Fluorescence Polarization Measurements Research Articles

Medicinal plant constituents interact with membrane-acting local anesthetics

Specific constituents in medicinal plants have been suggested to act on lipid bilayers and modify the physicochemical properties of biomembranes as well as amphiphilic membrane-active drugs. The interactions between selected phytochemicals and local anesthetics were studied at a membrane lipid level to verify the possibility that their concomitant use may influence local anesthesia. Biomimetic lipid bilayer membranes were prepared as unilamellar vesicle suspensions by the injection method using different phospholipids and cholesterol to mimic the lipid composition of neuronal membranes. They were treated at 37 °C for 30 min with apple flavonoid phloretin, chili pepper capsaicinoid capsaicin, harmala alkaloid tetrahydroharman and local anesthetic lidocaine separately or in combination, followed by measuring fluorescence polarization to determine their induced membrane fluidity changes. Lidocaine increased the fluidity of biomimetic membranes at clinically-relevant concentrations. In contrast, phloretin (10 – 50 µM) and tetrahydroharman (10 – 500 nM) decreased the membrane fluidity, but capsaicin (50 – 100 µM) increased. Phloretin (25 µM) and tetrahydroharman (˜15 nM) inhibited or counteracted the membrane-fluidizing effects of lidocaine (0.05 – 1 mg/mL), whereas capsaicin (50 µM) potentiated. Such membrane interactions were also found in bupivacaine. Local anesthetics mechanistically act on neuronal membranes besides voltage-gated sodium channels. The antagonistic or synergistic membrane interactions with medicinal plant constituents suggest the potentially beneficial or adverse effects that the counteraction by phloretin would successfully discontinue anesthesia after the treatment and the cooperation with capsaicin should prolong the duration of nerve block, while the inhibition by tetrahydroharman might reduce the anesthetic efficacy in certain patients. The membrane interactivity will be also an experimental clue to discover the drug leads for anesthetic adjuncts.

Insertion of pH-sensitive bola-type copolymer into liposome as a “stability anchor” for control of drug release

How to design intelligent carriers for delivering drugs to the target accurately and releasing drug timely with the help of a certain environmental stimulus is still a challenge in tumor treatment. In this work, pH-sensitive bola-type triblock copolymers, composed of poly(2-(diisopropylamino) ethylmethacrylate) (PDPA) and methoxy-poly(ethyleneglycol) (mPEG), were synthesized. Liposomes containing these copolymers (Liposome@Bola) have been prepared by simply mixing the copolymer with phospholipids and cholesterol. From the fluorescence polarization measurement, the stability of Liposome@Bola was found to be increased a lot comparing to the pure liposome. As a result, the doxorubicin (DOX) leakage of the former was restrained in neutral environment. However, when pH decreased from 7.4 to 6.0, DOX released percentage had been increased 30–60 points, which was heavily depend on the phospholipid composition. Furthermore, the size effects of PEG and PDPA segments were also investigated. These results indicated the synthesized bola-type copolymers improved the pH-controllability of drug release of liposome, i.e., increased the difference between the release amount under pH 7.4 and pH 6.0. The bola-type copolymer exhibited a good potential application in smartly controlling drug delivery system.

Fluorescence polarization measurement system using a liquid crystal layer and an image sensor.

The detection system which enables simultaneous fluorescence polarization (FP) measurement of multiple samples was proposed and proven by a proof-of-concept experiment on the viscosity dependence of FP of fluorescein sample in water-ethylene glycol solution and another experiment on the FP immunoassay of prostaglandin E2 sample. The measurement principle of FP is based on the synchronization between the orientation of the liquid crystal molecules and the sampling frequency of a CCD. This report is the first description of the simultaneous FP measurement of multiple samples. This system has a great potential for equipment miniaturization and price reduction as well as providing simultaneous FP measurement of multiple samples.

