Staff Publications

Staff Publications

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

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

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

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

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Cu2+ or Fe3+ Terpyridine/Aptamer Conjugates : Nucleoapzymes Catalyzing the Oxidation of Dopamine to Aminochrome
Biniuri, Yonatan ; Albada, Bauke ; Wolff, Mariusz ; Golub, Eyal ; Gelman, Dmitri ; Willner, Itamar - \ 2018
ACS Catalysis 8 (2018)3. - ISSN 2155-5435 - p. 1802 - 1809.
binding - catalyst - DNA - kinetic model - metal complex - molecular dynamics
A concept to tailor catalytic nucleic acid structures is introduced. The method involves the covalent conjugation of catalytically active metal ion complexes to sequence-specific ligand-binding nucleic acids (aptamers) yielding hybrids termed "nucleoapzymes" that act as enzyme-mimicking nucleic acid based structures. The concentration of the substrate by the aptamer binding site, in close proximity to the metal ion complex catalytic site, models the active site structures of native enzymes and yields catalytic systems. The possibility to tether the catalytic sites to the 3′- or 5′-ends of the aptamer, to internal bases associated with the aptamer sequence, or the feasibility to introduce arbitrary flexible nucleic acid chains between the metal ion complex catalyst and the aptamer binding site provides a rich arsenal of diverse nucleoapzymes for each chemical transformation. The synthesis and characterization of Cu2+-terpyridine nucleoapzymes and of Fe3+-terpyridine nucleoapzymes that catalyze the oxidation of dopamine to aminochrome by H2O2 is presented. One of the Cu2+-terpyridine nucleoapzymes reveals a 60-fold catalytic enhancement in comparison to the separated catalyst/aptamer units. Similarly, one Fe3+-terpyridine nucleoapzyme reveals a 140-fold catalytic enhancement in comparison to the separated catalyst/aptamer units. The different Cu2+-terpyridine nucleoapzymes reveal different activities, dominated by the relative spatial configurations of the catalytic site with respect to the dopamine (substrate) binding site. Molecular dynamics simulations were used to probe the association of the dopamine substrate to the different nucleoapzymes and to rationalize the experimental catalytic performance of the nucleoapzymes in terms of their computed structures. The nucleoapzyme concept bridges homogeneous catalysis with the binding properties of nucleic acids to yield catalysts operating in aqueous media.
Biofunctionalized nanoporous aluminum oxide culture chips : for capture and growth of bacteria
Debrassi, A. - \ 2016
University. Promotor(en): Han Zuilhof; Willem de Vos; Tom Wennekes. - Wageningen : Wageningen University - ISBN 9789462576179 - 218 p.
aluminium oxide - porous media - unimolecular films - immobilization - bacteria - binding - antibodies - aluminiumoxide - poreus medium - unimoleculaire films - immobilisatie - bacteriën - binden - antilichamen

Porous aluminum oxide (PAO) is a nanostructured material used for various biotechnological applications, including the culturing microorganisms and other types of cells. The ability to chemically modify the PAO surface and tailor its surface properties is a promising way to expand and refine its applications. The immobilization of biomolecules on PAO that specifically interact with and bind to target bacteria would enable the capture and subsequent growth of bacteria on the same surface, and this was the ultimate goal of the research presented in this thesis.

After a general introduction to the overall subject of this thesis, presented in Chapter 1, the most commonly used and recent methods to prepare glycosurfaces are reviewed and compared on their merits and drawbacks in Chapter 2. Although there are a great number of techniques, the main challenge that still remains is to develop an accessible, reproducible and inexpensive approach that produces well-defined and stable glycosurfaces using as few steps as possible. The most used analytical techniques for the characterization of glycosurfaces and several applications of these surfaces in the binding, capture, and sensing of bacteria and bacterial toxins were also discussed in Chapter 2.

