Male C57BL/6 mice weighing 21~23 g were housed under a 12-h light/dark cycle in a temperature-controlled room (22~24℃). Mice were given access to standard chow and water ad libitum.

The tubers of Dioscorea batatas were harvested in May at the Okcheon County in North Chungcheong Province, South Korea, and were stored at 4℃ until used. For isolation of the proteins, whole tubers were used. Glycoproteins were isolated from Dioscorea batatas according to the procedure of previously reported [7,20]. In brief, tubers were peeled and homogenized and subjected to hydrophobic chromatography on a phenyl-Toyopearl 650 M column (Tosoh, Japan) equilibrated with 50 mM Tris-HCl buffer (pH 7.0) containing ammonium sulfate to give 25% saturation. Elution was performed by a decrease of ammonium sulfate concentration from 25 to 0% in 50 mM Tris-HCl buffer (pH 7.0). The fractions yielding absorbance at 280 nm were pooled, dialyzed against distilled water, and lyophilized in a freeze dryer. The lyophilized proteins were subjected to an anion-exchange chromatography on a HiTrap Q HP column (GE Healthcare).

RAW 264.7 cells (ATCC TIB71) were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were then cultured in the presence of 5% CO₂ at 37℃.

RAW 264.7 cells were stimulated with GDB plus FN-γ for 24 h. Culture supernatants were collected and the accumulation of NO₂^- in culture supernatants was as an indicator of NO production in the medium as previously described [21]. The isolated supernatants were mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, and 2% phosphoric acid) and incubated at room temperature for 10 min. Using NaNO₂ to generate a standard curve, nitrite production was measured by an O.D. reading at 550 nm.

Total RNA was isolated with Tri Reagent (Molecular Researh Center, Cincinnati, OH, USA). The forward and reverse primer sequences are as follows: iNOS: 5'-CTG CAG CAC TTG GAT CAG GAA CCT G-3', 5'-GGG AGT AGC CTG TGT GCA CCT GGA A-3'; and β-actin: 5'-TGG AAT CCT GTG GCA TCC ATG AAA C-3', 5'-TAA AAC GCA GCT CAG TAA CAG TCC G-3'. Equal amounts of RNA were reverse transcribed into cDNA with oligo(dT)15 primers. PCR was performed with cDNA and each primer. Samples were heated to 94℃ for 5 min and cycled 30 times at 94℃ for 1 min, 55℃ for 1.5 min, and 94℃ for 1 min, after which an additional extension step at 72℃ for 5 min was included. PCR products were electrophoresed in 8% polyacrylamide gel followed by staining in ethidium bromide. The iNOS and β-actin primers produce amplified products at 311 and 349 bp, respectively.

Whole cell lysates were separated by 10% SDS-PAGE, then electro-transferred to nitrocellulose membranes (Amersham International, Buckinghamshire, UK). The membranes were preincubated for 1 hr at room temperature in Tris-buffered saline, pH 7.6 containing 0.05% Tween-20 and 3% bovine serum albumin. The nitrocellulose membranes were incubated with specific antibodies. Immunoreactive bands were then detected by incubation with conjugates of antirabbit IgG with horseradish peroxidase and enhanced chemiluminescence reagents (Amersham). Antibodies against, p38, phospho-p38 (Thr180/Tyr182), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), JNK, phospho-SAPK/JNK (Thr183/Tyr185), and β-actin were obtained from Cell Signaling Biotechnology, Inc (MA, USA). Goat anti rabbit IgG-HRP antibody was from purchased from Santa Cruz Biotechnology, Inc (CA, USA).

RAW264.7 cells were cultured on slide glass and treated with GDB (25 µg/ml) and IFN-γ (10 ng/ml) for 24 h. Morphological change and iNOS expression were analyzed using immunofluorescence staining. The slide was fixed with 4% paraformaldehyde for 10 min and washed with 0.01 M phosphate buffered saline (PBS, pH 7.4). The sample was blocked with 3% BSA in 1X PBS/0.03% Triton X-100 in a humidified chamber for 1 h at room temperature and incubated with rabbit anti-iNOS (1:200 dilutions in 1X PBS/0.03% Triton X-100) at 4℃ in a humidified chamber overnight. The slide was washed, hybridized with secondary antibodies conjugated with fluorescein isothiocyanate (FITC) for 1 h at room temperature in the dark. The slide was then washed and mounted with GEL/MOUNT solution containing anti-fading agents and was observed by laser scanning confocal microscopy (FV300, Olympus, Japan).

RAW 264.7 cells were grown at 5×10⁵ cells/ml in petri-dish and then treated with GDB for 2 h. Nuclear extracts were prepared as earlier described [22]. Briefly, cells were lysed with hypotonic buffer (10 mM HEPES, 1.5 mM MgCl₂, pH 7.5) and nuclei were pelleted by centrifugation at 3,000×g for 5 min. Nuclear lysis was performed using a hypertonic buffer (30 mM HEPES, 1.5 mM MgCl₂, 450 mM KCl, 0.3 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 µg/ml of aprotinin, and 1 µg/ml of leupeptin). Following lysis, the samples were centrifuged at 14,500×g for 15 min, and supernatant was retained for use in the DNA binding assay. The double-stranded oligonucleotides were end-labeled with [γ-³²P]-ATP. Nuclear extracts (5 µg) were incubated with poly (dI-dC) and the [³²P]-labeled DNA probe in binding buffer (100 mM KCl, 30 mM HEPES, 1.5 mM MgCl₂, 0.3 mM EDTA, 10% glycerol, 1 mM DTT, 1 mM PMSF, 1 µg/ml of aprotinin, and 1 µg/ml of leupeptin) for 10 min. DNA binding activity was separated from free probe using a 5% polyacrylamide gel in 0.5X TBE buffer. Following electrophoresis, the gel was dried and subjected to autoradiography.

