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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    Multi-host disease management: The why and the how to include wildlife
    Portier, Julien ; Ryser-Degiorgis, Marie Pierre ; Hutchings, Mike R. ; Monchâtre-Leroy, Elodie ; Richomme, Céline ; Larrat, Sylvain ; Poel, Wim H.M. Van Der; Dominguez, Morgane ; Linden, Annick ; Santos, Patricia Tavares ; Warns-Petit, Eva ; Chollet, Jean Yves ; Cavalerie, Lisa ; Grandmontagne, Claude ; Boadella, Mariana ; Bonbon, Etienne ; Artois, Marc - \ 2019
    BMC Veterinary Research 15 (2019)1. - ISSN 1746-6148
    Coordination - Decision-making framework - Emerging infectious diseases - Europe - Integrated management - Policy making - Proportionate management - Risk assessment - Wildlife - Zoonosis

    In recent years, outbreaks caused by multi-host pathogens (MHP) have posed a serious challenge to public and animal health authorities. The frequent implication of wildlife in such disease systems and a lack of guidelines for mitigating these diseases within wild animal populations partially explain why the outbreaks are particularly challenging. To face these challenges, the French Ministry of Agriculture launched a multi-disciplinary group of experts that set out to discuss the main wildlife specific concepts in the management of MHP disease outbreaks and how to integrate wildlife in the disease management process. This position paper structures the primary specific concepts of wildlife disease management, as identified by the working group. It is designed to lay out these concepts for a wide audience of public and/or animal health officers who are not necessarily familiar with wildlife diseases. The group's discussions generated a possible roadmap for the management of MHP diseases. This roadmap is presented as a cycle for which the main successive step are: step 1-descriptive studies and monitoring; step 2-risk assessment; step 3-management goals; step 4-management actions and step 5-assessment of the management plan. In order to help choose the most adapted management actions for all involved epidemiological units, we integrated a decision-making framework (presented as a spreadsheet). This tool and the corresponding guidelines for disease management are designed to be used by public and health authorities when facing MHP disease outbreaks. These proposals are meant as an initial step towards a harmonized transboundary outbreak response framework that integrates current scientific understanding adapted to practical intervention.

    Bioremediation : An Overview on Current Practices, Advances, and New Perspectives in Environmental Pollution Treatment
    Hlihor, Raluca Maria ; Gavrilescu, Maria ; Tavares, Teresa ; Favier, Lidia ; Olivieri, Giuseppe - \ 2017
    BioMed Research International 2017 (2017). - ISSN 2314-6133
    Blood–brain barrier transport and neuroprotective potential of blackberry-digested polyphenols : an in vitro study
    Figueira, Inês ; Tavares, Lucélia ; Jardim, Carolina ; Costa, Inês ; Terrasso, Ana P. ; Almeida, Andreia F. ; Govers, Coen ; Mes, Jurriaan J. ; Gardner, Rui ; Becker, Jörg D. ; McDougall, Gordon J. ; Stewart, Derek ; Filipe, Augusto ; Kim, Kwang S. ; Brites, Dora ; Brito, Catarina ; Brito, M.A. ; Santos, Cláudia N. - \ 2017
    European Journal of Nutrition (2017). - ISSN 1436-6207 - p. 1 - 18.
    Blackberry - Brain endothelial cells - In vitro digestion - Microarrays - Neuronal cells
    Purpose: Epidemiological and intervention studies have attempted to link the health effects of a diet rich in fruits and vegetables with the consumption of polyphenols and their impact in neurodegenerative diseases. Studies have shown that polyphenols can cross the intestinal barrier and reach concentrations in the bloodstream able to exert effects in vivo. However, the effective uptake of polyphenols into the brain is still regarded with some reservations. Here we describe a combination of approaches to examine the putative transport of blackberry-digested polyphenols (BDP) across the blood–brain barrier (BBB) and ultimate evaluation of their neuroprotective effects. Methods: BDP was obtained by in vitro digestion of blackberry extract and BDP major aglycones (hBDP) were obtained by enzymatic hydrolysis. Chemical characterization and BBB transport of extracts were evaluated by LC–MSn. BBB transport and cytoprotection of both extracts was assessed in HBMEC monolayers. Neuroprotective potential of BDP was assessed in NT2-derived 3D co-cultures of neurons and astrocytes and in primary mouse cerebellar granule cells. BDP-modulated genes were evaluated by microarray analysis. Results: Components from BDP and hBDP were shown to be transported across the BBB. Physiologically relevant concentrations of both extracts were cytoprotective at endothelial level and BDP was neuroprotective in primary neurons and in an advanced 3D cell model. The major canonical pathways involved in the neuroprotective effect of BDP were unveiled, including mTOR signaling and the unfolded protein response pathway. Genes such as ASNS and ATF5 emerged as novel BDP-modulated targets. Conclusions: BBB transport of BDP and hBDP components reinforces the health benefits of a diet rich in polyphenols in neurodegenerative disorders. Our results suggest some novel pathways and genes that may be involved in the neuroprotective mechanism of the BDP polyphenol components.
    Solidarity economy as a counterpoint to classical economics : Possibilities of changes
    Teixeira Coriolano, Luzianeide Menezes ; Tavares, Jean Max ; Ateljevic, Irena - \ 2016
    Tourismos 11 (2016)2. - ISSN 1790-8418 - p. 1 - 21.
    Human development - solidarity economy - Tourism - Transmodernsociety

