Non-Uniform Membrane Probe Distribution in Resonance Energy Transfer: Application to Protein–Lipid Selectivity

Biological membranes are, at the molecular level, quasi-two dimensional systems. Membrane components are often distributed non-uniformly in the bilayer plane, as a consequence of lipid phase separation/domain formation or local enrichment/depletion of particular lipid species arising form favorable/unfavorable lipid–membrane protein interactions. Due to its explicit dependence on donor–acceptor distance or local acceptor concentration, resonance energy transfer (RET) has large potential in the characterization of membrane heterogeneity. RET formalisms for the basic geometric arrangements relevant for membranes have now been known for several decades. However, these formalisms usually assume uniform distributions, and more general models are required for the study of membrane lateral heterogeneity. We present a model that addresses the possibility of non-uniform acceptor (e.g., lipid probe) distribution around each donor (e.g., protein) in a membrane. It considers three regions with distinct local acceptor concentration, namely, an exclusion zone, the membrane bulk, and, lying in between, a region of enhanced probability of finding acceptors (annular region). Numerical solutions are presented, and convenient empirical fitting functions are given for RET efficiency as a function of bulk acceptor surface concentration, for several values of the model parameters. The usefulness of the formalism is illustrated in the analysis of experimental data.     

RET receptor signaling: Function in development,metabolic disease,and cancer

The RET proto-oncogene encodes a receptor tyrosine kinase whose alterations are responsible for various human cancers and developmental disorders, including thyroid cancer, non-small cell lung cancer, multiple endocrine neoplasia type 2, and Hirschsprung’s disease. RET receptors are physiologically activated by glial cell line-derived neurotrophic factor (GDNF) family ligands that bind to the coreceptor GDNF family receptor α (GFRα). Signaling via the GDNF/GFRα1/RET ternary complex plays crucial roles in the development of the enteric nervous system, kidneys, and urinary tract, as well as in the self-renewal of spermatogonial stem cells. In addition, another ligand, growth differentiation factor-15 (GDF15), has been shown to bind to GFRα-like and activate RET, regulating body weight. GDF15 is a stress response cytokine, and its elevated serum levels affect metabolism and anorexia-cachexia syndrome. Moreover, recent development of RET-specific kinase inhibitors contributed significantly to progress in the treatment of patients with RET-altered cancer. This review focuses on the broad roles of RET in development, metabolic diseases, and cancer.     

Mechanisms of Fluorescence Quenching in Donor—Acceptor Labeled Antibody—Antigen Conjugates

The cyanine dyes Cy5 and Cy5.5 are presented as a new long wavelength-excitable donor-acceptor dye pair for homogeneous fluoroimmunoassays. The deactivation pathways responsible for the quenching of the fluorescence of the antibody-bound donor are elucidated. Upon binding of the donor dye to the antibodies at low dye/protein ratios, its fluorescence quantum yield rises to unity. Higher dye/protein ratios lead to progressive aggregation of the dyes, which results in quenching of monomer fluorescence due to resonance energy transfer (RET) from the monomers to the nonfluorescent dimers. The dependence of the quenching efficiency on the labeling ratio is described quantitatively by assuming a Poisson distribution of the dyes over the antibodies. The maximum fluorescence intensity per antibody is obtained at a labeling ratio of 4. Upon formation of the antibody-antigen complex, electron transfer and RET to the antigen-bound acceptor dye occur. Steady-state and time-resolved fluorescence measurements reveal that approximately 50% of the donor quenching is due to RET, while the residual quenching effect is caused by the static quenching process.     

Glucose-Sensitive Nanoassemblies Comprising Affinity-Binding Complexes Trapped in Fuzzy Microshells

