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.     

Functional analysis of the Lactococcus lactis usp45 secretion signal in the secretion of a homologous proteinase and a heterologous α-amylase

The ups45 gene encodes the major extracellular protein from Lactococcus lactis. The deduced sequence of the 27 residue leader peptide revealed the tripartite characteristics of a signal peptide. This leader peptide directed the efficient secretion of the homologous proteinase (PrtP) in L. lactis, indicating that the putative signal peptide of PrtP can be replaced by the 27 residue Usp45 leader peptide. In addition, the 27 residue leader peptide could be used to secrete the Bacillus stearothermophilus α-amylase, encoded by the amyS gene. Fusion of the usp45 promoter region and various parts of the leader sequence to an amyS gene devoid of its signal sequence, showed that in Escherichia coli the first 19, 20, and 27 residues of the Usp45 leader are able to direct α-amylase secretion. In L. lactis the shorter signal peptides did not result in secretion of α-amylase, providing experimental evidence for the hypothesis that gram-positive bacteria require a longer signal peptide for secretion than gram-negative organisms.     

Construction and characterization of the chimeric monoclonal antibody E48 for therapy of head and neck cancer

Data from an ongoing clinical radioimmunoscintigraphy trial indicate that^99mTc-labeled monoclonal antibody (mAb) E48 is highly capable of selectively targeting squamous cell carcinoma of the head and neck (HNSCC). The percentage of the injected dose per gram of tumor tissue was found to be high, rendering mAbE48 a promising candidate mAb for therapeutic purposes. We now describe the construction of a chimeric (moouse/human) mAb E48 by recombinant DNA technology. The genes encoding the variable domains of the heavy and light chain were cloned and ligated into experession vectors containing the human 1 heavy-chain gene and the human k lightchain gene respectively. Biological properties of the resulting chimeric mAb E48 were compared to the murine form in vitro and in vivo. The reactivities of chimeric (c)mAb and murine (m)mAb E48 with HNSCC, as assessed by immunohistochemical staining as well as immuno-blotting were shown to be similar. The affinity constant appeared to be 0.9×10¹⁰ M^–1 and 1.6×10¹⁰ M^–1 for the mmAb and cmAb respectively. The biodistribution of both antibodies was tested by simultaneous injection into nude mice bearing human HNSCC xenografts. cmAb E48 was found to be cleared more rapidly from the blood than mmAb E48, resulting in a 30% lower tumor uptake but similar tumor to non-tumor ratios, 3 days after injection. Moreover, it was shown that cmAb E48 is highly capable of lysing HNSCC targets in ADCC assays in vitro, whereas the mmAb appeared to be almost incative. These data indicate that cmAb E48 has potential as a targeting agent for the eradication of HNSCC in man.     

Studies on well-coupled Photosystem-I-enriched subchloroplast vesicles. Electron transfer by b- and c-type cytochromes in relation to the origin of the ‘slow’ electric potential component

Cytochrome redox changes and electric potential generation are kinetically compared during cyclic electron transfer in Photosystem-I-enriched and Photosystem-II-depleted subchloroplast vesicles (i.e., stroma lamellae membrane vesicles) supplemented with ferredoxin using a suitable electron donating system. In response to a single-turnover flash, the sequence of events is: (1) fast reduction of cytochrome b-563 (t_0.5 ≈ 0.5 ms) (2) oxidation of cytochrome c-554 (t_0.5 ≈ 2 ms), (3) slower reduction of cytochrome b-563 (t_0.5 ≈ 4 ms), (4) generation of the ‘slow’ electric potential component (t_0.5 ≈ 15–20 ms), (5) re-reduction of cytochrome c-554 (t_0.5 ≈ 30 ms) and (6) reoxidation of cytochrome b-563t_0.5 ≈ 90 ms). Per flash two cytochrome b-563 species turn over for one cytochrome c-554. These b-563 cytochromes are reduced with different kinetics via different pathways. The fast reductive pathway proceeds probably via ferredoxin, is insensitive to DNP-INT, DBMIB and HQNO and is independent on the dark redox state of the electron transfer chain. In contrast, the slow reductive pathway is sensitive to DNP-INT and DBMIB, is strongly delayed at suboptimal redox poising (i.e., low $\text{NADPH}\text{NADP}{}^{\mathrm{+}}$ ratio) and is possibly coupled to the reduction of cytochrome c-554. Each reductive pathway seems obligatory for the generation of about 50% of the slow electric potential component. Also cytochrome c-559_LP (LP, low potential) is involved in Photosystem-I-associated cyclic electron flow, but its flash-induced turnover is only observed at low preestablished electron pressure on the electron-transfer chain. Data suggest that cyclic electron flow around Photosystem I only proceeds if cytochrome b-559_LP is in the reduced state before the flash, and a tentative model is presented for electron transfer through the cyclic system.     

