Objective:To construct the prokaryotic expression vector pET28a(+)-comE, to get the highest expression of comE in E. coli BL21(DE3), and to obtain the purified ComE fusion protein. Methods: ComE gene was amplified by PCR with specific primers from genome of Streptococcus mutans UA159. After digested by two restriction endonucleases BamH Ⅰand Xho Ⅰ, the gene segment was inserted into vector pET28a(+). Then the recombinant vector was transformed into E. coli BL21(DE3). The protein expression was induced by IPTG and the result was confirmed by Western blot. The solubility of the fusion protein was examined by 12% SDS-PAGE, and the expression conditions were optimized. Finally the fusion protein was purified by a two-step purification procedure utilizing Ni2+ chelating column and S75 size exclusion column. Results: The recombinant plasmid was confirmed by PCR, restriction endonucleases digestion and sequence analysis, which showed that the inserted segment was identical to comE gene reported by GenBank without nucleotide mutation. The fusion protein which was confirmed by Western blot can be expressed in E. coli BL21(DE3) and existed both in supernatant and precipitation. The maximum expression product amount was obtained when the A600 value of E. coli BL21(DE3) was 0.4, IPTG concentration was 0.10 mmol/L and induction time was 4 h. Conclusion: The recombinant plasmid is constructed and the ComE fusion protein is purified by the optimum conditions.

Objective To assess the effect of rhein on diabetic nephropathy in rats with type 2 diabetes mellitus. Methods Diabetes was induced by high-lipids and high-sucrose diets (20% sucrose, 10% lard, 2.5% cholesterol, 1% cholic acid and 66.5 % regular chow, w/w) for 1 month and then intraperitoneal injection with 25 mg/kg streptozoticin(STZ) in female inbred Wistar rats. Rhein was continuously given 100 mg?kg-1?day-1 for 6 months 1 week after STZ injection in rhein-prevented group, and 1 month after STZ injection in rhein-treated group. Blood and urinary biochemistry and renal morphology were evaluated at divers times. Results Rhein ameliorated hypertriglyceridemia and hypercholesterolemia, but had few effect on the level of plasma glucose in both rhein-prevented group and rhein-treated group. Excretion of urinary protein reduced markedly and stable-state plasma glucose (SSPG) level decreased in both groups as compared with diabetic group. Examination of kidney sections from both rhein-prevented group and rhein-treated group showed that the structural lesions of glomeruli including mesangial expansion, diffuse glomerulosclersis and thickening of glomerular basement membrane, as well as artery injury such as arteriolar hyalinosis, atherosclerosis were markedly improved as compared with diabetic group. Morphometry of glomeruli from both rhein-prevented group and rhein-treated group showed that glomerular grow and mesangial hypertrophy occurred in diabetic group were ameliorated. The accumulation of fibronectin examined by immunostaining in glomeruli was reduced in both groups. Conclusion Therapeutic intervention with rhein can halt the progression of diabetic nephropathy and prevent the development of glomerulosclerosis and vascular injuries in this animal model of type 2 diabetes mellitus.

Objective To investigate vagal effects on atrial fibrillation(AF)thresholds at different sites of pulmonary veins after autonomic denervation.Methods In 10 mongrel dogs,the cervical vagal trunks were isolated bilaterally and decentralized from Oct 2004 to May 2005.The ansae subclaviae were exposed bilaterally as they exited from the stellate ganglia,doubly ligated,and cut.Local rapid electrical stimulation(S1S1=80 ms,impulse duration 0.5 ms)was performed to right appendage(RAA)、left appendage(LAA)、left atrium(LA)and different sites of 4 pulmonary veins.AF threshold was determined at each site in the baseline state,during bilateral cervical vagal stimulation(VNS,12.5 Hz,impulse duration 0.5 ms,stimulation voltage 5～8 V)and during infusion of atropine.Results During VNS,a lower AF threshold was observed at all sites,especially at distal of 4 pulmonary veins(P0.05).Conclusion Vagal nerve not only contributes to the maintenance of AF originating from pulmonary veins,but also greatly promotes the initiation of AF.

