尿激酶原-RGDS双功能分子——构建、表达及性质研究

利用定点突变及DNA重组技术 ,构建了在尿激酶原K区C 端的β 发夹区插入了精氨酸 甘氨酸 天冬氨酸 丝氨酸 (RGDS)片段的尿激酶原嵌合体基因 ,并利用昆虫杆状病毒表达系统通过感染Sf9细胞对嵌合体尿激酶原进行了高效表达 .用尿激酶原单抗亲和柱纯化表达产物 ,获得初步纯化的嵌合体蛋白 .对嵌合体蛋白进行血小板膜结合实验表明 ,此嵌合体具有依赖于钙离子的结合活化血小板膜的活性 .用生色底物Chromozym U测定嵌合体的酰胺解活性 ,结果显示 ,纤溶酶激活后的嵌合体比活为 62 0 0 0IU/mg,与文献报道的纤溶酶激活的尿激酶原比活 65 3 5 5IU/mg相近 .纤溶酶激活后的嵌合体激活纤溶酶原的反应符合米氏方程 ,其Km 值为 0 .97μmol/L ,与天然尿激酶的Km 值 1.64μmol/L相近 .嵌合体还显示了较强的体外抑制血小板聚集活性 .这些结果表明 ,此尿激酶原 RGDS嵌合体有可能成为一种新的双功能溶栓药物 .     

PRGDWR-尿激酶原双功能嵌合分子的性质研究

为赋予单链尿激酶型纤溶酶原激活剂(single chain urokinase-type plasminogen activator, scu-PA, 尿激酶原)以抗血小板聚集的功能,在scu-PA的kringle区118位Gly与119位Leu之间插入PRGDWR序列（insert mutant B,InB）.利用甲醇酵母（Pichia pastoris）进行分泌表达,经金属离子螯合亲和层析与S强阳离子交换层析,得到纯蛋白.实验测定InB对人工合成底物S-2444的酰胺解活性为5 900 IU/mg,动力学常数为：K_m,S-2444^InB=56.8 μmol·L^-1,k_cat,S-2444^InB=0.33 s^-1;水解天然底物plasminogen的动力学常数为：K_m,plg^InB=0.397 μmol·L^-1,k_cat,plg^InB=0.0164 s^-1.InB激活plasminogen的反应在有fibrin存在条件下InB的活性为无fibrin条件下的46.3％.该突变体在体外激活plasminogen的活性与同一系统表达的野生型scu-PA基本相同.该突变体表现出较强的抗血小板聚集活性,IC₅₀＝12.7 μmol·L^-1,而野生型scu-PA无此功能.实验表明scu-PA的K区插入突变体InB是一种极具潜力的双功能溶栓分子.     

对德氏乳杆菌DM8909菌株的临床药代动力学研究

本文对大连医科大学微生态学研究所研制的阴道乳杆菌活菌制剂进行药代动力学研究 ,目的是为了探讨德氏乳杆菌活菌阴道制剂在正常志愿者阴道中的变化情况 ,确定临床观察的给药剂量、间隔时间和给药方法。现将有关试验情况报告如下 :1　材料与方法1 .1　药代动力学试验研究对象　为 1 0名志愿者进行阴道乳杆菌活菌动态研究。1 .2　药品来源　定菌生 DM890 9由大连医科大学科达药业有限公司提供 ,批号 961 0 2 0。1 .3　试验设计　分 3个剂量组 ,单次阴道给药 ,低剂量组 ( 0 .2 5× 1 0 6CFU/粒 )、中剂量组 ( 0 .2 5×1 0 7CFU/粒 ) ;高剂…     

细胞药代动力学研究进展

经典的药物代谢动力学理论是建立在血浆药物浓度测定的基础上,常难以真实有效地预测体内药物的药效。很多药物必须穿透多重生物屏障,与细胞内的靶点相结合才能发挥药效。因此药代动力学研究迫切需要从“宏观”的血浆药物浓度深入到“微观”的细胞/ 亚细胞水平。综述细胞药代动力学研究领域取得的进展,重点介绍细胞药代动力学理论的提出、技术体系的建立及其在药物研发、筛选、临床方面的应用。     

