Parvalbumin in fish skin-derived gelatin: is there a risk for fish allergic consumers?

The major allergen parvalbumin was purified from cod muscle tissues, and polyclonal antibodies were raised towards it. The antibodies were tested for specificity and an enzyme-linked immunosorbent assay (ELISA) was developed using these antibodies. The ELISA was applied to measure parvalbumin in cod skin, the starting material for fish gelatin made from deep sea, wild fish. The ELISA was sufficiently sensitive (LLOQ = 0.8 ng ml(-1) in extracts, corresponding to 0.02 µg of parvalbumin per g of tissue), and did not cross-react with common food constituents. Fish gelatin, wine and beer, matrices for the potential use of this ELISA, did not cause disturbance of the assay performance. The data show that the parvalbumin content in cod muscle tissue is 6.25 mg g(-1), while the skins contained considerably less, 0.4 mg g(-1). Washing of the skins, a common industrial procedure during the manufacturing of fish gelatin, reduced the level of parvalbumin about 1000-fold to 0.5 µg g(-1), or 0.5 ppm. From 95 commercial lots of fish gelatin it is shown that 73 are below 0.02 µg g(-1) parvalbumin. From the other 22 lots, the one with the highest concentration contained 0.15 µg g(-1) of parvalbumin. These levels are generally assumed to be safe for fish-allergic individuals.

Increasing stability by pre-bending the nails in elastic stable intramedullary nailing: a biomechanical analysis of a synthetic femoral spiral fracture model.

Elastic stable intramedullary nailing (ESIN) is generally acknowledged to be the treatment of choice for displaced diaphyseal femoral fractures in children over the age of three years, although complication rates of up to 50% are described. Pre-bending the nails is recommended, but there are no published data to support this. Using synthetic bones and a standardised simulated fracture, we performed biomechanical testing to determine the influence on the stability of the fracture of pre-bending the nails before implantation. Standard ESIN was performed on 24 synthetic femoral models with a spiral fracture. In eight cases the nails were inserted without any pre-bending, in a further eight cases they were pre-bent to 30° and in the last group of eight cases they were pre-bent to 60°. Mechanical testing revealed that pre-bending to 60° produced a significant increase in the stiffness or stability of the fracture. Pre-bending to 60° showed a significant positive influence on the stiffness compared with unbent nails. Pre-bending to 30° improved stiffness only slightly. These findings validate the recommendations for pre-bending, but the degree of pre-bend should exceed 30°. Adopting higher degrees of pre-bending should improve stability in spiral fractures and reduce the complications of varus deformity and shortening.

Biomechanical analysis of a synthetic femur spiral fracture model: Influence of different materials on the stiffness in flexible intramedullary nailing.

BACKGROUND: Flexible intramedullary nail fixation of dislocated diaphyseal femur fractures has gained wide acceptance for children and adolescents with open physes. Studies with a special emphasis on complications reveal frequent problems regarding stability, usually in complex fracture types such as spiral fractures and in older children weighing >40kg. This biomechanical study analyses how much the material of the nails influences the stiffness in a synthetic bone model. METHODS: Twenty-four composite grafts (Sawbones®, 4th generation, medullar canal of 10mm) with an identical spiral fracture were used in three configurations of eight grafts. Elastic stable intramedullary nailing was performed in a retrograde C-shaped manner with two nails of equal size (2×3.5mm). Close contact of the fragments could be achieved. We compared Group A (steel nails) with Group B and C (two types of titanium nails). All specimens underwent 4-point bending, torsion and axial compression in the 0° and 9° positions, and the results were analysed. FINDINGS: Group A (steel nails) revealed a significantly higher stiffness in all directions than Group B. Apart from compression in the 9° position this steel nail fixation showed significant higher stiffness than titanium nails of Group C. Comparing Group B and C did not show an systematic difference. INTERPRETATION: In this biomechanical study with composite artificial bones the use of steel Nails demonstrated the highest stiffness in our model when compared to two different titanium nail configurations. Apart from in cases of known allergy or planned MRI-examinations our results and data from the literature question the use of titanium nails.

