The final step of bacterial denitrification, the two-electron reduction of N(2)O to N(2), is catalyzed by a multi-copper enzyme named nitrous oxide reductase. The catalytic centre of this enzyme is a tetranuclear copper site called CuZ, unique in biological systems. The in vitro reconstruction of the activity requires a slow activation in the presence of the artificial electron donor, reduced methyl viologen, necessary to reduce CuZ from the resting non-active state (1Cu(II)/3Cu(I)) to the fully reduced state (4Cu(I)), in contrast to the turnover cycle, which is very fast. In the present work, the direct reaction of the activated form of Pseudomonas nautica nitrous oxide reductase with stoichiometric amounts of N(2)O allowed the identification of a new reactive intermediate of the catalytic centre, CuZ degrees , in the turnover cycle, characterized by an intense absorption band at 680 nm. Moreover, the first mediated electrochemical study of Ps. nautica nitrous oxide reductase with its physiological electron donor, cytochrome c-552, was performed. The intermolecular electron transfer was analysed by cyclic voltammetry, under catalytic conditions, and a second-order rate constant of (5.5 +/- 0.9) x 10(5) M(-1 )s(-1) was determined. Both the reaction of stoichiometric amounts of substrate and the electrochemical studies show that the active CuZ degrees species, generated in the absence of reductants, can rearrange to the resting non-active CuZ state. In this light, new aspects of the catalytic and activation/inactivation mechanism of the enzyme are discussed.

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy), a drug of abuse commonly consumed at rave parties, is often taken in a polydrug abuse scenario, ethanol being one of the most associated drugs. Both MDMA and ethanol are mainly metabolized in the liver with formation of toxic metabolites. Our working hypothesis is that ethanol can modify the metabolism of MDMA through the cytochrome P450 system, and that this effect may be further potentiated by hyperthermia, a well-known consequence of MDMA abuse. To investigate these putative interactions we used primary rat hepatocyte cultures, which were exposed to 300 mM ethanol, 1.6 mM MDMA and the combination of both, at normothermic (36.5 degrees C) and hyperthermic (40.5 degrees C) conditions. After 24 h, the levels of MDA, HMA and HMMA in the cell culture medium were quantified by GC/MS. In addition, we repeated the same experimental design preceded by 1 h incubation with 0.18 mu M ketoconazole or 150 mu M diallyl sulphide (CYP3A and CYP2E1 inhibitors, respectively), to evaluate the putative role of these isoenzymes in the observed effects. The results obtained showed that ethanol exposure increases the formation of some MDMA metabolites such as HMA (1.8 times increase) and MDA (1.5 times increase). This effect was markedly increased under hyperthermic conditions (HMA, MDA and HMMA formation increased 10,6 and 16 times, respectively) and is mediated, at least partially, by CYP3A and CYP2E1. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

A phase-change thermochromic system was designed through the reversible transformation of the 4-substituted flavylium dye 4-(2-carboxyphenyl)-7-diethylamino-4'-dimethyl-amino-1-benzopyrylium into its leuco form, in the presence of a developer (ethyldiisopropylamine) and a suitable solvent (e.g., acetonitrile, n-pentadecanonitrile). The leuco form of the flavylium-based dye is a spirolactone species whose ring opens at low temperature (below the solvent melting point) to form the blue flavylium cation. Decarboxylation of the lactone to give 4-phenyl-7-diethylamino-4'-dimethylamino-1-benzopyrylium was observed upon irradiation of the system with UV light, erasing the thermochromic effect.

