Effect of Spacer Length and Solvent on the Concentration-Driven Aggregation of Cationic Hydrogen-Bonding Donor Polythiophenes.

Aggregation of cationic isothiouronium polythiophenes with alkoxy-spacers of different lengths at the 3-position of the thiophene ring was studied in solvents of different polarities. Hydrogen-bonding capacity was assessed by steady-state absorption and fluorescence spectroscopy, whereas the aggregation in aqueous solutions was studied by electron paramagnetic resonance spectroscopy, using paramagnetic probes of different polarities. The two polymers displayed similar features in respect to conformation, effect of cosolvents on aggregation, unstructured absorption-fluorescence spectra, Stokes shifts when aggregated, solvatochromic effect, and self-quenching concentration. However, these polymers also showed different specific interactions with water, Stokes shifts in water, effect of the solvent on the extent of dominant state of the S1 level, and also different inner cavities and hydrophobic-hydrophilic surface area in aqueous solution aggregates. Water maximized the difference between the polymers concerning the effect of specific increases in concentration, whereas the presence of 1,4-dioxane generated almost identical effects on both polymers.

A comparison between interactions of triglyceride oil and mineral oil with proteins and their ability to reduce cleanser surfactant-induced irritation.

OBJECTIVE: Skin irritation in personal cleansing has been correlated with surfactant binding with stratum corneum proteins. Polar and non-polar oils are increasingly being used in cleansing formulations which contain high (10-15%) level of anionic and non-ionic surfactants. However, the effects of oils in modulating skin damage from a cleansing product have not been studied in any detail. The objectives of this study are to determine whether low-viscosity polar and non-polar oils differ in their ability to reduce surfactant-induced skin irritation and, if so, how it might be related to their interactions with proteins. METHODS: Surfactant-induced skin irritation was measured by a 14-day in vivo cumulative patch irritation test. The methodology was similar to the well-known soap chamber test. Surfactant interactions with the water-soluble protein, bovine serum albumin (BSA), in the presence of oils were measured by conductometric titration. The effects of low-viscosity polar and non-polar oils on stratum corneum protein dynamics in the sulfhydryl group region were studied by electron paramagnetic resonance (EPR) using the covalently bound spin-label 3-maleimido-proxyl (5-MSL). EPR measurements were performed with stratum corneums obtained from discarded skins of 3- to 4-week-old female pigs. Simulation of the complex spectra provided insights on the environment and mobility of the protein-bound spin label. RESULTS: Addition of 1% polar sunflower seed oil (viscosity 42 centipoise) reduced in vivo irritation of 1% sodium lauryl ether sulphate with two ethoxylate/cocamidopropyl betaine (SLES/CAPB) by 20%, whereas 1% non-polar mineral oil (viscosity 15 centipoise) had no effect. Polar oil glyceryl trioleate (a major component in sunflower seed oil) at 10% level reduced surfactant binding to BSA protein in water by 40%, whereas the non-polar oil dodecane (a major component of mineral oil) at a similar level did not have any effect. The mobility of the spin label in a dry corneum was very low and was increased significantly with the addition of water and glycerol trioleate but less so with mineral oil. CONCLUSION: Sunflower seed oil reduces surfactant-induced in vivo skin irritation more than mineral oil. This is possibly due to stronger interaction of polar oil with proteins, thus protecting it from surfactant binding.

Antiviral properties against HIV of water soluble copper carbosilane dendrimers and their EPR characterization.

We describe here the use of anionic carbosilane dendrimers to obtain new copper complexes. UV-Vis and a computer aided analysis of the EPR spectra provided information about the coordination modes of copper depending on the nature of the dendrimer and about the geometry and structure of the complexes in solution. Some of these metallo-dendrimers have been tested "in vitro" as antiviral compounds in the inhibition of HIV infection in pre and post-infection treatment.

Kinetics of amyloid and prion fibril formation in the absence and presence of dense shell sugar-decorated dendrimers.

