Reconstituted low density lipoprotein: a vehicle for the delivery of hydrophobic fluorescent probes to cells.

Previous studies have shown that the cholesteryl ester core of plasma low density lipoprotein (LDL) can be extracted with heptane and replaced with a variety of hydrophobic molecules. In the present report we use this reconstitution technique to incorporate two fluorescent probes, 3-pyrenemethyl-23, 24-dinor-5-cholen-22-oate-3 beta-yl oleate (PMCA oleate) and dioleyl fluorescein, into heptane-extracted LDL. Both fluorescent lipoprotein preparations were shown to be useful probes for visualizing the receptor-mediated endocytosis of LDL in cultured human fibroblasts. When normal fibroblasts were incubated at 37 degrees C with either of the fluorescent LDL preparations, fluorescent granules accumulated in the perinuclear region of the cell. In contrast, fibroblasts from patients with the homozygous form of familial hypercholesterolemia (FH) that lack functional LDL receptors did not accumulate visible fluorescent granules when incubated with the fluorescent reconstituted LDL. A fluorescence-activated cell sorter was used to quantify the fluorescence intensity of individual cells that had been incubated with LDL reconstituted with dioleyl fluorescein. With this technique a population of normal fibroblasts could be distinguished from a population of FH fibroblasts. The current studies demonstrate the feasibility of using fluorescent reconstituted LDL in conjunction with the cell sorter to isolate mutant cells lacking functional LDL receptors.     

Discovery of the potential neuraminidase inhibitors from Polygonum cuspidatum by ultrafiltration combined with mass spectrometry guided by molecular docking

Neuraminidase is an important target in the treatment of the influenza A virus. Screening natural neuraminidase inhibitors from medicinal plants is crucial for drug research. This study proposed a rapid strategy for identifying neuraminidase inhibitors from different crude extracts (Polygonum cuspidatum, Cortex Fraxini, and Herba Siegesbeckiae) using ultrafiltration combined with mass spectrometry guided by molecular docking. Firstly, the main component library of the three herbs was established, followed by molecular docking between the components and neuraminidase. Only the crude extracts with numbers of potential neuraminidase inhibitors identified by molecular docking were selected for ultrafiltration. This guided approach reduced experimental blindness and improved efficiency. The results of molecular docking indicated that the compounds in Polygonum cuspidatum demonstrated good binding affinity with neuraminidase. Subsequently, ultrafiltration-mass spectrometry was employed to screen for neuraminidase inhibitors in Polygonum cuspidatum. A total of five compounds were fished out, and they were identified as trans-polydatin, cis-polydatin, emodin-1-O-β-D-glucoside, emodin-8-O-β-D-glucoside, and emodin. The enzyme inhibitory assay showed that they all had neuraminidase inhibitory effects. In addition, the key residues of the interaction between neuraminidase and fished compounds were predicted. In all, this study could provide a strategy for the rapid screening of the potential enzyme inhibitors from medicinal herbs.     

Electrophoretic mobility measurements of fluorescent dyes using on-chip capillary electrophoresis

We present an experimental study of the effect of pH, ionic strength, and concentrations of the electroosmotic flow (EOF)-suppressing polymer polyvinylpyrrolidone (PVP) on the electrophoretic mobilities of commonly used fluorescent dyes (fluorescein, Rhodamine 6G, and Alexa Fluor 488). We performed on-chip capillary zone electrophoresis experiments to directly quantify the effective electrophoretic mobility. We use Rhodamine B as a fluorescent neutral marker (to quantify EOF) and CCD detection. We also report relevant acid dissociation constants and analyte diffusivities based on our absolute estimate (as per Nernst-Einstein diffusion). We perform well-controlled experiments in a pH range of 3-11 and ionic strengths ranging from 30 to 90 mM. We account for the influence of ionic strength on the electrophoretic transport of sample analytes through the Onsager and Fuoss theory extended for finite radii ions to obtain the absolute mobility of the fluorophores. Lastly, we briefly explore the effect of PVP on adsorption-desorption dynamics of all three analytes, with particular attention to cationic R6G.     

Changes in the fluorescence characteristics of N-(1-pyrene) maleimide bound to the intestinal brush-border membranes by neuraminidase treatment

The effects of neuraminidase treatment on the dynamic properties of the porcine intestinal brush-border membranes have been examined by using a fluorogenic thiol reagent, N-(1-pyrene)maleimide (NPM). Desialylation of the membranes by treatment with neuraminidase resulted in changes in the fluorescence parameters of NPM-labeled membranes, i.e. a decrease of the fluorescence lifetime and a suppression of the temperature-dependent decrease of the fluorescence intensity. These results suggest that the environmental properties around NPM-labeled SH groups in the membrane proteins were modified by neuraminidase treatment. Perturbation of the microenvironment around NPM-labeled SH groups associated with desialylation by the enzyme treatment was also determined by measuring the increase of fluorescence anisotropy and decrease of quenching efficiency with acrylamide or CH3COOTl of the complex. Based on the results, it is suggested that the dynamic properties of the conformation around NPM-labeled SH groups in the membrane proteins are sensitively influenced by neuraminidase treatment.     

