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Polysaccharide extracted from red seaweed Bangia fusco-purpurea (BFP) is a novel sulfated galactan, differed from agarans and carrageenans in fine structure. In this study, in vitro fermentation characteristics of BFP by human gut microbiota and its protective effect on lipopolysaccharide (LPS)-induced injury in Caco-2 cells were investigated. Our results showed that BFP was mainly degraded at transverse colon for 18 h fermentation by gut microbiota with reduced molecular weight. Meanwhile, BFP fermentation was associated with increased short-chain fatty acids (SCFAs) as compared to control group, especially acetic acid was increased to 129.53 ± 0.24 from 82.14 ± 0.23 mmol/L, and butyric acid was up to 1.56 ± 0.004 from 0.62 ± 0.01 mmol/L. Furthermore, BFP promoted abundances of Bacteroidetes and Firmicutes, while decreased numbers of Proteobacteria. The up-regrated beneficial differential metabolites were SCFAs, L-proline, arginine, folic acid, pyridoxamine, thiamine, etc. (p 0.05), and their related metabolic pathways mainly included mTOR, arginine biosynthesis, and vitamin metabolism. Notably, BFP fermentation products at transverse colon significantly restored cell viability of LPS-treated Caco-2 cells from 73.79 ± 0.48 % to 93.79-99.64 %, which might be caused by increased beneficial differential metabolites (e.g., SCFAs). Our findings suggest that BFP has prebiotic potential and can enhance gut health.

Chondroitinase plays an important role in structural and functional studies of chondroitin sulfate (CS). In this study, a new member of chondroitinase B of PL6 family, namely ChSase B6, was cloned from marine bacterium Microbulbifer sp. ALW1 and subjected to enzymatic and structural characterization. The recombinant ChSase B6 showed optimum activity at 40 °C and pH 8.0, with enzyme kinetic parameters of Km and Vmax against chondroitin sulfate B (CSB) to be 7.85 µg/mL and 1.21 U/mg, respectively. ChSase B6 demonstrated thermostability under 60 °C for 2 h with about 50% residual activity and good pH stability under 4.0–10.0 for 1 h with above 60% residual activity. In addition, ChSase B6 displayed excellent stability against the surfactants including Tween-20, Tween-80, Trion X-100, and CTAB. The degradation products of ChSase B6-treated CSB exhibited improved antioxidant ability as a hydroxyl radical scavenger. Structural analysis and site-directed mutagenesis suggested that the conserved residues Lys248 and Arg269 were important for the activity of ChSase B6. Characterization, structure, and molecular dynamics simulation of ChSase B6 provided a guide for further tailoring for its industrial application for chondroitin sulfate bioresource development.

α-L-Rhamnosidase has attracted lots of attention due to its industrial potential applications. The applicability of α-L-rhamnosidase, however, was limited by their low ligand affinity on the industrial scale. In order to improve the affinity of α-L-rhamnosidase for industrial use, we investigated the variation of its affinity by amino acid replacement. Particularly, the enzyme affinity of a α-L-rhamnosidase from Aspergillus niger JMU-TS528 (rRha1) was measured with the semi-conservative amino acid (homology between 30% -80%) replaced. As a result, the enzyme affinity of the two mutants, R404S and N578D, were increased by 1.45-fold and 2.3-fold, respectively, showing that these two mutants could be the promising candidates for industrial use. To test if these mutations bring negative effect on the enzyme properties, we also determined the other enzymatic properties of these mutants and showed no negative effect. To understand the improvement of enzyme affinity, the conformational flexibility of (α/α)6-barrel catalytic domain were examined by molecular dynamics (MD) simulation, and demonstrated that the conformations of these mutants are more flexible, which could influence the affinity of substrates to the enzyme and hence the enzyme activity. This work not only enhanced the enzyme affinity of a α-L-rhamnosidase, making rRha1 a promising candidate for industrial processes, but also provided an effective technical strategy for improving affinity of other enzymes.

