Effect of ion-exchange chromatography on removal of fibronectin in von Willebrand factor / 中国输血杂志

【Objective】 To study the removal effect of fibronectin(Fn) from von willebrand factor(vWF) by ion-exchange chromatography through processing human coagulation factor Ⅷ chromatographic washing products, in order to select a method that can effectively reduce Fn without compromising the activity yield. 【Methods】 In a multi-batch process development experiment, Fractogel® EMD TMAE(M) strong anion filler produced by Merck(Germany) was used to conduct chromatography to investigate vWF ristomycins titer (vWF: RCof), vWF recovery, protein content and Fn content. 【Results】 During the development of vWF pilot purification process, the content of Fn in the samples can be effectively reduced by ion-exchange chromatography, with removal rate more than 87%, titer recovery of vWF more than 80%, and no significant change in other quality indexes. 【Conclusion】 The use of ion-exchange chromatography to purify vWF can effectively reduce the content of Fn, which has positive significance for developing new product process and improving the product quality of blood products manufacturers.

Clinical analysis of gamma-ray fractionated stereotactic radiotherapy for intracranial metastatic polycystic tumors / 中华放射肿瘤学杂志

Objective To analyze the clinical efficacy of gamma-ray fractionated stereotactic radiotherapy (FSRT) in the treatment of intracranial metastatic polycystic tumors.Methods Forty cases with 61 metastatic polycystic tumors were selected from 189 patients with 373 intracranial metastatic tumors admitted to our hospital from 2013 to 2015.All cases received gamma-ray FSRT.The isodose line at 50％ was defined as the prescription dose.The prescription dose was ranged from 40 to 48 Gy/10-12f.The survival rate was calculated by Kaplan-Meier method.The single factor analysis was performed by Log-rank method.Results The median follow-up time was 21months (range:6-39 months).The median survival time was 15.3 months.The 6-month,1-and 2-year local control rate was 93％,82％ and 79％,respectively.The 1-and 2-year survival rate was 63％ and 30％.Single factor analysis demonstrated that the volume of cysts and the volume of lesions were not significantly correlated with local control rate (P=0.17 and 0.48).Conclusion Gamma-ray FSRT can be adopted to treat intracranial metastatic polycystic tumors,which yields similar clinical efficacy to metastatic solid tumors.It deserves wide application in clinical practice due to high local control rate and safety.

Isolation, identification and structural characterization of secondary metabolites from amarine sponge-derived rare actinobacterium Dermacoccus sp. X4 / 生物工程学报

We isolated and identified the symbiotic and adnascent microorganisms from an unidentified sponge collected from 10-meter-deep seawater of the Paracel Islands in China. A total of 16 strains were obtained and identified. Through bacteriostatic activity assay, one of the strains, Dermacoccus sp. X4, was found to effectively inhibit the growth of Staphylococcus aureus. Subsequently, its secondary metabolites were purified by silica gel partition, octadecylsilane (ODS) reverse phase, Sephadex™LH-20 size exclusion, and C18 reverse phase chromatography. Using liquid chromatography, mass spectrometry, and nuclear magnetic resonance, three of the purified compounds were structurally characterized to be one 3-(4-hydroxybenzyl) hexahydropyrrolo [1,2-a]pyrazine-1,4-dione and two indole acid glycerides. This is the first report about indole acid glyceride isolated from microbial secondary metabolites, enriching marine drug candidate resources.

Dye decolorization by bacterial laccase Lac15 / 生物工程学报

We screened for laccase from a marine metagenomic library and obtained a bacterial laccase Lac15 and studied its decolorization ability. Using synthetic azo dyes and anthraquinonic dyes as substrates, we investigated the dye decolorization ability of recombinant Lac15 (rLac15). The purified rLac15 had better decolorization ability towards the azo dyes than the anthraquinonic dyes. When incubated at 45 degrees C and pH 8.5 for 1 h with methylsyringate as the mediator, 20 U/L of rLac15 could decolorize 95% of 100 micromol/L Acid Red 6B (AR-6B), 93% of Reactive Blue 194 (M-2GE), 76% of Reactive Brilliant Orange (K-7R) and 66% of Reactive Blue 171 (KE-R). The decolorization ability of rLac15 decreased with the dye concentration increasing. However, more than 80% of M-2GE and AR-6B were degraded even when the dye concentration was up to 200 micromol/L. At room temperature, rLac51 exhibited significant decolorization ability, with 96% of AR-6B, 86% of M-2GE, 66% of K-7R and 66% of KE-Rdegraded within 24 h at 25 degrees C. rLac15 has the potential of industrial applications.