Purification and substrate specificity of transglutaminases from blood and Streptoverticillium mobaraense.

A procedure for a fast and simple purification of bovine plasma transglutaminase was developed, which resulted in a homogeneous enzyme preparation. Two different procedures were developed for the purification of pig erythrocyte transglutaminase, both of which resulted in partial purification. Both enzymes were used in cross-linking reactions of alpha-lactalbumin, beta-lactoglobulin, bovine serum albumin, casein, hemoglobin, glycinin, and myosin. The substrate specificity was compared to that of bacterial transglutaminase isolated from Streptoverticillium mobaraense. The bacterial transglutaminase caused cross-linking of a wider range of proteins and, thus, exhibited a lower substrate specificity than the blood transglutaminases. In addition, differences exist in the necessity of the addition of reducing agents. These differences allow specific applications of blood and bacterial transglutaminases at protein cross-linking in single or complex protein systems.

Peroxidase-mediated cross-linking of a tyrosine-containing peptide with ferulic acid.

The tyrosine-containing peptide Gly-Tyr-Gly (GYG) was oxidatively cross-linked by horseradish peroxidase in the presence of hydrogen peroxide. As products, covalently coupled di- to pentamers of the peptide were identified by LC-MS. Oxidative cross-linking of ferulic acid with horseradish peroxidase and hydrogen peroxide resulted in the formation of dehydrodimers. Kinetic studies of conversion rates of either the peptide or ferulic acid revealed conditions that allow formation of heteroadducts of GYG and ferulic acid. To a GYG-containing incubation mixture was added ferulic acid in small aliquots, therewith keeping the molar ratio of the substrates favorable for hetero-cross-linking. This resulted in a predominant product consisting of two ferulic acid molecules dehydrogenatively linked to a single peptide and, furthermore, two ferulic acids linked to peptide oligomers, ranging from dimers to pentamers. Also, mono- and dimers of the peptide were linked to one molecule of ferulic acid. A mechanism explaining the formation of all these products is proposed.

Quantification of major peanut allergens Ara h 1 and Ara h 2 in the peanut varieties Runner, Spanish, Virginia, and Valencia, bred in different parts of the world.

BACKGROUND: The serology of peanut allergy seems to be different in various parts of the world. We analyzed the composition of 13 samples of three varieties of peanut in order to compare their allergenic nature. METHODS: Peanut cultivars that are commonly processed in the West were analyzed for protein content, protein composition, and Ara h 1 and Ara h 2 content by biochemical methods. IgE-binding properties were analyzed by ELISA using serum from patients with documented peanut allergy. RESULTS: Total protein contents were comparable for all tested samples (24-29%), and proteins were extractable to the same extent. SDS-PAGE patterns differed slightly, but all major bands were visible in all samples (molecular masses of approximately 14100 kDa under reducing conditions). Ara h 1 and Ara h 2 were quantified by SDS PAGE densitometry and were expressed as percentage of the total protein content. Ara h 1 was in the range 12-16%, whereas Ara h 2 was 5.9-9.3%. In view of the analytic uncertainty of this determination, the content of both Ara h 1 and Ara h 2 was not significantly different between the tested samples. In an IgE-binding inhibition ELISA, the affinities of the peanut proteins for peanut-specific IgE were measured. Minor differences were observed between the tested samples, with the most potent IgE-binding sample having a two times higher ability to bind IgE than the weakest IgE-binding sample. CONCLUSIONS: The results suggest that peanuts of different varieties and from different parts of the world contain similar proteins, including Ara h I and Ara h 2. Consequently, the IgE-binding properties are similar to a great extent. This indicates that differences in the serology of peanut allergy may not originate from differences in the allergen composition of the peanut.

Soy glycinin: influence of pH and ionic strength on solubility and molecular structure at ambient temperatures.

