Microcalorimetry of the intestinal mucus: Hydrogen bonding and self-assembly of mucin.

The effect of mucin hydrogen bonding on the structure of intestinal mucus has been studied with micro-differential scanning mirocalorimetry (µ-DSC), supported by spectroscopy. The experiments were performed in water-dimethyl sulfoxide (DMSO) solutions, using either water-DMSO mixtures of an appropriate DMSO content or water as blanks, as to isolate the effects of the solvent to hydrogen bonding. When using matched water-DMSO blanks, thermal events at low temperatures are linked to the negation of mucin-DMSO interactions, while events at higher temperatures are linked to the break-up of hydrogen bonds connecting the sugars of the individual macromolecules. When using a matched solvent as blank, alterations in Cp, such as increases at 10% and 15% DMSO, have been linked to the break-up and creation of quaternary structures. In the case of water as blank, a monotonic but not linear decrease in enthalpy, hence extent of hydrogen bonding, is observed. The above are complemented by UV spectroscopy: A blue shift of the conjugated aminoacids in the presence of DMSO suggests that the inherent stability of mucin is not only due to steric volume exclusions, but also due to extensive hydrogen bonding on behalf of the sugar moieties.

IgE versus IgG4 epitopes of the peanut allergen Ara h 1 in patients with severe allergy.

BACKGROUND: Development and maintenance of tolerance to food allergens appears to be associated with alterations in antigen specific IgE and IgG4 responses. Previous studies have focused only on comparing IgE and IgG4 linear epitope recognition patterns but take no account of conformational epitopes. OBJECTIVE: The aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. METHODS: Polyclonal sera from three individual patients, suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library. Resulting epitope-mimicking sequences were aligned for identification of consensus sequences and localised on the surface of the Ara h 1 molecule by a computer-based algorithm. RESULTS: All epitope-mimicking sequences identified were found to correspond to conformational epitopes. Each individual patient had his/her own distinct IgE as well as IgG4 epitope recognition profile, though some important IgE epitopes were common to all patients. In general the IgG4 epitope pattern was more heterogeneous than the IgE pattern, did not coincide with IgE epitopes and had a lower affinity than IgE. CONCLUSIONS: This study demonstrated the usefulness of the phage-display technology in distinguishing between the epitope pattern of IgE and IgG4, giving detailed information on fine specificity and affinity. Competitive immuno-screening of phage-display random peptide libraries could be a future valuable tool to study the balance and dynamics of the IgE and IgG4 epitope recognition repertoire and provide a diagnostic tool giving information on the associated allergic phenotype.

Effect of roasting on the allergenicity of major peanut allergens Ara h 1 and Ara h 2/6: the necessity of degranulation assays.

BACKGROUND: Peanuts are often consumed after roasting, a process that alters the three-dimensional structure of allergens and leads to Maillard modification. Such changes are likely to affect their allergenicity. OBJECTIVE: We aimed to establish the effect of thermal treatment mimicking the roasting process on the allergenicity of Ara h 1 and a mix of 2S albumins from peanut (Ara h 2/6). METHODS: Ara h 1 and Ara h 2/6 were purified from raw peanuts and heated in a dry form for 20 min at 145°C in the presence (R+g) or absence (R-g) of glucose, and soluble proteins were then extracted. Sera obtained from 12 well-characterized peanut-allergic patients were used to assess the IgE binding and degranulation capacities of the allergens. RESULTS: Extensive heating at low moisture resulted in the hydrolysis of both Ara h 1 and Ara h 2/6. However, in contrast to Ara h 2/6, soluble R+g Ara h 1 formed large aggregates. Although the IgE-binding capacity of R+g and R-g Ara h 1 was decreased 9000- and 3.6-fold, respectively, compared with native Ara h 1, their capacity to elicit mediator release was increased. Conversely, both the IgE-binding capacity and the degranulation capacity of R-g Ara h 2/6 were 600-700-fold lower compared with the native form, although the presence of glucose during heating significantly moderated these losses. CONCLUSIONS AND CLINICAL RELEVANCE: Extensive heating reduced the degranulation capacity of Ara h 2/6 but significantly increased the degranulation capacity of Ara h 1. This observation can have important ramifications for component-resolved approaches for diagnosis and demonstrates the importance of investigating the degranulation capacity in addition to IgE reactivity when assessing the effects of food processing on the allergenicity of proteins.

