The evaluation of the oxidative state of native-LDL: three methods compared

LDL-oxidation is considered a contributing factor to the development of atherosclerotic lesions. However, to utilise the oxidative state of LDL as a marker of cardiovascular risk, reliable analytical methods for its detection must be defined. We have compared three methods for their capacity to evaluate the difference in the oxidation state of isolated LDL subjected to either dialysis (D-LDL) or gel filtration (F-LDL) to remove EDTA. Their susceptibility to oxidation promoted by Cu(2+) was monitored by following the time course of conjugated diene (CD) and lipid hydroperoxide (ROOH) accumulation. The relative electrophoretic mobility (REM) of the same LDL samples was evaluated by capillary electrophoresis. As measured by all three methods, F-LDL are less prone to oxidation than D-LDL when added with CuSO(4). REM of F-LDL and D-LDL significantly differs already before the addition of the metal catalyst, whereas CD and ROOH contents become significantly different only after it. Besides confirming that a rapid centrifugation followed by gel filtration is a more convenient procedure than dialysis to remove EDTA during LDL isolation, our study suggests the REM of isolated-LDL as the biochemical marker of choice in the evaluation of its oxidative state.     

Nonintrusive Field Experiments Show Different Plant Responses to Warming and Drought Among Sites,Seasons, and Species in a North–South European Gradient

We used a novel, nonintrusive experimental system to examine plant responses to warming and drought across a climatic and geographical latitudinal gradient of shrubland ecosystems in four sites from northern to southern Europe (UK, Denmark, The Netherlands, and Spain). In the first two years of experimentation reported here, we measured plant cover and biomass by the pinpoint method, plant ¹⁴C uptake, stem and shoot growth, flowering, leaf chemical concentration, litterfall, and herbivory damage in the dominant plant species of each site. The two years of approximately 1°C experimental warming induced a 15% increase in total aboveground plant biomass growth in the UK site. Both direct and indirect effects of warming, such as longer growth season and increased nutrient availability, are likely to be particularly important in this and the other northern sites which tend to be temperature-limited. In the water-stressed southern site, there was no increase in total aboveground plant biomass growth as expected since warming increases water loss, and temperatures in those ecosystems are already close to the optimum for photosynthesis. The southern site presented instead the most negative response to the drought treatment consisting of a soil moisture reduction at the peak of the growing season ranging from 33% in the Spanish site to 82% in The Netherlands site. In the Spanish site there was a 14% decrease in total aboveground plant biomass growth relative to control. Flowering was decreased by drought (up to 24% in the UK and 40% in Spain). Warming and drought decreased litterfall in The Netherlands site (33% and 37%, respectively) but did not affect it in the Spanish site. The tissue P concentrations generally decreased and the N/P ratio increased with warming and drought except in the UK site, indicating a progressive importance of P limitation as a consequence of warming and drought. The magnitude of the response to warming and drought was thus very sensitive to differences among sites (cold-wet northern sites were more sensitive to warming and the warm-dry southern site was more sensitive to drought), seasons (plant processes were more sensitive to warming during the winter than during the summer), and species. As a result of these multiple plant responses, ecosystem and community level consequences may be expected.     

Increase in therapeutic index of doxorubicin and vinblastine by aptameric oligonucleotide in human T lymphoblastic drug-sensitive and multidrug-resistant cells

Aptameric GT oligomers are a new class of potential anticancer molecules that inhibit the growth of human cancer cell lines by binding to specific nuclear proteins. We demonstrated that an aptameric GT oligonucleotide increased the therapeutic index of doxorubicin and vinblastine in T lymphoblastic drug-sensitive and multidrug-resistant (MDR) cells. The doxorubicin ID50 decreased 6.5-fold by coadministration of 1 microM GT to CCRF-CEM cells and by 24-fold by coadministration of 0.75 microM GT to CEM-VLB300 cells. In CEM-VLB300 cells, the vinblastine ID50 decreased 11-fold by coadministration of 0.5 microM GT. Control CT sequence did not potentiate the drugs in either CCRF-CEM or CEM-VLB300 cells. The ability of GT to bind to specific nuclear proteins in cancer cells related to the increase in the therapeutic index of doxorubicin and vinblastine. No cooperation was detected by the administration of GT oligomer together with doxorubicin to rat differentiated thyroid FRTL-5 cells and to normal human lymphocytes. These cells did not show binding of GT to the specific nuclear proteins, and they were not sensitive to the cytotoxic action of the GT sequence. Drug potentiation by GT not involving normal human lymphocytes might be exploited to develop a more selective treatment of drug-sensitive and MDR tumors.     