Site-directed mutagenesis and sulfhydryl PEGylation of lysostaphin / 生物工程学报

The purpose of this paper is to establish sulfhydryl site-directed PEGylation method for lysostaphin and to evaluate effects of mutagenesis and modification of amino acid residue within putative linker on enzyme activity. On the basis of structural analysis of lysostaphin, amino acid 133-154 of tentative linker between the N-terminal and C-terminal domain were chosen as the candidate residues for site-directed mutagenesis to cysteine. Subsequently, sulfhydryl site-directed PEGylation was performed by reacting PEG-maleimide reagent with the newly introduced cysteine residue of the mutant lysostaphin. The Cys-mutant and PEG-modified proteins were both purified, and their enzymatic activity were further PEGylated lysostaphins. The mono-PEGylated lysostaphins were separated from unmodified lysostaphins through highly efficient one step method with Ni(2+)-NTA column chromatography. However, both Cys-mutant and PEGylated lysostaphin only retained partial activities of the wild-type enzyme. It suggests that sulfhydryl site-directed PEGylation modification of the tentative linker between the N-terminal and C-terminal domain may affect the catalytic activity of lysostaphin.

Cloning and characterization of a beta-glucosidase from marine metagenome / 生物工程学报

In the present study, through a functional strategy, a metagenome library of the marine microbes from the surface water of the South China Sea was screened for beta-glucosidase and six positive clones were obtained. One of these clones, pSB47B2, was subcloned and further analysed in sequence. The result showed that there was an open reading frame for a novel beta-glucosidase, which was nominated as bgl1B. Using pET22b(+) as vector and Escherichia coli BL21(DE3) as host, Bgl1B was overexpressed recombinantly with high yield obtained and substantial enzymatic activity detected. The recombinant protein (rBgllB) was purified by Ni-NTA affinity chromatography and further biochemically characterized. The results indicated that, with pNPG as substrate, the optimum pH and temperature for the hydrolytic activity of rBgl1B were about 6.5 and 40 degrees C respectively. Under the optimum conditions, rBgl1B hydrolyzed pNPG with an activity up to 39.7 U/mg, Km and Vmax being 0.288 mmol/L and 36.9 micromol/min respectively. In addition, rBgl1B could also hydrolyze cellobiose, with a Km of 0.173 mmol/L and a Vmax of 35 micromol/min. However, we did not detect evident hydrolytic activity of rBgl1B to lactose, maltose, sucrose, and CMC. The enzymatic activity of rBgl1B to pNPG was stimulated to certain degrees by low concentration of Ca2+ or Mn2+, whereas it exhibited significant tolerance against high Na+. Distinguished from most of the beta-glucosidases derived from fungi, which display the highest activities under acidic conditions, rBgl1B exhibited relatively higher activity and stability at pH between 7.0 and 9.0.

Study on the molecular mechanism of C-phycocyanin from Spirulina platensis induced apoptosis in HeLa cells / 中国药理学通报