Potential drug-like inhibitors of Group 1 influenza neuraminidase identified through computer-aided drug design

Pandemic (H1N1) influenza poses an imminent threat. Nations have stockpiled inhibitors of the influenza protein neuraminidase in hopes of protecting their citizens, but drug-resistant strains have already emerged, and novel therapeutics are urgently needed. In the current work, the computer program AutoGrow is used to generate novel predicted neuraminidase inhibitors. Given the great flexibility of the neuraminidase active site, protein dynamics are also incorporated into the computer-aided drug-design process. Several potential inhibitors are identified that are predicted to bind to neuraminidase better than currently approved drugs.     

Sol-gel column technology for capillary isoelectric focusing of microorganisms and biopolymers with UV or fluorometric detection

The sol-gel surface modification is used for capillary isoelectric focusing of microorganisms and biopolymers. The coating strongly decreases the electroosmotic flow so that it enables the use of the short capillaries down to 100 mm in the separation length. The examples of capillary isoelectric focusing of the low-molecular-mass pI markers and mixed cultures of microbial populations of Escherichia coli, Candida albicans, Staphylococcus epidermidis, and Enteroccocus faecalis with UV detection are shown. It is possible to quantify bacterial cells according to their peak areas; the minimum detectable number of microbial cells was 5 x 10(2)-1 x 10(3). The compatibility of sol-gel capillaries with sensitive fluorometric detection of fluorescent pI markers together with fluorescein labeled proteins is demonstrated.     

First direct fluorescence polarization assay for the detection and quantification of spirolides in mussel samples

In 2009, we achieve the first inhibition FP assay to detect imine cyclic toxins. In the present paper we propose a new FP assay for direct quantify spirolides. This new method has resulted in significant improvement of sensitivity, rapidity and accessibility. In the method design, nicotinic acetylcholine receptor from Torpedo marmorata membranes labelled with a derivative of fluorescein was used. Spirolides, 13-desmethyl spirolide C (13-desMeC) and 13,19-didesmethyl spirolide C (13,19-didesMeC) were extracted and purified from cultures of the Alexandrium ostenfeldii dinoflagellate. Data showed the decrease of FP when toxin concentration was increased. Thus, a relationship between the FP units and the spirolides amount present in a sample was obtained. This direct assay is a reproducible, simple and very sensitive method with a detection limit about 25 nM for 13-desMeC and 150 nM for 13,19-didesMeC. The procedure was used to measure spirolides in mussel samples using an extraction and clean up protocol suitable for the FP assay. Results obtained show that this method is able to quantify 13-desMeC in the range of 50–350 μg kg⁻¹ meat. Other liposoluble toxins did not interfere with the assay, proving a specific method. Moreover, the matrix do not affect in the range of toxin concentrations that involving risk of spirolides intoxication.     

荧光素二苯甲酰酯的晶体结构与水解性质的研究

以荧光素和苯甲酰氯为原料制备了荧光素二苯甲酰酯, 并用X射线单晶法测定了晶体和分子结构。晶体空间群为P21/C, a=0.8459(2), b=1.8221(5),c=1.7491(8)nm; β=103.79(3)°; V=2.618nm3, Z=4。酯水解和酯酶水解实验表明其水解速度明显低于荧光素的二醋酸酯、二丙酸酯、二丁酸酯和二已酸酯。根据酯链上苯环的空间位阻和共轭效应, 以及质谱分析结构对这一特性作了解释。     

Novel Dual‐Site‐Binding Neuraminidase Inhibitor from Virtual Screening by Pharmacophore and Molecular Dynamics Methods

Neuraminidase is a significant anti‐influenza target that plays crucial role in virus replication cycle. The discovery of 150‐cavity in Group‐1 neuraminidase provides us a novel mentality of designing inhibitor which can bind with both conserved site and 150‐cavity. In order to discover novel dual‐site‐binding inhibitors, a 3D chemical‐feature‐based pharmacophore model was established to cover dual‐site in neuraminidase. The dual‐site‐binding model was consistent in predicting the binding conformation of Group‐1 neuraminidase inhibitor and applied for virtual screening of Specs database. Compound 4 (ZINC05790048) that aligned well to the model was selected after multiple filtrations for molecular dynamics simulations, indicating improved binding energy with neuraminidase. It can sever as the lead compound for a novel series of inhibitors.     