阿魏酸及其不同配伍药代动力学比较研究

本文主要探讨阿魏酸及其不同配伍给药在大鼠体内的代谢动力学规律。将32只SD大鼠,分为阿魏酸组50 mg/kg、阿魏酸+川芎嗪(50 mg/kg+30 mg/kg)组、阿魏酸+延胡索乙素(50 mg/kg+20 mg/kg)组、阿魏酸+川芎嗪+延胡索乙素(50 mg/kg+30 mg/kg+20 mg/kg)组,分别灌胃给药,采用高效液相色谱法测定各组大鼠血浆中阿魏酸的浓度,DAS2.0程序计算药代动力学参数。结果表明:川芎嗪、延胡索乙素均可延长阿魏酸在大鼠体内的作用时间,增加阿魏酸在大鼠体内的吸收。     

草鱼体内双氟沙星药代动力学研究

为研究双氟沙星（Difloxacin,DIF）在草鱼（Ctenopharynodon idellus）体内的药代动力学以及在各组织中的残留量,采用高效液相色谱法测定在15℃水温状态下单次给草鱼灌喂20 mg/kg剂量的双氟沙星后,得出双氟沙星在各组织以及血液中的药时曲线均都符合二室开放性模型,双氟沙星能够在草鱼体内快速吸收,并且血液及各组织中均有分布,双氟沙星在草鱼体内的药物动力学方程为C=5.056e-0.012t+19.041e-0.011t,其中双氟沙星在血液、肌肉、肝脏、肾脏中吸收半衰期（T_1/2α）分别为0.176h、0.562h、4.562h和1.477h,消除半衰期分别为（T_1/2β）69.492h、65.303h、218.412h和163.937h,总体消除率（CL）分别为0.495、11.181、10.789和7.102 L/（h·kg）,药时曲线下面积（AUC）分别为81.550、1277.55、807.470和1432.150 μg/（L·h）。根据相关规定肌肉中双氟沙星最大残留量300 μg/kg为标准,建议休药期26d以上。     

生物技术药物药代动力学研究的方法学和实验设计

生物技术药物由于自身具有的特点 ,使得其在体内的药代动力学机制比传统药物更为复杂。近年来 ,对于这类药物在体内的代谢机制的研究日趋增加 ,研究方法也日趋成熟。概述了生物技术药物的药代动力学研究方法以及实验设计的特点。     

肌注和口服恩诺沙星在大菱鲆体内的药代动力学比较

在水温(16±0.6)℃条件下, 以20 mg/kg剂量给健康大菱鲆静注、肌注和口服恩诺沙星后, 用高效液相色谱法测定药物浓度,采用DAS2.0药动学软件对血药浓度进行分析,比较了肌注和口服两种给药方式下恩诺沙星在大菱鲆(Scophthalmus maximus)体内的药代动力学差异。结果显示,肌注和口服恩诺沙星后,在大菱鲆体内的代谢过程均符合一级吸收二室开放模型, 表达方程为C肌注=10.237e-0.702t+6.151e-0.01t-16.388e-25.796t和C口服=3.701e-0.072t+3.534e-0.007t-7.235e-0.364t。与口服给药后药代动力学参数比较, 肌注给药后的t1/2Ka(0.027h)、tmax(0.5h)、t1/2α(0.987h)和t1/2β(68.003h)均小于口服给药(1.904h、4h、9.621h和99.137h),且Cmax(21.7172μg/mL)和F(88.57%)均大于口服给药(5.3594μg/mL、66.42%)。结果表明, 肌注恩诺沙星在大菱鲆体内的吸收、消除均快于口服给药, 且比口服给药吸收完全。在试验条件下, 最佳给药方案为:肌注给药, 按鱼体重每次给药19.05 mg/kg,2天一次, 建议连续给药2-3次;口服给药,按鱼体重每次给药13.92mg/kg,1天一次, 建议连续给药3-5次, 建议休药期分别不低于30d和45d。       

