Tri- and Pentamethine Cyanine Dyes for Fluorescent Detection of α-Synuclein Oligomeric Aggregates

The pathogenesis of Parkinson’s disease that is the second most common neurodegenerative disease is associated with formation of different aggregates of α-synuclein (ASN), namely oligomers and amyloid fibrils. Current research is aimed on the design of fluorescent dyes for the detection of oligomeric aggregates, which are considered to be toxic and morbific spices. Fluorescent properties of series of benzothiazole trimethine and pentamethine cyanines were characterized in free state and in presence of monomeric, oligomeric and fibrilar ASN. The dyes with wide aromatic systems and bulky phenyl and alkyl substituents that are potentially able to interact with hydrophobic regions of oligomeric aggregates were selected for the studies. For majority of studied dyes noticeable changes in fluorescence characteristics were shown in the presence of fibrillar or oligomeric ASN, while the dyes slightly responded on the presence of monomeric protein. For pentamethine cyanine SL-631 and trimethine cyanine SH-299 certain specificity to oligomeric aggregates over fibrils was observed. Using these dyes at 10^?6 M concentration permits the detection of oligomeric ASN in the concentrations range of at least 0.2–2 microM. Pentamethine cyanine SL-631 is proposed as dye for fluorescent detection of oligomeric aggregates of ASN, while trimethine cyanine SH-299 is shown to be a sensitive probe both on oligomeric and fibrillar ASN. It is proposed that wide aromatic system of SL-631 pentamethine dye molecule could better fix on the less dense and structured oligomeric formation, while less bulky and more “crescent-shape” molecule of trimethine dye SH-299 could easier enter into the groove of beta-pleated structure.     

Domain Formation in DODAB–Cholesterol Mixed Systems Monitored via Nile Red Anisotropy

The effect of the cholesterol (ch) on liposomes composed of the cationic lipid dioctadecyldimethylammonium bromide (DODAB) was assessed by studying both the steady-state and time-resolved fluorescence anisotropy of the dye Nile Red. The information obtained combined with analysis of the steady-state emission and fluorescence lifetime of Nile Red (NR) for different cholesterol concentrations (5–50%) elucidated the presence of “condensed complexes” and cholesterol-rich domains in these mixed systems. The steady-state fluorescence spectra were decomposed into the sum of two lognormal emissions, emanating from two different states, and the effect of temperature on the anisotropy decay of Nile Red for different cholesterol concentrations was observed. At room temperature, the time-resolved anisotropy decays are indicative of NR being relatively immobile (manifest by a high r _∞ value). At higher temperature, rotational times ca. 1 ns were obtained throughout and a trend in increasing hindrance was seen with increase of Ch content.     

A Colorimetric and Ratiometric Fluorescent Chemosensor for Fluoride Based on Proton Transfer

N-Phenyl-N’-(3-quinolinyl)urea (1) has been developed as a highly selective colorimetric and ratiometric fluorescent chemosensor for fluoride ion based on a proton transfer mechanism. Evidences for the mechanism were provided by UV-vis and fluorescence titration and especially ¹H and ¹⁹F NMR experiments. The sensor gave the largest ratiometric fluorescent response reported so far (R_max/R_min = 2620) to fluoride. Taking H⁺ as the “recovering reagent”, the sensor can be reversibly “used” and “recovered” for several cycles with only a slight decay of the response ability.     

Fluorescence Investigation of Interactions Between Novel Benzanthrone Dyes and Lysozyme Amyloid Fibrils

A series of novel fluorescent benzanthrone dyes have been tested for their ability to identify and characterize fibrillar aggregates of lysozyme prepared by protein denaturation in concentrated ethanol solution (F_eth) or acidic buffer (F_ac). Quantitative parameters of the dye association with native and fibrillar protein have been derived from the results of fluorimetric titration. The binding characteristics proved to be different for F_eth- and F_ac-bound benzanthrones, highlighting the dye sensitivity to the distinctions in fibril morphology. By comparing the dye preference to fibrillar protein aggregates, AM2, A8 and A6 were selected as the most prospective amyloid tracers. Based on the analysis of red edge excitation shifts and fluorescence lifetimes of the amyloid-bound dyes it was assumed that surface grooves or dry “steric zipper” interface are potential fibril binding sites for the novel fluorophores.     

