Recombinant expression of black sesame polyphenol oxidase and its enzymatic properties / 生物工程学报

To investigate the enzyme properties of the black sesame polyphenol oxidase (BsPPO), a synthesized Bsppo gene was cloned into the vector pMAL-c5x and expressed in E. coli. Subsequently, the MBP fusion label in the recombinant protein was removed by protease digestion after affinity purification. The synthesized Bsppo gene contained 1 752 bp which encodes 585 amino acids with a deduced molecular weight of 65.3 kDa. Transformation of the recombinant vector into E. coli BL21(DE3) resulted in soluble expression of the fusion protein MBP-BsPPO. The enzymatic properties of the recombinant BsPPO was investigated after MBP fusion tag excision followed by affinity purification. The results demonstrated that the optimal temperature and pH for BsPPO was 25°C and 4.0, respectively. BsPPO exhibited a good stability under low temperature and acidic environment. Low-intensity short-term light exposure increased the activity of BsPPO. Cu²⁺ could improve the activity of BsPPO while Zn²⁺ and Ca²⁺ showed the opposite effect. BsPPO could catalyze the oxidation of monophenols, diphenols, and triphenols, and exhibited good catalytic activity on l-tyrosine and vanillic acid. Moreover, BsPPO exhibited high catalytic activity on black sesame metabolites, including 2-methoxy cinnamic acid, indole-3-carboxylic acid and phloretin. These results may serve as a basis for further characterization of BsPPO.

Expression and characterization of recombinant wheat quiescin sulfhydryl oxidase and its effect on bread quality / 生物工程学报

Wheat quiescin sulfhydryl oxidase was expressed in Escherichia coli for developing a new biological flour improver. The synthesized wqsox gene was constructed into the vector pMAL-c5x and expressed in E. coli, then the expression conditions of recombinant protein was optimized. The MBP fusion label in recombinant protein was removed by protease digestion after affinity purification. Moreover, enzymatic properties of the purified wQSOX and its effect on bread quality were investigated. The synthesized wqsox gene contained 1 359 bp and encoded 453 amino acids with a deduced molecular weight of 51 kDa. The constructed recombinant vector pMAL-c5x-wqsox could successfully express soluble recombinant protein MBP-wQSOX in E. coli Rosetta gamiB(DE3), and the optimal induced expression conditions for recombinant protein were 25 °C, 0.3 mmol/L IPTG and 6 h. MBP fusion tag was cut out by factor Xa protease and wQSOX was prepared after affinity purification. wQSOX could catalyze the oxidation of DTT, GSH and Cys, accompanying the production of H2O2, and exhibited the highest substrate specificity for DTT. Furthermore, enzymatic properties results demonstrated that the optimal temperature and pH for wQSOX catalyzing oxidation of DTT was 50 °C and 10.0, respectively, and wQSOX presented a good stability under high temperature and alkaline environment. The addition of wQSOX with 1.1 U/g flour significantly (P<0.05) increased 26.4% specific volume of the bread, and reduced 20.5% hardness and 24.8% chewiness of bread crumb compared to the control, indicating a remarkable ability to improve the quality of bread.

Influences on induction and maturation of mouse bone marrow-derived dendritic cells by varying combinations of cytokines / 中华微生物学和免疫学杂志

