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A cautionary note on the use of split-YFP/BiFC in plant protein-protein interaction studies
Horstman, A. ; Nougalli Tonaco, I.A. ; Boutilier, K.A. ; Immink, R.G.H. - \ 2014
International Journal of Molecular Sciences 15 (2014). - ISSN 1661-6596 - p. 9628 - 9643.
bimolecular fluorescence complementation - bifc-based fret - living cells - ternary complexes - visualization - arabidopsis - assay - expression - fragments - cloning
Since its introduction in plants 10 years ago, the bimolecular fluorescence complementation (BiFC) method, or split-YFP (yellow fluorescent protein), has gained popularity within the plant biology field as a method to study protein-protein interactions. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. The major drawback of BiFC is that the fluorescent protein halves are prone to self-assembly independent of a protein-protein interaction event. To circumvent this problem, several modifications of the technique have been suggested, but these modifications have not lead to improvements in plant BiFC protocols. Therefore, it remains crucial to include appropriate internal controls. Our literature survey of recent BiFC studies in plants shows that most studies use inappropriate controls, and a qualitative rather than quantitative read-out of fluorescence. Therefore, we provide a cautionary note and beginner’s guideline for the setup of BiFC experiments, discussing each step of the protocol, including vector choice, plant expression systems, negative controls, and signal detection. In addition, we present our experience with BiFC with respect to self-assembly, peptide linkers, and incubation temperature. With this note, we aim to provide a guideline that will improve the quality of plant BiFC experiments.
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
Ratio of mutated versus wildtype coat protein sequences in Pepino mosaic virus determines nature and severity of yellowing symptoms on tomato plants
Hasiów-Jaroszewska, B. ; Paeleman, A. ; Ortega-Parra, N. ; Borodynko, N. ; Byczyk, J. ; Czerowniec, A. ; Thomma, B.P.H.J. ; Hanssen, I.M. - \ 2013
Molecular Plant Pathology 14 (2013)9. - ISSN 1464-6722 - p. 923 - 933.
genomic rna - greenhouse tomatoes - isolate - replication - population - fragments - chlorosis - variants - models - crops
Recently, Pepino mosaic virus (PepMV) infections causing severe yellowing symptoms in tomato plants have been reported in glasshouse tomato crops. When studying this phenomenon in commercial glasshouses, two different types of yellowing symptoms, occurring in adjacent plants, were distinguished: interveinal leaf yellowing and yellow mosaics. After several weeks, the interveinal leaf yellowing symptoms gradually disappeared and the plant heads became green again, with yellow mosaic patterns on the leaves as an intermediate stage. The sequencing of multiple isolates causing interveinal leaf yellowing identified two point mutations, occurring in positions 155 and 166 of the coat protein (CP), as unique to the yellowing pathotype. Site-directed mutagenesis of infectious clones confirmed that both CP mutations are determinants of the interveinal leaf yellowing symptoms. Sequencing of CP clones from plants or plant parts with the yellow mosaic symptoms resulted in a mixture of wild-type and mutated sequences, whereas sequencing of CP clones from the green heads of recovered plants resulted in only wild-type sequences. Yellow mosaic symptoms could be reproduced by inoculation of an artificial 1:1 mixture of RNA transcripts from the wild-type and mutated infectious clones. These results show that the ratio of mutated versus wild-type sequences can determine the nature and severity of symptom development. The gradual recovery of the plants, which coincides with the disappearance of the yellowing mutations, suggests that selection pressure acts to the advantage of the wild-type virus. Experiments with wild-type and mutated infectious clones showed that reverse mutation events from mutant to wild-type occur and that the wild-type virus does not have a replicative advantage over the mutant. These results suggest that reverse mutation events occur, with subsequent selection pressure acting in favour of the wild-type virus in the growing plant parts, possibly related to a lower long-distance movement efficiency of the mutant.
