Investigating the effects of erythrosine B on amyloid fibril formation derived from lysozyme.

Formation of amyloid fibrils has been associated with at least 30 different protein aggregation diseases. The 129-residue polypeptide hen lysozyme, which is structurally homologous to human lysozyme, has been demonstrated to exhibit amyloid fibril-forming propensity in vitro. This study is aimed at exploring the influence of erythrosine B on the in vitro amyloid fibril formation of hen lysozyme at pH 2.0 and 55°C using ThT binding assay, transmission electron microscopy, far-UV circular dichroism absorption spectroscopy, 1-anilinonaphthalene-8-sulfonic acid fluorescence spectroscopy, and synchronous fluorescence study. We found that lysozyme fibrillogenesis was dose-dependently suppressed by erythrosine B. In addition, our far-UV CD and ANS fluorescence data showed that, as compared with the untreated lysozyme control, the α-to-ß transition and exposure of hydrophobic clusters in lysozyme were reduced upon treatment with erythrosine B. Moreover, it could be inferred that the binding of erythrosine B occurred in the vicinity of the tryptophan residues. Finally, molecular docking and molecular dynamics simulations were further employed to gain some insights into the possible binding site(s) and interactions between lysozyme and erythrosine B. We believe the results obtained here may contribute to the development of potential strategies/approaches for the suppression of amyloid fibrillogenesis, which is implicated in amyloid pathology.

Delayed luminescence of erythrosine in biological tissue and photodynamic therapy dosimetry.

The features of delayed fluorescence (DF) and phosphorescence in erythrosine stained healthy and cancer-diseased mammary gland tissues of BYRB-line mice were investigated in vitro. Thermoactivated delayed fluorescence (TDF), triplet-triplet annihilation (TTA) and originated from singlet-triplet annihilation (STA) delayed fluorescence are investigated as competing channels of radiative relaxation of the triplet states of erythrosine. The dominant role of diffusive-mobile molecular oxygen in deactivation of triplet-excited long-live states of the dye molecules in cells is determined. The previously not described phenomenon of light quenching of DF under pulsed laser irradiation (light quenching of DF, LQDF) in stained tissue was revealed. LQDF is increased if the energy of excited pulses is rise and their sequence period is decreased. The DF depletion disappears if time interval between pulses in the series is >5s. This phenomenon is due two process competition: fast consumption of singlet oxygen by oxidation of cell organelles right after laser pulse excitation and slow diffusive recovery of oxygen concentration in the pause between the pulses. Statistically valid distinction between DF characterization as well as LQDF erythrosine extent in healthy and pathological tissues was established. The use of this phenomenon will greatly simplify the determination of radiation "dose" in photodynamic therapy (PDT) directly during the treatment session.

Spectroscopic investigations on the interactions of AgTiO2 nanoparticles with lysozyme and its influence on the binding of lysozyme with drug molecule.

Binding of lysozyme with AgTiO2 nanoparticles was analyzed by using absorption, fluorescence, time resolved and synchronous fluorescence measurements. In the presence of AgTiO2 nanoparticles, the fluorescence intensity of lysozyme was decreased. Static type of binding was confirmed through lifetime and ground state absorption measurements. From the fluorescence quenching data, the binding constant and the number of binding sites were found to be 1.5×10(4)M(-1) and 1.03, respectively. From the synchronous fluorescence spectroscopic measurements, tryptophan residue in lysozyme was found to have interaction with the nanoparticles. Further, the influence of AgTiO2 nanoparticles on the binding strength of lysozyme with a drug molecule was analyzed through fluorescence quenching methods. The presence of nanoparticles decreases the binding capability of drug with protein. Overall, the observed results will provide basic insights on the utilization of nanoparticles in drug delivery applications.

Real-time histological imaging of kidneys stained with food dyes using multiphoton microscopy.

