Objective To explore the role of TRAIL receptors expression on peripheral blood mononuclear cell (PBMC) in the apoptosis of PBMC,and the relation of the expression of TRAIL receptors with the severity of liver damage in chronic hepatitis B patients.Methods The expressions of DR4,DRS,DcR1 and DcR2 in 55 patients and 30 cases of control were assayed by RT-RCR and floweytometery.The relationship of the expression of TRAIL receptors with the severity of liver damage were analized.Results The level of DcR1 in the PBMC of chronic hepatitis B patients was much lower than that of control group (P＜0.05).There was close relation between the expression of DcR1 and liver damage (P＜0.05).The level of DcR1 was negatively correlated with ALT but positively correlated with serum albumin.Conclusion The expression level of DcR1 on PBMC in chronic hepatitis B patients was down-regulated,which may contribute to the increased apoptosis of PBMC in chronic B hepatitis patients.The expression of DcR1 can reflect the degree of liver injury in chronic hepatitis B patients to some degree.

AIM: To explore the effect of HBV transfection on apoptosis induced by TWEAK and the regulatory effect of IFN-γ on the apoptosis process.METHODS: BEL-7402-pCDNA3/1.1HBV hepatoma cell line was used as cell model in this experiment, in which BEL-7402 was stably transfected with pCDNA3/1.1HBV and its corresponding empty vector BEL-7402-pCDNA3 was used as control. The effect of HBV transfection on TNF-like weak inducer of apoptosis(TWEAK) induced hepatoma cell apoptosis was determined by CCK-8. The apoptosis before and after HBV transfection induced by TWEAK and the effect of IFN-γ on apoptosis induced by TWEAK in HBV transfected hepatoma cells were detected by TUNEL and caspase 3 activity detection kit.RESULTS: BEL-7402-pCDNA3/1.1HBV and BEL-7402-pCDNA3 hepatoma cell line were successfully established and identified. Cell viability of hepatoma cells was increased after HBV transfection. The apoptosis rate was much higher after HBV transfection compared to the empty vector control and empty cell control. The apoptosis rate induced by TWEAK was much higher in BEL-7402-pCDNA3/1.1HBV cells when exposed to IFN-γ.CONCLUSION: After HBV infection, TWEAK might be involved in the clearance of HBV infected hepatocyte by apoptosis and inflammatory factor such as IFN-γ.

Objective To explore the effects of environmental enrichment (EE) on learning and memory ability and the expression of brain-derived neurotrophic factor (BDNF) and synaptophysin in hippocampus of neonatal rats with hypoxic-ischemic brain damage(HIBD).Methods Forty Wistar neonatal male rats aged 7 days were randomly divided into EE intervention for 6 hours(6 h EE) group (n =10),EE intervention for 12 hours (12 h EE) group (n =10),model group (n =10) and sham group (n =10).The first 3 groups were performed with HIBD.The 6 h EE and 12 h EE group received EE stimuli for 6 h and 12 h respectively,once a day for 14 days.Learning and memory of the rats were tested by using Morris water maze.The expression levels of BDNF and synaptophysin in hippocampus were determined with Western blot.Results The escape latency of all groups gradually reduced with the increase of training days,but there was no significant difference in the escape latency among the 4 groups (F =0.237,P ＞ 0.05).The rats in the 6 h EE group,12 h EE group and model group spent less time in the target quadrant and showed a significant reduction of BDNF and synaptophysin(6 h EE group:0.529 ± 0.038,0.889 ± 0.027;12 h EE group:0.660 ± 0.034,1.114 ± 0.037;model group:0.225 ± 0.015,0.672 ± 0.057) in the hippocampus compared with the sham group (0.803 ± 0.026,1.347 ± 0.092) (all P ＜ 0.01).In the 6 h EE group and 12 h EE group,the rats significantly increased the time spent in target quadrant and aggrandized the expression of BDNF and synaptophysin in hippocampus compared with the model group.Moreover,the 12 h EE group had a better performance than the 6 h EE group in the space exploration and the expression of BDNF and synaptophysin.Conclusion EE is helpful for improving learning and memory ability in neonatal rats with HIBD,which may be associated with up-regulating the expression of BDNF and synaptophysin in hippocampus.

