Purification and biochemical characterization of a novel thermophilic exo-ß-1, 3-glucanase from the thermophile biomass-degrading fungus Thielavia terrestris Co3Bag1

Background: The aim of this work was to purify and characterize exo-ß-1,3-glucanase, namely, TtBgnA, from the thermophilic fungus Thielavia terrestris Co3Bag1 and to identify the purified enzyme. Results: The thermophilic biomass-degrading fungus T. terrestris Co3Bag1 displayed ß-1,3-glucanase activity when grown on 1% glucose. An exo-ß-1,3-glucanase, with an estimated molecular mass of 129 kDa, named TtBgnA, was purified from culture filtrates from T. terrestris Co3Bag1. The enzyme exhibited optimum activity at pH 6.0 and 70°C and half-lives (t1/2) of 54 and 37 min at 50 and 60°C, respectively. Substrate specificity analysis showed that laminarin was the best substrate studied for TtBgnA. When laminarin was used as the substrate, the apparent KM and Vmax values were determined to be 2.2 mg mL-1 and 10.8 U/mg, respectively. Analysis of hydrolysis products by thin-layer chromatography (TLC) revealed that TtBgnA displays an exo mode of action. Additionally, the enzyme was partially sequenced by tandem mass spectrometry (MS/MS), and the results suggested that TtBgnA from T. terrestris Co3Bag1 could be classified as a member of the GH-31 family. Conclusions: This report thus describes the purification and characterization of TtBgnA, a novel exo-ß-1,3-glucanase of the GH-31 family from the thermophilic fungus T. terrestris Co3Bag1. Based on the biochemical properties displayed by TtBgnA, the enzyme could be considered as a candidate for potential biotechnological applications.

Protective effects of laminarin on vascular endothelium in rats with chronic inflammation induced by LPS / 中国药理学通报

Aim To observe the effect of laminarin L01 on the expression of eNOS and iNOS in aorta of rats with chronic inflammation induced by LPS. Methods Chronic inflammatory rat models were prepared by tail vein injection low dose LPS(0.4 mg·kg-1) once a week for four weeks. The rats were randomly divided into five groups. After the first injection of LPS, the DXM group was intraperitoneally injected with dexam-ethasone (10 mg·kg-1). L01 high,medium and low dose groups were intraperitoneally injected with L01 (50,30,10 mg·kg-1). The LPS group was injected intraperitoneally with equal volume of normal saline once a day. Another control group, only injection of normal saline, a total of four weeks. After the last administration,the number of whole white blood cells (WBC) was counted. ELISA was used to measure the hs-CRP in serum. The expressions of eNOS,iNOS and COX-2 mRNA were detected by RT-PCR. Results After four weeks of administration of L01, the number of WBC and the level of serum hs-CRP in chronic in-flammatory rats were significantly decreased. The ex-pression of eNOS was up-regulated, and iNOS and COX-2 expressions were down-regulated. Conclusions Laminarin L01 may regulate the expression and re-lease of endothelium-derived relaxing factor stimulated by LPS,and improve the endothelium-dependent dias-tolic function of aorta, thus protecting the damage of vascular endothelium.

Induction Study of Laminarin Sulfate LAMS-2 on Human Colon Cancer Cell Line LOVO Apoptosis through a Mitochondrial Pathway / 中国药学杂志

OBJECTIVE: To investigate the effects and mechanisms of LAMS-2 on the proliferation and apoptosis of human colon cancer cell line LOVO. METHODS: LOVO cells were treated with different concentrations of LAMS-2,3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay and morphology observation were performed with Hoechst 33258 dye to determine the effects of LAMS-2 on the proliferation and apoptosis of LOVO cells. Flow cytometry (FCM) was used to detect the level of reactive oxygen species (ROS), mitochondrion permeability transition pore (MPTP) and mitochondrial membrane potential (△ψm). Laser scanning confocal microscope (LSCM) was used to analyze intracellular calcium ion concentration, Western blot was performed to analyze the expressions of Cyt-C, caspase-9 and caspase-3. Spectrophotometer was applied to quantify the activity of caspase-9 and caspase-3. RESULTS: LAMS-2 inhibited LOVO cell proliferation and induced apoptosis, the apoptosis morphology was obvious. LAMS-2 treatment increased the intracellular level of ROS and Ca2+; activated intracellular MPTP and decreased △ψm; it also induced the release of Cyt-C and the activation of caspase-9 and caspase-3, increased the activity of caspase-9 and caspase-3. CONCLUSION: LAMS-2 can effectively inhibit the proliferation of LOVO cells and induce apoptosis through a mitochondria-mediated pathway. Keywords:

Role of Dectin-1 in the production of IL-10 and TNF-α by rat tracheal epithelial cells stimulated with heat-treated Candida glabrata / 中国感染控制杂志