We have developed a real-time imaging technique for diagnosis of kidney diseases which is composed of two steps, staining renal cells safely with food dyes and optical sectioning of living renal tissue to obtain histological images by multiphoton microscopy (MPM). Here, we demonstrated that the MPM imaging with food dyes, including erythrosine and indigo carmine, could be used as fluorescent agents to visualize renal functions and structures such as glomerular bloodstreams, glomerular filtration, and morphology of glomeruli and renal tubules. We also showed that the kidneys of IgA nephropathy model-mice stained with the food dyes presented histopathological characteristics different from those observed in normal kidneys. The use of the food dyes enhances the quality of tissue images obtained by MPM and offers the potential to contribute to a clinical real-time diagnosis of kidney diseases.

Identification properties of a recombinant class I hydrophobin rHGFI.

Hydrophobins fulfill various functions in fungal growth and morphology. These proteins can self-assemble at hydrophilic/hydrophobic interfaces and form amphipathic membranes. Based on their physical properties and hydropathy patterns, hydrophobins are divided into two classes (I and II). In order to identify the recombinant class I hydrophobin rHGFI, the different properties between rHGFI and the typical class II hydrophobin rHFBI were investigated. In contrast to rHGFI, no rodlet structure was observed on rHFBI coated mica surface, and the membranes formed on siliconized glass surfaces by rHFBI were not robust enough to resist treatment with 60% ethanol and 2% hot SDS. In contrast, the membranes formed by rHGFI on siliconized glass surfaces were so strong that could resist hot detergent and alcohol solution washing. Moreover, self-assembly of rHFBI at the water-air interface was not accompanied by a change in secondary structure. Meanwhile, ß-sheet structures dramatically increased after rHGFI self-assembled at water-air interface, which could cause the fluorescence intensity of Thioflavin T increased and Congo Red and CD absorption spectra shift. Water-insoluble erythrosin B dispersion prepared with rHGFI and rHFBI were both stable for more than one month, which indicated that the interaction between erythrosin B and rHGFI/rHFBI was strong. This might promote rHGFI and rHFBI to be considered as potential dispersing agents to stabilize water-insoluble erythrosin B.

The influence of dead time related distortions on live cell fluorescence lifetime imaging (FLIM) experiments.

Recent developments in the field of fluorescence lifetime imaging microscopy (FLIM) techniques allow the use of high repetition rate light sources in live cell experiments. For light sources with a repetition rate of 20-100 MHz, the time-correlated single photon counting (TCSPC) FLIM systems suffer serious dead time related distortions, known as "inter-pulse pile-up". The objective of this paper is to present a new method to quantify the level of signal distortion in TCSPC FLIM experiments, in order to determine the most efficient laser repetition rate for different FLT ranges. Optimization of the F -value, which is the relation between the relative standard deviation (RSD) in the measured FLT to the RSD in the measured fluorescence intensity (FI), allows quantification of the level of FI signal distortion, as well as determination of the correct FLT of the measurement. It is shown that by using a very high repetition rate (80 MHz) for samples characterized by high real FLT's (4-5 ns), virtual short FLT components are added to the FLT histogram while a F -value that is higher than 1 is obtained. For samples characterized with short real FLT's, virtual long FLT components are added to the FLT histogram with the lower repetition rate (20-50 MHz), while by using a higher repetition rate (80 MHz) the "inter-pulse pile-up" is eliminated as the F -value is close to 1.

Mast cells in common wolffish Anarhichas lupus L.: ontogeny, distribution and association with lymphatic vessels.

