Establishment of a simple assay in vitro for hepatitis C virus NS3 serine protease based on recombinant substrate and single—Chain protease

AIM: To establish a simple and convenient assay in vitro for the Hepatitis C virus NS3 serine protease based on the recombinant protease and substrate, and to evaluate its feasibility in screening the enzyme inhibitors. METHODS: Based on the crystallographic structure of hepatitis C virus (HCV) serine protease, a novel single-chain serine protease was designed, in which the central sequence of cofactor NS4A was linked to the N-terminus of NS3 serine protease domain via a flexible linker GSGS. The fusion gene was obtained by two-step PCR that was carried out with three primers and then cloned into the prokaryotic expression vector pQE30, and the recombinant clone was verified by DNA sequencing. The single-chain recombinant protease was expressed when the E.coli was induced with IPTG and the expression conditions were optimized to produce large amount of soluble protease. The recombinant substrate NS5ab that covers the cleavage point NS5A/B was also expressed in E.coli. Both of the protease and substrate were purified by using Ni-NTA agarose metal affinity resin, then they were mixed together in a specific buffer, and the mixture was analyzed by SDS-PAGE. The cleavage system was used to evaluate some compounds for their inhibitory activity on serine protease. RESULTS: The single-chain recombinant protease was over-expressed as soluble protein when the E.coli was induced at a low dosage of IPTG (0.2 mM) and cultured at a low temperature (15 degrees ). The protease was purified by using Ni-NTA agarose metal affinity resin (the purity is over 95 %). The recombinant substrate NS5ab was expressed in an insoluble form and could refold successfully after purification and dialysis. A simple and convenient assay in vitro was established, in which the purified single-chain serine protease could cleave the recombinant substrate NS5ab into two fragments that were visualized by SDS-PAGE. PMSF had an effect on inhibiting activity of serine protease, while EDTA had not. CONCLUSION: A simple and convenient assay in vitro for hepatitis C virus NS3 serine protease is based on recombinant substrate NS5ab and single-chain serine protease. This assay can be used in screening of enzyme inhibitors.     

Effects of somatostatin and octreotide on portal-systemic collaterals in portal hypertensive rats

BACKGROUND/AIMS: Somatostatin and its long-acting analogue, octreotide, have been used to cease variceal bleeding with uncertain mechanisms. This study investigated whether somatostatin and octreotide have a direct vasoconstrictive effect on the portal-systemic collaterals of portal-hypertensive rats and potentiate the vasoconstriction induced by endothelin-1 in these vascular beds. METHODS: The vascular responses of collateral vessels to graded concentrations of somatostatin (10(-9)-10(-5) mol/l), octreotide (10(-10)-10(-6) mol/l), norepinephrine (10(-9)-10(-5) mol/l) and vehicle (Krebs solution) were evaluated in perfused collateral vascular beds of rats with portal hypertension induced by partial portal vein ligation. In addition, the perfusion pressure changes of collateral vessels to endothelin-1 (10(-8) mol/l) in the presence of vehicle(control), somatostatin (10(-6) mol/l) and octreotide (10(-6) mol/l) were tested. RESULTS: Compared with the vehicle group, norepinephrine significantly increased the perfusion pressure of collateral vessels at concentrations between 10(-7) and 10(-5) mol/l. In contrast, neither somatostatin nor octreotide significantly changed the perfusion pressure. Somatostatin and octreotide significantly enhanced the endothelin-1-induced vasoconstrictive effect on the collaterals. CONCLUSIONS: Somatostatin and octreotide exert no direct vasoconstrictive effect on the collateral vessels of portal hypertensive rats. In the presence of endothelin-1, somatostatin and octreotide exert a local vasoconstrictive effect on these vascular beds.     