Hydroxy and Methoxy Substituted Thiacarbocyanines for Fluorescent Detection of Amyloid Formations

In present paper series of trimethine cyanines modified in 5,5′- or 6,6′- position with hydroxy- or methoxy- substituents is studied for their ability to interact selectively with fibrillar formations. Processes of dye aggregation that accompany this interaction were also investigated. Meso-methyl trimethynecyanines with 5,5′- methoxy (7519) and hydroxy (7515) substituents strongly (up to 40 times) increase fluorescence intensity in the presence of fibrillar insulin, and also give noticeable fluorescent response on the presence of various aggregated proteins (lysozyme, β-lactoglobulin, α-synuclein A53T). 7519 and 7515 dyes can be used for fluorometric detection of fibrillar insulin at concentrations of approximately 1.5–120 microg/ml. For meso-ethyl substituted dye 7514 the ability to form H- and J-aggregates upon interaction with insulin fibrils was suggested. The model of the H- and J-aggregate packing in the protein fibrillar structure has been proposed.     

Spectroscopic properties of DNA-acridine orange biopolymer films

Using electronic absorption, luminescence, and vibrational spectroscopy, we study the properties of DNA-acridine orange biopolymer films. The conformational structure of DNA molecules in the film is examined at different levels of the relative humidity, and the state of dye molecules incorporated into the structure of the biopolymer is estimated. These structures were formed according to the “guest-host” principle and the dye molecule acts as the “guest” and the “host” is the intercalative DNA binding sites. In such system dye molecules demonstrate high luminescence capability and are the uniform optical centers and their fluorescence spectrum doesn’t depend on excitation wavelength.     

Fluorescence Study of the Membrane Effects of Aggregated Lysozyme

The last decade has seen unprecedented upsurge of interest in the structural and toxic properties of particular type of protein aggregates, amyloid fibrils, associated with a number of pathological states. In the present study fluorescence spectroscopy technique has been employed to gain further insight into the membrane-related mechanisms of amyloid toxicity. To this end, erythrocyte model system composed of liposomes and hemoglobin was subjected to the action of oligomeric and fibrillar lysozyme. Acrylamide quenching of lysozyme fluorescence showed that solvent accessibility of Trp₆₂ and Trp₁₀₈ increases upon the protein fibrillization. Resonance energy transfer measurements suggested the possibility of direct complexation between hemoglobin and aggregated lysozyme. Using the novel squaraine dye SQ-1 it was demonstrated that aggregated lysozyme is capable of inhibiting lipid peroxidation processes. Fluorescent probes pyrene, Prodan and diphenylhexatriene were employed to characterize the membrane-modifying properties of hemoglobin and lysozyme. Both oligomeric and fibrillar forms of lysozyme were found to exert condensing influence on lipid bilayer structure, with the membrane effects of fibrils being less amenable to modulation by hemoglobin.     

Electric birefringence study of an amyloid fibril system: The short end of the length distribution

In this article, a system of amyloid fibrils, based on the protein β-lactoglobulin, is studied by transient electric birefringence. Single pulses of an electric field were applied to the solution, and the initial rise and subsequent decay of birefringence analysed. The decay takes place on a range of relaxation times, and therefore contains information about the length distribution of fibrils in the system. The information can be extracted using theories of the electric polarisability of polyelectrolyte rods, since the fibrils are an example of these. Despite the long-standing complications of such theories, useful quantitative information about the system can still be obtained. Using the Fixman model of polyelectrolyte polarisability, we obtain a measurement of the short end of the length distribution which shows the fibril concentration as a function of length rising linearly from 0.02-2 μm. The short end of the length distribution was unobtainable in our previous study using rheo-optics (S.S. Rogers et al., Macromolecules 38, 2948 (2005)), but reasonable agreement between the two techniques shows they are complementary.     