The aggregation behavior of the amyloid peptide Aß(1-28) and the prion peptide PrP(185-208) - both responsible for neurodegenerative disorders - was analyzed in the absence and in the presence of poly(propylene imine) (PPI) dendrimers at generation 5 (G5) with a dense shell of maltose and maltotriose units. Thioflavin T (ThT) fluorescence assay and circular dichroism (CD) experiments indicated that fibril formation is enhanced at low dendrimer concentration, while it is prevented at relatively high dendrimer concentrations. Computer aided EPR analysis by means of the selected spin probe 4-octyl-dimethylammonium,2,2,6,6-tetramethyl-piperidine-1-oxyl bromide (CAT8) further demonstrated this behavior, but also provided detailed information on the mechanism of fibril formation and on the different behavior of the differently decorated dendrimers. The CAT8 radicals were progressively trapped at the peptide interphase when peptide aggregates were formed, also monitoring pre-fibrillar structures. At later time, a phase separation of the CAT8 radicals monitors the formation of further supramolecular structures where the probes become squeezed among fibrillar aggregates. The addition of small amounts of dendrimers promotes the formation of peptide fibrils breaking them and providing a larger amount of ends that serve as sites of replications. Conversely, a high amount of dendrimers allows the peptides to well separate from each other such preventing their aggregation. EPR results also indicate that the perturbation played by PPI(G5)-Maltose are more effective onto PrP(185-208) than onto Aß(1-28), while PPI(G5)-Maltotriose is less effective towards PrP(185-208) in both promoting aggregation and preventing it by changing the dendrimer concentration. These results provide useful information about the mechanism and interactions which regulate the ability of macromolecules like the dendrimers to favor, prevent or cure neurodegenerative diseases.

Spectroscopic and theoretical study of the electronic structure of curcumin and related fragment molecules.

The low volatility and thermal instability made the photoelectron (PE), electron transmission (ET), and dissociative electron attachment (DEA) spectroscopy measurements on curcumin (a potent chemopreventive agent) unsuccessful. The filled and empty electronic structure of curcumin was therefore investigated by exploiting the PES, ETS, and DEAS results for representative fragment molecules and suitable quantum-mechanical calculations. On this basis, a reliable pattern of the vertical ionization energies and electron attachment energies of curcumin was proposed. The pi frontier molecular orbitals (MOs) are characterized by sizable interaction between the two phenol rings transmitted through the dicarbonyl chain and associated with a remarkably low ionization energy and a negative electron attachment energy (i.e., a largely positive electron affinity), diagnostic of a stable anion state not observable in ETS. The lowest energy electronic transitions of half-curcumin and curcumin and their color change by alkalization were interpreted with time-dependent density functional theory (DFT) calculations. For curcumin, it is shown that loss of a phenolic proton occurs in alkaline ethanolic solution.

Effect of humidity on the supramolecular structure of cotton, studied by quantitative spin probing.

The effect of water content on the physicochemical properties of the amorphous regions in cotton were investigated by measuring the electron paramagnetic resonance (EPR) of TEMPOL nitroxide radicals, deposited in cotton at different loadings, as a function of the relative humidity (RH) and temperature. Three different components contribute differently to the experimental EPR spectra, corresponding to (a) mobile radicals absorbed in the bulk amorphous region, (b) slow moving radicals adsorbed on the crystallite surfaces in cotton, and (c) aggregated radicals. These components were analyzed by means of computer-aided simulations of the line shapes and simplified line width methods. Polarity and mobility parameters were extracted from the analysis of the spectra. For all loadings and temperatures, the polarity suddenly dropped when the water content fell below approximately 3 wt %, i.e., when water was removed from the bulk amorphous regions. At the lowest loading (2 x 10(-5) mol kg(-1)), the spectra were independent of the RH, and only mobile radicals were observed. At intermediate loading (10(-4)-10(-3) mol kg(-1)) both mobile (fast) and adsorbed (slow) moving radicals were present, the fraction of which depended on the RH. The mobility of the adsorbed and mobile radical signals was smaller at higher loadings, indicating microdomains of different character. The temperature dependence of the rotational correlation times provided the activation energies, which were much lower than in liquids. An equilibrium exists between the mobile and the adsorbed radicals. The temperature dependence of the equilibrium constant, K, gave the enthalpy and the entropy of the adsorption process. At low RH, the enthalpy and the entropy values indicated a simple adsorption process. At 10(-3) mol kg(-1), the values were independent of the RH, but at low loadings the values increased with the increase in the RH, which suggested a displacement of adsorbed water by the radicals at high water content. At loadings above 10(-3) mol kg(-1), signals from radicals strongly interacting via spin exchange were observed, which are assigned to aggregated radicals; simulation of the spectra gave an activation energy of 13 kJ mol(-1) for the spin exchange process. These effects are rationalized on the basis of microdomains of different character within cotton, reflecting the variation in pore sizes (0.5-8 nm) and the relaxation behavior of the cellulose chains.