Interaction between tumor cells and immune cells determined highly heterogeneous microenvironments across patients, leading to substantial variation in clinical benefits from immunotherapy. Somatic gene mutations were found not only to elicit adaptive immunity but also to influence the composition of tumor immune microenvironment and various processes of antitumor immunity. However, due to an incomplete view of associations between gene mutations and immunophenotypes, how tumor cells shape the immune microenvironment and further determine the clinical benefit of immunotherapy is still unclear. To address this, we proposed a computational approach, inference of mutation effect on immunophenotype by integrated gene set enrichment analysis (MEIGSEA), for tracing back the genomic factor responsible for differences in immunophenotypes. MEIGSEA was demonstrated to accurately identify the previous confirmed immune-associated gene mutations, and systematic evaluation in simulation data further supported its performance. We used MEIGSEA to investigate the influence of driver gene mutations on the infiltration of 22 immune cell types across 19 cancers from The Cancer Genome Atlas. The top associated gene mutations with infiltration of CD8 T cells, such as CASP8, KRAS and EGFR, also showed extensive impact on other immune components; meanwhile, immune effector cells shared critical gene mutations that collaboratively contribute to shaping distinct tumor immune microenvironment. Furthermore, we highlighted the predictive capacity of gene mutations that are positively associated with CD8 T cells for the clinical benefit of immunotherapy. Taken together, we present a computational framework to help illustrate the potential of somatic gene mutations in shaping the tumor immune microenvironment.

We found that ascophyllan significantly inhibited the fibrillation of human insulin and was the most effective among the sulfated polysaccharides tested. Gel-filtration analysis suggested that ascophyllan was capable of forming a complex with insulin through a weak interaction. Secondary structure transition from native α-helix to β-sheet predominant structure of insulin under the fibrillation conditions was suppressed in the presence of ascophyllan. Interestingly, ascophyllan attenuated insulin fibril-induced hemolysis of human erythrocytes. Moreover, ascophyllan attenuated insulin amyloid-induced cytotoxicity on rat pheochromocytoma PC12 cells and reduced the level of intracellular reactive oxygen species. This is the first report indicating that a sulfated polysaccharide, ascophyllan, can suppress the insulin amyloid fibril formation and inhibit the fibril-induced detrimental bioactivities.

In this study, a sulfated polysaccharide (BFP) was isolated from the edible red alga Bangia fusco-purpurea. Gel-filtration and thin layer chromatographically analyses suggested that BFP was a homogenous polysaccharide. The chemical structural analysis revealed that BFP mainly consisted of galactose together with a small amount of uronic acid, mannose, and glucose. Its molecular mass was estimated to be 133.18 kDa by high-performance liquid chromatography (HPLC) analysis. BFP inhibited α-amylase and α-glucosidase in a concentration-dependent manner. The IC50 values of BFP against α-amylase and α-glucosidase were estimated to be 1.26 ± 0.11 mg/mL and 1.34 ± 0.07 mg/mL, respectively. Kinetic analyses suggested that BFP showed competitive and non-competitive inhibition against α-amylase and α-glucosidase, respectively. Circular dichroism spectral and fluorescence spectral analyses suggested that BFP affects the conformational structures of these enzymes, which may lead to the inhibition of the enzymatic activities. Abbreviations: Ara: D-arabinose; AnGal: anhydro-L-galactose residues; CD spectroscopy: Circular Dichroism spectroscopy; DNS: dinitrosalicylic acid; FT-IR: fourier transform infrared spectra; Fuc: L-fucose; Gal: D-galactose; Glc: D-glucose; GlcA: D-Glucuronic acid; HPLC: high performance liquid chromatography; Man: D-mannose; pNPG: p-nitrophenyl-α-D-glucoside; TFA: trifluoroacetic acid; TLC: thin-layer chromatography; PMP: 1-phenyl-3-methyl-5-pyrazolone; Xyl: D-xylose

A sulfated polysaccharide ascophyllan inhibited α-glucosidase in a concentration dependent manner, and more than 90% activity was inhibited at 1.0 mg/mL. The inhibitory activity was much higher than that of acarbose.No significant inhibitory effect of ascophyllan on α-amylase was observed up to 10.0 mg/mL. Ascophyllan HS, a commercially available ascophyllan preparation showed even higher inhibitory effect on α-glucosidase than ascophyllan. Interestingly, ascophyllan and ascophyllan HS induced the secretion of glucagon-like peptide-1 (GLP-1) from human intestinal NCI-H716 cell line in a concentration dependent manner (10~100 ng/mL). The oral glucose tolerance tests revealed that after continuous 8-week ingestion of ascophyllan HS at 100 mg/day, the glucose area under the curve values of the ascophyllan HS ingested group were significantly lower than placebo ingested group. Serum glycosylated hemoglobin (HbA1c) level in ascophyllan HS ingested group tended to decrease after 8-week ingestion, whereas no significant change was observed in placebo ingested group. This is the first report indicating that ascophyllan can induce the secretion of GLP-1 from human intestinal cell line (NCI-H716), besides the potent inhibitory effect on α-glucosidase.Furthermore, clinical trial suggested that ascophyllan HS may be a practically applicable blood glucose controlling agent in humans., International Journal of Biological Macromolecules, 125, pp.453-458; 2019

