Fluorescence lifetime distribution of 1,8-anilinonaphthalenesulfonate (ANS) in reversed micelles detected by frequency domain fluorometry.

The fluorescence emission decay of ANS (1,8-anilinonaphthalenesulfonate) in reversed AOT (sodium bis-(2-ethyl-1-hexy)sulfosuccinate) micelles at different water contents was investigated by frequency domain fluorometry. The whole ANS emission decay in reversed AOT micelles could not be fitted in terms of discrete lifetime values, i.e., mono-exponential and bi-exponential models. Better fits were obtained when using continuous unimodal Lorentzian lifetime distributions. This was interpreted as arising from the reorientation processes of water molecules around the excited state of ANS or probe exchange among different probe locations, occurring on a time scale longer than fluorophore lifetime. The dependence of ANS fluorescence anisotropy on the emission wavelength was consistent with the existence of a great emission heterogeneity especially for inverted micelles having reduced H2O/AOT molar ratio. Finally, the observation that the distribution width decreases with increasing temperature and/or micelle size suggested that fast processes of water dipolar reorganization around the fluorophore are facilitated under these conditions.     

Properties influencing fluorophore lifetime distributions in lipid bilayers

The fluorescence lifetime of the membrane fluorophore 1,6-diphenyl-1,3,5-hexatriene has been analyzed according to the distributional approach in a number of lipid bilayer systems. The systems included vesicles of 16:0/18:1-phosphatidylcholine (POPC), egg phosphatidylcholine (EYPC), microsomal phospholipids, and also intact microsomal membranes. With increasing complexity of composition, an increasingly broader width was found in the major component of a bimodal Lorentzian fluorescence lifetime distribution. In order to explain these findings, we propose a model based on environmental heterogeneity and environmental sampling, where the environment is defined as the lipid molecules immediately surrounding the fluorophore. Environmental heterogeneity is thought of as arising from organizational, compositional, and solvent factors. Environmental sampling pertains to the ability of a fluorophore to detect environments in a system and is a function of the fluorophore lifetime and the lipid dynamics. If the fluorescence lifetime is sufficiently short, the fluorophore will only sample a particular environment, and great compositional complexity will mean that each fluorophore in an ensemble will decay to the ground state with a different time. This appears to explain why in our results with DPH a narrow width is obtained for POPC, where vesicles are composed of a single phospholipid molecular species, compared to EYPC and microsomal phospholipid vesicles having complex molecular species composition. This model should serve as a basis for understanding the interrelationships of environmental complexity and lipid dynamics in membranes.     

Fluorescence quenching in model membranes: phospholipid acyl chain distributions around small fluorophores

Fluorescence quenching in lipid bilayers is treated by a new approach based on calculation of the probability distribution of quenching and nonquenching acyl chains around a fluorophore. The effect of acyl lattice site dependence (i.e., correlations of phospholipid sister chain occupancy of neighbor sites) was modeled by use of Monte Carlo simulations of acyl chain occupancy. This explicit accounting of site occupancy correlation was found to fit observed quenching behavior better than did a model wherein phospholipid quenchers are considered to be independent. A key aspect of this approach is to evaluate the rate for quenching in a bilayer composed of pure quenching lipid. In order to evaluate this quenching rate, and also to provide a strong test of the calculated probability distributions, we synthesized lipids with both acyl chains labeled with a quenching moiety (Br or nitroxide), as well as the more usual single-chain quenchers. The fluorescence of tryptophan octyl ester (TOE), and of the 1,6-diphenyl-1,3,5-hexatriene (DPH) derivatives trimethylammonium-DPH (TMA-DPH) and 1-lauroyl-2-(DPH-propionyl)phosphatidylcholine (DPH-PC), was examined. We obtained consistent results with all the fluorophores and quenchers indicating that up to 18 neighboring acyl sites can contribute to quenching, corresponding to two shells of acyl sites on a hexagonal lattice. Calculated discrete distributions of fluorescence intensities were converted into fluorescence lifetimes and compared with Gaussian and Lorentzian continuous lifetime distributions. The fluorescence quenching theory presented here may be used to explain quantitatively the heterogeneity of fluorophore environments in multicomponent membranes.     