Heat Shock Protein 90 Associates with the Per-Arnt-Sim Domain of Heme-free Soluble Guanylate Cyclase: IMPLICATIONS FOR ENZYME MATURATION

Heat shock protein 90 (hsp90) drives heme insertion into the β1 subunit of soluble guanylate cyclase (sGC) β1, which enables it to associate with a partner sGCα1 subunit and mature into a nitric oxide (NO)-responsive active form. We utilized fluorescence polarization measurements and hydrogen-deuterium exchange mass spectrometry to define molecular interactions between the specific human isoforms hsp90β and apo-sGCβ1. hsp90β and its isolated M domain, but not its isolated N and C domains, bind with low micromolar affinity to a heme-free, truncated version of sGCβ1 (sGCβ1(1-359)-H105F). Surprisingly, hsp90β and its M domain bound to the Per-Arnt-Sim (PAS) domain of apo-sGC-β1(1-359), which lies adjacent to its heme-binding (H-NOX) domain. The interaction specifically involved solvent-exposed regions in the hsp90β M domain that are largely distinct from sites utilized by other hsp90 clients. The interaction strongly protected two regions of the sGCβ1 PAS domain and caused local structural relaxation in other regions, including a PAS dimerization interface and a segment in the H-NOX domain. Our results suggest a means by which the hsp90β interaction could prevent apo-sGCβ1 from associating with its partner sGCα1 subunit while enabling structural changes to assist heme insertion into the H-NOX domain. This mechanism would parallel that in other clients like the aryl hydrocarbon receptor and HIF1α, which also interact with hsp90 through their PAS domains to control protein partner and small ligand binding interactions.

Tagging Glycoproteins with Fluorescently Labeled GDP-Fucoses by Using α1,3-Fucosyltransferases.

Fucose-containing glycans mediate a variety of biological processes, but there is little information on reaction processes and mechanisms mediated by fucosyltransferases. We recently reported on fluorescently labeled GDP-β-L-fucose-ATTO 550, which enabled monitoring of α1,3-fucosyltransferase activity. Here we present an extension to the previously described results, based on the synthesis of a fluorescein-isothiocyanate (FITC)-labeled and two carboxyfluorescein-labeled (FAM-labeled) NDP-β-L-fucose derivatives, and applied all four compounds in labeling of different glycoproteins with the aid of four different fucosyltransferases. The labeling processes were analyzed by in-gel fluorescence and fluorescence polarization measurements. Comparison with the ATTO-labeled sugar revealed that the FITC-labeled fucose was the best of these substrates, and that the bacterial enzyme HP-FucT tolerated the fluorescent substrates better than human fucosyltransferases.

Fluorescence polarization-based method with bisulfite conversion-specific one-label extension for quantification of single CpG dinucleotide methylation.

To quantify the methylation at individual CpG dinucleotide sites in large biological or clinical samples, we developed a bisulfite conversion-specific one-label extension (BS-OLE) method using visualization by fluorescence polarization (FP) measurement of methylation at single CpG sites in small amounts of genomic DNA. Genomic DNA was treated with sodium bisulfite to convert unmethylated cytosine to uracil leaving 5-methylcytosine unaltered, and BS-PCR was used to generate DNA template containing target CpG sites. BS-OLE uses a BS-primer hybridized immediately upstream of the target CpG site being examined and then fluorescent dCTP or dUTP is incorporated into the methylated (CpG) or unmethylated (TpG) form of the target site through single-nucleotide chain extension, yielding an FP ratio between the fluorescent dCTP- and dUTP-incorporated products as a measure of methylation. This provides stable estimates of the methylation level of human genomic DNA and of a 250-bp plasmid DNA segment containing a single TCGA TaqI cleavage site, in accordance with the results of a combined bisulfite restriction analysis method. We used BS-OLE to measure dose-dependent DNA hypomethylation in human embryonic kidney 293T cells treated with the DNA methyltransferase inhibitor 5-aza-dC. BS-OLE is well suited to high-throughput multi-sample applications in biological and medical studies.

