Javier Varela

Peroxisomal Copper,Zinc Superoxide Dismutase (Characterization of the Isoenzyme from Watermelon Cotyledons)

The biochemical and immunochemical characterization of a superoxide dismutase (SOD, EC 1.15.1.1) from peroxisomal origin has been carried out. The enzyme is a Cu,Zn-containing SOD (CuZn-SOD) located in the matrix of peroxisomes from watermelon (Citrullus vulgaris Schrad.) cotyledons (L.M. Sandalio and L.A. del Rio [1988] Plant Physiol 88: 1215–1218). The amino acid composition of the enzyme was determined. Analysis by reversed-phase high-performance liquid chromatography of the peroxisomal CuZn-SOD incubated with 6 M guanidine-HCI indicated that this enzyme contained a noncovalently bound chromophore group that was responsible for the absorbance peak of the native enzyme at 260 nm. The amino acid sequence of the peroxisomal CuZn-SOD was determined by Edman degradation. Comparison of its sequence with those reported for other plant SODs revealed homologies of about 70% with cytosolic CuZn-SODs and of 90% with chloroplastic CuZn-SODs. The peroxisomal SOD has a high thermal stability and resistance to inactivation by hydrogen peroxide. A polyclonal antibody was raised against peroxisomal CuZn-SOD, and by western blotting the antibody cross-reacted with plant CuZn-SODs but did not recognize either plant Mn-SOD or bacterial Fe-SOD. The antiSOD-immunoglobulin G showed a weak cross-reaction with bovine erythrocytes and liver CuZn-SODs, and also with cell-free extracts from trout liver. The possible function of this CuZn-SOD in the oxidative metabolism of peroxisomes is discussed.

Vicilin and convicilin are potential major allergens from pea

Background Allergic reactions to pea (Pisum sativum) ingestion are frequently associated with lentil allergy in the Spanish population. Vicilin have been described as a major lentil allergen.

Immunoglobulin E recognition patterns to purified Kiwifruit (Actinidinia deliciosa) allergens in patients sensitized to Kiwi with different clinical symptoms

Background Green kiwifruit allergy is on the rise. However, no surveys testing purified major kiwi allergens have been carried out in a large population, including both kiwi‐sensitized [skin prick test (SPT)‐positive] and truly kiwi‐allergic patients.

High-level synthesis in Escherichia coli of shortened and full-length human acidic fibroblast growth factor and purification in a form stable in aqueous solutions

A highly efficient expression for human acidic fibroblast growth factor (aFGF) has been assembled to direct the synthesis of both shortened and native full-length aFGF. The full-length aFGF-154 form of the protein had not been produced before in Escherichia coli by genetic engineering, and is obtained with its initiator methionine removed. The high production of the aFGF allows one to circumvent the use of reversed-phase chromatography (RPC) during the purification procedure. Here, it is shown that RPC, routinely used to obtain pure preparations of recombinant aFGF, modifies its chemical and physical properties in an unfavorable manner.

Recombinant lipid transfer protein Tri a 14: a novel heat and proteolytic resistant tool for the diagnosis of baker's asthma

Background Bakers asthma is an important occupational allergic disease. Wheat lipid transfer protein (LTP) Tri a 14 is a major allergen associated with wheat allergy. No panel of wheat recombinant allergens for component‐resolved diagnosis of bakers asthma is currently available.

Transport of Pru p 3 across gastrointestinal epithelium – an essential step towards the induction of food allergy?

Since intestinal absorption of food protein can trigger an allergic reaction, the effect of plant food allergen on intestinal epithelial cell permeability and its ability to cross the epithelial monolayer was evaluated.

