Delivery of Functionality in Complex Food Systems: Physically Inspired Approaches from Nanoscale to Microscale,Wageningen 18–21 October 2009

The Wageningen Delivery of Functionality symposium covered all aspects involved with food structural design to arrive at high-quality foods which meet demanding customer expectations and regulatory requirements. The symposium integrated aspects from the structural organization of foods at molecular and supramolecular scales to dedicated techniques required to describe and visualize such structures, the gastro-intestinal events and how to model these in a laboratory setting, and finally the impact those food structures and ingredients have on the consumer’s physiology and on the human perception. As an interdisciplinary platform, bringing together more than 160 researchers from academia and industry, the symposium meanwhile fulfills an important role in the food science community.     

Association of vinculin to the platelet cytoskeleton during thrombin-induced aggregation

Vinculin is a protein generally believed to be involved in membrane-cytoskeleton interaction, and its presence in platelets has been verified earlier. Here we show that in resting bovine platelets, vinculin is not associated with the Triton-insoluble cytoskeletal fraction but becomes incorporated into it during the thrombin-induced activation process. The incorporation starts around the same time as the release reaction and only after the shape change and the first phase of aggregation have taken place. Its time course parallels the cytoskeletal association of actin and certain other contractile proteins. Vinculin is a minor component of platelet cytoskeleton and only about 10% of the total platelet vinculin becomes incorporated into the Triton X-100 residue.     

Localization of two human homologs, HHR6A and HHR6B, of the yeast DNA repair gene RAD6 to chromosomes Xq24-q25 and 5q23-q31.

The chromosomal localizations of two closely related human DNA repair genes, HHR6A and HHR6B, were determined by in situ hybridization with biotinylated probes. HHR6A and HHR6B (human homolog of yeast RAD6) encode ubiquitin-conjugating enzymes (E2 enzymes), likely to be involved in postreplication repair and induced mutagenesis. The HHR6B gene was assigned to human chromosome 5q23-q31, whereas the HHR6A gene was localized on the human X chromosome (Xq24-q25). This latter assignment was confirmed with an X-specific human-mouse/hamster somatic cell hybrid panel. Southern blot analysis points to an X and an autosomal localization of HHR6A and HHR6B, respectively, in the mouse. The potential involvement of these genes in human genetic disorders is discussed.     

Detection of Plasmid Transfer from Pseudomonas fluorescens to Indigenous Bacteria in Soil by Using Bacteriophage phiR2f for Donor Counterselection

The transfer of a genetically marked derivative of plasmid RP4, RP4p, from Pseudomonas fluorescens to members of the indigenous microflora of the wheat rhizosphere was studied by using a bacteriophage that specifically lyses the donor strain and a specific eukaryotic marker on the plasmid. Transfer of RP4p to the wheat rhizosphere microflora was observed, and the number of transconjugants detected was approximately 10 transconjugants per g of soil when 10 donor cells per g of soil were added; transfer in the corresponding bulk soil was slightly above the limit of detection. All of the indigenous transconjugants which we analyzed contained a 60-kb plasmid and were able to transfer this plasmid to a Nx RpP. fluorescens recipient strain. The indigenous transconjugants were identified as belonging to Pseudomonas spp., Enterobacter spp., Comamonas spp., and Alcaligenes spp.     

Characterization of caulobacters isolated from wastewater treatment systems.

Caulobacters are generally assumed to be found only in environments of low organic content; however, we readily isolated strains from a variety of sewage treatment system designs and locations, and 33 distinct strains were characterized. Most were morphologically similar, having the crescent-shaped cell body, short stalk, and hexagonally packed, paracrystalline surface (S) layer characteristic of several Caulobacter crescentus laboratory strains. Upon closer examination, they were distinguishable on the basis of protein band profiles on polyacrylamide gel electrophoresis, gross colony characteristics, or holdfast composition or by DNA restriction fragment length polymorphism analysis with flagellin and S-layer gene probes. Most of the isolates contained one or more high-molecular-weight plasmids and were resistant to a number of antibiotics, characteristics generally not shared with caulobacters isolated from other sources. Six of the 33 strains were retained because they did not fit the typical isolate profile; these strains are overrepresented in our collection compared with their relative proportion in wastewater treatment systems. By colony hybridization and restriction fragment length polymorphism analysis, all of these and one typical isolate showed less homology than the others to the surface array gene of a laboratory strain (C. crescentus CB15), and three hybridized less strongly with the flagellin gene from the same strain. In sum, although the strains were distinguishable, caulobacters from the wastewater treatment systems we examined were relatively homogenous, were similar to characterized laboratory strains, and, with exceptions, could probably be reliably detected as a group by gene probes derived from C. crescentus strains.     