A Fast Algorithm for Wave Propagation from a Plane or a Cylindrical Surface

The newly developed Taylor-Interpolation-FFT (TI-FFT) algorithm dramatically increases the computational speeds for millimeter wave propagation from a planar (cylindrical) surface onto a “quasi-planar” (“quasi-cylindrical”) surface. Two different scenarios are considered in this article: the planar TI-FFT is for the computation of the wave propagation from a plane onto a “quasi-planar” surface and the cylindrical TI-FFT is for the computation of wave propagation from a cylindrical surface onto a “quasi-cylindrical” surface. Due to the use of the FFT, the TI-FFT algorithm has a computational complexity of O(N ² log₂  N ²) for an N × N computational grid, instead of N ⁴ for the direct integration method. The TI-FFT algorithm has a low sampling rate according to the Nyquist sampling theorem. The algorithm has accuracy down to −80 dB and it works particularly well for narrow-band fields and “quasi-planar” (“quasi-cylindrical”) surfaces.     

A Spectroscopic Study of 2-[4′-(Dimethylamino)phenyl]-benzothiazole Binding to Insulin Amyloid Fibrils

The spectroscopic properties of 2-[4′-(dimethylamino)phenyl]-benzothiazole (BTA-2) in solution and in the presence of amyloid fibrils were investigated using absorption and fluorescence spectroscopy. Solution studies show that BTA-2 forms micelles in aqueous solutions, but that the dye can be solvated upon the addition of acetonitrile (CH₃CN). BTA-2 binds to amyloid fibrils in solution leading to a characteristic blue-shift in the emission spectrum and an increase in fluorescence intensity. However, in solutions with increasing CH₃CN concentration, there was a marked decrease in binding of the BTA-2 to fibrils. Studies demonstrating the effect of BTA-2 concentration on binding were performed. A comparison with the standard amyloid fluorescent marker, thioflavin T (ThT), showed that BTA-2 is more fluorescent, making it an excellent dye to label amyloid samples.     

Influence of one-dimensional “defects” on the new-phase nucleation dynamics near the first-order transition in magnets

The evolution of a new-phase nucleus near the first-order spin-reorientation phase transition in magnets has been investigated. A strong influence of one-dimensional magnetic anisotropy “defects” on the nucleation dynamics has been shown. The conditions of the localization of the new-phase nucleus in the region of the magnetic anisotropy “defect” have been determined.     

Energy migration in DNA evaluated by the sensitized fluorescence of an intercalation dye

An equation is obtained to calculated the efficiency of energy migration along a biopolymer with transfer of excitation to a fluorescence dye using excitation and absorption spectra. With the aid of bromoethydium intercalating into DNA-phage it is shown that in a solution at room temperature energy is transferred to it only from one or two neighboring nucleotides. The low efficiency of energy migration in relation to DNA under these conditions is explained by the low probability of each event of “cold” and “hot” migration. Institute of Cell Biophysics of the Russian Academy of Sciences, Pushchino, 142292, Moscow Region, Russia. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 65, No. 5, pp. 794–798, September–October, 1998.     