Objective To investigate the effect of Dectin-1 on the release of inflammatory factors interleukin-10 (IL-10) and tumor necrosis factor-α (TNF-α) in rat tracheal epithelial cells (RTECs) stimulated with heat-treated Candida glabrata (C.glabrata).Methods RTECs cultivated in vitro were randomly divided into three groups,including control group(RTECs + sterile normal saline),fungal stimulation group(RTECs + heat-treated C.glabrata),and inhibitor intervention group(RTECs + laminarin + heat-treated C.glabrata),cells were harvested after incubation for 0,2,4,6 hours respectively,cell survival rate was determined by MTT method,expression of Dectin-1 was analyzed by Western Blot,expression of IL-10 and TNF-α were detected by enzyme-linked immunosorbent assay (ELISA).Results Heat-treated C.glabrata destroyed cell structure and reduced cell survival rate.At the beginning of the culture (0 h),cell survival rate,expressions of Dectin-1,IL-10 and TNF-α among three groups were all not significantly different(all P＞0.05).After incubation for 2,4,6 hours,expressions of Dectin-1,IL-10 and TNF-α in fungal stimulation group and inhibitor intervention group were both significantly higher than control group;expressions of Dectin-1,IL-10,and TNF-α in inhibitor intervention group was lower than fungal stimulation group(all P＜0.05).The expressions of IL-10 in inhibitor intervention group at 0 h and 2 h was not significantly different,expressions of Dectin-1,IL-10,and TNF-α in fungal stimulation group and inhibitor intervention group at different incubation periods were significantly different(all P＜0.05).Conclusion Dectin-1 is an important receptor for RTECs to recognize the heat-treated C.glabrata,it induces the release of IL-10 and TNF-α,and mediate the occurrence of inflammation.

Potentiation of Innate Immunity by beta-Glucans

beta-Glucans have been known to exhibit antitumor activities by potentiating host immunity by an unknown mechanism. The C-type lectin dectin-1, a beta-glucan receptor, is found on the macrophage and can recognize various beta-glucans. Previously, we demonstrated the presence of beta-glucan receptor, dectin-1, on the Raw 264.7 cells as well as on murine mucosal organs, such as the thymus, the lung, and the spleen. In order to investigate immunopotentiation of innate immunity by beta-glucan, we stimulated a murine macrophage Raw 264.7 cell line with beta-glucans from Pleurotus ostreatus, Saccharomyces cerevisiae, and Laminaria digitata. Then, we analyzed cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 by reverse transcription-polymerase chain reaction (RT-PCR). In addition we analyzed gene expression patterns in beta-glucan-treated Raw 264.7 cells by applying total mRNA to cDNA microarray to investigate the expression of 7,000 known genes. When stimulated with beta-glucans, the macrophage cells increased TNF-alpha expression. When co-stimulation of the cells with beta-glucan and lipopolysaccharide (LPS), a synergy effect was observed by increased TNF-alpha expression. In IL-6 expression, any of the beta-glucans tested could not induce IL-6 expression by itself. However, when co-stimulation occurred with beta-glucan and LPS, the cells showed strong synergistic effects by increased IL-6 expression. Chip analysis showed that beta-glucan of P. ostreatus increased gene expressions of immunomodulating gene families such as kinases, lectin associated genes and TNF-related genes in the macrophage cell line. Induction of TNF receptor expression by FACS analysis was synergized only when co-stimulated with beta-glucan and LPS, not with beta-glucan alone. From these data, beta-glucan increased expressions of immunomodulating genes and showed synergistic effect with LPS.

Study on the mechanism of laminarin sulfate in the prevention of experimental atherosclerosis / 中国海洋药物

Objective To explore the possible immunological mechanism of laminarin sulfate in the prevention of experimental atherosclerosis. Methods Serum soluble interleukin 2 receptor (sIL-2R) , circulating immuno-complex, sub units of T lymphocyte, inter leukin-6(IL-6) , in-terleukin-8(IL-8), tumor necrosis factor-a (TNF-a) and lipid metabolism were determined by ELISA, RIA in rats and quails. Results The lipid metabolism and immunologic function were prominent disturbance in animals after feeding with high-lipid food. However, Laminarin sulfate has obvious regulating effects on above-mentioned index. Conclusions The mechanism of laminarin sulfate in the prevention of atherosclerosis might be closely related to the regulation of the disturbance of lipid metabolism and to the regulation of the immunologic function of the body.

Inhibitory effect of laminarin (acidic polysaccharide) J201A on the proliferation of Human Lung Fibroblasts and its related mechanism of action / 中国海洋药物

To study the inhibitory effects of laminarin (acidic polysaccharide) J201A on the proliferation of human lung fibroblasts (HLF) and its related mechanism of action. Methods: Effect of J201A on the proliferation of HLF was evaluated by MTT assay, and the effects on cell cycle and synthesis of proteins of HLF were assessed by flow cytometry. The existence of J201A receptors on HLF was confirmed by fluorescent staining assay. Results:J201 A inhibited the synthesis of proteins and the proliferation of HLF at G0/G1 stage of cell cycle and J201A receptors existed on HLF. Conclusion:J201A exerted its antifibrotic activity by inhibiting HLF at G0/G1 stage of cell cycle and the synthesis of proteins.