尿激酶原变体Glu151-Glu154-mscu-PA的构建及其动力学性质

利用寡核苷酸介导的定点突变方法 ,将重组人尿激酶原 ( recombinant single chain uroki-nase- type plasminogen activator,rscu- PA)中 1 51位赖氨酸 ( Lys1 51 )突变为谷氨酸 ( Glu1 51 ) ,1 54位精氨酸 ( Arg1 54)突变为谷氨酸 ( Glu1 54) ,得到尿激酶原变体基因 ( mscu- PA) .尿激酶原变体和未突变的重组尿激酶原均在 E.coli中获得表达 ,超声后所得包涵体经体外变复性并得到纯化 .结果表明 ,尿激酶原变体对纤溶酶 ( plasmin)的敏感性比未突变的重组尿激酶原低约 40 % ,转变纤溶酶原 ( Glu- plasminogen)为纤溶酶的活性基本相同 .两种产物经纤溶酶活化后 ,分别得到了双链尿激酶 ( rtcu- PA)和双链尿激酶变体 ( mtcu- PA) .它们对人工合成的发色底物 S2 4 4 4反应的动力学基本一致 ,对 Glu- plasminogen的催化反应的米氏动力学常数 Km 基本一致 ,但 mtcu- PA的 Kcat仅为rtcu- PA的 80 % .酪蛋白降解系统 ( caseinolytic system)实验表明 ,在纤维蛋白和纤溶酶原存在的情况下 ,尿激酶原变体较未突变尿激酶原能加快酪蛋白的降解 ,说明 mtcu- PA对纤维蛋白有一定的亲和性     

阿魏酸及其不同配伍药代动力学比较研究北大核心CSCD

本文主要探讨阿魏酸及其不同配伍给药在大鼠体内的代谢动力学规律。将32只SD大鼠,分为阿魏酸组50 mg/kg、阿魏酸+川芎嗪(50 mg/kg+30 mg/kg)组、阿魏酸+延胡索乙素(50 mg/kg+20 mg/kg)组、阿魏酸+川芎嗪+延胡索乙素(50 mg/kg+30 mg/kg+20 mg/kg)组,分别灌胃给药,采用高效液相色谱法测定各组大鼠血浆中阿魏酸的浓度,DAS2.0程序计算药代动力学参数。结果表明:川芎嗪、延胡索乙素均可延长阿魏酸在大鼠体内的作用时间,增加阿魏酸在大鼠体内的吸收。     

An improved protein decoy set for testing energy functions for protein structure prediction

We have improved the original Rosetta centroid/backbone decoy set by increasing the number of proteins and frequency of near native models and by building on sidechains and minimizing clashes. The new set consists of 1,400 model structures for 78 different and diverse protein targets and provides a challenging set for the testing and evaluation of scoring functions. We evaluated the extent to which a variety of all-atom energy functions could identify the native and close-to-native structures in the new decoy sets. Of various implicit solvent models, we found that a solvent-accessible surface area-based solvation provided the best enrichment and discrimination of close-to-native decoys. The combination of this solvation treatment with Lennard Jones terms and the original Rosetta energy provided better enrichment and discrimination than any of the individual terms. The results also highlight the differences in accuracy of NMR and X-ray crystal structures: a large energy gap was observed between native and non-native conformations for X-ray structures but not for NMR structures.     

Simulation,construction and characterization of a multi-functional thrombolytic agent with anti-thrombosis activity

Prourokinase (scu-PA),a thrombolytic agent,was inserted between Glyl 18 and Ilel 19 with foreign anti-thrombosis functional motif (Lys-Gly-Asp-Trp-motif) to construct a multi-functional chimeric molecule.The molecular model of a chimera was simulated and pre-dicted.The recombinant chimeric protein was expressed by the baculovirus-insect cell expression system and puri-fied by affinity chromatography.The physico-chemical characteristics of the chimeric molecule were assayed.The thrombolytic activity was determined to be 90000 IU/mg of fibrinolytic special activity by the fibrin-plate method.The anti-thrombosis activities were also assayed with IC50 of 9.6 μM by an inhibition test of ADP-induced platelet aggregation.     