Biofunctionalization of surfaces in general requires a stepwise approach, in which it is very important to have a stable monolayer as the first step. At the beginning of this research it was known that various functional groups were able to react with (porous) aluminum oxide, but there was no comprehensive study comparing the stability of these modified surfaces under the conditions that are important for microbiological applications. In Chapter 3, the PAO surface was modified with various functional groups known to react with PAO (carboxylic acid, α-hydroxycarboxylic acid, alkyne, alkene, phosphonic acid, and silane), and the stability of these modified surfaces was assessed over a range of pH and temperatures that are relevant for microbial growth. Silane and phosphonate-modified PAO surfaces with a hydrophobic monolayer proved to be the most stable ones, but the phosphonate modification was both more easily applied and reproducible. This modification was stable for at least two weeks in buffer solutions with pH values between 4 and 8, and at temperatures up to 40 °C. Only at elevated temperatures of 60 °C and 80 °C under hydrolytic conditions it was observed that the stability of the same monolayer on PAO decreased gradually. As a proof-of-principle for the biofunctionalization and bacterial capture on this PAO phosphonate monolayer, an alkyne-terminated monolayer was biofunctionalized via a CuAAC click reaction with an azido-mannoside and the binding and growth of Lactobacillus plantarum was successfully demonstrated.

In Chapter 4 various approaches to install reactive groups onto the phosphonate-modified PAO surface were developed, creating a (bio)functionalization “tool-box”. PAO surfaces presenting different terminal reactive groups were prepared, such as azide, alkyne, alkene, thiol, isothiocyanate, and N-hydroxysuccinimide (NHS), starting from a single, straightforward and stable initial modification with a bromo-terminated phosphonic acid. These reactive surfaces were then used to immobilize (bio)molecules, including carbohydrates and proteins. Fluorescently labeled bovine serum albumin (BSA) was covalently immobilized on the PAO surface as a proof-of-principle, and it was shown that a range of bacteria could still grow on the BSA-functionalized PAO surface.

With a PAO (bio)functionalization tool-box in hand, the successful proof-of-principle mannoside-dependent binding and growth of L. plantarum on PAO (Chapter 3) was further investigated and expanded upon (Chapter 5). The parameters involved in the preparation of these surfaces and in the binding with L. plantarum were investigated in more detail in Chapter 5, such as the nature of the spacer connected to the mannoside derivative and the presence of soluble carbohydrates and bovine serum albumin (BSA) in the medium. The surfaces with the azido-mannoside with the long hydrophobic spacer showed the best binding of L. plantarum when compared to a long PEG-based hydrophilic spacer and a short hydrophobic one. The presence of a soluble a-glucoside did not prevent the binding of the bacteria to the mannose-presenting PAO, and similar results were obtained when BSA was present. Additionally, a mutant strain of L. plantarum that does not have the mannose-specific adhesion was not able to bind to the mannose-presenting PAO. When taken together, this proves that the mannoside–adhesin interaction is the main mechanism of binding the bacteria to the mannose-biofunctionalized PAO in this system.

In Chapter 6, the NHS-terminated PAO developed in Chapter 4 was used for the immobilization of antibodies against Escherichia coli. After an extensive optimization of the modification chemistry of the surfaces and the incubation conditions, commercially available anti-E. coli antibodies were immobilized on the PAO surface. Binding and washing experiments indeed demonstrated increased binding of E. coli on the antibody-presenting PAO surfaces, providing avenues for testing other bacteria such as Lactobacillus rhamnosus GG widely used in probiotic formulations worldwide.

In Chapter 7, the most important achievements of this project are discussed, together with additional ideas and recommendations for further research. Most notably some preliminary results are presented on the immobilization of two antibodies against L. rhamnosus GG: anti-L. rhamnosus GG, against the whole bacterial cell, and anti-SpaC, against only the SpaC part of the pili present on the cell surface of L. rhamnosus GG. Anti-L. rhamnosus GG antibody showed promising but not yet optimal increased binding of L. rhamnosus GG. Finally, some reflections on PAO and its (bio)functionalization are provided in the context of a risk analysis and technology assessment.

Comparing foam and interfacial properties of similarly charged protein–surfactant mixtures
Lech, F.J. ; Meinders, M.B.J. ; Wierenga, P.A. ; Gruppen, H. - \ 2015
Colloids and Surfaces. A: Physicochemical and Engineering Aspects 473 (2015). - ISSN 0927-7757 - p. 18 - 23.