Interaction of Anthracycline 3'-azido-epirubicin with Calf Thymus DNA via Spectral and Molecular Modeling Techniques.

Anthracycline antibiotics are extensively applied to clinical antitumor therapy. The binding mode and mechanism of a new anthracycline 3'-azido-epirubicin (AEPI) with calf thymus deoxyribonucleic acid (ctDNA) were investigated employing multiple spectroscopy techniques in Tris-HCl buffer solution (pH 7.4). Effect of pH on the interaction was provided to determine the proper environment for whole research. Iodide quenching studies and fluorescence polarization measurement indicated that ctDNA quenched the fluorescence of AEPI significantly via intercalation binding mode. The binding constants and binding sites for the interaction were calculated. From binding constant dependence on the temperature, static quenching mechanism of AEPI by ctDNA was confirmed based on the Stern-Volmer equation. Additionally, the thermodynamic parameters for the reaction revealed that the van der Waals force and hydrogen bonding were the main acting forces in the binding process. Molecular modeling result indicated that the hydrogen bonding played a major role in the binding of AEPI to ctDNA.

Highly sensitive naphthalimide-based fluorescence polarization probe for detecting cancer cells.

Fluorescence polarization (FP)-based signal is a self-referencing fluorescence signal, and it is less dependent on dye concentration and environmental interferences, which makes FP measurement an attractive alternative sensing technology to fluorescence intensity-based detection. However, most of the fluorescence polarization probes were constructed by introducing fluorescein, rhodamine, and cyanine dyes, which have relatively shorter excited-state lifetimes compared with BODIPY and naphthalimide dyes. Herein, a first naphthalimide based fluorescence polarization probe (BIO) was designed and synthesized for selective and direct detection of cancer cells. The relatively longer excited-state lifetimes and high photostability of naphthalimide makes BIO more sensitive and accuracy in quantitative determination of HeLa cells in homogeneous solution without cell lysis and further separation steps. The detection limit of BIO for HeLa cells was about 85 cells mL(-1), the linear range was from 2.5 × 10(2) cells mL(-1) to 1 × 10(6) cells mL(-1) and the response time is no more than 25 min. Moreover, due to the relatively high photostability of naphthalimide, BIO was particularly suitable for live cell imaging under continuous irradiation with confocal microscopy, and the specific interaction of BIO with CD44-overexpressing cell lines was clearly visualized. Importantly, this BIO based sensing platform offers a direct and real-time tool for cancer cell diagnosis when complemented with the use of naphthalimide-based fluorescence polarization probe.

Fluorescence life-time imaging and steady state polarization for examining binding of fluorophores to gold nanoparticles.

Nanocomposites as multifunctional agents are capable of combing imaging and cell biology technologies. The conventional methods used for validation of the conjugation process of nanoparticles (NPs) to fluorescent molecules such as spectroscopy analysis and surface potential measurements, are not sufficient. In this paper we present a new and highly sensitive procedure that uses the combination of (1) fluorescence spectrum, (2) fluorescence lifetime, and (3) steady state fluorescence polarization measurements. We characterize and analyze gold NPs with Lucifer yellow (LY) surface coating as a model. We demonstrate the ability to differentiate between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes. We suggest the approach for neuroscience applications where LY is used for detecting and labeling cells, studying morphology and intracellular communications. Histograms of Fluorescence lifetime imaging (FLIM) of free LY dye (Left) in comparison to the conjugated dye to gold nanoparticles, LY-GNP (Middle) enable the differentiation between LY-GNP (the conjugated complex) and a mixture of coated NP and free dyes (Right).

The transition from single molecule to ensemble revealed by fluorescence polarization.