    In the current context of social arrhythmia promoted by the culture of excess, consumerism and individualism the purpose of this article is to discuss the need to promote changes in tourism and society towards a more human development, based on the pillars of the solidarity economy. The article extends discussions on the solidarity economy into the context of classical economics, with new offers and demands that are focused not on capital accumulation only, but on the rights and development of human beings. To achieve this goal, we analyse four key foundations of the solidarity economy - solidarity, social equality, cooperation and sharing. The original contribution of the paper is to present cutting-edge ideas that show a counterpoint to capitalism and consumerism; one that is excluded from global tourism's contradictory path by a form of production that values social relations, and quality of life in community experiences and community tourism.

    The harmonized INFOGEST in vitro digestion method : From knowledge to action
    Egger, Lotti ; Ménard, Olivia ; Delgado-Andrade, Cristina ; Alvito, Paula ; Assunção, Ricardo ; Balance, Simon ; Barberá, Reyes ; Brodkorb, Andre ; Cattenoz, Thomas ; Clemente, Alfonso ; Comi, Irene ; Dupont, Didier ; Garcia-Llatas, Guadalupe ; Lagarda, María Jesús ; Feunteun, Steven Le; Janssen Duijghuijsen, Lonneke ; Karakaya, Sibel ; Lesmes, Uri ; Mackie, Alan R. ; Martins, Carla ; Meynier, Anne ; Miralles, Beatriz ; Murray, B.S. ; Pihlanto, Anne ; Picariello, Gianluca ; Santos, C.N. ; Simsek, Sebnem ; Recio, Isidra ; Rigby, Neil ; Rioux, Laurie Eve ; Stoffers, Helena ; Tavares, Ana ; Tavares, Lucelia ; Turgeon, Sylvie ; Ulleberg, E.K. ; Vegarud, G.E. ; Vergères, Guy ; Portmann, Reto - \ 2016
    Food Research International 88 (2016)Par B. - ISSN 0963-9969 - p. 217 - 225.
    Dairy proteins - Harmonized IVD protocol - In vitro digestion - Inter-laboratory trial - Mass spectrometry - Peptides

    Within the active field of in vitro digestion in food research, the COST Action INFOGEST aimed to harmonize in vitro protocols simulating human digestion on the basis of physiologically inferred conditions. A harmonized static in vitro digestion (IVD) method was recently published as a primary output from this network. To validate this protocol, inter-laboratory trials were conducted within the INFOGEST network. A first study was performed using skim milk powder (SMP) as a model food and served to compare the different in-house digestion protocols used among the INFOGEST members. In a second inter-laboratory study applying the harmonized protocol, the degree of consistency in protein hydrolysis was investigated. Analysis of the hydrolyzed proteins, after the gastric and intestinal phases, showed that caseins were mainly hydrolyzed during the gastric phase, whereas β-lactoglobulin was, as previously shown, resistant to pepsin. Moreover, generation of free amino acids occurred mainly during the intestinal phase.The study also showed that a few critical steps were responsible for the remaining inter-laboratory variability. The largest deviations arose from the determination of pepsin activity. Therefore, this step was further clarified, harmonized, and implemented in a third inter-laboratory study.The present work gives an overview of all three inter-laboratory studies, showing that the IVD INFOGEST method has led to an increased consistency that enables a better comparability of in vitro digestion studies in the future.