The mean±S.D. was determined for each treatment group in a given experiment. When significant differences occurred, treatment groups were compared to the vehicle control using a Dunnett's two-tailed t test [23].

The glycoproteins (GDB) isolated from Dioscorea batatas were analyzed by SDS-PAGE and produced a single band at 30 kD (Fig. 1A). To investigate macrophage activation by GDB, we treated RAW264.7 macrophage cells with GDB (50 µg/ml) in the presence of IFN-γ (10 ng/ml) and analyzed iNOS expression by immunofluorescence staining (Fig. 1B). GDB plus IFN-γ strongly stimulated macrophages to produce iNOS and induced morphological changes. To analyze the effect of GDB plus IFN-γ on iNOS gene expression further, we performed Western blot analysis and RT-PCR. GDB plus IFN-γ significantly induced iNOS gene expression, whereas GDB plus IFN-γ alone did not (Fig. 1C, D).

To investigate macrophage activation by GDB, we analyzed the effects of GDB on nitrite levels, which is produced from NO in RAW264.7 cells. RAW264.7 cells were treated with IFN-γ (1, 2.5, 5, and 10 ng/ml) in the presence or absence of GDB (25 µg/ml) for 24 h. Supernatants were then isolated and analyzed for nitrite generation. GDB (25 µg/ml) plus IFN-γ (1, 2.5, 5, and 10 ng/ml) significantly induced nitrite generation in a dose-dependent manner, whereas IFN-γ alone did not (Fig. 2A). LPS (200 ng/ml) was used as a positive control. The synergistic induction of nitrite by GDB (1, 10, 25, and 50 µg/ml) plus IFN-γ (10 ng/ml) was further confirmed in RAW264.7 cells (Fig. 2B). GDB (1, 10, 25, and 50 µg/ml) alone did not increase the nitrite generation.

Most cellular responses to extracellular stimuli are mediated by kinase and phosphatase cascades. The MAPKs are one of the most important kinase families in inflammatory cells, and its members have been strongly conserved through evolution, suggesting that they play vital roles in intracellular signaling. Thus, since MAPKs play important roles in macrophage activation, we analyzed the effects of GDB on MAPK activity by immunoblotting. When cells were treated with GDB (1, 10, and 25 µg/ml) for 30 min, we found that phosphorylation of ERK1/2, and especially of SAPK/JNK, was markedly induced (Fig. 3A). ERK1/2 and SAPK/JNK phosphorylations were induced by IFN-γ (10 ng/ml), and the phosphorylation was not further activated by GDB. The phosphorylation of p38 kinase was not strongly induced by GDB or IFN-γ. However, interestingly cotreatment of RAW264.7 cells with GDB and IFN-γ strongly induced p38 phosphorylation (Fig. 3). This synergistic induction of p38 by GDB and IFN-γ corresponds well with the activation of iNOS gene expression. These results suggest that GDB activates macrophages by activating signal transduction pathways, such as, p38, ERK1/2, and SAPK/JNK.

We further investigated whether the p38 kinase pathway is involved in GDB and IFN-γ-induced nitrite generation. We specifically blocked the p38 kinase pathway and then monitored nitrite production when RAW 264.7 cells were challenged with GDB plus IFN-γ. SB203580, a bicyclic imidazole inhibitor of p38, inhibited GDB plus IFN-γ-induced nitrite production (Fig. 4A). However, PD98059, a specific inhibitor of MEK-1, did not. LPS-induced nitrite production was also inhibited by SB203580 but not by PD98059 (Fig. 4A). However, GDB plus IFN-γ-induced nitrite production was not inhibited by polymyxin B (an LPS inhibitor), whereas LPS-induced nitrite generation was strongly inhibited by polymyxin B (Fig. 4B). These results suggest that p38 kinase pathway is importantly involved in the regulation of iNOS expression induced by GDB plus IFN-γ.

To investigate the mechanism whereby GDB plus IFN-γ activate macrophages, we analyzed the effect of GDB on NF-κB, AP-1, and Oct 1, whose binding sites are located in the promoter of iNOS gene. EMSA showed that treating RAW264.7 cells with GDB plus IFN-γ for 2 h did induce NF-κB DNA binding activity, whereas GDB alone dose-dependently induced NF-κB DNA binding activity (Fig. 5A). Both AP-1 and Oct1 were found to have small basal DNA binding activities and to be moderately induced by GDB (Fig. 5B, C). The specificity of bindings was demonstrated by competition assays using ³²P-unlabeled κB or mutant κB probes (data not shown). These results suggest that GDB activates NF-κB, which is known to play an important role in the activation and production of cytokines in macrophages.