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A method is reported for the determination of acaricides (amitraz, bromopropylate, coumaphos and fluvalinate) from honey by gas chromatography mass spectrometry after a new fast solid phase micro-extraction, SPME, procedure. Six different fibers were assessed for micro-extraction purpose studying the following variables: (i) SPME coating, (ii) extraction temperature, (iii) extraction time, (iv) desorption conditions and (v) agitation conditions. The new ultrasonic bath technology providing different sonication frequencies (35 and 130 kHz) and different working modes (Sweep, Standard and Degas) was studied and optimized for speeding up the acaricide micro-extraction. The best extraction results were achieved with the polyacrylate fiber. The extraction process was done in 30 min using the ultrasonic bath at 130 kHz in the Standard mode. Quality parameters of the proposed method show a good precision (<11%) and detection and quantitation limits lower than 6 and 15 ng/g, respectively, except for fluvalinate. Eleven Portuguese commercial honey samples were analyzed with the developed method in order to assess the performance of the method with real samples and to determine whether the concentration of acaricides in honey exceed their maximum residue levels (MRLs). Acaricide residues detected were lower than those established by the legislation. (c) 2006 Elsevier B.V. All rights reserved.

This work describes the construction and evaluation of lactate sol-gel biosensors to accomplish the determination of lactate in pharmaceutical products. Lactate oxidase was incorporated in a porous sol-gel film placed onto a platinum-based electrode. Acid and basic catalysis were assessed. When coupled to a sequential injection system (SIA) the biosensor, based on (3-aminopropyl)trimethoxysilane, 2-(3,4-epoxycyclohexyl)ethyl-trimethoxysilane, deionised water, polyethylene glycol 6000 and acid catalyst, presented a range of linearity of 5x10(-5) to 5x10(-3)M. The analytical usefulness of the developed biosensor was evaluated through analysis of commercial pharmaceutical products containing lactate with a sampling rate of 40 samples h(-1). The enzyme remained active for at least 30 days, enabling about 700 determinations without sensitivity decrease.

New findings in sample treatment based on high-intensity focused ultrasound (HIFU) for protein digestion after polyacrylamide gel electrophoresis separation are presented. The following variables were studied: (i) sample volume; (ii) sonotrode diameter; (iii) previous protein denaturation; (iv) cooling; (v) enzyme concentration; and (vi) protein concentration. Results showed that positive protein identification could be done after protein separation by gel electrophoresis through peptide mass fingerprint (PMF) in a volume as low as 25 mu L. The time needed was less than 2 min and no cooling was necessary. The importance of the sonotrode diameter was negligible. On the other hand, protein denaturation before sonication was a trade-off for the success of procedure here described. The protein coverage was raised from 5 to 30%, and the number of peptides matching the proteins was also increased in a percentage ranging 10-100% when the classical overnight treatment is compared with the proposed HIFU procedure. The minimum amount of protein that can be identified using the HIFU sample treatment by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was 0.06 mu g. The lower concentration of trypsin successfully used to obtain an adequate protein digestion was 3.6 mu g/mL. (c) 2006 Elsevier B.V. All rights reserved.

The periplasmic nitrate reductase from Cupriavidus necator (also known as Ralstonia eutropha) is a heterodimer that is able to reduce nitrate to nitrite. It comprises a 91 kDa catalytic subunit (NapA) and a 17 kDa subunit (NapB) that is involved in electron transfer. The larger subunit contains a molybdenum active site with a bis-molybdopterin guanine dinucleotide cofactor as well as one [4Fe-4S] cluster, while the small subunit is a di-haem c-type cytochrome. Crystals of the oxidized form of this enzyme were obtained using polyethylene glycol 3350 as precipitant. A single crystal grown at the High Throughput Crystallization Laboratory of the EMBL in Grenoble diffracted to beyond 1.5 A at the ESRF (ID14-1), which is the highest resolution reported to date for a nitrate reductase. The unit-cell parameters are a = 142.2, b = 82.4, c = 96.8 A, beta = 100.7 degrees, space group C2, and one heterodimer is present per asymmetric unit.

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We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using 13C-detection experiments.

We report the 98% assignment of the apo-form of an orange protein, containing a novel Mo-Cu cluster isolated from Desulfovibrio gigas. This protein presents a region where backbone amide protons exchange fast with bulk solvent becoming undetectable. These residues were assigned using C-13-detection experiments.

The contribution of decameric vanadate species to vanadate toxic effects in cardiac muscle was studied following an intravenous administration of a decavanadate solution (1mM total vanadium) in Sparus aurata. Although decameric vanadate is unstable in the assay medium, it decomposes with a half-life time of 16 allowing studying its effects not only in vitro but also in vivo. After 1, 6 and 12h upon decavanadate administration the increase of vanadium in blood plasma, red blood cells and in cardiac mitochondria and cytosol is not affected in comparison to the administration of a metavanadate solution containing labile oxovanadates. Cardiac tissue lipid peroxidation increases up to 20%, 1, 6 and 12h after metavanadate administration, whilst for decavanadate no effects were observed except 1h after treatment (+20%). Metavanadate administration clearly differs from decavanadate by enhancing, 12h after exposure, mitochondrial superoxide dismutase (SOD) activity (+115%) and not affecting catalase (CAT) activity whereas decavanadate increases SOD activity by 20% and decreases (-55%) mitochondrial CAT activity. At early times of exposure, 1 and 6h, the only effect observed upon decavanadate administration was the increase by 20% of SOD activity. In conclusion, decavanadate has a different response pattern of lipid peroxidation and oxidative stress markers, in spite of the same vanadium distribution in cardiac cells observed after decavanadate and metavanadate administration. It is suggested that once formed decameric vanadate species has a different reactivity than vanadate, thus, pointing out that the differential contribution of vanadium oligomers should be taken into account to rationalize in vivo vanadate toxicity.

