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      What Are the Proteolytic Enzymes of Honey and What They Do Tell Us? A Fingerprint Analysis by 2-D Zymography of Unifloral Honeys

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          Abstract

          Honey is a sweet and healthy food produced by honeybees ( Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.

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          Most cited references14

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          Royalactin induces queen differentiation in honeybees.

          The honeybee (Apis mellifera) forms two female castes: the queen and the worker. This dimorphism depends not on genetic differences, but on ingestion of royal jelly, although the mechanism through which royal jelly regulates caste differentiation has long remained unknown. Here I show that a 57-kDa protein in royal jelly, previously designated as royalactin, induces the differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybees. Surprisingly, it also showed similar effects in the fruitfly (Drosophila melanogaster). Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase, which was involved in the decreased developmental time, and increased the titre of juvenile hormone, an essential hormone for ovary development. Knockdown of epidermal growth factor receptor (Egfr) expression in the fat body of honeybees and fruitflies resulted in a defect of all phenotypes induced by royalactin, showing that Egfr mediates these actions. These findings indicate that a specific factor in royal jelly, royalactin, drives queen development through an Egfr-mediated signalling pathway.
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            Major royal jelly protein 3 modulates immune responses in vitro and in vivo.

            We have recently shown that royal jelly has potent antiallergic properties in a mouse model of immediate hypersensitivity. However, it is still unclear which components of royal jelly exhibit antiallergic activity. In this study, we have screened for antiallergic factors in royal jelly based on inhibition of IL-4 production by anti-CD3 stimulated spleen cells derived from OVA/alum-immunized mice. Using a series of column chromatographies, we purified a 70 kDa glycoprotein, major royal jelly protein 3 (MRJP3), that suppresses IL-4 production. In in vitro experiments, MRJP3 suppressed the production of not only IL-4 but also that of IL-2 and IFN-gamma by T cells concomitant with inhibition of proliferation. The MRJP3-mediated suppression of IL-4 production was also evident when lymph node cells from OVA/alum-immunized mice were stimulated with OVA plus antigen presenting cells. We next examined the purified suppressive factor on OVA/alum-induced allergic responses in mice. Interestingly, in spite of the antigenicity of MRJP3 itself as an extraneous foreign protein, intraperitoneal administration of MRJP3 inhibited serum anti-OVA IgE and IgG1 levels in immunized mice. In addition, heat-treated soluble MRJP3 treatment reduced its antigenicity while maintaining its inhibitory effects on antibody responses to OVA. These results indicate that MRJP3 can exhibit potent immunoregulatory effects in vitro and in vivo. Furthermore, considering the intriguing immunomodulatory effects of MRJP3, it may be of clinical significance to design MRJP3-derived antiallergic peptides by identifying the associated polypeptide regions.
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              Multivariate correlation between color and mineral composition of honeys and by their botanical origin.

              The mineral content and color characteristics of 77 honey samples were analyzed. Eighteen minerals were quantified for each honey. Multiple linear regression (MLR) was used to establish equations relating the colorimetric CIELAB coordinates to the mineral data. The results obtained shown that lightness (L) was significantly correlated with S, Ca, Fe, As, Pb, and Cd for the dark honey types (avocado, heather, chestnut, and honeydew). For the light and brown honey types (citrus, rosemary, lavender, eucalyptus, and thyme), C(ab) and b showed the lower correlation with the mineral content of the honeys; their regression functions involve a few independent variables (Mg and Al for b and only Al for C(ab)). Furthermore, by means of application of linear discriminant analysis to the mineral content, it was possible to obtain a model that classifies the honeys by their lightness. The prediction ability of the built model, determined with the test set method, was 85%.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS One
                PLoS ONE
                plos
                plosone
                PLoS ONE
                Public Library of Science (San Francisco, USA )
                1932-6203
                2012
                7 November 2012
                : 7
                : 11
                : e49164
                Affiliations
                [1 ]Department of Biology, Defence and Agro-Forestal Biotechnology and Centre of Bioproteomics, University of Basilicata, Potenza, Italy
                [2 ]Department of Animal Production Sciences, University of Basilicata, Potenza, Italy
                Aligarh Muslim University, India
                Author notes

                Competing Interests: The authors have declared that no competing interests exist.

                Conceived and designed the experiments: RR PR. Performed the experiments: ML TP MCP. Analyzed the data: RR PR. Contributed reagents/materials/analysis tools: AMP GM. Wrote the paper: RR PR. Performed the zymographic and electrophoretic analysis: ML TP MCP. Performed the palynological analysis: AMP GM. Performed MALDI-ToF mass spectrometry and the bioinformatics analysis: RR LM. Final approval of the version to be published: RR PR.

                Article
                PONE-D-12-10507
                10.1371/journal.pone.0049164
                3492327
                23145107
                94ded1cf-ae11-4c06-a916-514139f60ae9
                Copyright @ 2012

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                History
                : 13 April 2012
                : 4 October 2012
                Page count
                Pages: 17
                Funding
                This study was supported by grants from University of Basilicata (Fondo Ateneo, 2008) for the research in food and in part by the project 2010/R/35 of PR on “The molecular basis of nutritional intervention in Multiple Sclerosis”, funded by the Italian Foundation for Multiple Sclerosis (FISM)] for the development of bidimensional zymography (2-DZ). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
                Categories
                Research Article
                Agriculture
                Agricultural Production
                Biology
                Biochemistry
                Enzymes
                Proteins
                Developmental Biology
                Morphogenesis
                Sexual Differentiation
                Cell Differentiation
                Proteomics
                Spectrometric Identification of Proteins
                Zoology
                Entomology

                Uncategorized
                Uncategorized

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