The formation of Eu(III) nanoparticles in borosilicate sol-gels and the glass formation heat treatment effect on those particles were studied using luminescence techniques. The presence of the particles was observed using transmission electron microscopy (TEM) images followed by analysis with energy dispersive X-ray spectroscopy (EDS). These experiments showed the presence of particles with a large quantity of europium and chlorine and only small amounts of oxygen with sizes ranging from 30 to 100 nm. Heat treatment at 400, 600, and 800 degrees C lead to glass samples in which those particles were no longer observed. Steady-state and time-resolved luminescence techniques allowed a detailed study of Eu(III) photophysics in sot-gel and glass samples. In sol-gel matrices, the (5)D(0) -> (7)F(0) transition is very weak, hinting at Eu(III) species experiencing a rather symmetric crystal field. The (5)D(0) -> (7)F(2) transition intensity is not very strong, which according to a Judd-Ofelt analysis indicates low interaction with the anions present in the sol-gel matrices. This picture reverses after heat treatment, indicating a replacement of chloride anions with oxygen as preferential ligands of Eu(III). Time-resolved luminescence shows in a more detailed way these aspects. Sol-gel samples display nonexponential kinetics, which are attributed to Eu(III) species present in the nanoparticles surface (bound to oxygen) and Eu(III) in the core of the nanoparticles (bound to chloride). Glass samples display single-exponential luminescence decays, in which the decay constant approaches the values calculated for the radiative rate constant with Judd-Ofelt analysis. It is concluded that, in sol-gel, mechanisms like electron-phonon coupling suppress the Eu(III) luminescence, which disappear as soon as the nanoparticles are disrupted after heat treatment.

Substitution of the phenyl group in 2-hydroxychalcones by a 4-pyridine unit dramatically changes the network of chemical reactions of this compound: trans-chalcone-type (Ct), cis-chalcone-type (Cc), and a hemiketal (hydroxy-4-pyridinechromene) (B) and their protonated forms are formed, but the presence of a flavylium-type cation could not be detected even at very acidic pH Values. Moreover, whereas in 2-phenyl-2-benzopyrylium compounds B and Cc are generally elusive species whose kinetic processes in aqueous solutions occur oil the sub-second time-scale, in the present compound these species equilibrate on a timescale four orders of magnitude lower. Complete characterization of the equilibrium and kinetics of the reaction network could thus be achieved by (1)H NMR spectroscopy and UV/Vis spectrophotometry. ne network of chemical reactions exhibits cis-trans photoisomerization, as well as photochromism between the hemiketal and the chalcone-type species. The irradiation of Ct in MeOH/ H(2)O (1:1) at 365 nm produces B almost quantitatively through two Consecutive photochemical reactions: Ct -> Cc photoisomerization followed by Cc -> B photo ring Closure with a global quantum yield of 0.02. On the other hand, irradiation of B at 254 nm leads to it photostationary state composed by 80% Ct and 20%, B. with a quantum yield of 0.21.

There are numerous reports of coumarin ester derivatives, in particular phosphate esters, as photocleavable cages in biological systems. Despite the comprehensive analysis of the photocleavage mechanism, studies of 4-methylcoumarin caged phosphates and/or nucleotides were always performed at constant pH. In this work. we present the study of the pH effect on the photochemistry of (7-diethylaminocoumarin-4-yl)methyl phosphate (DEACM-P). Fluorescence and photocleavage quantum yields, as well as the fluorescence decay times were measured as a function of the pH. It was found that the pH produces significant changes in the overall photochemical quantum yield of DEACM-P, and the observed changes are complementary to those obtained from the fluorescence quantum yield. Deprotonation of DEACM-HPO(4)(-) to yield DEACM -PO(4)(2-), produces a decrease in the photochemical quantum yield (from 0.0045 to 0.0003) and an increase in the fluorescence quantum yield (from 0.072 to 0.092). Moreover, from the analysis of the decay times, we have also found that hydroxyl ion is not only relevant, but it is mechanistically involved in the photoreaction of DEACM-HPO(4)(-).

We show photorheology in aqueous solutions of weakly entangled wormlike micelles prepared with cetyltrimethylammonium bromide (CTAB), salicylic acid (HSal), and dilute amounts of the photochromic multistate compound trans-2,4,4'-trihydroxychalcone (Ct). Different chemical species of Ct are associated with different colorations and propensities to reside within or outside CTAB micelles. A light-induced transfer between the intra- and intermicellar space is used to alter the mean length of wormlike micelles and hence the rheological properties of the fluid, studied in steady-state shear Bow and in dynamic rheological measurements. Light-induced changes of fluid rheology are reversible by a the relaxation process. at relaxation rates which depend on pH and which are consistent with photochromic reversion rates measured by UV-vis absorption spectroscopy. Parameterizing viscoelostic rheological states by their effective relaxation time tau(c) and corresponding response modulus G(c), we find the light and dark states of the system to fall onto a characteristic state curve defined by comparable experiments conducted without photosensitive components. These reference experiments were prepared with the same concentration of CTAB, but different concentrations of HSal or sodium salicylote (NaSal), and tested at different temperatures.