    Soil-pore water distribution of silver and gold engineered nanoparticles in undisturbed soils under unsaturated conditions
    Tavares, D.S. ; Rodrigues, S.M. ; Cruz, N. ; Carvalho, C. ; Teixeira, T. ; Carvalho, L. ; Duarte, A.C. ; Trindade, T. ; Pereira, E. ; Römkens, P.F.A.M. - \ 2015
    Chemosphere 136 (2015). - ISSN 0045-6535 - p. 86 - 94.
    Chemical availability - Risk assessment - Silver/gold nanoparticles - Soil contamination - Soil distribution

    Release of engineered nanoparticles (ENPs) to soil is well documented but little is known on the subsequent soil-pore water distribution of ENPs once present in soil. In this study, the availability and mobility of silver (Ag) and gold (Au) ENPs added to agricultural soils were assessed in two separate pot experiments. Pore water samples collected from pots from day 1 to 45 using porous (

    Testing single extraction methods and in vitro tests to assess the geochemical reactivity and human bioaccessibility of silver in urban soils amended with silver nanoparticles
    Cruz, N. ; Rodrigues, S.M. ; Tavares, D. ; Monteiro, R.J.R. ; Carvalho, L. ; Trindade, T. ; Duarte, A.C. ; Pereira, E. ; Romkens, Paul - \ 2015
    Chemosphere 135 (2015). - ISSN 0045-6535 - p. 304 - 311.
    Bioavailability - Engineered silver nanoparticles - Geochemical reactivity - In vitro bioaccessibility - Soil pollution

    To assess if the geochemical reactivity and human bioaccessibility of silver nanoparticles (AgNPs) in soils can be determined by routine soil tests commonly applied to other metals in soil, colloidal Ag was introduced to five pots containing urban soils (equivalent to 6.8mgAgkg-1 soil). Following a 45days stabilization period, the geochemical reactivity was determined by extraction using 0.43M and 2M HNO3. The bioaccessibility of AgNPs was evaluated using the Simplified Bioaccessibility Extraction Test (SBET) the "Unified BARGE Method" (UBM), and two simulated lung fluids (modified Gamble's solution (MGS) and artificial lysosomal fluid (ALF)).The amount of Ag extracted by 0.43M and 2M HNO3 soil tests was

