利用表面活性剂介导的方法制备AgrC蛋白脂质体

膜蛋白AgrC是金黄色葡萄球菌双组分信号转导系统的感受激酶. 其信号转导机制的阐明对于解决细菌耐药问题具有重要意义. 目前膜蛋白研究的主要瓶颈是难以获得大量高纯度和功能稳定的蛋白. 将目标蛋白表达在大肠杆菌体内,利用表面活性剂将其从细胞膜上溶解,纯化,这一系列步骤容易引起膜蛋白不稳定和功能损失. 本文报道了用表面活性剂介导的方法将膜蛋白AgrC镶嵌到脂质体上,即形成蛋白脂质体. 脂质体和蛋白脂质体的结构、形貌以及平均粒径分别用透射电镜和动态光散射仪表征. 蔗糖密度梯度离心的结果表明蛋白重构效率达80%. 硫醇试剂标记实验确定AgrC的细胞质域在脂质体中取向于内侧. 体外磷酸化实验表明AgrC蛋白在脂质体中的自我磷酸化活性远远高于表面活性剂中的活性,且其自我磷酸化活性在2周内几乎没有损失. 蛋白脂质体的构建不仅解决了膜蛋白的不稳定性问题,也为体外研究AgrC蛋白的结构、功能和信号转导机制提供了新的思路.     

肽受体跨膜镶嵌的人工细胞膜的构建:细菌组氨酸蛋白激酶跨膜区多肽在合成肽脂囊泡上的镶嵌

选择agr I型金黄色葡萄球菌ATCC 6538组氨酸蛋白激酶AgrC蛋白六个跨膜区中与跨膜信号转导过程最密切的一条肽链为受体, 将此多肽采用超声法镶嵌到人工细胞膜上, 采用透射电镜和原子力显微镜对其形态进行了分析, 发现大部分囊泡及镶嵌体系的单个形态为均一球状, 直径大小在50～200 nm范围内. 考察悬浮液pH、体系温度及浓度等因素对其稳定性的影响, 发现在pH为7.0, 温度不超过30 ℃, 多肽和N^＋C₅Ala2C₁₆的物质的量之比为1∶100时, 镶嵌体系的稳定性最强. 同时, 通过圆二色谱、差示扫描量热分析等手段对镶嵌模型进行表征, 分析发现多肽分子能稳定地插入双层膜囊泡中, 且能主动地插入形成镶嵌体系.     

微量量热法研究黄连中小檗碱类生物碱对金黄色葡萄球菌生长代谢的影响

采用微量量热法研究中药黄连中具有抑菌作用的3种小檗碱类生物碱成分对金黄色葡萄球菌生长代谢的影响．在不同给药条件下,以表达功率．时间曲线（热谱曲线）的特征参数生长速率常数k、半数抑菌浓度（IC50）、最大输出功率（Pmax）及达峰时间（tp）和总产热量（Qt）为指标,对3种小檗碱类生物碱成分抑制金黄色葡萄球菌生长代谢程度进行客观地量化评价．结果表明,k、Pmax和Qt均随3种小檗碱类生物碱成分浓度的增加而相应的减小;tp随3种小檗碱类生物碱成分浓度的增加而相应的延长;其IC50分别为小檗碱101．4μg／mL、巴马汀241．0μg／mL和药根碱792．3μg／mL,即抑菌活性大小顺序为小檗碱〉巴马汀〉药根碱．初步构效关系研究提示,位于苯环的C2、C3上的亚甲二氧基或甲氧基较羟基更有助于提高其抑菌活性．     

五种西佛碱配合物对金黄色葡萄球菌代谢抑制的热化学研究

用微量热法研究了五种西佛碱金属配合物对金黄色葡萄球菌代谢抑制作用。发现不同西佛碱金属配合物对金黄色葡萄球菌的作用效果不同 ,其药效为 :Cu( ) - L>Cu( ) - OVG>Co( ) - L>Zn( ) - OVG>Zn( ) - L,半抑制浓度 HIC依次增大。不同金属配合物作用下 ,金黄色葡萄球菌代谢热谱有所不同 ,说明不同西佛碱金属配合物对金黄色葡萄球菌作用的方式不同。     

石墨烯基环糊精传感器的构建及对组氨酸对映体的电位识别

以具备手性识别作用的环糊精聚合物为手性选择剂, 以石墨烯为手性放大材料, 通过层层修饰的方法构建了石墨烯基环糊精聚合膜电位识别传感器. 由所构建的传感器测得的组氨酸对映体的氧化峰电位存在明显的差异, 电位差值近100 mV, 且不同比例的组氨酸对映体混合溶液的氧化峰电位值与对映体过量值呈现良好的线性关系. 据此, 建立了一种通过电位同时识别组氨酸对映体两组分的新方法.     