This study describes the relationship between the solubility of glycinin, a major soy protein, and its structural properties at a quaternary, tertiary, and secondary folding level under conditions representative for food products. When the ionic strength is lowered from 0.5 to 0.2 or 0.03, the basic polypeptides shift more to the exterior of the glycinin complex, as determined at pH 7.6 by labeling solvent-exposed lysines, supported by the study of the proteolytic action of clostripain on glycinin. This structural reorganization caused the pH of minimal solubility to shift to higher values. Ultracentrifugational analysis shows that at pH 7.6 and an ionic strength of 0.5 glycinin forms hexameric complexes (11S), whereas at pH 3.8 and at an ionic strength of 0.03 glycinin exists as trimers (7S). Intermediate situations are obtained by modulation of pH and ionic strength. The observed quaternary dissociation correlates with an increased amount of nonstructured protein at a secondary level and with changes in tertiary folding as determined using circular dichroism. Tryptophan fluorescence shows no significant structural changes for different ionic strengths but demonstrates a more tightly packed fluorophore environment when the pH is lowered from 7.6 to 3.8.

Heat denaturation of soy glycinin: influence of pH and ionic strength on molecular structure.

The 7S/11S glycinin equilibrium as found in Lakemond et al. (J. Agric. Food Chem. 2000, 48, xxxx-xxxx) at ambient temperatures influences heat denaturation. It is found that the 7S form of glycinin denatures at a lower temperature than the 11S form, as demonstrated by a combination of calorimetric (DSC) and circular dichroism (CD) experiments. At pH 7.6, at which glycinin is mainly present in the 11S form, the disulfide bridge linking the acidic and the basic polypeptides is broken during heat denaturation. At pH 3.8, at which glycinin has dissociated partly into the 7S form, and at pH 5.2 this disruption does not take place, as demonstrated by solubility and gel electrophoretic experiments. A larger exposure of the acidic polypeptides (Lakemond et al., 2000) possibly correlates with a higher endothermic transition temperature and with the appearance of an exothermic transition as observed with DSC. Denaturation/aggregation (studied by DSC) and changes in secondary structure (studied by far-UV CD) take place simultaneously. Generally, changes in tertiary structure (studied by near-UV CD) occur at lower temperatures than changes in secondary structure.

Isolation and characterization of patatin isoforms.

Patatin has, so far, been considered a homogeneous group of proteins. A comparison of the isoforms in terms of structural properties or stability has not been reported. A method to obtain various isoform fractions as well as a comparison of the physicochemical properties of these pools is presented. Patatin could be separated in four isoform pools, denoted A, B, C, and D, representing 62%, 26%, 5%, and 7% of the total amount of patatin, respectively. These isoforms differed in surface charge, resulting in a different behavior on anion exchange chromatography, isoelectric focusing, native polyacrylamide gel, and capillary electrophoresis. All isoforms of the patatin family contained proteins with two molecular masses of approximately 40.3 and 41.6 kDa, respectively. The size of this difference in the molar mass (1300 Da) is on the order of one carbohydrate moiety. Despite the biochemical differences given above, no variations in the structural properties nor in the thermal conformational stability could be observed using far-ultraviolet circular dichroism, infrared, and fluorescence spectroscopy.

Comparison of different immunochemical methods for the detection and quantification of hazelnut proteins in food products.

