Effects of wheat germ agglutinin on human gastrointestinal epithelium: insights from an experimental model of immune/epithelial cell interaction.

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed, among many others, by human gastrointestinal epithelial and immune cells. WGA is a toxic compound and an anti-nutritional factor, but recent works have shown that it may have potential as an anti-tumor drug and as a carrier for oral drugs. To quantitate the toxicity threshold for WGA on normal epithelial cells we previously investigated the effects of the lectin on differentiated Caco2 cells, and showed that in the micromolar range of concentrations WGA could alter the integrity of the epithelium layer and increase its permeability to both mannitol and dextran. WGA was shown to be uptaken by Caco2 cells and only approximately 0.1% molecules were observed to cross the epithelium layer by transcytosis. Here we show that at nanomolar concentrations WGA is unexpectedly bioactive on immune cells. The supernatants of WGA-stimulated peripheral blood mononuclear cells (PBMC) can alter the integrity of the epithelium layer when administered to the basolateral side of differentiated Caco2 cells and the effects can be partially inhibited by monoclonal antibodies against IL1, IL6 and IL8. At nanomolar concentrations WGA stimulates the synthesis of pro-inflammatory cytokines and thus the biological activity of WGA should be reconsidered by taking into account the effects of WGA on the immune system at the gastrointestinal interface. These results shed new light onto the molecular mechanisms underlying the onset of gastrointestinal disorders observed in vivo upon dietary intake of wheat-based foods.

Isolation and identification of two lipid transfer proteins in pomegranate (Punica granatum).

Lipid transfer proteins (LTPs) are a family of low molecular mass (7-9 kDa) polypeptides, the members of which share 35-95% sequence homology. These proteins are widely distributed throughout the plant kingdom and are receiving attention for their biochemical characteristics and biological activity. LTPs are indeed studied in different research fields varying from allergy to food technology, and numerous molecules belonging to this class are progressively being identified and investigated. Proteins from pomegranate juice were fractioned by cation exchange chromatography and analyzed by SDS-PAGE. Two proteins were identified as putative LTPs on the basis of their molecular weights and their electrophoretic behaviors under reducing and nonreducing conditions. Finally, proteins were purified and characterized by mass spectrometry. This analysis confirmed that the two polypeptides are LTPs on the basis of an amino acid sequence common to LTPs from other plant sources and cysteine content. The two proteins, named LTP1a and LTP1b, showed similar molecular masses but different immunological profiles when immunodetected with rabbit antibodies specific for Pru p 3 and human IgE from a patient suffering from pomegranate allergy. The demonstration of the existence of two immunologically unrelated LTPs in pomegranate confirms the variability and the complexity of the plant LTP family. This should be taken into account when the role of these proteins as elicitors of allergies to fruits is investigated and could help to explain the contradictory literature data on pomegranate allergy.

Full-fledged proteomic analysis of bioactive wheat amylase inhibitors by a 3-D analytical technique: Identification of new heterodimeric aggregation states.

Wheat proteinaceous alpha-amylase inhibitors (alpha-AIs) are increasingly investigated for their agronomical role as natural defence molecules of plants against the attack of insects and pests, but also for their effects on human health. The wheat genomes code for several bioactive alpha-AIs that share sequence homology, but differ in their specificity against alpha-amylases from different species and for their aggregation states. Wheat alpha-AIs are traditionally classified as belonging to the three classes of tetrameric, homodimeric and monomeric forms, each class being constituted by a number of polypeptides that display different electrophoretic mobilities. Here we describe a proteomic approach for the identification of bioactive alpha-AIs from wheat and, in particular, a 3-D technique that allows to best identify and characterize the dimeric fraction. The technique takes advantage of the thermal resistance of alpha-AIs (resistant to T > 70 degrees C) and consists in the separation of protein mixtures by 2-D polyacrylamide/starch electrophoresis under nondissociating PAGE (ND-PAGE, first dimension) and dissociating (urea-PAGE or U-PAGE second dimension) conditions, followed by in-gel spontaneous reaggregation of protein complexes and identification of the alpha-amylase inhibitory activity (antizymogram, third dimension) using enzymes from human salivary glands and from the larvae of Tenebrio molitor coleopter (yellow mealworm). Dimeric alpha-AIs from Triticum aestivum (bread wheat) were observed to exist as heterodimers. The formation of heterodimeric complexes was also confirmed by in vitro reaggregation assays carried out on RP-HPLC purified wheat dimeric alpha-AIs, and their bioactivity assayed by antizymogram analysis. The present 3-D analytical technique can be exploited for fast, full-fledged identification and characterization of wheat alpha-AIs.