A systematic study of the electrochromic (EC) behavior of electropolymerized poly[M(salen)] films (M = Ni, Cu, and Pd) was performed by spectroelectrochemistry. Color contrast between oxidized and reduced states, stability under square wave potential cycling, coloration efficiency, and switching rate were evaluated. Five polymers were selected to assemble solid-state EC cells in a symmetrical configuration (electrode/poly[M(salen)] film/opaque electrolyte/poly[M(salen)] film/electrode). The best EC performance was found for poly[Pd(3-Mesalen)], poly[1], with 38% of initial diffuse reflectance variation and loss of 50% after 6769 cycles. (C) 2010 The Electrochemical Society. [DOI: 10.1149/1.3457474] All rights reserved.

Flexible, transparent and self-supporting electrolyte films based on poly(trimethylene carbonate)/poly(ethylene oxide) (p(TMC/PEO) interpenetrating networks doped with LiClO(4) were prepared by the solvent casting technique. These novel solid polymer electrolyte (SPE) systems were characterized by measurements of conductivity, cyclic voltammetry, differential scanning calorimetry and thermogravimetry. The incorporation of solid electrolytes as components of electrochromic devices can offer certain operational advantages in real-world applications. In this study, all-solid-state electrochromic cells were characterized, using Prussian blue (PB) and poly-(3,4-ethylenedioxythiophene)-poly(styrenesulfonate) (PEDOT) as complementary electrochromic compounds on poly(ethyleneterphthalate) (PET) coated with indium tin oxide (ITO) as flexible electrodes. Assembled devices with PET/ITO/PB/SPE/PEDOT/ITO/PET "sandwich-like" structure were assembled and successfully cycled between light and dark blue, corresponding to the additive optical transitions for PB and PEDOT electrochromic layers. The cells required long cycle times (>600 s) to reach full color switch and have modest stability towards prolonged cycling tests. The use of short duration cycling permitted the observation of changes in the coloration-bleaching performance in cells with different electrolyte compositions. (C) 2009 Elsevier Ltd. All rights reserved.

We report a spectroscopic study of the network of reactions of a flavylium dye encapsulated in the one-dimensional channels of zeolite L. The positively charged 7,4'-dihydroxyflavylium (AH(+)) is easily incorporated and remains stable in zeolite L channels. Once encapsulated, the flavylium exhibits a red shift in the excitation spectrum comparative to aqueous solutions. Moreover, contrary to the observed behavior in water, no excited state proton transfer takes place in the loaded crystals, corroborating the encapsulation of AH(+). The trans-chalcone (Ct) form from the same flavylium network could also be encapsulated inside the zeolite L, using toluene with 20% triethylamine as solvent and K(+) as counter ion of the negative framework of the zeolite. The encapsulation of Ct is confirmed by changes on the excitation spectrum and by a blue shift in the emission. The encapsulated Ct was shown to generate AH(+) when the Ct-loaded crystals were suspended in water, which proves that isomerization, tautomerization and dehydration reactions take place inside the zeolite L.

