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.

Protein S binding in relation to the subunit composition of human C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) circulates in plasma either as a free protein or in a bimolecular complex with the vitamin K-dependent protein S. The major form of C4BP is composed of 7 identical, disulfide-linked 70 kDa subunits (alpha-chains), the arrangement of which gives the C4BP molecule a spider-like appearance. Recently, we identified a unique 45 kDa subunit (beta-chain) in C4BP. We have now isolated a subpopulation of C4BP, which does not bind protein S. This C4BP species, which had a molecular weight slightly lower than that of the predominant form, was found to lack the beta-chain. Another lower molecular weight form of C4BP was also purified. It contained the beta-chain and was efficient in binding protein S. Its subunit composition was judged to comprise six alpha-chains and one beta-chain. These results indicate C4BP in plasma to be heterogeneous at a molecular level vis-a-vis subunit composition and/or protein S binding ability and provide support for the concept that the beta-chain of C4BP contains the single protein S binding site.

Importance of the alpha 3-fragment of complement C4 for the binding with C4b-binding protein.

The human regulatory complement component C4b-binding protein (C4BP) is a multimeric plasma protein, which regulates the classical pathway of the complement system. C4BP functions as a cofactor to factor 1 in the degradation of C4b and accelerates the decay rate of the C4b2a complex. Previously, we have demonstrated that monoclonal antibodies (C4-2 and 9) directed against the alpha'-chain of C4b inhibit the binding of C4b to C4BP. In order to identify the structural domain of C4b that binds C4BP, proteolytic fragments of C4 were generated with trypsin and Staphylococcus aureus V8 protease. Sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and amino acid sequence analysis of the proteolytic fragments reactive with the anti-C4 mAb's revealed that the residues Ala738-Arg826 of the alpha 3-fragment of C4b are important for the interaction with C4BP.

The region Ser333-Arg356 of the alpha-chain of human C4b-binding protein is involved in the binding of complement C4b.

Human C4b-binding protein (C4BP) functions as a cofactor to factor I in the degradation of C4b and accelerates the decay rate of the C4b2a complex. In this study we describe a monoclonal antibody directed against the alpha-chain of C4BP that inhibits the binding of C4b to C4BP. In order to identify the structural domain of the alpha-chain of C4BP that interacts with C4b, tryptic fragments of C4BP were generated. Amino acid sequence analysis of the fragments revealed that the residues Ser333-Arg356 of the alpha-chain of C4BP contain the epitope of this antibody, and as a consequence, that this part of the alpha-chain of C4BP is likely to be involved in the interaction with C4b.

Separation of different forms of the fourth component of human complement by fast protein liquid chromatography.

Disruption of the thiolester in native C4 yields a 'C4b-like C4' molecule (iC4) that functionally resembles C4b and is therefore probably accompanied by conformational changes in the C4 molecule. In most purified C4 preparations, iC4 and C4b are present to a variable extent. In this study we evaluated the use of fast protein liquid chromatography (FPLC) to resolve and isolate these various forms of C4. C4 was purified from fresh human plasma in a 4-step procedure that included barium citrate adsorption, polyethylene glycol 6000 (PEG) precipitation, Q-Sepharose Fast Flow and mono Q ion exchange chromatography. The final preparation appeared to be homogeneous on SDS-PAGE and under reducing conditions consisted of three bands that corresponded to the intact alpha, beta and gamma chains of C4. In some preparations the alpha' chain of C4b was also observed. On a Mono Q column the purified C4 preparations could be separated into three peaks that by hemolytic assay and SDS-PAGE were characterized as representing native C4, and monomeric and dimeric iC4 (or monomeric and dimeric C4b). Finally, the apparent KA of the various forms of C4 for C4b-binding protein (C4BP) was investigated. The monomeric iC4 and C4b species demonstrated similar C4BP binding affinity with an apparent KA of 5.6-6.4 x 10(8) M-1, whereas their dimeric forms demonstrated a higher affinity for C4BP with an apparent KA: 0.9-2.3 x 10(9) M-1. Binding of native C4 to C4BP was undetectable.

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.

Identification of different forms of human C4b-binding protein lacking beta-chain and protein S binding ability.

