γ-glutamyltranspeptidase was detected from the cultured mycelia of Cordyceps sinensis (CSGT). Km and Vmax of CSGT was 2.54×10-4 mol/L and 0.1808 mol/L·min respectively when L-glutamic acid 5-(4-nitroanilide) (GpNA) and glycyglycine was used as its substrate. CSGT was stable from pH 8.0 to 11.0 and at or below 20℃. It was optimally active at pH 9.0～10.0 and 30℃. A series of reducing reagents could activate CSGT, and metal cations such as Zn2+, Cu2+, Hg2+ , Mn2+ inhibited strongly activity of the enzyme, but K+, Ca2+, Mg2+ and Na+ at high concentrations had no effect on its activity, indicating that its active center could contain -SH.

OBJECTIVE：To improve the synthesis of aromatic nitriles.METHOD:Aromatic aldehydes were converted into aromatic nitriles by the ammonium chloride,copper powder,oxygen system in pyridine.RESULTS:Ten aromatic nitriles were synthesized by this method.CONCLUSION:Compared with literatures,this method has following merits:reagents are very convenient and not expensive,operation is simple and the yield is good.

BACKGROUND:The structure of tissue engineering carrier affects the bio-action of cells greatly.OBJECTIVE: To investigate the biological characteristics of bone marrow stem cells (MSCs) in different concentrations of alginate combined with de-antigen bone xenograft (DBX).DESIGN: Observational trial.SETTING: PLA Institute of Trauma and Orthopedics, the Fourth Military Medical University of Chinese PLA.MATERIALS: Alginate, calcium chloride, MSCs, bone xenograft.METHODS: Bovine cancellous bone was out into cubes, which were degreased, deproteinized and then lyophilized.Cubes in pore size within 300-500 μm were selected for use after ethylene oxide sterilization. The purified sodium alginate was dissolved in DMEM cell culture medium of concentrations as different as 0.5%, 2%, 8% and 16%; 1×1012 L-1 induced MSCs were blended with isopyknic alginate-DMEM and compounded with DBX at a status of 0.5 Mpa negative pressure for 5 minutes in order to make a cell suspension fully fill into the pores of the cancellous bone. Then alginate was crosslinked with 50 g/L calcium gluconic acid for 30 seconds. The complex was put into a CO2 incubator and cultured for 4 days. The gel compound and cell growth in the pores of the complex were grossly observed with an inverted microscope. Status of cell growth in the complex with different concentrations of alginate was observed with scanning electron microscopeMAIN OUTCOME MEASURES: Compound status of alginate and bone xenograft, cell growth status and matrix secretion in compound carries.RESULTS: When the concentration of alginate was 0.25% or 1%, alginate was equally combined in DBX, while that of 4% and 8% only combined on the surface of cancellous bone. After in vitro cultured for 4 days, alginate of 0.25% were broken off from DBX surface. But alginate of 1% was equally combined with DBX pores with cells secreting well in alginate. Development of cells in alginate of 4% was restricted and no cells were seen in alginate of 8%.CONCLUSION: Alginate of 1% is suitable for constructing the carrier of bone tissue engineering with bone xenograft.

Objective To investigate the early diagnostic value of EBV-DNA load in Plasma and peripheral blood mononuclear cells (PBMCs) for Children′s Primary Infectious Mononucleosis(IM).Methods The plasma and PBMCs samples were collected from children of 67 cases with primary IM (IM groups) and 75 healthy cases (H groups) during April, 2015 to March, 2016 in our hospital. The EBV-DNA load in PBMCs and plasma samples were quantified by Real-time PCR. Statistical differences between two groups were analyzed by using Mann-Whitney Test. Receiver operating characteristic curves (ROC) were analyzed to assess the early diagnostic value of EBV-DNA load for primary IM.Results Levels of EBV-DNA in the samples of plasma were significantly higher in the IM groups (median, 380 IU/ml) compared to the H groups (Z=-9.332,P<0.01), and levels of EBV-DNA in the PBMCs samples were also significantly higher (median, 2.51×105 IU/ml, Z=-9.194,P<0.01). According to ROC curve analysis, the AUC of EBV-DNA load in plasma was 0.908, the most appropriate cut-off value was 12.5 IU/ml (sensitivity: 83.6% and specificity: 94.7%),the AUC of EBV-DNA load in PBMCs was 0.944, the most appropriate cut-off value was 2.19×104 IU/ml (sensitivity: 89.6% and specificity: 82.7%), the AUC of combined testing(plasma and PBMCs) was 0.967(sensitivity: 98.3% and specificity: 99.0%).Conclusions Combined testing of plasma and PBMCs have better clinical value, it may be useful for the diagnosis of early stage of Children′s Primary Infectious Mononucleosis.

