药物与细胞膜相互作用的ppKCE动力学参数

本文在区段-区段动力学毛细管电泳（ppKCE）的理论基础上,以兔红细胞膜、人红细胞膜为受体,首次同时测得了表征药物与细胞膜间结合快慢的正向结合速率常数kon、反向解离速率常数koff、以及配体与受体间相互作用的结合常数Kb。实验中考察了迁移时间及峰高的稳定性,pH值对药物与细胞膜相互作用动力学参数的影响。该方法为评价药物疗效、毒性及药物动力学研究提供一种新的方法。     

毛细管电泳测定主客分子相互作用的结合常数

在２５℃下,用毛细管区带电泳测定了主管体分子（配体和溶质分子）β－环糊精（β－ＣＤ）和心得舒（ａｌｐｒｅｎｏｌｏ）在ｐＨ值为２．５,浓度为１００ｍｍｏｌ／Ｌ的磷酸盐缓冲溶液下的结合常数,并对４种求解方法既非线性拟合法,双倒数法,ｙ－倒数法和ｘ－倒数法的结果进行了比较,所得值分别是３０７．２、４０８．４、３３１．４和３４３．１Ｌ／ｍｏｌ,同时获得了结合物的迁移率。该方法可用于测定结合比为１：１的各种     

利用表面上的小分子控制细胞黏附

细胞黏附是重要的生理过程,多细胞生物体中大部分种类的细胞都依赖于在表面的黏附而进行其正常生理活动。细胞的黏附需要固定在表面的有机分子（例如蛋白质或多肽）作配体。我们利用表面小分子模拟蛋白质或多肽作为配体,通过与细胞膜上受体结合,促进细胞黏附到表面。聚乙二醇（PEG）可以抵抗细胞在表面的黏附,我们利用含有PEG的表面小分子来调节细胞黏附。细胞表面的受体与胞外基质表面的配体结合是一个动态过程,在适宜时间和空间发生的时候,细胞就会产生运动和迁移,细胞的迁移也是重要的生理过程。本文主要介绍近年来利用小分子的表面化学和微纳米结构控制细胞在表面的黏附和迁移。     

基于雌激素β受体结构雌激素类化合物的三维定量结构-活性关系与分子对接研究

雌激素类化合物由于其对人和野生动物健康的负面影响而受到广泛关注.雌激素受体存在α和β两种亚型,由于雌激素β受体（ERβ）与α受体（ERα）两者结合腔中的氨基酸序列存在明显差异,因此配体化合物在与雌激素β受体和α受体的结合活性和模式上也可能存在较大差别.本文以50个与雌激素β受体结合的化合物为研究对象,应用比较分子相似性指数分析（COMSIA）的三维定量结构-活性关系（3D-QSAR）分析方法研究化合物结构与活性之间的关系,比较了原子契合和基于受体结构两种分子叠合方式对模型质量的影响,建立了相关性显著、预测能力强的定量活性预测模型（R^2=0.961,qLOO^2=0.671,R^2Pred=0.722）,并结合分子对接方法揭示了影响化合物活性的分子结构特征和分子机理.     

微流控纸芯片法研究去甲肾上腺素与牛血清白蛋白相互作用

建立了一种基于微流控纸芯片测定牛血清白蛋白（BSA）与去甲肾上腺素结合常数的快速检测方法。以纸芯片为分析平台,在样品加载区键合BSA;重酒石酸去甲肾上腺素（NE-B）与Fe³⁺发生氧化还原反应,产物Fe²⁺与显色剂邻二氮菲在纸芯片上进行显色,通过手机拍照,Photoshop软件分析显色强度。以去甲肾上腺素浓度倒数为横坐标,显色强度RGB值的倒数为纵坐标进行线性拟合,计算出药物与蛋白的结合常数K_b。结果表明：该方法日内重复性相对标准偏差（RSD）为0.55%,日间重复性RSD为0.76%;在1.5～5.5 mmol/L范围内,去甲肾上腺素与BSA的K_b为1598.33 L/mol。该方法可用于还原性药物与BSA的作用常数测定,具有样品用量少、成本低廉、检测速度快的特点。     