The morphology, ontogeny and tissue distribution of mast cells were studied in common wolffish(Anarhichas lupus L.) at the larval, juvenile and adult life stages using light and electron-microscopy and immunohistochemistry. Fish were sampled at 1 day, 1, 2, 3, 4, 8 and 12 weeks post-hatching in addition to 6 and 9 months and 2 years and older. From 8 weeks post-hatching, mast cells in common wolffish mainly appeared as oval or rounded cells 8-15 mm in diameter with an eccentrically placed, ovoid nucleus and filled with cytoplasmic granules up to 1.2 mm in diameter. Granules were refractile and eosinophilic to slightly basophilic in H&E and stained bright red with Martius-scarlet-blue and purple with pinacyanol erythrosinate in formalin-fixed tissues. Mast cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 1 day to 4 weeks post-hatching, immature mast cell containing only a few irregularly sized cytoplasmic granules were observed by light and electron-microscopy in loose connective tissue of cranial areas. From 1 day post-hatching, these cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 12 weeks post-hatching, mast cells showed a primarily perivascular distribution and were particularly closely associated with lymphatic vessels and sinuses. Mast cells were mainly located at the peripheral border of the adventitia of arteries and veins, while they were in intimate contact with the endothelium of the lymphatic vessels. Numerous mast cells were observed in the intestine. A stratum compactum, as described in salmonids, was not observed in wolffish intestine,nor were mast cells confined to a separate layer, a stratum granulosum. Lymphatic vessels consisting of endothelium, intimal connective tissue and a poorly developed basal lamina were observed in the intestine. Scanning electron microscopy was used to compare the structure and localization of intestinal mast cells of common wolffish and rainbow trout. Scanning electron microscopy also revealed endothelial surface features and confirmed the existence of three distinctly different types of vessels in the wolffish intestine. Rainbow trout mast cell granules appeared as intact globular structures while empty vacuoles were observed in common wolffish. Mast cells were closely associated with lymphatic vessels in common wolffish, but not in rainbow trout.

In vitro studies on erythrosine-based photodynamic therapy of malignant and pre-malignant oral epithelial cells.

Photodynamic Therapy (PDT) involves the administration of a tumor localizing photosensitizing agent, which upon activation with light of an appropriate wavelength leads to the destruction of the tumor cells. The aim of the present study was to determine the efficacy of erythrosine as a photosensitizer for the PDT of oral malignancies. The drug uptake kinetics of erythrosine in malignant (H357) and pre-malignant (DOK) oral epithelial cells and their susceptibility to erythrosine-based PDT was studied along with the determination of the subcellular localization of erythrosine. This was followed by initial investigations into the mechanism of cell killing induced following PDT involving both high and low concentrations of erythrosine. The results showed that at 37 °C the uptake of erythrosine by both DOK and H357 cells increased in an erythrosine dose dependent manner. However, the percentage of cell killing observed following PDT differed between the 2 cell lines; a maximum of ~80% of DOK cell killing was achieved as compared to ~60% killing for H357 cells. Both the DOK and H357 cell types exhibited predominantly mitochondrial accumulation of erythrosine, but the mitochondrial trans-membrane potential (ΔΨ(m)) studies showed that the H357 cells were far more resistant to the changes in ΔΨ(m) when compared to the DOK cells and this might be a factor in the apparent relative resistance of the H357 cells to PDT. Finally, cell death morphology and caspase activity analysis studies demonstrated the occurrence of extensive necrosis with high dose PDT in DOK cells, whereas apoptosis was observed at lower doses of PDT for both cell lines. For H357 cells, high dose PDT produced both apoptotic as well as necrotic responses. This is the first instance of erythrosine-based PDT's usage for cancer cell killing.

Actin filament dynamics in the actomyosin VI complex is regulated allosterically by calcium-calmodulin light chain.

The contractile and enzymatic activities of myosin VI are regulated by calcium binding to associated calmodulin (CaM) light chains. We have used transient phosphorescence anisotropy to monitor the microsecond rotational dynamics of erythrosin-iodoacetamide-labeled actin with strongly bound myosin VI (MVI) and to evaluate the effect of MVI-bound CaM light chain on actin filament dynamics. MVI binding lowers the amplitude but accelerates actin filament microsecond dynamics in a Ca(2+)- and CaM-dependent manner, as indicated from an increase in the final anisotropy and a decrease in the correlation time of transient phosphorescence anisotropy decays. MVI with bound apo-CaM or Ca(2+)-CaM weakly affects actin filament microsecond dynamics, relative to other myosins (e.g., muscle myosin II and myosin Va). CaM dissociation from bound MVI damps filament rotational dynamics (i.e., increases the torsional rigidity), such that the perturbation is comparable to that induced by other characterized myosins. Analysis of individual actin filament shape fluctuations imaged by fluorescence microscopy reveals a correlated effect on filament bending mechanics. These data support a model in which Ca(2+)-dependent CaM binding to the IQ domain of MVI is linked to an allosteric reorganization of the actin binding site(s), which alters the structural dynamics and the mechanical rigidity of actin filaments. Such modulation of filament dynamics may contribute to the Ca(2)(+)- and CaM-dependent regulation of myosin VI motility and ATP utilization.

