定点突变法确定人体CYP2A6和CYP2A13香豆素代谢的关键氨基酸

【目的】通过比较人体主要尼古丁代谢酶CYP2A6和CYP2A13野生型及突变体酶蛋白对香豆素7-羥化反应的催化动力学特征,确定影响两者催化活性差异的关键氨基酸残基,提供建立评估人群尼古丁代谢酶基因多态性与吸烟行为及癌症易感性生物标记的依据。【方法】运用定点突变和Sf9昆虫杆状病毒蛋白表达体系产生CYP2A6和CYP2A13在300,301以及369位点氨基酸互换的突变体蛋白质,测定系列香豆素浓度下的各个酶蛋白对香豆素7-羥化的催化反应活性,获得动力学参数并与野生型酶蛋白进行比较分析。【结果】与野生型CYP2A6相比较,其Ile300→Phe突变体催化效率(Kcat=Vmax/Km)显著下降而Gly301→Ala突变体催化效率显著上升(1.64和8.02相对于野生型3.56,P<0.01)。与之相对应,CYP2A13的Phe300→Ile突变体催化效率则明显上升而Ala301→Gly突变体催化效率显著下降(1.03和0.06相对于野生型的0.27,P<0.05)。这些酶催化特性的改变,主要是由于Vmax的改变所致。369位的氨基酸残基,无论是Ser还是Gly对CYP2A6或CYP2A13的香豆素7-羥化反应催化活性均无明显的影响。【结论】300和301位的氨基酸是影响CYP2A6及CYP2A13对香豆素7-羥化反应催化活性的关键残基,而369位氨基酸不影响此活性。

Critical Amino Acid Residues for Nicotine 5'-Hydroxylation in Human CYP2A Enzymes

Objective： We have continued previous work in which we demonstrated that #117 and #372 amino acids contributed to the high activities of human CYP2A13 in catalyzing 4-methylnitrosamino-1-（3-pyridyl）-1-butanone（NNK） and aflatoxin BI（AFB1） carcinogenic activation. The present study was designed to identify other potential amino acid residues that contribute to the different catalytic characteristics of two CYP2A enzymes, CYP2A6 and CYP2A13, in nicotine metabolism and provide insights of the substrate and related amino acid residues interactions. Methods： A series of reciprocally substituted mutants of CYP2A6lle^300→ Phe, CYP2A6Gly^301aAla, CYP2A6Ser^369 → Gly, CYP2A13Phe^300→ Ile, CYP2A13Ala^301 → Gly and CYP2A13Gly^369 → Set were generated by site-directed mutagenesis/baculovirus-Sf9 insect cells expression. Comparative kinetic analysis of nicotine 5＇hydroxylatin by wild type and mutant CYP2A proteins was performed. Results：All amino acid residue substitutions at 300, 301 and 369 caused significant kinetic property changes in nicotine metabolism. While CYP2A6Ile^300→ Phe and CYP2A6Gly^301→Ala mutations had notable catalytic efficiency increases compared to that for the wild type CYP2A6, CYP2A13Phe^300→Ile and CYP2A13Ala^301→Gly replacement introduced remarkable catalytic efficiency decreases. In addition, all these catalytic efficiency alterations were caused by Vmax variations rather than Km changes. Substitution of #369 residue significantly affected both Km and Vmax values. CYP2A6Ser^369 → Gly increase the catalytic efficiency via a significant Km decrease versus Vmax enhancement, while the opposite effects were seen with CYP2A13Gly^369 → Ser. Conclusion：#300, #301 and #369 residues in human CYP2A6/13 play important roles in nicotine 5＇ -oxidation. Switching #300 or #301 residues did not affect the CYP2A protein affinities toward nicotine, although these amino acids are located in the active center. Set369 to Gly substitution indirectly affected nicotine binding by creating more space and conformational flexibility for the nearby residues, such as Leu^370 which is crucial for many hydroxylations.

Attitudes and Practices of Health Care Providers Regarding the Management of Uncomplicated Malaria in Abidjan, Cote d＇lvoire

Malaria is a major public health problem in the Cote d＇Ivoire where it is the leading cause of morbidity and mortality. The objective of this study was to assess the knowledge, attitudes and practices of health care providers on the diagnosis and therapeutic procedures used to ensure the management of uncomplicated malaria in the health district of Abidjan. A cross-sectional study was conducted from November 2008 to December 2008 among 169 caregivers who treated 1,691 cases of uncomplicated malaria. Results： The knowledge of the caregivers of the national guidelines regarding malaria was sufficient （89%）. A diagnosis of uncomplicated malaria was established only on clinical arguments （70%）. The percentage of patients who were prescribed artemisinin-based combination therapy （ACT） was 86%. However, 25% of the antimalarials drugs that were prescribed to patients （oral quinine, artemisinin derivatives monotherapy, and herbal medicine） did not meet the recommendations on first choice treatment as suggested by the national program against malaria. These results indicate sufficient knowledge of the national guidelines for the treatment of malaria by health care providers as evidenced by acceptable prescriptions for ACTs despite an incomplete knowledge of the guidelines.