Antibody-based approaches in Alzheimer's research: safety, pharmacokinetics, metabolism, and analytical tools

High morbidity, enormous socioeconomic costs, and lack of specific treatments emphasize the importance of research on protective therapies against Alzheimer's disease. The efficacy of anti-amyloid immunization strategies has been demonstrated preclinically, prompting the design of clinical studies. However, the detailed mechanisms of action of therapeutic antibodies, especially their influence on the complex amyloid β peptide (Aβ) metabolism and various Aβ-equilibria present both within and outside the CNS, are far from being clear. Furthermore, physiological Aβ metabolism is poorly understood and the analytical tools to characterize and quantify treatment effects on Aβ metabolism are suboptimal. Thus, the design of immunization strategies with optimized benefit-to-risk ratios for patients is subjected to significant obstacles. Indeed, an active immunization trial with Aβ was discontinued because of severe adverse effects. Anti-Aβ immunization protocols designed to attain high blood levels of antibodies bear the potential to induce brain inflammation and/or hemorrhage, thus directing the biomedical research towards development of more predictable therapies for minimizing the risk of adverse effects. The focus of this review is to summarize current knowledge of Aβ metabolism under physiological and antibody-based therapeutic conditions and to introduce a promising approach, namely the passive immunization using antibody fragments, which are characterized by entirely different pharmacokinetic and pharmacodynamic properties compared with conventional monoclonal antibodies.     

原发性胆汁性肝硬化患者临床指标变化与肝脏病理分期的回顾性分析

目的通过肝活检检测原发性胆汁性肝硬化（PBC）患者的病理分期,并对比血生化、自身抗体等指标进一步明确各期的生化特点,便于指导临床。方法所有患者采静脉血检查肝功,自身抗体,免疫球蛋白,所有患者进行肝脏活检,分析其肝脏病理分期。结果 43例患者血清抗线粒体抗体（AMA）及AMA-M2阳性为31例（72.1%）。27例（62.8%）ANA阳性,37例（86.1%）患者血清IgM水平升高。均有肝功能指标的明显异常,以GGT及ALP升高最明显。结论对胆酶增高而原因未明的肝病患者,早期自身免疫抗体及肝脏病理检查对原发性胆汁性肝硬化的诊断及治疗具有临床指导意义。     

Parrots as key multilinkers in ecosystem structure and functioning

Mutually enhancing organisms can become reciprocal determinants of their distribution, abundance, and demography and thus influence ecosystem structure and dynamics. In addition to the prevailing view of parrots (Psittaciformes) as plant antagonists, we assessed whether they can act as plant mutualists in the dry tropical forest of the Bolivian inter‐Andean valleys, an ecosystem particularly poor in vertebrate frugivores other than parrots (nine species). We hypothesised that if interactions between parrots and their food plants evolved as primarily or facultatively mutualistic, selection should have acted to maximize the strength of their interactions by increasing the amount and variety of resources and services involved in particular pairwise and community–wide interaction contexts. Food plants showed different growth habits across a wide phylogenetic spectrum, implying that parrots behave as super‐generalists exploiting resources differing in phenology, type, biomass, and rewards from a high diversity of plants (113 species from 38 families). Through their feeding activities, parrots provided multiple services acting as genetic linkers, seed facilitators for secondary dispersers, and plant protectors, and therefore can be considered key mutualists with a pervasive impact on plant assemblages. The number of complementary and redundant mutualistic functions provided by parrots to each plant species was positively related to the number of different kinds of food extracted from them. These mutually enhancing interactions were reflected in species‐level properties (e.g., biomass or dominance) of both partners, as a likely consequence of the temporal convergence of eco‐(co)evolutionary dynamics shaping the ongoing structure and organization of the ecosystem. A full assessment of the, thus far largely overlooked, parrot–plant mutualisms and other ecological linkages could change the current perception of the role of parrots in the structure, organization, and functioning of ecosystems.     

