Towards a surrogate system to express human lipid binding TCRs.

BACKGROUND: Previously we reported that natural nut lipids were necessary for sensitization and that natural killer T cells (NKTs) must play a critical role in the development of food allergic responses. A major bottleneck in further understanding the interaction of nut lipids with the cells of the human immune system is the lack of well-characterized lipid responsive human cell lines. OBJECTIVE: In the present study, we engineered human T cell receptor (TCR) sequences TRAV10 and TRBV25 responsive to α-GalCer into a stable murine iNKT hybridoma and surrogate human T cell lines. RESULTS: The murine hybridoma system has shown to be problematic. To overcome this limitation, the expression of human TCR α/ß sequences has been achieved driven by a bidirectional promoter on a plasmids or a lentivirus system, employing stable DC cell lines as lipid presenting cells, and a stable T cell line as a surrogate system. Further, a commercial human Jurkat T cell line containing an inducible secreted luciferase reporter construct was shown to be functional and can be used for a transient expression of human TCRs in a lipid screening program. The transfection efficiencies were improved using the lentivirus polycistronic constructs containing the P2A sequence in a TCR αß/γδ null cell line (Jurkat 76). CONCLUSIONS: The results suggest that the mis-pairing of the endogenous α/ß TCR during ER folding in the presence of the new human TCR sequences could be impairing the functionality of the TCR lipid receptors. The surrogate systems presented here are important first steps in the establishment of human cell-specific lipid responsive libraries for the study of natural lipid substances.

Use of humanized rat basophilic leukemia reporter cell lines as a diagnostic tool for detection of allergen-specific IgE in allergic patients: time for a reappraisal?

The interaction between allergens and specific IgE is at the heart of the allergic response and as such lies at the center of techniques used for diagnosis of allergic sensitization. Although serological tests are available, in vivo tests such as double-blind placebo-controlled food challenges (DBPCFC) and skin prick test (SPT) associated to the patients' clinical history are still the main guides to clinicians in many practices around the world. More recently, complex protein arrays and basophil activation tests, requiring only small amounts of whole blood, have been developed and refined, but are yet to enter clinical practice. Similarly, the use of rat basophilic leukemia (RBL) cell lines for detection of allergen-specific IgE has been made possible by stable transfection of the human FcεRI α chain into this cell line more than 20 years ago, but has not found widespread acceptance among clinicians. Here, we review the perceived limitations of diagnostic applications of humanized RBL systems. Furthermore, we illustrate how the introduction of reporter genes into humanized RBL cells is able to overcome most of these limitations, and has the potential to become a new powerful tool to complement the armamentarium of allergists. A demonstration of the usefulness of humanized RBL reporter systems for elucidation of complex IgE sensitization patterns against wheat proteins and a section on the use of fluorescence-based reporter systems in combination with allergen arrays close the review.

Chemical reaction versus vibrational quenching in low energy collisions of vibrationally excited OH with O.

Quantum scattering calculations are reported for state-to-state vibrational relaxation and reactive scattering in O + OH(v = 2 - 3, j = 0) collisions on the electronically adiabatic ground state (2)A'' potential energy surface of the HO2 molecule. The time-independent Schrödinger equation in hyperspherical coordinates is solved to determine energy dependent probabilities and cross sections over collision energies ranging from ultracold to 0.35 eV and for total angular momentum quantum number J = 0. A J-shifting approximation is then used to compute initial state selected reactive rate coefficients in the temperature range T = 1 - 400 K. Results are found to be in reasonable agreement with available quasiclassical trajectory calculations. Results indicate that rate coefficients for O2 formation increase with increasing the OH vibrational level except at low and ultralow temperatures where OH(v = 0) exhibits a slightly different trend. It is found that vibrational relaxation of OH in v = 2 and v = 3 vibrational levels is dominated by a multi-quantum process.

Diabetic neuropathy: Molecular approach a treatment opportunity.