In vitro digestibility of beta-casein and beta-lactoglobulin under simulated human gastric and duodenal conditions: a multi-laboratory evaluation.

Initially the resistance to digestion of two cow's milk allergens, beta-casein, and beta-lactoglobulin (beta-Lg), was compared using a "high-protease assay" and a "low-protease assay" in a single laboratory. The low-protease assay represents an alternative standardised protocol mimicking conditions found in the gastrointestinal tract. For the high-protease assay, both proteins were incubated with either pepsin or pancreatin and digestion monitored by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and reverse phase-high performance liquid chromatography. The low-protease assay involved gastroduodenal digestion in the presence or absence of phosphatidylcholine (PC). Both beta-casein and beta-Lg were susceptible to hydrolysis by pepsin and pancreatin in the high-protease assay. In contrast, the kinetics of beta-casein digestion in the low-protease assay were slower, beta-Lg being pepsin resistant. During duodenal digestion, beta-Lg was gradually degraded and addition of PC slowed digestion. Subsequently, the reproducibility of the low-protease assay was assessed in 12 independent laboratories by visual assessment of the gels and densitometric analysis: the inter- and intra-laboratory variability was affected by sampling and electrophoresis method employed. The low-protease assay was shown to be reproducible. Future studies will extend these findings using a broader panel of proteins.

Digested Ara h 1 has sensitizing capacity in Brown Norway rats.

BACKGROUND: Food allergies are a public health issue of growing concern, with peanuts in particular being associated with severe reactions. The peanut allergen, Ara h 1, belongs to the cupin plant food allergen family, which, unlike other structural families, appears to be broken down rapidly following gastrointestinal digestion. OBJECTIVE: Using Ara h 1 as a model allergen, the ability of digested protein to sensitize has been investigated. METHODS: Ara h 1 was purified from whole roasted peanuts. Intact Ara h 1 was digested in an in vitro model, simulating the human gastrointestinal digestion process. Digestion products were analysed for peptide sizes and their ability to aggregate. Brown Norway (BN) rats, used as an animal model, were immunized with purified intact Ara h 1 or the gastrointestinal digestion products thereof. The sensitizing capacity was evaluated by analyses of specific antibody (IgG1, IgG2a and IgE) responses and ability to trigger mediator release of rat basophilic leukaemia (RBL)-2H3 cells. RESULTS: The present study showed that Ara h 1 was broken down, resulting in peptide fragments of sizes<2.0 kDa, of which approximately 50% was in aggregated complexes of Mr up to 20 kDa. Ara h 1 digesta were shown to have sensitizing capacity in BN rats, being capable of inducing specific IgG and IgE antibodies. The IgE response was functional, having the capacity to induce specific degranulation of RBL cells. CONCLUSION: From this study, it can be concluded that lability of a food allergen to gastrointestinal digestion does not necessarily abrogate its allergenic sensitizing potential.

The effect of thermal processing on the IgE reactivity of the non-specific lipid transfer protein from apple, Mal d 3.