A new design for glucose monitoring with "smart" materials based on self assembly, competitive binding, and resonance energy transfer (RET) is presented. The basic transduction principle is changing RET efficiency from fluorescein isothiocyanate (FITC) to tetramethylrhodamine isothiocyanate (TRITC), as FITC-dextran is displaced from TRITC-Concanavalin A (Con A) with the addition of glucose. Nanoscale fabrication by self-assembly of Con A/dextran into multilayer films, followed by polymer multilayers. The advantages of this approach include physical localization and separation of sensing molecules from the environment via entrapment of the biosensorelements in a semi-permeable polymeric shell, and only functional molecules are included in the sensors. To realize these nanostructures, dissolvable resin microparticles were coated with FITC-dextran+TRITC-Con A multilayers, followed by polyelectrolyte multilayers, and the core particles were then dissolved to yield hollow capsules. The nanoassembly process was studied using microbalance mass measurements, fluorescence spectroscopy, confocal fluorescence microscopy, and zeta-potential measurements. The key findings are that the specific binding between Con A and dextran can be used to deposit ultrathin multilayer films, and these exhibit changing RET in response to glucose. Fluorescence spectra of a microcapsules exhibited a linear, glucose-specific, 27% increase in the relative fluorescence of FITC over the 0-1800 mg/dL range. These findings demonstrate the feasibility of using self-assembled microcapsules as optical glucose sensors, and serve as a basis for work toward better understanding the properties of these novel materials.     

Observation of rotational energy transfer in iodine by two-photon time delayed spectroscopy

The temporal evolution for individual rovibrational levels of the B state of the iodine molecule is observed by two-photon delayed spectroscopy. In particular the rotational energy transfer (RET) process due to collisions of the molecules in the B state with molecules in the ground state is studied. Thermal averaged rate constants for pairs of rotational levels of the v=27 vibrational state are measured. The results are discussed in terms of the dynamics of the collision process and numerical fits to the most important parametric fitting laws for RET are presented.     

Effect of laser intensity and of lower-state rotational energy transfer upon temperature measurements made with laser-induced fluorescence

4 –air flame, with OH at 2000 K. We calculate the ratio of LIF intensities that would be induced by doubled dye-laser light near 283 nm, by means of the A←X, 1←0, P₁(7), and Q₂(11) transitions in OH. Here we show that the ratio of LIF signals from those two transitions, and thus the deduced temperature, is sensitive to laser intensity. That is caused mainly by the competition between laser-pumping of molecules out of the lower rotational state and of rotational energy transfer (RET) collisions into that state. A-state collisional effects are normally important, but are minimized here by assuming that they are the same for both transitions. The laser spectral intensity dependence of the fluorescence ratio depends heavily upon the value of the RET coefficients within the X-state. While RET reduces the sensitivity of the observed signal to the laser spectral intensity, the conversion of a measured fluorescence ratio to temperature is particularly difficult. That is because RET rates, and quenching rates, can be a function of local conditions and of the rotational state being populated. Two different models are used to demonstrate these effects, and both predict large effects upon temperature. Received: 19 February 1998/Revised version: 16 June 1998     

State-specific rotational energy transfer in OH (A 2Σ+, v′=0) by some combustion-relevant collision partners

State-to-state rotational energy transfer (RET) co-efficients were determined for inelastic collisions of OH (A ²⁺, v=0) with N₂, CO₂, and H₂O at 300 K. The experimental procedure described previously allows the direct evaluation of state-specific RET coefficients from time-resolved laser-induced fluorescence (LIF) measurements without any assumptions on the RET. The results show strikingly different RET behaviour for the three collision partners. The data can serve as a basis for a comparison with dynamic collision models.A. Jörg is now with IBM Corporation, Frankfurt, Fed. Rep. Germany     

Effects of Metallic Silver Particles on Resonance Energy Transfer Between Fluorophores Bound to DNA

We examined the effects of metallic silver island films on resonance energy transfer (RET) between a donor and acceptor bound to double helical DNA. The donor was 4',6-diamidino-2-phenylindole (DAPI) and the acceptor was propidium iodide (PI). Proximity of the labeled DNA to the silver particles resulted in a dramatic increase in RET as seen from the emission spectra and the donor decay times. Proximity to silver particles results in an increase of the Förster distance from 35 Å an to an apparent value of 166 Å. These results suggest a new type of DNA hybridization assays based on RET over distances much longer than the free-space Forster distance.     