Conformational substates of myoglobin detected by extrinsic dynamic fluorescence studies

The extent of conformational substates of two apomyoglobins, i.e., sperm whale and tuna apomyoglobin, was investigated by examining the fluorescence decay in the frequency domain of the extrinsic fluorophore TNS [6-(p-toluidino)-2-naphthalenesulfonic acid] bound to the heme binding site. Data analysis was performed in terms of a continuous, unimodal lifetime distribution having a Lorentzian shape. The results were compared with those for the free fluorophore in an isotropic nonviscous solvent. The incorporation of TNS into the protein matrix resulted in a broadening of the lifetime distribution due to the microenvironmental heterogeneity generated by structural fluctuations. The larger width of lifetime distribution observed for TNS bound to tuna apomyoglobin was related to a more extended conformational space accessible to the fluorophore in this protein compared to sperm whale myoglobin. A temperature increase from 15 to 40 degrees C produced a further broadening of the lifetime distributions of TNS bound to both proteins. This result can be explained by assuming the existence of conformational substates at high energy content or separated by high energy barriers, which are not populated at low temperature. The overall picture emerging from the reported data is that the lifetime distributions of TNS bound to apomyoglobins are determined largely by the number of conformational substates accessible to the protein matrix and, to a lesser extent, by the interconversion rates among these states.     

Cholesterol oxidase as a structural probe of biological membranes: its application to brush-border membrane

Cholesterol present in intact brush-border membrane vesicles made from rabbit small intestine is a poor substrate for cholesterol oxidase (EC 1.1.3.6, from Nocardia sp. and Nocardia erythropolis). It becomes susceptible to oxidation by the enzyme only after the addition of detergent, e.g., Triton X-100, in quantities sufficient to disrupt the membrane. This is also true for cholesterol present in bilayers of small unilamellar phosphatidylcholine or phosphatidylserine vesicles made by ultrasonication. The data presented here on intestinal brush-border membrane are in good agreement with results reported on other biological membranes, e.g., from erythrocytes and vesicular stomatitis virus, but are somewhat different from those on rat intestinal brush-border membrane. Our results on phospholipid bilayers agree well with published work on model membranes. From the work presented we conclude that, with our present understanding, cholesterol oxidase can hardly be used to probe the distribution of cholesterol in biological membranes. A prerequisite for using the enzyme successfully as such a probe would be the understanding of the factors controlling the interaction of the enzyme with its substrate cholesterol. The question under which conditions cholesterol oxidase could be useful for probing the distribution and preferred location of cholesterol in biological membranes is discussed.     

Molecular organization and dynamics of the outer membrane of Salmonella thyphimurium mutant strains detected by frequency domain fluorometry

The fluorescence decay of 1,6-diphenyl-1,3,5-hexatriene (DPH) in the outer membrane bilayer of two mutant strains of Salmonella thyphimurium, i.e., SH 5014 and SH 6261, at different temperatures was analyzed in terms of continuous Lorentzian lifetime distributions. The results were compared with those obtained for the free fluorophore in an isotropic nonviscous solvent. The incorporation of DPH in the outer membrane fragments resulted in a broadening of the lifetime distribution which was attributed to the microenvironmental heterogeneity of the membrane bilayer for the extrinsic fluorophore. The differences observed between the two types of membrane bilayers were interpreted in terms of a different molecular organization and, to a lesser extent, in terms of a different fluidity. The comparison between the DPH lifetime distributions obtained using two different excitation wavelengths, i.e., 280 and 350 nm, suggested that the structural organization of the membrane domains, which are richest in proteins, is almost identical in the two examined mutant strains. This observation indicates that the different susceptibility of the two mutant strains toward phagocytosis and complement-mediated lytic action may depend on the molecular organization and dynamics of the lipid regions far from those containing proteins.     