In this study, we found that a sulfated polysaccharide isolated from the brown alga Ascophyllum nodosum, ascophyllan, showed suppressive effects on stimulated RAW264.7 cells. Ascophyllan significantly inhibited expression of inducible nitric oxide synthase mRNA and excessive production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW264.7 cells in a dose-dependent manner without affecting the viability of RAW264.7 cells. Ascophyllan also reduced the elevated level of intracellular reactive oxygen species (ROS) in LPS-stimulated RAW264.7 cells. Furthermore, preincubation with ascophyllan resulted in concentration-dependent decrease in ROS production in phorbol 12-myristate-13-acetate-stimulated RAW264.7 cells. Our results suggest that ascophyllan can exhibit anti-inflammatory effects on stimulated macrophages mainly through the attenuation of NO and ROS productions.

In this study, we evaluated the structural characteristics and novel biological activity of polysaccharide purified from red seaweed Bangia fusco-purpurea (BFP). Methylation, GC/MS, and NMR analyses suggested that the proposal repeating structure of BFP was →3)-β-D-Galp-(1→, →3)-β-D-Galp6S-(1 → 4)-α-D-Galp-(1→, →4)-α-D-Galp-(1 → 4)-α-L-AnGalp-(1 → 3)-β-D-Galp-(1→, and →4)-α-D-Galp-(1 → at a molar ratio of 13: 1: 1: 1. Interestingly, BFP exhibited significant cell migration- and tube formation-promoting activities toward human umbilical vein endothelial cells (HUVECs) in a concentration-dependent manner via increasing the N-cadherin expression and decreasing the E-cadherin expression. Furthermore, ERK and p38 mitogen-activated protein kinase (MAPK) specific inhibitors exhibited potent inhibitory effects on BFP-induced cell migration but not JNK MAPK inhibitor, suggesting ERK and p38 MAPK signaling pathways were mainly involved in BFP-induced cell migration. Moreover, vascular endothelial growth factor (VEGF) receptor tyrosine kinase inhibitor significantly inhibited BFP-induced cell migration and tube formation in HUVECs, suggesting VEGF receptors of HUVECs were involved in the pro-angiogenesis activity of BFP. This is the first report that a sulfated polysaccharide possessing a pro-angiogenic effect was obtained from red seaweed. Our findings are expected to promote the practical use of red seaweed B. fusco-purpurea and its polysaccharide in the development of the in vitro and ex vivo vascular endothelial cell-based cell therapy products.

This study aimed to improve the thermostability of arylsulfatase from Pseudoalteromonas carrageenovora. A total of 10 single-site mutants were chosen using the PoPMuSiC program, and two mutants of K253N and P314T showed enhanced thermal stability. By saturation mutagenesis and thermostability analysis, K253H and P314T were the best mutants at the two sites. Combinational mutations of K253H, P314T and H260L were subsequently introduced, and the best mutant of K253H/H260L was selected. Thermal inactivation analysis showed the half-life (t1/2) value at 55°C for K253H/H260L was 7.7-fold that of the wild-type enzyme (WT), meanwhile this mutant maintained the specific enzyme activity. Structure modeling demonstrated that the additional hydrogen bonds, optimization of surface charge-charge interactions, and increasing of hydrophobic interaction could account for the improved thermostability imparted by K253H/H260L.