Fluorescence polarization measurements in the condensed phase provide rich information on rotational dynamics and interactions between macromolecules. An important parameter in these studies is the limiting polarization or po which is the emission polarization in the absence of molecular rotation. Here we explore how molecular number averaging affects the observed value of po. Using a simple mathematical model we show that for a collection of fluorescent dipoles (1–50 molecules) the fluorescence polarization (p) increases with the number of molecules (N) due to the progressive onset of photo-selection with a relation of the form p = po(1 − N−β). This concept is demonstrated experimentally using single molecule polarization measurements of perylene diimide dye molecules in a rigid polymer matrix where it is shown that the average emission polarization increases significantly when the number of molecules per averaging window is increased from 1 to 10 molecules. These results suggest that the definition of limiting polarization needs to be refined in the quasi-single molecule regime. Moreover, these results pave a new way for measuring clustering of molecules from single cluster polarization histograms.

G143 蛍光偏光法によるマイクロ流路内の流体温度計測手法の開発(OS-12:先端計測による熱工学の学際的展開(1))

A method that can measure the fluid temperature in microscopic scale by measuring the fluorescence polarization is described in this study. To understand the basic characteristics and advantage of the method, the effects of the fluid temperature, fluid viscosity, measurement time, and pH of the solution on the measured fluorescence polarization degree are discussed. The results showed that fluorescence polarization is considerably less sensible to the measurement time and pH compared to the fluorescence intensity demonstrating the stability of the temperature measurement. The correlations between the fluid temperature and the inverse value of P measured by two photo detectors (photomultiplier and CCD camera) both showed a linear relationship. This agreed with the theoretical characteristic, and confirmed the feasibility of this method.

Effect of Kolavenic Acid on the Structure of Human Serum Albumin

The effect of Kolavenic acid (KA), an active component isolated from the genus Polyalthia, on the structure of human serum albumin (HSA) was investigated by fluorescence polarization, synchronous fluorescence, three-dimensional (3D) fluorescence, and absorption spectroscopy in combination with molecular modeling techniques under physiological conditions. The synchronous and absorption fluorescence spectra confirmed that KA has an effect on the microenvironment around HSA in aqueous solution. The two-dimensional (2D) and 3D fluorescence spectra showed that KA could quench the intrinsic fluorescence of HSA and make its conformation change. Fluorescence polarization measurements provided useful information on the relaxation time and aggregation behavior of the complex formed between HSA and KA, and indicated that the presence of KA caused changes in the fluidity and microviscosity of HSA. The binding constants and thermodynamic parameters for KA-HSA systems were obtained under different temperatures (298, 308, and 318 K). Molecular docking showed that the KA moiety bound to the hydrophobic cavity of HSA, and there were three hydrogen-bonding interactions between KA and the Lys195 and Asp451 residues. Fluorescent displacement measurements confirmed that KA bound to HSA at site II. In addition, the binding mechanism of KA and HSA was revealed by the physicochemical parameters at the molecular level. The results showed that the interaction between KA and HSA was strong, indicating that KA may be stored and transferred by serum albumin.

Aptamer Displacement Screen for Flaviviral RNA Methyltransferase Inhibitors

RNA-protein interactions are vital to the replication of the flaviviral genome. Discovery focused on small molecules that disrupt these interactions represent a viable path for identification of new inhibitors. The viral RNA (vRNA) cap methyltransferase (MTase) of the flaviviruses has been validated as a suitable drug target. Here we report the development of a high-throughput screen for the discovery of compounds that target the RNA binding site of flaviviral protein NS5A. The assay described here is based on displacement of an MT-bound polynucleotide aptamer, decathymidylate derivatized at its 5′ end with fluorescein (FL-dT10). Based on the measurement of fluorescence polarization, FL-dT10 bound to yellow fever virus (YFV) MTase in a saturable manner with a Kd = 231 nM. The binding was reversed by a 250-nucleotide YFV messenger RNA (mRNA) transcript and by the triphenylmethane dye aurintricarboxylic acid (ATA). The EC50 for ATA displacement was 1.54 µM. The MTase cofactors guanosine-5′-triphosphate and S-adenosyl-methionine failed to displace FL-dT10. Analysis by electrophoretic mobility shift assay (EMSA) suggests that ATA binds YFV MTase so as to displace the vRNA. The assay was determined to have a Z′ of 0.83 and was successfully used to screen a library of known bioactives.