Diabetic neuropathy (DN) encompasses a group of clinical or subclinical manifestations involving a dysfunction in the peripheral nervous system. The cause of the dysfunction is the development of microvascular complications related to diabetes, a disease that affects about 381 million people worldwide. Approximately 50% of patients currently diagnosed with diabetes are expected to manifest DN in the next 10 years. The diagnosis can be made clinically by establishing a good patient history and delving into the symptoms to rule out other etiologies. Treatment of DN focuses on glycemic control and the use of medications to reduce pain, including NSAIDs, antidepressants and antiepileptic drugs. The pathogenesis is of multifactorial origin, associated with various metabolic, vascular, inflammatory and neurodegenerative disorders. The three fundamental cellular alterations participating in the development of DN are chronic inflammation, endothelial dysfunction and oxidative stress. Since the combination of all three is capable of giving rise to nerve ischemia and direct axonal injury, these factors play a key role in the development of polyneuropathy. However, neuronal and microvascular changes do not occur in the same way in all patients with DN, some of whom have no detectable blood abnormalities.

Specific berenil-DNA interactions: an approach for separation of plasmid isoforms by pseudo-affinity chromatography.

Small molecules, like some antibiotics and anticancer agents that bind DNA with high specificity, can represent a relevant alternative as ligands in affinity processes for plasmid DNA (pDNA) purification. In the current study, pDNA binding affinities of berberine, berenil, kanamycin, and neomycin were evaluated by a competitive displacement assay with ethidium bromide using a fluorimetric titration technique. The binding between pDNA and ethidium bromide was tested in different buffer conditions, varying the type and the salt concentration, and was performed in both the absence and presence of the studied compounds. The results showed that the minor groove binder berenil has the higher pDNA binding constant. Chromatographic experiments using a derivatized column with berenil as ligand showed a total retention of pDNA using 1.3M ammonium sulfate in eluent buffer. A selective separation of supercoiled and open circular isoforms was achieved by further decreasing the salt concentration to 0.6M and then to 0M. These results suggest a promising application of berenil as ligand for specific purification of pDNA supercoiled isoform by pseudo-affinity chromatography.

Effect of O-linked glycosylation on the antigenicity, cellular uptake and trafficking in dendritic cells of recombinant Ber e 1.

Ber e 1, a major Brazil nut allergen, has been successfully produced in the yeast Pichia pastoris expression system as homogenous recombinant Ber e 1 (rBer e 1) with similar physicochemical properties and identical immunoreactivity to its native counterpart, nBer e 1. However, O-linked glycans was detected on the P.pastoris-derived rBer e 1, which is not naturally present in nBer e 1, and may contribute to the allergic sensitisation. In this study, we addressed the glycosylation differences between P. pastoris-derived recombinant Ber e 1 and its native counterparts. We also determined whether this fungal glycosylation could affect the antigenicity and immunogenicity of the rBer e 1 by using dendritic cells (DC) as an immune cell model due to their role in modulating the immune response. We identified that the glycosylation occurs at Ser96, Ser101 and Ser110 on the large chain and Ser19 on the small polypeptide chain of rBer e 1 only. The glycosylation on rBer e 1 was shown to elicit varying degree of antigenicity by binding to different combination of human leukocyte antigens (HLA) at different frequencies compared to nBer e 1 when tested using human DC-T cell assay. However, both forms of Ber e 1 are weak immunogens based from their low response indexes (RI). Glycans present on rBer e 1 were shown to increase the efficiency of the protein recognition and internalization by murine bone marrow-derived dendritic cells (bmDC) via C-type lectin receptors, particularly the mannose receptor (MR), compared to the non-glycosylated nBer e 1 and SFA8, a weak allergenic 2S albumin protein from sunflower seed. Binding of glycosylated rBer e 1 to MR alone was found to not induce the production of IL-10 that modulates bmDC to polarise Th2 cell response by suppressing IL-12 production and DC maturation. Our findings suggest that the O-linked glycosylation by P. pastoris has a small but measurable effect on the in vitro antigenicity of the rBer e 1 compared to its non-glycosylated counterpart, nBer e 1, and thus may influence its applications in diagnostics and immunotherapy.

Mating type and the genetic basis of self-fertility in the model fungus Aspergillus nidulans.