Alginate is a naturally acidic polysaccharide consisting alternately of β-d-mannuronic acid and α-l-guluronic acid with 1, 4-glycosidic linkages and is derived from brown seaweeds. Herein, the effect of alginate on the promotion of macrophage phagocytosis and the corresponding molecular mechanisms were investigated in murine RAW264.7 cells. Alginate could enhance the intracellular phagocytosis of gold nanoparticles (AuNPs), fluorescent microspheres and immunoglobulin G (IgG)-opsonized Staphylococcus aureus (S. aureus). Moreover, alginate increased Toll-like receptor 4 (TLR4) expression and activated the Akt/nuclear factor-κB (NF-κB) and p38 mitogen-activated protein kinase (MAPK) signalling pathways. Alginate-promoted phagocytosis was suppressed by the addition of inhibitors of TLR4, NF-κB and p38 MAPK and by TLR4 gene knockdown, indicating the involvement of these key components. This work is the first to propose that alginate promotes phagocytosis via upregulating TLR4 expression and stimulating the Akt/NF-κB and p38 MAPK signalling pathways, which may contribute to the capacity of alginate to activate macrophages.

A library of Pseudoalteromonas carrageenovora arylsulfatase mutants was constructed by introducing random mutagenesis using error-prone PCR. After screening, one mutant strain was obtained whose arylsulfatase had improved thermal stability. Protein sequence analysis revealed one amino acid substitution of H260L. The mutant arylsulfatase (named H260L) retained higher residual activity than wild-type enzyme (named WT) after incubation at 45, 50, 55 and 60°C for 60min. Thermal inactivation analysis showed that the half-life (t1/2) value at 55°C for H260L was 40.6min, while that of WT was 9.1min. When p-nitrophenyl sulfate was used as a substrate, the optimal reaction temperature and pH for the mutant enzyme were 55°C and pH 8.0, respectively. H260L was stable over the pH range of 6.0-9.0. Inhibition assay with EDTA indicated that metal ions play an important role during the catalytic process of the mutant enzyme. The desulfation ratio against agar of Gracilaria lemaneiformis was 82%.

In this study, a low-molecular-weight saccharide fragment (LMWAs-L) was prepared from alginate lyase (EC 4.2.2.3) hydrolyzed ascophyllan by ultra-filtration separation method. LMWAs-L was a homogeneous saccharide fraction with an average molecular weight of 6.96 kDa. Enzymolysis process optimization experiments revealed that the optimum process parameters for preparing LMWAs-L were the enzyme concentration 0.02 U/mL, initial pH 6.8, and enzymolysis temperature 43 °C. After optimization, the yield of LMWAs-L was increased to 9.74% higher than that without optimization. Interestingly, LMWAs-L exhibited stronger enhancing activities on the proliferation and migration of human skin fibroblasts cells in vitro and better antibacterial activities as compared to native ascophyllan at the same mass concentration. Our study establishes a simple way to prepare low-molecular-weight saccharide with beneficial bioactivities from ascophyllan efficiently. This is the first report to reveal that ascophyllan and its low-molecular-weight saccharide have the potentials to be developed as natural biological dressing and antibacterial agents.

Ascophyllan HS is a commercially available preparation of the edible brown alga Ascophyllum nodosum containing ascophyllan, a sulfated polysaccharide with diverse beneficial biological activities. In this study, the effects of ascophyllan HS were evaluated in a severe intranasal Streptococcus pneumoniae infection mouse model. The control untreated mice started to die on day 7 and 80% had died by day 14 post-infection. Continuous oral administration of ascophyllan HS before and after bacterial infection resulted in a remarkable increase in survival rate, with 90% of the low (167 mg/kg body weight/day) and 100% of the high (500 mg/kg body weight/day) dose ascophyllan HS-treated mice surviving at day 14 post-infection. Histopathological observation of the lungs of the infected mice revealed the induction of typical pneumonia features in the alveolar spaces of the untreated control mice, such as extensive infiltration of inflammatory cells, edema, and fibrin deposition. In contrast, notable levels of lung injuries or alterations were not observed in the ascophyllan HS-treated mice, and only a minor lesion was observed in one mouse. Furthermore, bacterial burdens in the lungs were significantly reduced in the ascophyllan HS-treated mice as compared to the control mice at day 4 post-infection. Significantly higher levels of IL-12 were detected in the serum of ascophyllan HS-treated mice than that of control mice measured at the end of the infection experiment (day 14). These results suggest that orally administered ascophyllan HS exerts a therapeutic effect on S. pneumoniae infection by activating the host defense systems. This is the first report of the therapeutic effect of an orally administered seaweed polysaccharide preparation on S. pneumoniae infection. Our findings suggest that ascophyllan HS has the potential to be developed as nutraceuticals and pharmaceuticals applicable for humans as well as a safe and promising therapeutic agent against S. pneumoniae infection.