Plant lectins as carriers for oral drugs: is wheat germ agglutinin a suitable candidate?

Wheat germ agglutinin (WGA) is a plant protein that binds specifically to sugars expressed also by gastrointestinal epithelial cells. WGA is currently investigated as an anti-tumor drug and as a carrier for oral drugs. Information on whether it can cross the gastrointestinal epithelium and on its possible effects on the integrity of the epithelial layer is however scanty or lacking, and herein we address these issues. Differentiated Caco2 cells have been used as a model of polarized intestinal epithelium. WGA concentration at both the apical and the basolateral side of the epithelium has been quantified using a sensitive ELISA assay (sensitivity threshold 0.84 nM). Trans epithelial electrical resistance (TEER) has been measured to evaluate the integrity of the epithelium upon treatments with WGA. (3)H-Mannitol (182.2 Da) and FITC-dextran (3000 Da) have been used to measure the permeability of the epithelium. Cell viability has been measured by the MTT, by 7-AAD uptake, and Annexin-V binding assays. Up to a concentration of 5.6 microM, approximately 0.1% of intact WGA molecules only could cross the epithelial layer. WGA perturbed the integrity of the epithelium and increased the permeability of the tissue in a dose- and time-dependent manner. WGA did not induce cell death but increased the permeability of individual cells to 7-AAD which is normally not uptaken by viable cells. These data allowed us to define a toxicity threshold for WGA on epithelial cells. WGA suitability as a carrier for oral drugs can therefore be evaluated on a rational basis.

Anti-tumour potential of a gallic acid-containing phenolic fraction from Oenothera biennis.

A phenolic fraction purified form defatted seeds of Oenothera biennis promoted selective apoptosis of human and mouse bone marrow-derived cell lines following first-order kinetics through a caspase-dependent pathway. In non-leukemia tumour cell lines, such as human colon carcinoma CaCo(2) cells and mouse fibrosarcoma WEHI164 cells, this fraction inhibited (3)H-thymidine incorporation but not cell death or cell cycle arrest. Human peripheral blood mononuclear cells showed low sensitivity to treatment. Single bolus injection of the phenolic fraction could delay the growth of established myeloma tumours in syngeneic animals. HPLC and mass spectrometry analysis revealed that the fraction contains gallic acid. However, the biological activity of the fraction differs from the activity of this phenol and hence it should be attributed to other co-purified molecules which remain still unidentified.

Studies on the joint cytotoxicity of Wheat Germ Agglutinin and monensin.

Wheat Germ Agglutinin (WGA) cytotoxicity has been studied using two human leukemia cell lines, Molt3 and K562, and human peripheral blood mononuclear cells (PBMC). In spite of similar binding at the cell surface, WGA was found to promote cell death to a different extent in Molt3, K562 and PBMC and to induce different death events leading to apoptosis in Molt3 and either apoptosis and necrosis in K562 cells and PBMC. In Molt3 but not in K562 cells, WGA cytotoxicity could be potentiated 66-200 fold by 50 nM monensin, a carboxylic ionophore that perturbs the intracellular trafficking of endocytosed molecules. Synergism between the cytotoxic activities of WGA and monensin was demonstrated in Molt3 cells by comparing non toxic, or slightly toxic, doses of WGA and monensin alone or in combination. These results show that the cytotoxic effect of WGA is dependent on internalisation events which may differ among the cell lines used. WGA and monensin can enter the human diet being a component of wheat germ and an antibiotic used for zootechnic reasons in the bioindustry, respectively. These data reveal the synergistic effect between two dietary molecules, otherwise per se toxic at much higher concentrations, with possible implications for human and animal health.