sodium dodecyl-sulfate - bovine serum-albumin - air-water interfaces - beta-lactoglobulin - titration calorimetry - binding - sds - adsorption - rheology - layers
The foam stability of protein–surfactant mixtures strongly depends on the charge of the protein and the surfactant, as well as on their mixing ratio. Depending on the conditions, the mixtures will contain free proteins, free surfactants and/or protein–surfactant complexes. To be able to compare different protein–surfactant mixtures, generic knowledge about the occurrence of each of these states and their relative contribution to foam stability is essential. In this work, the foam stability and interfacial properties of bovine serum albumin (BSA) mixed with sodium dodecyl sulphate (SDS) as well the binding of SDS to BSA as are studied at different molar ratios (MR). A comparison is made with ß-lactoglobulin (BLG) mixed with SDS. Both proteins and SDS are negatively charged at pH 7. The foam stability in the presence of small amounts (up to MR 1) of SDS is half the value of the pure protein solutions. The foam stability for both protein surfactant mixtures reaches a minimum at MR 20. A further increase of the MR leads to an increase of foam stability. The foam stability of BLG–SDS at MR >20 follows the foam stability of pure SDS solutions at equivalent concentrations, while BSA–SDS mixtures have an offset and begin to increase from MR >50. This behaviour was also reflected in the surface pressure and complex dilatational elastic moduli, and could be linked to the binding of the surfactant to the proteins. Both proteins bind SDS at high and low affinity binding sites. BSA's high affinity binding sites have a binding stoichiometry of 5.5 molSDS/molprotein, and BLG's high affinity binding site has a stoichiometry of 0.8 molSDS/molprotein (determined by isothermal titration calorimetry). Binding to the low affinity binding sites, occurs with a binding ratio, leading to an accumulation of free surfactants. While the basic mechanisms underlying the foam properties of mixed systems are not explained in detail by this approach, the foam stability plots of both protein surfactant mixtures could be superimposed using the concentration of free SDS.
The heat shock transcription factor PsHSF1 of Phytophthora sojae is required for oxidative stress tolerance and detoxifying the plant oxidative burst
Sheng, Yuting ; Wang, Yonglin ; Meijer, H.J.G. ; Yang, Xinyu ; Hua, C. ; Ye, Wenwu ; Tao, Kai ; Liu, Xiaoyun ; Govers, F. ; Wang, Yuanchao - \ 2015
Environmental Microbiology 17 (2015)4. - ISSN 1462-2912 - p. 1351 - 1364.
signal-transduction - in-vivo - pathogen - infestans - expression - sequence - defense - laccase - binding - yeast
In the interaction between plant and microbial pathogens, reactive oxygen species (ROS) rapidly accumulate upon pathogen recognition at the infection site and play a central role in plant defence. However, the mechanisms that plant pathogens use to counteract ROS are still poorly understood especially in oomycetes, filamentous organisms that evolved independently from fungi. ROS detoxification depends on transcription factors (TFs) that are highly conserved in fungi but much less conserved in oomycetes. In this study, we identified the TF PsHSF1 that acts as a modulator of the oxidative stress response in the soybean stem and root rot pathogen Phytophthora sojae. We found that PsHSF1 is critical for pathogenicity in P.¿sojae by detoxifying the plant oxidative burst. ROS produced in plant defence can be detoxified by extracellular peroxidases and laccases which might be regulated by PsHSF1. Our study extends the understanding of ROS detoxification mechanism mediated by a heat shock TF in oomycetes.
Fluorescent Lectins for Local in Vivo Visualization of Peripheral Nerves
KleinJan, G.H. ; Buckle, T. ; Willigen, D.M. van; Oosterom, M.N. van; Spa, S.J. ; Kloosterboer, H.E. ; Leeuwen, F.W.B. van - \ 2014
Molecules 19 (2014)7. - ISSN 1420-3049 - p. 9876 - 9892.
nervous-system - sentinel node - immunohistochemical localization - radical prostatectomy - sulfate proteoglycan - axonal-transport - guided surgery - identification - carcinoma - binding
Damage to peripheral nerves caused during a surgical intervention often results in function loss. Fluorescence imaging has the potential to improve intraoperative identification and preservation of these structures. However, only very few nerve targeting agents are available. This study describes the in vivo nerve staining capabilities of locally administered fluorescent lectin-analogues. To this end WGA, PNA, PHA-L and LEL were functionalized with Cy5 (lambda(ex) (max) 640 nm; lambda(em) (max) 680 nm). Transfer of these imaging agents along the sciatic nerve was evaluated in Thy1-YFP mice (n = 12) after intramuscular injection. Migration from the injection site was assessed in vivo using a laboratory fluorescence scanner and ex vivo via fluorescence confocal microscopy. All four lectins showed retrograde movement and staining of the epineurium with a signal-to-muscle ratio of around two. On average, the longest transfer distance was obtained with WGA-Cy5 (0.95 cm). Since WGA also gave minimal uptake in the lymphatic system, this lectin type revealed the highest potential as a migration imaging agent to visualize nerves.
