A high molecular arabinogalactan from Ribes nigrum L.: influence on cell physiology of human skin fibroblasts and keratinocytes and internalization into cells via endosomal transport |
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Authors: | Janina Zippel Dirk Pappai |
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Institution: | a University of Münster, Institute for Pharmaceutical Biology and Phytochemistry (IPBP), Hittorfstraße 56, D-48149 Münster, Germany b University of Münster, Dermatology Department, Von-Esmarch-Str. 58 D-48149 Münster, Germany |
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Abstract: | An arabinogalactan protein (F2) was isolated in 1.5% yield from the seeds of Ribes nigrum L. (Grossulariaceae) by aqueous extraction and a one-step anion exchange chromatography on DEAE-Sephacel with 24% galactose, 43% arabinose, and 20% xylose as main carbohydrate residues. Methylation analysis revealed the presence of a 1,3-/1,3,6-galactose backbone, side chains from arabinose in different linkages, and terminal xylose residues. The polysaccharide which turned out to be an arabinogalactan protein had a molecular weight of >106 Da and deaggregated under chaotropic conditions. The cellular dehydrogenase activities (MTT and WST-1 tests) of human skin cells (fibroblasts, keratinocytes) as well as the proliferation rate of keratinocytes (BrdU incorporation ELISA) were significantly stimulated by the polymer at 10 and 100 μg/mL. F2 had no influence on differentiation status of keratinocytes and did not exhibit any cytotoxic potential (LDH test). The biological activity of F2 was not dependent on the high molecular weight. Influence of the polysaccharide on the gene expression of specific growth factors, growth factor receptors, signal proteins and marker proteins for skin cell proliferation, and differentiation by RT-PCR could not be shown. Gene array investigations indicated an increased expression of various genes encoding for catabolic enzymes, DNA repair, extracellular matrix proteins, and signal transduction factors. Removal of terminal arabinose residues by α-l-arabinofuranosidase did not influence the activity toward skin cells, while the treatment with β-d-galactosidase yielded an inactive polysaccharide. The FITC-labeled polysaccharide was incorporated in a time-dependent manner into human fibroblasts (laser scanning microscopy) via endosomal transport. This internalization of the polysaccharide was inhibited by Cytochalasin B. |
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Keywords: | AEC anion exchange chromatography BrdU bromodeoxyuridine FCS fetal calf serum FITC fluoresceinisothiocyanate GPC gel permeation chromatography HaCaT human adult low calcium high temperature keratinocyte cell line HPAEC-PAD high pressure anion exchange chromatography with pulsed-amperometric detection LDH lactate dehydrogenase pNHDFs primary normal human dermal fibroblasts pNHEK primary normal human epidermal keratinocytes RPS raw polysaccharide RT-PCR real-time polymerase chain reaction |
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