Review article: the diagnosis and management of food allergy and food intolerances.

BACKGROUND: Adverse reactions to food include immune mediated food allergies and non-immune mediated food intolerances. Food allergies and intolerances are often confused by health professionals, patients and the public. AIM: To critically review the data relating to diagnosis and management of food allergy and food intolerance in adults and children. METHODS: MEDLINE, EMBASE and the Cochrane Database were searched up until May 2014, using search terms related to food allergy and intolerance. RESULTS: An estimated one-fifth of the population believe that they have adverse reactions to food. Estimates of true IgE-mediated food allergy vary, but in some countries it may be as prevalent as 4-7% of preschool children. The most common food allergens are cow's milk, egg, peanut, tree nuts, soy, shellfish and finned fish. Reactions vary from urticaria to anaphylaxis and death. Tolerance for many foods including milk and egg develops with age, but is far less likely with peanut allergy. Estimates of IgE-mediated food allergy in adults are closer to 1-2%. Non-IgE-mediated food allergies such as Food Protein-Induced Enterocolitis Syndrome are rarer and predominantly recognised in childhood. Eosinophilic gastrointestinal disorders including eosinophilic oesophagitis are mixed IgE- and non-IgE-mediated food allergic conditions, and are improved by dietary exclusions. By contrast food intolerances are nonspecific, and the resultant symptoms resemble other common medically unexplained complaints, often overlapping with symptoms found in functional disorders such as irritable bowel syndrome. Improved dietary treatments for the irritable bowel syndrome have recently been described. CONCLUSIONS: Food allergies are more common in children, can be life-threatening and are distinct from food intolerances. Food intolerances may pose little risk but since functional disorders are so prevalent, greater efforts to understand adverse effects of foods in functional disorders are warranted.

Thermodynamic analysis of the binding of aromatic hydroxamic acid analogues to ferric horseradish peroxidase.

Peroxidases typically bind their reducing substrates weakly, with K(d) values in the millimolar range. The binding of benzhydroxamic acid (BHA) to ferric horseradish peroxidase isoenzyme C (HRPC) [K(d) = 2.4 microM; Schonbaum, G. R. (1973) J. Biol. Chem. 248, 502-511] is a notable exception and has provided a useful tool for probing the environment of the peroxidase aromatic-donor-binding site and the distal heme cavity. Knowledge of the underlying thermodynamic driving forces is key to understanding the roles of the various H-bonding and hydrophobic interactions in substrate binding. The isothermal titration calorimetry results of this study on the binding of aromatic hydroxamic acid analogues to ferric HRPC under nonturnover conditions (no H(2)O(2) present) confirm the significance of H-bonding interactions in the distal heme cavity in complex stabilization. For example, the binding of BHA to HRPC is enthalpically driven at pH 7.0, with the H-bond to the distal Arg38 providing the largest contribution (6.74 kcal/mol) to the binding energy. The overall relatively weak binding of the hydroxamic acid analogues to HRPC is due to large entropic barriers (-11.3 to -37.9 eu) around neutral pH, with the distal Arg38 acting as an "entropic gate keeper". Dramatic enthalpy-entropy compensation is observed for BHA and 2-naphthohydroxamic acid binding to HRPC at pH 4.0. The enthalpic loss and entropic gain are likely due to increased flexibility of Arg38 in the complexes at low pH and greater access by water to the active site. Since the Soret absorption band of HRPC is a sensitive probe of the binding of hydroxamic acids and their analogues, it was used to investigate the binding of six donor substrates over the pH range of 4-12. The negligible pH dependence of the K(d) values corrected for substrate ionization suggests that enthalpy-entropy compensation is operative over a wide pH range. Examination of the thermodynamics of binding of ring-substituted hyrazides to HRPC reveals that the binding affinities of aromatic donors are highly sensitive to the position and nature of the ring substituent.

Expression, purification, and characterization of recombinant purine nucleoside phosphorylase from Escherichia coli.