Molecular interaction studies of peptides using steady-state fluorescence intensity. Static (de)quenching revisited.

Protein-protein interactions, as well as peptide-peptide and peptide-protein interactions are fields of study of growing importance as molecular-level detail is avidly pursued in drug design, metabolic regulation and molecular dynamics, among other classes of studies. In membranes, this issue is particularly relevant because lipid bilayers potentiate molecular interactions due to the high local concentration of peptides and other solutes.However, experimental techniques and methodologies to detect and quantify such interactions are not abundant. A reliable, fast and inexpensive alternative methodology is revisited in this work.Considering the interaction of two molecules, at least one of them being fluorescent, either intrinsically (e.g. Trp residues) or by grafting a specific probe, changes in their aggregation state may be reported, as long as the fluorophore is sensitive to local changes in polarity, conformation and/or exposure to the solvent. The interaction will probably lead to modifications in fluorescence intensity resulting in a decrease ('quenching') or enhancement ('dequenching'). Although the presented methodology is based on static quenching methodologies, the concept is extended from quenching to any kind of interference with the fluorophore.Equations for data analysis are shown and their applications are illustrated by calculating the binding constant for several data-sets.     

Dynamics of lipid chain attached fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) in negatively charged membranes determined by NMR spectroscopy

We have determined the average location and dynamic reorientation of the fluorophore 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) attached to a C12 sn-2 chain of a phosphatidylserine (PS) analogue (C12-NBD-PS) in zwitterionic phosphatidylcholine (PC) and negatively charged phosphatidylserine (PS) host membranes. (1)H magic angle spinning nuclear Overhauser enhancement spectroscopy indicates a highly dynamic reorientation of the aromatic molecule in the membrane. The average location of NBD is characterized by a broad distribution function along the membrane director with a maximum indicating the location of the probe in the lipid/water interface of the lipid membrane. This behavior can be explained by a backfolding of the sn-2 chain towards the aqueous phase. Small differences in the distribution profiles of the NBD group along the membrane normal between PC and PS host membranes were found: in a PC host membrane, the NBD distribution has its maximum in the glycerol region; in a PS host membrane, NBD resides mostly in the upper chain region. These differences may be accounted for by packing differences in the PC versus PS host membranes. As seen by (2)H NMR order parameters, PS bilayers show a much higher packing density compared to PC membranes. Consequently, backfolding of the sn-2 chain with the NBD group attached causes a larger decrease of molecular order of the sn-1 chain in PS than in PC membranes. The broad distributions obtained for lipid chain attached NBD molecules reflect the motional freedom and molecular disorder in the liquid-crystalline lipid membrane.     

Cholesterol Reporter Molecules

Cholesterol is a major constituent of the membranes in most eukaryotic cells where it fulfills multiple functions. Cholesterol regulates the physical state of the phospholipid bilayer, affects the activity of several membrane proteins, and is the precursor for steroid hormones and bile acids. Cholesterol plays a crucial role in the formation of membrane microdomains such as "lipid rafts" and caveolae. However, our current understanding on the membrane organization, intracellular distribution and trafficking of cholesterol is rather poor. This is mainly due to inherent difficulties to label and track this small lipid. In this review, we describe different approaches to detect cholesterol in vitro and in vivo. Cholesterol reporter molecules can be classified in two groups: cholesterol binding molecules and cholesterol analogues. The enzyme cholesterol oxidase is used for the determination of cholesterol in serum and food. Susceptibility to cholesterol oxidase can provide information about localization, transfer kinetics, or transbilayer distribution of cholesterol in membranes and cells. The polyene filipin forms a fluorescent complex with cholesterol and is commonly used to visualize the cellular distribution of free cholesterol. Perfringolysin O, a cholesterol binding cytolysin, selectively recognizes cholesterol-rich structures. Photoreactive cholesterol probes are appropriate tools to analyze or to identify cholesterol binding proteins. Among the fluorescent cholesterol analogues one can distinguish probes with intrinsic fluorescence (e.g., dehydroergosterol) from those possessing an attached fluorophore group. We summarize and critically discuss the features of the different cholesterol reporter molecules with a special focus on recent imaging approaches.     