Aim To investigate the influence and molecular mechanism of C-phycocyanin(CPC) from Spirulina platensis on apoptosis of HeLa cells in vitro.Methods Firstly,the effect of purified CPC on proliferation of HeLa cells in vitro was determined by MTT assay,and then electron microscope was exploited to observe the characteristic apoptotic features of cells treated with CPC.Subsequently,genomic DNA changes of HeLa cells were observed by agarose electrophoresis.Flow cytometric analysis revealed the influence of different concentrations of CPC on cell cycle of HeLa cells.The expressions of apoptosis related genes of CPC treated HeLa cells were determined by immunohistochemistry analysis.In addition,the activities of caspases and the release of cytochrome c from mitochondria into the cytosol were detected.Results Compared with control cells untreated with CPC,a significant decreased in the numbers of HeLa cells in survival treated with CPC and concentration dose effects existed.CPC could induce characteristic apoptotic features including cell shrinkage,membrane blebbing,microvilli loss,chromatin margination and condensation into dense granules or blocks.DNA of HeLa cells treated with CPC showed fragmentation pattern(DNA ladder of oligomers of 180~200 bp) typical for apoptotic cells.HeLa cells treated with different concentrations of CPC demonstrated an increasing percentage of cells in sub-G0/G1 phase.In addition,CPC could promote the expression of pro-apoptotic gene(Fas and ICAM-1);meanwhile,held back the anti-apoptotic gene(Bcl-2) expression,and then facilitated the transduction of tumoral apoptosis signals that resulted in the apoptosis of HeLa cells in vitro.In CPC treated HeLa cells,CPC treatment of HeLa cells also resulted in activation of caspases and release of cytochrome C from mitochondria into the cytosol.Conclusion C-phycocyanin from Spirulina platensis can induce the apoptosis of HeLa cells in vitro.By virtue of the promotion of the apoptosis signals transduction in HeLa,CPC realizes its antitumor activities.

Effects of the fucoidan from Cladosiphon okamuranusa on Sar-coma_(180)-bearing mice / 中国海洋药物

Objective Effects of fucoidan from Cladosiphon okamuranusa (CF)on mice bearing Siso were investigated. Methods Different concentrations of CF were given to mice bearing tumor by ig. The tumor weight, spleen index, thymus index and serum MDA content, serum SOD activity were detected by physical and biochemical methods. Results The growth of S180 tumor was inhibited significantly by CF in different concentrations. The inhibitory rate ranged from 27. 83% to 33. 97%. The highest inhibition rate occurred in the medium dosage. The spleen index, thymus index and leukocyte counts were not influenced obviously. The content of serum MDA was decreased remarkably and the activity of serum SOD was enhanced notably. Conclusion The mechanism of anti -tumor effects of CF may relate with its antioxidation.

Spontaneous perforation of sigmoid colon(a report of 11 cases) / 中国普通外科杂志

Objective To study the causes, diagnosis and treatment of the spontaneous perforation of sigmoid colon(SPSC). Methods The clinical data of 11 patients with SPSC admitted into our hospital from January. 1984 to September. 2000 were analyzed retrospectively. Results All the 11 cases of SPSC were proved by operation. Among them, 4 cases had chronic constipation history, 1 case complicated with mild proctoptoma before operation and the other 6 cases had no special records. 9 cases were misdiagnosed as perforation of acute appendicitis, perforation of upper gastroenteric tract, perforation of colon carcinoma, inflammatory perforation or fecal mass perforation. 2 cases recovered after repairing operation; of 9 cases underwent colostomy and other procedure, 7 cases recovered and colostomy closure was done 2-3 months after operation. 2 cases died after operation. Conclusions The occurrence of SPSC is closely related to the configuration and position of the patient's sigmoid colon. Increase of intra-abdominal pressure and intra-intestinal pressure are the predisposing causes of the disease. This disease has no special clinical manifestation. The key to diagnosis is a full knwledge of the disease before operation. The main treatment is operation. The mode of operation is determined according to patient's age, general condition, severity of abdominal pollution, attacking time. The best mode of operation is to repair and close the perforation or to resect and anastomose the bowel and colostomy.

Clinical study of the effect of actovegin in the prevention of acute irradiation oropharyngeal mucositis / 实用口腔医学杂志

0.05). Regarding to the grade of irradiation mucositis, the majority of patients in the treatment group were at stage Ⅰ-Ⅱ,while those in the control at stage Ⅲ-Ⅳ(P0.05).No adverse drug reaction was observed in both groups during irradiation process.Conclusion:Actovegin can postpone the development of irradiation oropharyngeal mucositis and decrease the incidence of gradeⅢ and Ⅳmucositis.

