δdi 4s

Concentrations of urinary and plasma GAGs and KS were measured by tandem mass spectroscopy as described elsewhere (16 (link)). Briefly, the chromatographic system consists of an HP1100 system (Agilent Technologies) and a Hypercarb column (Thermo Electron). The API-4000 mass spectrometer (Applied Biosystems) was equipped with a turbo ionspray ion source. Disaccharides of keratan, heparan, dermatan sulfate were used as standards [Galß1,4GlcNAc(6S), ΔDiHS-0S, ΔDiHS-NS, ΔDi-4S, ΔDi-6S (Seikagaku). Chondrosine was used as internal standard (Glycosyn). Urine or plasma was centrifuged, and the supernatants were digested overnight with 1 mU of chondroitinase b, 1mU heparitinase, and 1mU keratanase II (Seikagaku). Recovered samples were analyzed by the LC-MS/MS system and normalized by creatinine concentration in urine. Total GAGs refer to total glycosaminoglycans or the additive sum of all the GAGs measured (HS, DS and KS). Disaccharide GAG concentrations were calculated by Analyst 1.5.1 software (AB SCIEX). Each sample was measured in triplicate with three injections for each sample.

Dried fins (without skin and cartilage) of Isurus oxyrinchus and Prionace glauca and raw fin (without skin) of Prionace glauca were kindly provided by Mrs. T. Mano and T. Wada (Nihon Pharmaceutical Co. Ltd.). Deep-sea elasmobranchs (sharks and rays) were collected by H. Tejima through gill net fisheries at the mouth of Tokyo Bay off Kanaya, Chiba, Japan (35.17°N, 139.79°E; 200–300 m depths). Fins from those elasmobranchs were kindly provided by H. Tejima, before being processed as food materials. Actinase E was from Kaken Pharmaceutical Co., Ltd., Tokyo, Japan. Chondroitinase ABC (ChaseABC) from Proteus vulgaris, chondroitinase ACII (ChaseACII) from Arthrobacter aurescens, unsaturated disaccharides (ΔDi-0S, ΔDi-4S, ΔDi-6S, ΔDiUA-2S, ΔDi-diS_E, ΔDi-diS_B, ΔDi-diS_D, ΔDi-TriS) and CS-E were purchased from Seikagaku Corp., Tokyo, Japan. Dialysis membrane for desalting was from Spectrum Medical (Los Angelis, CA, USA).

A CFU-GM assay was performed as described previously [17 (link)]. A colony-forming cell assay for rat cells (MethoCult GFR3774, StemCell Technology) is recommended for the detection and quantification of rat CFU-GM progenitors in bone marrow samples. Briefly, 0.3 mL of cell suspension (2,500 cells/mL for SCE-UCBCs, 1 x 10⁵ cells/mL for UCB-MNCs, and 1 x 10⁶ cells/mL for adult MNCs) was added to 3 mL of MethoCult. Using a 2.5 mL syringe, 1.1 mL of the MethoCult mixture was dispensed into each of two wells of a 6-well plate and cultured. After 10 days, the cultures were photographed using a stereo microscope (M165FC, Leica), and the number of colonies in each of the two wells was counted using a Photoshop counting tool (Adobe) and then averaged. The percentage of colony-forming cells was calculated as the number of colonies divided by the number of seeded cells. The experiments were repeated 3 times (n = 3).
UCB-MNCs were cultured with PBS, Chase ABC (0.2 U/mL, Seikagaku Corporation), CS-A (from whale cartilage, 80% of which consists of ΔDi-4S (A-unit), 100 μg/mL, Seikagaku Corporation), or CS-E (from squid cartilage, 60–65% of which consists of ΔDi-di4, 6S (E-unit), 100 μg/mL, Seikagaku Corporation) (Fig 5). Data are shown as the percentage of the control (PBS). The experiments were repeated 4 times and statistically analyzed.