BACKGROUND: Anti-virus therapy is not suitable for the patients who have asymptomatic human immunodeficiency virus (HIV); otherwise, it is significant for early intervention of Chinese herbs to cure asymptomatic HIV.OBJECTIVE: To observe the interventional effects of fuzheng paidu pian on level of CD4+T lymphocytes and changes of plasma viral vector expression in early phase of asymptomatic HIV infection.DESIGN : Observational contrast study.SETTING: Institute of Acquired Immunodeficiency Syndrome (AIDS), Henan College of Traditional Chinese Medicine.PARTICIPANTS: A total of 69 asymptomatic HIV patients, who were infected by paid blood, were randomly selected from some countryside in November 2005. There were 39 males and 30 females. Their ages ranged from 30 to 61 years and the mean age was (43±8) years, and the mean course was (13±3) years. Inclusion criteria: ① coincident with diagnostic criteria of asymptomatic HIV infection published by National AIDS committee (modified in 2005); ②from 18 to 65 years old; ③ 200/mm3 ≤CD4+T lymphocyte count ＜ 600/mm3; ④ Patients did not treat with antiviral and other drugs; ⑤ All patients provided the confirmed consent. Exclusion criteria: ① severe liver and renal malfunction,combining with cardio-cerebral vascular disease and severe primary diseases of lung and hematopoietic system,mental diseases; ② primary immunodeficiency, secondary immunodeficiency induced by hormone and radiation therapy, hematologic disease; ③ pregnant or lactational women. Another 25 asymptomatic HIV patients were randomly selected.and regarded as the blank group. Inclusion and exclusion criteria were as the same as those in the treatment group.METHODS: Patients in the treatment group were treated with fuzheng paidu pian (consisting of huangqi, raw baizhu,fangfeng, baihuashe shecao, ganca o, etc.; Aolinte Pharmaceutical Factory of Henan Institute of Traditional Chinese Medicine; batch number: 050601) by using warm boiled water, 5 tablets once (eqUal to 6.6 g raw materials) for three times a day. Three months were regarded as a course and the treatment was performed four courses in total. Patients in the blank group were not treated with any drugs.MAIN OUTCOME MEASURES: ① Values of CD4+, CD8+ and CD4+/CD8+ in the treatment group before and after treatment;CD4+T lymphocyte count in the blank group after 6 months; ② Viral vector was detected by using reverse transcription polymerase chain reaction (RT-PCT) in the treatment group.RESULTS: All 69 patients in the treatment group and 25 patients in the blank group were involved in the final analysis. ①CD4+ value was decreased in the blank group after 6 months, and there was significant difference before and after 6 months based on α =0.05 level (P ＜ 0.05).However, CD4+ value was increased in the treatment group. ② Viral vector was randomly detected from 23 patients in the treatment group. As compared with before treatment, plasma viral vector was decreased in 9 patients at 6 months after treatment and in 12 patients at 1 year after treatment. The effective rate was 91.30%.CONCLUSION: Fuzheng paidu pian can improve immunological function of asymptomatic HIV patients and decrease viral vector in some cases during early interventional period.

Aging ; physiology ; Animals ; Catalase ; metabolism ; Circadian Rhythm ; Free Radicals ; metabolism ; Lipid Peroxides ; metabolism ; Male ; Melatonin ; metabolism ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase ; metabolism ; Thymus Hormones ; pharmacology

Background and purpose:The American College of Surgeons Oncology Group (ACOSOG) Z0011 trial has been described as practice-changing of sentinel lymph node (SLN) positive breast cancers. The goal of this study was to determine the exportability of the criteria deifned by Z0011 trial for selecting patients who are eligible for avoiding axillary lymph node dissection (ALND) after a positive sentinel lymph node biopsy (SLNB) result in China.Methods:We collected 194 breast cancer patients with positive SLNs and classiifed them into two groups according to Z0011 criteria. One group was consisted of patients who were eligible for omitting ALND and with SLNB only. The other one was ALND group. Then the patients of SLNB group were compared with cohort included in the Z0011 trial and the ALND group of this study separately.Results:Seventy-seven patients were potentially eligible for omitting ALND and 117 patients were still needed to have ALND. Compared with Z0011 cohort, the SLNB group in this research had signiifcantly more T1 stage and ER positive tumors (P<0.05). More patients in SLNB group had positive LNs with the number less than 3 and negative NSLNs (P<0.05). The ALND group had more T2 and T3 stage tumors than the SLNB group but the difference was not signiifcantly. The number of positive LNs in ALND group was higher than the SLNB group signiifcantly (P<0.05). More patients in the ALND group had positive non-sentinel lymph node (NSLN) than the SLNB group and the difference was also signiifcantly (P<0.05).Conclusion:Applied to the SLN positive patients in this study, Z0011 criteria could make patients with better prognosis and lower risk than Z0011 cohort be selected and let these patients avoiding ALND more safely.