组氨酸/乳状液膜复合净化材料对甘油三酯的清除研究

依据甘油三酯在体内的代谢过程,将组氨酸作为模拟酶,与液体石蜡-Span 80乳状液膜体系复合,制备具有双重选择性的复合血液净化材料,并对其主要影响因素进行了研究.对三硬酯酸甘油酯的清除实验表明,该复合体系在30min内,即可有效清除目标物,表观清除率可达0.03.     

铒离子及其阳离子卟啉配合物对金黄色葡萄球菌生长的抑制和刺激作用

用微量热法研究了Er³⁺、TMP{TMP=5，10，15，20-四(4-甲氧基苯基)卟啉}及其阳离子铒卟啉配合物[Er(TMP)(H₂O)₃]Cl对金黄色葡萄球菌生长作用的生物热动力学特征，求算了在Er³⁺、TMP和[Er(TMP)(H₂O)₃]Cl作用下，金黄色葡萄球菌生长的热动力学参数。实验结果表明：TMP对金黄色葡萄球菌生长的作用表现为单向的抑制作用，而Er³⁺和[Er(TMP)(H₂O)₃]Cl对金黄色葡萄球菌的生长有双向调节作用。在低浓度下，Er³⁺对金黄色葡萄球菌生长的刺激作用强于[Er(TMP)(H₂O)₃]Cl；在较高浓度下，[Er(TMP)(H₂O)₃]Cl对金黄色葡萄球菌生长的抑制作用显著强于Er³⁺。对金黄色葡萄球菌的抗菌活性为：[Er(TMP)(H₂O)₃]Cl>>TMP>Er³⁺。对相互作用机理做了初步探讨。     

聚乙烯醇与葡激酶的相互作用及其对葡激酶二级结构的影响

在生理条件下, 使用凝胶过滤色谱、荧光光谱、差示扫描量热分析、傅里叶变换红外光谱(FTIR)和葡激酶的纤溶活性分析研究了聚乙烯醇(PVA)和葡激酶的相互作用及其对葡激酶高级结构的影响. 凝胶过滤色谱研究结果表明, PVA与葡激酶之间形成了复合物; 荧光光谱和差示扫描量热分析结果提示, 葡激酶与PVA之间的相互作用没有破坏葡激酶的高级结构; 进一步使用红外光谱法结合可增强分辨率的傅里叶去卷积技术和高斯曲线拟合技术, 用于对葡激酶与PVA复合物冻干粉中葡激酶酰胺Ⅰ带的定量分析发现, 复合物冻干粉葡激酶分子中易导致蛋白质变性的分子间β-折叠组分含量明显减少. 纤溶活性分析结果进一步确认, PVA与葡激酶的相互作用未影响葡激酶的活性, 并对蛋白质的活性起保护作用.     

跨膜Ca~(2+)梯度对重组腺苷酸环化酶活力及构象的影响

本文将分离并部分纯化的腺苷酸环化酶催化亚基(ACc)重组于具有或不具有跨膜Ca~(2+)梯度的大豆磷脂脂质体上.酶活性的测试结果表明,酶活性中心一侧Ca~(2+)浓度低于另一侧(500倍)的脂酶体(类似生理条件)酶活性最高;在Ca~(2+)浓度梯度相反的情况下酶活性最低.外加Ca~(2+)载体A23187消除脂酶体两侧的Ca~(2+)梯度可导致前者酶活力降低而后者酶活力升高.不具有Ca~(2+)浓度梯度的脂酶体酶活性介于上述两种脂酶体之间. 蛋白内源荧光及KI对其淬灭效率的测试结果均反映上述几种脂酶体中ACc构象的差异.     

Ce4+对不同培养时期红豆杉细胞p-ERK-like的影响

为了探究培养生长不同时期的红豆杉细胞对Ce4 应答差异的机制,采用West-Blot免疫印迹法考察了1 mmol.L-1Ce4 对培养不同时期的红豆杉细胞的细胞外信号调节蛋白激酶磷酸化(p-ERK-like)的表达变化。结果发现,悬浮培养的红豆杉细胞基础p-ERK-like的表达在整个培养周期中呈"M"型变化,分别在培养第五天和第二十一天出现两个高峰,而培养第十一天达到低谷,仅为参比值的77%。在培养的不同时间点应用Ce4 诱导后,细胞的最大p-ERK-like表达量以及到达最大量所需的时间表现出显著的差异,培养第十一天的细胞经Ce4 诱导后其p-ERK-like表达速率达高峰。结果表明,处于不同培养时期的红豆杉细胞其p-ERK-like表达存在显著差异,这可能是导致不同培养时期细胞对Ce4 产生不同应答效应的一个因素。     

