Genetic and antigenic characterization of the surface lipoprotein P48 of Mycoplasma bovis

The presence of a membrane lipoprotein homologous to the P48 of Mycoplasma agalactiae was investigated in different Mycoplasma bovis isolates selected by geographical locations and biological properties. Its potential as a diagnostic tool was also discussed. The presence of a specific signal observed in all M. bovis field isolates probed with a rabbit antiserum raised against the M. agalactiae recombinant P48 demonstrated that this protein is structurally and antigenically conserved within the M. bovis cluster. No signal was detected when testing six different mycoplasma species found in cattle. The p48 gene was identified by PCR approach and partially sequenced. Full length gene sequence was obtained by direct bacterial chromosome sequencing. Five UGAs were selectively mutated into UGG and the full length mutated gene, lacking the signal peptide, was cloned and expressed in Escherichia coli. The purified recombinant antigen (r-P48) was evaluated as a potential marker of infection using a panel of 86 well-characterized sera from experimentally and naturally infected cattle. Specific IgM antibodies were detected within 6-9 days after experimental infection followed by an IgG response lasting from the third/fourth week after contact. Although antibody titers were well below those observed in sheep or goats infected with M. agalactiae, results suggest that M. bovis r-P48 can be used as a specific marker of infection.     

One- and two-dimensional electrophoretic identification of IgE-binding polypeptides of Lupinus albus and other legume seeds

The prevalence of food allergies in the world population requires integrated approaches to identify new potential allergens, especially those of plant origin. The aim of this work was the allergen in vitro analysis of Lupinus albus seed proteome, a promising food protein source, and the assessment of IgE cross-reactivities with other more diffused legume species. A combination of one- and two-dimensional gel electrophoresis and immunoblotting analyses with specific IgGs for band identification and lupin-sensitized patients' circulating IgEs for allergenicity studies has been used. Two lupin proteins, namely, conglutin gamma and 11S globulin basic subunits, strongly reacted with all patients' sera. Also, cross-reactivities with the homologous polypeptides of other legume species were observed. Otherwise, no reaction at all was detected with a 2S-type lupin protein. This global electrophoretic approach has allowed the identification of a new potential lupin allergen and confirmed the cross-reactivity among the legume 11S globulin basic subunits.     

Bioaccessibility and antioxidant activity stability of phenolic compounds from extra-virgin olive oils during in vitro digestion

The impact of an in vitro procedure that mimics the physiochemical changes occurring in gastric and small intestinal digestion on the bioaccessibility and antioxidant activity of phenols from 10 extra-virgin olive oil samples was assessed. Extra-virgin olive oil phenols were totally extracted in the aqueous phase, which reproduces gastric fluids during the digestion procedure. A linear bioaccessibility model, based on tyrosol behavior in model oil samples, was used to estimate the bioaccessibility index (BI%) of extra-virgin olive oil phenols. The BI% varied amongst samples from a maximum of 90% to a minimum of 37%, thus indicating that only a fraction of phenols can be considered bioaccessible. The specific antioxidant activity of olive oil phenols proved to be negatively affected by the digestion procedure. By computing a principal component analysis, it was possible to show that differences in the potential bioactive effect of extra-virgin olive oil samples were related to different phenolic profiles.     

In vitro activity of the essential oil of Cinnamomum zeylanicum and eugenol in peroxynitrite-induced oxidative processes

The essential oil obtained from the bark of Cinnamomum zeylanicum Blume (Lauraceae) and three of its main components, eugenol, (E)-cinnamaldehyde, and linalool (representing 82.5% of the total composition), were tested in two in vitro models of peroxynitrite-induced nitration and lipid peroxidation. The essential oil and eugenol showed very powerful activities, decreasing 3-nitrotyrosine formation with IC50 values of 18.4 microg/mL and 46.7 microM, respectively (reference compound, ascorbic acid, 71.3 microg/mL and 405.0 microM) and also inhibiting the peroxynitrite-induced lipid peroxidation showing an IC50 of 2.0 microg/mL and 13.1 microM, respectively, against 59.0 microg/mL (235.5 microM) of the reference compound Trolox. On the contrary, (E)-cinnamaldehyde and linalool were completely inactive.     

