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The present study investigated immune stimulatory effects of Cladosiphon okamuranus-derived fucoidan to activate murine macrophage-like cell line RAW264, and the functional relationship with zymosan, a Saccharomyces cerevisiae-derived β-glucan. The production of nitric oxide (NO) and tumor necrosis factor-α (TNF-α) in RAW264 cells were remarkably enhanced in the presence of 10 μg/mL fucoidan, and the stimulatory effects of fucoidan were maximally augmented in combinational treatment with 500 ng/mL zymosan, whereas any TLR ligands had no those effects. Confocal microscopic analyses suggested that fucoidan bound on plasma membrane, and it was estimated that some cell surface molecules acted as receptor for fucoidan because cytochalasin D, an inhibitor of phagocytosis, did not affect the immune enhancing activities, whereas methyl-β-cyclodextrin (MβCD), a general agent for disruption of lipid rafts, diminished that. Furthermore, it was revealed that the additive effects of zymosan on the immune activation with fucoidan was thought to be mediated by dectin-1 based on the results with dectin-1-knockdown RAW264 cells. All of results suggested that fucoidan and some kinds of β-glucan would cooperatively reinforce the activity of innate immune cells via interactive receptor crosstalk., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The liquid chromatography-multiple reaction monitoring-tandem mass spectrometry (LC-MRM-MS/MS) method using (13)C stable isotope-labeled dipeptides was newly developed to simultaneously determine the absorption of three antihypertensive peptides (Val-Tyr, Met-Tyr, and Leu-Tyr) into blood of spontaneously hypertensive rats in one run-in assay. After extracting (13)C-labeled peptides in blood sample with a C(18) cartridge, the extract was applied to a (13)C monoisotopic transition LC-MRM-MS/MS system with D-Val-Tyr included as internal standard. An excellent separation of each dipeptide in LC was achieved at the elution condition of 5-100% methanol in 0.1% formic acid at a flow rate of 0.25 ml/min. The (13)C-labeled peptides ionized by electron spray were detected in the positive ion mode within 15 min. The established method showed high reproducibility with less than 10% coefficient of variation as well as high accuracy of more than 85%. After the administration of a mixture containing the three (13)C-labeled dipeptides to rats at each dose of 30 mg/kg, we could successfully determine the intact absorption of each (13)C-labeled peptide with the maximal absorption amount of 1.1 ng/ml plasma for Val-Tyr by the proposed LC-MRM-MS/MS method., (Copyright © 2011 Elsevier Inc. All rights reserved.)