An extracellular agarase from marine bacterium Stenotrophomonas sp. NTa was purified to homogeneity. By size exclusion chromatography and SDS-PAGE analysis, the enzyme was determined to be a homodimer with monomeric molecular mass of 89.0 kDa. The optimal temperature and pH of strain NTa agarase were 40 °C and 10.0, respectively. It exhibited striking stability across a wide pH range of 5.0-11.0. Agarase from Stenotrophomonas sp. NTa had a relatively good resistance against the detected inhibitors, detergents and urea denaturant. The Km and Vmax for agar were 11.3mg/ml and 25.4 U/mg, respectively. Thin layer chromatography analysis, mass spectrometry, and enzyme assay using p-nitrophenyl-α/β-D-galactopyranoside revealed that strain NTa agarase was a β-agarase that degraded agarose into neoagarobiose, neoagarotetraose and neoagarohexaose as the predominant products, as well as a small amount of 3,6-anhydro-α-L-galactose. This is the first to present evidence of agarolytic activity in strain from genus Stenotrophomonas.

The anti-inflammatory properties of porphyrans (D1-D4) obtained from four discolored nori (Pyropia yezoensis) with different growth backgrounds were studied to examine possible variations in their bioactivities. Elution profiles of the porphyrans on Sepharose 4B indicated that D2-porphyran had relatively lower-molecular-size porphyrans than the other porphyrans. Inhibitory activities of the four porphyrans against nitric oxide (NO) and tumor necrosis factor-α (TNF-α) secretion by lipopolysaccharide (LPS)-stimulated RAW264.7 cells were different, whereas no significant differences were observed in the sulfate and anhydrogalactose levels. D2-porphyran showed the highest inhibitory activity against NO and TNF-α secretion by LPS-stimulated RAW264.7 cells, whereas D3- and D4-porphyrans had almost no activity. All porphyrans were efficiently degraded by free radical generated with ascorbate and hydrogen peroxide. The free-radical degradation resulted in a significant increase in the inhibitory activities of the four porphyrans against NO and TNF-α secretion, with varying rates depending on the porphyrans. The ability of D2-porphyran to suppress the receptor activator of nuclear factor κB ligand (RANKL)-induced osteoclastogenesis in RAW264.7 cells was also significantly enhanced after degradation. Our results suggest that molecular size is an important factor affecting the anti-inflammatory activity of porphyrans, and radical degradation might be a promising procedure to obtain active low-molecular-size porphyrans.

We previously found that ascophyllan, a sulfated polysaccharide isolated from brown seaweed Ascophyllum nodosum, exhibited antitumor activity in sarcoma-180 tumor-bearing mice. In this study, we found that ascophyllan inhibited the migration and adhesion of B16 melanoma cells by reducing the expression of N-cadherin and enhancing the expression of E-cadherin in a concentration-dependent manner. Transwell invasion assay revealed that ascophyllan suppressed the invasion ability of B16 cells. It also inhibited the expression of matrix metalloprotease-9 (MMP-9) mRNA and the secretion of MMP-9 protein in B16 cells, a process that may involve the extracellular signal-regulated kinase (ERK) signaling pathway. Furthermore, ascophyllan administered intraperitoneally at 25 mg/kg showed anti-metastatic activity in a mouse model of metastasis induced by intravenous injection of B16 cells, and the number of lung surface metastatic nodules in ascophyllan-treated mice was significantly reduced compared to that in the untreated control mice. Since splenic natural killer cell activity enhanced in the mice injected with ascophyllan intraperitoneally, we suggest that ascophyllan may exhibit in vivo anti-metastatic activity on B16 melanoma cells through activation of the host immune system in addition to a direct action on cancer cells.