Recombinant purine nucleoside phosphorylase (PNPase) from Escherichia coli was prepared in high yield in order to facilitate its use in coupled assays to measure the kinetics of phosphate-liberating enzymes. The E. coli enzyme was overexpressed in E. coli by inserting the genomic fragment containing the deoD gene downstream of the isopropyl beta-d-thiogalactoside-inducible promotor of pSE380 expression vector. The recombinant protein was purified to approximately 90% homogeneity and with a yield of approximately 9000 units of activity/L of culture, using an efficient one-column procedure. A continuous spectrophotometric assay coupling P(i) release to the phosphorolysis of the nucleoside analogue 7-methylinosine (m(7)Ino) was recently described. Here, we report the steady-state kinetic parameters of the recombinant E. coli PNPase catalyzed reaction with m(7)Ino and P(i) as substrates and compare these parameters with those of a bacterial PNPase commercially available for use in coupled assays. Under the assay conditions described, the recombinant E. coli protein is active at higher pH values and is stable up to a temperature of approximately 55 degrees C and following multiple freeze-thaw cycles. It is activated by high ionic strength but loses some activity following dialysis or concentration under pressure. Finally, a new procedure for the synthesis of m(7)Ino from inosine is described which is safe and cost effective, making the use of this methylated nucleoside in PNPase-coupled P(i) assays more attractive.

The polysaccharide, PGG-glucan, enhances human myelopoiesis by direct action independent of and additive to early-acting cytokines.

beta-Glucans stimulate leukocyte anti-infective activity, enhance murine hematopoietic recovery following bone marrow injury and mobilize murine progenitor cells from bone marrow. This study evaluated the in vitro hematopoietic potential of the beta-glucan, PGG-glucan, on human bone marrow mononuclear cells (BMMC) and CD34+ BMMC compared with protein cytokines. In the presence of submaximal concentrations of recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF; 0.5 ng/ml), PGG-glucan significantly increased BMMC myeloid colony formation comparable to the increase observed with either interleukin-3 (rhIL-3) or stem cell factor (rhSCF). Moreover, the addition of PGG-glucan to cultures containing GM-CSF + IL-3 or GM-CSF + SCF significantly augmented granulocyte-macrophage colony production above baseline, demonstrating that PGG-glucan acts independently of those early-acting cytokines and can enhance their activity in an additive manner. Anti-PGG-glucan monoclonal antibody specifically abrogated the growth-enhancing effect of added PGG-glucan in a saturable manner and other control carbohydrate polymers failed to affect colony formation. Further, PGG-glucan was not associated with induction of IL-6, GM-CSF production and removal of accessory cells by CD34+ cell isolation did not alter the PGG-glucan effect. These data demonstrate that PGG-glucan acts on committed myeloid progenitors to enhance human hematopoietic activity by a mechanism of direct action independent of IL-3 or SCF and independent of secondary cytokine stimulation.

Identifying groups involved in the binding of prephenate to prephenate dehydrogenase from Escherichia coli.

Site-directed mutagenesis was used to investigate the importance of Lys178, Arg286, and Arg294 in the binding of prephenate to the bifunctional enzyme chorismate mutase-prephenate dehydrogenase. From comparison of the kinetic parameters of wild-type enzyme and selected mutants, we conclude that only Arg294 interacts specifically with prephenate. The R294Q substitution reduces the enzyme's affinity for prephenate without affecting V/Et of the dehydrogenase reaction or the kinetic parameters of the mutase reaction. Arg294 likely interacts with the ring carboxylate at C-1 of prephenate since the dissociation constants for a series of inhibitors missing the ring carboxyl group were similar for wild-type and R294Q enzymes. The pH dependencies of log (V/KprephenateEt) and of pKi for hydroxyphenyllactate show that the wild-type dehydrogenase possesses a group with a pK of 8.8 that must be protonated for binding prephenate to the enzyme. None of the three conserved residues is this group since its titration is observed in the V/KprephenateEt profiles for the mutants K178Q, R286A, and R294Q. This group is also seen in the pH-rate profiles of the binding of two substrate analogues, hydroxyphenyllactate and deoxoprephenate. Their only common structural feature at C-1 is the side chain carboxylate, indicating that the protonated residue (pK 8.8) must interact with prephenate's side chain carboxylate. Gdn-HCl-induced denaturation was conducted on wild-type and selected mutant proteins. Unfolding of the wild-type enzyme proceeds through a partially unfolded dimer which dissociates into unfolded monomers. The order of stability is wild-type = R294Q > K178Q > R286A > K178R. The least unstable mutants have reduced mutase and dehydrogenase activities.

