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.     

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.     

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。     

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。     

四君子汤对脾虚大鼠肝、心肌、胃黏膜和骨骼肌细胞线粒体损伤的修复作用

背景:脾与线粒体具有相关性,中医脾主运化不仅仅是指食物在胃肠的消化吸收,更重要的是线粒体的生物氧化产能过程。目的:分析经典健脾益气方剂四君子汤对脾虚大鼠肝、心肌、胃黏膜和骨骼肌细胞线粒体损伤的修复作用。设计:随机对照观察。单位:广州中医药大学第一附属医院二内科和广州中医药大学。材料:实验于2004-06/12在广州中医药大学第一附属医院内科实验室和广州中医药大学测试中心完成。选择SD大鼠40只,由广州中医药大学动物中心提供。小承气汤由厚朴、枳实、大黄(比例3∶3∶2)组成;四君子汤由党参、白术、茯苓、甘草(比例2∶2∶2∶1)组成,均由广州中医药大学第一附属医院药剂科提供,并由该院制剂室制成100%煎剂。方法:大鼠饲养1周后按随机数字表法分成4组,即正常对照组、脾虚模型组、自然复健组和四君子汤组。除正常对照组外,其余3组均制作大鼠长期脾虚模型。①正常对照组大鼠常规喂养,生理盐水3mL/(次·只)灌胃,隔日1次,共34周。②脾虚模型组大鼠小承气汤3mL/(次·只)灌胃,隔日1次,同时隔日喂食,共34周。③自然复健组喂养方法前26周同脾虚模型组,26周后改为常规饲养,共34周。④四君子汤组喂养方法前26周同脾虚模型组;26周后改为常规饲养,同时用四君子汤灌胃8周,4mL/(次·只),1次/d。于实验34周末麻醉断头处死大鼠后,迅速取出骨骼肌、肝脏、胃黏膜、心肌组织,用双缩脲法测定线粒体悬液的蛋白量,并根据组织质量计算出每克组织的线粒体含量;应用透射电镜观察线粒体形态。主要观察指标:各组大鼠的骨骼肌、肝、心肌、胃黏膜组织线粒体含量及超微形态。结果:40只大鼠全部进入结果分析,无脱失。①实验34周末各组大鼠骨骼肌、肝、心肌、胃黏膜组织线粒体含量比较:脾虚模型组大鼠各组织线粒体含量均显著低于正常对照组(P<0.01);自然复健组显著高于脾虚模型组(P<0.05～0.01);四君子汤组大鼠各组织线粒体含量最高,除显著高于脾虚模型组和自然复健组外(P<0.05～0.01),并显著高于正常对照组(P<0.01)。②实验34周末各组大鼠骨骼肌、肝、心肌、胃黏膜组织线粒体形态比较:脾虚模型组大鼠心肌细胞线粒体高度肿胀;肝细胞线粒体致密质粒减少或消失,嵴断裂;骨骼肌细胞线粒体数量减少,线粒体变小,线粒体膜结构破坏;胃壁细胞线粒体减少,线粒体内部结构不清,胃主细胞线粒体峭断裂。自然复健组大鼠各组织的线粒体形态改变较轻,四君子汤组与正常对照组接近。结论:四君子汤具有提高脾虚大鼠骨骼肌、肝、心肌、胃黏膜组织细胞线粒体含量,修复线粒体损伤的作用。     

Polymeric gene carrier for insulin secreting cells: poly(L-lysine)-g-sulfonylurea for receptor mediated transfection.

Ex vivo transfer of therapeutic genes to cells is one of the potential strategies to prolong the life span of cell transplants. However, relatively safe non-viral carriers have not been extensively investigated due to their lower transfection efficiency. In this study, poly(L-lysine)-g-sulfonylurea varying SU content (PLL-SU) was synthesized to promote gene delivery efficacy to an insulin secreting cell line, RINm5F, which is known to express sulfonylurea receptor (SUR). The polymer formed complexes with a model reporter gene of pCMV-Luc (DNA) and the size of resulting particles was around 100 nm. The transfection efficiency of a polymer synthesized with 5 mol% of SU in the reaction feed (PLL-SU5%) to RINm5F cell was at least 5 times higher than that of PLL. The cytotoxicity of PLL-SU5%/DNA complex was equivalent to that of PLL/DNA complex. PLL-SU5% showed less transfection efficiency than PLL to NIH3T3 and HepG2 cells which are SUR negative. In RINm5F cells, the addition of free SU decreased the transfection efficiency of PLL-SU5%/DNA complex, suggesting that the complex shares the same receptors for SU. The PLL-SU5%/DNA complex seems to be internalized via SUR-mediated endocytosis pathway as suggested by vacuolar ATPases inhibition by Bafilomycin A1. It is noted that RINm5F cells treated with PLL-SU5%/DNA complex secreted more insulin than control, untreated cells, suggesting the insulinotropic effect of SU in PLL-SU5%. In conclusion, PLL-SU may be useful for transfer of therapeutic genes into insulin secreting cells.