Sexual reproduction occurs in two fundamentally different ways: by outcrossing, in which two distinct partners contribute nuclei, or by self-fertilization (selfing), in which both nuclei are derived from the same individual. Selfing is common in flowering plants, fungi, and some animal taxa. We investigated the genetic basis of selfing in the homothallic fungus Aspergillus nidulans. We demonstrate that alpha and high-mobility group domain mating-type (MAT) genes, found in outcrossing species, are both present in the genome of A. nidulans and that their expression is required for normal sexual development and ascospore production. Balanced overexpression of MAT genes suppressed vegetative growth and stimulated sexual differentiation under conditions unfavorable for sex. Sexual reproduction was correlated with significantly increased expression of MAT genes and key genes of a pheromone-response MAP-kinase signaling pathway involved in heterothallic outcrossing. Mutation of a component MAP-kinase mpkB gene resulted in sterility. These results indicate that selfing in A. nidulans involves activation of the same mating pathways characteristic of sex in outcrossing species, i.e., self-fertilization does not bypass requirements for outcrossing sex but instead requires activation of these pathways within a single individual. However, unlike heterothallic species, aspects of pheromone signaling appeared to be independent of MAT control.

A theoretical study of H(2) dissociation on (sq.rt(3) x sq.rt(3))R30 degrees CO/Ru(0001).

We have studied the influence of preadsorbed CO on the dissociative adsorption of H(2) on Ru(0001) with density functional theory calculations. For a coverage of 1/3 ML CO, we investigated different possible reaction paths for hydrogen dissociation using nudged elastic band and adaptive nudged elastic band calculations. One reaction path was studied in detail through an energy decomposition and molecular orbital type of analysis. The minimum barrier for H(2) dissociation is found to be 0.29 eV. At the barrier the H-H bond is hardly stretched. Behind this barrier a molecular chemisorption minimum is present. Next, the molecule overcomes a second barrier, with a second local chemisorption minimum behind it. To finally dissociate to chemisorbed atoms, the molecule has to overcome a third barrier. To move along the reaction path from reactants to products, the hydrogen molecule needs to rotate, and to significantly change its center-of-mass position. The procedure of mapping out reaction paths for H(2) reacting on low-index surfaces of bare metals (computing two-dimensional elbow plots for fixed impact high-symmetry sites and H(2) orientations parallel to the surface) does not work for H(2)+CO/Ru. The first barrier in the path is recovered, but the features of the subsequent stretch to the dissociative chemisorption minimum are not captured, because the molecule is not allowed to change its center-of-mass position or to rotate. The dissociative chemisorption of H(2) on CO/Ru(0001) is endoergic, in contrast to the case of H(2) on bare Ru(0001). The zero-point energy corrected energies of molecularly and dissociatively chemisorbed H(2) are very close, suggesting that it may be possible to detect molecularly chemisorbed H(2) on (sq.rt(3) x sq.rt(3))R30 degrees CO/Ru(0001). The presence of CO on the surface increases the barrier height to dissociation compared with bare Ru(0001). Based on an energy decomposition and molecular orbital analysis we attribute the increase in the barrier height mainly to an occupied-occupied interaction between the bonding H(2) sigma(g) orbital and the (surface-hybridized) CO 1pi orbitals, i.e., to site blocking. There is a small repulsive contribution to the barrier from the interaction between the H(2) molecule and the Ru part of the CO covered Ru surface, but it is smaller than one might expect based on the calculations of H(2) interacting with a clean Ru surface, and on calculations of H(2) interacting with the CO overlayer only. Actually, the analysis suggests that the Ru surface as a subsystem is (slightly) more reactive for the reaction path studied with CO preadsorbed on it than without it. Thus, the results indicate that the influence of CO on H(2) dissociation on Ru is not only a simple site-blocking effect, the electronic structure of the underlying Ru is changed.

Use of Humanized RBL Reporter Systems for the Detection of Allergen-Specific IgE Sensitization in Human Serum.

Determination of allergen-specific immunoglobulin E (IgE) levels in human blood samples is an important diagnostic technology for the assessment of allergic sensitization. The presence of specific IgE in human serum samples can be measured by sensitizing humanized rat basophil leukemia (RBL) cell lines with diluted serum and measuring cellular activation after challenge with the suspected allergens. This has been traditionally performed by measuring the levels of ß-hexosaminidase released upon RBL degranulation. Here, we describe the use of two recently developed humanized RBL reporter cell lines, which offer higher sensitivity and are amenable to high-throughput scale experiments.

Use of Humanized Fluorescent Reporter Cell Line RBL NFAT-DsRed for the Detection of Allergen-Specific IgE in Patient Sera Using Allergen Microarrays.