AIM To investigate the effects of trifluoperazine on naloxone precipitated withdrawal symptoms in morphine dependent rats and mice, and its pharmacological mechanisms. METHODS\ Naloxone precipitated tests in morphine dependent rats and mice were used. RESULTS\ Trifluoperazine(2～20 mg?kg -1 ) dose dependently inhibited naloxone precipitated withdrawal jumping, wet dog shakes, paw tremor and weight loss in morphine dependent mice. With ip trifluoperazine (5～20 mg?kg -1 ), most of positive withdrawal symptoms, including jumping, wet dog shakes, defeacation, weight loss, teeth chattering, salivation, diarrhea, ptosis and irritating, induced by naloxone in morphine dependent rats were significantly reduced. Apomorphine (2～8 mg?kg -1 ), a mixed DA 1/DA 2 receptor agonist, did not affect inhibition of trifluoperazine on naloxone precipitated withdrawal symptoms in morphine dependent mice. However, nifedipine(5～20 mg?kg -1 ), a L type voltage sensitive calcium channel blocker, enhanced a pharmacological action of trifluoperazine against naloxone precipitated symptoms in morphine dependent mice. CONCLUSION\ Trifluoperazine attenuates naloxone precipitated withdrawal symptoms in morphine dependent rats and mice by inhibiting the activity of post receptor calmodulin, but it does not antagonizes DA 2 receptor, in central nervous system.

AIM:To explore the effect of HBV transfection on apoptosis induced by TWEAK and the regulatory effect of IFN-? on the apoptosis process.METHODS:BEL-7402-pCDNA3/1.1HBV hepatoma cell line was used as cell model in this experiment,in which BEL-7402 was stably transfected with pCDNA3/1.1HBV and its corresponding empty vector BEL-7402-pCDNA3 was used as control. The effect of HBV transfection on TNF-like weak inducer of apoptosis(TWEAK) induced hepatoma cell apoptosis was determined by CCK-8. The apoptosis before and after HBV transfection induced by TWEAK and the effect of IFN-? on apoptosis induced by TWEAK in HBV transfected hepatoma cells were detected by TUNEL and caspase 3 activity detection kit.RESULTS:BEL-7402-pCDNA3/1.1HBV and BEL-7402-pCDNA3 hepatoma cell line were successfully established and identified. Cell viability of hepatoma cells was increased after HBV transfection. The apoptosis rate was much higher after HBV transfection compared to the empty vector control and empty cell control. The apoptosis rate induced by TWEAK was much higher in BEL-7402-pCDNA3/1.1HBV cells when exposed to IFN-?.CONCLUSION:After HBV infection,TWEAK might be involved in the clearance of HBV infected hepatocyte by apoptosis and inflammatory factor such as IFN-?.

Objective To discuss the application of microsurgery in the treatment of male infertility.Methods From March 2007 to March 2012,there were totally 853 infertile men received microsurgical treatments in our department.Among them,344 patients with unilateral or bilateral varicocele underwent microsurgical varicocelectomy,60 underwent vasovasostomy (VV) and 192 underwent vastoepidystomy (VE)in microsurgical methods due to obstructive azoospermia.257 non-obstructive azoospermia (NOA) patients were performed microdissection of testicular sperm extraction (MD-TESE),at the same time,pathologic examination was done.Results ①For the varicocele patients,the pre-operative sperm density was (10 ±6) × 106/ml,the progressive sperm percentage was (16 ± 9)％.The post-operative density was (15 ± 8) ×106/ml,the progressive sperm percentage was (28 ± 14)％.The natural pregnant rate was 10.8％ (37/344).②In 60 patients undergone VV,the patent rate was 80.0％ (48/60),the natural pregnant rate was 35.0％ (21/60).In 192 VE patients,the patent rate was 53.1％ (102/192),the natural pregnant rate was 19.8％ (38/192).③In 257 NOA patients,the testicular volume,sperm retrieval rate of MD-TESE was significantly higher than that of conventional testicular sperm extraction (60.3％ vs.38.1％).Conclusion The microsurgery techniques in male infertility treatments could have some advantages such as explicit effects and decreased injuries.

Objective To explore the expression of early growth response gene-1 (EGR-1) in T cells that were positive for Tax protein of human T-cell leukemia virus type 1 (HTLV-1) and its possible reg-ulatory mechanism .Methods A series of expression structures carrying the regulatory elements of EGR-1 in different length and luciferase reporter genes were constructed .TaxP cells were transfected with the con-structs containing reporter genes and cultured with 5μmol/L of NF-κB inhibitor BAY 11-7082 or equal vol-ume of DMSO.After cultured for 24 hours the cells were collected to test the luciferase activity .BAY 11-7082 or equal volume of DMSO was added into the supernatant of TaxP cell culture to test the expression of EGR-1 protein by Western blot after 24 hours of culture .Tax and its mutants M22 and M47 were transfected into 293 T cells respectively to test the expression of EGR-1 protein by Western blot after 24 hours of culture . Results The expression structures carrying the regulatory elements of EGR-1 in different length and their mutants followed by luciferase reporter genes were successfully constructed .The luciferase activity in the cells transfected with the constructs containing the elements E 1 and E2 were higher than that transfected with E3, DelE and MutE, but the reporter gene expressions were decreased with the interference of BAY 11-7082 (P<0.01).However, there were no significant changes with the luciferase activity in the cells transfected by elements E3, DelE and MutE.Western blot analysis indicated that the expression of EGR-1 protein was significantly decreased with the interference of BAY 10-7082 .The expression of EGR-1 protein in M22 mu-tants-transfected 293 T cells were decreased significantly in comparison with those by wild type tax-and M47-transfected cells .Conclusion NF-κB was the key nuclear factor in regulating the expression of EGR-1 pro-tein in Tax-positive T cells .

Objective To evaluate effects of ursolic acid (UA) on interleukin?33 (IL?33) expression in HaCaT cells induced by interferon?γ(IFN?γ), and to explore their mechanism. Methods Some HaCaT cells were treated with UA at different concentrations(0, 0.1, 1, 5, 10, 20, 40 and 80μmol/L)for 24, 48 and 72 hours separately. Then, methyl thiazolyl tetrazolium(MTT)assay was conducted to evaluate cell proliferative activity. A cell model of inflammation was established by culture of HaCaT cells with the presence of 200μg/L IFN?γ. Some HaCaT cells were classified into several groups to be treated with IFN?γ(200μg/L)and UA(10 and 15μmol/L)alone or in combination (firstly treated with IFN?γ followed by UA treatment), and those receiving no treatment served as the blank control group. Reverse transcription PCR (RT?PCR) was performed to detect mRNA expressions of IL?6 and IL?33, and Western?blot analysis to measure IL?33 protein expression after 12?hour culture. The expressions of extracelluar signal?regulated kinase 1/2(ERK1/2)and phosphorylated ERK1/2(p?ERK1/2)were also measured by Western?blot analysis after 5?and 60?minute treatments with IFN?γand UA alone or in combination. Results MTT assay showed that the treatments with 5-20μmol/L UA for 24 hours had no effects on cell proliferative activity, while 40-80μmol/L UA could significantly inhibit it at 24, 48 and 72 hours (all P < 0.05). Thus, 10 and 15 μmol/L were chosen as the concentrations of UA for further study. After the treatment with 200μg/L IFN?γ, there was a significant increase in the expressions of IL?33 mRNA(0.812 ± 0.036 vs. 0.412 ± 0.021), IL?6 mRNA(0.947 ± 0.091 vs. 0.595 ± 0.030)and IL?33 protein(1.317 ± 0.119 vs. 0.147 ± 0.036)in HaCaT cells compared with the blank control group(all P<0.05). Compared with the IFN?γgroup, the IFN?γ+10?μmol/L UA group and IFN?γ+15?μmol/L UA group both showed significantly decreased expressions of IL?33 mRNA(0.447 ± 0.042 and 0.438 ± 0.028 respectively, both P<0.05), IL?6 mRNA(0.437 ± 0.099 and 0.350 ± 0.075 respectively, both P<0.05)and IL?33 protein(0.923 ± 0.058 and 0.564 ± 0.113 respectively, both P<0.05). There were no significant differences in IL?33 mRNA expression between the IFN?γ+10?or 15?μmol/L UA group and blank control group(P>0.05), while IL?33 protein expression was significantly lower in the IFN?γ+15?μmol/L UA group than in the IFN?γ+10?μmol/L UA group(P<0.05). The p?ERK1/2 protein expression significantly increased in HaCaT cells treated with IFN?γ for 5 and 60 minutes compared with the blank control group, but significantly decreased in the IFN?γ+15?μmol/L UA group compared with the IFN?γgroup(0.458 ± 0.053 vs. 0.941 ± 0.042 at 5 minutes, 0.302 ± 0.054 vs. 0.509 ± 0.032 at 60 minutes, both P < 0.05). However, no significant differences were observed in the total ERK1/2 protein expression between the IFN?γ+15?μmol/L UA group and IFN?γgroup at 5 or 60 minutes. Conclusion UA can suppress IL?33 expression in HaCaT cells induced by IFN?γ, likely by regulating expressions of the ERK signaling pathway?related proteins.

We purified a sarcosine oxidase from Bacillus sp. strain BSD-8 isolated from soil. We purified the enzyme by ammonium sulfate precipitation, DEAE-cellulose, Toyopearl hydrophobic and Sephadex G-75 molecular sieve chromatography and characterized the purified sarcosine oxidase. This sarcosine oxidase was a flavin enzyme containing a noncovalently bound flavin with the subunit molecular mass of 51 kDa. The optimal temperature for this enzyme was 60 degrees C and it showed its highest activity at pH 8.5. It was stable in the pH range of 8.0-10.0 and at the temperature of 60 degrees C. Estimated by Lineveaver-Burk plots, the K(m) of the enzyme was 3.1 mmol/L. Ag+, Hg2+, SDS and Tween 80 dramatically inhibted the enzyme activity, whereas Tween 20 and Triton X-100 had no effect on enzyme activity. The thermostability of this enzyme was better than reported sarcosine oxidases, and it could be applied in enzymatic measuring of creatinine.
Bacillus ; enzymology ; isolation & purification ; Bacterial Proteins ; chemistry ; isolation & purification ; metabolism ; Chemical Precipitation ; Enzyme Stability ; Sarcosine Oxidase ; chemistry ; isolation & purification ; metabolism ; Soil Microbiology

Objective To investigate the influence of extracellular high mobility group box 1 (HMGB1) on viral replication in HTLV-1 infected T cells.Methods HMGB1 in culture supernatants of adult T-cell leukemia virus 1 (HTLV-1) virus-negative cell:Jurkat,MOLT4 cells and HTLV-1 virus-positive cells:MT2,MT4,was detected by ELISA;The HTLV-1 long terminal repeat reporter gene (pHTLV-1-LTR-luc) was transfected into MT2 cells by Tfx-50-mediated transfection,and 0.25,0.50,0.75 μg/ml of HMGB1 polyclonal antibody(HMGB1 PcAb) and its isotype control rabbit IgG antibodies,0.03,0.1,0.3 μg/ml rhHMGB1 and its control PBS,were added into culture supernatant respectively,then luciferase activity was detected after 48 h;Similarly,0.25 μg/ml HMGB1 PcAb and the isotype control antibody,0.3 μg/ml rhH-MGB1 and the control PBS were added to the culture supernatant of MT2 cell,the viral gene,pol1,pol2,gag,env,etc,were performed by real-time PCR.Results Culture supernatant HMGB1 levels has no significant difference between HTLV-1 positive cells MT2 and MT4 and the other two virus-negative T cell lines;Compared with isotype control antibody group,the culture supernatant,to which is added 0.25 μg/ml HMGB1 PcAb,can significantly inhibit the HTLV-1-LTR transcriptional activity and suppress the expressions of the viral gene pol1,pol2,gag,env.Compared with the control PBS,0.3 μg/ml rhHMGB1 significantly promotes the transcriptional activity of the HTLV-1-LTR and the expressions of the viral gene pol1,pol2,gag,env.Conclusion The extracellular HMGB1 can promote viral replication of HTLV-1 infected T cells.