Assessing the susceptibility of amylose-lysophosphatidylcholine complexes to amylase by the use of iodine
Ahmadi-Abhari, S. ; Woortman, A.J.J. ; Hamer, R.J. ; Loos, K. - \ 2014
Starch-Stärke 66 (2014)5-6. - ISSN 0038-9056 - p. 576 - 581.
wheat-starch - chain-length - inclusion complexes - fatty-acids - in-vivo - digestion - binding - digestibility - microscopy - property
The formation of amylose-lysophosphatidylcholine (LPC) inclusion complexes renders amylose less susceptible to amylase digestion. In order to better understand this phenomenon on a structural level, the complexation of 9% wheat starch suspensions with 0, 2, 3, and 5% exogenous LPC was developed in RVA. Amylose-LPC inclusion complexes were isolated after 15, 30, 60, 120, and 240min in vitro digestion of the wheat starch suspensions to quantify the amount of non-complexed amylose by spectrophotometry. The samples were dissolved in DMSO containing 0.5% LiBr and exposed to iodine. In addition, parts of the digesta were defatted and subjected to the same procedure to elucidate the total amount of amylose that remained undigested. In this way, more insight was obtained into the protective effect of amylose-LPC complex formation on digestion of starch. This study confirms that the amylose susceptibility to amylolysis decreases in the presence of LPC. Higher LPC concentrations not only induced the formation of more amylose inclusion complexes but also resulted in more stable complexes which remained undigested as well as longer amylose chains after enzyme hydrolysis, due to the presence of LPC inside the amylose helix. In addition, a higher melting enthalpy of the amylose-LPC complexes in the digesta demonstrates the protective effect of LPC during enzyme hydrolysis.
Bispecific antibody generated with sortase and click chemistry has broad anti-influenza virus activity
Wagner, K. ; Kwakkenbos, M.J. ; Claassen, Y.B. ; Maijoor, K. ; Bohne, M. ; Sluijs, K.F. van der; Witte, M.D. ; Zoelen, D.J. van; Cornelissen, A.H.M. ; Beaumont, T. ; Bakker, A.Q. ; Ploegh, H.L. ; Spits, H.G. - \ 2014
Proceedings of the National Academy of Sciences of the United States of America 111 (2014)47. - ISSN 0027-8424 - p. 16820 - 16825.
influenza-a-viruses - memory b-cells - staphylococcus-aureus - human igg1 - proteins - site - fab - binding - design - heterodimerization
Bispecific antibodies have therapeutic potential by expanding the functions of conventional antibodies. Many different formats of bispecific antibodies have meanwhile been developed. Most are genetic modifications of the antibody backbone to facilitate incorporation of two different variable domains into a single molecule. Here, we present a bispecific format where we have fused two full-sized IgG antibodies via their C termini using sortase transpeptidation and click chemistry to create a covalently linked IgG antibody heterodimer. By linking two potent anti-influenza A antibodies together, we have generated a full antibody dimer with bispecific activity that retains the activity and stability of the two fusion partners.
Lactate racemase is a nickel-dependent enzyme activated by a widespread maturation system
Desguin, B. ; Goffin, P. ; Viaene, E. ; Kleerebezem, M. ; Martin-Diaconescu, V. ; Maroney, M.J. ; Declercq, J.P. ; Soumillion, P. ; Hols, P. - \ 2014
Nature Communications 5 (2014). - ISSN 2041-1723
lactobacillus-plantarum - lactic-acid - racemization - binding - dehydrogenase - purification - proteins - bacteria - growth - model
Racemases catalyse the inversion of stereochemistry in biological molecules, giving the organism the ability to use both isomers. Among them, lactate racemase remains unexplored due to its intrinsic instability and lack of molecular characterization. Here we determine the genetic basis of lactate racemization in Lactobacillus plantarum. We show that, unexpectedly, the racemase is a nickel-dependent enzyme with a novel a/ß fold. In addition, we decipher the process leading to an active enzyme, which involves the activation of the apo-enzyme by a single nickel-containing maturation protein that requires preactivation by two other accessory proteins. Genomic investigations reveal the wide distribution of the lactate racemase system among prokaryotes, showing the high significance of both lactate enantiomers in carbon metabolism. The even broader distribution of the nickel-based maturation system suggests a function beyond activation of the lactate racemase and possibly linked with other undiscovered nickel-dependent enzymes.