We recently reported on the use of caged nucleotides to attain full control of enzymatic polymerization of RNA solely by light. In the absence of light no RNA formation was possible due to the efficient caging by the coumarin moiety: after irradiation, caged ATP was released with quantitative precision and RNA polymerization was resumed. As photolabile protecting group [7-(diethylamino)coumarin-4-yl]methyl] (DEACM) was used due to its high absorbance in the visible region of the spectrum, fast deprotection kinetics and absence of radical intermediates. However, the 7-diethylamino-4-hydroxymethylcoumarin photo-product (DEACM-OH) was shown to inhibit the transcription reaction for concentrations higher than 30 mu M [5]. This inhibition has been associated with poor water solubility, which is commonly dealt with via cumbersome chemical modifications of the protecting moiety. To overcome inhibition, we evaluated the use of molecular scavengers to sequester DEACM-OH formed after irradiation. Determination of association constants of coumarin with beta-cyclodextrins allowed the assessment of its capability to remove free coumarin molecules from solution. The influence of beta-cyclodextrin in transcription reaction was also assessed. Results show that beta-cyclodextrin can be successfully used as scavenger as it increases the DEACM-OH threshold concentration for inhibition, amplifying the efficiency of light controlled in vitro transcription. (C) 2010 Elsevier B.V. All rights reserved.

This work aimed at the development of targeted drug delivery systems using nanoparticles fused with antibodies. The antibody anti-human {CD8} was coupled onto {PLGA} nanoparticles, and the ability of these particles to specifically target cells expressing {CD8} was studied. The obtained particles were found to be of spherical shape exhibiting a size between 350 and 600 nm. In vitro experiments with different cellular cultures {(TE671}, {CHO} and {HEK293)} using unmodified nanoparticles containing rhodamine have shown that particles were present on their surface within 48 h of incubation. In vitro tests using {anti-CD8} conjugated nanoparticles in {CHO} cell cultures indicated that all transfected cells which express {CD8} show these particles on their surface within 1h of incubation. These results demonstrated that, in a shorter time, the produced particles can target cells expressing {CD8} on their surface which offers the ability to reduce drug side effects. The antibody-coupled nanoparticles represent a promising approach to improve the efficacy of active targeting for lymphoblastic leukaemia therapy.

This study reports the comparison of fluorimetric techniques (fluorescence microscopy and spectrofluorimetry on a 96-well format) for the on-bead screening of combinatorial libraries of affinity ligands for chromatographic separations. Two solid-phase libraries of synthetic ligands based on distinct scaffolds were synthesized by combinatorial chemistry. The libraries comprising ligands representing different hydrophobic/hydrophilic properties and sizes were tested for binding to randomly selected biomolecules (labelled with a fluorophore). Fluorescence microscopy was revealed to be a reliable and reproducible technique for the detection of lead ligands which strongly bound the target biomolecule. Results obtained by fluorescence intensity measurements in a 96-well format were less consistent, mainly due to challenges related with the accurate dispensing of the solid support.

This paper has its starting point in the background analysis of the Lithuanian energy sector after closing down the only Lithuanian nuclear power plant in 2010. Based on the hypothesis that one of the main governance failures in this sector leading to weak industry level strategies is the lack of participatory debate and sufficient linkages between the different actors involved in the dynamic of the energy sector in Lithuania, this paper proposes industry level foresight as an instrument of long term planning. Foresight exercises could become an important instrument for reorienting energy sector policy, building new networks and linkages among the different actors, bringing new stakeholders into the strategic debate, exploring future opportunities State investment (including R&D), etc. The primary objective of this paper is therefore the design of a foresight exercise on energy sector with the aim of producing a long term strategy for this sector. The secondary objective is to address a topic on how to select foresight methods at industry level. The argument is that a better understanding of the fundamental attributes of foresight methods and their linkages to the core phases of a foresight process can provide useful insights as to how the selection of methods is carried out. The method applied in this paper is dual: firstly, the synthesis of the academic literature on the selection of foresight methods is carried out; secondly, the comparative case study analysis of three foresight cases in the Baltic Sea Region (Poland, Finland and Russia) is applied. Case study analysis allows to explore the usage of foresight methods at industry level in the Baltic Sea Region and to understand if there are any similarities in the approach, also to explore success factors and weaknesses. The analysis in this paper is comprised of four main parts. The first part provides a background analysis on the energy sector in Lithuania and justification for the foresight exercise. Second part describes the underlying frameworks and definitions in the field of foresight research. The third part develops a comparative analysis of case studies of industry level foresight. The third part provides recommendations for energy sector foresight methodology in Lithuania. The paper combines concepts and frameworks from literature (such as the Foresight Process and the Foresight Diamond) with comparative practical case study analysis. The results can be utilised by lecturers and students to describe and understand better the use of foresight methods at industry level, and by practitioners of foresight to better inform decisions during the design of more coherent methodological frameworks; as well as by the energy sector stakeholders in Lithuania and other countries.