The food colorant erythrosine is a promiscuous protein-protein interaction inhibitor.

Following our observation that erythrosine B (FD&C Red No. 3) is a relatively potent inhibitor of the TNF-R-TNFα and CD40-CD154 protein-protein interactions, we investigated whether this inhibitory activity extends to any other protein-protein interactions (PPI) as well as whether any other approved food colors possess such inhibitory activity. We found erythrosine, a poly-iodinated xanthene dye, to be a non-specific promiscuous inhibitor of a number of PPIs within the tumor necrosis factor superfamily (TNF-R-TNFα, CD40-CD154, BAFF-R-BAFF, RANK-RANKL, OX40-OX40L, 4-1BB-4-1BBL) as well as outside of it (EGF-R-EGF) with a remarkably consistent median inhibitory concentration (IC(50)) in the 2-20 µM (approximately 2-20mg/L) range. In agreement with this, erythrosine also showed cellular effects including clear cytotoxic effects around this concentration range (IC50≈50 µM). Among the seven FDA-approved food colorants, only erythrosine showed consistent PPI inhibitory activity in the sub-100 µM range, which might also explain (at least partially) why it also has the lowest approved acceptable daily intake (ADI) (0.1 mg/kg body weight/day). Among a number of xanthene structural analogs of erythrosine tested for activity, rose Bengal, a food colorant approved in Japan, showed similar, maybe even more pronounced, promiscuous inhibitory activity, whereas fluorescein was inactive and gallein, phloxine, and eosin were somewhat active in some of the assays.

Characterization of erythrosine B binding to bovine serum albumin and bilirubin displacement.

The interaction of crythrosine B (ErB), a commonly used dye for coloring foods and drinks, with bovine scrum albumin (BSA) was investigated both in the absence and presence of bilirubin (BR) using absorption and absorption difference spectroscopy. ErB binding to BSA was reflected from a significant red shift of 11 nm in the absorption maximum of ErB (527 nm) with the change in absorbance at lamdamax. Analysis of absorption difference spectroscopic titration results of BSA with increasing concentrations of ErB3 using Benesi-Hildebrand equation gave the association constant, K as 6.9 x 10(4) M(-1). BR displacing action of ErB was revealed by a significant blue shift in the absorption maximum, accompanied by a decrease in absorbance difference at lamdamax in the difference spectrum of BR-BSA complex upon addition of increasing concentrations of ErB. This was further substantiated by fluorescence spectroscopy, as addition of increasing concentrations of ErB to BR-BSA complex caused a significant decrease in fluoresccnce at 510 nm. The results suggest that ErB binds to a site in the vicinity of BR binding site on BSA. Therefore, intake of ErB may increase the risk of hyperbilirubinemia in the healthy subjects.

Resonance Rayleigh scattering spectra, non-linear scattering spectra of tetracaine hydrochloride-erythrosin system and its analytical application.