To improve the thermostability of α-L-rhamnosidase (r-Rha1), an enzyme previously identified from Aspergillus niger JMU-TS528, multiple arginine (Arg) residues were introduced into the r-Rha1 sequence to replace several lysine (Lys) residues that located on the surface of the folded r-Rha1. Hinted by in silico analysis, five surface Lys residues (K134, K228, K406, K440, K573) were targeted to produce a list of 5 single-residue mutants and 4 multiple-residue mutants using site-directed mutagenesis. Among these mutants, a double Lys to Arg mutant, i.e. K406R/K573R, showed the best thermostability improvement. The half-life of this mutant's enzyme activity increased 3 h at 60 °C, 23 min at 65 °C, and 3.5 min at 70 °C, when compared with the wild type. The simulated protein structure based interaction analysis and molecular dynamics calculation indicate that the thermostability improvement of the mutant K406R-K573R was possibly due to the extra hydrogen bonds, the additional cation-π interactions, and the relatively compact conformation. With the enhanced thermostability, the α-L-rhamnosidase mutant, K406R-K573R, has potentially broadened the r-Rha1 applications in food processing industry.

Alginate from marine seaweeds is receiving continuous attention owing to its wide physiological activities. Herein, we sought to elucidate possible effects of alginate-derived polyguluronate (PG) and unsaturated guluronate oligosaccharide (GOS) on antibacterial activities of macrophages. Our results showed that, in contrast to PG, GOS markedly increased the phagocytosis of IgG-opsonized Escherichia coli and Staphylococcus aureus and further inhibited the survival of intracellular bacteria in macrophages. In line with this, GOS treatment resulted in the enhanced expression of Fcγ receptors on macrophages. In addition, GOS activated NF-κB pathway, induced TNF-α secretion, and elevated the expression of inducible nitric oxide synthase and the production of nitric oxide. Meanwhile, GOS stimulated the production of reactive oxygen species in macrophages. Moreover, guluronate trimer to hexamer (G3-G6) in GOS exhibited significant activity that increased the bacterial phagocytosis of macrophages, with the pentamer (G5), displaying the highest activity. Finally, our in vivo results further confirmed that GOS but not PG significantly improved bacterial clearance in murine acute peritonitis. In conclusion, GOS enhances antibacterial activities of macrophages via modulating signaling pathways related to innate immunity, suggesting that GOS might be a promising therapeutic candidate to improve the host defense against bacterial infection.

Antioxidant activities of sulfated polysaccharide ascophyllan from Ascophyllum nodosum was investigated in vitro by various assays, and compared with those of fucoidan. A chemiluminescence (CL) analysis using a luminol analog, L-012, showed that ascophyllan scavenges superoxide, and the activity is greater than fucoidan. However, in the presence of 10μg/ml of ascophyllan or 10μg/ml and 100μg/ml of fucoidan, slightly enhanced CL-responses were observed. Since EDTA-treatment resulted in disappearance of the enhancement effects, it was suggested that metal ions especially iron ions in the polysaccharides might be involved in this phenomenon. In fact, metal element analysis revealed that ascophyllan and fucoidan inherently contain iron and other metal elements. EDTA-treatment resulted in significant increase in Fe2+-chelating activities of these polysaccharides. In an electron spin resonance (ESR)-spin trapping analysis in which direct UV-radiation to hydrogen peroxide was used as a source of hydroxyl radical, ascophyllan and fucoidan showed potent hydroxyl radical scavenging activity with similar extent. Reducing power of ascophyllan was stronger than that of fucoidan. Our results indicate that ascophyllan can exhibit direct and potent antioxidant activity., International Journal of Biological Macromolecules, 59, pp.305-312; 2013

A time-course analysis of reactive oxygen species (ROS) generation in fertilized eggs of the devil stinger (Inimicus japonicus) from 0 h post-fertilization (hpf) to the early larval stage indicated that the ROS level was highest in the 22 hpf embryo, and declined thereafter. Phorbol myristate acetate (PMA) had no effect on ROS generation by the 22 hpf embryo, whereas PMA significantly increased larval ROS generation, suggesting that the ROS generation mechanisms of the 22 hpf embryo and larva are different at least in terms of PMA-responsiveness. Our results suggest the presence of a specific ROS generation system in devil stinger embryo which can be transitionally activated during embryogenesis.