The periplasmic sensor domains encoded by genes gsu0582 and gsu0935 are part of methyl accepting chemotaxis proteins in the bacterium Geobacter sulfurreducens (Gs). The sensor domains of these proteins contain a heme-c prosthetic group and a PAS-like fold as revealed by their crystal structures. Biophysical studies of the two domains showed that nitric oxide (NO) binds to the heme in both the ferric and ferrous forms, whereas carbon monoxide (CO) binds only to the reduced form. In order to address these exogenous molecules as possible physiological ligands, binding studies and resonance Raman (RR) spectroscopic characterization of the respective CO and NO adducts were performed in this work. In the absence of exogenous ligands, typical RR frequencies of five-coordinated (5c) high-spin and six-coordinated (6c) low-spin species were observed in the oxidized form. In the reduced state, only frequencies corresponding to the latter were detected. In both sensors, CO binding yields 6c low-spin adducts by replacing the endogenous distal ligand. The binding of NO by the two proteins causes partial disruption of the proximal Fe-His bond, as revealed by the RR fingerprint features of 5cFe-NO and 6cNO-Fe-His species. The measured CO and NO dissociation constants of ferrous GSU0582 and GSU0935 sensors reveal that both proteins have high and similar affinity toward these molecules (Kd ≈ 0.04−0.08 μM). On the contrary, in the ferric form, sensor GSU0582 showed a much higher affinity for NO (Kd ≈ 0.3 μM for GSU0582 versus 17 μM for GSU0935). Molecular dynamics calculations revealed a more open heme pocket in GSU0935, which could account for the different affinities for NO. Taken together, spectroscopic data and MD calculations revealed subtle differences in the binding properties and structural features of formed CO and NO adducts, but also indicated a possibility that a (5c) high-spin/(6c) low-spin redox-linked equilibrium could drive the physiological sensing of Gs cells.

The effect of kefir grains on the proteolysis of major milk proteins in milk kefir and in a culture of kefir grains in pasteurized cheese whey was followed by reverse {phase-HPLC} analysis. The reduction of kappa-, alpha-, and beta-caseins {(CN)}, alpha-lactalbumin {(alpha-LA)}, and beta-lactoglobulin {(beta-LG)} contents during 48 and 90 h of incubation of pasteurized milk {(100mL)} and respective cheese whey with kefir grains (6 and 12 g) at 20 degrees C was monitored. Significant proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins was observed. The effect of kefir amount (6 and 12 {g/100mL)} was significant for {alpha-LA} and alpha- and {beta-CN.} {alpha-Lactalbumin} and {beta-CN} were more easily hydrolyzed than {alpha-CN.} No significant reduction was observed with respect to {beta-LG} concentration for 6 and 12 g of kefir in {100mL} of milk over 48 h, indicating that no significant proteolysis was carried out. Similar results were observed when the experiment was conducted over 90 h. Regarding the cheese whey kefir samples, similar behavior was observed for the proteolysis of {alpha-LA} and {beta-LG:} {alpha-LA} was hydrolyzed between 60 and 90% after 12h (for 6 and 12 g of kefir) and no significant {beta-LG} proteolysis occurred. The proteolytic activity of lactic acid bacteria and yeasts in kefir community was evaluated. Kefir milk prepared under normal conditions contained peptides from proteolysis of {alpha-LA} and kappa-, alpha-, and beta-caseins. Hydrolysis is dependent on the kefir:milk ratio and incubation time. {beta-Lactoglobulin} is not hydrolyzed even when higher hydrolysis time is used. Kefir grains are not appropriate as adjunct cultures to increase {beta-LG} digestibility in whey-based or whey-containing foods.