    Soil and water conservation strategies in Cape Verde (Cabo Verde in Portuguese) and their impacts on livelihoods: an overview from the Ribeira Seca Watershed
    Baptista, I. ; Fleskens, L. ; Ritsema, C. ; Querido, A. ; Ferreira, A.D. ; Tavares, J. ; Reis, E.A. ; Gomes, S. ; Varela, A. - \ 2015
    Land 4 (2015)1. - ISSN 2073-445X - p. 22 - 44.
    Severe land degradation has strongly affected both people’s livelihood and the environment in Cape Verde (Cabo Verde in Portuguese), a natural resource poor country. Despite the enormous investment in soil and water conservation measures (SWC or SLM), which are visible throughout the landscape, and the recognition of their benefits, their biophysical and socioeconomic impacts have been poorly assessed and scientifically documented. This paper contributes to filling this gap, by bringing together insights from literature and policy review, field survey and participatory assessment in the Ribeira Seca Watershed through a concerted approach devised by the DESIRE project (the “Desire approach”). Specifically, we analyze government strategies towards building resilience against the harsh conditions, analyze the state of land degradation and its drivers, survey and map the existing SWC measures, and assess their effectiveness against land degradation, on crop yield and people’s livelihood. We infer that the relative success of Cape Verde in tackling desertification and rural poverty owes to an integrated governance strategy that comprises raising awareness, institutional framework development, financial resource allocation, capacity building, and active participation of rural communities. We recommend that specific, scientific-based monitoring and assessment studies be carried out on the biophysical and socioeconomic impact of SLM and that the “Desire approach” be scaled-up to other watersheds in the country.
    Linkage mapping in the oilseed crop Jatropha curcas L. reveals a locus controlling the biosynthesis of phorbol esters which cause seed toxicity
    King, A.J. ; Montes, L.R. ; Clarke, J.G. ; Affleck, J. ; Li, Y. ; Witsenboer, H. ; Vossen, E. van der; Linde, P. van der; Tripathi, Y. ; Tavares, E. ; Shukla, P. ; Rajasekaran, T. ; Loo, E.N. van; Graham, I.A. - \ 2013
    Plant Biotechnology Journal 11 (2013)8. - ISSN 1467-7644 - p. 986 - 996.
    transcriptome analysis - genetic diversity - biofuel plant - genome - oil - markers - qtl - construction - sequence - maps
    Current efforts to grow the tropical oilseed crop Jatropha curcas L. economically are hampered by the lack of cultivars and the presence of toxic phorbol esters (PE) within the seeds of most provenances. These PE restrict the conversion of seed cake into animal feed, although naturally occurring ‘nontoxic’ provenances exist which produce seed lacking PE. As an important step towards the development of genetically improved varieties of J. curcas, we constructed a linkage map from four F2 mapping populations. The consensus linkage map contains 502 codominant markers, distributed over 11 linkage groups, with a mean marker density of 1.8 cM per unique locus. Analysis of the inheritance of PE biosynthesis indicated that this is a maternally controlled dominant monogenic trait. This maternal control is due to biosynthesis of the PE occurring only within maternal tissues. The trait segregated 3 : 1 within seeds collected from F2 plants, and QTL analysis revealed that a locus on linkage group 8 was responsible for phorbol ester biosynthesis. By taking advantage of the draft genome assemblies of J. curcas and Ricinus communis (castor), a comparative mapping approach was used to develop additional markers to fine map this mutation within 2.3 cM. The linkage map provides a framework for the dissection of agronomic traits in J. curcas, and the development of improved varieties by marker-assisted breeding. The identification of the locus responsible for PE biosynthesis means that it is now possible to rapidly breed new nontoxic varieties.
    Quality controlled logistics in vegetable supply chain networks: how can an individual batch reach an individual consumer in hte optimal state?
    Schouten, R.E. ; Kooten, O. van; Vorst, J.G.A.J. van der; Marcelis, W.J. ; Luning, P.A. - \ 2012
    In: Proceedings of the 18th Internatioanl Congress on Science and Horticulture for people (IHC2010). - Leuven : ISHS - ISBN 9789066054943 - p. 45 - 52.
    Western-European consumers have become demanding on product availability in retail outlets and vegetable attributes such as quality, integrity, safety. When (re)designing vegetable supply chain networks one has to take these demands into consideration, next to traditional efficiency and responsiveness requirements. In post¬harvest research, much attention has been paid to quality decay modelling and the development of Time-Temperature Indicators to individually monitor the temperature conditions of vegetables throughout distribution. This paper discusses opportunities to use time-dependent product quality information in supply chain/logistics decision making to improve the design of vegetable supply chain networks. If product quality in each step of the supply chain can be predicted, product flows based on availability predictions can be controlled and better chain designs can be established. A case is presented to illustrate the value of this innovative concept of Quality Controlled Logistics through a Dutch tomato chain.
    Mycorrhizal associations in agroforestry systems
    Carvalho, A.M.X. de; Castro Tavares, R. de; Cardoso, I.M. ; Kuyper, T.W. - \ 2010
    In: Soil biology and agriculture in the tropics / Bion, P., Springer Berlin Heidelberg (Soil Biology 21) - ISBN 9783642050756 - p. 185 - 208.
    Agroforestry systems can be a viable alternative to the preservation of natural resources while contributing to sustainable food production in the tropics. These perennial systems promote beneficial biological interactions between micro-organisms and plant species, especially those formed by arbuscular mycorrhizal fungi and roots. Mycorrhizal fungi increase the soil volume explored by the roots, increase nutrient absorption by the plants, protect the root system against pathogens, toxic elements and certain heavy metals, help the formation and maintenance of soil structure, increase the input of soil carbon, and contribute to the maintenance of biodiversity. Agroforestry systems have the potential to maximize the benefits associated with AMF, which in turn could mitigate negative interactions between trees and annual crops. This beneficial impact between agroforestry management and mycorrhizal action may be depicted as a particular form of symbiosis, and deserves more study
    Complexity in quantitative food webs
    Banasek-Richter, C. ; Bersier, L.F. ; Cattin, M.F. ; Baltensperger, R. ; Gabriel, J.P. ; Merz, Y. ; Ulanowicz, R.E. ; Tavares, A.F. ; Williams, D.D. ; Ruiter, P.C. de; Winemiller, K.O. ; Naisbit, R.E. - \ 2009
    Ecology 90 (2009)6. - ISSN 0012-9658 - p. 1470 - 1477.
    trophic interactions - community stability - sampling effort - scale - networks - descriptors - connectance - adaptation - ecosystems - patterns
