BACKGROUND: The application of deacetylated chitin (chitosan, DC) prepared by traditional technique was limited because of its lower degree of deacetylation, impurity and relative lower molecular mass.OBJECTIVE: To improve the traditional technique for extraction and preparation of DC from the rustae f oratosquila oratoria.DESIGN: Contrasting observation.SETTING: Shandong Institute of Biopharmaceutics.MATERIALS: The experiment was carried out in the Shandong Key Laboratory for iopharmaceutics, Shandong Institute of Biopharmaceutics from February 2003 to January 2004. Experimental nstrument: Mettler AE240 electronic analytical balance and ZK-40AB electrothermal vacuum drying oven were rovided by Mettler-Toledo Instruments Co., Ltd. And ShangHai Shuli Instruments Co.,Ltd., respectively. Xperimental materials: oratosquila oratoria was purchased from Jinan aquatic products market.METHODS: ①C as traditionally prepared from chitin by decoloring with strong oxidant and deacetylating with concentrated alkaline liquor under high temperature. Chitin was extracted from crustae of shrimp and crab fter decalcification with diluted acid and deproteinization with diluted base. ②The improved preparation echnique: The crustae of oratosquila oratoria was decalcificated and decolorized with diluted acid, and as deproteinized and decolorized with diluted base at room temperature, and then the chitin was obtained fter the second decalcification and decolorization with diluted acid. The crude DC was obtained from hitin after deacetylation and decolorization with concentrated ase at 55-65 ℃. The crude DC was issolved n diluted acid and filtered. The filtrate was collected, precipitated with dilute base. After filtering, he sediments were retained, washed with water and dried. Then pure DC was obtained.MAIN OUTCOME MEASURES: he comparison between improved technique and traditional technique; Color, dissolvability in diluted cid, ield, degree of deacetylation and dynamic viscosity of DC prepared by different techniques.RESULTS: ①echnique comparison: Compared with traditional technique, the reaction of improved preparation technique as milder, and the deproteinization was at room temperature; it did not need an isolated decolorization;deacetylation was at low temperature, and had a refine process. ②Detection indexes of DC: he yield of DC prepared by improved technique was little lower than that by traditional technique (15%, 7% respectively). The degree of deacetylation and dynamic viscosity were both higher than those by raditional technique (＞ 95%,＜ 70%; ＞ 120mPa·s,＜ 80 mPa·s , respectively). The color was white or lmost white, which was better than that by traditional technique (yellow or gray). The DC could ompletely issolve in dilute acid, and the dissolvability was better than that by traditional technique (dissolved artly in dilute acid).CONCLUSION: Compared with the traditional technique, the improved technique was mpler and the reaction conditions were milder. And also the quality of obtained DC was obviously improved.

Olive oil is one of the important byproducts in agriculture. It is rich in oleuropein, which can be hydrolyzed into several bioactive phenolic compounds, including hydroxytyrosol (HT).There are many literatures have been confirmed that HT has significant pharmacological effects in the anti-inflammatory, anti-virus, regulation of metabolic disorders and treatment of degenerative diseases. However, HT has severe instability in vivo and in vitro. It is a challenge how to improve its stability through structural transformation or utilization of pharmaceutical means to processing. This paper will focus on biological activity of HT and its stability improvement, and provid some new ideas for expanding the research and promoting the development of HT in the field of Medicine.

Objective To develop a gel permeation chromatography method for determination of content and molecular weight ( Mw ) of Mussel Polysaccharide.Methods Using GPC method, the sample was separated with TSK-gel GMPWXL(7.8 mm ×300 mm) chromatography column which was set at 35℃.The mobile phase was 0.05 mol/mL NaNO3(including 0.05%Na2N3) and the flow rate was 0.6 mL/min.The detector was RID-20AT. Results The average molecular weight of the polysaccharide of Mytilus coruscus was 1 261 411 and the average content was 88.6%by using of the calibration curves of dextrans.The average molecular weight of the polysaccharide of Mytilus edulis was 1 244 062 and the average content was 87.4%. Conclusion The method established in this paper is simple and rapid, accurate and reproducible, which can be used for the quality control of Mussel Polysaccharide.

Objective To construct a eukaryotic expression vector in Pichia pastoris containing human fibrinogen gene, in order to achieve high level secretory expression in extracellular.Methods Expression plasmid,pGAPZαA-FGB-FGG-FGA-AOX1,was constructed by inserting the synthesized sequence encoding human fibrinogen(FGA, FGB,FGG) and then introduced into Pichia pastoris SMD1168H by electroporation.Transformants were availably screened by Zeocin resistance,the expression of recombinant protein was identified by SDS-PAGE and Western blot analysis, the protein yield was tested by ELISA assay.After ultrafiltration and purification, the biological activity of protein was detected.Results The crude yield of human fibrinogen in Pichia pastoris supernatant reached 15 mg/L in flask and the biological aggregation activity was determined.Conclusion The human fibrinogen gene was obtained and successfully expressed in Pichia pastoris and the active products were secreted into the medium.

BACKGROUND:Tissue engineering provides new ideas and approaches for repair of cartilage defects.
OBJECTIVE:To develop a complete set of solutions for construction of tissue engineered cartilagein vitro, with chondrocytes as seed cels and cross-linked sodium hyaluronate as scaffold materials.
METHODS: New Zealand rabbit articular chondrocytes were isolated, counted, and then cultured and passaged to prepare cellsuspension. Toluidine blue staining, RT-PCR and immunocytochemistry were exerted to evaluate the cultured cels. Chondrocytes were seeded and co-cultured with cross-linked sodium hyaluronate scaffold for 21 days. Then, RNA was isolated for RT-PCR, and frozen sections were prepared for morphological observation and immunohistochemistry study.
RESULTS AND CONCLUSION:The chondrocytes could adhere to the cross-linked sodium hyaluronate scaffold and aggregate, growing between fibers or adhering to the scaffold in a monolayer manner. The transcripts of cartilage specific aggrecan gene and colagen type II alpha 1 gene and cartilage specific protein colagen type II were expressed in cel-scaffold complexes to maintain the phenotype of chondrocytes. Cel-scaffold complexes co-cultured in vitro can form cartilage extracelular matrix, by which tissue engineered cartilage is expected to be obtained.

Objective To construct a prokaryotic expression vector in BL21 to secretorily expressα-Cyclodextrin Glycosyltransferase(α-CGTase). Methods α-CGT gene was amplified from Bacillus macerens genome by PCR.pET26b and α-CGT gene were connected after digested with Nco I, Xho I respectivly, and then transformed into Escherichia coli BL21 strain.α-CGTase was expressed in fermentation culture medium and AA-2G was prepared by using α-CGTase, VC and starch.Results α-CGTase was expressed secretorily and the enzyme activity was up to 120 U/mL.AA-2G was prepared by the biotransformation of VC and starch using α-CGTase which proved to be correct by HPLC.Conclusion AA-2G was prepared by using self-madeα-CGTase, after optimized the preparation conditions the yield of AA-2G was 17.46 g/L, and the conversion rate reached 58.2%(mg/mg).