BACKGROUND: Non-specific lipid transfer proteins (LTPs) are involved in allergy to fresh and processed fruits. We have investigated the effect of thermal treatment and glycation on the physico-chemical and IgE-binding properties of the LTP from apple (Mal d 3). METHODS: Mal d 3 was purified from apple peel and the effect of heating in the absence and presence of glucose investigated by CD spectroscopy, electrospray and MALDI-TOF mass spectrometry. IgE reactivity was determined by RAST and immunoblot inhibition, SPT and basophil histamine release test. RESULTS: The identity and IgE reactivity of purified Mal d 3 was confirmed. Mild heat treatment (90 degrees C, 20 min) in the absence or presence of glucose did not alter its IgE reactivity. More severe heat treatment (100 degrees C, 2 h) induced minor changes in protein structure, but a significant decrease in IgE-binding (30-fold) and biological activity (100- to 1000-fold). Addition of glucose resulted in up to four glucose residues attached to Mal d 3 and only a 2- and 10-fold decrease of IgE-binding and biological activity, respectively. CONCLUSIONS: Only severe heat treatment caused a significant decrease in the allergenicity of Mal d 3 but glycation had a protective effect. The presence of sugars in fruits may contribute to the thermostability of the allergenic activity of LTP in heat-processed foods.

Swelling behavior of the tomato cell wall network.

The swelling of tomato pectin and isolated tomato pericarp cell wall material was investigated in aqueous media under different ionic conditions, pH, and external osmotic stress. Conditions were chosen to include those that would be encountered in vivo. Swelling in these systems was strongly influenced by the polyelectrolyte nature of the polymer and the extent of cross-linking with divalent counterions.

Maillard reaction kinetics in model preservation systems in the vicinity of the glass transition: experiment and theory.

Rates of reactant consumption for the Maillard reaction between lysine and glucose were measured for a noncrystallizing trehalose-sucrose-water matrix in the glass transition region. At temperatures above the glass transition temperature (T(g)), the consumption rates showed Arrhenius temperature dependence with activation energies of 135 and 140 kJ mol(-1) for lysine and glucose, respectively. Finite reaction rates were observed for glassy samples that were faster than that of one of the nonglassy samples. A comparison of experimental results with predicted diffusion-controlled reaction rate constants indicated that the reaction was reaction-controlled at temperatures above T(g) and approached the diffusion-influenced regime in the glassy state. The needs for further research on reactant diffusivity, the theory of the orientation dependence of reactivity, and a detailed understanding of the reaction mechanism and kinetics were identified.

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin.

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.

Specific degradation of pectins via a carbodiimide-mediated Lossen rearrangement of methyl esterified galacturonic acid residues.

A specific, chemical degradation of the methyl esterified galacturonic acid residues of pectins is described. These residues are converted, with hydroxylamine, to hydroxamic acids, and then, with a carbodiimide, to isoureas; the latter undergo a Lossen rearrangement on alkaline hydrolysis. The isocyanates formed are hydrolysed to 5-aminoarabinopyranose derivatives, which spontaneously ring open to give 1,5-dialdehydes. The latter are reduced, in situ, to avoid peeling reactions, with sodium borohydride to give substituted arabitol residues. Thus, overall, partially esterified pectins are specifically cleaved to generate a series of oligogalacturonic acids bearing an arabitol residue as aglycone. Analysis of oligomers so generated discloses the pattern of contiguous nonesterification in a variety of pectins of differing degrees of esterification. Other potential applications are described.

Investigating the nature of branching in pectin by atomic force microscopy and carbohydrate analysis.

Atomic force microscopy (AFM) has been used to investigate the nature of the long branches attached to pectin which were described in a previous report [Round, A. N.; MacDougall, A. J.; Ring, S. G.; Morris, V. J. Carbohydr. Res. 1997, 303, 251-253]. Analysis of the AFM images and comparison with neutral sugar and linkage analyses of the two pectin fractions suggest that the distribution and total amount of branches observed do not correspond with the pattern of neutral sugar distribution. It is thus postulated that the long chains consist of polygalacturonic acid, attached via an as yet undetermined linkage to the pectin backbone, with the neutral sugars present as short, undetected branches. This explanation would have important implications for the nature of 'in situ' pectin networks within plant cell walls and models of gelation in commercial extracted pectin, and the existence of significant branching will markedly influence the viscosity of extracted pectins.

Observations on the crystallization of oligogalacturonates.