    Food webs depict who eats whom in communities. Ecologists have examined statistical metrics and other properties of food webs, but mainly due to the uneven quality of the data, the results have proved controversial. The qualitative data on which those efforts rested treat trophic interactions as present or absent and disregard potentially huge variation in their magnitude, an approach similar to analyzing traffic without differentiating between highways and side roads. More appropriate data are now available and were used here to analyze the relationship between trophic complexity and diversity in 59 quantitative food webs from seven studies (14-202 species) based on recently developed quantitative descriptors. Our results shed new light on food-web structure. First, webs are much simpler when considered quantitatively, and link density exhibits scale invariance or weak dependence on food-web size. Second, the "constant connectance'' hypothesis is not supported: connectance decreases with web size in both qualitative and quantitative data. Complexity has occupied a central role in the discussion of food-web stability, and we explore the implications for this debate. Our findings indicate that larger webs are more richly endowed with the weak trophic interactions that recent theories show to be responsible for food-web stability.
    Molecular detection of Papaya meleira virus in the latex of Carica papaya by RT-PCR
    Araujo, M.M.M. de; Tavares, E.T. ; Silva, F.R. da; Marinho, V.L.D. ; Souza, M.T. - \ 2007
    Journal of Virological Methods 146 (2007)1-2. - ISSN 0166-0934 - p. 305 - 310.
    A RT-PCR assay was developed for early and accurate detection of Papaya meleira virus (PMeV) in the latex from infected papayas. The meleira disease is characterized by an excessive exudation of more fluidic latex from fruits, leaves and stems. This latex oxidises and gives the fruit a ¿sticky¿ texture. In the field, disease symptoms are seen almost exclusively on fruit. However, infected plants can be a source of virus for dissemination by insects. Primers specific for PMeV were designed based on nucleotide sequences of the viral dsRNA obtained using a RT-RAPD approach. When tested for RT-PCR amplification, one of these primers (C05-3¿) amplified a 669-nucleotide fragment using dsRNA obtained from purified virus particles as a template. The translated sequence of this DNA fragment showed a certain degree of similarity to the amino acid sequence of RNA-dependent RNA polymerases from other dsRNA viruses. When used as the single primer in two RT-PCR kits available commercially, primer C05-3¿ also amplified the DNA fragment from papaya latex of infected, but not from healthy plants. The RT-PCR-based method developed in this study could simplify early plant disease diagnosis, assist in monitoring the dissemination of the pathogen within and between fields, and assist in guiding plant disease management.
    Natural occurence of Wolbachia-infected and uninfected Trichogramma species in tomato fields in Portugal
    Gonçalves, C.I. ; Huigens, M.E. ; Verbaarschot, P.G.H. ; Duarte, S. ; Mexia, A. ; Tavares, J. - \ 2006
    Biological Control 37 (2006)3. - ISSN 1049-9644 - p. 375 - 381.
    sex-ratio chromosome - biological-control - parthenogenesis - sequences - wasps - gene - mass
    Minute egg parasitoids of the genus Trichogramma (Hymenoptera; Trichogrammatidae) are promising candidates for biological control of lepidopteran pests in tomato in Portugal. This certainly applies to native Trichogramma strains that have thelytokous reproduction, i.e., produce only daughters. In Trichogramma wasps, thelytoky is mostly induced by the intracellular bacterium Wolbachia. In this study, we carried out a field survey of native Trichogramma species in four locations in Ribatejo, the main processing tomato region of Portugal, and determined the prevalence of Wolbachia in those species. Five Trichogramma species were found to emerge from lepidopteran eggs collected in the field, namely Trichogramma bourarache, Trichogramma cordubensis, Trichogramma evanescens, Trichogramma pintoi, and Trichogramma turkestanica. T. evanescens and T. pintoi were by far the dominating species representing, respectively, 64.9 and 26.4% of the trichogrammatids collected. Total natural parasitism rates of the collected lepidopteran eggs by Trichogramma wasps ranged from 28.2 to 64.6%. Three Trichogramma species were found to be infected with Wolbachia, namely T. cordubensis, T. evanescens, and T. turkestanica. All the wasp broods belonging to T. cordubensis were infected, whereas low infection rates were found in T. evanescens (0.9% of the broods) and T. turkestanica (4.5% of the broods). The latter represents the first record of a Wolbachia infection in T. turkestanica. Sequencing of the Wolbachia surface protein, wsp, revealed this Wolbachia infection to be related to other Wolbachia infections in Trichogramma wasps. As Wolbachia-infected thelytokous strains exist for T. evanescens, the most abundant Trichogramma species naturally occurring in the tomato fields of the Ribatejo region, this species offers interesting and powerful options for biological control of lepidopteran pests in processing tomato in this region.
    Non-purified anti-peptide sera generate tissue specific artefacts in immunohistochemical staining of Arabidopsis thaliana
    Tavares, R. ; Vidal, J. ; Lammeren, A.A.M. van; Kreis, M. - \ 2002
    Plant Science 162 (2002). - ISSN 0168-9452 - p. 309 - 314.
    AtSK theta, a plant homologue of SGG/GSK-3 marks developing tissues in Arabidopsis thaliana
    Tavares, R. ; Vidal, J. ; Lammeren, A.A.M. van; Kreis, M. - \ 2002
    Plant Molecular Biology 50 (2002)2. - ISSN 0167-4412 - p. 261 - 271.
    The Arabidopsis thaliana AtSK sub-family of serine threonine protein kinases groups 10 homologues of SHAGGY/GSK-3. Previous results obtained with different plant members of the SHAGGY/GSK-3 family strongly suggest that these proteins are involved in cell differentiation and stress responses. In order to gain further insight into the biological functions of this family in A. thaliana, polyclonal antibodies were raised against specific domains of the AtSKtheta protein. The antibodies were purified and used in immunolocalization studies in various tissues of A. thaliana. Our results show that the protein is located in the cell nuclei of various developing organs. Differential protein localization profiles were found in some of the observed tissues, notably during gametophyte and embryo development. Based on this protein location pattern, and on what is known about the mammalian members of the GSK-3 family, we suggest that AtSKtheta may have a role in the regulation of transcription factors.
    Structural studies on metal-containing enzymes: T4 endonuclease VII and D. gigas formate dehydrogenase
    Raaijmakers, H.C.A. - \ 2001
    Wageningen University. Promotor(en): N.C.M. Laane; D. Suck. - S.l. : S.n. - ISBN 9789058084125 - 85
    enzymen - röntgenkristallografie - desulfovibrio - oxidoreductasen - wolfraam - enzymes - x ray crystallography - desulfovibrio - oxidoreductases - tungsten