Two-dimensional electrophoresis and western-blotting analyses with anti Ara h 3 basic subunit IgG evidence the cross-reacting polypeptides of Arachis hypogaea, Glycine max, and Lupinus albus seed proteomes

The allergenicity of seed storage proteins, the major components of edible legume seeds, may cause serious reactions in both children and adult population. Updated methodologies for evaluation of the activity of these proteins are needed. In this paper we used two-dimensional (2D) electrophoretic techniques to investigate the immuno-cross-reactivities of anti Ara h 3 basic subunit IgG to the seed proteomes of three legume species, namely, peanut, soybean, and lupin. The seed proteins, extracted with two different procedures, were separated by 2D electrophoresis, and the electrophoretic maps were analyzed by Western blot. In peanut proteome the antibodies strongly reacted with the 23 kDa polypeptides, corresponding to Ara h 3 basic isoforms, the antigen they were raised to, and three unidentified acidic polypeptides near 45 kDa. Remarkable cross-reactivities with lupin and soybean Ara h 3 homologous polypeptides and nonrelated proteins, namely, lupin conglutin gamma and soybean Bg7S, were detected. Therefore, these proteins may be regarded as new putative allergens. The present findings show the potentiality of 2D electrophoresis in the identification of food allergens and open the way to the traceability of the new cross-reacting proteins in the food chain.     

Phenolic constituents and antioxidant properties of Xanthosoma violaceum leaves

An extract of Xanthosoma violaceum leaves was subjected to a polyphenol profile determination, including total polyphenols, and antioxidant activity evaluation. Analysis of the extract resulted in the isolation of a new flavone C-glycoside, apigenin 6-C-beta-D-glucopyranosyl-8-C-beta-D-apiofuranoside (1), as well as known flavone C-glycosides, including vitexin (2), isovitexin (3), isovitexin 4'-O-rhamnopyranoside (4), apigenin 6-C-[beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranoside] (5), and apigenin 6,8-diC-beta-D-glucopyranoside (6). The antioxidant activity of the extract was assessed by means of two different in vitro tests: bleaching of the stable 1,1-diphenyl-2-picrylhydrazyl radical (DPPH test) and peroxidation induced by the water-soluble radical initiator 2,2'-azobis(2-amidinopropane) hydrochloride, on mixed dipalmitoylphosphatidylcholine/linoleic acid unilamellar vesicles (LP-LUV test). In both tests used, the extract and a fraction II showed a significant antioxidant/free-radical scavenging effect (fraction II, EC(50) = 11.6 microg/mL) in comparison to alpha-tocopherol (EC(50) = 10.1 microg/mL).     

IgE-Mediated Cross-Reactivity among Leguminous Seed Proteins in Peanut Allergic Children

The immunological cross-reactivity among major protein- and oil-crops, including lupin, lentil, pea, peanut, kidney bean and soybean, has been studied by a combination of in vitro and in vivo experimental approaches: SDS-PAGE separations of legume protein extracts and immuno-blot revelations with 12 peanut-sensitive subjects’ sera, Immuno-CAP and Skin Prick tests on the same subjects. The immuno-blotting data showed a wide range of IgE-binding responses both displayed by one subject towards different plant extracts and among subjects. Differences were both quantitative and qualitative. The prevalent responses of most subjects’ sera were seen with peanut polypeptides, as expected, as well as with various polypeptides of the other legumes, the most recurrent of which were the basic subunits of the 11S globulins. The distribution of in vivo responses generally paralleled those obtained by in vitro approaches with strong responses elicited by peanut, lentil and pea protein extracts, especially by most sensitive subjects, thus providing a consistent overall set of results. In this work, the comparison of various approaches has allowed us to get an overall broad picture of the immunological cross-reactivities among proteins of widely used different seed species and to hypothesize the role of most conserved specific polypeptides.     

Properties of lupine proteins relevant to their nutritional performance

Composition, solubility, bound sugar content and quality, subunit composition and structure of the storage proteins of seeds ofLupinus albus are discussed. Aminoacid composition is also given for each protein, the various protein classes and the whole flour. These data allow for the characterization of the molecules of the various storage proteins and their contributions to the nutritional properties of the seed.In vitro digestibility (by mammalian endopeptidases) is reported and is less than for animal proteins. Possible causes, at the molecular level, for this behaviour and possible means to overcome these effects are examined. The relationships between the above data are considered in view of the nutritional performance of the proteins and of the genetical, agronomic and technological approaches most suited to improve the nutritional quality of lupine seeds as a protein source.