The presence of allergen-specific IgE (sIgE) in human sera can be determined by measuring the binding of sIgE to solid phase-bound preparations containing the allergens to be tested. These can be complex extracts, purified or recombinant allergens, or peptides. Older methods, such as the IgE CAP test, only allow sIgE measurements to multiple allergens in individual measurements. Newer technologies such as the ImmunoCAP® ISAC test allows semiquantitative testing of sIgE to over a hundred allergens on a protein array. Allergen arrays have higher numerical power, allowing testing to many allergens at the same time, using only a small amount of serum. We have previously demonstrated how allergen arrays can be used in combination with purified peripheral blood basophils, introducing a clinically relevant readout. Here, we describe a protocol and materials that allow the testing of sIgE with multiple allergens in array format, using a humanized fluorescent IgE reporter system (RBL NFAT-DsRed).

Development of a protein microarray-based diagnostic chip mimicking the skin prick test for allergy diagnosis.

Protein microarrays have been successfully used for detection of allergen-specific IgE in patient sera. Here, we demonstrate proof-of-concept of a solid-phase technique coupling the high-throughput potential of protein microarrays with the biologically relevant readout provided by IgE reporter cells, creating a novel allergic sensitization detection system. Three proteins (κ-casein, timothy grass pollen extract, polyclonal anti-human IgE) were printed onto three different polymer-coated surfaces (aldehyde-, epoxy- and NHS ester-coated). ToF-SIMs analysis was performed to assess printed protein stability and retention during washing steps. NFAT-DsRed rat basophil leukemia cell attachment and retention during washing steps was assessed after treatment with various extracellular matrix proteins. NFAT-DsRed IgE reporter cells were sensitized with serum of an allergic donor, incubated on the printed slides, and cell activation determined using a microarray laser scanner. NFAT DsRed IgE reporter cell binding was significantly increased on all polymer surfaces after incubation with fibronectin and vitronectin, but not collagen or laminin. All surfaces supported printed protein stability during washing procedure, with epoxy- and NHS ester-coated surfaces showing best protein retention. Cell activation was significantly higher in NHS ester-coated slides after timothy grass pollen extract stimulation appearing a suitable substrate for further development of an automated allergy diagnosis system.

Validation of the Spanish, Portuguese and French versions of the Lupus Damage Index questionnaire: data from North and South America, Spain and Portugal.

We have previously developed and validated a self-administered questionnaire, modelled after the Systemic Lupus International Collaborating Clinics Damage Index (SDI), the Lupus Damage Index Questionnaire (LDIQ), which may allow the ascertainment of this construct in systemic lupus erythematosus (SLE) patients followed in the community and thus expand observations made about damage. We have now translated, back-translated and adapted the LDIQ to Spanish, Portuguese and French and applied it to patients followed at academic and non-academic centres in North and South America, Portugal and Spain while their physicians scored the SDI. A total of 887 patients (659 Spanish-speaking, 140 Portuguese-speaking and 80 French-speaking patients) and 40 physicians participated. Overall, patients scored all LDIQ versions higher than their physicians (total score and all domains). Infrequent manifestations had less optimal clinimetric properties but overall agreement was more than 95% for the majority of items. Higher correlations were observed among the Spanish-speaking patients than the Portuguese-speaking and French-speaking patients; further adjustments may be needed before the Portuguese and French versions of the LDIQ are applied in community-based studies. The relationship between the LDIQ and other outcome parameters is currently being investigated in a different patient sample.

Characterization of human FcεRIα chain expression and gene copy number in humanized rat basophilic leukaemia (RBL) reporter cell lines.