眼镜蛇咬伤患者早期肝肾功能、心肌酶的变化

目的观察眼镜蛇咬伤患者早期肝、肾功能及心肌酶的改变,以探讨其临床意义。方法对30例眼镜蛇咬伤患者入院时即抽取静脉血2.0 ml,分离血清,按常规方法行肝、肾功能及心肌酶的测定,并与健康体检者进行比较。结果与对照组比较,眼镜蛇咬伤患者早期肝功能及心肌酶指标显著升高(P0.05);肾功能检测结果差异无显著性(P0.05)。结论通过对眼镜蛇咬伤患者肝、肾功能及心肌酶的测定,了解患者肝脏、肾脏、心肌损害程度,对指导临床治疗具有一定意义。     

The effect of circadian rhythm on pharmacokinetics and metabolism of the Cdk inhibitor,roscovitine, in tumor mice model

Roscovitine is a selective Cdk-inhibitor that is under investigation in phase II clinical trials under several conditions, including chemotherapy. Tumor growth inhibition has been previously shown to be affected by the dosing time of roscovitine in a Glasgow osteosarcoma xenograft mouse model. In the current study, we examined the effect of dose timing on the pharmacokinetics, biodistribution and metabolism of this drug in different organs in B6D2F1 mice. The drug was orally administered at resting (ZT3) or activity time of the mice (ZT19) at a dose of 300?mg/kg. Plasma and organs were removed at serial time points (10, 20 and 30?min; 1, 2, 4, 6, 8, 12 and 24?h) after the administration. Roscovitine and its carboxylic metabolite concentrations were analyzed using HPLC-UV, and pharmacokinetic parameters were calculated in different organs. We found that systemic exposure to roscovitine was 38% higher when dosing at ZT3, and elimination half-life was double compared to when dosing at ZT19. Higher organ concentrations expressed as (organ/plasma) ratio were observed when dosing at ZT3 in the kidney (180%), adipose tissue (188%), testis (132%) and lungs (112%), while the liver exposure to roscovitine was 120% higher after dosing at ZT19. The metabolic ratio was approximately 23% higher at ZT19, while the intrinsic clearance (CL_int) was approximately 67% higher at ZT19, indicating faster and more efficient metabolism. These differences may be caused by circadian differences in the absorption, distribution, metabolism and excretion processes governing roscovitine disposition in the mice. In this article, we describe for the first time the chronobiodistribution of roscovitine in the mouse and the contribution of the dosing time to the variability of its metabolism. Our results may help in designing better dosing schedules of roscovitine in clinical trials.     

湖南八大公山黄杉种群结构和群落特征研究

利用样方法对湖南八大公山黄杉(Pseudotsuga sinensis Dode)群落进行调查,分析黄杉群落的物种组成、区系特征、径级结构和物种多样性。结果显示,群落内共有维管植物132种,隶属于48科89属。群落垂直结构明显,分为乔木层、灌木层和草本层,黄杉是乔木层的优势种,另有檵木(Loropetalum chinense(R.Br.)Oliver)、马尾松(Pinus massoniana Lamb)和油桐(Vernicia fordii(Hemsl.)Airy Shaw)等主要伴生种,檵木为灌木层优势种,两色鳞毛蕨(Dryopteris setosa(Thunb.)Akasawa)为草本层优势种。群落区系组成以泛热带分布和北温带分布为主,具有明显的亚热带向温带过渡的性质。种群年龄结构可分为稳定型向衰退型过渡和衰退型2个类型,样方中幼苗和幼树极度缺失,中龄和老龄个体居多。黄杉、钩栲针阔混交林的物种多样性高于黄杉纯林,其中灌木层的多样性指数最高,乔木层的物种多样性指数最低。群落冠层郁闭度高造成的幼苗缺失可能是影响其濒危的主要原因,建议采取人为干扰创造林窗、加快繁殖技术研究等措施扩大其种群数量。