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.     

Epileptic seizures attributed to cerebral hyperperfusion after percutaneous transluminal angioplasty and stenting of the internal carotid artery

Cerebral hyperperfusion syndrome as a complication of carotid endarterectomy (CEA) has been widely reported in the surgical literature. It may occur within hours to 3 weeks after CEA and is characterized by symptoms ranging from headaches, fits, confusion, focal neurological signs to intracerebral hemorrhage. Although percutaneous transluminal angioplasty (PTA) and stenting are increasingly performed as an alternative to CEA in the treatment of carotid artery stenosis, few cases of cerebral hyperperfusion injury following carotid stenting have been reported. We describe 2 cases of cerebral hyperperfusion syndrome following PTA and stenting for high-grade internal carotid artery (ICA) stenosis. Both cases involved a lesion of 95% in severity. The first case was a 73-year-old man who developed generalized convulsion 7 h following stenting to the left ICA. The second case was an 80-year-old woman who developed recurrent right periorbital headache and confusion 16 h after stenting to the right ICA, followed by left upper limb seizure 14 days later. Both patients fully recovered without any intracerebral hemorrhage or infarction. To our knowledge, this is the first report of cerebral hyperperfusion injury after carotid stenting without associated intracranial hemorrhage and with full recovery. In the patient with neurological symptoms following carotid stenting, it is important to consider cerebral hyperperfusion syndrome as a differential diagnosis to embolic or hemorrhagic stroke since early recognition and meticulous control of blood pressure may prevent progression to cerebral hemorrhage and death.     

增氧呼吸器在模拟缺氧条件下对血氧饱和度和心率的影响

目的:探讨在模拟缺氧条件下增氧呼吸器对人体血氧饱和度和心率的影响. 方法:16名受试者在低压舱中(模拟海拔5000 m)进行运动负荷试验,随机分为2组(n=8):A组佩戴使用增氧呼吸器,B组不使用仪器. 分别检测血氧饱和度(SaO2)和心率. 结果:与B组比较,A组SaO2值明显增加(P<0.05),心率的变化则没有显著性差异. 运动负荷结束后3 min及5 min心率恢复速度两组之间的差异有统计学意义(P<0.05). 结论:增氧呼吸器能有效地促进高原习服过程,提高和改善低氧条件下人体的劳动能力.     

昼夜节律对PEMFs暴露小鼠血液流变学及红细胞脆性的影响

目的: 探讨昼夜节律对脉冲电磁场暴露小鼠血液流变学的影响.方法: 将30只雄性小鼠随机分为3组(n=10): 对照组、磁场A组和磁场B组.两磁场组饲养于强度为8×10-4 T的脉冲电磁场(pulsed electromagnetic fields,PEMFs)环境中(f=15 Hz),暴磁时间均为6 h/d(磁场A组为白天10∶00～16∶00,磁场B组为夜间22∶00～4∶00).对照组饲养于线圈中,但不暴磁.10 d后统一摘右眼取血,对血液流变学指标及红细胞脆性进行测定.结果: 磁场组与对照组比较,血液流变学特性以及红细胞脆性的差异均有统计学意义(P<0.05);两磁场组(A,B)之间比较,血液流变学各指标和开始溶血NaCl溶液浓度的差异具有显著性意义(P<0.05),而完全溶血的浓度差异不明显(P>0.05).结论: PEMFs可以改善小鼠血液流变特性,降低红细胞脆性,白天暴磁效果优于夜间,提示昼夜节律对其生物效应有影响.     

鼻泪管逆行插入硅胶管治疗慢性泪囊炎20例

目的：探讨鼻泪管逆行插入硅胶管治疗慢性泪囊炎的临床疗效。 方法：泪道内逆行插入由甘肃酒泉人民医院眼科高顺义研制的泪道再通硅胶管。 结果：随访1～2a，治愈16例，4例失败。 结论：鼻泪管逆行插入硅胶管术具有颜面部无切口，术后面部不遗留疤痕，创伤小，简便易行等优点。     