Hazelnuts are widely used in the food industry owing to their nutritive value and taste. The amount of hazelnut present in a recipe is usually considered as a mark of quality. On the other hand, contamination of foods that normally do not contain hazelnuts is a threat for patients with a hazelnut allergy. For this reason, the availability of a method for the detection and quantification of hazelnuts in foods would be desirable. The objective of this study was to develop a method for the detection and quantification of minor amounts of hazelnut protein in food products that is potentially applicable for the food industry. Several immunochemical methods, e.g., immunoblotting and enzyme-linked immunosorbent assay (ELISA), were developed with antibodies from both hazelnut-sensitized patient sera and the sera of rabbits hyperimmunized with hazelnut protein. Immunoblotting appeared to be non-specific when the sera of patients were used as a source of antibodies. Using immunopurified antibodies from rabbits immunized with hazelnuts, immunoblotting became specific, but the sensitivity of this method was limited. Inhibition of IgE binding is a generally used test in clinical laboratories to establish contamination with hazelnuts. This approach is sensitive and specific, but not readily accessible for the food industry since patient serum is needed. Similar results in terms of sensitivity and specificity were obtained with a sandwich ELISA constructed with an immunopurified antibody from rabbits sensitized to hazelnuts. No substantial cross-reactivity with other nuts, legumes or other food constituents was observed, and concentrations as low as 5 ng/ml, corresponding to 1 ppm in food products, were detected. In a field test, several consumer products regarded to be free of hazelnuts were shown to contain traces of hazelnut. This sandwich ELISA constructed with immunopurified antibodies from rabbits sensitized with hazelnut protein is a sensitive and specific method to detect and quantify hazelnut and is useful in detecting trace contamination with hazelnut in various consumer products. Since this test does not require serum from patients, it is appropriate for use in the food industry.

Heat-induced conformational changes of Ara h 1, a major peanut allergen, do not affect its allergenic properties.

Ara h 1, a major peanut allergen was isolated, and its structure on secondary, tertiary, and quaternary level at ambient temperature was investigated using spectroscopic and biochemical techniques. Ara h 1 appeared to be a highly structured protein on a secondary level, possesses a clear tertiary fold, and is present as a trimeric complex. Heat treatment of purified Ara h 1 results in an endothermic, irreversible transition between 80 and 90 degreesC, leading to an increase in beta-structures and a concomitant aggregation of the protein. Ara h 1 from peanuts that were heat-treated prior to the purification procedure exhibited a similar denatured state with an increased secondary folding and a decreased solubility. The effect of heat treatment on the in vitro allergenic properties of Ara h 1 was investigated by means of a fluid-phase IgE binding assay using serum from patients with a clinically proven peanut allergy. Ara h 1 purified from peanuts heated at different temperatures exhibited IgE binding properties similar to those found for native Ara h 1, indicating that the allergenicity of Ara h 1 is heat-stable. We conclude that the allergenicity of Ara h 1 is unaffected by heating, although native Ara h 1 undergoes a significant heat-induced denaturation on a molecular level, indicating that the recognition of conformational epitopes of Ara h 1 by IgE either is not a dominant mechanism or is restricted to parts of the protein that are not sensitive to heat denaturation.

Purification and Characterization of a [beta]-D-Xylosidase and an Endo-Xylanase from Wheat Flour.

A [beta]-D-xylosidase and an endo-xylanase were purified from European wheat (Triticum aestivum) flour. The [beta]-D-xylosidase had a molecular weight of approximately 64,000 and an isoelectric point of 5.5. It hydrolyzed p-nitrophenyl-[beta]-D-xylopyranoside and xylo-oligosaccharides and released D-xylose units from wheat arabinoxylan and oat spelts xylan. An endo-xylanase with a molecular weight of approximately 55,000 was also obtained and it consisted of a number of isoforms with isoelectric points between 4.0 and 5.0. The action of the isolated endo-xylanase depended on the degree of substitution of the polysaccharide. Unbranched polymers were preferentially hydrolyzed. Since xylo-oligosaccharides were not hydrolyzed, the enzyme appeared to need at least five or more consecutive unsubstituted xylose units. Finally, an [alpha]-L-arabinofuranosidase that hydrolyzed p-nitrophenyl-[alpha]-L-arabinofuranoside was partially purified.

Identification and characterization of a novel arabinoxylanase from wheat flour.