Mechanism of Isoflavone Adsorption from Okara extracts onto Food-Grade Resins
Méndez Sevillano, D. ; Jankowiak, L. ; Gaalen, T.L.T. van; Wielen, L.A.M. van der; Hooshyar, N. ; Goot, A.J. van der; Ottens, M. - \ 2014
Industrial & Engineering Chemistry Research 53 (2014)39. - ISSN 0888-5885 - p. 15245 - 15252.
defatted soybean flakes - dietary fiber - by-product - soy milk - polyphenols - selection - caffeine - binding
Okara is a byproduct of the soy milk industry containing valuable phytochemicals, called isoflavones, among other components (i.e., proteins, sugars, fibers, etc.). As a waste product, okara is an interesting source material for obtaining valuable chemicals, and knowledge of the behavior of such components in their complex matrix is a key step for design of a purification process. Six commercially available macroporous polymeric resins are investigated to measure and model the equilibrium properties of the adsorption of isoflavones, proteins, and total solids onto these resins. A new model is evaluated in which adsorption of isoflavones onto a protein layer is proposed describing the system isoflavones–resin XAD 4 better than a linear isotherm model. Parameters for both the linear model and the bilayer model are regressed and reported with their accuracy and correlated to the hydrophobicity of each of the isoflavones.
GtfA and GtfB are both required for protein O-glycosylation in Lactobacillus plantarum
Lee, I.C. ; Swam, I.I. van; Tomita, S. ; Morsomme, P. ; Rolain, T. ; Hols, P. ; Kleerebezem, M. ; Bron, P.A. - \ 2014
Journal of Bacteriology 196 (2014)9. - ISSN 0021-9193 - p. 1671 - 1682.
complete genome sequence - lactic-acid bacteria - escherichia-coli - campylobacter-jejuni - acidophilus ncfm - epithelial-cells - surface protein - rhamnosus gg - glycoproteins - binding
Acm2, the major autolysin of Lactobacillus plantarum WCFS1, was recently found to be O-glycosylated with N-acetylhexosamine, likely N-acetylglucosamine (GlcNAc). In this study, we set out to identify the glycosylation machinery by employing a comparative genomics approach to identify Gtf1 homologues, which are involved in fimbria-associated protein 1 (Fap1) glycosylation in Streptococcus parasanguinis. This in silico approach resulted in the identification of 6 candidate L. plantarum WCFS1 genes with significant homology to Gtf1, namely, tagE1 to tagE6. These candidate genes were targeted by systematic gene deletion, followed by assessment of the consequences on glycosylation of Acm2. We observed a changed mobility of Acm2 on SDS-PAGE in the tagE5E6 deletion strain, while deletion of other tagE genes resulted in Acm2 mobility comparable to that of the wild type. Subsequent mass spectrometry analysis of excised and in-gel-digested Acm2 confirmed the loss of glycosylation on Acm2 in the tagE5E6 deletion mutant, whereas a lectin blot using GlcNAc-specific succinylated wheat germ agglutinin (sWGA) revealed that besides Acm2, tagE5E6 deletion also abolished all but one other sWGA-reactive, protease-sensitive signal. Only complementation of both tagE5 and tagE6 restored those sWGA lectin signals, establishing that TagE5 and TagE6 are both required for the glycosylation of Acm2 as well as the vast majority of other sWGA-reactive proteins. Finally, sWGA lectin blotting experiments using a panel of 8 other L. plantarum strains revealed that protein glycosylation is a common feature in L. plantarum strains. With the establishment of these enzymes as protein glycosyltransferases, we propose to rename TagE5 and TagE6 as GtfA and GtfB, respectively.
Live imaging of baculovirus infection of midgut epithelium cells: a functional assay of per os infectivity factors (PIFs)
Mu, J.F. ; Lent, J.W.M. van; Smagghe, G. ; Wang, Q. ; Chen Xinwen, ; Vlak, J.M. ; Oers, M.M. van - \ 2014
Journal of General Virology 95 (2014)Pt 1. - ISSN 0022-1317 - p. 2531 - 2539.