Several laboratories have created rat basophil leukemia (RBL) cell lines stably transfected with the human high affinity IgE receptor (FcεRIH). More recently, humanized RBL cell lines saw the introduction of reporter genes such as luciferase (RS-ATL8) and DsRed (RBL NFAT-DsRed). These reporters are more sensitive than their parental non-reporter humanized RBL cell lines. However, no studies so far have addressed the levels of FcεRIH surface expression on humanized RBL cell lines. This is a critical parameter, as it determines the ability of these cells to be efficiently sensitized with human IgE, hence it should affect the sensitivity of the cell assay-a critical parameter for any diagnostic application. Our purpose was to assess and compare the levels of expression of the transfected FcεRIH chain in humanized RBL cell lines. We compared surface levels of FcεRIαH by flow cytometry, using a fluorescently labelled monoclonal antibody (CRA-1/AER-37) and determined receptor numbers using calibration microspheres. FcεRIαH copy numbers were assessed by qPCR, and the sequence verified. Transfection with FcεRIγH cDNA was assessed for its ability to increase FcεRIαH expression in the NFAT-DsRed reporter. While both SX-38 and RS-ATL8 expressed about 500.000 receptors/cell, RBL 703-21 and NFAT-DsRed had approximately 10- to 30-fold lower FcεRIαH expression, respectively. This was neither related to FcεRIH gene copy numbers, nor to differences in steady state mRNA levels, as determined by qPCR and RT-qPCR, respectively. Instead, FcεRIαH surface expression appeared to correlate with the co-expression of FcεRIγH. Stable transfection of NFAT-DsRed cells with pBJ1 neo-huFcεRI gamma, which constitutively expresses FcεRIγH, increased FcεRIαH chain expression levels. Levels of FcεRIαH surface expression vary greatly between humanized RBL reporter cell lines. This difference will affect the sensitivity of the reporter system when used for diagnostic purposes.

Affinity partitioning of plasmid DNA with a zinc finger protein.

The affinity isolation of pre-purified plasmid DNA (pDNA) from model buffer solutions using native and poly(ethylene glycol) (PEG) derivatized zinc finger-GST (Glutathione-S-Transferase) fusion protein was examined in PEG-dextran (DEX) aqueous two-phase systems (ATPSs). In the absence of pDNA, partitioning of unbound PEGylated fusion protein into the PEG-rich phase was confirmed with 97.5% of the PEGylated fusion protein being detected in the PEG phase of a PEG 600-DEX 40 ATPS. This represents a 1322-fold increase in the protein partition coefficient in comparison to the non-PEGylated protein (Kc = 0.013). In the presence of pDNA containing a specific oligonucleotide recognition sequence, the zinc finger moiety of the PEGylated fusion protein bound to the plasmid and steered the complex to the PEG-rich phase. An increase in the proportion of pDNA that partitioned to the PEG-rich phase was observed as the concentration of PEGylated fusion protein was increased. Partitioning of the bound complex occurred to such an extent that no DNA was detected by the picogreen assay in the dextran phase. It was also possible to partition pDNA using a non-PEGylated (native) zinc finger-GST fusion protein in a PEG 1000-DEX 500 ATPS. In this case the native ligand accumulated mainly in the PEG phase. These results indicate good prospects for the design of new plasmid DNA purification methods using fusion proteins as affinity ligands.

siRNA as a molecular tool for use in Aspergillus niger.

Gene silencing using siRNA has been examined in the industrially-important fungus, Aspergillus niger. Protoplasts of an A. niger strain containing a single genomic copy of the Escherichia coli uidA gene, encoding beta-glucuronidase (GUS), under control of the A. niger glaA promoter at the same genomic locus, were exposed to siRNA targeted against the uidA gene. Down-regulation of uidA mRNA and GUS activity by siRNA was observed in mycelia that developed from the protoplasts. The down-regulation was transient and was not carried over to conidiation. We concluded that gene silencing by siRNA provides a relatively quick method for analysis of gene function in A. niger.

Biotechnologies in new high-throughput food allergy tests: why we need them.

The increase in prevalence of food allergies generates a need for more accurate and reliable quantitative allergy testing in order to help diagnosis. In this short review, we briefly outline the history of food allergy testing and extend our comments to current multiplex techniques. Particular emphasis is given to new developments in the protein microarray area, where the use of recent advances in biotechnology has the potential to produce high-throughput devices with improved clinical significance.

Impact of the native-state stability of human lysozyme variants on protein secretion by Pichia pastoris.

We report the secreted expression by Pichia pastoris of two human lysozyme variants F57I and W64R, associated with systemic amyloid disease, and describe their characterization by biophysical methods. Both variants have a substantially decreased thermostability compared with wild-type human lysozyme, a finding that suggests an explanation for their increased propensity to form fibrillar aggregates and generate disease. The secreted yields of the F57I and W64R variants from P. pastoris are 200- and 30-fold lower, respectively, than that of wild-type human lysozyme. More comprehensive analysis of the secretion levels of 10 lysozyme variants shows that the low yields of these secreted proteins, under controlled conditions, can be directly correlated with a reduction in the thermostability of their native states. Analysis of mRNA levels in this selection of variants suggests that the lower levels of secretion are due to post-transcriptional processes, and that the reduction in secreted protein is a result of degradation of partially folded or misfolded protein via the yeast quality control system. Importantly, our results show that the human disease-associated mutations do not have levels of expression that are out of line with destabilizing mutations at other sites. These findings indicate that a complex interplay between reduced native-state stability, lower secretion levels, and protein aggregation propensity influences the types of mutation that give rise to familial forms of amyloid disease.

