Structure,function, and regulation of renal organic anion transporters

Renal elimination of anionic drugs, xenobiotics, and toxins is necessary for the survival of mammalian species. This process is mediated by vectorial transport from blood to urine through the cooperative functions of specific transporters in the basolateral and apical membranes of the proximal tubule epithelium. The first step of this process is the extraction of organic anions from the peritubular blood plasma into proximal tubule cells largely through the organic anion transporter (OAT) pathway. Therefore, the OAT pathway is one of the major sites for body drug clearance/detoxification. As a result, it is also the site for drug-drug interaction and drug-induced nephrotoxicity. To maximize therapeutic efficacy and minimize toxicity, the structure-function relationships of OATs and their regulation must be defined. The recent cloning and identification of OATs have paved the way for such investigations. This review summarizes the available data on the general properties of OATs, focusing in particular on the recent progress made from the author's laboratory as well as from other's, on the molecular characterization of the structure-function relationships of OATs and their regulatory mechanisms.     

山药苦瓜复方制剂降低2型糖尿病大鼠血糖和血脂的实

背景药食两用中药山药、苦瓜和甘蔗纤维等降糖和降脂作用明显,但作用机制仍不十分清楚.目的观察山药苦瓜复方制剂对2型糖尿病大鼠血糖、血脂、血胰岛素水平及抗感染能力的影响.设计随机对照动物实验.单位广东医学院科技开发中心、药理教研室、实验动物中心、中心实验室.材料选用SPF级雌性SD大鼠80只,鼠龄4个月.二甲双胍(吉林省东北亚药业股份有限公司,批号040126).总胆固醇、高密度脂蛋白胆固醇、三酰甘油试剂盒购于北京中生生物技术股份有限公司;Surestep Life scan血糖仪及试纸(美国强生公司);胰岛素放射免疫试剂盒(上海海军医学研究所);UV-3010紫外分光光度计(日本岛津);山药苦瓜复方制剂由广东医学院科技开发中心提供,批号040321,混悬液,由山药、苦瓜和甘蔗渣膳食纤维等成分经水提而成,其中生药含量1 kg/L.方法实验中的动物饲养和标本采集于2004-06/2005-12在广东医学院实验动物中心和科技开发中心实验室完成,指标的检测在广东医学院药理研究室和中心实验室完成.①随机选取20只为正常对照组,每天灌胃生理盐水5 mL/kg.其余60只大鼠灌胃脂肪乳剂5 mL/kg,1次/d,连续4周后,大鼠禁食12 h,腹腔注射2g/L链脲佐菌素30 mg/kg.正常对照组腹腔注射等体积柠檬酸缓冲液.3 d后测随机血糖,2周后重复测血糖,以血糖水平高于13.5 mmol/L或尿糖＞++以上且持续2周为造模成功(n=48).②将48只大鼠按随机摸球法分为3组模型组,二甲双胍组和山药苦瓜复方制剂组,每组16只.模型组灌胃脂肪乳剂5 mL/kg;二甲双胍组和山药苦瓜复方制剂组灌胃脂肪乳剂5 mL/kg后分别灌胃二甲双胍1 mg/kg和含生药1 kg/L的山药苦瓜复方制剂5 mL/kg.正常对照组灌胃生理盐水5 mL/kg.各组均1次/d,连续6周.③在实验结束前1天测随机血糖.禁食12 h用放射免疫法测定血浆胰岛素水平,用分光光度法测定血浆总蛋白、白蛋白水平,按相应试剂盒说明测定三酰甘油、总胆固醇、高密度脂蛋白胆固醇水平.④计量资料差异比较采用方差分析,先用Levene'stest对数据进行方差齐性检验,如方差齐用Bonferroni检验,如方差不齐则用Tamhane's T2检验.主要观察指标山药苦瓜复方制剂对2型糖尿病大鼠血糖、胰岛素、血脂和血浆蛋白水平的影响.结果造模失败脱失12只,模型组和二甲双胍组在实验过程有4,2只动物死亡,最终62只进入结果分析.①空腹血糖和血浆胰岛素测定结果正常对照组大鼠空腹血糖水平明显低于其他3组(t=2.673～4.224,＜0.05～0.01),血胰岛素水平明显高于其他3组(t=3.780～5.824,P＜0.05～0.01).模型组大鼠空腹胰岛素水平明显低于山药苦瓜复方制剂组(t=2.825,P＜0.05),空腹血糖水平明显高于二甲双胍组和山药苦瓜复方制剂组(t=3.906,3.056,P＜0.05).二甲双胍组大鼠胰岛素水平明显低于山药苦瓜复方制剂组(t=3.014,P＜0.05),但空腹血糖水平与山药苦瓜复方制剂组相近(P＞0.05).②血脂水平测定结果正常对照组大鼠血浆总胆固醇和三酰甘油水平明显低于其他3组,高密度脂蛋白胆固醇水平明显高于其他3组(t=2.521～4.892,P＜0.05～0.01).模型组大鼠血浆总胆固醇水平明显高于二甲双胍组和山药苦瓜复方制剂组(t=2.466,2.512,P＜0.05),三酰甘油水平明显高于山药苦瓜复方制剂组(t=2.612,P＜0.05),高密度脂蛋白胆固醇水平明显低于山药苦瓜复方制剂组(t=3.688,P＜0.05).二甲双胍组大鼠血浆三酰甘油水平明显高于山药苦瓜复方制剂组(t=2.620,P＜0.05).③血浆蛋白水平测定结果各组大鼠血浆总蛋白水平相近(P＞0.05);正常对照组大鼠血浆白蛋白水平明显高于模型组和二甲双胍组(t=3.773,3.104,P＜0.05),与山药苦瓜复方制剂组相近(P＞0.05),白蛋白与球蛋白比值明显高于其他3组(t=2.830～3.056,P＜0.05).模型组大鼠血浆白蛋白水平和白蛋白与球蛋白比值明显低于山药苦瓜复方制剂组(t=2.604,3.808,P＜0.05).结论山药苦瓜复方制剂可降低2型糖尿病模型大鼠血糖、血脂水平,升高胰岛素水平,提高抗感染能力.     