一种测定功能蛋白与其配体结合参数的简单方法

以羰基二咪唑法分别将牛血清白蛋白(BSA)和β2-肾上腺素受体(β2-AR)键合在大孔硅胶上,合成两种色谱固定相。采用直接进样法测定BSA与奥美拉唑、普萘洛尔和盐酸异丙嗪的结合常数及结合位点数,β2-AR与盐酸异丙肾上腺素、沙丁胺醇和去甲肾上腺素的结合常数及结合位点数。方法可用于功能蛋白与其配体分子相互作用的研究。     

多肽对钙调素亲和力的预测

根据前文提出的钙调素可结合多肽的钙调素结合部位的模型,并综合钙调素可结合多肽的报道,提出一种简单方法来预测多肽的钙调素结合部位和其复合物的解离常数,研究了多肽的疏水性、形成α螺旋结构的倾向、碱性等因素对解离常数的影响.为了进一步检验模型和预测方法,设计合成了模型肽,合成的模型肽与钙调素的复合物的解离常数的实测值与预测值相符.     

一种测定功能蛋白与其配体结合参数的简单方法

以羰基二咪唑法分别将牛血清白蛋白(BSA)和β2-肾上腺素受体(β2-AR)键合在大孔硅胶上,合成两种色谱固定相。采用直接进样法测定BSA与奥美拉唑、普萘洛尔和盐酸异丙嗪的结合常数及结合位点数,β2-AR与盐酸异丙肾上腺素、沙丁胺醇和去甲肾上腺素的结合常数及结合位点数。方法可用于功能蛋白与其配体分子相互作用的研究。     

酪氨酸激酶受体多肽片段在SPR传感芯片表面的磷酸化及其与下游蛋白的相互作用

胰岛素样生长因子-1受体(IGF-1R)信号转导通路与各种正常生理活动及多种疾病发生密切相关.将IGF-1R来源的未磷酸化多肽序列LYASVNPEY固定于表面等离子体共振(SPR)生物传感器专用的CM5芯片表面,以含有相应蛋白激酶的细胞裂解液进行处理,利用SPR生物传感器和抗酪氨酸磷酸化抗体PY20进行酪氨酸磷酸化水平检测.结果表明:PY20能灵敏地检测多肽酪氨酸磷酸化水平变化,即能够区分不同孵育时间以及不同成分的细胞裂解液所引起的多肽酪氨酸磷酸化水平差异;研究了IGF-1R来源多肽的酪氨酸磷酸化对多肽-蛋白间相互作用的影响,证实了只存在磷酸化的多肽与其下游的胰岛素受体底物(IRS-1)相互作用,测定了此磷酸化的多肽与IRS-1的亲和常数(KA)为 (2.02 ± 0.09)×108 L mol-1、结合速率常数(ka)为(2.30±0.15)×106 L mol-1s-1以及解离速率常数(kd)为(1.14±0.13)×10-2 s-1.这种新型的模拟受体的多肽酪氨酸磷酸化研究方法可用于受体磷酸化信号通路的研究以及基于这些信号通路的药物筛选研究.     

胰高血糖素样肽-1受体胞外区域突变体复合物的动力学研究

采用分子动力学模拟方法研究了胰高血糖素样肽-1(GLP-1)与GLP-1受体(GLP-1R)胞外区域的相互作用.结果表明,配体的结合导致受体的构象发生改变,Loop2区域的氨基酸Pro90和Trp91以及C末端的Glu128向配体移动.根据保守位点突变受体(P73A,V81L,Y88A,P90A和W91A)后所得多肽模拟数据,发现Loop2区域在突变体中的结构和柔性均发生了明显变化,Trp91和Tyr88的突变将导致配体亲和力丧失.研究结果证明,P73A突变型受体和野生型受体分别与配体相互作用后,二者数值差别不大,因此Pro73不是关键残基;V81L突变体则会导致配体亲和力的丧失.该结果为GLP-1药物设计提供了重要理论依据.     