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Nowadays the control of pesticides in honey is an issue of primary health importance as consequence of the increasing content of these chemicals in the aforementioned matrix. This poisoning has led to the worldwide increasing loss of bees since 1995. From Europe to Canada, scientist, beekeepers and chemical companies disagree about the reasons that have led to colony losses higher than 50% in some areas. This problem has become a public health issue due to the high honey worldwide consumption. The presence of pesticides in honey has been directly related to bees' mortality by some researchers through pesticide presence in (1) pollen, (2) honeycomb walls, (3) own bees and (4) honey. In this work we describe the actual state-of-the-art for pesticides determination in honey along with a review in this subject focused on sample treatments and instrumentation. Finally, future trends are also commented. (c) 2006 Elsevier B.V. All rights reserved.

Fast (120 s) and high-throughput (more than six samples at once) in-gel trypsin digestion of proteins using sonoreactor technology has been achieved. Successful protein identification was done by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, MALDI-TOF-MS. Specific identification of the adenylylsulphate reductase alfa subunit from a complex protein mixture from Desulfovibrio desulfuricans ATCC 27774 was done as a proof of the methodology. The new sample treatment is of easy implementation, saves time and money, and can be adapted to online procedures and robotic platforms.

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A combination of spectroscopy and density functional theory (DFT) calculations has been used to evaluate the pH effect at the CuZ site in Pseudomonas nautica (Pn) nitrous oxide reductase (N2OR) and Achromobacter cycloclastes (Ac) N2OR and its relevance to catalysis. Absorption, magnetic circular dichroism, and electron paramagnetic resonance with sulfur K-edge X-ray absorption spectra of the enzymes at high and low pH show minor changes. However, resonance Raman (rR) spectroscopy of PnN2OR at high pH shows that the 415 cm-1 Cu-S vibration (observed at low pH) shifts to higher frequency, loses intensity, and obtains a 9 cm-1 18O shift, implying significant Cu-O character, demonstrating the presence of a OH- ligand at the CuICuIV edge. From DFT calculations, protonation of either the OH- to H2O or the mu4-S2- to mu4-SH- would produce large spectral changes which are not observed. Alternatively, DFT calculations including a lysine residue at an H-bonding distance from the CuICuIV edge ligand show that the position of the OH- ligand depends on the protonation state of the lysine. This would change the coupling of the Cu-(OH) stretch with the Cu-S stretch, as observed in the rR spectrum. Thus, the observed pH effect (pKa approximately 9.2) likely reflects protonation equilibrium of the lysine residue, which would both raise E degrees and provide a proton for lowering the barrier for the N-O cleavage and for reduction of the [Cu4S(im)7OH]2+ to the fully reduced 4CuI active form for turnover.

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The compound 7,4'-dihydroxy-5-methoxyflavylium (dracoflavylium) was identified as the major red colorant in samples of the resin "dragon's blood", extracted from the tree Dracaena draco. The complex network of reversible chemical reactions that dracoflavylium undergoes in aqueous solution is fully described; for the first time, all the equilibrium constants that enable a complete characterisation of the system have been obtained (K'(a)=1.6 x 10(-4), K-a1 = 1.0 x 10(-4), K-a2 = 3.2 x 10(-8), K-Ct1 = 1.0 x 10(-7), K-Ct2 = 1.3 x 10(-10)). It is concluded that the red colour is due to a stable quinoid base, A, which is the major species at pH 4-7. It is further shown that this compound does not fit the commonly accepted definitions of anthocyanidin nor 3-deoxyanthocyanidin. Similarly to synthetic flavylium salts, the natural compound 7,4'-dihydroxy-5-methoxyflavylium gives rise to several species (multistate system) reversibly interconverted by external stimuli, such as pH.

An ultrasonic bath, an ultrasonic probe and a sonoreactor were used to speed up the kinetics of the reactions involved in each step of the sample handling for in-gel protein identification by peptide mass fingerprint, PMF, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The following steps were successfully accelerated using ultrasonic energy: gel washing, protein reduction, and protein alkylation. As a result, a reduction comprising 80% to 90% of the total time involved in the classic approach was achieved. In addition the sample handling was also drastically simplified. The number of peptides identified and the protein sequence coverage obtained for the new procedure were comparable to those obtained with the traditional sample treatment for the following protein standards: glycogen phosphorylase b, BSA, ovalbumin, carbonic anhydrase, trypsin inhibitor and alpha-lactalbumin. Finally, as a proof of the procedure, specific proteins were identified from complex protein mixtures obtained from three different sulphate- reducing bacteria: Desulfovibrio, desulfuricans G20, Desulfuvibrio gigas NCIB 9332, and Desulfuvibrio desulfuricans ATCC 27774. Copyright (c) 2007 John Wiley & Sons, Ltd.

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