Dependence of the constants of binding for nanomarkers of the fluorescein family with human serum albumin on Ph

The effective constants of binding for probes of the fluorescein family (fluorescein and its halogen derivatives, eosin and erythrosin) with human serum albumin (HSA) at different pH were determined. It was found that the introduction of halogen groups into the structural formula of fluorescein changes the character of the dependence on pH of the effective constant of binding of a nanomarker with HSA: the nonlinear dependence of the effective constants of binding with its protein typical for fluorescein, eosin, and erythrosine is characterized by an almost linear reduction with increasing pH dependence of the effective constants of their binding with human serum albumin. It was shown that the presence of more electronegative atoms in the structural formula of nanomarker leads to decrease of values of effective constants of nanomarker binding with HSA.     

金纳米粒子-荧光素体系的光谱特性

纳米粒子具有量子尺寸效应和表面效应等许多特有的性质 [1] ,在光吸收、医药及新材料等方面具有广阔的应用前景 .纳米粒子具有较高的比表面能且带有电荷 ,当光子与其接近时 ,实际上是光子与纳米粒子的界面电子发生了作用 [2 ,3 ] .基于此建立的共振散射 (RS)光谱技术已成为一种高灵敏度和高选择性的分析技术 ,是研究生物化学和液相纳米粒子特性的良好手段 [4～ 9] .我们 [2 ,3 ] 研究发现 ,较大粒径纳米粒子和界面的形成是导致散射光增强的根本原因 ;金、银等液相纳米粒子产生 RS效应和 RS峰等 .荧光猝灭 (FQ)效应已用于分析化学和蛋白…     

Comparison of micro- and mesoporous inorganic materials in the uptake and release of the drug model fluorescein and its analogues

The uptake of the three species of the drug model fluorescein (fluorescein sodium salt (FNa), fluorescein free acid (F), and fluorescein diacetate (FDA)) by zeolite NaX and the mesoporous zeotype MCM-41 was investigated as well as their release rates into solutions at pH 7 and pH 4.5. UV/Vis analysis was carried out at a wavelength of 490 nm. Uptakes of the sodium salt of 9 % for zeolite X and 14 % for MCM suggest little penetration of the pores. The use of ethanol as the loading solvent for F resulted in little uptake for both zeolitic materials due to the successful competition of the ethanol for binding sites. Use of acetone (weaker proton acceptor) as loading solvent significantly improved the uptake of F to 17 % and 12 % for zeolite X and MCM, respectively, whilst the uptake of FDA in acetone increased still further to 22 % and 17 % for zeolite X and MCM, respectively. Generally there was a large initial release of the fluorescein analogues from the surface of the zeolites with very little further increase over time. The prescence of an esterase enzyme in the release medium of FDA tripled the release from MCM to 15 % but left the release from zeolite X unaffected at 6 %. The results obtained show that uptake of fluorescein and its analogues is dependent on the loading solvent used, the amount released is influenced by not only the solvent but the pH and the presence of enzymes in the release medium.     

Thermal lens technique to study the effect of pH on electronic energy transfer in organic dye mixtures

The effect of pH on the fluorescence efficiency of fluorescein is evaluated using thermal lens technique. Fluorescence efficiency increases as the sample becomes more and more alkaline. But when fluorescein is mixed with rhodamine B fluorescence quenching of fluorescein takes place with the excitation of rhodamine B. The electronic energy transfer in this mixture is investigated using Optical Parametric Oscillator as the excitation source. The effect of pH on the efficiency of energy transfer in fluorescein-rhodamine B mixture is presented.     

Proximity Ligation‐Based Fluorogenic Imaging Agents for Neuraminidases

Reagents to visualize and localize neuraminidase activity would be valuable probes to study the role of neuraminidases in normal cellular processes as well as during viral infections or cancer development. Herein, a new class of neuraminidase‐imaging probes that function as proximity ligation reagents by releasing a highly reactive fluorophore that tags nearby cellular material is described. It is further demonstrated that it is possible to create an influenza virus‐specific reagent, which can specifically detect influenza virus infections in mammalian cells. These reagents have potential use as specific histological probes independent of viral antigenicity and, therefore, offer some advantages over commonly used anti‐neuraminidase antibodies.     