脂质体介导基因转染人精子膜蛋白PH20DNA疫苗质粒向小鼠骨骼肌导入

目的:精子膜蛋白PH-20分布在精子表面,在受精过程中起重要作用,可作为免疫避孕候选疫苗.采用脂质体介导基因转染法将pcDNA3.1( )-hPH20DNA疫苗质粒导入小鼠骨骼肌中,检测目的基因hPH-20在体内的表达水平.方法:实验于2006-06/2007-02在郑州大学解剖学实验室完成.①清洁级健康BALB/C小鼠12只,随机数字表法分为重组质粒组、空载体组、生理盐水组,4只,组,实验过程中对动物的处置符合动物伦理学标准.已构建好的pcDNA3.1( )-hPH20重组质粒由本室保存.②实验方法:质粒提取后,用精胺盐酸去除内毒素,鲎试剂榆测晕阳性代表去除成功.取少量质粒行琼脂糖电泳鉴定纯度,用紫外分光光度计测量质粒DNA的含量.每只小鼠于股四头肌分点注射利多卡因预处理3 d.重组质粒组将脂质体包裹的peDNA3.1( )-hPH20 DNA缓慢注射于股四头肌内,200μg/只.空载体组单纯将pcDNA3.1( )缓慢注射到股四头肌内,200 μ g/只.生理盐水组于相同部位缓慢注射等鼍生理盐水.重组质粒组分别于注射后第4,7,11,16天以断颈法各处死1只小鼠,空载体组、生理盐水组小鼠均于注射后第11天同法处死.迅速取股内侧肌肉30mg,研磨成粉末状,RT-PCR法检测目的基因hPH-20 mRNA在体内的表达.结果:①提取质粒琼脂糖凝胶电泳检测结果:提取的质粒成功去除内毒素后,琼脂糖凝胶电泳显示质粒纯度较高,绝大部分处于超螺旋状态,有利于体内转基因的表达.紫外分光光度计测量质粒DNA的含量(A 260/A280)为1.8～2.0.②目的基因hPH-20mRNA体内表达RT-PCR检测:重组质粒组注射后第4天即可检测到1530 bp特异性条带,第7天表达增强,第11天达到高峰,第16天表达下降.空载体组、生理盐水组各时间点均未见特异性条带.结论:脂质体介导基因转染法能够高效将peDNA3.1( )-hPH20 DNA疫苗质粒导入小鼠骨骼肌中,且hPH-20基因于注射后11 d呈峰值表达.     