溶剂诱导相变萃取-分光光度法测定血管紧张素转化酶活性

在传统体外测定血管紧张素转化酶(ACE)活性方法的基础上, 以猪肺ACE粗提物为酶源, 采用溶剂诱导相变萃取法(SIPSE)萃取酶反应产物马尿酸(HA), 用分光光度法快速定量测定HA以评估ACE活性或样品对ACE的抑制活性. 以卡托普利为模型验证了此方法, 并分别测定了中药复方天麻钩藤饮及其含有的11味中药对ACE的抑制活性. 结果表明, 猪肺ACE粗提物即可满足ACE抑制活性测定的需要; 与传统液液萃取法相比, SIPSE对HA具有更高的选择性及萃取能力, 萃取率为(97.47±0.55)%(n=3); 通过计算校正, 溶剂诱导相变萃取-分光光度法能简便、 快速且准确地测定成分复杂体系的ACE抑制活性.     

First Determination of Glycidyl Ester Species in Edible Oils by Reverse-Phase Ultra-Performance Liquid Chromatography Coupled with an Evaporative Light-Scattering Detector

In this work, a new ultra-performance liquid chromatograph-evaporative light-scattering detector (UPLC-ELSD) method for quantitation of glycidyl esters (GE) contents in edible oils is presented. The method features complete separation of five GE species within 20 min by a C18 column and gradient elution with a mobile phase consisting of 85% and 2.5% methanol aqueous solutions. The coefficients of regression (R²) were all ≥0.9999 for the linear-quadratic regression curves of GE species in a concentration range of 5~80 μg/mL. The intraday and interday recoveries (%) of GE species in solvent were in a range of 81.3~107.3%, and the intraday and interday coefficients of variation (CVs, %) were all ≤8.6%. The average recovery (%) of GE species spiked in extra-virgin olive oil samples ranged from 88.3~107.8% and the intermediate precision (CV, %) of ≤14% indicated acceptable accuracy and precision. The method exhibited limit of quantification (LOQ) for each GE species (0.6 μg glycidol equivalents/g oil). The method was applied to determine GE concentrations of six commercial oil samples, and total glycidol equivalents were consistent with data obtained by GC-MS method. This UPLC-ELSD method could be adopted for precursory screening and research purposes to improve food safety when MS detectors are unavailable.     

The use of zinc dimethacrylate functionalized graphene as a reinforcement in rubber composites

Zinc dimethacrylate functionalized graphene (Z‐GE), as reinforcing nanofiller for natural rubber (NR), was synthesized by liquid‐phase exfoliation and in situ method. The morphology and structure of Z‐GE were characterized to confirm the exfoliation and functionalization of GE. The NR/Z‐GE composites were prepared and investigated by mechanical analysis, crosslinked network analysis and the analysis of thermal conductivity. The results showed that the tensile strength, tear strength and modulus at 300% strain of NR/Z‐GE‐20 composites (contents 1.400 phr GE) were increased by 142%, 76% and 231% as compared with the pure NR, respectively. And the thermal conductivity of NR/Z‐GE‐30 composites is enhanced by 39% as that of the pure NR. This significant improvement is attributed to the formation of covalent crosslinked network and ionic crosslinked network and efficient interfacial interaction between GE and NR matrix. This method provides a new insight into the fabrication of multifunctional GE composites and enlarges its potential applications in high performance GE‐based rubber composites. Copyright © 2015 John Wiley & Sons, Ltd.     

