How Do Amphiphiles Form Ion-Conducting Channels in Membranes? Lessons from Linear Oligoesters

THOMAS M. FYLES

ACCOUNTS OF CHEMICAL RESEARCH

The X-ray crystal structures of biological ion channels are exquisitely complex, but not all natural products capable of forming ion-conducting channels are equally elaborate. Examples such as the peptides gramicidin or alamethicin or the polyene antibiotics amphotericin and nystatin clearly form well-defined channels without requiring a massive protein superstructure. These molecules form the starting point for a supramolecular chemistry challenge: how to create synthetic compounds and systems that catalyze the translocation of ionic species across bilayer membranes mimicking naturally occurring channels. Over the past three decades, supramolecular chemists have developed numerous examples of systems with transport rates and efficiencies that rival natural channels. As the field developed, researchers discovered many compounds that are functional for ion transport but bear very little resemblance to any imagined architectures of ion channels. We and others have followed these lead compounds extensively in a quest to focus on the mechanisms such simple compounds use to achieve their function. These compounds show all the hallmarks of ion channels including high activity, ion specificity, regular time-dependent conductance changes, and in some cases higher-order phenomena such as voltage-dependent activity.

Comparative Properties and Methods Of Preparation Of Lipid Vesicles (Liposomes)

Francis Szoka, Jr and Demetrios Papahadjopoulos

Ann. Rev. Biophys. Bioeng. 1980. 9:467-508

In this review we point out the salient points in vesicle preparation and the relative advantages of each type of vesicle. Our aim is to acquaint the reader with the pitfalls in liposome preparations, the lipid compositions that can be used, methods of preparation, size distributions of the resulting vesicles, efficiencies of encapsulation of the aqueous space, and methods to characterize the resulting vesicles.

Time-resolved nonlinear fluorescence spectroscopy using femtosecond multiphoton excitation and single-photon timing detection

Andreas Volkmer, David A Hatrick and David J S Birch

Meas. Sci. Technol. 8 (1997) 1339–1349. Printed in the UK

Abstract: We have developed a time-correlated single-photon timing nonlinear fluorometer for recording the fluorescence decay times and rotational correlation times of molecular probes using 120 fs regeneratively amplified Ti:Sapphire laser excitation via simultaneous non-resonant absorption of two or more near infrared photons. A microchannel plate photomultiplier giving 70 ps impulse response is used for detection. Studies on 1,6-diphenylhexatriene, rhodamine 6G and p-terphenyl in propylene glycol demonstrate two- and three-photon induced fluorescence characteristics. The radiative properties for one- and multiphoton excitation were found to be identical. However, the time-zero anisotropy observed for multiphoton excitation was larger than for one-photon excitation, indicating an increased degree of orientation of excited molecules after multiphoton absorption. The results reveal the potential of multiphoton-induced fluorescence anisotropy in the study of the structure and dynamics of microheterogeneous systems (i.e. biomembranes, porous matrices etc) by selecting the excitation wavelength and class of probe molecule.

Bilayer heating in magnetite nanoparticle–liposome dispersions via fluorescence anisotropy

Geoffrey D. Bothun, Matthew R. Preiss
Journal of Colloid and Interface Science 357 (2011) 70–74

Temperature measurements have been made within magnetite (Fe3O4) nanoparticle–liposome dispersions subjected to electromagnetic field at radiofrequency (RF) heating based on the fluorescence anisotropy of diphenylhexatriene (DPH) embedded within the bilayer. Incorporating cholesterol within dipalmitoylphosphatidylcholine (DPPC) bilayers broadened the anisotropy window associated with lipid melting. Cryogenic transmission electron microscopy showed that the dispersions contained magnetoliposomes with nanoparticle aggregates at both low and high encapsulation densities. RF heating results demonstrated the ability to measure the temperature of the ML bilayer with on/off RF cycles using DPH anisotropy. These measurements reflected the temperature of the bulk aqueous phase, which is consistent with previous work showing rapid heat dissipation from a nanoparticle surface during RF heating and a negligible difference between surface and bulk temperature.