An endogenous wheat (Triticum aestivum) flour endoxylanase was purified to homogeneity from a crude wheat flour extract by ammonium sulfate precipitation and cation-exchange chromatography. The 30-kD protein had an isoelectric point of 9.3 or higher. A sequence of 19 amino acids at the NH2 terminus showed 84.2% identity with an internal sequence of 15-kD grain-softness protein, friabilin. High-performance anion-exchange chromatography and gel-permeation analysis of the hydrolysis products indicated the preferential hydrolysis of highly branched structures by the enzyme; wheat arabinoxylan and rye (Secale cereale) arabinoxylan (high arabinose to xylose ratios) were hydrolyzed more efficiently by this enzyme than oat (Avena sativa) spelt xylan (low arabinose to xylose ratios). The release of the hydrolysis products as a function of time suggested that the endoxylanolytic activity was associated with the release of arabinose units from the polysaccharides, suggesting that the enzyme action is similar to that by endoxylanases from Ceratocystis paradoxa, Aspergillus niger, and Neurospora crassa. Although the enzyme released arabinose from arabinoxylan, it did not hydrolyze p-nitrophenyl-alpha-L-arabinofuranoside. From the above, it follows that the enzyme, called arabinoxylanase, differs from most microbial endoxylanases and from an endoxylanase purified earlier from wheat flour.

The Specific-Stress-Free housing system has positive effects on productivity, health, and welfare of pigs.

An experiment was conducted to determine the health, welfare, and growth performance of pigs housed under optimal climatic conditions in a Specific-Stress-Free (SSF) housing system. This system was compared to a conventional housing system with the same climatic conditions. Two identical experimental rooms with five pens each were used. In each room five litters were used for the experiments. The SSF pigs were not mixed or transported, whereas the pigs in the conventional housing system were mixed at weaning and mixed and transported at 25 kg. Average daily gain for the SSF pigs was higher (P < .05) both for the rearing period and for the finishing period (P < .01). Live weight at 143 d was, therefore, higher in the SSF group (95.09 kg vs 84.8 kg, P < .001). Clinical signs were hardly seen in the SSF group, but in the control group high levels of aggression after mixing caused ear, skin, and tail lesions. Cortisol concentration of the saliva was lower in SSF pigs after weaning (P < .01). Seven and 21 d after mixing, the SSF pigs had a higher response to an intradermal injection of phytohemagglutinin (P < .001) than the control pigs. In conclusion, production performance, health, and welfare are improved when pigs are kept in an SSF housing system where they are not mixed or transported.

Growth and oesophagogastric lesions in finishing pigs offered pelleted feed ad libitum.

The growth rates of 458 finishing pigs between 25 kg and 107 kg liveweight, which were offered finely ground pelleted feed ad libitum, were determined and their stomachs were examined at slaughter. Two herds were involved and a macroscopical examination of the mucosal lesions in the pars oesophagea revealed a prevalence of 75 per cent of the 274 pigs in herd A and 89 per cent of the 184 pigs in herd B with hyperkeratosis of the pars oesophagea, and approximately 11 per cent of the pigs in both herds with extensive erosions and/or ulceration; on average the pigs with extensive lesions gained 50 to 75 g/day less weight than the pigs with no lesions in the pars oesophagea. There was no difference between the prevalence of the oesophagogastric lesions of different severity between barrows and gilts, but there was evidence for differences between litters.

Individual differences in cell-mediated and humoral immunity in pigs.

Previous experiments displayed consistent individual behavioural differences in pigs. Some showed a more active behavioural response (aggressive and resistant; so-called A/R pigs), other a more passive behavioural response (non-aggressive and non-resistant; so-called NA/NR pigs). Moreover, these behavioural coping strategies were associated with different behavioural, physiological and endocrine responses under stress conditions. In the present study we selected 32 A/R and 32 NA/NR individuals and tested their immune reactivity in reaction to stress using several cell-mediated (CMI) and humoral immunological tests. Active A/R pigs had a higher in vivo and in vitro CMI to nonspecific and specific antigens, while after stress CMI reduced more in A/R than in NA/NR pigs. In contrast, humoral immunity was highest in NA/NR pigs. Furthermore, some serologically typed swine lymphocyte antigen (SLA) class I haplotypes were not equally distributed between A/R and NA/NR pigs. In general, these findings show that measurement of immune reactivity is an important tool to define how animals cope with environmental demands.