Influence of PEGylation with linear and branched PEG chains on the adsorption of glucagon to hydrophobic surfaces
Pinholt, C. ; Bukrinsky, J.T. ; Hostrup, S. ; Frokjaer, S. ; Norde, W. ; Jorgensen, L. - \ 2011
European Journal of Pharmaceutics and Biopharmaceutics 77 (2011)1. - ISSN 0939-6411 - p. 139 - 147.
poly(ethylene glycol)-modified lysozyme - protein adsorption - polyethylene-glycols - silica - conformation - fluorescence - association - molecules - fragments - peptide
PEGylation has proven useful for prolonging the plasma half lives of proteins, and since approval of the first PEGylated protein drug product by the FDA in 1990, several PEGylated protein drug products have been marketed. However, the influence of PEGylation on the behavior of proteins at interfaces is only poorly understood. The aim of this work was to study the effect of PEGylation on the adsorption of glucagon from aqueous solution to a hydrophobic surface and to compare the effects of PEGylation with a linear and a branched PEG chain, respectively. The 3483 Da peptide glucagon was PEGylated with a 2.2 kDa linear and a branched PEG chain, respectively, and the adsorption behaviors of the three proteins were compared using isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. PEGylation decreased the number of glucagon molecules adsorbing per unit surface area and increased the initial adsorption rate of glucagon. Furthermore, the results indicated that the orientation and/or structural changes of glucagon upon adsorption were affected by the PEGylation. Finally, from the isothermal titration calorimetry and the reflectometry data, it was observed that the architecture of the PEG chains had an influence on the observed heat flow upon adsorption as well as on the initial rate of adsorption, respectively.
A Broad Set of Different Llama Antibodies Specific for a 16kDa Heat Shock Protein of Mycobacterium tuberculosis
Trilling, A.K. ; Ronde, H. de; Noteboom, L. ; Houwelingen, A.M.M.L. van; Roelse, M. ; Srivastava, S.K. ; Haasnoot, W. ; Jongsma, M.A. ; Kolk, A. ; Zuilhof, H. ; Beekwilder, J. - \ 2011
PLoS ONE 6 (2011)10. - ISSN 1932-6203 - 10 p.
b-cell - monoclonal-antibodies - rapid detection - in-vitro - antigen - single - fragments - resistance - epitopes - binding
Background Recombinant antibodies are powerful tools in engineering of novel diagnostics. Due to the small size and stable nature of llama antibody domains selected antibodies can serve as a detection reagent in multiplexed and sensitive assays for M. tuberculosis. Methodology/Principal Findings Antibodies for Mycobacterium tuberculosis (M. tb) recognition were raised in Alpaca, and, by phage display, recombinant variable domains of heavy-chain antibodies (VHH) binding to M. tuberculosis antigens were isolated. Two phage display selection strategies were followed: one direct selection using semi-purified protein antigen, and a depletion strategy with lysates, aiming to avoid cross-reaction to other mycobacteria. Both panning methods selected a set of binders with widely differing complementarity determining regions. Selected recombinant VHHs were produced in E. coli and shown to bind immobilized lysate in direct Enzymelinked Immunosorbent Assay (ELISA) tests and soluble antigen by surface plasmon resonance (SPR) analysis. All tested VHHs were specific for tuberculosis-causing mycobacteria (M. tuberculosis, M. bovis) and exclusively recognized an immunodominant 16 kDa heat shock protein (hsp). The highest affinity VHH had a dissociation constant (KD) of 4×10-10 M. Conclusions/Significance A broad set of different llama antibodies specific for 16 kDa heat shock protein of M. tuberculosis is available. This protein is highly stable and abundant in M. tuberculosis. The VHH that detect this protein are applied in a robust SPR sensor for identification of tuberculosis-causing mycobacteria.
Is spatial structure the key to promote plant diversity in Mediterranean forets plantations?
González-Moreno, P. ; Quero, J.L. ; Poorter, L. ; Bonet, F.J. ; Zamora, R. - \ 2011
Basic and Applied Ecology 12 (2011)3. - ISSN 1439-1791 - p. 251 - 259.