Exploring the Cause of Oseltamivir Resistance Against Mutant H274Y Neuraminidase by Molecular Simulation Approach

Oseltamivir (Tamiflu) is the preferred anti-viral drug employed to fight the flu virus in infected individuals. The principal target for this drug is a virus surface glycoprotein, neuraminidase (NA), which facilitates the release of nascent virus and thus spreads infections. Until recently, only a low prevalence of neuraminidase inhibitors (NAIs) resistance (<1?%) had been detected in circulating viruses. However, there have been reports of significant numbers of A (H1N1) influenza strains with a H274Y neuraminidase mutation that was highly resistant to the NAI, oseltamivir. In this study, we highlight the effect of point mutation-induced oseltamivir resistance in H1N1 subtype neuraminidases by molecular docking and molecular dynamics simulation approach. Our results suggested that wild-type NA could be more indispensable for the oseltamivir binding, as characterized by minimum number of H-bonds, high flexibility and largest binding affinity than mutant-type NA. This study throws light on the possible effects of drug-resistant mutations on the large functionally important collective motions in biological systems.     

Frequency-domain measurement of the photodegradation process of fluorescein

The frequency-domain technique is applied to measure the photodegradation rate of fluorescein in aqueous solutions. The illuminating light is modulated, and the changes in fluorescence from the illuminated region are detected synchronously. A constant flow rate is imposed on the fluorescein solution to control the mass transport of fluorescein into the illuminated region. The fluorescence response is described by a model that assumes that photodegradation occurs from the triplet excited state. The predictions of the model are consistent with the observed variations in the fluorescence response with flow rate, modulation frequency and incident power. We discuss in this article how the dependence of the model parameters on experimental conditions can be used to infer the photodegradation rate as well as some of the details of the photodegradation mechanism. The results are consistent with the known mechanism of photodegradation of fluorescein. The frequency-domain technique gives a photodegradation rate of 53 s(-1) in an air-saturated solution and 37 s(-1) in solutions purged with argon gas.     

荧光素和苯磺酰氯间反应机理研究

在N,N-二甲基甲酰胺存在下,荧光素和苯磺酰氯间的反应经历串联两步:首先荧光素被苯磺酰氯酯化,然后生成的氯离子亲核进攻芳碳,取代苯磺酰氧基,后者是良好的离去基团。反应的结果是氯取了荧光素的酚羟基,反应机理由本工作找到的关键中间体证实。     

Fluorimetric determination of fluorescein at the femtomole level with a self-ordered ring of a sessile droplet on glass slide support

A method for fluorimetric microscopic determination of trace amounts of fluorescein at the femtomole level has been developed. Since the solvent evaporates from the droplet on a hydrophobic-treated glass slide, an outward capillary flow of the solvent from the interior of the droplet occurs. The resulting outward capillary flow then carries the solute to the perimeter of the droplet spot, where the solute accumulates to form a fluorescent self-ordered ring (SOR). Depending on the volume of the droplet of fluorescein solution, SORs of different sizes with an outer diameter less than 1.2 mm and a ring belt width less than 24 μm can be obtained. Data analysis for a digitally imaged SOR using a charge-coupled device (CCD) camera showed that the fluorescein molecules across the fluorescent SOR belt section follow a Gaussian distribution; the maximum fluorescent intensity at central ring belt (I _max) was found to be proportional to fluorescein content. When a 0.1-μL droplet was spotted on the solid support, fluorescein in a range of 0.6–120 femtomol (or 6.2–1200.0 nM) can be detected, and the limit of detection can reach 62 attomol (or 0.6 nM). With the present method, the contents of fluorescein in fluorescein sodium injections were satisfactorily detected with recoveries of 95–105% and RSD of 3.5 and 4.2%, respectively. The text was submitted by the authors in English.     

Binding of a natural anthocyanin inhibitor to influenza neuraminidase by mass spectrometry

The binding of a natural anthocyanin to influenza neuraminidase has been studied employing mass spectrometry and molecular docking. Derived from a black elderberry extract, cyanidin-3-sambubiocide has been found to be a potent inhibitor of sialidase activity. This study reveals the molecular basis for its activity for the first time. The anthocyanin is shown by parallel experimental and computational approaches to bind in the so-called 430-cavity in the vicinity of neuraminidase residues 356–364 and 395–432. Since this antiviral compound binds remote from Asp 151 and Glu 119, two residues known to regulate neuraminidase resistance, it provides the potential for the development of a new class of antivirals against the influenza virus without this susceptibility.     

荧光黄与蛋白质相互作用的研究

研究了在人体生理条件下,荧光黄与人血清白蛋白和人体γ球蛋白间的相互作用。求出了荧光黄与蛋白质的结合常数及结合位置,并根据热力学参数确定了它们之间的主要作用力类型。认为”相分配模型“是对荧光黄与蛋白质相互作用模式的最好描述。