Allergy to kiwi in patients with baker's asthma: identification of potential cross-reactive allergens

BACKGROUND Bakers asthma is a frequent IgE-mediated occupational disorder mainly provoked by inhalation of cereal flour. Allergy to kiwifruit has being increasingly reported in the past few years. No association between both allergic disorders has been described so far. METHODS Twenty patients with occupational asthma caused by wheat flour inhalation were studied. Kiwi allergens Act d 1 and Act d 2 were purified by cation-exchange chromatography. Wheat, rye, and kiwi extracts, purified kiwi allergens, and model plant glycoproteins were analyzed by IgE immunodetection, enzyme-linked immunosorbent assay (ELISA), and inhibition ELISAs. RESULTS Kiwifruit ingestion elicited oral allergy syndrome in 7 of the 20 patients (35%) with bakers asthma. Positive specific IgE and skin prick test responses to this fruit were found in all these kiwi allergic patients, and IgE to Act d 1 and Act d 2 was detected in 57% and 43%, respectively, of the corresponding sera. Actinidin Act d 1 and bromelain (harboring cross-reactive carbohydrate determinants) reached above 50% inhibition of the IgE binding to wheat and/or kiwi extracts. CONCLUSIONS A potential association between respiratory allergy to cereal flour and allergy to kiwifruit has been disclosed. Cross-reactive carbohydrate determinants and thiol-proteaseshomologous to Act d 1 are responsible for wheat-kiwi crossreactivity in some patients.

Allergenic Characterization of New Mutant Forms of Pru p 3 as New Immunotherapy Vaccines

Nowadays, treatment of food allergy only considered the avoidance of the specific food. However, the possibility of cross-reactivity makes this practice not very effective. Immunotherapy may exhibit as a good alternative to food allergy treatment. The use of hypoallergenic molecules with reduced IgE binding capacity but with ability to stimulate the immune system is a promising tool which could be developed for immunotherapy. In this study, three mutants of Pru p 3, the principal allergen of peach, were produced based on the described mimotope and T cell epitopes, by changing the specific residues to alanine, named as Pru p 3.01, Pru p 3.02, and Pru p 3.03. Pru p 3.01 showed very similar allergenic activity as the wild type by in vitro assays. However, Pru p 3.02 and Pru p 3.03 presented reduced IgE binding with respect to the native form, by in vitro, ex vivo, and in vivo assays. In addition, Pru p 3.03 had affected the IgG4 binding capacity and presented a random circular dichroism, which was reflected in the nonrecognition by specific antibodies anti-Pru p 3. Nevertheless, both Pru p 3.02 and Pru p 3.03 maintained the binding to IgG1 and their ability to activate T lymphocytes. Thus, Pru p 3.02 and Pru p 3.03 could be good candidates for potential immunotherapy in peach-allergic patients.

Oxygen-Linked Electron Transfer and Energy Conversion in Rhodospirillum Rubrum

Pigmented Rbodospirillum rubrum cells from dark chemotrophic cultures contain several pathways for the transfer of electrons from reduced substrates to O2. In order of decreasing H+ -translocating efficiency, they are: (i) a cytochrome (oxidase) pathway that is inhibited by low concentrations of KCN and by inhibitors of the cytochrome b.ci complex, but not by CO; (ii) a CO sensitive or alternative (oxidase) pathway that is partly blocked by inhibitors of the cytochrome b.c 1 complex; and (iii) a third pathway that operates in the presence of CO plus antimycin A and that is absent in the presence of CO plus myxothiazol and in a mutant which lacks rhodoquinone. In addition, a significant fraction of the O2 uptake activity remains when H+ translocation is completely blocked by inhibitors of electron transfer or mutations. Since the rate of respiratory electron transfer appears to be limited at the substrate level, it is difficult to make a direct estimation of the contribution of each pathway to the final rate of respiration. However, from the relative energy-transducing efficiency of the cytochrome and the CO sensitive pathways (as measured by the H+/O ratios in O2 pulses) and the final cell yields of C-limited cultures of wild type and cytochrome-oxidase deficient strains, it seems that the contribution of the cytochrome pathway to the energy-conserving O2 uptake of pigmented chemotrophic R. rubrum, growing on malic and glutamic acids as carbon sources, is close to 80%.