磁弹性无线传感器检测不同液体介质中的金黄色葡萄球菌

研制了磁弹性金黄色葡萄球菌无线传感器,用于检测不同液体介质中的金黄色葡萄球菌。取0.2mL一定浓度菌液加到含2mL无菌液体介质的检测玻璃管中,磁弹性传感器共振频率随细菌的生长而改变。通过改变牛肉膏和蛋白胨的浓度,得出传感器在含有2×105cells/mL金黄色葡萄球菌的培养基CM2-2中共振频率响应最大。结果表明,此传感器可以测定的金黄色葡萄球菌浓度范围在CM2-2中是3×103～2×107cells/mL,在牛奶中是1×104～2×107cells/mL,检出限分别是1×103cells/mL和1×104cells/mL。传感器共振频移大小与金黄色葡萄球菌的浓度呈线性相关关系,相关系数在牛奶中是0.98,在培养基中是0.99。     

Highly Stable,Amide‐Bridged Autoinducing Peptide Analogues that Strongly Inhibit the AgrC Quorum Sensing Receptor in Staphylococcus aureus

Blocking quorum sensing (QS) pathways has attracted considerable interest as an approach to suppress virulence in bacterial pathogens. Toward this goal, we recently developed analogues of a native autoinducing peptide (AIP‐III) signal that can inhibit AgrC‐type QS receptors and attenuate virulence phenotypes in Staphylococcus aureus. Application of these compounds is limited, however, as they contain hydrolytically unstable thioester linkages and have only low aqueous solubilities. Herein, we report amide‐linked AIP analogues with greatly enhanced hydrolytic stabilities and solubilities relative to our prior analogues, whilst maintaining strong potencies as AgrC receptor inhibitors in S. aureus. These compounds represent powerful tools for the study of QS.     

Fluorescent Bioconjugate Based on Gold Nanoparticles for the Determination of Staphylococcus aureus

A practical method to determine Staphylococcus aureus using bioconjugated gold nanoparticles is reported. The protocol uses gold nanoparticles stabilized by tetramethylrhodamine isothiocyanate-labeled streptavidin followed by functionalization with biotinylated anti-S. aureus antibodies. The streptavidin-stabilized gold nanoparticles were obtained by titration and analyzed by ultraviolet–visible spectroscopy and transmission electron microscopy. The obtained fluorescent bioconjugate selectively linked to the surface of S. aureus in samples contaminated with the microorganism, as demonstrated by confocal micrographs. The biorecognition process was performed by mixing the fluorescent bioconjugate with the sample. Bacterial dilutions from 1 × 108 to 1 × 102 cell/ml of S. aureus were determined, obtaining sensitivity values of 1 × 105 cell/ml by photoluminescence and 1 × 102 cell/ml by bioimpedance. This methodology represents a useful bioanalytical approach for the determination of S. aureus.     

Microcalorimetric investigation of the effect of berberine alkaloids from Coptis chinensis Franch on Staphylococcus aureus growth

The inhibitory effects of three berberine alkaloids (BAs) from rhizome of Coptis chinensis Franch, a traditional Chinese medicinal (TCM) herb, on Staphylococcus aureus growth were investigated by mi- crocalorimetry. The power–time curves of S. aureus with and without BAs were acquired; meanwhile the extent and duration of inhibitory effects on the metabolism were evaluated by studying the growth rate constant (k), half inhibitory ratio (IC50), maximum heat-output power (Pmax), peak time of maximum heat-output power (tp) and total heat production (Qt). The value of k of S. aureus in the presence of the three BAs decreased with the increasing concentrations of BAs. Moreover, Pmax was reduced and the value of tp increased with increasing concentrations of the three drugs. The inhibitory activity varied with different drugs. The values of IC50 of the three BAs are respectively, 101.4 μg/mL for berberine, 241.0 μg/mL for palmatine and 792.3 μg/mL for jateorrhizine. The sequence of antimicrobial activity of the three BAs is: berberine > palmatine > jateorrhizine. It is suggested that the functional group me- thylenedioxy or methoxyl at C2 on the phenyl ring could possibly improve antimicrobial activity more strongly than hydroxyl at C2 on the phenyl ring.     