Ascophyllan isolated from the brown alga Ascophyllum nodosum is a fucose-containing sulfated polysaccharide, which has similar but distinct characteristic monosaccharide composition and entire chemical structure to fucoidan. In this study, we examined the effects of ascophyllan, fucoidan isolated from A. nodosum (A-fucoidan), and fucoidan from Sigma (S-fucoidan) as a representative fucoidan derived from other source (Fucus vesiculosus) on mouse macrophage cell line RAW264.7 cells. No significant cytotoxic effects of ascophyllan and A-fucoidan on RAW264.7 cells were observed up to 1000μg/ml, while S-fucoidan showed cytotoxic effect in a concentration-dependent manner. Ascophyllan induced extremely higher level of nitric oxide (NO) production from RAW264.7 cells than those induced by fucoidans over the concentration range tested (0-200μg/ml). Reverse transcription polymerase chain reaction (RT-PCR) and western blot analysis revealed that expression level of inducible NO synthase (iNOS) in ascophyllan-treated RAW264.7 cells was much higher than the levels detected in the cells treated with fucoidans. Furthermore, the activities of ascophyllan to induce the secretion of tumor necrosis factor-α (TNF-α) and granulocyte colony-stimulating factor (G-CSF) from RAW264.7 cells were also greater than those induced by fucoidans especially at lower concentration range (3.1-50μg/ml). The activities of ascophyllan to induce NO and cytokine production in mouse peritoneal macrophages were also stronger than those of fucoidans. Electrophoretic mobility shift assay (EMSA) using infrared dye labeled nuclear factor-kappa B (NF-κB) and AP-1 consensus sequences suggested that ascophyllan can strongly activate these transcription factors. Marked increase in the nuclear translocation of p65, and the phosphorylation and degradation of IκB-α were also observed in ascophyllan-treated RAW264.7 cells. Analysis using mitogen-activated protein (MAP) kinase inhibitors and western blot analysis suggested that c-Jun N-terminal kinase (JNK) and p38 MAP kinase are mainly involved in ascophyllan-induced NO production., Nitric Oxide, 25(4), pp.407-415; 2011

A putative cDNA of α-l-rhamnosidase was PCR-cloned from Aspergillus niger JMU-TS528 and further extracellular over-expressed in Pichia pastoris GS115. The activity of the recombinant α-l-rhamnosidase r-Rha1 was 711.9U/mL, eightfold higher than the native α-l-rhamnosidase from A. niger JMU-TS528. r-Rha1 is a N-glycosylated protein of 90kDa and possesses broad substrate specificities by hydrolyzing α-1,2, α-1,3 α-1,4, and α-1,6 linkages to β-d-glucosides. This is the first report presenting that α-l-rhamnosidase showed activity on four kinds of glucosidic linkages. Compared with other previously characterized α-l-rhamnosidases, r-Rha1 showed a good thermostability and wide range of pH-stability with the optimum pH of 5.0 and temperature of 60°C. r-Rha1 activity was not greatly affected by representative metal ions and other detected effectors and showed excellent tolerance abilities against glucose and ethanol. These beneficial characteristics of r-Rha1 suggest that r-Rha1 should be considered a potential new biocatalyst for food and drug industrial applications.

Alginate has notably diverse pharmacological activities. The present study investigated the anti-inflammatory activity of the guluronate oligosaccharides prepared by oxidative degradation (GOS-OD) from alginate. GOS-OD significantly attenuated the production of nitric oxide (NO), prostaglandin E2 (PGE2), and reactive oxygen species (ROS), the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2, and the secretion of pro-inflammatory cytokines in lipopolysaccharide (LPS)-activated murine macrophage RAW 264.7 cells. Moreover, GOS-OD potently decreased the binding of LPS to the cell surface and LPS-induced Toll-like receptor 4 (TLR4) and cluster of differentiation (CD) 14 expression. Additionally, GOS-OD could remarkably inhibit the LPS-induced activation of nuclear factor (NF)-κB and mitogen-activated protein (MAP) kinase pathways in RAW 264.7 cells. These results indicate that GOS-OD may reduce the LPS-stimulated inflammatory responses through blocking the activation of NF-κB and MAP kinases, suggesting that GOS-OD may be considered as a potential nutraceutical for inflammation.

Splenic natural killer (NK) cell activity against YAC-1 cells increased in mice intraperitoneally injected with ascophyllan. Ascophyllan enhanced the cytotoxicity of RAW264.7 cells toward YAC-1 cells in a concentration-dependent manner. The cytotoxicity of ascophyllan-stimulated RAW264.7 cells as to YAC-1 cells was suppressed with N(G)-nitro-L-arginine methyl ester hydrochloride (L-NAME), an inhibitor of nitric oxide (NO) synthase, suggesting the involvement of NO in the cytotoxicity of ascophyllan-stimulated RAW264.7 cells.