Mixed Micellization of Dimeric (Gemini) Surfactants and Conventional Surfactants

R. G. Alargova, I. I. Kochijashky, M. L. Sierra, K. Kwetkat, and R. Zana

Journal of Colloid and Interface Science 235, 119–129 (2001)

The aqueous solutions of mixtures of various conventional surfactants and dimeric anionic and cationic surfactants have been investigated by electrical conductivity, spectrofluorometry, and time-resolved fluorescence quenching to determine the critical micelle concentrations and the micelle aggregation numbers in these mixtures. The following systems have been investigated: 12- 2-12/DTAB, 12-2-12/C12E6, 12-2-12/C12E8, 12-3-12/C12E8, Dim3/ C12E8, and Dim4/C12E8 (12-2-12 and 12-3-12Ddimethylene-1,2- and trimethylene-1,3-bis(dodecyldimethylammonium bromide), respectively; C12E6 and C12E8 Dhexa- and octaethyleneglycol monododecylethers, respectively; Dim3 and Dim4Danionic dimeric surfactants of the disodium sulfonate type, Scheme 1; DTABD dodecyltrimethylammonium bromide). For the sake of comparison the conventional surfactant mixtures DTAB/C12E8 and SDS/C12E8 (SDSDsodium dodecylsulfate) have also been investigated (reference systems). Synergism in micelle formation (presence of a minimum in the cmc vs composition plot) has been observed for the Dim4/C12E8 mixture but not for other dimeric surfactant/nonionic surfactant mixtures investigated. The aggregation numbers of the mixed reference systems DTAB/C12E8 and SDS/C12E8 vary monotonously with composition from the value of the aggregation number of the pure C12E8 to that of the pure ionic component. In contrast, the aggregation number of the dimeric surfactant/C12E8 mixtures goes through a minimum at a low value of the dimeric surfactant mole fraction. This minimum does not appear to be correlated to the existence of synergism in micelle formation. The initial decrease of the aggregation number of the nonionic surfactant upon addition of ionic surfactant, up to a mole fraction of ionic surfactant of about 0.2 (in equivalent per total equivalent), depends little on the nature the surfactant, whether conventional or dimeric. The results also show that the microviscosity of the systems containing dimeric surfactants is larger than that of the reference systems.

Vesicle-to-Micelle Transformation in System Containing Dimeric Surfactants

B.K. Paul, N. Guchhait
J. Colloid Interface Sci., 363 (2011), pp. 529–539

Insolubility of lipids in Triton X-100: physical origin and relationship to sphingolipid/cholesterol membrane domains (rafts)

Erwin London, Deborah A. Brown

Biochimica et Biophysica Acta 1508 (2000) 182-195

The insolubility of lipids in detergents is a useful method for probing the structure of biological membranes. Insolubility in detergents like Triton X-100 is observed in lipid bilayers that exist in physical states in which lipid packing is tight. The Triton X-100-insoluble lipid fraction obtained after detergent extraction of eukaryotic cells is composed of detergent-insoluble membranes rich in sphingolipids and cholesterol. These insoluble membranes appear to arise from sphingolipid- and cholesterol-rich membrane domains (rafts) in the tightly packed liquid ordered state. Because the degree of lipid insolubility depends on the stability of lipid-lipid interactions relative to lipid-detergent interactions, the quantitative relationship between rafts and detergent-insoluble membranes is complex, and can depend on lipid composition, detergent and  temperature. Nevertheless, when used conservatively detergent insolubility is an invaluable tool for studying cellular rafts and characterizing their composition. ß 2000 Elsevier Science B.V. All rights reserved.

Chiral analysis of baclofen by alpha-cyclodextrin-modified capillary electrophoresis and laser-induced fluorescence detection

Mei-Tsu Chiang, Sarah Y. Chang, Chen-Wen Whang

Electrophoresis 2001, 22, 123-127

Lipopolysaccharide Identification with Functionalized Polydiacetylene Liposome Sensors

Marianne Rangin and Amit Basu

J. AM. CHEM. SOC. 2004, 126, 5038-5039

Lipopolysaccharides (LPS) are complex glycolipids embedded within the outer membrane of Gram negative bacteria.1 Each cell contains over 2 million copies of LPS.1b LPS consists of a conserved lipidated disaccharide, known as Lipid A, which is attached to a core oligosaccharide fragment. The core region is extended by additional glycosylation to provide the O-specific antigen or polysaccharide region. The identity of these terminal sugars vary for different bacterial species and serotypes. Sensors which are capable of detecting and identifying different types of LPS can be used to develop devices for bacterial diagnostics.

A high-throughput fluorescence polarization anisotropy assay for the 70N domain of replication protein A

Souza-Fagundes EM, Frank AO, Feldkamp MD, Dorset DC, Chazin WJ, Rossanese OW, Olejniczak ET, Fesik SW.

Anal Biochem. 2012 Feb 15;421(2):742-9. doi: 10.1016/j.ab.2011.11.025. Epub 2011 Dec 1.