Use of site-directed mutagenesis to identify residues specific for each reaction catalyzed by chorismate mutase-prephenate dehydrogenase from Escherichia coli.

Site-directed mutagenesis was performed on the bifunctional enzyme chorismate mutase-prephenate dehydrogenase in order to identify groups important for each of the two reactions. We selected two residues for mutagenesis, Lys37 and His131, identified previously by differential peptide mapping to be essential for activity [Christendat, D., and Turnbull, J. (1996) Biochemistry 35, 4468-4479]. Kinetic studies reveal that K37Q exhibits no mutase activity while retaining wild-type dehydrogenase activity, verifying that Lys37 plays a key role in the mutase. By contrast His131 is not critical for the dehydrogenase; H131A is a reasonably efficient catalyst exhibiting 10% dehydrogenase and 30% mutase activity compared to the wild-type enzyme. Chemical modification of H131A by diethyl pyrocarbonate further inactivated the dehydrogenase, suggesting that a different histidine is now accessible to modification. To identify this group, the protein's remaining eight histidines were changed to alanine or asparagine. A single substitution, H197N, decreased the dehydrogenase activity by 5 orders of magnitude while full mutase activity was retained. In H197N, the Michaelis constants for prephenate and NAD+ and the mutant's elution profile from Sepharose-AMP were similar to those of wild-type enzyme, indicating that catalysis rather than substrate binding is altered. Log V for the dehydrogenase reaction catalyzed by H197N is pH-independent and is in contrast to wild-type enzyme, which shows a decrease in activity at low pH and pK of about 6.5. We conclude that His197 is an essential catalytic residue in the dehydrogenase reaction.

Functional and structural properties of the mitochondrial outer membrane receptor Tom20.

Tom20 is an outer mitochondrial membrane protein that functions as a component of the import receptor complex for cytoplasmically synthesized mitochondrial precursor proteins. The human homologue, hTom20, consists of an N-terminal membrane anchor region predicted between aa5-25 and a soluble cytosolic domain from aa30 to 145. To analyze the properties of hTom20, we have expressed several truncations of the cytosolic domain as fusion proteins with glutathione S-transferase. Our studies reveal that the cytosolic region of hTom20 is a monomeric protein in solution containing two domains which are involved in different functions of the receptor. The N-terminal region is involved in membrane binding (aa30-60) and recognition of the cleavable matrix targeting signals (aa50-90). In addition, we have demonstrated that the receptor recognizes the alpha-helical state of the matrix targeting signal. The dissociation constant for this interaction in the presence of a detergent which induces this secondary structure is 0.6 microM, one-fifth the value in the absence of detergent. In aqueous solution, the region between aa30 and 60 is loosely folded and stabilized against proteolytic cleavage by interaction with detergents or a matrix targeting signal. Our work further shows that the remainder of the cytosolic domain of hTom20, aa60-145, is a compactly folded globular domain containing a region (aa90-145) that is critical for the recognition of proteins bearing internal signal sequences such as the uncoupling protein and porin.

Characterization of the N-terminal targeting signal binding domain of the mitochondrial outer membrane receptor, Tom20.

hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion.

A continuous spectrophotometric assay for aspartate transcarbamylase and ATPases.