The preliminary metabolic profile of total diterpene acid (TDA) isolated from Pseudolarix kaempferi was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the predominant metabolite in plasma, urine, bile and feces after both oral and intravenous administrations to rats using HPLC-UV and HPLC-ESI/MS(n), and demethoxydeacetoxypseudolaric acid B (DDPB), a metabolite proposed to be the glucoside of PC2 (PC2G), as well as pseudolaric acid C (PC), pseudolaric acid A (PA), pseudolaric acid A O-beta-D glucopyranoside (PAG), pseudolaric acid B O-beta-D glucopyranoside (PBG) and deacetylpseudolaric acid A (DPA) originated from TDA could also be detected. It was demonstrated by tests that the metabolism of TDA is independent of intestinal microflora, and neither of pepsin and trypsin is in charge of metabolism of TDA, TDA is also stable in both pH environments of gastric tract and intestinal tract. The metabolites of TDA in whole blood in vitro incubation were found to be PC2, DDPB and PC2G, which demonstrated that the metabolic reaction of TDA in vivo is mainly occurred in blood and contributed to be the hydrolysis of plasma esterase to ester bond, as well as the glucosylation reaction. These results clarified the metabolic pathway of TDA for the first time, which is of great significance to the in vivo active form and acting mechanism research of P. kaempferi.

The metabolic profile of pseudolaric acid B (PB) was investigated by using in vivo and in vitro tests. Pseudolaric acid C2 (PC2) was identified as the specific metabolite of PB in plasma, urine, bile and feces using HPLC and HPLC-ESI/MS(n) after both oral and intravenous administration to rats, and almost no prototype was detected in all kinds of samples. The metabolic behaviors of PB orally administered in rats treated with antibiotics to eliminate intestinal microflora were identical with those in untreated rats, demonstrating that the metabolism of PB is independent of intestinal microflora. PB was stable in 48 h respective incubation with artificial gastric juice and artificial intestinal juice, suggesting that neither pepsin nor trypsin is in charge of metabolism of PB, and also demonstrating that PB is stable in both pH environments of gastric tract and intestinal tract. In vitro research on metabolism of PB in rat liver microsomes incubation revealed that little PB was metabolized and that the proposed metabolites were the demethoxy and demethoxydecarboxy products of the prototype. The amount of metabolites was extremely low compared with the prototype, indicating that liver microsomes are not responsible for the metabolism of PB either. PB was gradually metabolized into PC2 during 1 h in whole blood incubation in vitro, and the metabolic process showed dynamically dependent manner with incubation time. Once absorbed into blood, PB was quickly metabolized into PC2, accordingly, little prototype was detected in all kinds of samples. The metabolism was attributed to the rapid hydrolysis of C-19 ester bond by plasma esterase. These results clarified the metabolic pathway of PB for the first time, which was of great significance to identify the in vivo active form and interpret acting mechanism of the active compounds of P. kaempferi.

Objective To explore the optimal power of experimental pancreatic endoscopic ultrasonography (EUS)-guided radiofrequency ablation (RFA) therapy.Methods Six healthy live minipigs were randomly divided into two groups according to random number table,which received RFA under EUS or in laparotomy.Each pig in each group was randomly allocated to received RFA at different power (5 W,10 W,15 W).The impedance was monitored and the time for each therapy was recorded.The width diameter of coagulation necrosis from RFA was measured by EUS and gross pathology.Results RFA was completed in all pigs smoothly with stable life signs and without complications.Gross and micro pathologic observation confirmed formation of coagulation necrosis lesions.The width diameter of lesions from EUS-RFA was similar to that from surgery RFA,both having a maximum value at 10 W power (11 nun VS 10 mm).Conclusion The optimal power of experimental pancreatic EUS-RFA in pig is 10W,which should be further explored for the clinical purpose.

Objective To evaluate the pattern of lateral cervical metastases and to investigate the risk factors for lateral cervical lymph node metastases in paoiuary thyroid carcinoma patients with clinical negative lateral neck lymph node.Methods 73 patients with paoiuary thyroid carcinoma who underwent prophylactic lateral neck dissections(level Ⅱ-Ⅵ or level Ⅱ-ⅣandⅥ) were reviewed retrospectively on their medical records paoiuary thyroid carcinoma.None of patients in this study had a clinically positive lymph node.Neck dissection specimens were obtained for histological analysis for node metastasis with respect to the individual neck levels.Results Occult metastases in lateral neck were observed in 12(16.4 ％)patients.9.6 ％,0,13.6 ％,9.6 ％,0,4.8 ％ and 42.4 ％ patients had histologically positive lymph nodes in levels Ⅱa,Ⅱb,Ⅲ,Ⅳ,Va,Vband Ⅵ respectively.In multivariate analysis,lymphatic metastases in level Ⅵ was associated with lateral neck metastasis(OR=7.3,P=0.020)in cN0 patients with paoiuary thyroid carcinoma.Conclusion Levels Ⅲ,Ⅱa and Ⅳwere the most common stages showing occult lymph node metastases.Prophylactic lateral neck dissections may be omitted in the treatment of cN0 PTC patients ff level Ⅵ lymphatic metastases are not found on histological exam.