pine plantations - seed dispersal - fragments - restoration - recruitment - vegetation - patterns - distance - spain - edge
Mediterranean forest plantations are currently under an intense debate related to their ecological function, sustainability and future performance. In several Mediterranean countries, efforts are directed to convert pine plantations into mixed and more diverse forests. This research aims to evaluate the effect of the spatial configuration of pine plantations on regeneration and plant diversity in order to facilitate plantation management towards more diversified stands. Spatial characteristics of plantations (proximity to different vegetation types, fragmentation and internal patch structure) were related to abundance of seedlings of an ecologically important broadleaved species, Holm Oak (Quercus ilex L.), and the Shannon diversity index of the community. Q. ilex seedling abundance and plant diversity in pine plantation patches are favoured by the proximity to oak patches located uphill. Fragmentation affected only plant diversity, with smaller patches having more diversity. The internal structure of the pine patch influenced both regeneration of Q. ilex and diversity. Pine patches with lower pine tree density were characterized by higher diversity and less Q. ilex regeneration confirming that internal structure affects species differently. From a management perspective, the process of conversion of Mediterranean pine plantations to mixed oak–pine forests could be facilitated by (1) having the seed source uphill from the plantation, (2) increasing the fragmentation of plantations and (3) promoting the internal heterogeneity of plantations to create a diverse range of light environments matching the different requirements of species
Postdispersal seed predation and seed viability in forest soils: implications for the regeneration of tree species in Ethiopian church forests
Wassie Eshete, A. ; Bekele, T. ; Sterck, F.J. ; Teketay, D. ; Bongers, F. - \ 2010
African Journal of Ecology 48 (2010)2. - ISSN 0141-6707 - p. 461 - 471.
europaea ssp-cuspidata - tropical rain-forest - germination ecology - northern ethiopia - prunus-africana - fragments - distance - rodents - edge - microhabitat
Almost all dry Afromontane forests of Northern Ethiopia have been converted to agricultural, grazing or scrub lands except for small fragments left around churches ('Church forests'). Species regeneration in these forests is limited. We investigated (i) how intense postdispersal seed predation was in church forest, and if this seed predation varied with species and/or habitat, and (ii) for how long tree seeds maintained their viability while buried in forest soil. In the seed predation experiment, we monitored seeds of six tree species in four habitats for a period of 14 weeks (the peak seeding season). In the seed viability experiment, we assessed seed viability of five species in four habitats after being buried 6, 12, or 18 months. Ninety-two percent of the tree seeds were predated within 3.5 months. Predation was mainly dependent on species whereas habitat had a weaker effect. Seed viability decreased sharply with burial time in soil for all species except for Juniperus. To minimize seed availability limitation for regeneration of such species in the forest, the standing vegetation needs to be persistently managed and conserved for a continuous seed rain supply. Additional seed sowing, and seed and seedling protection (by e.g. animal exclosures) may increase successful regeneration of important species in these forests
Genetic variables of various manifestations of osteochondrosis and their correlations between and within joints in Dutch warmblood horses
Grevenhof, E.M. van; Schurink, A. ; Ducro, B.J. ; Weeren, P.R. van; Tartwijk, J.M.F.M. van - \ 2009
Journal of Animal Science 87 (2009)6. - ISSN 0021-8812 - p. 1906 - 1912.
german coldblood horses - limb joints - prevalence - fragments
Osteochondrosis (OC) is an important orthopedic developmental disorder in many horse populations. A review of the literature revealed widely variable heritability estimates for the disorder. We estimated the genetic variables (heritabilities and genetic correlations) of various manifestations of OC. Femoropatellar, tarsocrural, and metacarpophalangeal and metatarsophalangeal joints of 811 randomly selected yearlings from the Royal Warmblood Studbook of the Netherlands, descending from 32 representative stallions, were scored for OC at 28 predilection sites. At each site, OC was scored in 5 categories, distinguishing between flattened bone contours and fragments. At the animal level, the overall heritability of OC was 0.23, the heritability of flattened bone contours was 0.08, and the heritability of fragments was 0.22. At the joint level, heritability was greatest in the tarsocrural joints, intermediate in the metacarpophalangeal and metatarsophalangeal joints, and least in the femoropatellar joints. The heritability estimates for the contralateral joint homologs were very similar. The genetic correlation between the tarsocrural and femoropatellar joint was strong, whereas correlations between the metacarpophalangeal and metatarsophalangeal and other joints were moderate. The genetic correlation between flattened bone contours and fragments at the animal level was 0.80. Scoring OC on a 5-point categorical scale resulted in greater heritability on the observed scale than when analyzing OC as a binary trait. Our results suggest that selection against OC could best be performed by taking into account the OC status of all 4 joints, the femoropatellar, the tarsocrural, and the metacarpophalangeal and metatarsophalangeal joints, and discerning between flattened bone contours and fragments.