A new continuous coupled uv-spectrophotometric assay is described for two phosphate-releasing enzymes, aspartate transcarbamylase and ATPase of herpes simplex virus (HSV). Phosphate release is coupled to the phosphorolysis of the nucleoside analog 7-methylinosine (m7Ino) catalyzed by purine nucleoside phosphorylase. When this reaction is monitored at 291 nm, the coupled assay can readily detect 10 nmol Pi released/min. Our method offers advantages over a recently reported continuous assay devised for measuring aspartate transcarbamylase activity using the nucleoside analog methylthioguanosine (MESG) as the linking substrate. In contrast to MESG, m7Ino is easily and inexpensively synthesized and is also commercially available. The spectrophotometric signal at 291 nm, produced by the difference in the extinction coefficients between nucleoside substrate and the base product, is significant over a much wider pH range than the signal difference between MESG and its phosphorolysis product at 360 nm. Saturation curves for aspartate and carbamyl phosphate and pH rate profiles have been reproduced using the purine nucleoside phosphorylase/m7Ino coupled assay. Initial velocity patterns constructed over micromolar to millimolar concentrations of aspartate and carbamyl phosphate yielded four kinetic parameters simultaneously. To further illustrate the application of this coupled assay, kinetic parameters were determined for the DNA-dependent ATPase reaction of HSV helicase-primase.

Ligand-induced conformational change in the ferrichrome-iron receptor of Escherichia coli K-12.

Ferrichrome-iron is actively transported across the outer membrane of Escherichia coli by the TonB-dependent receptor FhuA. To obtain FhuA in a form suitable for secondary-structure analyses, a hexahistidine tag was inserted into a surface-located site and the recombinant protein was purified by metal chelate chromatography. Functional studies indicated that the presence of the hexahistidine tag did not interfere with FhuA localization or with ligand-binding activity. Ferrichrome protected lysine 67 but not lysine 5 of purified recombinant FhuA from trypsinolysis. Results from trypsin digestion were interpreted as a conformational change in FhuA which had occurred upon ferrichrome binding, thereby preventing access of trypsin to lysine 67. Circular dichroism and Fourier transform infrared spectroscopy revealed a predominance of beta-sheet structure for the purified protein. In the presence of ferrichrome, FhuA exhibited a secondary structure and a thermostability which were similar to FhuA without ligand. The addition of ferrichrome to purified FhuA reduced the ability of certain anti-FhuA monoclonal antibodies to bind to the receptor. All antibodies which could in this manner discriminate between FhuA and FhuA bound to ferrichrome had their determinants within a loop which is toward the N-terminus and which is exposed to the periplasm. These data indicate that the binding of ferrichrome induces a structural change that is propogated across the outer membrane and results in an altered conformation of a periplasmically exposed loop of FhuA. It is proposed that by such an alteration of FhuA conformation, TonB is triggered to energize the active transport of the bound ligand across the outer membrane.

Small scale biosynthesis and purification of gram quantities of chorismic acid.

This paper details improvements in the biosynthetic method of purification of chorismic acid using Klebsiella pneumoniae 62-1. New growth and accumulation conditions yielded 4 L of accumulation medium containing approximately 0.5 g/L of chorismate from 2 L of bacterial growth. Improvements in the handling and extraction procedures produced yields of approximately 2 g of 75 to 90% chorismic acid. A new recrystallization procedure yielded chorismic acid 97% pure by weight (using epsilon 275 = 2630 M-1 cm-1), and 99.8% pure by enzymatic conversion. These results represent a three- to five-fold increase in yield over average published values.

Ionization of amino acid residues involved in the catalytic mechanism of aspartate transcarbamoylase.

The chemical and kinetic mechanisms of the reaction catalyzed by the catalytic trimer of aspartate transcarbamoylase have been examined. The variation of the kinetic parameters with pH indicated that at least four ionizing amino acid residues are involved in substrate binding and catalysis. The pH dependence of K(ia) for carbamoyl phosphate and the K(i) for N-(phosphonoacetyl)-L- aspartate revealed that a protonated residue with a pK value of 9.0 is required for the binding of carbamoyl phosphate. However, the variation with pH of K(i) for succinate, a competitive inhibitor of aspartate, and for cysteine sulfinate, a slow substrate, showed that a single residue with a pK value of 7.3 must be protonated for binding these analogues and, by inference, aspartate. The profile of log V against pH displayed a decrease in reaction rate at low and high pH, suggesting that two groups associated with the Michaelis complex, a deprotonated residue with a pK value of 7.2 and a protonated group with a pK value of 9.5, are involved in catalysis. By contrast, the catalytically productive form of the enzyme-carbamoyl phosphate complex, as illustrated in the bell-shaped pH dependence of log (V/K)(asp), is one in which a residue with a pK value of 7.0 must be protonated while a group with a pK value of 9.1 is deprotonated. This interpretation is supported by the results from the temperature dependence of the V and V/K profiles and from the pH dependence of pK(i) for the aspartate analogues.(ABSTRACT TRUNCATED AT 250 WORDS)

The contribution of threonine 55 to catalysis in aspartate transcarbamoylase.