A sulfonamide based glucose-responsive hydrogel with covalently immobilized glucose oxidase and catalase.

A new glucose-sensitive hydrogel, based on sulfonamide chemistry with covalently conjugated glucose oxidase and catalase, was synthesized and tested. The pH-induced full swelling transition of the gel occurred in the range of pH 6.5 approximately 7.5. In a glucose concentration range of 0-300 mg/dl in an isotonic phosphate buffered saline solution (pH 7.4), the pH inside the gel varied from pH 7.4 to 7.2. At the same glucose concentration range, the gel showed reversible glucose dependent swelling without hysteresis from 12 to 8, expressed in water (g)/polymer (g).     

Adriamycin loaded pullulan acetate/sulfonamide conjugate nanoparticles responding to tumor pH: pH-dependent cell interaction, internalization and cytotoxicity in vitro.

The cytotoxicity of adriamycin (ADR)-loaded and pH-sensitive nanoparticles made of pullulan acetate (PA) and sulfonamide (sulfadimethoxine; SDM) (PA/SDM) conjugate to a breast tumor cell line (MCF-7) was investigated to test the feasibility of the nanoparticles in targeting acidic tumor extracellular pH (pH(e)). At pH 6.8, ADR loaded PA/SDM nanoparticles showed cytotoxicity in the cell culture experiment, comparable to that of free ADR at the same ADR concentrations, while the relative cytotoxicity at pH 7.4 was low at the tested concentration range. This pronounced cytotoxicity of the nanoparticles at low pH was attributed to the accelerated release of ADR triggered by pH, enhanced interaction with cells, and internalization. At pH 6.8 and 6.4, the PA/SDM nanoparticles aggressively bounded to MCF-7 cells, probably due to interactions of the cells with hydrophobized nanoparticle surfaces caused by SDM deionization. A confocal laser microscopic study revealed intracellular localization of the drug-loaded nanoparticles. Based on these findings, the pH-sensitive nanoparticles deserve further investigation with an in vivo animal model as a targeted carrier of pH(e).     

Poly( -histidine)–PEG block copolymer micelles and pH-induced destabilization

Poly(L-histidine)-poly(ethylene glycol) diblock copolymers (polyHis-b-PEG) were prepared and used for the construction of polymeric micelles responding to local pH changes in the body. PolyHis was synthesized by ring opening polymerization of L-histidine N-carboxyanhydride, the imidazole amine group of which was protected by the dinitrophenyl group. The resulting polymer (M(n): 5,000 g/mole) was coupled to poly(ethylene glycol) (M(n): 2,000 g/mole) via an amide linkage using the dicyclohexyl carbodiimide and N-hydroxysuccinimide-mediated reaction. The block copolymer in dimethyl sulfoxide formed polymeric micelles on diafiltration against a borate buffer at pH 8. Dynamic light scattering and atomic force microscopy showed the micelles were spherical, diameter approximately 114 nm, with a unimodal distribution. The critical micelle concentration (CMC) at pH 8.0 was 2.3 mg/l. The CMC increased markedly on decreasing the pH of the diafiltration medium below 7.2. Micelles prepared at pH 8.0 were gradually destabilized below pH 7.4, as evidenced by a slight increase in light transmittance, an alteration in size distribution, and a decrease in the pyrene fluorescence intensity. It was concluded that the ionization of the polyHis block forming the micelle core determined the pH-dependent CMC and stability. After further optimization of the pH-sensitivity, pH-sensitive micelles are expected to have application for solid tumor treatment, exploiting the fact that most solid tumors have an acidic extracellular pH.     