Effects of soil type, management type and soil amendments on the survival of the potato brown rot bacterium Ralstonia solanacearum
Messiha, N.A.S. ; Bruggen, A.H.C. van; Franz, E. ; Janse, J.D. ; Schoeman-Weerdesteijn, M.E. ; Termorshuizen, A.J. ; Diepeningen, A.D. van - \ 2009
Applied Soil Ecology 43 (2009)2-3. - ISSN 0929-1393 - p. 206 - 215.
gradient gel-electrophoresis - organic farming systems - weed hosts - biovar 2 - race-3 - fate - communities - fragments - severity - climates
Potato brown rot disease (Ralstonia solanacearum) is a serious economic problem in Egypt, partly due to an European Union requirement that potatoes for export to the EU should be grown in so-called pest free area's (PFA's), where fields are tested and infested fields are put under quarantine measures. To investigate pathogen survival and to determine the time required to keep infested fields in quarantine, the survival of R. solanacearum race 3 biovar 2 was tested in soils differing in origin (Dutch versus Egyptian soils), soil type (sand versus clay), and management type (organic versus conventional). All eight soils were tested at moderate (15 °C) and elevated temperatures (28 °C). Also the effects of artificial fertilizer and organic (compost and cow manure) amendments on survival of R. solanacearum were tested. In all soils, with and without amendments, the pathogen dropped below the detection limit (102 CFU g-1 d.w. soil) within 5 months. At both temperatures, all Egyptian soils showed a significantly faster decline in pathogen density than the Dutch soils. The decline in colony forming units of R. solanacearum per gram of soil was faster in sandy soils than in clay soils from both countries. Management effects on decline of R. solanacearum were smaller and less consistent: for some soils, organic management resulted in a significantly shorter 50%-reduction-time and/or greater decline rate than conventional management, for other soils the differences were not significant. Survival periods at 15 °C were longer than at 28 °C in Dutch soils, but not in Egyptian soils, where survival was slightly shorter at the lower temperature. Amendments with NPK fertilizer to the conventional soils and with cow manure to the organic soils enhanced the decline rate of R. solanacearum in these soils. The decline rate of the pathogen was negatively correlated with total soluble organic matter and positively with bacterial diversity. In conclusion, the overriding factors determining survival of R. solanacearum in soil may be the production of toxic concentrations of ammonia on the one hand, and availability of substrate in combination with microbial competition on the other hand. The sandy desert soils of Egypt are very suitable for production of export potatoes because the pathogen would survive for only a relatively short period in those soils, if it were accidentally introduced. Addition of ammonia-producing amendments can reduce populations of R. solanacearum, whereas compost addition and organic management do not necessarily result in an enhanced decline of the pathogen
Prevalence of various radiographic manifestations of osteochondrosis and their correlations between and within joints in Dutch Warmblood horses
Grevenhof, E.M. van; Ducro, B.J. ; Weeren, P.R. van; Tartwijk, J.M.F.M. van; Belt, A.J. van den; Bijma, P. - \ 2009
Equine Veterinary Journal 41 (2009)1. - ISSN 0425-1644 - p. 11 - 16.