Evolution of parasitoid host preference and performance in response to an invasive host acting as evolutionary trap

The invasion of a novel host species can create a mismatch in host choice and offspring survival (performance) when native parasitoids attempt to exploit the invasive host without being able to circumvent its resistance mechanisms. Invasive hosts can therefore act as evolutionary trap reducing parasitoids'' fitness and this may eventually lead to their extinction. Yet, escape from the trap can occur when parasitoids evolve behavioral avoidance or a physiological strategy compatible with the trap host, resulting in either host‐range expansion or a complete host‐shift. We developed an individual based model to investigate which conditions promote parasitoids to evolve behavioral preference that matches their performance, including host‐trap avoidance, and which conditions lead to adaptations to the unsuitable hosts. The model was inspired by solitary endo‐parasitoids attacking larval host stages. One important aspect of these conditions was reduced host survival during incompatible interaction, where a failed parasitization attempt by a parasitoid resulted not only in death of her offspring but also in host killing. This non‐reproductive host mortality had a strong influence on the likelihood of establishment of novel host–parasitoid relationship, in some cases constraining adaptation to the trap host species. Moreover, our model revealed that host‐search efficiency and genetic variation in host‐preference play a key role in the likelihood that parasitoids will include the suboptimal host in their host range, or will evolve behavioral avoidance resulting in specialization and host‐range conservation, respectively. Hence, invasive species might change the evolutionary trajectory of native parasitoid species, which is important for predicting biocontrol ability of native parasitoids towards novel hosts.     

Safety,biodistribution, pharmacokinetics,and immunogenicity of <Superscript>99m</Superscript>Tc-labeled humanized monoclonal antibody BIWA 4 (bivatuzumab) in patients with squamous cell carcinoma of the head and neck