Energy transfer in the A2Σ+ state of OH following v’ = 1 excitation in a low pressure CH4/O2-flame

2 Σ⁺(v’=1) level of OH. Measurements were performed in a laminar premixed flame at 10 Torr total pressure. The low pressure allowed the spatial variation of the effective quenching rate to be determined through the flame front. In addition, the dependence of the quenching rate on rotational quantum number was measured by exciting a series of rotational lines in the range N’=0–16. The results show that the total quenching rate decreases only 17% through the flame front, in the region where OH can be detected. Nevertheless, the absolute value of the quenching rate Q is required if absolute concentrations are to be determined from LIF-signals. The variation both of Q and of the rotational relaxation rate with excited rotational quantum state must be known for quantification of LIF-temperature measurements via the Boltzmann relation. Finally, the rotational and vibrational energy transfer (RET, VET), was investigated by recording the spectrally and temporally resolved fluorescence. For all excited rotational lines, efficient RET to neighbouring rotational states was observed, but only very little VET. Total RET rates were determined from the difference between the time-resolved broadband (total fluorescence) and narrowband (fluorescence from the laser excited level) curves. The experimental results were compared with simulations using a dynamic model, which describes the energy transfer for flame conditions. With the available input data (temperature, major species concentrations and collision-partner specific RET cross sections), good agreement was obtained. Received: 3 February 1997/Revised version: 3 September 1997     

Voice quality changes following phonatory-respiratory effort treatment (LSVT) versus respiratory effort treatment for individuals with Parkinson disease.

Perceptual ratings of hoarseness and breathiness were used to assess the efficacy of two intensive methods for treating dysarthrophonia in individuals with idiopathic Parkinson disease. One method emphasized phonatory-respiratory effort (the Lee Silverman Voice Treatment, LSVT) and the other emphasized respiratory effort alone (RET). Perceptual ratings were performed by two expert listeners based on random order presentation of the patients' pretreatment and posttreatment recordings of the "Rainbow Passage." The listeners were blinded to the patients and their treatment group. Statistically significant pretreatment to posttreatment improvement in hoarseness and breathiness was observed in the LSVT group but not in the RET group. The present findings are consistent with acoustic and physiologic findings reported previously, providing further evidence for the efficacy of the LSVT.     

A scaling formalism for the representation of rotational energy transfer in OH (A 2Σ+) in combustion experiments

In order to predict spectra and temporal decays of the OH radical measured by Laser-Induced Fluorescence (LIF), the collisional energy transfer between different quantum states must be taken into account. At the elevated temperatures relevant for combustion studies, the large number of interacting quantum states and corresponding state-to-state energy transfer coefficients precludes the experimental measurement of all necessary information for the appropriate mixtures of colliders. Therefore, a scaling procedure has been devised which allows the representation of the matrix of Rotational Energy Transfer (RET) coefficients on the basis of measured data with four scaling coefficients. The scaling coefficients have been determined by comparison of the calculated RET rates with available measured data. The mathematical formalism for the scaling law - the ECS-EP law - is based on the Energy Corrected Sudden (ECS) law and includes an Exponential Power law (EP) for the representation of the basis coefficients.     

Direct Visualization of Vesicle Maturation and Plasma Membrane Protein Trafficking

Internalization and intracellular trafficking of membrane proteins are now recognized as essential mechanisms that contribute to a number of cellular processes. Current methods lack the ability to specifically label the plasma membrane of a live cell, follow internalization of labeled membrane molecules, and conclusively differentiate newly formed membrane-derived vesicles from pre-existing endocytic or secretory structures in the cytoplasm. Here, we detail a visualization method for surface biotinylation of plasma membrane-derived vesicles that allows us to follow their progress from membrane to cytosol at specific time points. Using the transmembrane receptor RET as a model, we demonstrate how this method can be applied to identify plasma membrane-derived vesicle maturation, determine RET’s presence within these structures, and monitor RET’s recycling to the cell surface. This method improves on static and less discriminatory methods, providing a tool for analysis of real-time vesicle trafficking that is applicable to many systems.     