Biomimetic design of nanopatterned membranes

The biomimetic approach copying the supramolecular building principle of many archaeal cell envelopes (i.e., a plasma membrane with associated S-layer proteins) has resulted in stable lipid membranes with excellent reconstitution properties for transmembrane proteins. This is a particular challenge as one-third of all proteins in an organism are membrane proteins like pores, ion channels, or receptors. At S-layer supported lipid membranes, spatial well-defined domains on the S-layer protein interact noncovalently with lipid head groups within the lipid membrane resulting in a nanopatterning of a few anchored and scores of diffusional free-lipid molecules. In addition, no impact on the hydrophobic core region and on the function of reconstituted integral proteins has been determined. Among others, particularly S-layer stabilized membranes can be used for structure-function studies on reconstituted integral proteins and also in the membrane protein-based molecular nanotechnology, e.g., in the design of biosensing devices (e.g., lipid chip or lab-on-a-chip), or for receptor or ion channel-based high-throughput screening.     

The analysis of space use patterns.

In this paper we describe and test a new method for characterizing the space use patterns of individual animals on the basis of successive locations of marked individuals. Existing methods either do not describe space use in probabilistic terms, e.g. the maximum distance between locations or the area of the convex hull of all locations, or they assume a priori knowledge of the probabilistic shape of each individual's use pattern, e.g. bivariate or circular normal distributions. We develop a method for calculating a probability of location distribution for an average individual member of a population that requires no assumptions about the shape of the distribution (we call this distribution the population utilization distribution or PUD). Using nine different sets of location data, we demonstrate that these distributions accurately characterize the space use patterns of the populations from which they were derived. The assumption of normality is found to result in a consistent and significant overestimate of the area of use. We then describe a function which relates probability of location to area (termed the MAP index) which has a number of advantages over existing size indices. Finally, we show how any quantities such as the MAP index derived from our average distributions can be subjected to standard statistical tests of significance.     

Shotgun lipidomics reveals the temporally dependent, highly diversified cardiolipin profile in the mammalian brain: temporally coordinated postnatal diversification of cardiolipin molecular species with neuronal remodeling

Large-scale neuronal remodeling through apoptosis occurs shortly after birth in all known mammalian species. Apoptosis, in large part, depends upon critical interactions between mitochondrial membranes and cytochrome c. Herein, we examined the hypothesis that the large-scale reorganization of neuronal circuitry after birth is accompanied by profound alterations in cardiolipin (CL) content and molecular species distribution. During embryonic development, over 100 CL molecular species were identified and quantitated in murine neuronal tissues. The embryonic CL profile was notable for the presence of abundant amounts of relatively short aliphatic chains (e.g., palmitoleic and oleic acids). In sharp contrast, after birth, the CL profile contained a remarkably complex repertoire of CL molecular species, in which the signaling fatty acids (i.e., arachidonic and docosahexaenoic acids) were markedly increased. These results identify the rapid remodeling of CL in the perinatal period with resultant alterations in the physical properties of the mitochondrial membrane. The complex distribution of aliphatic chains in the neuronal CL pool is separate and distinct from that in other organs (e.g., heart, liver, etc.), where CL molecular species contain predominantly only one major type of aliphatic chain (e.g., linoleic acid). Analyses of mRNA levels by real-time quantitative polymerase chain reactions suggested that the alterations in CL content were due to the combined effects of both attenuation of de novo CL biosynthesis and decreased remodeling of CL. Collectively, these results provide a new perspective on the complexity of CL in neuronal signaling, mitochondrial bioenergetics, and apoptosis.     