Reducing dust using the electrocautery pencil with suction combined with the infusion catheter in mastectomy

The dust from the vaporized tissue released during a mastectomy presents a hazard to the patients and the operating room personnel. More dust has been noted using the conventional electrocautery pencil in dissecting breast tissue than with the metal knife used in the past. It is very important to reduce the hazardous dust released during mastectomy. For this study 80 patients undergoing mastectomy for breast cancer from March to June 2001 were divided into two groups: 1) those whose dissections were performed with a combination of an electrocautery pencil and suction with an intravenous infusion catheter (40 cases) and 2) those whose dissections were performed with the conventional method in which the electrocautery pencil was handled by the surgeon and the metal suction tube was used separately by an assistant (40 cases). During mastectomy the personal air sampler was affixed to the operator's neck to collect the dust from the vaporized tissue. The concentrations of the total dust were significantly lower in the combined electrocautery-suction method (mean 5.56 +/- 3.26 microg/m3) than in the conventional method (mean 34.81 +/- 4.83 microg/m3) during mastectomy (P < 0.05). Although the operating time and blood loss were less in the combined method than in the conventional method this difference was not statistically significant (P > 0.05). The combined method of using the electrocautery pencil for dissecting breast tissue along with the intravenous infusion catheter reduced the concentrations of the total dust from the vaporized tissue plume. Furthermore this method reduces the hazards of dust to the surgeons and operating room personnel. Additionally the cost of this combined method is lower than that of the conventional method.     

酒依赖患者的个性特点分析与护理对策

目的探讨酒依赖病人的个性特点和相应的护理干预。方法采用中科院心理研究所修订的MMPI—R问卷，对121例酒依赖病人进行个性分析。结果酒依赖病人个性特点为被动依赖、对人疏远、自我为中心、情绪不稳、自控力差、易激惹、低自尊、有反社会行为、焦虑、记忆力减退等。结论针对酒依赖病人个性特点，恰当的护理干预，能使病人早日脱瘾，全面康复。     

湖南省农村实施欧盟生态旱厕项目健康教育效果分析

目的在农村中推广应用生态旱厕、粪便无害化处理与卫生行为干预相结合的模式。方法在项目实施之初,采用随机抽样方法对项目地区进行基线调查;然后,在项目实施的同时,开展知识传播、技能培训、行为干预等一系列健康教育活动。结果项目实施后,调查对象中使用生态旱厕已经达到68.4%,厕所设有洗手设施、家庭设有专用洗澡间、使用安全自来水三项指标分别达到66.8%、51.4%、56.9%;饭前便后洗手、食物隔离、不吸烟、不乱倒生活污水、不乱扔生活垃圾等健康行为形成率分别达到82.7%、83.9%、43.4%、86.0%和66.0%;农村居民及小孩腹泻患病率分别下降到3.0%和3.4%。结论欧盟生态旱厕项目的实施,促进了农村环境卫生面貌的改观和村民健康行为的形成,降低了与粪水相关疾病的患病率,探索了一套在国际卫生项目中开展健康教育工作的模式。     

非体外循环下冠状动脉搭桥术的临床应用

目的分析非体外循环下冠状动脉搭桥术（OPCABG）的优点及疗效。方法对2002年2月～2005年6月所行33例OPCABG患者的临床资料进行总结，患者术前心功能Ⅱ～Ⅲ级，左室射血分数34％-68％，均在非体外循环、心脏跳动状态下完成冠状动脉搭桥术。结果手术患者无一例死亡，术后心绞痛症状均消失，心功能得到不同程度改善。结论OPCABG是一种安全可行、效果良好的治疗冠心病的方法。     

医院发展要重视高技能人才的培养

本文通过对10余所医院高学历人才队伍现状的分析，探讨了医院在人才培养中的误区，提出医院要发展，在重视学历教育的同时，更应重视高技能人才的培养。     

均匀设计优化二元溶剂分散法制备莪术油纳米粒的研究

目的优化二元溶剂分散法制备莪术油纳米粒（ZTO-NP）的制备工艺技术条件，探讨其可行性。方法以形态、粒径、包封率为指标，采用均匀设计，考察分散剂浓度、载体材料用量、有机相体积和搅拌时间等影响因素，筛选出比较理想的制备工艺。结果最佳工作条件为：聚氧乙烯-聚氧丙烯共聚物（普朗尼克F68）0．1％，乳酸-乙醇酸共聚物（PLGA）0．01g，搅拌时间4h，丙酮体积1mL。所制得的纳米粒为圆整的类球形粒子，表面光滑，平均粒径为233．1nm（PDI值为0．047），zeta电位为-22．1mV，包封率为70．01％，载药量为57．55％。结论二元溶剂分散法可用于薮术油类液体药物纳米粒的制备。