Cloning, Overexpression, and Characterization of a Thermostable, Organic Solvent-Tolerant Laccase from Bacillus pumilus ARA and Its Application to Dye Decolorization.

A thermostable and organic solvent-tolerant bacterial laccase from Bacillus pumilus ARA has been expressed heterologously and characterized, which shows potential decolorization capacity to various types of industrial synthetic dyes. The optimal temperature and pH were 85 °C and 3.5, respectively, while the purified recombinant laccase B.P.Lacc was stable under 55-75 °C and pH 5.0-8.0 conditions. The apparent kinetic parameters K m and V max of B.P.Lacc for ABTS as the substrate were 0.33 mM and 32.4 U/mg, respectively. Ethanol (1%, v/v) and methanol (2%, v/v) could stimulate the enzyme activity. The recombinant laccase retained over 95% of its initial activity in 10% (v/v) methanol. The optimal expression conditions for the laccase production of B.P.Lacc in LB medium were obtained: induction temperature of 25 °C, 0.4 mM Cu2+, and 1.0 mM IPTG added into the culture. After 5 h, the final laccase production was 1283 U/mL. Moreover, the laccase activity increased to 4822 U/mL after follow-up 2 h stationary cultivation, with about a 3.76-fold increase. The purified B.P.Lacc was able to efficiently decolorize synthetic dyes combined with mediators. Adding 1.0 mM ABTS, more than 90% of BRRB was decolorized by the enzyme, whether at pH 4.0 or pH 7.9. The outstanding enzymatic properties suggested that B.P.Lacc may be suitable for a wide application in future biodegradation fields.

Protective effect of hyperoside against hydrogen peroxide-induced dysfunction and oxidative stress in osteoblastic MC3T3-E1 cells.

Oxidative stress can induce apoptosis and decrease activities of osteoblasts. Hyperoside (HYP) is a potent antioxidant derived from Chinese herb. This study aims to evaluate the protective effects provided by HYP to osteoblastic MC3T3-E1 cells. MC3T3-E1 cells were pre-treated with HYP for 24 h before being treated with 0.3 mM hydrogen peroxide (H2O2) for 24 h. Cell viability, flow cytometric analysis and mRNA expression of alkaline phosphatase (ALP), collagen I (COL-I) and osteocalcin (OCN) in MC3T3-E1 cells were examined. We next examined apoptosis-related and mitogen-activated protein kinase (MAPK) related proteins in HYP and H2O2 groups. HYP over the dose of 40 µmol/L could obviously increase the MC3T3-E1 cell viability at 24 h and 48 h (p < .05). HYP significantly (p < .05) increased mRNA expression of ALP, COL-I and OCN than H2O2 group. Moreover, HYP decreased the apoptosis rate and apoptosis-related proteins that induced by H2O2. In addition, HYP decreased the production of phosphorylated Jun N-terminal kinase (JNK) and p38 levels of osteoblastic MC3T3-E1 cells induced by H2O2. These results demonstrated that the protective effect provided by HYP to osteoblastic MC3T3-E1 cells was mediated, at least in part, via inhibition of MAPK signalling pathway and oxidative damage of the cells.

Macrophages and T lymphocytes are the predominant cells in intimal arteritis of resected renal allografts undergoing acute rejection.

BACKGROUND: In acute renal allograft rejection, T-cell-mediated processes have been generally regarded as dominant. Although there is recent evidence that macrophages play important roles in acute vascular rejection, less is known about the exact proportion of immunocytes in the intimal arteritis of renal allografts with unfavorable outcomes. METHODS: By immunohistochemical staining using nine primary antibodies, we classified the proportions of infiltrating immunocytes in intimal arteritis and glomerulitis in five allografts resected because of acute irreversible graft failure. RESULTS: All five allografts had features of antibody-mediated rejection based on criteria established by the Banff classification. In intimal arteritis, CD3+ T-lymphocytes accounted for 30.6±13.3% of the immunocytes and macrophages for 40.4±9.2%. 45.4% of the T-cells were CD8+ cytotoxic T-cells. Neutrophils were also present but accounted for a relatively low proportion of the cells (8.8±8.6%). B-cells and plasma cells all accounted for <5% of the immunocytes. NK cells were readily detected (4.2±4.2%). When we compared types of arteritis, the CD15+ neutrophils accounted for as many as 27.8±15.1% of immunocytes in V3 vasculitis and only 1.0±1.4% in V2 vasculitis. CD3+ T-lymphocytes accounted for 25.8±7.3% of immunocytes in V3 vasculitis and 41.5±7.9% in V2 vasculitis. In glomerulitis, the immunocytes were mainly macrophages (53.1±9.1%) and neutrophils (34.6±9.9%). CONCLUSION: Macrophages and T-lymphocytes accounted for the highest percentage of immunocytes in the intimal arteritis of irreversible renal failure associated with antibody-mediated rejection. 45.4% of the T-cells were CD8+ cytotoxic T-cells. Neutrophils and NK cells were also present in these lesions. The proportion of neutrophils in V3 vasculitis was much higher than in V2 vasculitis. These observations suggest that besides macrophages and T-lymphocytes, neutrophils may also play a role in the more severe arterial lesion. To our knowledge this is the first report of this observation. Macrophages and neutrophils were the main inflammatory cells in the glomerulitis of acute rejection.