荒酸二甲酯的水相合成

用氰氨化钙、碳酸钙和二硫化碳等为原料，硫酸二甲酯为甲基化试剂，采用水相合成工艺制得荒酸二甲酯，总收率72％，纯度99％。     

聚乙二醇化天花粉蛋白注射液中天花粉蛋白的HPLC测定

建立了HPLC法检测聚乙二醇化天花粉蛋白注射液中天花粉蛋白的含量。采用硅胶基TSK-G3000SW凝胶柱，0．02mol／L磷酸二氢钠溶液-0．15mol／L氯化钠溶液-95％乙醇（45：45：10）为流动相，检测波长222nm。天花粉蛋白在5～50μg／ml范围内线性关系良好。平均回收率为100．0％，RSD为1．8％。     

针刺对颈椎病所致椎动脉血流动力学紊乱的影响

目的 评价针刺疗程对椎动脉血流速度的影响。方法 对32例椎动脉血流动力学紊乱的颈椎病患者，用TCD检测针刺颈夹脊穴的即刻与近期效应。结果 近期效应明显优于即刻效应（TCD参数Vp、Vm、Vd均P＜0．01）。结论 针刺颈夹脊穴对调节颈椎病所致椎动脉血流动力学紊乱的效应具有持续性和积累性。     

Effect of Micronutrient Supplementation on the Growth of Preschool Children in China

Objective To study the relationship between micronutrient supplementation and children growth. Methods A double-blind, placebo-controlled trial was conducted in 156 growth retarded preschool children. They were randomly assigned to five groups: supplemental control (S-control,n=28), zinc supplementation(+Zn, 3.5mg Zn/day, n=34), zinc and calcium supplementation (+ZnCa,3.5mg Zn+250mg Ca/day, n=37), zinc and calcium and vitamin A supplementation (+ZnCaVA,3.5mgZn +250mgCa+200gVA/day, n=28), Calcium and vitamin A supplementation (+CaVA,250mgCa + 200gVA/day, n=29). Another 34 children with normal height were selected as normal control (N-control). Supplementation continued for twelve months. Results The height gain in +Zn group (7.84cm per year) and +ZnCa group (7.70 cm per year) was significantly higher than that in S-control group (6.74 cm per year, P＜0.05); The weight gain in +ZnCaVA group (2.55kg per year)and +CaVA group (2.57 kg per year) was also significantly higher than that in S-control group (2.19kg per year, P＜0.05); The average days of illness in each supplementation group were lower than that in S-control (13 days per year compared with 23 days per year). No significant difference was observed on bone age. Conclusion Zinc and Zinc+Calcium supplementation can improve the height gain,and vitamin A can improve weight gain in growth retarded preschool children, but do not affect the maturity of bone. Micronutrient supplementation can lower the morbidity of these children.     

Magnetic resonance imaging of the entorhinal cortex and hippocampus in mild cognitive impairment and Alzheimer's disease

OBJECTIVES: To explore volume changes of the entorhinal cortex (ERC) and hippocampus in mild cognitive impairment (MCI) and Alzheimer's disease (AD) compared with normal cognition (NC); to determine the powers of the ERC and the hippocampus for discrimination between these groups. METHODS: This study included 40 subjects with NC, 36 patients with MCI, and 29 patients with AD. Volumes of the ERC and hippocampus were manually measured based on coronal T1 weighted MR images. Global cerebral changes were assessed using semiautomatic image segmentation. RESULTS: Both ERC and hippocampal volumes were reduced in MCI (ERC 13%, hippocampus 11%, p<0.05) and AD (ERC 39%, hippocampus 27%, p<0.01) compared with NC. Furthermore, AD showed greater volume losses in the ERC than in the hippocampus (p<0.01). In addition, AD and MCI also had cortical grey matter loss (p< 0.01) and ventricular enlargement (p<0.01) when compared with NC. There was a significant correlation between ERC and hippocampal volumes in MCI and AD (both p<0.001), but not in NC. Using ERC and hippocampus together improved discrimination between AD and CN but did not improve discrimination between MCI and NC. The ERC was better than the hippocampus for distinguishing MCI from AD. In addition, loss of cortical grey matter significantly contributed to the hippocampus for discriminating MCI and AD from NC. CONCLUSIONS: Volume reductions in the ERC and hippocampus may be early signs of AD pathology that can be measured using MRI.