A detailed rate equation model for the simulation of energy transfer in OH laser-induced fluorescence

A rate equation model (the LASKIN program packet) has been developed for the detailed computation of energy transfer in Laser-Induced Fluorescence (LIF). Calculations of this type are necessary for analysis of the influence of energy transfer processes [e.g., electronic quenching and Rotational Energy Transfer (RET)] on the fluorescence signal. The model has been utilized to examine linear LIF in the OHA ²⁺-X ² (0, 0) band. Available data on quenching, RET and spontaneous emission rates for the (A, v = 0) state have been reviewed, and models for the state-specific RET and quenching rates have been developed. The accuracy of the calculations has been confirmed by comparison with experimental data, and the LASKIN program has been applied to the analysis of potential error sources in the widely applied two-line LIF temperature-measurement technique. Extensions of the model to the examination of saturated LIF, OHA–X (1, 0) and (3, 0) excitation and LIF of other species (e.g., O₂ and NO) are discussed.     

Voice Quality Changes Following Phonatory-Respiratory Effort Treatment (LSVT®) Versus Respiratory Effort Treatment for Individuals with Parkinson Disease

Perceptual ratings of hoarseness and breathiness were used to assess the efficacy of two intensive methods for treating dysarthrophonia in individuals with idiopathic Parkinson disease. One method emphasized phonatory-respiratory effort (the Lee Silverman Voice Treatment, LSVT®) and the other emphasized respiratory effort alone (RET). Perceptual ratings were performed by two expert listeners based on random order presentation of the patients' pretreatment and posttreatment recordings of the “Rainbow Passage.” The listeners were blinded to the patients and their treatment group. Statistically significant pretreatment to posttreatment improvement in hoarseness and breathiness was observed in the LSVT® group but not in the RET group. The present findings are consistent with acoustic and physiologic findings reported previously, providing further evidence for the efficacy of the LSVT®.     

Classical Limit of Relativistic Moments Associated with Boltzmann–Chernikov Equation: Optimal Choice of Moments in Classical Theory

Journal of Statistical Physics - The moment system associated with the Boltzmann equation is largely used in many applications and is the main ingredient of Rational Extended Thermodynamics (RET)....     

Numerical analysis of quantitative measurement of hydroxyl radical concentration using laser-induced fluorescence in flame

The aim of the present work is to quantitatively measure the hydroxyl radical concentration by using LIF(laserinduced fluorescence) in flame.The detailed physical models of spectral absorption lineshape broadening,collisional transition and quenching at elevated pressure are built.The fine energy level structure of the OH molecule is illustrated to understand the process with laser-induced fluorescence emission and others in the case without radiation,which include collisional quenching,rotational energy transfer(RET),and vibrational energy transfer(VET).Based on these,some numerical results are achieved by simulations in order to evaluate the fluorescence yield at elevated pressure.These results are useful for understanding the real physical processes in OH-LIF technique and finding a way to calibrate the signal for quantitative measurement of OH concentration in a practical combustor.     

A History of Resolution Enhancement Technology

Resolution enhancement techniques (RETs) have enabled the adoptin of optical lithography well below the wavelength of the exposing light. Examination of the history of RET and recent developments shows that only 3 of the 4 independent variables available for wavefront engineering have been utilized. This suggest that, with full utilization of polarization and other electromagnetic effects, optical lithography will be the manufacturing technology for ICs for the foreseeable future.     

Structural modification of an EGFR inhibitor that showed weak off-target activity against RET leading to the discovery of a potent RET inhibitor

Here, we describe the structural optimization of a known EGFR inhibitor (compound 1) that showed weak off-target activity against RET. Twenty-six analogs of 1 were synthesized. SAR analysis led to the discovery of several compounds that showed considerable potency against the RET-dependent thyroid cancer cell line TT. Kinase inhibitory potency was then measured for the most active compound (2u) in the cellular assay. The results showed that 2u is a potent RET inhibitor with an $\hbox {IC}_{50}$ value of 7 nM.     

Resolution enhancement techniques for optical lithography and optical imaging theory

Production of a fine pattern is necessary to get a high integration degree of integrated circuits. The conventional methods which utilize high numerical aperture and short wavelength exposure are limited by designing and manufacturing of a practical lens and make the focus depth narrow. Resolution enhancement techniques (RETs) have, therefore, been required and proposed. This paper introduces a phase-shifting mask, a typical RET, points out the problems and inconsistencies of conventional optical imaging theory and explains the image formation concept of expansion of plane waves. Essentially using this concept, an attempt is also made to describe some other typical RETs with potential.