The interaction between erythrosine (ET) and tetracaine hydrochloride (TA) was studied by resonance Rayleigh scattering (RRS), frequency doubling scattering (FDS) and second-order scattering (SOS) combining with absorption spectrum. In a weak acidic medium of Britton-Robinson (BR) buffer solution of pH 4.5, erythrosine reacted with tetracaine hydrochloride to form 1:1 ion-association complex. As a result, the new spectra of RRS, SOS and FDS appeared and their intensities enhanced greatly. The maximum peaks of RRS, SOS and FDS were at 342 nm, 680 nm and 380 nm, respectively. The intensities of the three scattering were directly proportional to the concentration of TA in the range of 0.008-4.2 microg mL(-1) for RRS, 0.027-4.2 microg mL(-1) for SOS and 0.041-4.2 microg mL(-1) for FDS. The methods had very high sensitivities and good selectivities, and the detection limits were 0.003 microg mL(-1) for RRS, 0.008 microg mL(-1) for SOS and 0.012 microg mL(-1) for FDS, respectively. Therefore, a new method was developed to determinate trace amounts of TA. The recovery for the determination of TA in blood serum and urine samples was between 97.0% and 103.8%. In this study, mean polarizability was calculated by AM1 quantum chemistry method. In addition, the reasons for the enhancement of scattering spectra and the energy transfer between absorption, fluorescence and RRS were discussed.

Dye induced quenching of firefly luciferase-luciferin bioluminescence.

The quenching of firefly bioluminescence (BL) in presence of xanthene dyes and tetratolylporphyrin was investigated. The BL intensity was quenched with an altered decay pattern in presence of xanthene dyes and tetratolylporphyrin. The electronic absorption spectra indicate that there is no significant interaction occurring between the dyes and the BL components in the ground state. The BL quenching decay rate and fluorescence quenching studies of luciferin by the dyes suggest an energy transfer through an exciplex, involving oxyluciferin, in the excited state and the dyes, in the ground state. The bimolecular quenching rate constant (K(q)) values obtained from fluorescence studies varied between 7.7 x 10(12) and 19.8 x 10(12)M(-1)s(-1). The magnitude of the bimolecular quenching rate constants confirmed the complex formation between dye and excited oxyluciferin. The exciplex subsequently undergoes a non-radiative decay to the ground state via a combination of heavy atom induced and Förster-type energy transfer. The decay rate constants in presence and in absence of dyes vary between 7.47 x 10(-4) and 7.6 x 10(-2)s(-1). In the presence of dyes the effective decay rate constants (k(eff)) increased while the lifetime of light emitting species decreased. The kinetic studies in presence of singlet oxygen scavengers, like beta-carotene and NaN(3), prove that there is no significant quenching of the firefly BL due to the formation of singlet oxygen.

Sonodynamic effect of erythrosin B on sarcoma 180 cells in vitro.

The ultrasonically induced cytotoxic effect of erythrosin B (EB) on isolated sarcoma 180 cells was investigated. The tumor cells were suspended in an air-saturated phosphate buffered saline and exposed to ultrasound at 1.93 MHz in a standing-wave mode for up to 60 s in the presence and absence of EB. The rate of cell damage induction by ultrasound was enhanced by 4-5 times with 160-microM EB, while no cell damage was observed with EB alone. This enhancement was significantly inhibited by histidine. Sonochemical generation of active oxygen species in the presence of EB, measured by ESR spectroscopy, was also inhibited by histidine. These results indicate the involvement of a sonochemical mechanism.

The mast cell function-associated antigen and its interactions with the type I Fcepsilon receptor.

Rat mucosal-type mast cells of the RBL-2H3 line express a glycoprotein termed the MAst cell Function-associated Antigen (MAFA). When MAFA is clustered by its specific monoclonal antibody G63, secretion normally triggered by aggregating these cells' type I Fcepsilon receptor (FcepsilonRI) is substantially inhibited. The nature of MAFA-FcepsilonRI interactions giving rise to this inhibition remains unclear. Rotational diffusion of a membrane protein is a sensitive probe of its involvement in intermolecular interactions. We have therefore studied by time-resolved phosphorescence anisotropy the rotational behavior of both MAFA and FcepsilonRI as ligated by various reagents involved in FcepsilonRI-induced degranulation and MAFA-mediated inhibition thereof. From 4 to 37 degrees C, the rotational correlation times (mean +/- SD) of FcepsilonRI-bound, erythrosin-conjugated IgE resemble those observed for MAFA-bound, erythrosin-conjugated G63 Fab, 82 +/- 17 and 79 +/- 31 micros at 4 degrees C, respectively. Clustering the FcepsilonRI-IgE complex by antigen or by anti-IgE increases the phosphorescence anisotropy of G63 Fab and slows its rotational relaxation. Lateral diffusion of G63 Fab is also slowed by antigen clustering of the receptor. Taken together, these results indicate that unperturbed MAFA associates with clustered FcepsilonRI. They are also consistent with its interaction with the isolated receptor.