Replication protein A (RPA) interacts with multiple checkpoint proteins and promotes signaling through the ATR kinase, a key regulator of checkpoint pathways in the mammalian response to DNA damage. In cancer cells, increased DNA repair activity contributes to resistance to chemotherapy. Therefore, small molecules that block binding of checkpoint proteins to RPA may inhibit the DNA damage response and, thus, sensitize cancer cells to DNA-damaging agents. Here we report on the development of a homogeneous, high-throughput fluorescence polarization assay for identifying compounds that block the critical protein-protein interaction site in the basic cleft of the 70N domain of RPA (RPA70N). A fluorescein isothiocyanate (FITC)-labeled peptide derived from the ATR cofactor, ATRIP, was used as a probe in the binding assay. The ability of the assay to accurately detect relevant ligands was confirmed using peptides derived from ATRIP, RAD9, MRE11, and p53. The assay was validated for use in high-throughput screening using the Spectrum collection of 2000 compounds. The FPA assay was performed with a Z' factor of ≥ 0.76 in a 384-well format and identified several compounds capable of inhibiting the RPA70N binding interface.

Production of reactive oxygen species in endothelial cells under different pulsatile shear stresses and glucose concentrations

L. K. Chin, J. Q. Yu, Y. Fu, T. Yu, A. Q. Liua and K. Q. Luo

Lab Chip, 2011, 11, 1856

A hemodynamic Lab-on-a-chip system was developed in this study. This system has two unique features: (1) it consists of a microfluidic network with an array of endothelial cell seeding sites for testing them under multiple conditions, and (2) the flow rate and the frequency of the culture medium in the microchannel are controlled by a pulsation free pump to mimic the flow profile of the blood in the blood vessel under different physiological conditions. The investigated physiological conditions were: (1) the resting condition in a normal shear stress of 15 dyne cm 2 with a normal heart rate of 70 bpm, (2) an exhaustive exercise condition with a high shear stress of 30 dyne cm 2 and a fast heart rate of 140 bpm, and (3) a constant high shear stress of 30 dyne cm 2. Two chemical conditions were investigated (10 mM and 20 mM glucose) to mimic hyperglycemic conditions in diabetes patients. The effects of various shear stresses either alone or in combination with different glucose concentrations on endothelial cells were examined using the developed hemodynamic Lab-on-a-chip system by assessing two parameters. One is the intracellular level of reactive oxygen species (ROS) determined by a fluorescent probe, H2DCFDA. Another is the mitochondrial morphology revealed with a fluorescent dye, MitoTracker Green FM. The results showed that ROS level was elevated nearly 4-fold after 60 min of exhaustive exercise. We found that the pulsatile nature of the fluid was the determination factor for causing ROS generation in the cells as almost no increase of ROS was detected in the constant shear stress condition. Similarly, much higher level of ROS was detected when 10 mM glucose was applied to the cells under normal or high pulsatile shear stresses compared with under a static condition. These results suggest that it is necessary to use pulsatile shear stress to represent the physiological conditions of the blood flow, and demonstrate the advantage of utilizing this newly developed hemodynamic Lab on-a-chip system over the conventional non-pulsatile system in the future shear stress related studies.

Evaluation of the Probe 2‘,7‘-Dichiorofluorescin as an Indicator of Reactive Oxygen Species Formation and Oxidative Stress

Carl P. LeBel, Harry Ischiropoulos, and Stephen C. Bondy

Chem. Res. Toxicol. 1992, 5, 227-231

The use of dichlorofluorescin (DCFH) as a measure of reactive oxygen species was studied in aqueous media. Hydrogen peroxide oxidized DCFH to fluorescent dichlorofluorescein (DCF), and the oxidation was amplified by the addition of ferrous iron. Hydrogen peroxide-induced DCF formation in the presence of ferrous iron was completely inhibited by deferoxamine and partially inhibited by ethylenediaminetetraacetic acid, but was augmented by diethylenetriaminepentaacetic acid. Iron-peroxide-induced oxidation of DCFH was partially inhibited by catalase but not by horseradish peroxidase. Nonchelated iron-peroxide oxidation of DCFH was partially inhibited by several hydroxyl radical scavengers, but was independent of the scavenger concentration, and this suggests that free hydroxyl radical is not involved in the oxidation of DCFH in this system. Superoxide anion did not directly oxidize DCFH. Data suggest that H2O2-Fe2+-derived oxidant is mainly responsible for the nonenzymatic oxidation of DCFH. In addition, peroxidase alone and oxidants formed during the reduction of H202 by peroxidase oxidize DCFH. Since DCFH oxidation may be derived from several reactive intermediates, interpretation of specific reactive oxygen species involved in biological systems should be approached with caution. However, DCFH remains an attractive probe as an overall index of oxidative stress in toxicological phenomena.