Robust simplifications of multiscale biochemical networks

Background

Angiogenesis is a process by which new capillaries are formed from pre-existing blood vessels in physiological (e.g., exercise, wound healing) or pathological (e.g., ischemic limb as in peripheral arterial disease, cancer) contexts. This neovascular mechanism is mediated by the vascular endothelial growth factor (VEGF) family of cytokines. Although VEGF is often targeted in anti-angiogenic therapies, there is little knowledge about how its concentration may vary between tissues and the vascular system. A compartment model is constructed to study the VEGF distribution in the tissue (including matrix-bound, cell surface receptor-bound and free VEGF isoforms) and in the blood. We analyze the sensitivity of this distribution to the secretion rate, clearance rate and vascular permeability of VEGF.

Results

We find that, in a physiological context, VEGF concentration varies approximately linearly with the VEGF secretion rate. VEGF concentration in blood but not in tissue is dependent on the vascular permeability of healthy tissue. Model simulations suggest that relative VEGF increases are similar in blood and tissue during exercise and return to baseline within several hours. In a pathological context (tumor), we find that blood VEGF concentration is relatively insensitive to increased vascular permeability in tumors, to the secretion rate of VEGF by tumors and to the clearance. However, it is sensitive to the vascular permeability in the healthy tissue. Finally, the VEGF distribution profile in healthy tissue reveals that about half of the VEGF is complexed with the receptor tyrosine kinase VEGFR2 and the co-receptor Neuropilin-1. In diseased tissues, this binding can be reduced to 15% while VEGF bound to the extracellular matrix and basement membranes increases.

Conclusion

The results are of importance for physiological conditions (e.g., exercise) and pathological conditions (e.g., peripheral arterial disease, coronary artery disease, cancer). This mathematical model can serve as a tool for understanding the VEGF distribution in physiological and pathological contexts as well as a foundation to investigate pro- or anti-angiogenic strategies.     

Multiple conformational states in myoglobin revealed by frequency domain fluorometry

The tryptophanyl fluorescence decays of two myoglobins, i.e., sperm whale and tuna myoglobin, have been examined in the frequency domain with an apparatus which utilizes the harmonic content of a mode-locked laser. Data analysis was performed in terms of continuous distribution of lifetime having a Lorentzian shape. Data relative to sperm whale myoglobin, which possesses two tryptophanyl residues, i.e., Trp-A-5 and -A-12, provided a broad lifetime distribution including decay rates from a few picoseconds to about 10 ns. By contrast, the tryptophanyl lifetime distribution of tuna myoglobin, which contains only Trp-A-12, showed two well-separated and narrow Lorentzian components having centers at about 50 ps and 3.37 ns, respectively. In both cases, the chi 2 obtained from distribution analysis was lower than that provided by a fit using the sum of exponential components. The long-lived components present in the fluorescence decay of the two myoglobins do not correspond to any of those observed for the apoproteins at neutral pH. The tryptophanyl lifetime distribution of sperm whale apomyoglobin consists of two separated Lorentzian components centered at 2.25 and 5.4 ns, whereas that of tuna apomyoglobin consists of a single Lorentzian component, whose center is at 2.19 ns. Acidification of apomyoglobin to pH 3.5 produced a shift of the distribution centers toward longer lifetimes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A device to study the effects of stretch gradients on cell behavior

Mechanical forces are key regulators of cell function with varying loads capable of modulating behaviors such as alignment, migration, phenotype modulation, and others. Historically, cell-stretching experiments have employed mechanically simple environments (e.g., uniform uniaxial or equibiaxial stretches). However, stretch distributions in vivo can be highly non-uniform, particularly in cases of disease or subsequent to interventional treatments. Herein, we present a cell-stretching device capable of subjecting cells to controllable gradients in biaxial stretch via radial deformation of circular elastomeric membranes. By including either a defect or a rigid fixation at the center of the membrane, various gradients are generated. Capabilities of the device were quantified by tracking marked positions of the membrane while applying various loads, and experimental feasibility was assessed by conducting preliminary experiments with 3T3 fibroblasts and 10T1/2 cells subjected to 24 h of cyclic stretch. Quantitative real-time PCR was used to measure changes in mRNA expression of a profile of genes representing the major smooth muscle phenotypes. Genes associated with the contractile state were both upregulated (e.g., calponin) and downregulated (e.g., α-2-actin), and genes associated with the synthetic state were likewise both upregulated (e.g., SKI-like oncogene) and downregulated (e.g., collagen III). In addition, cells aligned with an orientation perpendicular to the maximal stretch direction. We have developed an in vitro cell culture device that can produce non-uniform stretch environments similar to in vivo mechanics. Cells stretched with this device showed alignment and altered mRNA expression indicative of phenotype modulation. Understanding these processes as they relate to in vivo pathologies could enable a more accurately targeted treatment to heal or inhibit disease, either through implantable device design or pharmaceutical approaches.     