german coldblood horses - fragments - dissecans - metatarsophalangeal - metacarpophalangeal - fetlock
Reasons for performing study: Osteochondrosis (OC) is the most important orthopaedic developmental disorder in horses and may manifest in several different forms. No detailed study on the prevalence and/or interrelation of these forms is available, even though these data are a prerequisite for conclusive genetic studies. Objectives: To assess the prevalence of the various manifestations of OC as detected radiographically and to evaluate possible relationships between their occurrence within the same joint and between different joints. Methods: The FP (femoropatellar), TC (tarsocrural) and MCP/MTP (metacarpophalangeal/metatarsophalangeal) joints of 811 yearlings selected randomly, descending from 32 representative stallions, were radiographed and scored for the presence and grade of osteochondrotic lesions. Results were compared at the sire, animal, joint and predilection site levels. Results: In the FP joint, the percentage of animals showing normal joint contours in all sites was 60.7%. For the TC joint and the combined MCP/MTP joints, these figures were 68.6 and 64.6%, respectively. For all joints combined, the percentage dropped to 30.5%. Sedation improved detection of OC lesions in the FP joint. There was a high correlation between the right and left joints. The correlation between flattened bone contours and fragments was considerably less. Conclusions: Scoring on a detailed scale is necessary to achieve good insight into the prevalence of OC. Observations on the right and left joints can be combined in further analyses, whereas flattened bone contours and fragments should be evaluated as statistically different disorders. Potential relevance: This study provides insight into the prevalences of various manifestations of OC and their relationships, within and between joints. These results form the basis for detailed quantitative and/or molecular genetic studies that should lead to the establishment of breeding indices and/or genetic marker sets for OC.
Quantification and characterization of enzymatically produced hyaluronan with fluorophore-assisted carbohydrate electrophoresis
Kooy, F.K. ; Muyuan Ma, ; Beeftink, H.H. ; Eggink, G. ; Tramper, J. ; Boeriu, C.G. - \ 2009
Analytical Biochemistry 384 (2009)2. - ISSN 0003-2697 - p. 329 - 336.
polyacrylamide-gel electrophoresis - synthase - oligosaccharides - microanalysis - acid - fragments - sulfate - assay - face
Hyaluronan (HA) is a polysaccharide with high-potential medical applications, depending on the chain length and the chain length distribution. Special interest goes to homogeneous HA oligosaccharides, which can be enzymatically produced using Pasteurella multocida hyaluronan synthase (PmHAS). We have developed a sensitive, simple, and fast method, based on fluorophore-assisted carbohydrate electrophoresis (FACE), for characterization and quantification of polymerization products. A chromatographic pure fluorescent template was synthesized from HA tetrasaccharide (HA4) and 2-aminobenzoic acid. HA4-fluor and HA4 were used as template for PmHAS-mediated polymerization of nucleotide sugars. All products, fluorescent and nonfluorescent, were analyzed with gel electrophoresis and quantified using lane densitometry. Comparison of HA4- and HA4-fluor-derived polymers showed that the fluorophore did not negatively influence the PmHAS-mediated polymerization. Only even-numbered oligosaccharide products were observed using HA4-fluor or HA4 as template. The fluorophore intensity was linearly related to its concentration, and the limit of detection was determined to be 7.4 pmol per product band. With this assay, we can now differentiate oligosaccharides of size range DP2 (degree of polymerization 2) to approximately DP400, monitor the progress of polymerization reactions, and measure subtle differences in polymerization rate. Quantifying polymerization products enables us to study the influence of experimental conditions on HA synthesis
The 'habitat backbone' as strategy to conserve pioneer species in dynamic port habitats: lessons from the natterjack toad ( Bufo calamita ) in the Port of Antwerp (Belgium)
Snep, R.P.H. ; Ottburg, F.G.W.A. - \ 2008
Landscape Ecology 23 (2008)10. - ISSN 0921-2973 - p. 1277 - 1289.
urban landscape - populations - persistence - fragments
Biodiversity conservation in economic areas like ports has recently become more important in the European Union due to a stricter interpretation of nature protection laws. In this study we develop a planning and design strategy¿the `habitat backbone¿¿ with which to support the long-term survival of pioneer species that occur in ports and have low dispersal abilities. For those species, long-term survival in port areas is uncertain because supply of their habitats (on vacant lots) is capricious and depends on land use dynamics. By gaining knowledge about spatial and temporal characteristics of these dynamics we were able to develop a solution to conserve such species. Our solution is based on the creation of permanent habitat¿defined as a `backbone¿¿ on (semi-) public land with an overall carrying capacity sufficient to support persistent populations. This best ensures long-term survival, and the backbone may also act as refugium. Satellite populations that emerge on adjacent vacant lots will thereby add to the persistence of the overall metapopulation. Management of permanent habitat is focused on retaining early-successional stages of vegetation. Implementing this strategy in the case of the natterjack toad in the Port of Antwerp taught us that realization of a habitat backbone is possible only if landowners, local governments and environmental NGOs cooperate. In the case at hand, such cooperation resulted in a plan that should ensure a coherent and persistent habitat network in which a chorus of some 1,400 natterjack toads could be accommodated¿ more than the number of toads currently observed.