BACKGROUND: Bone morphogenetic protein(BMP) is one of the most important cytokines that induce and promote seed cells to be transformed into osteocytes. Insoluble natural BMP can hardly affect the life of cultured seed cells. The expensive soluble recombinant BMP is also hard to work on the seed cells at the appropriate time and dose. Therefore, gene therapy technique provides us with a brand new idea of using gene-modified seed cells.OBJECTIVE: To transfect exogenous BMP-3 gene into the fibroblasts and screen the positive fibroblast clones that can express BMP-3 stably.DESIGN: Simple sample study.SETTING: Orthopaedic Research Institute, Xijing Hospital, Fourth Military Medical University of Chinese PLA.MATERIALS: The fibroblasts(NIH3T3) were kindly presented by Professor Situ Zhen-qiang of the Stomatological College of Fourth Military Medical University of Chinese PLA.METHODS: This experiment was conducted in the Key Laboratory of Chinese PLA, which belongs to the Orthopaedic Research Institute of Fourth Military Medical University. BMP-3 gene was transfected into the fibroblasts through lipofectamin. The transfected cells were screened by G418. The separated cloned cells were identified through immunohistochemistry. The positively stained cells were the clones of BMP-3 expressing fibroblasts.MAIT OUTCOME MEASURES: The screening concentration of NIH3T3 cells, screening of positive transfected cells, and expression of BMP-3 in screened cells.RESULTS: BMP-3 gene was successfully transfected into the fibroblasts. BMP-3 expressing fibroblast clones were creened and identified through immunohistochemistry. Fibroblast strains with stable BMP-3 expression were obtained.CONCLUSION: The transfection of BMP-3 gene eukaryonic expression vector into the fibroblasts and obtaining of fibroblast strains with BMP-3 expression have laid foundation for the usage of gene-modified seed cells in future research of bone tissue engineering.

Objective To validate the effect of anti-infective reconstituted bone xenograft (ARBX) in treating posttraumatic osteomyelitis by one-stage grafting in the adults.Methods With clinical application approval of Medical Command,Logistics Ministry of PLA,ARBX was used to treat 27 adult patients (29 lesions) with posttraumatic osteomyelitis by one-stage grafting after debridement since September 2001.The study analyzed 27 patients (29 grafts) who were followed up for average 26 months (12-63 months).Results The follow-up for average 26 months (12-63 months) in 27 patients showed that infection of 22 patients (24 lesions) was controlled and cured,except for three with failure to control the infection or with recurrence of infection,two with controlled infection but with postoperative nonunion.The infection control rate was 89.7% (26/29) and the cure rate was 82.8% (24/29) ,which were better than the results of traditional therapy.Conclusions ARBX has high osteoinductive activity and enhanced anti-infective capability,which enables it to be used as one-stage grafting to treat posttraumatic osteomyelitis in the adults.