Electron paramagnetic resonance investigations of free radicals in extra virgin olive oils.

Free radicals in olive oils were identified and quantified by EPR, by means of the spin-trapping technique making use of alpha-phenylbutylnitrone (PBN) as spin trap. The radical species were identified as PBN-trapped hydroxyl radicals (PBN-*OH) in the water microdroplets inside the fat medium. The largest radical concentration was 12.5 microM identical with 100%. The following were the relative concentrations of the radicals under different conditions: (1) Two oils, produced by continuous centrifugation, aged for 1 year, showed a 25-30% increase in the radicals compared to nonaged oils; 1-year-old oil, produced by pressure, did not differ from the nonaged oil. (2) Radical production was markedly reduced by N(2) bubbling; it was increased by heating, whereas it showed a biphasic pattern by air bubbling over time. (3) Radical concentration as a function of the UV irradiation time increased up to a maximum, after which it decreased and finally remained constant. The phenolic and oxygen contents were related to the radical content. This study demonstrates that the EPR technique is suitably applied to the detection of free radicals in olive oil and that storage, handling, and stress conditions of the oils significantly influence the radical concentration.

Study of the stability of a paramagnetic label linked to mesoporous silica surface in contact with rat mesothelial cells in culture.

Stable radicals detectable by electron paramagnetic resonance (EPR) may be use in the investigation of early events in cell-particle toxicity. Piperidine-N-oxyl derivatives (nitroxides), covalently linked to the surface of a high surface area silica (used as model solid for the technique), served as probes in the investigation of the effects of incubation of silica particles with mesothelial cells. A mesoporous silica (MCM-41), prepared by precipitation from a micellar solution, was the most appropriate silica-based particle for this purpose, as its channels allow direct contact with small molecules but not with macromolecules. The cytotoxicity of this amorphous silica is very low, allowing relatively high particle loading in the cell cultures. Both the high surface area of the sample and the large amount of inorganic material extracted from the cell culture provide enough material to run reasonably intense EPR spectra. Computer-aided analysis of the EPR spectra of silica-bound nitroxides provided information on the sensitivity of the labeled silica monitoring different environments, e.g., to follow the path of particles in a mammalian cell culture. Upon contact of the particles with mesothelial cells, the mean distance among the labels at the silica surface decreased as a consequence of the release of oxidizing and/or radical moieties from the cells.

Protective effect of darodipine, a calcium antagonist, on rat cardiomyocytes against oxygen radical-mediated injury.