Objective To investigate the influences of culture conditions on the in vitro induction and maturation of dendritic cells by using different combinations of cytokines. -ethods Mouse bone mar-row cells were isolated and cultured in media containing varying combinations of cytokines, including granu-locyte-macrophage colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and fms-like tyrosine kinase 3 ligand (Flt3L). After cultured at 37℃ for seven days, the attached bone marrow cells were collected and stained by fluorescence-labeled monoclonal antibodies (McAb) against CD11c, MHCⅡ and CD86 for flow cytometry analysis. In the parallel group, LPS was added on day 5 to a final concentration of 1μg/ml for DC maturation analysis by flow cytometry. Results In group Flt3L (20 ng/ml)/GM-CSF (20 ng/ml)/IL-4 (10 ng/ml), 90％ of bone marrow cells were CD11c-positive. Flt3L (100 ng/ml) could induce 88％ of bone marrow cells to express CD11c. Bone marrow cells positive for MHCⅡ accounted for 35. 4％ and 36. 1％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF, where both Flt3L and GM-CSF were used at a concentration of 20 ng/ml. After LPS stimulation, the positive rates of MHCⅡ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF were 58. 1％ and 59. 6％, which increased by 22. 7％ and 23. 5％, re-spectively. The percentages of CD86-positive bone marrow cells were 7. 1％ and 5. 5％ in group Flt3L/GM-CSF/IL-4 and group Flt3L/GM-CSF. Bone marrow cells positive for CD86 grew by 7. 1％ and 6. 2％ in group Flt3L (20 ng/ml) and group GM-CSF/IL-4 after LPS stimulation. Conclusions Flt3L and GM-CSF probably dominated the differentiation and maturation of bone marrow-derived dendritic cells with a synergis-tic effect. Combined usage of Flt3L and GM-CSF at the concentration of 20 ng/ml would be an optimal proto-col for DC research.

Exploration of Domestic Medical Electrical Equipment Reliability Promotion Method / 中国医疗器械杂志

The reliability of domestic medical equipment is one of the main factors that restrict the competitiveness of domestic medical devices. It is also an important factor that endangers the safety of patients and a blind spot in safety risk management. By analyzing the core elements of reliability and the situation of domestic medical device industry, this paper sorts out and analyzes the problems existing in the reliability of medical device industry, and puts forward the key points and problems to be solved to improve the reliability of domestic medical equipment products.

Study on Serum Levels of C1q and Their Clinical Significance in Children with Nephrotic Syndrome / 现代检验医学杂志

Objective To analyze serum levels of C1q in children with nephrotic syndrome (NS),and investigate the clinical significance and the relationship among the altered serum C1q levels and other lipid/lipoprotein and renal function parametersin children with NS inacute and remission phases.Methods Serum levels of C1q were measured in 78 NS children with acute phase,in 64NS children with remission and in 77 healthy control children.The other lipid/lipoprotein and renal function parameters were also analyzed in these children,including TP,ALB,TC,TG,LDL-C,HDL-C,Urea,Cr and Uric.Results Compared with the healthy control children [173.00(161.00～185.00)mg/L],children with NS inacute [203.50(183.75 ～ 223.75) mg/L] and remission phases [185.00 (161.00 ～ 202.00) mg/L] all had a significantly increasedserum levels of C1q.Compared with NS children in remission,those in acute phase showed a significantly increased C1q (P＜0.001).In all the NS children,the serum levels of C1q were positively correlated with the levels of TC (r=0.483,P＜0.001),TG (r=0.423,P＜0.001) and LDL-C (r=0.450,P＜0.001),while negatively correlated with the levels of TP (r=-0.276,P=0.001 ＜0.01) and ALB (r=-0.410,P＜0.001).Multiple linear regression analyses showed that serum levels of C1q were independently associated with serum TG levels (β=9.235,P＜0.001;adjusted R2 =0.215) after adjustment of other related factors.Conclusion Serum levels of C1q were significantly increased in NS children in association with their conditions and the levels of lipid/lipoprotein parameters,and may be function as anovel parameter for assessing the development of NS.

Comparison of two mouse models of alcoholic liver disease induced by oral ethanol gavage or Lierber-DeCarli ethanol liquid diet / 中国比较医学杂志

Objective To select a simple, stable and reliable mouse model of alcoholic liver disease. Methods The mouse models of alcoholic liver disease were induced by oral gavage ethanol or Lierber?DeCarli ethanol liquid diet for 8 weeks. The food intake and body weight were recorded. Pathological changes were examined using HE staining. Liver injury was assessed by the activities of serum ALT, AST, AKP and γ?GT, and serum and hepatic TC and TG. Results After modeling, both models showed significantly increased activities of serum ALT, AST, AKP, and contents of serum and hepatic TG (P<0?05), indicating the successful development of alcoholic steatohepatitis. However, oral ethanol gavage led to body weight loss and weak mental state. Ethanol liquid diet less affected the body weight and mental state. Ethanol liquid diet enhanced liver to?body weight ratio and serum TC, but oral gavage of ethanol did not. The changes of serum ALT, AST, serum and hepatic TG, and hepatic steatosis in the ethanol liquid diet models were more severe than those in the oral gavage ethanol models, suggesting that Lierber?DeCarli ethanol liquid diet led to more serious liver injury than oral gavage ethanol. Conclusions Lierber?DeCarli ethanol liquid diet model is better than oral gavage ethanol model, and is more suitable for studies on mechanisms and evaluation of hepato?protective drugs for alcoholic liver disease.