强力杀菌消毒剂杀灭微生物效果的试验观察

为了解含二氧化氯的强力杀菌消毒剂杀灭微生物的效果,在实验室用悬液定量杀菌试验对其进行了观察。结果,用含50mg/L二氧化氯的强力杀菌消毒剂溶液对大肠杆菌作用5min、对金黄色葡萄球菌作用10min、对白色念珠菌作用15min,用含300mg/K二氧化氯的消毒剂溶液对枯草杆菌黑色变种芽孢作用20min,杀菌率均为100％。另外,用含2500mg/L二氧化氯的强力杀菌消毒剂,按6ml/m~3喷雾作用15min,室内空气自然菌比对照组减少94.41％。     

中脑导水管周围灰质内催产素对痛阈和电针镇痛效应的影响

本工作以钾离子透入法引起大鼠甩尾反应的电流强度（ｍＡ）为痛反应指标，观察了中脑导水管周围灰质（ＰＡＧ）腹外侧区注射催产素（ＯＴ）和抗催产素血清（ＡＯＴＳ）对大鼠痛阈和电针镇痛效应的影响。结果表明，ＰＡＧ注射ＯＴ能增加大鼠痛阈和电针镇痛效应；注射ＡＯＴＳ以中和内源性的ＯＴ后，对大鼠痛阈虽克明显影响，但能显著降低电针期的停针后的电针镇痛效应。提示ＰＡＧ内生理水平的ＯＴ在电针镇痛中发挥一定作用。     

PCR产物固相杂交酶联显色观察白细胞介素-2联合干扰素-α治疗慢性丙型肝炎疗效

目的 建立一步法基因扩增产物固相杂交酶联免疫吸附方法并用于临床观察白细胞介素—2(IL—2)联合干扰素—α(IFN—α)治疗慢性丙型肝炎的疗效。方法 7例患者中有4例患者接受IL—2和IFN—α联合治疗，其中2例为IFN—α单独或联合抗病毒药物治疗后复发者，未接受过抗病毒药物治疗者2例，疗程为一年。余下的3例患者为单独使用IFN—α治疗者。同时采用一步法基因扩增产物固相杂交酶联免疫吸附方法检测血清丙型肝炎病毒核酸(HCVRNA)滴度评价疗效。结果 IL—2联合干扰素治疗对干扰素治疗后复发者仍有效，完全应答率为100％；对未接受过抗病毒药物治疗者也有效，完全应答率为50％。结论 白细胞介素—2联合干扰素可作为初次治疗及复发后再治疗的选择方案。而一步法基因扩增产物固相杂交酶联显色检测血清HCV—RNA滴度的方法可快速、特异、灵敏地反应出治疗过程中HCV—RNA量的变化，为临床治疗提供可靠的疗效判定指标。     

睡眠节律紊乱导致阿尔兹海默病相关机制的研究进展

认知功能损害患者睡眠障碍患病率高,表现形式多样,主要包括失眠、日间过度思睡、睡眠呼吸障碍、异态睡、不宁腿综合征、睡眠节律紊乱等。阿尔茨海默病(Alzheimer disease,AD)是最常见的认知损害类型。73%的中国汉族AD患者伴有睡眠障碍,其中53%伴有不同程度的睡眠节律紊乱。AD患者睡眠障碍在病程后期较为突出,因此睡眠节律紊乱一直被认为是AD相关神经退行性变的结果,如"日落现象"。但近期研究表明,睡眠节律紊乱很可能参与AD发生的始动环节。国外前瞻性随访研究发现,认知正常老年人群睡眠节律紊乱发生5年后更易发生AD。目前,关于睡眠节律紊乱通过何种途径促使神经系统退行性变发生的研究尚不深入。本文对睡眠节律紊乱引发AD相关病理、生物标志物变化进而导致AD发生的机制作一综述。