1. We used electrophysiological and electron spin resonance (e.s.r.) techniques to study the mechanism of the protective effect of darodipine on rat isolated cardiomyocytes exposed to an exogenous source of oxygen free radicals (OFR). 2. The L-type calcium current (I(Ca,L)), action potential and cell shortening were measured in patch-clamped cells in the whole-cell configuration. I(Ca,L) blockade by darodipine was concentration-dependent, peak current being reduced by 20% with 50 nM and by 58% with 100 nM darodipine. The lowest concentration of darodipine did not affect action potential or twitch profile. 3. Exposure to OFR-generating solution (5 mM dihydroxyfumarate, DHF) caused the appearance of electrophysiological alterations and/or spontaneous activity in 73% of cells (n=26) within 5 min; action potential duration (APD) was prolonged (195+/-16 ms vs 140+/-6 ms in the control) and maximum diastolic potential (MDP) was reduced (-59.5+/-2.6 mV vs -69.8+/-0.8 mV in the control) (P<0.05, n=25). 4. A 2 min pretreatment with 50 nM darodipine significantly reduced the incidence of these arrhythmogenic events following a 5 min exposure to OFR (36% of cells, n=14; P<0.05 vs nonpretreated cells). Pretreatment with darodipine also prevented APD prolongation caused by OFR (137+/-12 ms after DHF vs 117+/-6 ms before DHF n=14, not significant) but not the decrease of MDP (-63.4+/-2.5 mV after DHF vs -70.9+/-1.0 mV before DHF, P<0.05). 5. The e.s.r. spectra obtained from the DHF-DMPO solution in the absence of darodipine demonstrated the presence of two components corresponding to two DMPO adducts. The addition of darodipine (50-500 nM) led to a concentration-dependent decrease in intensity of the signals, the intensity of the DMPO-COO.- adduct being decreased more than that of the DMPO-OH. adduct. 6. Our results demonstrate that darodipine dose-depentently blocks I(Ca,L) in rat isolated cardiomyocytes. Furthermore it exerts protective effects against free-radical-induced electrophysiological alterations independently of its calcium antagonistic properties; this effect is possibly due to trapping of specific radical species.

Carbon centered radicals reduce the density of L-type calcium channels in rat cardiac membranes.

The carbon centered radicals generated by preincubation of cumene hydroperoxide with rat cardiac membranes dose dependently reduced the Bmax of [3H]nitrendipine binding, while KD was unchanged. The reduction induced by cumene hydroperoxide was prevented by 2,6-di-t-butlyl-4-methyl-phenol. The generation of OH did not influence the [3H]nitrendipine binding; also other oxidative agents (H2O2 and HClO4) did not modify this binding. Therefore the reduction in [3H]nitrendipine binding sites is not attributable to generic oxidative stress but to the formation of carbon centered radicals.

The macroscopic organization of reconstituted M13 coat protein-phospholipid systems. An EPR spectroscopy and polarizing microscope study.

The coat protein of the bacteriophage M13 in the alpha-helical state is reconstituted in macroscopically oriented systems of dioleoylphosphatidylcholine that are prepared by squeezing the reconstituted material between glass plates. The coat protein dramatically influences the macroscopic orientation of the multibilayers, as is investigated by polarizing microscopy and EPR spectroscopy of the cholestane spin label embedded in the bilayers. It is found that with increasing amounts of protein the spontaneous macroscopic orientation of the reconstituted system decreases. This effect is proposed to be due to an increase of the apparent viscosity of the lipid-protein systems with increasing amounts of protein. This is assumed to arise from a sticky effect of the C- and N-terminal protein parts that extend into the aqueous phase between the bilayers.

Do ethoxy radicals reduce 3H-nitrendipine binding in rat cardiac membranes?

The incubation of xanthine-oxidase (XOD) with rat cardiac membranes induced the formation of free radicals; they were identified by electron spin resonance spectroscopy (ESR) studies using 0.1 M 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) for spin-trapping. The following DMPO adducts were measured: DMPO-OH, which was produced during the first minute of incubation, DMPO-R radicals, which were present after 4 min of incubation and where the signal intensity remained constant for at least 20 min. The binding studies performed after 15 min incubation showed that XOD (1-50 mU) dose dependently reduced 3H-nitrendipine binding. This reduction was caused by a decrease in the density of binding sites while the affinity remained unchanged. These results suggest that ethoxy radical formation may be an important step in the regulation of L type calcium channels.

Electron spin resonance studies of dental composites: effects of irradiation time, decay over time, pulverization, and temperature variations.

