METHODS: Jejunal segments of anaesthetised piglets were infected with ETEC or ETEC heat-labile toxin (LT) and subsequently perfused for 8 hours with control or tea solutions containing green or black tea extract (BTE) or 3 different BTE subfractions containing small-size, large-size or no phenolics. Changes in intestinal fluid and electrolyte net absorption were measured. To assess the antisecretory effects of tea, BTE was incubated before or after administration of the secretagogue forskolin in rat jejunal tissue placed in Ussing chambers and Cl- secretion measured as changes in short-circuit current (I(SC)). RESULTS: Enterotoxigenic E. coli infection of piglet jejunal segments significantly reduced net absorption of fluid, Na+ and Cl- and increased net secretion of K+ compared with controls. Perfusion of the ETEC-infected segments with both 3 g/L green tea extract and BTE significantly inhibited these disturbances in fluid and electrolyte balance. The BTE subfraction rich in polymeric phenolics but not the other subfractions improved the fluid and electrolyte balance. Addition of forskolin to rat jejunal tissue induced a significant increase in I(SC). Pretreating but not posttreating the jejunal tissue with BTE inhibited the forskolin-induced increase in I(SC). CONCLUSIONS: Tea may inhibit net fluid and electrolyte losses involved in secretory diarrhoea from ETEC.
Comparison of gas-phase acidities of some carbon acids with their rates of hydron exchange in methanolic methoxide
DeTuri, V.F. ; Koch, H.F. ; Koch, J.G. ; Lodder, G. ; Mishima, M. ; Zuilhof, H. ; Abrams, N.M. ; Anders, C.E. ; Biffinger, J.C. ; Han, P. ; Kurland, A.R. ; Nichols, J.M. ; Ruminski, A.M. ; Smith, P.R. ; Vasey, K.D.J. - \ 2006
Journal of Physical Organic Chemistry 19 (2006)5. - ISSN 0894-3230 - p. 308 - 317.
proton-transfer reactions - methanolic sodium methoxide - bronsted correlation - isotope exchange - hydrocarbons - mechanism - solvolysis - solvation - ketones - benzyl
Hydron exchange reaction rates, k(exch)M(-1) s(-1), using methanolic sodium methoxide are compared with gas-phase acidities, Delta G(Acid)(0) kcal/mol, for four 9-YPhenylfluorenes-9-H-i, seven (YC6H4CH)-H-i(CF3)(2), seven YC6H4-(CHClCF3)-H-i, and (C6F5H)-H-i. Fourteen of the fluorinated benzylic compounds and pentafluorobenzene result in near unity experimental hydrogen isotope effects that suggest substantial amounts of internal return associated with the exchange process. Although the reactions of 9-phenylfluorene have experimental isotope effects that appear to be normal in value, they do not obey the Swain-Schaad relationship. This suggests that they occur with small amounts of internal return. The entropies of activation, Delta S-double dagger, are +12 to +14eu, for the benzylic compounds and different significantly from those for the 9-YPhenylfluorenes, Delta S-double dagger of -8 to - 12 eu. The Delta S-double dagger similar to 1 eu for the reactions of pentafluorobenzene falls between the other compounds. Density functional calculations using B3LYP/6-31+G(d,p) are reported for the reactions of CH3O-(HOCH3)(3) with C6F5H, C6H5CH(CF3)(2), C6H5CHClCF3, and 9-phenylfluorene. Copyright (c) 2006 John Wiley & Sons, Ltd.