Localization and quantification of extracellular polymers in methanogenic granular sludge

Summary Extracellular polymers were localized and quantitatively analysed in methanogenic granular sludge cultivated on either propionate or ethanol in laboratory upflow anaerobic sludge-blanket (UASB) reactors. Electron microscopical analysis of ultrathin sections of the two sludge types stained with ruthenium red revealed the presence of extracellular polymers with different densities and structures. For quantification, granular sludge from a large-scale UASB reactor at a liquid sugar plant was also included in this study. A three-step physical disintegration procedure was used to extract water-soluble extracellular material from the granules. After each disintegration step the extracts were analysed for polysaccharides and proteins. Cell damage and thus the contribution of intracellular proteins and polysaccharides was estimated simultaneously by the determination of free DNA and free ATP in the extracts. After two extraction steps, up to 3.5 mg polysaccharides/g organic material and 5.5 mg protein/g organic material were extracted, whereas no significant increase in DNA was detected. The role of extracellular polymers in granular stability is discussed. Offsprint requests to: A. J. B. Zehnder     

The amino terminal half of the MS2-coded lysis protein is dispensable for function: implications for our understanding of coding region overlaps.

We have asked whether genetic overlaps only evolve to provide extra coding capacity in genomes of restricted size. As a model system we have used the lysis gene of the RNA bacteriophage MS2. This gene overlaps with the distal part of the coat protein gene and with the proximal part of the replicase gene. Using recombinant DNA procedures we have determined whether either of the two overlaps codes for amino acids that are not essential for the function of the 75 amino acid long lysis protein. We find that the first 40 amino acids of the lysis protein are dispensable for function. Thus all of the genetic information essential to the synthesis of the active C-terminal peptide lies within the overlap with the replicase gene, whereas all dispensable residues are encoded in the overlap with the coat protein gene and in the intercistronic region. This suggests that the overlap with the coat protein gene is not required for extra coding capacity but serves to regulate the expression of the lysis gene. Comparative sequence analysis is consistent with this idea.     

Root growth dynamics of Brussels sprouts (Brassica olearacea var.gemmifera) and leeks (Allium porrum L.) as reflected by root length,root colour and UV fluorescence

Minirhizotron observations of roots of leeks and Brussels sprouts grown in the Wageningen Rhizolab were used to study the dynamics of root length. Day of appearance and the time of decay were assessed for individual root segments visible on the minirhizotron surface.A Brussels sprouts crop produced much more root length than leeks, but the average longevity of these roots was about half that of leek roots.To investigate whether root colour or UV fluorescence could be used as a quantitative index of root functionality or root age, changes in root colour (on a scale of greys) over time were measured with interactive image analysis. In both crops a gradual change towards black was found with ageing. Measurements of the intensity of the UV fluorescence showed that leek roots fluoresced more than Brussels sprouts roots. Over time, UV fluorescence decreased in Brussels sprouts roots but increased in leek roots. It is concluded that UV fluorescence cannot be used as a universal indicator of root age or root functionality, but in some plant species it may be used to separate (transparent) roots from the background with image analysis techniques.     

Characterization of In Vivo Reporter Systems for Gene Expression and Biosensor Applications Based on luxAB Luciferase Genes

Advances in genetic engineering methods have allowed the development of an increasing number of practical and scientific applications for bioluminescence with lux genes cloned from a variety of organisms. Bioluminescence derived from the shortened lux operon (luxAB genes) is a complex process, and applications seem to be proliferating in advance of an understanding of the underlying biochemical processes. In this report, we describe a two-phase kinetic behavior of the light emission which must be properly taken into account in any quantitative measurements of the bioluminescence signal. By using strains of Escherichia coli and Caulobacter crescentus, this behavior was characterized and interpreted in terms of the biochemistry underlying the bacterial luciferase mechanism. We show that the intensity profile of each of the two phases of the luminescence signal is responsive (and exhibits different sensitivities) to the concentration of added decanal and other components of the assay mix, as well as to the order of mixing and incubation times. This study illustrates the importance of appropriate protocol design, and specific recommendations for using the luxAB system as a molecular reporter are presented, along with versatile assay protocols that yield meaningful and reproducible signals.     

The differentiation of Staphylococcus aureus from other Micrococcaceae isolated from bovine mammary glands

Haemolysin production, the slide coagulase test and the tube coagulase test were assessed for their capability to differentiate Staphylococcus aureus among other Micrococcaceae in 199 isolates from udders of cows in herds with a low bulk milk somatic cell count. The API-Staph test was used as a reference.
Haemolysin production was less effective in identifying Staph. aureus among Micrococcaceae than a combination of other tests. Differences were found in the predictive values of results from diagnostic protocols in which the slide coagulase test was performed on all Micrococcaceae, or on β-haemolysin-negative Micrococcaceae only. Diagnostic protocols in which haemolysin production was combined with the results of the other tests resulted in excellent diagnostic performance and a reduction in diagnostic procedures. Recommendations for routine Staph. aureus identification in bovine mastitis bacteriology are given.