Abstract Four different DNA extraction methods were used to extract genomic DNA of the medicinal mushroom Lingzhi from its developing stage materials, such as mycelium, dry fruiting body, or sliced and spore powder or sporoderm‐broken spore powder. The DNA samples were analyzed using agarose gel electrophoresis, UV spectrophotometer, and PCR amplification. According to the average yields and purity of DNA, high salt concentrations and low pH methods were the best for DNA extraction. The mycelia and sporoderm‐broken spore powder yielded higher and purer DNA. The method developed could effectively eliminate the influence of the secondary metabolites to DNA extraction. The DNA samples extracted from the developed method could be successfully used for PCR applications. 相似文献
Milk protein of farm animals is difficult to isolate because of the presence of casein micelles, which are hard to separate from whey by using centrifugation or filtration. Insoluble casein micelles also create an obstacle for purification instruments to operate efficiently. The conventional method, to precipitate caseins by lowering pH to 4.6 and then recover the whey fraction for further purification using chromatography techniques, is not applicable to proteins having an isoelectric point similar to caseins. In addition, the acid condition used for casein removal usually leads to significantly poor yields and reduced biological activities. In this study, a novel method of precipitating caseins under neutral or weak acidic conditions is presented. The method employs a phosphate salt and a freeze–thaw procedure to obtain a casein-free whey protein fraction. This fraction contains more than 90% yield with little loss of bioactivity of the target protein, and is readily available for further chromatographic purification. This method was successfully applied to purify recombinant human factor IX and recombinant hirudin from the milk of transgenic pigs in the presented study. It is an efficient pretreatment approach prior to chromatographic purification of milk protein from farm animals and particularly of great value to collect those recombinants secreted from transgenic livestock. 相似文献
Earlier studies on fructose laurate ester products have shown that recombinant Pichia pastoris displaying Candida antarctica lipase B (CALB) on the cell surface acts as an efficient whole-cell biocatalyst for sugar ester production from fructose and lauric acid in an organic solvent. The effects of various reaction factors, including solvent composition, substrate molar ratio, enzyme dose, temperature and water activity, on esterification catalyzed by the CALB-displaying P. pastoris whole-cell biocatalyst were examined in the present study. Under the preferred reaction conditions, specifically, 5 mL organic solvent mixture of 2-methyl-2-butanol/DMSO (20% v/v), 2 mmol fructose with a lauric acid to fructose molar ratio of 2:1, 0.3 g whole-cell biocatalyst (1,264 U/g dry cell) with an initial water activity of 0.11, 1.2 g 4Å molecular sieve, reaction temperature of 55oC and 200 rpm stirring speed, the fructose mono laurate ester yield was 78% (w/w). The CALBdisplaying P. pastoris whole-cell biocatalyst exhibited good operational stability, with an evident increase, rather than decrease, in relative activity after the continuous recover and reuse cycle. The relative activity of the biocatalyst remained 50% higher than that of the first batch, even following reuse for 15 batches. Our results collectively indicate that the CALB-displaying P. pastoris whole-cell biocatalyst may be potentially utilized in lieu of free or immobilized enzyme to effectively produce non-ionic surfactants such as fatty acid sugar esters, offering the significant advantages of cost-effectiveness, good operational stability and mild reaction conditions.
It is well known that neutrophils play very important roles in the development of rheumatoid arthritis (RA) and interleukin (IL)-8 is a critical chemokine in promoting neutrophil migration. We previously showed that increased production of Cyr61 by fibroblast-like synoviocytes (FLS) in RA promotes FLS proliferation and Th17 cell differentiation, thus Cyr61 is a pro-inflammatory factor in RA pathogenesis. In this study, we explored the role of Cyr61 in neutrophil migration to the joints of RA patients.
Methods
RA FLS were treated with Cyr61 and IL-8 expression was analyzed by real-time PCR and ELISA. The migration of neutrophils recruited by the culture supernatants was determined by the use of a chemotaxis assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as a control. Arthritis severity was determined by visual examination of the paws and joint destruction was determined by hematoxylin-eosin (H&E) staining. Signal transduction pathways in Cyr61-induced IL-8 production were investigated by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay or chromatin immunoprecipitation (ChIP) assay.
Results
We found that Cyr61 induced IL-8 production by RA FLS in an IL-1β and TNF-α independent pathway. Moreover, we identified that Cyr61-induced IL-8-mediated neutrophil migration in vitro. Using a CIA animal model, we found that treatment with anti-Cyr61 mAb led to a reduction in MIP-2 (a counterpart of human IL-8) expression and decrease in neutrophil infiltration, which is consistent with an attenuation of inflammation in vivo. Mechanistically, we showed that Cyr61 induced IL-8 production in FLS via AKT, JNK and ERK1/2-dependent AP-1, C/EBPβ and NF-κB signaling pathways.
Conclusions
Our results here reveal a novel role of Cyr61 in the pathogenesis of RA. It promotes neutrophil infiltration via up-regulation of IL-8 production in FLS. Taken together with our previous work, this study provides further evidence that Cyr61 plays a key role in the vicious cycle formed by the interaction between infiltrating neutrophils, proliferated FLS and activated Th17 cells in the development of RA. 相似文献
Abstract The pharmacokinetics and toxicology of 2′,3′-dideoxy-β-L-5-fluorocytidine (β-L-FddC) and 2′,3′-dideoxy-β-L-cytidine (β-L-ddC) in mice was investigated. In addition, 2′,3′-dideoxy-β-L-5-azacytidine (β-L-5-aza-ddC) and its α-L-anomer (α-L-5-aza-ddC) were synthesized by coupling the silylated 5-azacytosine derivative with 1-O-acetyl-5-O-(tert-butyldimethylsilyl)-2,3-dideoxy-L-ribofuranose, followed by separation of the α-and β-anomers and were evaluated in vitro against HBV and HIV. β-L-5-aza-ddC was found to show significant anti-HBV activity at approximately the same level as 2′,3′-dideoxy-β-D-cytidine (ddC), which is a known anti-HBV agent. β-L-5-aza-ddC was not cytotoxic to L1210, P388, S-180, and CCRF-CEM cells up to a concentration of 100 μ. Conversely, the α-L-anomer was not active against HBV at the same concentration. 相似文献