Determination of toxic cyclic heptapeptides by liquid chromatography with detection using ultra-violet, protein phosphatase assay and tandem mass spectrometry

Microcystins, toxic cyclic heptapeptides and nodularin-R, a toxic cyclic pentapeptide, were determined using liquid chromatography (LC) with detection using photo-diode array ultra-violet (PDA-UV) and protein phosphatase (PP) assay. Positive fractions were analysed for toxins using collision-induced dissociation (CID) and tandem MS/MS experiments which were carried out simultaneously using electrospray ion-trap instrumentation. Reversed-phase liquid chromatography (LC) using an acetonitrile/water gradient was used for the LC-MS(2) determination of six microcystins standards and nodularin. The molecular related ion species, [M+H](+)([M+2H](2+) in the case of MC-RR), were used as the precursor ions for MS(2) experiments. Optimum calibration and reproducibility data were obtained for MC-LR using LC-MS(2); 0.1-5.0 microg/ml, r2 = 0.992 (n = 3); % RSD < or =7.3 at 0.25 microg MC-LR/ml (n = 3). The detection limit (S/N = 3) was better than 0.1 ng. Water samples for microcystin analysis were first screened using protein phosphatase (PP) assays and positives were concentrated using C-18 solid-phase extraction. The developed method was applied to examine a lake in Ireland contaminated by Microcystis sp. and MC-LR and MC-LA were identified.     

Analysis of dioxins and furans in environmental samples by GC-ion-trap MS/MS

The work aims to characterise PCDDs/Fs in environmental matrices by high resolution gas chromatography coupled to low resolution quadrupole ion-trap mass spectrometry (GC-QITMS/MS). In particular, the study was mainly focused in different environmental samples such as sewage sludge, soils, and sediments and in combustion residues as fly ashes. Previous experiences have already detected and quantified PCDDs/PCDFs in all matrices studied. Moreover, isomer-specific analysis of 2,3,7,8-PCDDs/Fs compounds corroborated good correlation between GC-QITMS/MS and a well-established technique such as high resolution mass spectrometry (HRMS). Finally, QC measures included the participation in intercalibration exercises. Successful results were achieved using GC-QITMS/MS in the analysis of fly ashes, soils and sediment materials. In general, GC-QITMS/MS constitutes an interesting alternative for routine analysis of dioxins in such as matrices.     

Agricultural landscapes as multi-scale public good and the role of the Common Agricultural Policy

During the last 50 years, the Common Agricultural Policy (CAP) has impacted the evolution of European agricultural landscapes by driving changes in land use and farming practices. We propose a typology characterising the scales relevant for agricultural landscapes management and argue that action is required on three scales: (1) a landscape oriented management at the farm level; (2) the coordination of land managers’ actions at the landscape level; and (3) the conservation of the diversity of agricultural landscapes in the EU. We provide evidence that until now the CAP has mainly focused on the first scale. We also illustrate how agricultural policy could encourage coordinated actions at the landscape- and EU-scales. In particular, we propose policy instruments to coordinate actions of individual land owners (e.g. collective bonus in agro-environmental contracts or support to environmental cooperatives (scale 2)). We also analyse how the recognition and transposition of the European Landscape Convention could promote trans-frontier landscape cooperation in order, not only to conserve high-quality rural landscapes, but also to ensure the conservation of the diversity of EU landscapes (scale 3). This paper provides a knowledge base to support an integrated CAP design in the direction of improved landscape management, as an important component of the EU project towards more sustainable agriculture.     

Rapid molecular prenatal diagnosis of ataxia-telangiectasia by direct mutational analysis

Mutations of the ataxia-telangiectasia-mutated (ATM) gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). This study reports the first A-T prenatal diagnosis performed in Spain by direct molecular analysis. The pregnant woman had a previous child suffering from A-T due to a deletion in the ATM gene. The ATM coding region was sequenced in the A-T patient and her parents. Then, a specific polymerase chain reaction (PCR) to detect the deletion was performed for prenatal diagnosis. Additionally, polymorphic HLA loci were examined in order to exclude the possible contamination by maternal DNA. In this family of Gypsy origin, we carried out a rapid molecular diagnosis of A-T. Then, a prenatal diagnosis was carried out, identifying the deletion in the fetal DNA. Additionally, we performed a population study in unrelated Spanish Gypsies and in unrelated controls, showing that the deletion described could be a hotspot in the Spanish Gypsy population. The size of the coding region and the genomic structure, together with the absence of hotspots, make the mutation screening of the ATM gene difficult. The ability to identify ATM mutations provides a tool that can be applied in confirmatory diagnosis, genetic counselling, carrier prediction and prenatal diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.     