Medium effects in the production and π0γ decay of ω-mesons in pA collisions in the GeV region

The ω resonance production and its π⁰γ decay in pA reactions close to threshold is considered within the Intranuclear Cascade (INC) model. The π⁰γ invariant-mass distribution shows two components which correspond to the ω decay “inside” and “outside” the nucleus, respectively. The “inside” component is distorted by medium effects, which introduce a mass shift as well as collisional broadening for the ω-meson and its decaying pion. The relative contribution of the “inside” component is analyzed in detail for different kinematical conditions and nuclear targets. It is demonstrated that a measurement of the correlation in azimuthal angle between the π⁰ and γ momenta allows to separate events related to the “inside”ω decay from different sources of background when uncorrelated π⁰'s and γ's are produced. Received: 2 April 2001 / Accepted: 5 June 2001     

Localized excitation effects in (E,E)-2,5-<Emphasis Type="Italic">bis</Emphasis>[2-(4-N,N-dipropylaminophenyl) ethylenyl]-3,6-dimethylpyrazine and generation of its different forms in solution

Dipole moments of (E,E)-2,5-bis[2-(4-N,N-dipropylaminophenyl)ethylenyl]-3,6-dimethylpyrazine (VS365) quadrupolar dye in 1,4-dioxane and cyclohexane in addition to its instantaneous fluorescence spectra and fluorescence lifetimes in dichloromethane (pure and with added trifluoroacetic acid) were measured. Quantumchemical calculations show that the π − π conjugated system is localized in the flattest part of the molecule due to disorder in the ground-state geometry of the dye molecule and that this part is the one responsible for light absorption. The effect of localized excitation of the dye causes a considerable change in its dipole moment Δ^aμ. Various fluorescent forms of the dye that feature considerably spaced emission spectra and different fluorescence lifetimes originate by adding trifluoroacetic acid to the solution of oligophenylenevinylene in dichloromethane.     

Biophysical Studies on the Interactions of a Classic Mitochondrial Uncoupler with Bovine Serum Albumin by Spectroscopic,Isothermal Titration Calorimetric and Molecular Modeling Methods

The interaction between a classic uncoupler (2,4-dinitrophenol, DNP) and bovine serum albumin (BSA) was investigated by fluorescence spectroscopy under the physiological conditions. The fluorescence quenching constants were calculated by the Stern-Volmer equation, and based upon the temperature dependence of quenching constants, it was proved that DNP caused a static quenching of the intrinsic fluorescence of BSA. Owing to the static quenching mechanism, different associative binding constants at various temperatures were determined and thus the thermodynamic parameters, namely enthalpy (ΔH = −21.12 kJ mol⁻¹) and entropy changes (ΔS = 23.51 J mol⁻¹ K⁻¹) could be calculated based on the binding constants. Moreover, the enthalpy and entropy changes are consistent with the “Enthalpy-Entropy Compensation” equation obtained from our previous work. The negative enthalpy and positive entropy indicated that the electrostatic interactions played a major role in DNP-BSA binding process. Site marker competitive displacement experiments were carried out by using fluorescence and isothermal titration calorimetry (ITC) methods. These results showed that DNP bound with high affinity to Sudlow’s site I (subdomain IIA) of BSA. The distance (r = 3.78 nm) between donor (BSA) and acceptor (DNP) was obtained according to the mechanism of fluorescence resonance energy transfer (FRET). Furthermore, the results of synchronous fluorescence and circular dichroism (CD) spectroscopic studies indicated that the microenvironment and the secondary conformation of BSA were altered. The above results were supported by theoretical molecular modeling methods.     

Different Conformation of Thiol Protease Inhibitor During Amyloid Formation: Inhibition by Curcumin and Quercetin

Cystatins are thiol proteinase inhibitors ubiquitously present in mammalian body and serve various important physiological functions. In the present study, we examined the effects of acid denaturation on newly identified thiol protease inhibitors from the lungs of Capra hircus (Goat) with a focus on protein conformational changes and amyloid fibril formation. Acid denaturation as studied by CD (Circular Dichroism) and fluorescence spectroscopy showed that purified inhibitor named GLC (Goat Lung Cystatin) populates three partly unfolded species, a native like state at pH?3.0, a partly unfolded intermediate at pH2.0, and unstructured unfolded state at pH?1.0, from each of which amyloid like fibrils grow as assessed by thioflavin T (ThT) spectroscopy. The result showed, native like structure formed at pH?3.0 is more responsive towards amyloid formation when compare to other conformation of proteins. Morphology of the protein species incubated for amyloid process was observed using transmission electron microscopy (TEM). Moreover, anti-fibrillogenic effects of curcumin and quercetin were analysed using ThT binding assay. Curcumin and quercetin produced a concentration dependent decline inThT fluorescence suggesting deaggregation of the fibrils. When added prior to amyloid fibril initiation 50 μM curcumin inhibited amyloid aggregation. However, more quercetin is needed to prevent the same extent of fibrillation. Implications for therapeutics in view of polyphenols as essential nutrients are suggested in lung diseases.     