5-aminolevulinic Acid - Mediated Photodynamic Therapy for Antimelanoma Treatment

Photodynamic therapy(PDT)is a novel approach for the anti-tumor treatment of photosensitive drugs and laser activation.In the study,5-aminolevulinic acid(ALA)was used as a photosensitizer to mediate PDT to inhibit the growth,metastasis and epithelial-mesenchymal transformation of melanoma cells.Firstly,the therapeutic effect of PDT was investigated through in vitro experiments.Human melanoma A375 cells cultured with ALA were treated with different light intensity,and the cell survival rates of A375 cells were measured by CCK8 method.Subsequently,the metastasis and invasion rates of A375 cells were detected by scratch test and Transwell test.Western blot was used to detect the expression of molecules related to epithelial mesenchymal transformation,such as E-cadherin,Snail,TGF-β,Smad2 and Smad3.In vitro results showed that A375 cells in the ALA-PDT treatment group showed weaker migration and invasion ability,while A375 cells in the control group showed higher migration and invasion rate(P<0.05).E-cadherin expression was significantly increased in the ALA-PDT treatment group(P<0.01),while expression of Snail,Smad2,Smad3 and TGF-βwere significantly decreased in the ALA-PDT treatment group(P<0.05).Therefore,ALA is a good photosensitizer that can inhibit the migration and invasion of melanoma,which also can inhibit the epithelial mesenchymal transformation of melanoma.

实践中的指导——在计算机系统电子档案领域中21 CFR 11适用性的解释

自从1997年8月以来，美国联邦法规第21号第11部分一直是人们讨论的话题。讨论不时地出现争议，为此美国食品及药物监督管理局(FDA)不得不再三给予这条法规新的解释。作为“面向21世纪的基于风险的现行优良药品制造标准(cGMP)”的一部分，FDA希望阐明它在“对工业界的指导，第11部分的适用范围和应用”中所述的状况。本文即是要说明这种解释在实际应用中的重要意义。

非ST段抬高急性冠脉综合征患者随机应用依诺肝素或普通肝素抗凝治疗的疗效和出血并发症：系统综述

背景：抗凝疗法已成为急性冠脉综合征（acute coronary syndrome，ACS）治疗指南推荐的标准疗法。但是，最近某些试验在ACS患者中对依诺肝素（enoxaparin）与普通肝素（unfractionated heparin）的应用进行了比较，发现这些抗凝疗法的疗效及安全性并不及既往试验结果。目的：有6项随机对照试验对依诺肝素与普通肝素治疗ACS患者进行了比较，对其终点（即全因死亡及非致死性心肌梗死）、输血与大出血进行系统评估。资料来源：从杜克临床研究所（Duke Clinical Research Institute）获取ESSENCE、A to Z及SYNERGY试验的原始数据。由TIMI 11B、ACUTEⅡ及INTERACT研究的主要研究人员提供各自的基线特征和事件发生频率。研究选取：在非ST段抬高ACS患者中比较依诺肝素与普通肝素的6项随机对照试验均人选进行分析。数据提取：从全部试验人群和随机分组前未接受抗凝治疗的亚人群中获取疗效终点和安全性终点。数据综合：应用随机效应经验性贝叶斯模型（random—effects empirical Bayes model），系统评估21946例患者的结果。依诺肝素与普通肝素30天死亡率无显著差异（3．0％比3．0％，优势比[odds ratio，OR]，1．00；95％可信区间[confidence interval，CI]，0．85～1．17）。在所有试验人群中，依诺肝素与普通肝素相比，30天死亡或非致死性心肌梗死（myocardial infarction，MI）联合终点显著下降，具有统计学差异（10．1％比11．0％；OR，0．91；95％CI，0．83～0．99；所需治疗例数107）。随机分组前未接受抗凝治疗的依诺肝素组患者30天死亡或MI联合终点亦显著下降，具有统计学差异（8．0％比9．4％；OR，0．81；95％CI，0．70～0．94；所需治疗例数72）。随机分组后第7天，总体安全人群或者随机分组前未接受抗凝治疗的人群输血（OR，1．01；95％CI，0．89～1．14）或大出血的发生率（OR，1．04；95％CI，0．83～1．30）无显著差异。结论：对近22000例各类ACS患者进行系统回顾发现，在预防死亡或MI联合终点方面依诺肝素较普通肝素更有效。

肌萎缩侧索硬化症病人大脑中央前回运动区磁共振波谱分析

目的 :1H MRS是否可确定ALS病人大脑皮质运动区神经元受损 ,是否适用于监测ALS病情。方法 :病例组包括 9例ALS病人。对照组包括 5例健康人 ,同时测定大脑皮质运动区的NAA、Cho、Cr。结果 :ALS病例组中NAA/Cr显著低于对照组 (P <0 0 5 ) ,Cho/Cr显著高于对照组 (P <0 0 1)。结论 :NAA/Cr下降和Cho/Cr升高 。