Objective To investigate the effects and mechanisms of hydrogen inhalation on serum levels of pro-inflammatory factors and intestinal injury in severe septic mice. Methods 176 male ICR mice were randomly divided into four groups: sham operation group, hydrogen control group ( sham + hydrogen inhalation ), model group ( severe sepsis model ) and hydrogen treatment group ( severe sepsis model+hydrogen inhalation ), with 44 mice in each group. Severe sepsis model was reproduced by cecal ligation and puncture ( CLP ). 2%hydrogen inhalation was given for 1 hour at 1 hour and 6 hours after sham or CLP operation. Twenty animals in each group were selected and observed for 7-day survival rate. Six animals in each group were selected and sacrificed at 6, 12, 24 and 48 hours after sham or CLP, the concentrations of tumor necrosis factor-α ( TNF-α), interleukins ( IL-6, IL-10 ) and high mobility group box 1 ( HMGB1 ) in serum were determined, the intestinal histopathological changes and scores were evaluated by light microscopy, and the activities of myeloperoxidase ( MPO ) and caspase-3 were determined. Results The 7-day survival rate of severe sepsis mice was 0; the 7-day survival rate was increased to 60% in hydrogen treatment group, with statistical significance in variables compared with model group ( P<0.05 ). Compared with sham operation group, the serum concentrations of TNF-α, IL-6, IL-10 and HMGB1 were obviously increased, the intestine were heavily injured along with higher histopathological scores, and the intestinal MPO and caspase-3 activities were significantly enhanced at different time points after CLP in model group ( all P<0.05 ). Compared with model group, the serum concentrations of TNF-α, IL-6 and HMGB1 were significantly decreased [ TNF-α( ng/L ):6 hours:110.34±9.28 vs. 440.55±25.78, 12 hours: 82.29±8.43 vs. 448.36±32.54, 24 hours: 79.68±9.04 vs. 346.42±22.24, 48 hours: 80.79±10.06 vs. 368.94±31.58; IL-6 ( ng/L ): 12 hours: 58.68±8.55 vs. 158.28±16.73, 24 hours: 46.98±7.58 vs. 146.74±18.02, 48 hours: 38.67±8.22 vs. 136.45±15.45; HMGB1 (μg/L ): 6 hours: 15.75±4.32 vs. 55.56±10.04, 12 hours:32.02±9.33 vs. 89.65±15.65, 24 hours: 35.87±8.54 vs. 86.44±20.33, 48 hours: 23.85±9.83 vs. 98.33±18.88, all P<0.05 ], the serum concentrations of IL-10 ( ng/L ) at 24 hours and 48 hours after CLP were obviously increased ( 24 hours:135.44±16.43 vs. 79.22±12.03, 48 hours:110.92±12.54 vs. 74.47±11.18, both P<0.05 ), the intestinal injury were ameliorated with decreased histopathological scores ( 12 hours: 1.70±0.06 vs. 3.23±0.44, 24 hours:2.12±0.31 vs. 4.51±0.58, 48 hours:2.03±0.42 vs. 4.27±0.58, all P<0.05 ), and the intestinal MPO and caspase-3 activities were significantly decreased [ MPO ( U/g ):6 hours:13.75±4.21 vs. 25.56±5.34, 12 hours:14.72±4.22 vs. 30.53±6.87, 24 hours:11.62±3.14 vs. 33.58±7.24, 48 hours:11.33±4.03 vs. 38.57±8.12;caspase-3 ( fluorescence intensity ): 6 hours: 0.37±0.07 vs. 0.69±0.23, 12 hours: 0.42±0.07 vs. 0.86±0.13, 24 hours: 0.53±0.11 vs. 1.36±0.23, 48 hours:0.50±0.08 vs. 1.48±0.15, all P<0.05 ] in hydrogen treatment group. Conclusion Hydrogen inhalation can down-regulate the systemic inflammatory response to ameliorate the intestinal injury, and it may improve the septic process and increase the survival rate of mice with severe sepsis.

Objective To evaluate the effect of sevoflurane anesthesia on activation of gliocytes in aged rat hippocampi.Methods One hundred and four male Wistar rats,weighing 600-650 g,were randomly divided into 2 groups (n =52 each) using a random number table:control group (group C) and sevoflurane group (group S).Group S inhaled 3.6％ sevoflurane for 2 h,and open reduction and internal fixation was performed after tibial fracture was induced.Y-maze test and fear conditioning test were carried out on days 1,3 and 7 after surgery.Six rats of each group were chosen on days 1,3 and 7 after surgery and hippocampi were removed to detect the activation of microglias and astrocytes (by immunofluorescence) and expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) (by Western blot).Results Compared with group C,the number of entries into each arm was significantly reduced,the duration of N arm stay was shortened,the percentage of time spent freezing induced by condition and the percentage of time spent freezing induced by context were decreased,and the activation of microglias and astrocytes was increased,and the expression of IL-6 and TNF-α was up-regulated in S groups (P ＜ 0.05).Conclusion Sevoflurane-induced postoperative cognitive dysfunction may be related to enhanced activation of gliocytes in aged rat hippocampi.

Objective To explore the molecular mechanism of the formulae for calming the liver and suppressing YANG in migraine rat model with syndrome of hyperactivity of the liver-YANG.Methods A rat model of migraine with hyperactivity of liver-YANG was established through electrical trigeminal ganglion stimulation and syndrome of oral administration of Fuzi decoction. The total proteins of the lymphocyte in the rats were separated by immobilized pH gradient-based 2-dimensional gel electrophoresis(2-DE), and the 2-DE image was analyzed by PDQuest 7.0 software. Matrix-assisted laser desorption/ionzation-time of flight mass spectrometry (MALDI-TOF-MS) and the SWISS-PORT and MSDB database were used to identify differential proteins.Results The formulae for calming the liver and suppressing YANG could also improve headache. Well-resolution and reproducible 2-DE patterns of rat lymphocyte from normal, model, and therapy tissues were obtained. Eleven of the total 13 differential protein spots were successfully identified by MALDI-TOF-MS. These proteins were alcohol dehydrogenase 3(ADH3), glycogen phosphorylase, ATP synthase D chain, annexin-3, ubiquitin, neutrophil defensin 4 precursor, melanoma-associated antigen E2, heat shock protein-27, annexin-A1, peroxirdoxin-Ⅱ, MU class glutathione S-transferase (Fragment)(GSH). Conclusion Differences occur in the expression of lymphocyte proteins in migraine rats with syndrome of hyperactivity of liver-YANG after treatment with the formulae for calming the liver and suppressing YANG, and the 11 identified protein spots may be associated with its mechanism.