Signaling at Crossroads: The Dialogue between PDEs and PKA is Spoken in Multiple Languages

Signaling at Crossroads: The Dialogue between PDEs and PKA is Spoken in Multiple Languages

Fluorescence Behavior of Single Guest Molecules in Nonpolar Oil Droplets Covered with Stabilizing Surfactants

By using confocal and wide-field microscopic techniques we have investigated fluorescence behaviors of a single perylenediimide derivative, N,N′-dipropyl-1,6,7,12-tetrakis(4-tert-butylphenoxy)-3,4,9,10-perylenetetracarboxydiimide (BP-PDI), in ca. 200–300 nm sized droplets of n-octane covered with surfactant molecules. The guest dye did not show distinguishable blinking in the droplets prepared with a nonionic surfactant, Triton X-100, while frequent blinking was observed in the droplets with a cationic surfactant, trimethylstearylammonium chloride (STAC). The survival time of the guest dye before the permanent photodegradation was much longer in the droplets with Triton X-100 than those with STAC. On the basis of the analysis with a model assuming the transition from the S1 state to the dark state (DS) and the permanent photodegradation during the dark state, it was deduced that the probability of the transition from the S1 state to the DS in the droplet with STAC, 7.2 × 10–6, was significantly larger than that with Triton X-100, 1.1 × 10–6. On the other hand, the probability of the photodegradation during the dark state was comparable between these two systems. These results indicate that the probability of transition from the S1 state to the DS essentially dominates the survival time of the guest dyes. Fluorescence polarization measurements also revealed that guest dyes frequently stay in the vicinity of polar interface in the droplets prepared with STAC.

Effects of water content and oil on physicochemical and microenvironmental properties of mixed surfactant microemulsions

In this report, phase behavior, conductivity, viscosity, dynamic light scattering, fluorescence lifetime, steady state fluorescence anisotropy and Fourier transform infrared spectroscopy (FTIR) techniques were employed for understanding of the physicochemical properties and microenvironment of water-in-oil microemulsion comprising equimolar (1:1) cetyltrimethylammonium bromide (C16TAB) and polyoxyethylene (20) cetyl ether (C16E20)/1-butanol/heptane or decane, with varying water content (ω) at 303K. Both conductivity and viscosity of these systems were increased with increase in ω in both oils. Droplet size was also increased with increase in ω and corroborated well with the conductance and viscosity measurements, and depends on oil chain length. The physicochemical changes in the microenvironment with increase in ω were presented by measuring the changes in decay time using a fluoroprobe (7-hydroxycoumarin). The effect of hydration on the microstructure of these systems was studied by polarized fluorescence measurements. FTIR measurements reveal three states of water molecules, viz. trapped, bound and bulk water, in water pool of these systems. Further, the interfacial composition and free energy of transfer of 1-butanol from oil to the interface were evaluated by the dilution method. Changes in interfacial composition as a function of ω corroborate well with FTIR results indicating bound and bulk water.

Generation of Recombinant Antibodies to Rat GABAA Receptor Subunits by Affinity Selection on Synthetic Peptides

The abundance and physiological importance of GABAA receptors in the central nervous system make this neurotransmitter receptor an attractive target for localizing diagnostic and therapeutic biomolecules. GABAA receptors are expressed within the retina and mediate synaptic signaling at multiple stages of the visual process. To generate monoclonal affinity reagents that can specifically recognize GABAA receptor subunits, we screened two bacteriophage M13 libraries, which displayed human scFvs, by affinity selection with synthetic peptides predicted to correspond to extracellular regions of the rat α1 and β2 GABAA subunits. We isolated three anti-β2 and one anti-α1 subunit specific scFvs. Fluorescence polarization measurements revealed all four scFvs to have low micromolar affinities with their cognate peptide targets. The scFvs were capable of detecting fully folded GABAA receptors heterologously expressed by Xenopus laevis oocytes, while preserving ligand-gated channel activity. Moreover, A10, the anti-α1 subunit-specific scFv, was capable of detecting native GABAA receptors in the mouse retina, as observed by immunofluorescence staining. In order to improve their apparent affinity via avidity, we dimerized the A10 scFv by fusing it to the Fc portion of the IgG. The resulting scFv-Fc construct had a Kd of ∼26 nM, which corresponds to an approximately 135-fold improvement in binding, and a lower detection limit in dot blots, compared to the monomeric scFv. These results strongly support the use of peptides as targets for generating affinity reagents to membrane proteins and encourage investigation of molecular conjugates that use scFvs as anchoring components to localize reagents of interest at GABAA receptors of retina and other neural tissues, for studies of receptor activation and subunit structure.