occlusion-derived virus - heliothis-virescens larvae - nucleopolyhedrovirus - binding - complex - replication - proteins - surface - genes - p74
The occlusion-derived viruses (ODVs) of baculoviruses are responsible for oral infection of insect hosts, whereas budded viruses (BVs) are responsible for the systemic infection within the host. The ODV membrane proteins play crucial roles in mediating virus entry into midgut epithelium cells to initiate infection and are important factors in host range determination. For Autographa californica multiple nucleopolyhedrovirus (AcMNPV), seven conserved ODV membrane proteins have been shown to be essential for oral infectivity and are called per os infectivity factors (PIFs). Information on the function of the individual PIF proteins in virus entry is limited, partly due to the lack of a good in vitro system for monitoring ODV entry. Here, we constructed a baculovirus with an enhanced green fluorescent protein (EGFP) fused to the nucleocapsid to monitor the entry of virus into primary midgut epithelium cells ex vivo by confocal fluorescence microscopy. The EGFP-labeled virus showed the same BV virulence and ODV infectivity as wild type virus. The ability to bind and enter host cells was then visualized for wild type AcMNPVs and viruses with mutations in P74 (PIF0), PIF1 or PIF2, showing that P74 is required for ODV binding, while PIF1 and PIF2 play important roles in entry of ODV after binding to midgut cells. This is the first live imaging of ODV entry into midgut cells and complements the genetic and biochemical evidence for the role of PIFs in the oral infection process.
Solid phase microextraction speciation analysis of triclosan in aqueous mediacontaining sorbing nanoparticles
Zielinska, K. - \ 2014
Environmental Chemistry 11 (2014)1. - ISSN 1448-2517 - p. 72 - 76.
nd-spme - samples - water - adsorption - products - binding - surface
Solid phase microextraction (SPME) is applied in the speciation analysis of the hydrophobic compound triclosan in an aqueous medium containing sorbing SiO2 nanoparticles (NPs). It is found that these NPs, as well as their complexes with triclosan, partition between the bulk medium and the solid phase poly(dimethylsiloxane) (PDMS). Furthermore, they appear to aggregate at the PDMS–water interface. The total triclosan concentration in the solid phase thus includes both the free and the NP-bound forms. Proper computation of the analyte concentration in the sample medium requires (i) consideration of the speciation of triclosan inside the solid phase and (ii) elimination of the effects of aggregation of NP complexes at the solid phase–bulk medium interface. Possible solutions include application of a protective membrane with pore size smaller than the NP diameter. This allows measurement of the free triclosan concentration, albeit at the cost of longer accumulation times and loss of kinetic information on the triclosan–NP complex.
RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication
Feenstra, F. ; Gennip, H.G.P. van; Water, S.G.P. van de; Rijn, P.A. van - \ 2014
PLoS One 9 (2014)3. - ISSN 1932-6203
polymerase-chain-reaction - intragenic recombination - insect cells - protein ns2 - fever virus - vp6 protein - viral-rna - segment - binding - core
Members of the Reoviridae family are non-enveloped multi-layered viruses with a double stranded RNA genome consisting of 9 to 12 genome segments. Bluetongue virus is the prototype orbivirus (family Reoviridae, genus Orbivirus), causing disease in ruminants, and is spread by Culicoides biting midges. Obviously, several steps in the Reoviridae family replication cycle require virus specific as well as segment specific recognition by viral proteins, but detailed processes in these interactions are still barely understood. Recently, we have shown that expression of NS3 and NS3a proteins encoded by genome segment 10 of bluetongue virus is not essential for virus replication. This gave us the unique opportunity to investigate the role of RNA sequences in the segment 10 open reading frame in virus replication, independent of its protein products. Reverse genetics was used to generate virus mutants with deletions in the open reading frame of segment 10. Although virus with a deletion between both start codons was not viable, deletions throughout the rest of the open reading frame led to the rescue of replicating virus. However, all bluetongue virus deletion mutants without functional protein expression of segment 10 contained inserts of RNA sequences originating from several viral genome segments. Subsequent studies showed that these RNA inserts act as RNA elements, needed for rescue and replication of virus. Functionality of the inserts is orientation-dependent but is independent from the position in segment 10. This study clearly shows that RNA in the open reading frame of Reoviridae members does not only encode proteins, but is also essential for virus replication
Interaction of flavan-3-ol derivatives and different caseins is determined by more than proline content and number of proline repeats
Bohin, M.C. ; Vincken, J.P. ; Westphal, A.H. ; Tripp, A.M. ; Dekker, Peter ; Hijden, H.T.W.M. ; Gruppen, H. - \ 2014
Food Chemistry 158 (2014). - ISSN 0308-8146 - p. 408 - 416.