Egg-matrix for large-scale single-step affinity purification of plant lectins with different carbohydrate specificities.

Hen eggs represent an easily available and inexpensive source of glycoproteins expressing a variety of sugars. Egg glycoproteins might therefore be exploited to purify by affinity chromatography carbohydrate-binding proteins (lectins) with different specificities. A method to generate an affinity matrix from hen eggs is described. The matrix was assayed for its ability to purify in a single step biologically active phytohemagglutinin, wheat germ agglutinin, lentil lectin, and peanut agglutinin. Milligrams of purified lectins per gram of matrix was obtained, with the only exception of peanut agglutinin that was not efficiently retained into the affinity column. Hen egg chromatography is a relatively simple, fast, and reproducible method to purify high amount of plant lectins.

Fully reversible procedure for silver staining improves densitometry of complex mixtures of biopolymers resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Due to its high sensitivity, silver staining is a widely popular method for the revelation of biopolymers separated by both native and denaturing electrophoresis. A step-by-step method for the destaining and restaining of overdeveloped/overloaded silver-stained bands is described that is applicable to both proteins and nucleic acids. The procedure significantly improves densitometric analysis of gels that have been silver stained with either commercial kits or solutions made in-house. The method permits reproducible densitometry of silver-stained gels and allows quantification of both main and minor components in complex mixture of molecules resolved on the same gel slab. All steps may be interrupted and are readily reversible, allowing for facile densitometric analyses and photographic recording under optimized conditions. Furthermore, common artifacts such as differential staining of the two gel surfaces, localized uneven yellow-ochre background, and the presence of fold marks and fingerprints can be easily removed.

Quantitative determination of dietary lectin activities by enzyme-linked immunosorbent assay using specific glycoproteins immobilized on microtiter plates.

An immunoenzymatic method for the quantitative determination of dietary lectin activities employing immobilized glycoproteins was studied. Lectins from wheat germ (WGA), peanut (PNA), and jack bean (ConA) were added to microtiter plates coated with ovalbumin or asialofetuin and quantified by enzyme-linked immunosorbent assay (ELISA) with lectin-specific antibodies. ELISA responses for lectin activity were dose-dependent in the concentration range 30-1000 ng/mL for WGA and 80-1000 ng/mL for both PNA and ConA. Inhibition assays carried out with different saccharides confirmed that the binding of lectins to immobilized glycoproteins was specific. The proposed method is specific and sensitive, allowing the quantitative determination of lectin activities on raw samples by simple dilution of the extracts. Examples of application to wheat germ and roasted peanut extracts are reported.

Effects of dietary wheat germ deprivation on the immune system in Wistar rats: a pilot study.

Bioactive molecules that can gain access to body tissues through the gastrointestinal tract may interact with immune regulatory circuits and effector functions. Among these are plant lectins, such as wheat germ (WG) agglutinin, which constitute common components of the human diet and target the immune system on a daily basis. Dietary bioactive molecules might be considered as immunomodulatory signals. To investigate the possible effects on the immune system of the long-term absence of such signals, two groups of rats were fed on a diet containing or deprived of WG. The WG-deprived diet induced a state of functional unresponsiveness in lymphocytes from primary and secondary lymphoid organs, as evaluated by in vitro stimulation with T cell mitogen phytohemoagglutinin (PHA) and B cell mitogen lypopolysaccarides (LPS). The unresponsive state of the immune cells could be reversed by injection of antigen emulsified in oil with inactivated mycobacteria (complete Freund's adjuvant, CFA) Dietary signals can thus interact with the immune system possibly influencing its shaping during ontogenesis.