Development of Nano Particle Sizing System Using Fluorescence Polarization

<p>In order to measure the sizes of nanoparticles with a wide size distribution in a solvent, we developed an optical microscopy system that allows for fluorescence polarization (FP) measurement and optical observation. This system allows the evaluation of nanoparticle sizes over a wide range, because the fluorescent signal intensity is independent of changes in the nanoparticle sizes. In this paper, we describe a fundamental experiment to verify the feasibility of using this system for different sizes of nanoparticles.</p>

Effects of lipid composition on physicochemical characteristics and cytotoxicity of vesicles composed of cationic and anionic dialkyl lipids

The behaviors of cat-anionic vesicles composed of dioctadecyldimethylammonium bromide (DODAB) and dihexadecyl phosphate (DHP) with varying lipid composition were investigated through the measurements of size, zeta potential and fluorescence polarization, morphological observations, determination of thermotropic phase behavior, cell viability assay, and examination of entrapment efficiency and colloid stability. DODAB is miscible with DHP in the bilayer domain, which expresses a non-ideal mixing characteristic. The DODAB-rich vesicles show a smaller particle size, higher positive zeta potential, lower main transition temperature, less angular structure, better storage stability, and higher encapsulation efficiency than the DHP-rich ones. Introduction of DODAB into DHP vesicles enhances the membrane fluidity in the ripple and liquid crystalline phases. The membrane fluidity of mixed DODAB-DHP vesicles with the near charge might have a significant effect on the survival of nontransformed human skin fibroblast Hs68 cells. The degree of the cytotoxicity of Hs68 cells is dominated mainly by the charge nature of DODAB-DHP vesicles with varying lipid composition. The results gathered provide necessary information for future drug/gene delivery applications.

Dynamics of Myosin XI: The Family Speed Demon

Myosin XI is the fastest known processive motor, and is implicated in organelle transport and propulsion of cytoplasmic streaming in plant cells. We studied the motility and dynamics of a recombinant myosin XI motor domain hybridized with the myosin Va lever arm with 6 calmodulin-bearing IQ motifs and the dimerization motif. Single molecule fluorescence tracking (FIONA) and polarization (polTIRF) measurements support the hand-over-hand stepping model for myosin XI. Its step size (∼34.5 nm) is slightly shorter than the helical pitch of actin filaments, suggesting an overall left-handed helical walking path. The path was confirmed by polTIRF azimuthal angles as well as bead motility on suspended actin filaments. The hybrid myosin XI showed a maximal velocity of at least 4 μm/s, which is ∼7-fold faster than its structurally similar cousin, myosin V, but they have similar run lengths of ∼1 μm. FIONA measurements showed faster myosin XI stepping rates on actin filaments as compared to bundled actin. The gliding filament assay gave 5-10-fold slower velocities than single molecule processive runs. PolTIRF experiments identified several classes of molecules, with different leading and trailing probe angles. These classes may represent placement of the bifunctional rhodmaine probe on different calmodulins. Compared with myosin V, myosin XI showed more variable localization between steps and more off-axis motion, which may relate to myosin XI's role in cytoplasmic streaming on bundled actin beams. Supported by NIH grant GM086352.