Stable recombinant alpaca antibodies for detection of Tulip virus X
Beekwilder, M.J. ; Houwelingen, A.M.M.L. van; Beckhoven, J.R.C.M. van; Speksnijder, A.G.C.L. - \ 2008
European Journal of Plant Pathology 121 (2008)4. - ISSN 0929-1873 - p. 477 - 485.
monoclonal-antibodies - fragments - immunoassay - proteins - extracts - domains
For detection of the plant pathogenic Tulip virus X (TuVX), a panel of six recombinant antibodies was identified. To this end, a repertoire of variable domains from heavy-chain immunoglobulins (VHH) was cloned from an alpaca, which had been immunized with TuVX. Binding domains were selected by phage display and panning on immobilized TuVX particles. Recombinant VHH antibodies were tested for sensitivity in a sandwich ELISA, and were demonstrated to be readily able to distinguish TuVX-infected tulip leaf material from uninfected leafs. No cross-reactivity of the VHH antibodies to related flexiviridae was observed. Recombinant VHHs maintained their reactivity upon storage at ¿20°C for over a year. The effect of incubation at higher temperatures for prolonged time was studied. Two out of three VHH proteins retained activity after several weeks of storage at 37°C
Diversity of ground beetles (Coleoptera: Carabidae) and spiders (Araneae) in roadside verges with grey hair-grass
Noordijk, J. ; Schaffers, A.P. ; Sykora, K.V. - \ 2008
European Journal of Entomology 105 (2008)2. - ISSN 1210-5759 - p. 257 - 265.
dispersal corridors - habitats - impact - netherlands - movements - fragments - abundance - survival - faunas - dunes
Roadside verges in densely populated areas are often a significant addition to the total semi-natural area and as such may contribute to the conservation of biodiversity. Furthermore, they can enhance the ecological cohesion of a region, especially when the existing nature reserves are small and/or highly fragmented. We investigated the occurrence of ground beetles and spiders in six highway verges with grey hair-grass vegetation in the Veluwe region, The Netherlands. Total species number in the verges was similar to the values found in nearby nature reserves with comparable vegetation, but the ground beetles tended to be more abundant in the reserves. Many stenotopic species were present in the verges, confirming the significant conservation value of this habitat. However, compared with the nature reserves, there were fewer species of stenotopic ground beetles and the stenotopic spiders were less abundant in the verges. From our knowledge of the biology and ecology of the species captured it seems likely that this is attributable to differences in "habitat quality". In the verges, species preferring bare sand are scarcer, and the weighted mean body length of ground beetles is shorter than in nature reserves. This suggests that the sward in the road verges is too dense and the suitable vegetation too patchy to sustain some of the stenotopic species. Habitat analyses support this contention: Compared with the nature reserves, the verges had proportionally less bare sand, more herb and tree cover and the vegetation patches were smaller. The strategy suggested to promote stenotopic species entails removing encroaching trees and shrubs from the verges in order to expand the nutrient-poor zone and (re-)create pioneer conditions. Subsequent management should aim at further improving the road verges as extensions of nutrient-poor habitat.
Phage display-selected single chain antibodies confer high levels of resistance against Tomato spotted wilt virus
Prins, M.W. ; Lohuis, H. ; Schots, A. ; Goldbach, R.W. - \ 2005
Journal of General Virology 86 (2005)7. - ISSN 0022-1317 - p. 2107 - 2113.