    Many biological processes require metal ions, and many of these metal-ion functions involve metalloproteins. The metal ions in metalloproteins are often critical to the protein's function, structure, or stability. This thesis focuses on two of these proteins, bacteriophage T4 endonuclease VII (EndoVII) and D. gigas fonnate dehydrogenase, which are studied by X-ray crystallography. The structure of EndoVII reveals how a magnesium or calcium ion is used to cleave several kinds of irregular but flexible DNA, while a zinc ion maintains the structural integrity of this DNase.

    The formate dehydrogenase contains a tungsten ion and a seleno-cysteine at the active site, that catalyses the oxidation of formate to carbon dioxide. The two released electrons are transferred through four [4Fe-4S] clusters before they can be handed over to another protein. Two of the [4Fe-4S] and the selenium have been overlooked by other techniques, but could be localised and identified by crystallography.

    Chapter 1 gives a general introduction on metals in biological systems, X-ray crystallography and also describes the biological background of both proteins.

    Chapter 2 presents the structure of the four-way DNA-junction resolving enzyme T4 endonuclease VII, and that of the inactive N62D mutant. The betterexpressed mutant was solved first, using seleno-methionine, mercury and gold derivatives. These mercury and gold derivatives bind to the sulphurs that also ligand the zinc. The wild-type was solved with help of a single mercury derivative since molecular replacement with the mutant structure failed.

    On its own, the EndoVII monomer would not represent a stable fold, as it exposes many hydrophobic residues to the solvent. But two monomers intertwine to form a dimer without this problem. In this dimer, the monomers are aligned head-to-tail; the N-terminus of one monomer interacts with the C-terminus of the other monomer and vice versa . The major dimerization element, unique to EndoVII, is the "four-helix-cross" domain, which consists of helix-2 and helix-3 from each monomer. It contains an extended hydrophobic core.

    Another feature is the "beta-finger", residues 38-56. Its stability depends critically on the zinc. This zinc ion is tetrahedrally co-ordinated to four cysteines, linking helix- I through residues C23 and C26 firmly to the N-terminal part of helix-2 (C58, C61). Indeed, interfering mutations inactivate the protein. Finally, the calcium ion, which marks the active site, is liganded to aspartate-40 and asparagine-62. Mutation studies show that these amino acids are essential for activity: The N62D mutant is completely inactive.

    The EndoVII structure has been docked to a "stacked-X" four-way DNA junction, one of its many substrates. This model is not refined, since both the DNA and the protein are known to be flexible and might undergo conformational changes. However, its overall features confirm experimental data: 1) The EndoVII dimer binds to the minor groove side of the four-way junction; 11) Basic residues on helix-2 can interact with phosphates on the exchanging strands and those on the C-terminal domain can interact with phosphates in the continuous strands, consistent with observed foot-printing patterns; 111) The C-terminus binds up to nine base pairs away from the junction, confirming the minimal length of two arms of the substrate; IV). The active sites do not cleave both the scissile phosphates simultaneously.

    Surprisingly, the N62D mutant shows a major rearrangement in the "four-helixcross" domain, when compared to the wild-type: helices-2 are translated by half a turn each, in opposite direction and the opening of the "bays", between each helix-2 and betafinger, is wider. These differences might be attributed to the point-mutation, which introduces an extra charge in the active site, to differences in crystallisation conditions, to the different pH employed, to crystal contacts or perhaps they are simply a sign of the intrinsic flexibility of EndoVII.

