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Institut für Ernährungsmedizin

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Browsing Institut für Ernährungsmedizin by Classification "630"

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    High-resolution proteomics reveals differences in the proteome of spelt and bread wheat flour representing targets for research on wheat sensitivities
    (2020) Afzal, Muhammad; Pfannstiel, Jens; Zimmermann, Julia; Bischoff, Stephan C.; Würschum, Tobias; Longin, C. Friedrich H.
    Wheat consumption can trigger celiac disease, allergic reactions and non-celiac wheat sensitivity (NCWS) in humans. Some people with NCWS symptoms claim a better tolerability of spelt compared to bread wheat products. We therefore investigated potential differences in the proteomes of spelt and bread wheat flour using nano LC–ESI–MS/MS on a set of 15 representative varieties for each of the two species. Based on the bread wheat reference, we detected 3,050 proteins in total and for most of them the expression was mainly affected by the environment. By contrast, 274 and 409 proteins in spelt and bread wheat, respectively, had a heritability ≥ 0.4 highlighting the potential to influence their expression level by varietal choice. We found 84 and 193 unique proteins for spelt and bread wheat, respectively, and 396 joint proteins, which expression differed significantly (p ≤ 0.05) when comparing both species. Thus, about one third of proteins differed significantly between spelt and bread wheat. Of them, we identified 81 proteins with high heritability, which therefore might be interesting candidates for future research on wheat hypersensitivities.
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    Rolle von SNARE-Proteinen bei der Mediatorfreisetzung humaner Mastzellen
    (2010) Frank, Simon P. C.; Lorentz, Axel
    Mast cells are involved in a variety of physiological and pathophysiological processes in the body. They posses a wide range of stored and de novo synthesized inflammatory mediators. Therefore they play an important role not only in allergic diseases - as it was originally assumed. The aim of the present work is to acquire a more detailed insight into the processes of mediator release of human intestinal tissue mast cells. To enable the release of histamine, proteases, cytokines, chemokines and other substances, it is necessary that secretory vesicles fuse with the plasma membrane. In this process so-called SNARE proteins are essentially involved. They are generally divided into vesicle membrane (v) - and target membrane (t) -SNAREs. The present work could show that a large number of SNAREs are expressed in human intestinal mast cells. The t-SNAREs SNAP-23, syntaxin-2, -3, -4, -6, Vti1b, and the v SNAREs VAMP-2, -3, -5, -7, -8 could be clearly detected, while SNAP-25, Syntaxin-1b and VAMP-4 were expressed only slightly. A subsequent visualization of these proteins by coupling with fluorescence-marked antibodies showed their localization in the cell. There is a clear arrangement of the t-SNAREs SNAP-23, syntaxin-3, -4 and -6 at the plasma membrane, whereas syntaxin-2, Vti1b, VAMP-3, -7 and -8 are distributed in the cytoplasm in resting cells. However, after activation of the cells for 15 minutes, this arrangement changed for VAMP-7 and -8 which moved in the direction of the plasma membrane, as well as Vti1b, which shifted after a prolonged activation phase of 2 hours also towards the cell edge. In activated mast cells colocalization of SNAP-23, syntaxin-3 and -4, VAMP-7 and -8, as well as Vti1b could be demonstrated. Using co-immunoprecipitation complex formation of SNAP-23 with syntaxin-4, VAMP-7 and -8 was confirmed. For the elucidation of the functional significance of individual SNARE proteins, respective SNAREs have been turned off. After transfection of human intestinal mast cells by using different transfection reagents failed, transfection of mast cells with siRNA by electroporation succeeded. However, it turned out that the electrically treated cells lost their ability to be stimulated. In contrast, blocking of the SNARE proteins achieved by using inhibitory antibodies was successful with no deterioration of cells. Interestingly enough it was revealed that a variety of SNARE proteins participate in degranulation processes in mast cells. The release of the prestored beta-hexosaminidase was significantly reduced by antibodies against SNAP-23, syntaxin-3, -4 and -6, VAMP-7 and -8 and Vti1b after a stimulation time of one hour. It could be noted that VAMP-7 seemed to play no role anymore in the case of a prolonged activation time of six hours. The same SNAREs were also responsible for the release of LTC4. The liberation of the cytokines and chemokines IL-5, IL-6, IL-8, MCP-1, MIP-1-alpha and MIP-1-beta showed an inconsistent involvement of SNARE proteins. However, inhibition of SNAP-23, syntaxin-3 and Vti1b resulted in almost complete blockage of the release of all measured cytokines. Furthermore, antibodies against syntaxin-6 caused a significant reduction in the secretion of IL-5, IL-8 and MCP-1 and a trend towards a reduction of IL 6, MIP-1-alpha and MIP-1-beta. To different degrees both of the v-SNAREs VAMP-7 and -8 seemed to account individually for the release of different cytokines. Since release of the chemokine MIP-1-beta was not affected neither by the blockade of VAMP-7 nor by VAMP-8, another non-studied member of the VAMP family could be involved. Inhibition of syntaxin-4 led only for IL-8 to a marked and significant reduction in the release. In summary, it was shown that the release of different mediators in human intestinal mast cells is catalyzed by various different SNAREs and thus the existence of specific carriers and a targeted transport of mediators are very likely. One explanation for the involvement of a number of SNARE proteins in secretion processes may be that while some SNAREs are responsible for building up intracellular structures which are necessary for the compound exocytosis, some others are in charge of the docking of vesicles at the plasma membrane. A detailed understanding of the mechanisms and the involvement of specific SNARE proteins in the release of various mediators of human mast cells is the basis for new forms of treatments for allergies and asthma. New therapeutic approaches might aim through the temporary deactivation of certain SNARE proteins to achieve an intended reduction of mediator release.