Effects of polysaccharide from spirulina platensis on the growth of HeLa cells / 中国海洋药物

Objective To investigate the effects of polysaccharide from Spirulina platensis (PSP) on the growth of HeLa cells. Methods The antiproliferation activities of PSP on HeLa cells were evaluated by MTT assay. The morphologic changes of HeLa cells were observed under light microscope. Cells division cycle was measured by flow cytometry. Results It was showed that with the increase of PSP concentration, the inhibited rate on HeLa cells increased and the survived rate declined; The HeLa cells exposed to PSP for 24～48h displayed significant morphologic changes; PSP had a specific effect on the HeLa cell division cycle, increased the number of cells in phase G 1. Conclusion These results indicated that the effects of PSP on HeLa cells may probably be attributed to increase of the number of cells in phase G 1.

Mass cultivation of EPA-rich marine microalgae Nannochloropsis oculata / 中国海洋药物

Industrial culture media for cultivation of marine microalgae Nannochloropsis oculata was optimized by using Random Design software. High eicosapentaenoic acid (EPA) content was obtained over 40% of total fatty acid, and high average specific growth rate of 0.32d -1 was obtained in cells grown in the selected Formula 1. Mass cultivation of this species was practiced in indoor open raceway ponds of 6.12 m 2 and 28.02 m 2, and in outdoor open pond of 83.50 m 2 during May and June in 1999. The average area yield of biomass were obtained at 2.77, 1.62 and 2.52 g /m 2?d, and in October, 1.09 g/ m 2?d was obtained in 83.5m 2 outdoor pond, and the harvest algae powder contained 49.50% of protein, 20.30% of total lipid and 3.00% of EPA of dry weight. With high temperature tolerant species during August in 2000, the average area yield of biomass were increased to 5.44?9.96 and 7.66 g/ m 2?d in above ponds, respectively and 2.50% of EPA of dry weight was contained in the powder. It is indicated that the industrialized cultivation of Nannochloropsis oculata rich in EPA is completely doable on practice, and the algal powder was a good raw material as single cell protein and development of EPA rich products.

Hypocalcemic effect of recombinant Saccharomyces cerevisiae expressing salmon calcitonin / 解放军医学杂志

Objective To investigate the effect of gavaged yAGA2-sCT, a recombinant Saccharomyces cerevisiae which expressed salmon calcitonin on its surface, on serum calcium concentration of rats. Methods The sCT gene was artificially synthesized in advance and cloned into the pYD1 vector, a kind of surface displaying plasmid, to yield pYD1-sCT. The recombinant plasmid pYD1-sCT was transformed into S. cerevisiae EBY100 by LiAC method and achieved yAGA2-sCT. The sCT protein, which was expressed on the surface of recombinant S. cerevisiae yAGA2-sCT, was indirectly labeled with FITC. Labeled yeast was immediately detected under the fluorescent microscope. Before being gavaged to rats, recombinant yeast was frozen and vacuum-dried. Lyophilized yeast yAGA2-sCT was administered to rats in dosage of 0.1g/kg, 0.5g/kg and 5.0g/kg, respectively, with 6 rats in each group. Positive drug group (200mU/kg calcitonin via hypodermic injection) and two conditional control groups (being gavaged 5g/kg yAGA2-sCT and 30?g/kg calcitonin, respectively) were acted as controls. The serum was collected from canthus venous plexus and the calcium concentration was immediately measured by automatic biochemical analyzer. Results The sCT protein was successfully expressed on the surface of recombinant yAGA2-sCT, and FITC-labeled yAGA2-sCT was observed under the fluorescent microscope. The recombinant S. cerevisiae yAGA2-sCT was able to reduce the serum calcium concentration of rats in a dose-dependent manner. In the three subgroups, the hypocalcemic effect of high-dosage subgroup, with 5g/kg lyophilized yAGA2-sCT, was the highest (P