Polymer Carriers for Anticancer Drugs Targeted to EGF Receptor

A novel actively targeted polymer carrier for anticancer drugs based on an N‐(2‐hydroxypropyl)methacrylamide copolymer (PHPMA) is proposed. An oligopeptide sequence GE7, attached to the polymer, is a specific ligand for the EGF receptor overexpressed on most tumor cells. Co‐attachment of selected chemotherapeutics will therefore lead to formation of tumor‐specific polymer therapeutics, further enhanced by the EPR effect. FACS measurements prove elevated binding activity of the fluorescently labeled PHPMA/GE7 conjugate in EGFR‐rich cells (FaDu, MCF‐7), compared to conjugates of scrambled peptides. Cell lines with low EGFR level (SW620, B16F10) bind the GE7 conjugate significantly less.

     

96孔板法用于高通量血管紧张素转化酶抑制剂体外检测

为在体外迅速检测血管紧张素转化酶抑制剂( ACEI)的抑制活性,选用96孔板,以呋喃丙烯酰三肽(FAPGG)为模拟底物,通过检测血管紧张素转化酶(ACE)酶解FAPGG生成N-[3-(呋喃)丙稀醇酰-2-苯丙氨酸( FAP)和双甘氨肽(GG)后340 nm处吸光值的下降衡量ACE的活性,采用加入ACEI前后ACE的活性变化衡量ACEI的活性.考察了不同缓冲体系、Cl-浓度、ACE酶活性(ACE酶浓度)、缓冲体系的pH值等对上述检测模型反应体系的影响,建立了高通量降血压肽活性体外检测方法,本方法最多可同时检测96个样品的ACE抑制活性,上机分析时间仅需10 s.不同批次活性平行测定的相对标准偏差均小于0.001％,p=0.667＞0.05,各测定结果无显著差异,重现性好,精密度较高.采用本方法测定了商品血管紧张素转化酶抑制剂Captopril的IC50值为16.19 nmol/L,与文献报道的测定结果一致.     

A sensitive LC–MS/MS method for simultaneous quantification of geniposide and its active metabolite genipin in rat plasma and its application to a pharmacokinetic study

Genipin (GP), an active metabolite of geniposide (GE), exhibits more potent pharmacological effects than its parent compound. In this paper, a sensitive LC‐MS/MS method was developed and fully validated for the simultaneous determination of GE and GP in rat plasma. We found that GP degraded rapidly in rat plasma at room temperature as a result of irreversible binding with the endogenous nucleophiles in plasma. GP was stable when the sample's pH was ≤4.0. The degradation of GP in rat plasma was well prevented by immediate addition of 5% glacial acetic acid to the freshly collected plasma. The detection was performed on a tandem mass spectrometer coupled with electrospray ionization source in negative mode. Quantification was conducted by multiple reaction monitoring of the transitions [M + CH₃COO]⁻ m/z 447.3 → 225.3 for GE and [M − H]⁻ m/z 225.2 → 123.1 for GP. The method exhibited high sensitivity (LLOQ 1 ng/mL for GE and 0.2 ng/mL for GP) by selecting the acetate adduct ions as the precursor ions for GE. The robust developed method was successfully applied to a pharmacokinetic study in rats after oral administration of GE.     

Analysis method of the angiotensin-I converting enzyme inhibitory activity based on micellar electrokinetic chromatography.

A micellar electrokinetic chromatography (MEKC) method was developed for estimating the angiotensin-I converting enzyme (ACE) inhibitory activity by separating the hippuric acid liberated in the ACE reaction mixture in the presence of an inhibitor, captopril. The hippuric acid was successfully separated and detected by MEKC with a 25 mM sodium dodecyl sulfate solution in a 25 mM phosphate-50 mM borate buffer at pH 7.0; the total analysis took about 5 min. A good linear relationship was observed between the inhibitor and the peak area of hippuric acid release. No significant difference in the ACE inhibitory activity (IC50) of captopril (an antihypertensive medicine) or autolyzed-mushrooms (functional foods) was observed between the conventional method and the MEKC method. The MEKC method was found to be a useful technique for a rapid assay of the ACE inhibitory activity.     