food proteins - binding - astringency - polymerization - precipitation - complexation - fluorescence - procyanidins - polyphenols - gallate
Interactions of Type A and B flavan-3-ol dimers (procyanidins) and several monomeric flavan-3-ols, with a-casein and ß-casein, were investigated. Binding affinities measured were related to the ligands structure, including several properties (e.g. intrinsic flexibility (number of rotatable bonds) and hydrophobicity), and to the amino-acid composition of the caseins. A monomeric flavan-3-ol esterified with gallic acid (EGCG) had a five to ten times higher affinity to caseins compared to the non-galloylated dimeric flavan-3-ols. In this case, the larger number of rotatable bonds in EGCG might be accountable for this difference. Comparing flavan-3-ol dimers, intrinsic flexibility did not consistently promote interactions, as procyanidin A1 displayed a higher affinity to a-casein than the supposedly more flexible B-type dimers investigated. Despite its higher content of proline, compared to a-casein, ß-casein did not always have a higher affinity for the ligands investigated (e.g. no interaction with procyanidin A1 detected). These results suggest that more factors than proline content and the number of proline repeats govern phenolic–casein interactions.
Identification and mapping of a novel dominant resistance gene, TuRB07 to Turnip mosaic virus in Brassica rapa
Jin, M. ; Lee, S.S. ; Ke, L. ; Kim, J.S. ; Seo, M.S. ; Sohn, S.H. ; Park, B.S. ; Bonnema, A.B. - \ 2014
Theoretical and Applied Genetics 127 (2014). - ISSN 0040-5752 - p. 509 - 519.
quantitative trait loci - chinese-cabbage - arabidopsis-thaliana - signal-transduction - linkage maps - host-defense - protein rx - binding - nucleotide - napus
The inheritance of resistance to Turnip mosaic virus in Brassica rapa was investigated by crossing the resistant line, VC1 with the susceptible line, SR5, and genotyping and phenotyping diverse progenies derived from this cross. Both a doubled haploid population, VCS3M-DH, an F2 and two BC1 (F1 × VC1 and F1 × SR5) populations were created. Population tests revealed that the resistance to the TuMV C4 isolate in B. rapa is controlled by a single dominant gene. This resistance gene, TuRB07 was positioned on the top of linkage group A06 of the B. rapa genome through bulk segregation analysis and fine mapping recombinants in three doubled haploid- and one backcross population using microsatellite markers developed from BAC end sequences. Within the region between the two closely linked markers flanking TuRB07, H132A24-s1, and KS10960, in the Chiifu reference genome, two genes encoding nucleotide-binding site and leucine-rich repeat proteins with a coiled-coil motif (CC-NBS-LRR), Bra018862 and Bra018863 were identified as candidate resistance genes. The gene Bra018862 is truncated, but the gene Bra018863 has all the domains to function. Furthermore, the analysis of structural variation using resequencing data of VC1 and SR5 revealed that Bra018863 might be a functional gene because the gene has no structural variation in the resistant line VC1 when compared with Chiifu, whereas at the other NBS-LRR genes large deletions were identified in the resistant line. Allelic differences of Bra018863 were found between VC1 and SR5, supporting the notion that this gene is a putative candidate gene for the virus resistance.
Nonlinear Amplification of a Supramolecular Complex at a Multivalent Interface
Hsu, S.H. ; Yilmaz, M.D. ; Reinhoudt, D.N. ; Velders, A.H. ; Huskens, J. - \ 2013
Angewandte Chemie-International Edition 52 (2013)2. - ISSN 1433-7851 - p. 714 - 719.
self-assembled monolayers - molecular printboards - chemistry - binding - membrane - nanostructures - organization - selection - discrete - peptides
Competition with a monovalent cyclodextrin host (blue cones) in solution drives the multivalent binding of a Eu3+ complex and a sensitizer molecule to cyclodextrin monolayers through a nonlinear self-assembly process. Adamantyl groups (light-blue spheres) are attached to the EDTA ligand (black) and the antenna molecule (orange), which has a carboxylate group for coordination to the Eu3+ ion (yellow or red in free or complexed form, respectively).