In this study, we investigated the bioactivity of ascophyllan in terms of reactive oxygen species (ROS) generation. In RAW264.7 cells, we found that ascophyllan induced ROS generation in a concentration-dependent manner, but with bell-shaped profile. Immunoblot analysis demonstrated that ascophyllan promoted the translocation of cytosolic subunits (p67phox and p47phox) of NADPH oxidase to the plasma membrane. Among mitogen activated protein (MAP) kinase inhibitors tested, JNK inhibitor showed the most potent inhibitory effect on ascophyllan-induced ROS production. Consistently, significant level of phosphorylated JNK MAP kinase was detected in ascophyllan-treated RAW264.7 cells. Our findings suggest for the first time that ascophyllan can stimulate macrophages to produce ROS through the activations of NADPH oxidase and JNK MAP kinase., International Journal of Biological Macromolecules, 52, pp.164-169; 2013

Heterocapsa circularisquama showed much higher toxic effects on short-necked clams than Chattonella marina. Clams exposed to H. circularisquama exhibited morphological changes concomitant with an accumulation of mucus-like substances in the gills, a profound reduction in filtration activity, and lysosomal destabilization in hemocytes. Chattonella marina was less effective than H. circularisquama, and Heterocapsa triquetra was almost harmless in all these criteria. These results suggest that H. circularisquama exerted its lethal effect on short-necked clams through gill tissue damage and subsequent induction of physiological stress.

The effects of cytotoxic lectins, modeccin and phytohemagglutinin (PHA) on mouse macrophage cell line RAW264.7 was studied by detecting the induction of inflammatory mediators. Results showed that modeccin induced the release of tumor necrosis factor-α (TNF-α) from RAW264.7 cells with a bell-shape concentration-dependent profile. PHA that showed no significant cytotoxicity on RAW264.7 cells up to 100,000 ng/ml induced much higher level of TNF-α than modeccin. PHA simultaneously induced the secretion of granulocyte colony stimulation factor (G-CSF) from RAW264.7 cells with even much higher level than that of TNF-α, whereas modeccin did not. Furthermore, PHA induced the secretion of nitric oxide (NO) in RAW264.7 cells, while no significant level of NO was detected in the modeccin-treated cells. NH₄Cl (a lysomotoropic agent) and cycloheximide (a ribosome inhibitor) strongly inhibited modeccin-induced TNF-α secretion, but no significant inhibitory effects of these reagents on the PHA-induced TNF-α secretion were observed. Contrary to modeccin-induced TNF-α secretion, even slightly increased TNF-α secretion was observed in PHA-treated cells in the presence of 10 mM NH₄Cl. In addition, the inhibition profiles of modeccin-induced TNF-α secretion by various kinase inhibitors were different from those of PHA. These results suggested that the action mode of modeccin to stimulate RAW264.7 cells leading to the secretion of inflammatory molecules, including TNF-α, is distinct from that of PHA. On the other hand, significantly increased translocation of activator protein-1 (AP-1), a crucial transcription factor involved in expression of inflammatory molecules, into nucleus was observed in RAW264.7 cells treated with PHA and modeccin., Acta biochimica et biophysica Sinica, 43(1), pp.52-60; 2011

An N-acetylgalactosamine (GalNAc)-specific Ca(2+)-dependent lectin (C-type lectin), isolated from the marine invertebrate Holothuroidea (Cucumaria echinata), CEL-I, showed potent mitogenic activity toward normal mouse spleen cells. The mitogenic activity of CEL-I, which reached a maximum at 100 microg/ml, was inhibited by GalNAc in a concentration-dependent manner. The mitogenic effect of CEL-I at 10 microg/ml on T cell- enriched splenocytes was at a similar level due to a well-known T cell mitogen, concanavalin A (Con A), at 10 microg/ml. Furthermore, CEL-I evoked a mitogenic response from nude mouse spleen cells, while no significant effects of Con A on this cell population were observed over a wide range of concentrations. These results suggest that CEL-I is a potent mitogenic lectin with the ability to stimulate both T and B cells.