Half-life estimation under indefinite “mother-daughter” relation

An algorithm has been proposed to build an estimate of the half-life of a “daughter” nucleus in case, when it is unknown, which nucleus is its “mother” (“indefinite start time”). For a decay of the “mother” at an instant t we can determine P—a probability of such a decay, if we assume that each “mother”, which has been decayed before t has equal chances to be “mother” of this “daughter”:

Optical and spectroscopic characterization of Nd-doped aluminosilicate fiber preforms made by the MCVD method using chelate delivery

4 F_3/2 level of Nd³⁺ in aluminosilicate optical fiber preforms after 807-nm excitation are reported in the temperature range from 15 °C to 500 °C for Nd₂O₃ concentrations in the range from 0.2 to [%wt]0.9 and Al₂O₃ concentrations of between [%wt]3.8 and [%wt]8. At room temperature it is observed that for concentrations of less than [%wt]0.6 Nd₂O₃ the fluorescence decay is approximately exponential; for the higher Nd₂O₃ concentrations the decay is increasingly bi-exponential. We have identified a “slow” (≈500 μs) and a “fast” (≈100 μs) decay time; the slow decay is attributed to isolated ions and the faster decay to energy transfer between homogeneously dispersed ions. The measured 1/e folding fluorescence decay time τ decreases slowly at a constant rate of -0.08±0.007 μs/K for [%wt]0.9 Nd₂O₃. It is shown that increasing the Al₂O₃ concentration tends to suppress this temperature quenching effect. A radiative decay time of 640 μs is obtained by Judd–Ofelt analysis. Received: 24 October 1997/Revised version: 2 February 1998     

Spectroscopic Investigations on the H-Type Aggregation of Coumarin 153 Dye Molecules: Role of Au Nanoparticles and γ- Cyclodextrin

Here, we study the formation of H-type aggregation of coumarin 153 (C153) dye molecule in presence of Au nanoparticles and the removal of dye aggregation in presence of γ- cyclodextrin (CD) due to confinement of dye molecules inside the nanocavity of γ- cyclodextrin (CD) using steady state and time resolved spectroscopy. Blue shifting of absorption band, photoluminescence (PL) band and the enhancement of decay time of C153 dye confirm the formation of H-aggregation. It is found that the concentrations of γ-CD and Au nanoparticles play an important role on H-type aggregation of dye. The rotational relaxation time of free C153 is 0.113 ns and the average relaxation time of C153 dye are 0.275 ns and 0.425 ns for 2 mM and 5 mM γ-CD confined systems, respectively, indicating the anisotropy increases due to confinement of dye. An associated type anisotropy decay of C153 dye is found at 20 mM concentration of CD may be due to formation of nanotubular aggregates of γ-CD.     

Search for efficient,near UV lasing dyes. III. Monocyclic and miscellaneous dyes

A potential new class of “monocyclic” lasing dyes is discussed and four families out of nine within the class are examined. The 2,6-diaminopyridines in acid are found to lase under flashlamp excitation but show low slope efficiencies and lower stabilities than the “bicyclic” dyes described previously. Other, miscellaneous dyes show oxygen quenching of laser action rather than enhancement and the best of these produces short pulses under long pulse excitation. This study was supported under contract SANL 284 from the Lawrence Livermore Laboratory.