transgenic tobacco - gene-sequences - rna-polymerase - plant cytosol - fv-antibody - expression - protein - fragments - cells - tospoviruses
Rational design of antibodies targeting essential viral proteins can complement the palette of antiviral resistance strategies. Here, stable and high expression of single-chain monoclonal antibodies targeting the nucleoprotein of the economically important plant virus Tomato spotted wilt virus, a protein that is involved in multiple steps in the viral infection cycle, is reported. High cytoplasmic expression levels of three selected phage display-derived anti-viral single-chain antibodies were established. Of these antibodies, two led to high levels of resistance against this plant virus. Protoplast experiments provided evidence that the two resistance-conferring antibodies may have a different mode of action and could be combined for higher durability of resistance in the field
Electrostatic interactions between immunoglobulin (IgG) molecules and a charged sorbent
Bremer, M.G.E.G. ; Duval, J.F.L. ; Norde, W. ; Lyklema, J. - \ 2004
Colloids and Surfaces. A: Physicochemical and Engineering Aspects 250 (2004)1-3. - ISSN 0927-7757 - p. 29 - 42.
colloidal particles - hydrophobic surfaces - adsorption - proteins - forces - reflectometry - polystyrene - antibodies - interfaces - fragments
The influence of electrostatic interactions on the adsorption of IgG is examined both theoretically and experimentally. The long-range interaction between IgG and the charged sorbent surface is treated in terms of the DLVO theory taking into account the possibility of charge- and potential regulation upon approach of IgG towards the surface. The relevant electrostatic and van der Waals interactions are a combination of interactions between IgG molecules and the sorbent surface (hetero-interaction part) and interactions between adsorbed and incoming IgG molecules (homo-interaction part). Experiments are performed on hydrophilic surfaces, namely negatively charged silica and positively charged amine-functionalised silica. It is concluded that electrostatic interactions have a strong influence on the adsorption behaviour. Excellent agreement was found between experiment and theory with respect to the dependence of the adsorption kinetics on pH and ionic strength. Furthermore, the steady-state adsorbed amount as a function of these variables is satisfactorily accounted for
Effects of Tylosin on Bacterial Mucolysis, Clostridium perfringens Colonization, and Intestinal Barrier Function in a Chick Model of necrotic Enteritis
Collier, C.T. ; Klis, J.D. van der; Deplancke, B. ; Anderson, D.B. ; Gaskins, G.R. - \ 2003
Antimicrobial Agents and Chemotherapy 47 (2003)10. - ISSN 0066-4804 - p. 3311 - 3317.
gradient gel-electrophoresis - broiler-chickens - resistance - fragments - community - sediments - growth - diets - flora - drugs
Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats. Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics. However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C. perfringens as well as the pathophysiological consequences of its growth. We report a study with a chick model of NE, which used molecular (16S rRNA gene [16S rDNA]) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C. perfringens. The effects of tylosin and pectin on mucolytic activity of the microbiota and C. perfringens colonization and their relationship to pathological indices of NE were of particular interest. The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C. perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function. The presence of pectin did not significantly affect the variables measured. Thus, it appears that tylosin can control NE through its modulation of C. perfringens colonization and the mucolytic activity of the intestinal microbiota.
On-farm impact of cattle slurry manure management on biological soil quality
Goede, R.G.M. de; Brussaard, L. ; Akkermans, A.D.L. - \ 2003
Netherlands Journal of Agricultural Science 51 (2003)1-2. - ISSN 0028-2928 - p. 103 - 133.
rundveemest - rundveedrijfmest - behandeling - plantenvoeding - kunstmeststoffen - biologische bodemactiviteit - melkveehouderij - nederland - bodemfauna - mestverwerking - cattle manure - cattle slurry - treatment - plant nutrition - fertilizers - soil fauna - biological activity in soil - dairy farming - netherlands - manure treatment - gradient gel-electrophoresis - maturity index - nitrogen - nematodes - fragments - emissions - dynamics - carbon
The effects of dairy cattle slurry management on soil biota, soil respiration and nitrogen (N) mineralization were evaluated in a farm trial across 12 farms and a field experiment on 2 farms located in a dairy farming area in the north of the Netherlands. The slurry management consisted of slit injection or surface application of slurry; the use or no use of additives [Euromestmix® (MX) and Effective Microbes® (EM)] and the type and level of inorganic N fertilization. Slit injection negatively affected epigeic earthworms whereas its effect on anecic and endogeic earthworms was absent or even positive. Enchytraeids were not affected in a consistent way, whereas numbers of nematodes indicative of nutrient- enriched conditions increased. Inorganic N fertilizer had similar effects. Bacterial diversity was not different among the treatments. Nitrifier diversity, however, was high at one of the farms in the field experiment, and was negatively affected by inorganic N fertilizer. The use of MX was usually associated with higher numbers of earthworms. EM affected numbers of earthworms and numbers of bacterial and plant-feeding nematodes, but only in specific combinations of field history, slurry type and slurry application method. We found no effects of EM on the composition of the microbial community. Soil respiration was increased when slurry was surface-applied. The calculated N mineralization by earthworms was in the order of 70–200 kg N ha -¹ year -¹. It was highest under farm-characteristic surface application of slurry with MX and lowest under farm-characteristic slit injection of slurry without additives. Compared with the N mineralization by earthworms, that by enchytraeids and nematodes was quantitatively insignificant. Negative treatment effects on earthworms led to corresponding reductions in calculated N mineralization.