Metabolic Profile of Dyskinetic Cerebral Palsy Based on Metabonomics / 中国康复理论与实践

Objective To analyze the metabolic profile of children with dyskinetic cerebral palsy by metabolomics, and its abnormal metabolic pathway. Methods The serum of 10 children with dyskinetic cerebral palsy (patient group) and 7 healthy children (control group) aged 6 to 12 years were collected at clinic from May to August, 2014. The serum samples were tested by the nuclear magnetic resonance spectrometer and the spectroscopies were discriminated by partial least squares-discriminant analysis. According to the human metabolome database, the final metabolites disturbed would be figured out. Results 15 chemical shifts were defined, and 6 of them, including 2.04 ppm, 2.12 ppm, 3.00 ppm, 3.24 ppm, 3.76 ppm, 6.50 ppm, were significantly different between 2 groups (P<0.05). The KEGG Pathway Database showed that the levels of taurine, fumarate, oxaloacete, pyruvate, citrate, aspartate, succinate, malate, cysteine decreased, and the levels of glutamate, 2-oxoglutarate, glutamine, leucine, alanine increased. The abnormal metabolism was found in taurine metabolism, glutamine me-tabolism and energy metabolism pathways. Conclusion Based on metabolomics, the metabolic profile of children with dyskinetic cerebral palsy was discriminated out successfully. The further research can focus on the small molecules found out.

Effects of peptide-based enteral nutrition on inflammatory response and immune function in intestinal mucositis rat / 中华临床营养杂志

Objective To evaluate the effects of peptide-based enteral nutrition (PBEN) on inflammatory response and immune function in rats with intestinal mucositis.Methods 48 Sprague-Dawley male rats were randomly divided into 6 groups (all n =8):basal feed group (BFG),PBEN group (PBENG),intact protein enteral nutrition group (IPENG),methotrexate (MTX) + BFG,MTX + PBENG,and MTX + IPENG.The rats in MTX + BFG,MTX + PENG,and MTX + IPENG were intraperitoneal injected with MTX 10 mg/kg on day 0 and day 6 to induce sustained intestinal injury.From day 1,BFG and MTX + BFG were fed with basal feed,PBENG and MTX + PBENG with PBEN,IPENG and MTX + IPENG with IPEN.The daily energy intake of each rat was 1.80 kJ/g body weight.All the rats were sacrificed on day 11.The pathological changes of intestinal tissue were observed with HE staining,the levels of tissue myeloperoxidase (MPO),nitric oxide (NO),inducible nitric oxide synthase (iNOS),the thymus index and spleen gland index of intestinal tissue were measured using colorimetry,and the serum levels of immunoglobulins IgG,IgA,and IgM were determined with enzyme-linked immunosorbent assay.Results There were no significant differences among BFG,PBENG,and IPENG in each index.Serious injury of intestinal mucosa was observed in MTX groups.Significant differences were noted in all indexes between MTX + BFG and BFG (all P ＜0.05).The mucosal damage score (Chiu score) and the level of MPO and iNOS in MTX + PBENG were significantly lower than those in MTX + BFG [2.3 ± 0.69vs.2.96 ± 0.75,P =0.003 ; (2.30 ± 0.42) U/g tissue vs.(2.98 ± 0.23) U/g tissue,P =0.040 ; (0.37 ±0.06) U/mg prot vs.(0.44 ±0.10) U/rag prot,P =0.030] ; the serum levels of IgG and IgA were significantly higher than those in MTX + BFG (P =0.015,P =0.021) ; however,the levels of NO and IgM were not significantly different between the 2 groups (P =0.597,P =0.160).There were no statistically significant differences between MTX + IPENG and MTX + BFG in terms of the indexes (all P ＞ 0.05).Conclusion PBEN can reduce the inflammation response and improve the immune function in intestinal mucositis rat.