Structural Variations in the Central Heterocyclic Scaffold of Tripartite 2,6-Difluorobenzamides: Influence on Their Antibacterial Activity against MDR Staphylococcus aureus

Five series of heterocyclic tripartite 2,6-difluorobenzamides, namely 1,2,3-triazoles, 1,2,4- and 1,3,4-oxadiazoles, analogs of reported model anti-staphylococcal compounds, were prepared. The purpose was to investigate the influence of the nature of the heterocyclic central scaffold on the biological activity against three strains of S. aureus, including two drug-resistant ones. Among the 15 compounds of the new collection, a 3-(4-tert-butylphenyl)-1,2,4-oxadiazole linked via a methylene group with a 2,6-difluorobenzamide moiety (II.c) exhibited a minimal inhibitory concentration between 0.5 and 1 µg/mL according to the strain. Subsequent studies on II.c demonstrated no human cytotoxicity, while targeting the bacterial divisome.     

Microcalorimetric Study of the Action of Yb^3＋ ion on the Growth of Staphylococcus aureus

A microcalorimetric method was used to evaluate the action of Yb^3 ions onthe growth metabolism of Staphylococcus aureus.The power-time curves of the growth metabolism of Staphylo.coccus aureus and the action of Yb^3 ions were obtained by us-ing stopped-flow method at 37 ℃. For evaluation of the action,the growth rate constants ( k1 and k2) for the log phase 1, log phase 2, and the total heat effect (Qtotal) for Staphylococcus aureus were determined. The results show that Yb^3 ions at low concentrations have the stimnlatory effect on Staphylococcus au-reus and that Yb^3 ions at higher concentration could inhibit its growth.     

Effects of Ulva sp. Extracts on the Growth,Biofilm Production,and Virulence of Skin Bacteria Microbiota: Staphylococcus aureus,Staphylococcus epidermidis,and Cutibacterium acnes Strains

Ulva sp. is known to be a source of bioactive compounds such as ulvans, but to date, their biological activity on skin commensal and/or opportunistic pathogen bacteria has not been reported. In this study, the effects of poly- and oligosaccharide fractions produced by enzyme-assisted extraction and depolymerization were investigated, for the first time in vitro, on cutaneous bacteria: Staphylococcus aureus, Staphylococcus epidermidis, and Cutibacterium acnes. At 1000 μg/mL, poly- and oligosaccharide fractions did not affect the growth of the bacteria regarding their generation time. Polysaccharide Ulva sp. fractions at 1000 μg/mL did not alter the bacterial biofilm formation, while oligosaccharide fractions modified S. epidermidis and C. acnes biofilm structures. None of the fractions at 1000 μg/mL significantly modified the cytotoxic potential of S. epidermidis and S. aureus towards keratinocytes. However, poly- and oligosaccharide fractions at 1000 μg/mL induced a decrease in the inflammatory potential of both acneic and non-acneic C. acnes strains on keratinocytes of up to 39.8%; the strongest and most significant effect occurred when the bacteria were grown in the presence of polysaccharide fractions. Our research shows that poly- and oligosaccharide Ulva sp. fractions present notable biological activities on cutaneous bacteria, especially towards C. acnes acneic and non-acneic strains, which supports their potential use for dermo-cosmetic applications.     

Effect of Different Pretreatment Methods on the Measurement of the Entrapment Efficiency of Liposomes and Countermeasures in Free Drug Analysis

Further investigation into the methods for the analysis of the entrapment efficiency of liposomes has been prompted by the urgent need to improve traditional methods which offer more risk to liposome leakage. The aim of this study was to investigate the suitability of hollow fiber centrifugal ultrafiltration (HF-CF-UF) coupled with HPLC as an alternative method for the routine determination of the entrapment efficiency of liposomes and to compare it with previously developed nonequilibrium procedures based on size-exclusion chromatography (SEC). By comparison, evaluation of the entrapped fraction with SEC resulted in 3 or 4 % lower entrapment values. More importantly, the free drug concentrations were three- to sixfold higher than with HF-CF-UF. In addition, we investigated the reasons for the different values obtained with SEC and a dynamic equilibrium theory was put forward based on this. In addition, we have developed and characterized an equilibrium method of separating free and liposomal drugs for liposomal formulations based on HF-CF-UF. The method was validated over the concentration range of 7.9–235 μg mL^?1 for indomethacin and 0.258–8.24 μg mL^?1 for vitamin A. Inter- and intra-day precision (RSD %) were ≤1.2 % for indomethacin and ≤1.8 % for vitamin A, respectively. The recoveries of both free drug and total drug were higher than 96.0 % with RSD ≤1.3 % (n = 5). Taken together, our improved method differs from those previously reported with regard to higher recovery, small sample volume, less laborious, and most importantly without damaging or disturbing liposomes. Validation results suggested that our method was sufficiently accurate and sensitive to be used to evaluate the entrapment efficiency of a liposome formulation without complicated pretreatment.