Rooting of microcuttings: Theory and practice
Klerk, G.J.M. de - \ 2002
In Vitro Cellular and Developmental Biology. Plant 38 (2002)5. - ISSN 1054-5476 - p. 415 - 422.
micropropagated plants - pericyclic topipotency - apple microcuttings - rorippa-sylvestris - cultivated invitro - aerial organs - stem slices - fragments - acid
Poor adventitious root formation is a major obstacle in micropropagation and in conventional propagation. This paper reviews recent progress in the understanding of adventitious root formation as a developmental process focusing on the role of plant hormones and on the effect of rooting conditions on plant performance. Since the discovery of the rhizogenic effect of auxin ca. 70 yr ago, no new broadly applicable rooting treatments have been developed. Recent research, though, may lead to new rooting procedures. Application of wounding-related compounds may be effective in difficult-to-root crops. Furthermore, by adapting conditions during the propagation phase, microcuttings with an enhanced capability to root may be produced. These conditions include elongation of stems (by etiolation or double-layer culture) and repeated subculture (rejuvenation; i.e. transition from adult to juvenile). Data are presented that show that during tissue culture maturation (transition from juvenile to adult) also occurs. The conditions during the in vitro rooting treatment may have a tremendous effect on performance after transfer ex vitro. In particular, accumulation of ethylene during in vitro rooting may have a devastating effect. Addition of stress-protecting compounds during propagation or rooting in vitro may enhance the performance ex vitro
Molecular characterization of transgenic shallots (Allium cepa L.) by adaptor ligation PCR (AL-PCR) and sequencing of genomic DNA flanking T-DNA borders
Zheng, S.J. ; Henken, G. ; Sofiari, E. ; Jacobsen, E. ; Krens, F.A. - \ 2001
Transgenic Research 10 (2001)3. - ISSN 0962-8819 - p. 237 - 245.
polymerase chain-reaction - mediated transformation - arabidopsis-thaliana - plasmid rescue - amplification - walking - plants - gene - junctions - fragments
Genomic DNA blot hybridization is traditionally used to demonstrate that, via genetic transformation, foreign genes are integrated into host genomes. However, in large genome species, such as Allium cepa L., the use of genomic DNA blot hybridization is pushed towards its limits, because a considerable quantity of DNA is needed to obtain enough genome copies for a clear hybridization pattern. Furthermore, genomic DNA blot hybridization is a time-consuming method. Adaptor ligation PCR (AL-PCR) of genomic DNA flanking T-DNA borders does not have these drawbacks and seems to be an adequate alternative to genomic DNA blot hybridization. Using AL-PCR we proved that T-DNA was integrated into the A. cepa genome of three transgenic lines transformed with Agrobacterium tumefaciens EHA105 (pCAMBIA 1301). The AL-PCR patterns obtained were specific and reproducible for a given transgenic line. The results showed that T-DNA integration took place and gave insight in the number of T-DNA copies present. Comparison of AL-PCR and previously obtained genomic DNA blot hybridization results pointed towards complex T-DNA integration patterns in some of the transgenic plants. After cloning and sequencing the AL-PCR products, the junctions between plant genomic DNA and the T-DNA insert could be analysed in great detail. For example it was shown that upon T-DNA integration a 66bp genomic sequence was deleted, and no filler DNA was inserted. Primers located within the left and right flanking genomic DNA in transgenic shallot plants were used to recover the target site of T-DNA integration