Manufactured silver and gold nanoparticles-induced apoptosis by caspase-pathway in human cell lines

Abstract

Metallic nanoparticles have emerged as an important class of nanomaterials for a wide range of industrial and medical applications. Because of the intensive commercial applications, risk assessment of these nanoparticles is of great importance. In the present study, the human hepatoma and leukemia cells were used to characterize the apoptotic effects of silver nanoparticles (4.7 and 42?nm) and gold nanoparticles (30?nm). Apoptotic cells were identified by chromatin condensation and flow cytometry analysis, using Annexin V/PI, TUNEL and caspase activation assays. Flow cytometry analysis showed that the three metallic nanoparticles induced apoptotic cell death in a concentration and time dependent-manner. Moreover, the three nanoparticles induced activation of caspase-3 and -7 in hepatoma and leukemia cells. Apoptotic effects were stronger after exposure of both cell lines with 4.7?nm silver nanoparticles than those obtained with 42?nm silver and 30?nm gold nanoparticles. In conclusion, silver (4.7 and 42?nm) and gold (30?nm) nanoparticles induced apoptosis in hepatoma and leukemia cells via the caspase dependent pathway. The smaller silver nanoparticles (4.7?nm) had a greater ability to induce apoptosis in both cell lines.     

Modelling the Ecological Vulnerability to Forest Fires in Mediterranean Ecosystems Using Geographic Information Technologies

Forest fires represent a major driver of change at the ecosystem and landscape levels in the Mediterranean region. Environmental features and vegetation are key factors to estimate the ecological vulnerability to fire; defined as the degree to which an ecosystem is susceptible to, and unable to cope with, adverse effects of fire (provided a fire occurs). Given the predicted climatic changes for the region, it is urgent to validate spatially explicit tools for assessing this vulnerability in order to support the design of new fire prevention and restoration strategies. This work presents an innovative GIS-based modelling approach to evaluate the ecological vulnerability to fire of an ecosystem, considering its main components (soil and vegetation) and different time scales. The evaluation was structured in three stages: short-term (focussed on soil degradation risk), medium-term (focussed on changes in vegetation), and coupling of the short- and medium-term vulnerabilities. The model was implemented in two regions: Aragón (inland North-eastern Spain) and Valencia (eastern Spain). Maps of the ecological vulnerability to fire were produced at a regional scale. We partially validated the model in a study site combining two complementary approaches that focused on testing the adequacy of model’s predictions in three ecosystems, all very common in fire-prone landscapes of eastern Spain: two shrublands and a pine forest. Both approaches were based on the comparison of model’s predictions with values of NDVI (Normalized Difference Vegetation Index), which is considered a good proxy for green biomass. Both methods showed that the model’s performance is satisfactory when applied to the three selected vegetation types.     

Evaluation of the Spanish hot dip galvanising sector as a source of polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans

A survey to estimate the polychlorinated dibenzo-p-dioxins (PCDDs) and polychlorinated dibenzofurans (PCDFs) emissions of Spanish hot dip galvanising sector was carried out during 2002. This investigation is the first presenting Spanish experimental data related to this industrial sector. Three different matrices: flue gas, ash and filter dust were tested to quantify the PCDD/Fs generated during the galvanising process. The organic source of PCDD/F formation could be from the insufficient degreasing o from inhibitors or additives used in the pickling steps such as quinoline, isoquinoline, 8-methylquinoline or polyether phosphoric acid. Low levels PCDD/Fs were achieved in air emissions when air control devices are used. On the contrary, filter dusts are highly contaminated; indicating that the absence of air control devices would increase the risk of fugitive emissions. Homologue profiles and Principal Component Analysis demonstrate there are differences in the formation mechanisms in the bath zone (ashes) compared to the stack location (filter dusts and air emissions), related to the de novo synthesis and reaction time. The annual PCDD/F emission to the atmosphere for this sector during 2002 has been estimated in 0.023g I-TEQ. The emission factor of plants with air control devices has been calculated at 0.030microg I-TEQ/ton of galvanised steel.     

Copper-algae interactions: Inheritance or adaptation?

This study evaluated the responses of wild, adult plants of Enteromorpha compressa, and their progeny, to various copper concentrations. Experiments were designed to test the hypotheses that: 1) individuals of E. compressa from Caleta Palito, a copper-enriched coastal locality, tolerate higher copper concentrations than those from a place with no history of copper pollution and 2) such copper tolerance is under genetic control and therefore, was an inherited character. Our results indicate that algae which inhabit a copper-enriched environment tolerate higher concentrations of copper than those from waters with low copper concentrations. On the other hand, our results suggest that generalizations regarding heritability of the tolerance to copper do not apply to the Chilean E. compressa, as no differences in growth or rhizoid production were found between the progeny from Caleta Palito and Caleta Zenteno. These findings are an indication that heritability and adaptation may represent alternative strategies used by different populations of the same algal species to tolerate copper.