Rationalising lysozyme amyloidosis: insights from the structure and solution dynamics of T70N lysozyme.

T70N human lysozyme is the only known naturally occurring destabilised lysozyme variant that has not been detected in amyloid deposits in human patients. Its study and a comparison of its properties with those of the amyloidogenic variants of lysozyme is therefore important for understanding the determinants of amyloid disease. We report here the X-ray crystal structure and the solution dynamics of T70N lysozyme, as monitored by hydrogen/deuterium exchange and NMR relaxation experiments. The X-ray crystal structure shows that a substantial structural rearrangement results from the amino acid substitution, involving residues 45-51 and 68-75 in particular, and gives rise to a concomitant separation of these two loops of up to 6.5A. A marked decrease in the magnitudes of the generalised order parameter (S2) values of the amide nitrogen atom, for residues 70-74, shows that the T70N substitution increases the flexibility of the peptide backbone around the site of mutation. Hydrogen/deuterium exchange protection factors measured by NMR spectroscopy were calculated for the T70N variant and the wild-type protein. The protection factors for many of backbone amide groups in the beta-domain of the T70N variant are decreased relative to those in the wild-type protein, whereas those in the alpha-domain display wild-type-like values. In pulse-labelled hydrogen/deuterium exchange experiments monitored by mass spectrometry, transient but locally cooperative unfolding of the beta-domain of the T70N variant and the wild-type protein was observed, but at higher temperatures than for the amyloidogenic variants I56T and D67H. These findings reveal that such partial unfolding is an intrinsic property of the human lysozyme structure, and suggest that the readiness with which it occurs is a critical feature determining whether or not amyloid deposition occurs in vivo.

The expression and processing of two recombinant 2S albumins from soybean (Glycine max) in the yeast Pichia pastoris.

Soybean seeds contain two 2S albumin storage proteins (AL1 and AL3) which may contribute to their industrial processing quality and allergenicity. We show that these proteins (AL1 and AL3) are well expressed by the methylotrophic yeast Pichia pastoris and that one of the secreted proteins (AL3) has a similar conformation and stability to that purified from soybean seeds. Further, we show that the subunits are post-translationally processed within the same loop region as the native protein but with some differences in the precise sites. This internal processing provides useful information on the endoproteolytic activity in P. pastoris. We also show that, similar to many plant allergens, the 2S albumins from soybean are stable to heat and chemical treatments.

A family 11 xylanase from Penicillium funiculosum is strongly inhibited by three wheat xylanase inhibitors.

Steady-state kinetic approaches were used to investigate the binding of a novel Penicillium funiculosum xylanase, XYNC, with three known xylanase inhibitor proteins from wheat (Triticum aestivum). The xylanase gene (xynC) was cloned from a P. funiculosum genomic library and the deduced amino acid sequence of XYNC exhibited high sequence similarity with fungal family 11 xylanases. xynC was overexpressed in P. funiculosum and the product (XYNC: M(r)=23.6 kDa; pI=3.7) purified and shown to efficiently degrade birchwood xylan [K(m)=0.47% w/v, Vmax=2540 micromol xylose min(-1) (mg protein)(-1) at pH 5.5 and 30 degrees C] and soluble wheat arabinoxylans [K(m)=1.45% w/v, Vmax=7190 micromol xylose min(-1) mg protein)(-1) at pH 5.5 and 30 degrees C]. The xylanase activity of XYNC was inhibited strongly by three xylanase inhibitor proteins from wheat; XIP-I, TAXI I and TAXI II. The inhibition for each was competitive, with very tight binding (K(i)=3.4, 16 and 17 nM, respectively) equivalent to free energy changes (deltaG degrees ) of -49, -45 and -45 kJ mol(-1). This is the first report describing a xylanase that is inhibited by all three wheat xylanase inhibitor proteins described to date.