Arsenate and phosphate adsorption in relation to oxides composition in soils: LCD modelling
Cui, Y. ; Weng, L. - \ 2013
Environmental Science and Technology 47 (2013)13. - ISSN 0013-936X - p. 7269 - 7276.
charge-distribution - humic substances - competitive adsorption - ion adsorption - heavy-metals - speciation - parameters - minerals - sorption - binding
The pH dependent solid-solution distribution of arsenate and phosphate in five Dutch agricultural soil samples was measured in the pH range 4–8, and the results were interpreted using the LCD (ligand and charge distribution) adsorption modeling. The pH dependency is similar for both oxyanions, with a minimum soluble concentration observed around pH 6–8. This pH dependency can be successfully described with the LCD model and it is attributed mainly to the synergistic effects from Ca adsorption. The solubility of phosphate is much lower than that of arsenate. This big difference cannot be sufficiently explained by the reduction of small amount of As(V) into As(III), neither by slow desorption/adsorption. The difference between phosphate and arsenate in their solid-solution distribution becomes larger with the increase of aluminum (hydr)oxides (Al-oxides) contribution to the total amount of metal (Al and Fe) (hydr)oxides. The influence of Al-oxides is much larger than its relative amount extracted from the soils. When Al-oxides account for >40% of the soil oxides, the whole adsorbents behave apparently similarly to that of pure Al-oxides. These results indicated that surface coating and substitution may have modified significantly oxyanion adsorption to Fe-oxides in soils, and how to account for this complexity is a challenge for geochemical modeling.
LysM effectors: secreted proteins supporting fungal life
Kombrink, A. ; Thomma, B.P.H.J. - \ 2013
PLoS Pathogens 9 (2013)12. - ISSN 1553-7366 - 4 p.
chitin-triggered immunity - plant chitinases - virulence factor - pathogen - diversification - recognition - fragments - infection - receptor - binding
Influence of soil humic and fulvic acid on the activity and stability of lysozyme and urease
Li, Y. ; Tan, W. ; Koopal, L.K. ; Wang, M. ; Liu, Fan ; Norde, W. - \ 2013
Environmental Science and Technology 47 (2013). - ISSN 0013-936X - p. 5050 - 5056.
isothermal titration calorimetry - insecticidal cry1ab protein - ionic-strength - prion protein - ph - adsorption - complexes - binding - substances - fractions
Humic substances (HS), including humic acids (HA) and fulvic acids (FA), are important components of soil systems. HS form strong complexes with oppositely charged proteins, which will lead to changes in the enzyme activity. The effect of soil HS on the activity and stability of two enzymes was investigated as a function of pH, ionic strength, and mass ratio HS/enzyme. Humic acid (JGHA) and fulvic acid (JGFA) are negatively charged, lysozyme is net positive at pH values below 10.4, and urease is net positive below pH 5.2 or net negative above pH 5.2. The enzyme activities in the HS-enzyme complexes were suppressed when the enzymes were oppositely charged to the HS. The largest activity suppression was observed around the mass ratio HS/enzyme where the HS-protein complex was at its isoelectric point (IEP). At the IEP strong aggregation of the complexes led to encapsulation of the enzyme. The ionic strength was important; an increase decreased complex formation, but increased aggregation. Due to the larger hydrophobicity of JGHA than JGFA, the reduction in enzyme activity was stronger for JGHA. The enzyme stability also decreased maximally at mass ratio around the IEP of the complex when HS and protein were oppositely charged. When urease and HS were both negatively charged no complexes were formed, but the presence of JGHA or JGFA improved the activity and stability of the enzyme.
A capture approach for supercoiled plasmid DNA using a triplex-forming oligonucleotide
Ruigrok, V.J.B. ; Westra, E.R. ; Brouns, S.J.J. ; Escudé, C. ; Smidt, H. ; Oost, J. van der - \ 2013
Nucleic Acids Research 41 (2013)10. - ISSN 0305-1048
lac repressor - padlock oligonucleotides - helix formation - sequence - binding - protein - replication - operators - association - biosensor
Proteins that recognize and bind specific sites in DNA are essential for regulation of numerous biological functions. Such proteins often require a negative supercoiled DNA topology to function correctly. In current research, short linear DNA is often used to study DNA-protein interactions. Although linear DNA can easily be modified, for capture on a surface, its relaxed topology does not accurately resemble the natural situation in which DNA is generally negatively supercoiled. Moreover, specific binding sequences are flanked by large stretches of non-target sequence in vivo. Here, we present a straightforward method for capturing negatively supercoiled plasmid DNA on a streptavidin surface. It relies on the formation of a temporary parallel triplex, using a triple helix forming oligonucleotide containing locked nucleic acid nucleotides. All materials required for this method are commercially available. Lac repressor binding to its operator was used as model system. Although the dissociation constants for both the linear and plasmid-based operator are in the range of 4 nM, the association and dissociation rates of Lac repressor binding to the plasmid-based operator are ~18 times slower than on a linear fragment. This difference underscores the importance of using a physiologically relevant DNA topology for studying DNA-protein interactions
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