Polymerization induced by UV-VIS light of composite dental materials produces a solid matrix within which terminal radicals of non-polymerized monomers remain trapped. Electron Spin Resonance (ESR) allowed three different types of radicals to be identified. The analysis of ten normally available commercial products gave information on: (1) the propagation of the conversion reaction as a result of exposure to light; (2) the time necessary for the decay of each type of radical; and (3) the variations with temperature and the effects of shattering on the materials under study. The presence of inorganic filling material slowed the process of polymerization, while it accelerated the decay of radicals. It was suggested that the nature of these processes depended on the composition of the base resin materials, whereas it did not depend on the sizes of the filler particles. Moreover, the complete propagation of the conversion reaction needed a period of light exposure greater than that currently suggested by the manufacturers. The structural stability and the resistance of the composites were confirmed by both the long period of decay and the high temperatures needed to overcome the potential barrier for starting the radical decay process. Finally, the composite shattering investigation indicated that particles removed by surface abrasion experience rapid radical decay, thus reducing the possibility of harmful effects on internal organs.

A small protein in model membranes: a time-resolved fluorescence and ESR study on the interaction of M13 coat protein with lipid bilayers.

Model membranes with unsaturated lipid chains containing various amounts of M13 coat protein in the alpha-helical form were studied using time-resolved fluorescence and ESR spectroscopy. The lipid-to-protein (L/P) ratios used were > 12 to avoid protein-protein contacts and irreversible aggregation leading to beta-polymeric coat protein. In the ESR spectra of the 12-SASL probe in dioleoyl phosphatidylcholine (DOPC) bilayers no second protein induced component is observed upon incorporation of M13 coat protein. However, strong effects are detected on the ESR lineshapes upon changing the protein concentration. The ESR lineshapes are simulated by assuming a fixed ratio between the parallel (D parallel) and perpendicular (D perpendicular) diffusion coefficients of 4, and an order parameter equal to zero. It is found that increasing the protein concentration from L/P infinity to L/P 15 results in a decrease of the rotational diffusion coefficient D perpendicular from 3.4 x 10(7) to 1.9 x 10(7) s-1. In the time-resolved fluorescence experiments with DPH-propionic acid as a probe, it is observed that increasing the M13 coat protein concentration causes an increase of the two fluorescent lifetimes, indicating an increase in bilayer order. Analysis of the time-resolved fluorescence anisotropy decay allows one to quantitatively determine the order parameters and , and the rotational diffusion coefficient D perpendicular of the fluorescent probe. The order parameters and increase from 0.34 to 0.55 and from 0.59 to 0.77, respectively, upon adding M13 coat protein to DOPC bilayers with an L/P ratio of 35.(ABSTRACT TRUNCATED AT 250 WORDS)

Changes in erythrocyte properties during the first hours of life: electron spin resonance of reacting sulfydryl groups.

In an attempt to clarify the mechanism by which the red cells (RBC) of newborn infants are protected against oxidative agents, electron spin resonance (ESR) assays were carried out using the nitroxide radical 4-maleimide-2,2,6,6-tetramethylpiperidinyl-1-oxyl (Mal-6), a sulfydryl-reacting agent. The ESR assays were performed in 24 samples of cord blood, 20 samples of blood from 4-day-old infants, and eight samples of 8-h-old infants. The analyses were carried out on whole blood and washed erythrocytes were resuspended in buffered saline. The same experiments were performed in 10 blood samples from healthy adults as controls. Whole blood, before and after removing the buffy coat, and cell-free plasma were also examined by ESR assay. Cell-free plasma and buffy coats proved not to be appreciably involved in the Mal-6 behavior. The data of the ESR spectroscopy demonstrated a significantly slower reaction rate in the samples of cord blood and in blood of 8-h-old infants, compared to that of 4-day-old infants and adults. No significant differences in Mal-6 behavior could be detected between cord blood and 4-day-old infant blood in the results of ESR assays performed in washed red cells. Chemical determination of RBC-reacting sulfydryl groups and the assays of glutathione also demonstrated the absence of differences between cord blood and blood of 4-day-old infants. The results of our investigation suggest that the RBC-sulfydryl-reacting groups are less involved in the detoxification of oxidative agents during the first hours of life than in the following days.(ABSTRACT TRUNCATED AT 250 WORDS)