Social rank and disease susceptibility in pigs.

Two experiments were carried out to investigate the inter-individual variation in immune reactivity and disease susceptibility of group housed pigs of different social status. The social status of the individual pig was determined by the outcome of social ranking fights and food competition tests. On Day 75 after the start of both experiments, all pigs were challenged with 0.5 ml of an Aujeszky disease virus (ADV) in each nostril. Data combined from both experiments showed that mortality and/or morbidity after the ADV challenge was highest among subordinates. In both experiments, a lymphocyte proliferation assay, using purified ADV as an antigenic stimulus, showed that dominant pigs had significantly higher counts per minute than subdominant and subordinate pigs. Kendall's partial correlations showed that morbidity had been associated with high values in haematological and clinicochemical blood parameters and not with social status of the individual pig. In each experiment, maternal derived antibodies against the ADV and the antibody level after the ADV challenge hardly differed between pigs of different social status. Major histocompatibility complex typing of class I and II by iso-electro focusing of all pigs in Experiment 2 showed that not all haplotypes were distributed equally among dominant, subdominant and subordinate pigs. The present work shows that there are large individual differences in immune reactivity and disease susceptibility which appear to be related to the social status of the individual pig in a stable social structure.

Effect of climatic stress on the immunological reactivity of weaned pigs.

Weaned pigs exposed daily to either unpredictable draught (experiment 1) or intermittent unpredictable draught (experiment 2) showed different lymphocyte blastogenic responses after mitogenic stimulation with phytohaemagglutinin (PHA) and concanavalin A (ConA). In both experiments PHA skin test responses were lower for draught exposed pigs than for control pigs and leucocyte numbers or profiles were altered compared to those of control pigs. Superoxide production and chemiluminescence of porcine granulocytes were similar for draught exposed and control animals. Furthermore, serum globulin content did not differ significantly between pigs in the experimental and control room. The strong increase in serum gamma-globulin after the Aujeszky Disease Virus (ADV)-challenge was the same for draught exposed and control pigs. The same held for the lymphocyte blastogenic response with ADV protein as antigenic stimulus. The present study shows the effects of climatic stress on immunological reactivity, which may reflect a homeostatic disturbance of the pig's immune system elicited by exposure to unpredictable draught.

Individual behavioral and physiological strategies in pigs.

Previous experiments demonstrated consistent individual behavioral differences in pigs. Some showed a more active behavioral response (so-called A/R pigs), others a more passive behavioral response (so-called NA/NR pigs). In the present study we selected 32 A/R and 32 NA/NR individuals and tested them individually in an open field at 3 (OF1) and 8 weeks of age (OF2). Individual response patterns were remarkably consistent between OF1 and OF2. While more A/R than NA/NR pigs made escape attempts, the A/R ones vocalized less, and were less inhibited to approach novel objects in OF1 and OF2, although they spent less time in exploring these objects than NA/NR pigs. Cortisol (CS) level after OF1 increased in A/R pigs but did not change in NA/NR ones, while CS level in OF2 remained constant in A/R pigs but decreased in NA/NR pigs. CS response to ACTH1-39 was measured at 3 and 8 weeks of age but did not differ between types. Basal CS level was higher in NA/NR than in A/R pigs and accompanied by adrenal hypertrophy. Mean heart rate (HR) was higher of A/R pigs compared to NA/NR ones in two backtests. HR of A/R pigs substantially increased (23.9 bpm = 15.5%) in reaction to the novel object in OF2, while HR of NA/NR ones only slightly increased (4.5 bpm = 2.9%), or even decreased (bradycardia). A/R pigs had more often heart deviations than NA/NR ones. The present study demonstrates that the two behavioral strategies of pigs are characterized by consistent differences in behavioral, physiological, and endocrine responses to conflict situations.