Preparation of polymer nanocomposites with enhanced mechanical properties using hybrid of graphene and partially wrapped multi-wall carbon nanotube as nanofiller

Triblock copolymer of poly(p-dioxanone) and polyethylene glycol end-capped with pyrene moieties ((Py-PPDO)₂-b-PEG) was synthesized and used as modifier for multi-wall carbon nanotubes (MWCNTs). Nano-aggregates ((Py-PPDO)₂-b-PEG@MWCNTs) with shish-kebab like partially wrapped morphology and very good stability were obtained by incorporating the copolymer with MWCNTs. The bare MWCNT sections of (Py-PPDO)₂-b-PEG@MWCNTs were able to induce π-π interactions with graphene (GE) and resulted in a novel GE/(Py-PPDO)₂-b-PEG@MWCNTs hybrid. The dispersity of GE in solution or polymer matrix was therefore greatly improved. The PCL nanocomposite films using GE/(Py-PPDO)₂-bPEG@MWCNTs as hybrid nanofiller exhibited obviously improved mechanical properties especially at very low hybrid nanofiller content. The influence of the nanofiller content and feed ratio of GE/MWCNTs on the mechanical properties of composites films was evaluated. When the feed ratio of GE to MWCNTs is 2:8 and the total loading of nanofiller is only 0.01 wt%, the tensile strength of the composite film increased by 163% and the elongation at break increased by 17% compared to those of neat PCL. These results can be attributed to fine dispersion of the nanofillers in PCL matrix and the hybrid interactions between GE and MWCNTs. Therefore, this work provides a novel method for preparing polymer nanocomposites with high mechanical performance and low nanofiller loading.     

EGFR/cell membrane chromatography-online-high performance liquid chromatography/mass spectrometry method for screening EGFR antagonists from Radix Angelicae Pubescentis

The intracellular kinase domains of the epidermal growth factor receptor (EGFR) in some tumor cells are significant targets for drug discovery. We have developed a new EGFR cell membrane chromatography (EGFR/CMC)-online-high performance liquid chromatography/mass spectrometry (HPLC/MS) method for screening anti-EGFR antagonists from medicinal herbs such as Radix Angelicae Pubescentis. In this study, the HEK293 EGFR cells with high expression of EGFR were used to prepare cell membrane stationary phase (CMSP) in the EGFR/CMC model. The retention fractions on the EGFR/CMC model were directly analyzed by combining a 10 port columns switcher with a HPLC/MS system online. As a result, osthole from Radix Angelicae Pubescentis was found to be the active component acting on EGFR like dasatinib as the control drug. There was a good relationship between their inhibiting effects on EGFR secretion and HEK293 EGFR cell growth in vitro. This new EGFR/CMConline-HPLC/MS method can be applied for screening anti-EGFR antagonists from TCMs, for instance, Radix Angelicae Pubescentis. It will be a useful method for drug discovery with natural medicinal herbs as a leading compound resource.     

Detection of single-base mutation by affinity capillary electrophoresis using a DNA-polyacrylamide conjugate

We have developed an affinity capillary electrophoresis (ACE) method for detection of gene point mutations using a DNA-polyacrylamide conjugate as a pseudostationary affinity phase. In this study, the target DNA was prepared by mixing two PCR products: the wild type of K-ras gene and its codon 12 point mutant. The ligand DNA was designed to be complementary to codons 11 and 12 of the wild type. The target DNA was denatured by the addition of formamide and by heating at 95 degrees C for 5 min, and then electrophoretically separated by difference in affinity to the pseudoimmobilized ligand DNA. The method successfully separated a mixture of the wild-type DNA and each of six codon 12 point mutants by the same ligand DNA. The limit of mutation detection was determined by mixing the wild-type DNA with decreasing concentrations of the mutant DNA. The lowest level of detection was 10% mutant DNA in a background of the wild type. The practicability of this method has been confirmed using a colorectal carcinoma cell line. This study is the first demonstration of detection of gene point mutation in polymerase chain reaction (PCR) products using ACE, and opens up a new possibility of CE-based gene diagnosis.