[Establishment and significance of the experimental minimal persistent inflammation models in allergic rhinitis guinea pigs].

OBJECTIVE: To develop an animal model of minimal persistent inflammation (MPI) in allergic rhinitis guinea pigs and to investigate its significance. METHODS: Sixty male Hartley guinea pigs were divided into four groups: group A (positive control group), B (MPI model group), C (negative group) and D (bland group) respectively, with fifteen animals in each group. Guinea pigs from group A, B and C were sensitized intraperitoneally by injection of suspension of ovalbumin (OVA) and aluminum hydroxide in 0.9% physiological saline. Then, repeated local booster sensitization with different concentration of OVA suspension (1% and 0.01%) or physiological saline into the nasal cavity of those guinea pigs were performed. For group D, physiological saline was used only. Symptoms (sneezing) of guinea pigs after antigen challenge were observed and the infiltration of eosinophils (EOS) together with the expression of intercellular adhesion molecule 1 (ICAM-1) in the nasal epithelial cells were also examined. RESULTS: When challenged with 1% OVA, the sneezing number of guinea pigs in group B was increased markedly than that in group D (P < 0.05). However, there was no difference between group B, A and C (P > 0.05). When challenged with 0.01% OVA, the symptom of sneezing almost disappeared in group B just like that in group D and there was no difference between the two groups (P > 0.05). Besides, there was still more EOS infiltrated in the nasal mucosa of guinea pigs in group B than that in group D (P < 0.05). There was no expression of ICAM-1 in nasal epithelium of guinea pigs in group D, nevertheless, ICAM-1 was found mildly expressed in group B. CONCLUSIONS: MPI models have been established successfully through long term challenge with lower density of OVA in the sensitized guinea pigs, which will provide us with a new method for further research in the mechanism and treatment of allergic rhinitis.

[Effects of minimal persistent inflammation on nasal mucosa of experimental allergic rhinitis in guinea pigs].

OBJECTIVE: To establish an animal model of minimal persistent inflammation (MPI) of allergic rhinitis in guinea pigs and to investigate the changes of nasal mucosa. The expression of transforming growth factor-beta1 (TGF-beta1) and matrix metalloproteinases-9 (MMP-9) were discussed. METHODS: Thirty male Hartley guinea pigs were randomly divided into two groups: MPI model group and control group randomly, with fifteen animals in each group. Guinea pigs from MPI model group were sensitized intraperitoneally by injection of suspension of ovalbumin (OVA) and aluminum hydroxide in 0.9% physiological saline. Then, repeated local booster sensitization with low concentration of OVA suspension into the nasal cavity was performed to establish MPI models. Alcian blue-periodic acid-Schiff (AB-PAS) staining and Masson's trichrome (MT) staining were used to determine the number of goblet cells and collagen deposition within the basement membrane of epithelium. The expression and distribution of TGF-beta1 and MMP-9 in nasal mucosa were estimated by double immunofluorescence under a confocal laser scan microscopy system. RESULTS: Compared with the control group, the increased goblet cells (t = 13.720, P < 0.05) in nasal epithelium together with the increased collagen fibrils (t = 4.542, P <0.05) within the basement membrane of epithelium were observed in the MPI model group. There was nearly no expression of TGF-beta1, in the control group and the expression of MMP-9 was only found in the epithelium cell. In contrast, there was significantly higher expression of TGF-beta1 and MMP-9 (t = 25.218, P <0.05) in nasal mucosa of MPI model group than that in control group. TGF-beta1 mainly expressed in the epithelium cell, the infiltrated inflammatory cell and extracellular matrix, while MMP-9 expressed in the epithelium cell and the infiltrated inflammatory cell. CONCLUSIONS: Long time MPI in allergic rhinitis resulted in some changes of tissue remodeling in nasal mucosa. TGF-beta1 and MMP-9 may play an important role in disease progression.

[Role of aquaporin 1 in the migration of eosinophils from asthmatic guinea pigs].

OBJECTIVE: To investigate the role of aquaporin 1 (AQP1) in the migration of eosinophils (EOS) and to determine if AQP-1 can be viewed as the chemotactic activity marker of EOS. METHODS: Asthma model of guinea pigs were developed and EOS were purified from both peripheral blood and bronchoalveolar lavage fluid (BALF). The smears of EOS were studied by in situ hybridization for determining AQP1 mRNA and immunofluorescence under laser scanning confocal microscope for determining AQP1 protein. RESULTS: AQP1 was found expressed in EOS both from peripheral blood and from BALF. Compared with the expression of AQP1 mRNA (mean grey value 109.200 +/- 5.756, x +/- s) and protein (average fluorescence intensity 279.926 +/- 11.293) in EOS from BALF, there was stronger expression of AQP1 mRNA (92.904 +/- 3.290) and protein (425.081 +/- 17.474) in EOS from peripheral blood. The difference both of AQP1 mRNA (t = 9.519, P < 0.05) and protein(t = 27.020, P < 0.05) were considered statistically significant respectively. CONCLUSIONS: AQP1 plays a crucial role in EOS movement. It is possible that EOS produce more AQP1 protein to accelerate its migration to inflammatory tissues under allergic disease and EOS with AQP1 highly expressed are activated. AQP1 can be viewed as the chemotactic activity marker of EOS.