    This dilemma is partly solved in chapter 3, which presents the crystal structure of wild-type EndoVII in a different space group, which contains less solvent. It crystallised in the same drop, so that differences observed between the two wild-type structures cannot be attributed to the mutation, pH or salt concentrations. Since the helical-cross region of this second structure is very similar to that of the mutant, rearrangements in this region must be seen as a consequence of intrinsic flexibility of EndoVII. The widening of the "bays", however, might still be a consequence of the mutation, different pH, absence of Ca 2+ or crystal packing. An investigation of the flexibility of EndoVII with TLS- refinement, i.e. anisotropic refinement of rigid bodies, provides only limited insight. However, it confirms that rotations along the axes of the helices 2 and 4 and along the beta-finger are a main source of flexibility and also that the C-terminus, helix-4, 5 and 6, behave as a rigid body.

    The high-resolution structure of the N62D mutant brings more clarity towards the reaction mechanism of the nuclease. This model contains important water molecules and reveals the position and orientation of 14 sulphate ions, which may indicate favoured phosphate (DNA) binding sites. Supported by new mutation data (Birkenbihl, unpublished), these sulphate and water positions, combined with the Ca 2+ positions in the wild-type structures, suggest a reaction-mechanism similar to those proposed for some other magnesium dependent nucleases.

    2 Asparagine-62, glutamate-65 and aspartate-40 are important to position Mg 2+ or Ca 2+ next to the scissile phosphate of the DNA substrate. Histidine-41 activates a water molecule, which in turn executes a nucleophilic attack on the phosphor atom. Histidine-43 stabilises this phosphate directly through a hydrogen bond. Unfortunately it is still unclear why the N62D shows no DNase activity at all; an aspartate would also be able to ligand/position a divalent cation. The extra charge that this mutation introduces in the active site might distort the geometry of the active site, and repel the DNA. A more attractive, albeit more speculative hypothesis, assumes that the amino group of asparagine-62 donates a hydrogen-bond to the phosphate, which would also stabilise the transition state.

    At present, there are no known proteins with significant sequence homology to EndoVII, though nucleases with structural similarities do exist. One group consists of magnesium-dependent nucleases, which have a similar geometry of liganding sidechains around the magnesium (or calcium) ion in the active site; e.g. the E. coli proteins RuvC (Ariyosi et al., 1994) and RNase H (Katayanagi et al., 1990). However, these nucleases have no resembling fold. Most likely, this just shows that magnesium-dependent nucleases need a certain geometry to function.

    A more interesting group shares a folding motif similar to the beta finger and helix-2: Serratia Nuclease (Miller et al., 1994), Ppol (Flick et al., 1998) and perhaps even Colicin E9 (Kleanthous et al., 1999). Asparagine-62 and histidine-41 are conserved between Serratia nuclease, Ppol and T4 endonuclease VII. Ppol has also been crystallised in complex with DNA. If one superimposes this with the EndoVII structure, it turns out that the Ca 2+ in EndoVII is buried deeper within the protein, but small rotations (10-20 degrees) along helix 2 and the beta-finger suffice to superimpose them. These two nucleases act on different substrates, and maybe the larger DNA junctions of EndoVII need a wider and deeper binding groove than the double stranded DNA of Ppol. However, it could also be the source of EndoVII's specificity; flexible DNA might impose this conformational change of EndoVII upon binding, readying the enzyme for cleavage, while the magnesium or calcium ion might be too far away if EndoVII approaches more rigid DNA. A structure of EndoVII in complex with DNA would solve these questions.

    Chapter 4 presents the major part of the determination of the 3D structure of the tungsten-containing formate dehydrogenasc (W-FDH) from Desulfovibrio gigas, one of the first tungsten-containing enzymes isolated from a mesophile. The large subunit (92 kDa) is structurally related to several tungsten- and molybdenumcontaining enzymes and X-ray structures have been determined for two of them. One of these, the periplasmic nitrate reductase (Dias et al, 1999), could be used to obtain a molecular replacement solution. But the quality of phasing was not sufficient to generate a clear, interpretable electron density map. Furthermore, the amino acid sequence of W-FDH has not yet been determined, what makes model building complicated. Multiple wavelength diffraction (MAD) measurements were undertaken at the absorption edges of W and Fe to define unambiguously the number, positions and identity of these anomalous scatterers and to improve the X-ray phases. The MAD-analysis revealed one W-atom with a Se-cys ligand and one [4Fe-4S] cluster bound to the large subunit, and three [4Fe-4S] clusters in the small subunit. The four [4Fe-4S] clusters are ca. 10 Å apart, creating a feasible electron transfer pathway, which connects the exterior of the protein to the W/Se site in the large subunit. Two of the four iron-sulphur clusters had not been predicted before by spectroscopic techniques (Almendra et al., 1999). A reinvestigation of the spectroscopic data was performed, but gave the same results as before. If these data were correct, this means that the [4Fe-4S] clusters are instable, and that only protein with fully occupied clusters crystallises.