Previous studies have shown the potential of murine and chimeric anti-CD44v6 monoclonal antibodies (MAbs) for radioimmunotherapy (RIT) of head and neck squamous cell carcinoma (HNSCC). A limitation of these MAbs, however, appeared to be their immunogenicity. Therefore, humanized monoclonal antibody BIWA 4 (bivatuzumab), with an intermediate affinity for CD44v6, was recently selected. As a prelude to RIT, we evaluated the safety, tumor-targeting potential, pharmacokinetics, and immunogenicity of technetium-99m-labeled BIWA 4 in patients undergoing operations for primary HNSCC in this study. Ten patients were treated at BIWA 4 dose levels of 25 mg (n=3), 50 mg (n=4), and 100 mg (n=3). Patients received 2 mg of 750 MBq 99mTc-BIWA 4, together with 23-, 48-, and 98-mg unlabeled BIWA 4, respectively. Radioimmunoscintigraphy (RIS) was performed within 1 h and after 21 h, and patients underwent surgery at 48 h after injection. Biodistribution of 99mTc-BIWA 4 was evaluated by radioactivity measurements in blood, bone marrow, and in biopsies of a surgical specimen obtained 48 h after injection. BIWA 4 concentration in blood was assessed by ELISA and high performance liquid chromatography and related to soluble CD44v6 levels in serum samples. The development of human anti-human antibody (HAHA) responses was determined. Administration of 99mTc-BIWA 4 was well tolerated by all patients and no HAHA responses were observed. A mean t1/2 in plasma of 54.8 +/- 11.5 h, 76.1 +/- 21.8 h, and 68.5 +/- 21.2 h was found for the 25-, 50-, and 100-mg dose group, respectively. No complex formation of BIWA 4 with soluble CD44v6 in blood was observed. RIS showed targeting of primary tumors and lymph node metastases in 8 of 10 and 1 of 5 patients, respectively. The highest tumor uptake and tumor to nontumor ratios were observed for the 50-mg dose group. Tumor uptake was 12.9 +/- 5.9, 26.2 +/- 3.1, and 15.4 +/- 1.9% of the injected dose (ID)/kg for the 25-, 50-, and 100-mg dose group, respectively, while the tumor to bone marrow ratios for these groups were 1.7 +/- 0.5, 3.2 +/- 1.1, and 2.0 +/- 0.6, respectively. CONCLUSION: 99mTc-BIWA 4 can safely be administered to patients with HNSCC, with absence of detectable HAHA responses. The 50-mg dose level showed the highest tumor uptake and tumor to nontumor ratios. These findings support the use of BIWA 4 for RIT studies in patients with HNSCC.     

Effect of enrichment on variation and results in the light/dark test

Several confounding factors may influence the outcome of an experiment and the extent of inter-individual variation. The aim of this study was to investigate if cage enrichment induces an effect on experimental mean values and on inter-individual variation in the light/dark paradigm using diazepam as the anxiolytic drug. The behaviour of 216 naive adult male mice of two different strains (BALB/c and C57BL/6) was studied. The animals were housed in groups of four in 'non-enriched', 'enriched' (nesting material) or 'super-enriched' (nest-box, nesting material, wooden gnawing stick and PVC tube) cages. After 5 weeks the animals were assigned to one of three treatments: control (no injection), sham (saline injection i.p.) or diazepam (1 mg/kg bw i.p.) and tested in the light/dark test for 5 min. Variation data were analysed using three different methods (mean absolute deviation, coefficient of variation and power analysis). The C57BL/6 mice scored higher than BALB/c mice in activity related measurements and showed a less 'emotional' behaviour profile in the pharmacological control situation of the light/dark test. In this study the anxiolytic effect of diazepam was clear in BALB/c mice but absent in C57BL/6 mice. Mice housed in enriched and super-enriched cages gained more weight than mice in non-enriched cages, although food intake was not affected. Generally, the strain of mouse had the greatest impact on both mean values and variation. However, there was no consistent increase for one particular strain. The choice of statistical method for analysing variation may influence the interpretation of within-group variability, but none of the methods showed any significant differences between standard and enriched conditions on variability in any of the parameters measured.     

Effects of environmental enrichment for mice: variation in experimental results

This study focused on the effects of different enriched environments for mice in a number of behavioral and physiological parameters in 2 routine laboratory testing procedures: potency testing for tetanus vaccine and stress-induced hyperthermia. The variability in the results was studied by calculating and analyzing mean absolute deviations. Mice from enriched conditions weighed more and consumed more food than mice from standard housing conditions. However, mice from enriched conditions lost more body weight after being housed individually. Other physiological parameters showed no differences. Mice from standard conditions were more active in an open field, suggesting a tendency to overrespond to various stimuli in a testing environment. Mice from enriched environments were more tranquil and easier to handle. The enrichment did not influence the variability in any of the parameters measured, although earlier results and results of other studies suggest that the effects on the variability in results are parameter dependent. When enrichment does not influence variability, there is no reason for not introducing cage enrichment and by doing so contributing to the animals' welfare.