Human C4b-binding protein (C4BP) is a multimeric regulatory component of the complement system that circulates in plasma either as a free protein or in a noncovalent complex with the vitamin K-dependent protein S. The major form of C4BP is composed of seven identical alpha-chains (70 kDa) and one beta-chain (45 kDa). C4BP was purified from human plasma after barium citrate adsorption using anti-C4BP monoclonal antibody affinity chromatography. C4BP-high and low Mr forms were both obtained from the barium citrate precipitate and the supernatant. C4BP-high and low forms from the barium citrate precipitate were separated by sodium dodecylsulfate polyacrylamide slab gel electrophoresis and extracted with Triton X-100. Both forms contained the beta-chain as was demonstrated on sodium dodecylsulfate polyacrylamide slab gel electrophoresis under reduced conditions after silver-staining and with Western-blotting using monoclonal antibodies specific for the beta-chain. The C4BP-high and low forms demonstrated similar protein S binding affinity (KA: 3.18 x 10(8) and 3.21 x 10(8) M-1, respectively) in a C4BP-protein S binding assay and a protein S ligand blot using a peroxidase-conjugated monoclonal anti-protein S antibody. The barium citrate supernatant contained two forms of C4BP-high and one form of C4BP-low. One form of C4BP-high did contain the beta-chain and was capable of protein S binding (KA: 4.35 x 10(8) M-1). The two other forms of C4BP lacked the beta-chain and were unable to bind protein S.

Expression and characterization of recombinant human protein S in heterologous cells--studies of the interaction of amino acid residues leu-608 to glu-612 with human C4b-binding protein.

Mouse C127 epithelioid cells were genetically engineered to produce biologically active gamma-carboxylated human protein S. A full length human protein S cDNA was cloned into a bovine papilloma virus (BPV) based shuttle vector under the transcriptional control of the Moloney murine sarcoma virus enhancer and the mouse metallothionein promoter. Stable expression was obtained in transfected C127 cells. Expression of gamma-carboxylated protein S was dependent on the presence of vitamin K in the culture medium. Protein sequence analysis showed that recombinant and plasma protein S have the same amino terminal sequence. Analysis of specific post-translationally modified amino acids shows that recombinant protein S is fully gamma-carboxylated and fully beta-hydroxylated. Immunoblotting analysis using polyclonal and monoclonal antibodies shows that recombinant protein S has a slightly higher molecular weight than plasma protein S. After N-Glycanase treatment, identical molecular weights are observed for recombinant and plasma protein S, indicating that the difference is caused by differences in the N-linked carbohydrate side chains. Recombinant protein S also demonstrates normal cofactor activity for activated protein C in a clotting assay. Binding studies with the complement component, C4b-binding protein (C4BP), shows that recombinant protein S binds to C4BP with the same apparent affinity as plasma protein S. Two variant molecules are also tested for their binding to C4BP. The first variant has a replacement of amino acid residue leu-608 by val and was designated B variant. The second variant has three alterations, at positions 609, 611 and 612 where the acidic amino acid residues asp, asp and glu were replaced by asn, asn and gln, respectively and this variant was designated C variant.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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.

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.

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.

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.

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.

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.

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.

[Mucosal lesions in the pars esophagus in swine: prevalence and the effect of stress]. / Slijmvliesveranderingen in de pars oesophagea bij varkens: prevalentie en de invloed van stress.

Morphological investigations of slaughterhouse material revealed a prevalence of 63% of the sows (n = 224) and 36% of the slaughter pigs (n = 209) with mucosal lesions in the pars oesophagea. The mucosal lesions were composed of severe hyperkeratosis, erosions or ulceration. Microscopic examination showed that hyperkeratosis is attended with parakeratosis. From experiments in a climate controlled pighouse it could be concluded that mixing of unfamiliar pigs resulted in higher prevalences of gastric lesions as compared with keeping the litter together until the end of the experiment (farrow to finish system). There was no relation between gastric lesions and climatic stressors or between gastric lesions and growth and sex of the pigs. When pigs, according to social ranking, were divided in high, middle and low rank it showed that more pigs in the middle-ranked group had gastric lesions. The serum pepsinogen was not related with gastric lesions. There was a tendency that plasma cortisol increased with severity of gastric lesions. There was a strong significant 'litter-effect', which can indicate a genetic predisposition for the development of gastric lesions. Maybe that the presence of mucosal lesions in the pars oesophagea can be used as an objective indicator for welfare in intensive pig husbandry.