Oligogalacturonates were produced by the limited enzymic hydrolysis of polygalacturonic acid and purified by ion-exchange chromatography. The fractions obtained were of limited polydispersity, determined by analytical ion-exchange chromatography. Oligomers with an average degree of polymerization of 10-15 were readily crystallized from aqueous salt solutions at neutral pH as single crystals. Crystal morphology of the salts examined, Na+, K+ and Ca2+ were characteristic of the salt. The wide-angle X-ray diffraction patterns obtained for the sodium salt were consistent with published fibre diffraction data of this salt form.

Phase behavior and component partitioning in low water content amorphous carbohydrates and their potential impact on encapsulation of flavors.

The compositions at which amorphous ethanol-maltose-water mixtures exhibit liquid-liquid separation have been determined in the temperature range from 20 to 80 degrees C. At water contents below approximately 20% w/w two phases were observed, with the maltose-rich phase slightly richer in water. Partition coefficients of organic nonelectrolytes ranging in hydrophobicity from 1, 2-ethanediol and 1,2-propanediol to benzyl alcohol and propyl acetate have been measured for octanol/sorbitol, benzyl alcohol/sorbitol, and 1-butanol/sorbitol mixtures. Linear correlations were found between the log partition coefficients in the various solvent systems. Replacing water with sorbitol results in more organic partitioning into the octanol. Replacing octanol with benzyl alcohol or 1-butanol also results in more organic partitioning into the hydrophobic phase. The results establish a relationship with partition coefficients for octanol/water mixtures, which are well studied experimentally and for which predictive approaches exist. The implications of these results for flavor retention and encapsulation are discussed.

Phase Separation of Plant Cell Wall Polysaccharides and Its Implications for Cell Wall Assembly.

Concentrated binary mixtures of polymers in solution commonly exhibit immiscibility, resolving into two separate phases each of which is enriched in one polymer. The plant cell wall is a concentrated polymer assembly, and phase separation of the constituent polymers could make an important contribution to its structural organization and functional properties. However, to our knowledge, there have been no published reports of the phase behavior of cell wall polymers, and this phenomenon is not included in current cell wall models. We fractionated cell walls purified from the pericarp of unripe tomatoes (Lycopersicon esculentum) by extraction with cyclohexane diamine tetraacetic acid (CDTA), Na2CO3, and KOH and examined the behavior of concentrated mixtures. Several different combinations of fractions exhibited phase separation. Analysis of coexisting phases demonstrated the immiscibility of the esterified, relatively unbranched pectic polysaccharide extracted by CDTA and a highly branched, de-esterified pectic polysaccharide present in the 0.5 N KOH extract. Some evidence for phase separation of the CDTA extract and hemicellulosic polymers was also found. We believe that phase separation is likely to be a factor in the assembly of pectic polysaccharides in the cell wall and could, for example, provide the basis for explaining the formation of the middle lamella.

Movement and metabolism of oligogalacturonide elicitors in tomato shoots.

We have studied the movement and metabolism of oligogalacturonides through shoots of tomato (Lycopersicon esculentum L. cv Rutgers). Oligomers of polygalacturonic acid were prepared by enzyme digestion and gel filtration. These were end-reduced with [(3)H]NaBH4, using an improved reaction method, to yield oligoalditols. The radiolabelled oligomer of degree of polymerisation 6 was supplied to tomato shoots through their transpiration stream. Analysis of the distribution of radiolabel in the plant, and TLC of radiolabelled material recovered from the plant revealed the following: a) material recovered from the plant could be identified as an oligogalacturonide from its behaviour on TLC and susceptibility to digestion with polygalacturonase; b) end-reduced oligogalacturonides moved freely through the plant and were not complexed to high-molecularweight compounds and immobilised; c) during passage through the plant, modifications to the oligogalacturonide occurred, presumably as a consequence of metabolism in the apoplastic space. We found evidence of i) esterification of the molecule, and ii) shortening of the oligogalacturonide chain. The results show that in the assay for protease-inhibitor-inducing factor using cut shoots, oligogalacturonide elicitors can move into the leaves and act directly on the cells producing protease inhibitor.