Establishment and assessment of a rat model of persistent intestinal mucosal injury induced by methotrexate / 中国实验动物学报

Objective To establish a rat model of persistent intestinal mucosal injury.Methods The rat model of persistent intestinal mucosal injury was induced by intraperitoneal injection of methotrexate.The food intake and body weight of all the rats were recorded.Pathological changes were observed using HE staining, the level of D-lactate and diam-ine oxidase in plasma, and myeloperoxidase and malondiadehyde in the intestinal tissue were measured by biochemistry. Results After modeling, the rat body weight and food intake were decreased.On day 4, the scores of mucosal damage, the levels of plasma D-lactate, DAO, MPO and MDA were significantly increased (P<0.05).On day 5, the intestinal damages of rats began to restore, and there was no significant difference among groups on day 6.The symptoms after the secondary injection were similar to those after the first injection, and the rats recovered gradually at day 12.Conclusions Intestinal mucosal injury in rats induced by 20 mg/kg MTX is an acute injury process, the course only lasts for 4-5 days. Intermittent injections twice of 10 mg/kg MTX can cause persistent intestinal mucosal injury in rats.This persistent injury model is more suitable for nutritional therapy evaluation in medium-and long-term studies of nutritional therapy.

Determination of Five Anthraquinones Constituents in Yanxiaodinaer Syrups / 中国药师

Objective:To establish the method for the determination of five anthraquinones constituents including aloe-emodin , rhein, emodin , chrysophanol and physcion in Yanxiaodinaer syrups. Methods: An HPLC method was used with a DIONEX Acclaim 120A C18 (250 mm × 4. 6 mm,5 μm) column. The mobile phase was acetonitrile-0. 1% phosphoric acid with gradient elution at flow rate of 1. 0 ml·min-1 . The detection wavelength was 254nm,the column temperature was 30℃and the injection volume was 10 μl. Results:The linear ranges of the five anthraquinones were 0. 018 7-0. 467 0 μg(r=0. 999 8),0. 020 2-0. 504 0 μg(r=0. 999 9), 0.013 0-0.390 6 μg(r=0.999 9),0.013 3-0.399 9 μg(r=0.999 9)and 0.009 3-0.277 8 μg(r=0.999 9), and the recoveries were 94. 30%(RSD=2. 68%,n=6),95. 49%(RSD=2. 50%,n=6),93. 70%(RSD=2. 42%,n=6),92. 52%(RSD=2. 40%,n=6) and 93. 72%(RSD=2. 54%,n=6), respectively. Conclusion:The method is accurate , sensitive and reproducible, which can be used as the basis for the quality control of Yanxiaodinaer syrups.

Diagnosis and treatment of unexpected gallbladder carcinoma in laparoscopic cholecystectomy / 中华肝胆外科杂志

Objective To explore the feasibility of radical cholecystectomy for early gallbladder car cinoma found during or after laparoscopic cholecystectomy.Methods A retrospective study was conducted on patients who received laparoscopic cholecystectomy between January 2007 to August 2013 and were diagnosed to have gallbladder cancer during or after the operation.There were 34 patients.In 29 patients intraoperative frozen section diagnosed gallbladder carcinoma.In 5 patients postoperative histopathological study diagnosed stage Ⅰ or Ⅱ gallbladder carcinoma.Results Surgery was conducted successfully on these 34 patients.In 20 patients with stage Ⅰ,Ⅱ and Ⅲ,the tumor had invaded the serosa,or into the liver with a depth of less than 2 cm,laparoscopic cholecystectomy alone or radical/extended radical cholecystectomy were carried out.In 9 patients,the laparoscopic surgery was converted to open surgery and these patients underwent cholecystectomy with resection of the adjacent liver segments/sections.In 5 patients who were diagnosed to have gallbladder carcinoma after laparoscopic cholecystectomy,they were re-operated with laparoscopic radical cholecystectomy.Conclusions Stage Ⅰ,Ⅱ and Ⅲ gallbladder carcinoma with tumor invasion into serosa,or patients with tumor invasion into the liver with a depth of less than 2 cm should undergo radical or extended radical cholecystectomy.Laparoscopic assisted radical or extended radical cholecystectomy could achieve the same operation as with open surgery but with better short-term results.There were less pain,smaller incisions,better scars and shorter hospitalization stay.