Properties of small molecules affecting insulin receptor function.

Small molecules with insulin mimetic effects and oral availability are of interest for potential substitution of insulin injections in the treatment of diabetes. We have searched databases for compounds capable of mimicking one epitope of the insulin molecule known to be involved in binding to the insulin receptor (IR). This approach identifies thymolphthalein, which is an apparent weak agonist that displaces insulin from its receptor, stimulates auto- and substrate phosphorylation of IR, and potentiates lipogenesis in adipocytes in the presence of submaximal concentrations of insulin. The various effects are observed in the 10(-5)-10(-3) M range of ligand concentration and result in partial insulin activity. Furthermore, analogues of the related phenol red and fluorescein molecules fully displace insulin from the IR ectodomain, however, without insulin agonistic effects. The interactions are further characterized by NMR, UV-vis, and fluorescence spectroscopies. It is shown that both fluorescence and UV-vis changes in the ligand spectra induced by IR fragments occur with Kd values similar to those obtained in the displacement assay. Nevertheless, insulin itself cannot completely abolish binding of the small molecules. Determination of the binding stoichiometry reveals multiple binding sites for ligands of which one overlaps with the insulin binding site on the receptor.

Binding of dystrophin's tandem calponin homology domain to F-actin is modulated by actin's structure.

Dystrophin has been shown to be associated in cells with actin bundles. Dys-246, an N-terminal recombinant protein encoding the first 246 residues of dystrophin, includes two calponin-homology (CH) domains, and is similar to a large class of F-actin cross-linking proteins including alpha-actinin, fimbrin, and spectrin. It has been shown that expression or microinjection of amino-terminal fragments of dystrophin or the closely related utrophin resulted in the localization of these protein domains to actin bundles. However, in vitro studies have failed to detect any bundling of actin by either intact dystrophin or Dys-246. We show here that the structure of F-actin can be modulated so that there are two modes of Dys-246 binding, from bundling actin filaments to only binding to single filaments. The changes in F-actin structure that allow Dys-246 to bundle filaments are induced by covalent modification of Cys-374, proteolytic cleavage of F-actin's C-terminus, mutation of yeast actin's N-terminus, and different buffers. The present results suggest that F-actin's structural state can have a large influence on the nature of actin's interaction with other proteins, and these different states need to be considered when conducting in vitro assays.

Differences in structural dynamics of muscle and yeast actin accompany differences in functional interactions with myosin.

We have used spectroscopic probes ErIA and IAEDANS attached to Cys374 to compare the structural dynamics of yeast actin filaments with that of muscle actin, to understand the structural basis of the less productive interaction of yeast actin with myosin. Time-resolved phosphorescence anisotropy (TPA) of ErIA and steady-state fluorescence of IAEDANS were measured. TPA indicated more rapid rotational motion and more restricted angular amplitude in yeast actin. The fluorescence spectrum was less intense and more red-shifted in yeast actin, suggesting more exposure of the probe to solvent. These results indicate that the two actins differ substantially in the conformational dynamics of the C-terminal region. Binding of myosin S1 induced significantly different spectroscopic changes in TPA and fluorescence of muscle and yeast actin. As a result, the spectroscopic differences between the two actins were decreased by the addition of S1. These results suggest that yeast actin is less effective at activating myosin because of larger changes required in the structure of actin upon strong myosin binding. These results provide insight into the relationship between actomyosin dynamics and function, and they provide a useful framework for structure-function analysis of mutant yeast actin.