Heavy-atom isotope effects and steady-state kinetic parameters were measured for the catalytic trimer of an active site mutant of aspartate transcarbamoylase, T55A, to assess the role of Thr 55 in catalysis. The binding of carbamoyl phosphate to the T55A mutant was decreased by 2 orders of magnitude relative to the wild-type enzyme whereas the affinities for aspartate and succinate were not markedly altered. This indicates that Thr 55 plays a significant role in the binding of CbmP. If, as had been suggested previously, Thr 55 assists in the polarization of the carbonyl group of CbmP, the carbon isotope effect for the T55A mutant should increase relative to that observed for the wild-type enzyme. However, the opposite is seen, indicating that Thr 55 is not involved in stabilizing the oxyanion in the transition state. Quantitative analysis of a series of 13C and 15N isotope effects suggested that the rate-determining step in the reaction catalyzed by T55A trimer may be a conformational change in the protein subsequent to formation of the Michaelis complex. Thus, Thr 55 may facilitate a conformational change in the enzyme that is a prerequisite for catalysis. An altered active site environment in the binary and Michaelis complexes with T55A trimer is reflected in the pH profiles for log V, log (V/K)asp, and pK(i) succinate, show a displacement in the pK values of ionizing residues involved in aspartate binding and catalysis relative to the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Steady-state kinetics and isotope effects on the mutant catalytic trimer of aspartate transcarbamoylase containing the replacement of histidine 134 by alanine.

A detailed kinetic analysis of the catalytic trimer of aspartate transcarbamoylase containing the active site substitution H134A was performed to investigate the role of His 134 in the catalytic mechanism. Replacement of histidine by alanine resulted in decreases in the affinities for the two substrates, carbamoyl phosphate and aspartate, and the inhibitor succinate, by factors of 50, 10, and 6, respectively, and yielded a maximum velocity that was 5% that of the wild-type enzyme. However, the pK values determined from the pH dependence of the kinetic parameters, log V and log (V/K) for aspartate, the pK(i) for succinate, and the pK(ia) for carbamoyl phosphate, were similar for both the mutant and the wild-type enzymes, indicating that the protonated form of His 134 does not participate in binding and catalysis between pH 6.2 and 9.2. 13C and 15N isotope effects were studied to determine which steps in the catalytic mechanism were altered by the amino acid substitutions. The 13(V/K) for carbamoyl phosphate exhibited by the catalytic trimer containing alanine at position 134 revealed an isotope effect of 4.1%, probably equal to the intrinsic value and, together with quantitative analysis of the 15N isotope effects, showed that formation of the tetrahedral intermediate is rate-determining for the mutant enzyme. Thus, His 134 plays a role in the chemistry of the reaction in addition to substrate binding. The initial velocity pattern for the reaction catalyzed by the H134A mutant intersected to the left of the vertical axis, negating an equilibrium ordered kinetic mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

The isolation and characterization of cyclic nucleotide phosphodiesterases from Morris hepatoma 5123tc(h) and rat liver.

Four main phosphodiesterase (PDE) forms were resolved and partially purified from rat liver and Morris hepatoma 5123tc(h). The activities of the high Km cyclic nucleotide PDE (form II) in hepatoma were markedly reduced compared to liver, while the activities of the low Km cAMP PDE (form III) and low Km cyclic nucleotide PDE (form IV) in hepatoma were markedly higher than those of liver. The partially purified low Km cAMP PDE's (forms III and IV) from liver showed non-linear Lineweaver-Burk plots, whereas the same enzyme forms in hepatoma displayed linear kinetics. Activation of low Km cGMP PDE activity by calmodulin was found with form I in liver whereas in hepatoma form II was responsive to calmodulin.