The relationship between Rac1 mRNA gene levels detected by FQ-RT-PCR and the metastasis in gastric carcinomas / 中华检验医学杂志

Objective To investigate the relationship between tumor metastasis-related Rac1 mRNA expression levels and gastric carcinomas metastasis, and to investigate the significance of Rac1 as a tumor marker for the evaluation of gastric carcinomas metastasis and distinguish between benign and malignant lesions. Methods This experiment used fluorescence quantitative RT-PCR TaqMan probe technology, chose Rac1 target gene fragment, which was cloned into the pET-20b (+) vector, constructed recombinant plasmid, and established the Rac1 mRNA fluorescence quantitative RT-PCR standard curve. And then the Rac1 mRNA levels were detected in 52 cases of gastric carcinoma tissues,52 cases of para-carcinoma tissues and 12 cases of benign gastric disease tissues. Its association with the metastasis of gastric carcinomas was analyzed. Results The positive rates and levels (median, P25-P75) of Rac1 mRNA were 100. 0% ,7.41 ×105 (3. 50 × 105-4. 36 × 106) copies/μl in gastric carcinomas, 46. 2% ,0(0-1.73 × 104) copies/μl in parscarcinoma tissues, 33. 3%, 0(0-3.55 × 103) copies/μl in benign tissues. The positive rates and leves(x2 =43.16,x2Xk-w = 64. 19, P ＜0. 01)among the three groups were significantly different. When the critical value of Rac1 mRNA level was 1.73 × 104 copies/μl determined by ROC, the sensitivity and specificity were 88. 5% and 100% respectively. When the critical value of Rac1 level was 7.49 × 105 copies/μl, the sensitivity and specificity were 57.9% and 78. 6% respectively. ConclusionThe Rac1 mRNA level detected with fluorescence quantitative RT-PCR has reference value to distinguish between benign and malignant lesions and evaluate gastric carcinoma metastasis.

ADR Case Reports of Our Hospital in 2008:An Analysis of 117 Cases / 中国药房

OBJECTIVE:To probe into the general regularity and characteristics of ADR in our hospital. METHODS:117 ADR reports were analyzed retrospectively in respect of the patient’s gender,age,dosage form,route of medication,medication combination,category of drugs,involved organs and system and clinical manifestation,ect. RESULTS:Of total 117 cases of ADR,female cases of 69 accounted for 58.97% while 48 male cases 41.03%. ADR cases were mainly found in the group aged from 41 to 50(23.93%). ADR was predominantly caused by intravenous drip infusion(86.32%). 46 kinds of drugs in 10 categories were involved,in which antimicrobial drug took dominate place including 20 kinds in 10 categories(58.97%). The most common clinical symptom represented as lesion of skin and its appendants(54.70%). 117 cases were cured and improved without death case. CONCLUSION:Great importance should be attached to rational use of drug and safety of drug use of special population.

Effect of Retroviral Vector-Mediated IL6/IL2 Fused Gene Transduction on B16 Melanoma Cells Adhesive and Metastatic Potential / 中国肿瘤生物治疗杂志

In order to examine the feasibility of cytokine fusion gene transfer into tumor cells, a retroviral vector (pLXSN) for human interleukin 6/interleukin 2 (IL6/IL2) fusion gene was constructed by using PCR and ligating the 2 genes with a synthesized flexible linker. The IL6/IL2 fused gene was introduced into B16 melanoma cell line mediated by retrovirus and the changes of adhesive and metastatic ability of fused gene-modified cells were detected. The B16-IL6/ IL2 cells showed efficient expression of both cytokines and decreased binding affinity with extracellular matrix (Laminin and Matrigel), accompanying with decreased experimental metastatic potentials. These data suggest that the mechanism involving the decreased metastases may relate with the changes of biological characteristics and immune stimulating activity of gene-modified cells.