    The formate dehydrogenase H (FDH-H) from E. coli catalyses the same reaction as W-FDH, but uses a molybdenum instead of tungsten. Both are liganded to two molybdopterin-cofactors and to a seleno-cysteine, so the question remains why W-FDH prefers tungsten to the more common molybdenum. The full structure will allow a comparison of the two enzymes in atomic detail, and perhaps, it will shed some light on this phenomenon.

    X-ray crystallography has been used to characterise the nature of metal-centres in proteins, their coordination geometry and even their identity. Sometimes, the way metal ions are bound to the protein already clarifies its role in the protein. In other cases it has to be supplemented with other studies before the role can be fully understood. Either way, crystallography provides a powerful tool for the study of metalloproteins.

    Almendra, M.J., Brondino, C.D., Gavel, 0., Pereira, A.S., Tavares, P., Bursakov, S., Duarte, R., Caldeira, J., Moura, J.J.G., Moura, 1. (1999) Biochemistry, 38 , 16366-16372
    Ariyosi, M., Vassylyev, D., Iwasaki, H., Shinagawa, H. and Morikawa, K. (1994) Cell, 78 , 1063-1072.
    Flick, K.E., Jurica, M.S., Monnart Jr, R.J. and Stoddard, B.L. (1998), Nature, 394 , 96-101.
    Katayanagi, M., Miyagawa, M., Matsushima, M., Ishikawa, M., Kanaya, S., Ikehara, M., Matsuzaki, M. and Morikawa, K. (1990) Nature, 347 , 306-309.
    Kleanthous, C., Kuhlmann, U.C., Pornmer, A.J., Ferguson, N., Radford, S.E., Moore, G.R., James, R. and Hemmings, A.M. (1999) Nature Struct. Biol., 6 , 243-252.
    Miller, M.D., Tanner, J., Alpaugh, M., Benedik, M.J. and Krause, K.L. (1994) Nature Struct Biol., 1 , 461-468.
    Atmospheric volatile organic compounds (VOC) at a remote tropical forest site in central Amazonia
    Kessermeier, J. ; Kuhn, U. ; Wolf, A. ; Andreae, P. ; Ciccioli, P. ; Brancaleoni, E. ; Frattoni, M. ; Ganzeveld, L.N. ; Guenther, J. ; Greenberg, J.P. ; Castro Vasconcellos, P. De; Tavares, T. ; Artaxo, P. - \ 2000
    Atmospheric Environment 34 (2000)24. - ISSN 1352-2310 - p. 4063 - 4072.
    boundary-layer - acetic-acids - photochemical smog - isoprene emission - aerosol formation - hydrocarbons - terpene - monoterpenes - photooxidation - chemistry
    According to recent assessments, tropical woodlands contribute about half of all global natural non-methane volatile organic compound (VOC) emissions. Large uncertainties exist especially about fluxes of compounds other than isoprene and monoterpenes. During the Large-Scale Biosphere/Atmosphere Experiment in Amazonia ¿ Cooperative LBA Airborne Regional Experiment 1998 (LBA-CLAIRE-98) campaign, we measured the atmospheric mixing ratios of different species of VOC at a ground station at Balbina, Amazonia. The station was located 100 km north of Manaus, SE of the Balbina reservoir, with 200¿1000 km of pristine forest in the prevailing wind directions. Sampling methods included DNPH-coated cartridges for carbonyls and cartridges filled with graphitic carbons of different surface characteristics for other VOCs. The most prominent VOC species present in air were formaldehyde and isoprene, each up to several ppb. Concentrations of methylvinyl ketone as well as methacroleine, both oxidation products of isoprene, were relatively low, indicating a very low oxidation capacity in the lower atmospheric boundary layer, which is in agreement with a daily ozone maximum of
    Plant homologues of SGG/GSK-3(Shaggy/Glycogen synthase kinase-3)
    Tavares, R. ; Dornelas, M. ; Tichtinsky, G. ; Laurent, F. ; Schwebel-Dugue, N. ; Picaud, A. ; Lammeren, A.A.M. van; Kreis, M. - \ 2000
    In: Abstr. 6th Int. Congr. of Plant Molecular Biology Quebec : - p. S05 - 27.
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