Applications of a parametric model for informative censoring

In this article, we explore the use of a parametric model (for analyzing survival data) which is defined to allow sensitivity analysis for the presence of informative censoring. The dependence between the failure and the censoring processes is expressed through a parameter delta and a general bias function B(t, theta). We calculate the expectation of the potential bias due to informative censoring, which is an overall measure of how misleading our results might be if censoring is actually nonignorable. Bounds are also calculated for quantities of interest, e.g., parameter of the distribution of the failure process, which do not depend on the choice of the bias function for fixed delta. An application that relates to systematic lupus erythematosus data illustrates how additional information can result in reducing the uncertainty on estimates of the location parameter. Sensitivity analysis on a relative risk parameter is also explored.     

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.     

A MILP-based flux alternative generation and NMR experimental design strategy for metabolic engineering

A mixed-integer linear program (MILP) is described that can enumerate all the ways fluxes can distribute in a metabolic network while still satisfying the same constraints and objective function. The multiple solutions can be used to (1) generate alternative flux scenarios that can account for limited experimental observations, (2) forecast the potential responses to mutation (e.g., new reaction pathways may be used), and (3) (as illustrated) design (13)C NMR experiments such that different potential flux patterns in a mutant can be distinguished. The experimental design is enabled by using the MILP results as an input to an isotopomer mapping matrices (IMM)-based program, which accounts for the network circulation of (13)C from a precursor such as glucose. The IMM-based program can interface to common plotting programs with the result that the user is provided with predicted NMR spectra that are complete with splittings and Lorentzian line-shape features. The example considered is the trafficking of carbon in an Escherichia coli mutant, which has pyruvate kinase activity deleted for the purpose of eliminating acetate production. Similar yields and extracellular measurements would be manifested by the flux alternatives. The MILP-IMM results suggest how NMR experiments can be designed such that the spectra of glutamate for two flux distribution scenarios differ significantly.     

Correlating Metal-Enhanced Fluorescence and Structural Properties in Ag@SiO2 Core-Shell Nanoparticles

Metal@silica concentric nanoparticles capable of metal-enhanced fluorescence (MEF) represent a powerful means to improve the brightness and stability of encapsulated organic fluorophores and are finding numerous applications in biology, analytical chemistry, and medical diagnostics. The rational design of MEF-enabled labels and sensors often involves comparing fluorescence enhancement factors (EF) between nanostructures having different structural properties (e.g., metal core diameter, silica shell thickness, extent of spectral overlap between plasmon band and fluorophore). Accurate determination of EFs requires the measurement of fluorescence emission intensity in the presence and absence of the plasmonic core while minimizing the impact of physical and chemical artifacts (e.g., signal variations due to scattering, adsorption, sedimentation). In this work, Ag@SiO₂@SiO₂ + x (where x is fluorescein, eosin, or rhodamine B) nanostructures were synthesized with excellent control of core size, silica spacer shell thickness and fluorophore concentration. Using UV-VIS spectrometry, spectrofluorimetry, time-resolved fluorometry, and transmission electron microscopy, we investigated the influence of these key structural factors on fluorescence emission intensity, and the results were used to develop a generalized methodology for the determination of fluorescence enhancement factors in Ag@SiO₂ core-shell nanoparticles. This methodology should be of general importance to designing MEF-enabled nanostructures, sensors, and related analytical techniques.