Colony Age of Trichoderma azevedoi Alters the Profile of Volatile Organic Compounds and Ability to Suppress Sclerotinia sclerotiorum in Bean Plants

Common bean (Phaseolus vulgaris L.) is one of the most important crops in human food production. The occurrence of diseases, such as white mold, caused by Sclerotinia sclerotiorum can limit the production of this legume. The use of Trichoderma has become an important strategy in the suppression of this disease. The aim of the present study was to evaluate the effect of volatile organic compounds (VOCs) emitted by Trichoderma azevedoi CEN1241 in five different growth periods on the severity of white mold in common bean. The in vitro assays were carried out in double-plate and split-plate, and the in vivo assays, through the exposure of the mycelia of S. sclerotiorum to the VOCs of T. azevedoi CEN1241 and subsequent inoculation in bean plants. Chemical analysis by gas chromatography coupled to mass spectrometry detected 37 VOCs produced by T. azevedoi CEN1241, covering six major chemical classes. The profile of VOCs produced by T. azevedoi CEN1241 varied according to colony age and was shown to be related to the ability of the biocontrol agent to suppress S. sclerotiorum. T. azevedoi CEN1241 VOCs reduced the size of S. sclerotiorum lesions on bean fragments in vitro and reduced disease severity in a greenhouse. This study demonstrated in a more applied way that the mechanism of antibiosis through the production of volatile compounds exerted by Trichoderma can complement other mechanisms, such as parasitism and competition, thus contributing to a better efficiency in the control of white mold in bean plants.     

Direct Visualization of the Action of Triton X-100 on Giant Vesicles of Erythrocyte Membrane Lipids

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.     

Neuroprotective and anticonvulsant effects of organic and conventional purple grape juices on seizures in Wistar rats induced by pentylenetetrazole

Epilepsy is the most common neurological disorder worldwide. Studies have shown that recurrent seizures may increase the concentration of reactive oxygen species, which can lead to oxidative stress and neuronal damage. These seizures result in substantial deleterious effects on an individual's health. Organic and conventional grape juices are rich in polyphenols, compounds with important antioxidant activity. However, these juices could have differences in their polyphenol content. The aim of this study was to investigate the neuroprotective and anticonvulsant effects of organic and conventional grape juice treatments in Wistar rats against pentylenetetrazole (a convulsant drug)-induced damage. In addition, we evaluated potential behavioral changes in rats treated with the juices and the polyphenolic profile of those samples. Animals (n=16 in each group) received treatment with saline, organic or conventional grape juice for 17 days. On the eighteenth day, behavioral changes were evaluated by an open field test. Afterwards, half of the rats from each group received pentylenetetrazole and were observed for 30 min to evaluate possible seizure characteristics. The animals were subsequently killed by decapitation and their hippocampus, cerebellum and cerebral cortex tissues were isolated. The results of this study showed that neither organic nor conventional grape juice altered the behavior parameters, and no statistical differences were observed in the seizure characteristics of the groups. Nevertheless, both juice types were able to protect from lipid and protein oxidative damage, decrease nitric oxide content and increase enzymatic (superoxide dismutase and catalase) and non-enzymatic (sulfhydryl protein) antioxidant defenses in brain tissues following pentylenetetrazole-induced seizures. In general, organic juice showed superior results in each test, probably due to its higher polyphenol content relative to conventional juice. These results indicate that grape juices can provide further insight into natural neuroprotective compounds and may lead to the development of new therapeutic strategies for epileptic patients.     

Toxicity of rhizomes of the invasive Hedychium coronarium (Zingiberaceae) on aquatic species

The production and release of chemical compounds by invasive plants can affect competitors and native species overall, destabilizing ecological interactions and harming ecosystem functioning. Hedychium coronarium is an invasive macrophyte common on Brazilian riparian areas that produces a wide variety of allelochemicals, but little is known about their effect on aquatic species. Here, we identified the major chemical compounds of the aqueous extract of H. coronarium rhizomes and assessed its toxicity, evaluating the growth inhibition of one alga (Raphidocelis subcapitata) and one macrophyte (Lemna minor), and the lethality of cladoceran (Ceriodaphnia silvestrii and Daphnia similis) and Chironomidae larvae (Chironomus sancticaroli). The majoritarian compounds of H. coronarium rhizomes were Coronarin D and Coronarin D Ethyl Ether. The aqueous extract was toxic for all tested species. We observed growth inhibition in R. subcapitata, as well as reduction in biomass in L. minor. