Aim To investigate the molecular mechanisms of differentiation in human leukemia HL-60 cells induced by diallyl disulfide(DADS)using suppression subtractive hybridization(SSH).Methods In our privious study,the subtractive cDNA library was constructed successfully and efficiently. 30 clones were randomly analyed with restriction enzyme.The inserts of cDNAs were analyzed by restrictive enzyme EcoR I.Positive clones were sequenced and the homology of resulting cDNA sequences were analyzed through bioinformatics software Blastn.Results 18 clones contained 100～600 bp cDNA inserts.10 differantiation genes were obtained and involved in cell cycle,signal transduction,metabolism and RNA binding.And 3 of 10 genes,p21,STAT1 and CAMTA1 were up-regulated and detected by RT-PCR,the results matched with SSH.Conclusion sThere are tight correlation between the differentiation induced by DADS and three-upregulated gene:p21,STAT1 and CAMTA1.

Aim Toresearchthemolecularmecha-nisms of DADS-induced apoptosis in human leukemia K562cells.Methods Cellviabilitywasmeasuredby MTT. Levels of DADS-induced ROS were measured by 2ˊ, 7ˊ-dichlorofluorescein diacetate ( DCFH-DA) fluo-rescence. DADS-induced mRNA levels of components of the NADPH oxidase were detected by Real-time PCR. The combination of protein Rac2 and p67phox was measured by immunoprecipitation assays. Flow cy-tometry methods were used to determine the percentage of apoptosis cells. DADS-induced Rac2 levels were measuredbyWesternblot.Results TheDADS-trea-ted K562 cells showed a dose-and time-dependent de-crease in cell viability and proliferation. There was sig-nificant up-regulation of the mRNA level of components of the NADPH oxidase complex in K562 cells after treatment with 6 mg·L-1 DADS for 6 h. Western blot results revealed that, compared with the control group, there was a significant up-regulation of Rac2 protein in K562 cells treated with 5. 0 and 10. 0 mg·L-1 DADS for 24h. And Rac2 combined with p67phox in DADS-induced apoptosis in K562 cells. PMA markedly in-creased the percentage of apoptotic cells, and DPI re-duced the percentage of apoptotic cells in DADS-in-duced K562 cells. Levels of DADS-induced ROS, also showed enhancement when exposed in PMA, but there was no DADS-induced ROS production evident when exposed in DPI in DADS induced K562 cells. Conclu-sions TheseresultsindicatethatNADPHoxidaseis the main source of DADS-induced ROS production. Diallyl disulfide induces apoptosis in human leukemia K562 cells through activation of NADPH oxidase.

Objective To determine the role of erythrocytes immunoadhering in the pathogenesis of the patients with mesangiopro-liferative glomerulonephntis (MsPGN). Methods The immunoadhering functions of erythroeytes and leukoeytcs were measured in 31 patients with no- IgAN,24 patients with IgAN and 30 normal children by rosette tests:RCR,RICR,TRR,TNR and TLR. Results 1. The immunoadhering functions of erythroeytes (RCR and TRR) of the patients with no- IgAN and IgAN were obviously decreased compared to those of control group,but RICR showed no significant difference;2. The immunoadhering functions of leukocytes (TNR and TLR ) of the patients with no- IgAN and IgAN were obviously decreased compared to those of control group;3 The degree of the decreased immunoadhering functions of erythroeytes was correlated respectively with that of the immunoadhering functions of leukoeytes. Conclusions The decrease of the function of erythroeyte and leukocyte immunoadhering plays an important role in the pathogenesis in patients with MsPGN. These rosette tests may be used in clinic.

Aim To study the effects of cycle arrest and molecular mechanism in human leukemia HL-60 cells induced by diallyl disulfide ( DADS ) . Methods Cell count, colony formation in soft agar experiments and flow cytometry analysis were employed to observe the DADS-induced cell growth inhibition and the effect of cycle arrest in HL-60 cells. The expressions of Chk1/2 and its downstream element in HL-60 cells were detected by Western blot. Results Cell count revealed that population doubling time increased to 35. 03 h and 71. 82 h, respectively, from 19. 14 h in HL-60 cells treated with 60 and 120 μmol·L-1 DADS ( P<0. 05 ) . Colony formation in soft agar experiments showed that colony formation inhibition rate of HL-60 cells exposed to 30, 60, 90 and 120μmol·L-1 DADS increased to 35. 06%, 62. 10%, 93. 79% and 99. 35%, respectively ( P<0. 05 ) . Flow cytometry a-nalysis exhibited that HL-60 cells treated with 60 and 120 μmol · L-1 DADS for 24 h and 48 h arrested in G2/M phase in a concentration-and time-dependent manner ( P <0. 05 ) . Western blot disclosed that the expression of p-Chk1 increased in a time-dependent manner ( P <0. 05 ); however, Chk1, Chk2 and p-Chk2 were not changed in HL-60 cells treated with 60μmol·L-1 DADS (P >0. 05). The expression of Cdc25C, CyclinB1 and CDK1 decreased after treated with 60 μmol·L-1 DADS in a time-dependent manner ( P<0. 05 ) , but the expression of 14-3-3 protein did not change ( P>0. 05 ) . Conclusion DADS can in-hibit the proliferation of HL-60 cells, and induce G2/M arrest through Chk1/Cdc25 C/CyclinB1/CDK1 path-way.

Abstract: Objective This study aimed to investigate the expression of CD73 in colonic tissues in Crohn's disease (CD) and its significance and possible mechanism of action. Methods Thirty male BALB/c mice were randomly divided into normal control group, model group and intervention group. The control group was fed normally, and the model group was treated with TNBS+40% alcohol enema to establish a mouse model of Crohn's disease induced by chronic inflammation. The intervention group was treated with AB-680 intraperitoneally on the second day of each enema based on the model group. Mice body weight, fecal traits and fecal occult blood were recorded for disease activity index (DAI) score of inflammatory bowel disease. The animals were sacrificed at 7th week, their colonic tissues were removed, weighed and measured. The tissue inflammation was observed by standard hematoxylin-eosin (HE) staining. Masson staining was used to measure the area of collagen in colon tissue of mice. CD73 was detected by fluorescence quantitative PCR and immunohistochemistry. The expression levels of TGF-β, TNF-α and IL-6 in colon tissue of mice were determined by ELISA. Results The DAI score was (0.10±0.16) in the control group, (2.80±0.79) in the model group, and (3.07±0.34) in the intervention group. Compared with the control group, the DAI scores of the model and intervention groups were increased significantly (P<0.05). Compared with the model group, the DAI score of the intervention group was significantly increased (P<0.05). HE staining showed that there was no inflammation in the colon of the control group, while the colon of the model group and the intervention group showed typical inflammatory manifestations such as edema and congestion, inflammatory cell infiltration, and mucosal ulcer. The area ratio of collagen in the control group was (4.95±0.82)%, in the model group was (24.62±1.46)%, and in the intervention group was (54.47±2.75)%. Compared with the control group, the area ratio of collagen in the model group and the intervention group was significantly increased (P<0.05). Compared with the model group, the area ratio of collagen in the intervention group was significantly increased (P<0.05). Compared with the control group, the expression of CD73 in colon tissue of the model group and the intervention group was significantly increased (P<0.05). Compared with the model group, the expression of CD73 in colon tissue of the intervention group was decreased (P<0.05). Compared with the control group, the expressions of TGF-β, TNF-α and IL-6 in the model group and the intervention group were significantly increased (P<0.05). Compared with the model group, the expression of TGF-β, TNF-α and IL-6 in the intervention group decreased (P<0.05). Conclusions CD73 is upregulated in colon tissue of CD mice, it can inhibit inflammatory reaction and improve fibrosis by up-regulating TGF-β expression. On the other hand, CD73 can aggravate the inflammatory response in CD intestinal inflammation and fibrosis by up-regulating the expression of IL-6 and TNF-α. Therefore, CD73 may play a bidirectional regulatory role in intestinal inflammation and fibrosis of CD.

Objective To study the efficacy of late course accelerated fractionation(LCAF) radio- therapy in the treatment of nasopharyngeal carcinoma(NPC).The end-po s were local control,radiation-in- duced complications,factors influencing survival.Methods From December 1995 to April 1998,178 NPC patients were admitted for radiation treatment.The radiation beam used was ~(60)Co?or 6 MV X-ray.For the first two-thirds of the treatment,two daily fractions of 1.2 Gy were given to the primary lesion ,with an interval of≥6 hours,5 days per week to a total dose of 48 Gy/40 fractions,over a period of 4 weeks.For the last one third of the treatment,i.e.beginning from the 5th week,an accelerated hyperfractionation schedule was carried out.The dose per fraction was increased to 1.5 Gy,2 fractions per day with an interval of≥6 hours,the total dose for this part of the protocol was 30 Gy/20 fractions over 2 weeks.Thus the total dose was 78 Gy in 60 fractions in 6 weeks.Results All patients completed the treatment.Acute mucosi- tis:none in 2 patients,Grade 1 in 43,Grade 2 in 78,Grade 3 in 52,and Grade 4 in 3 patients.Local control rate:the 5-year nasopharyngeal local control rate was 87.7%,and the cervical lymph node local control rate was 85.7%.The 5-year distant metastasis rate was 26.1%,and 5-year survivals was 67.9%. Sixteen patients had radiation-induced cranial nerve palsy.Conclusions With this treatment schedule, patient's tolerance is good,local control and 5 year survivals are better than control groups of conventional fractionation and hyperfractionation radiotherapy.Radiation-related late complication does not increase.Ran- domized clinical trials are being carried out to further confirm the efficacy of LCAF for nasopharyngeal carci- noma.

Chondroitinase has been used as an important tool in the study of the structure, function and distribution of glycosaminoglycans for many years. Recently, the enzyme has been reported to be a potential enzyme for chemonucleolysis, an established treatment for intervertebral disc protrasion. In this paper, a chondroitinase had been purified from the culture supernatant of Aeromonas sobria YH311 using a simple purification procedure of ammonium sulfate precipitation, QAE-Sephadex A50 ion exchange chromatography and Sephadex G-150 gel filtration. The immobilization of purified chondroitinase using sodium alginate or cellulose as carriers has also been studied. The chondroitinase obtained from Aeromonas sobria YH311 was purified 55-fold to 95.3% pure, the specific activity of the purified enzyme was 31.86u/mg and the yield was 37%. The molecular weight of chondroitinase from Aeromonas sobria YH311 was determined by SDS-PAGE to be 80kD, which was almost the same as those chondroitinase AC from Arthrobacter aurescens, Aeromonas liquefaciens and Flavobacterium heparinum. But its isoelectric point was 4.3 - 4.6, which was far lower than the microbial chondroitinase AC. After the immobilization on sodium alginate or cellulose, the properties of chondroitinase changed greatly. The optimum temperature and pH of the free enzyme were 50 degrees C and 7.0 respectively, and about 10% activity remained after heat treatment at 80 degrees C for 20 minutes, and 47% activity remained after two weeks storage at 4 degrees C. The chondroitinase immobilized on sodium alginate had the optimum temperature and pH of 40 degrees C and 7.0 respectively, about 50% activity remained after 80 degrees C heat treatment for 120 minutes and 50% remained after 30 days storage at 4 degrees C. The chondroitinase immobilized on cellulose had the optimum temperature and pH of 70 degrees C and 6.0 respectively, and more than 70% activity remained after heat treatment at 80 degrees C and 30 days storage at 4 degrees C. The yield of the immobilization was very low, with 18.56% for alginate and 18.86% for cellulose.
Aeromonas ; enzymology ; Chondroitinases and Chondroitin Lyases ; isolation & purification ; metabolism ; Enzyme Stability ; Enzymes, Immobilized ; metabolism ; Temperature

Background c-Myc,EZH2 and p27 were defined to modulate the behavior of prostate cancer with pro-tumoral or anti-tumoral effects and had ability in predicting prostate cancer progression,but the research of their co-expression value of prognosis is rarely.This study aimed to investigate the prognostic value of combining tri-marker together in patients with intermediate-risk prostate cancer after surgery.Methods Expression levels of c-Myc,EZH2 and p27 in 129 patients with intermediate-risk prostate cancer were assessed using immunohistochemistry in a semi-quantitative manner.The expression profiles of these three markers were analyzed and investigated for association with biochemical recurrence.Results In all,fifty of 129 cases experienced biochemical recurrence during a median follow-up time of 31 months (range,6-60 months).Of these relapse patients,one case without and 10 cases with any single positive marker were observed; 39 cases were detected with any two or all three positive markers (22 cases with any two and 17 cases with all three positive markers).Survival analysis showed that patients with over-expression of c-Myc or EZH2,and lower expression of p27 manifested significantly higher biochemical recurrence rates.Subsequent multivariate analysis revealed that c-Myc,EZH2 and p27 expression statuses showed potential in predicting relapse,respectively.Notably,combining three markers together as a "composite index" (0 or 1,vs.2 or 3 positive markers) provided powerful prognostic value (HR 6.57,95％ CI 3.02-14.31,P ＜0.001).There was a significant difference between the patient subgroups with 0 or 1 and those with 2 or 3 positive markers expression statuses,and tri-marker composite index was an independent risk factor for predicting relapse in patients with intermediate-risk prostate cancer after surgery.Conclusion Composite index of c-Myc,EZH2,and p27 can be valued as powerful prognosis parameter for intermediate-risk prostate cancer patients after the surgery,and postoperative adjuvant therapy can be adopted accordingly.

Objective To explore the nursing intervention Oil the rehabilitation of patients with coronary heart disease,help the patients build healthy daily habits.make their life better.Methods 118 patients who have the heart illness and were admitted to our hospital between July 2004 and December 2006 were selected and tlleir knowledge about the heart attack.their dailv life style and how they take the medicine were evaluated.Depend oil the result of the evaluatioil,the speeches about healthy life.scientific life style and how to take the medicine scientifically were given,and then the second evaluation Was conducted after having the speeches 6 months.1 year and 2years later.Results The patient'S knowledge about the decease was obviously increasing (average P

Objective To assist the office workers with pre-diabetes to get rid of the unhealthy lifestyle by living behaviour intervention and to analyze the effect on them.Methods Totally 50 patients were selected who meet the standards of diabetes in the early stage after having medical check up.Following measurements were carried out:formulating healthy diets and exercising plans purposefully,holding special lectures periodically and handing out brochures on health education.Results Unhealthy life behaviour was greatly improved after being intervened for one year.The differences of fasting blood glucose and the blood glucose two hours after a meal were statistically difference between pre-and post-intervention.Conclusions The living behaviour intervention can obviously lower the danger of pre-diabetes and improve the cases' life quality.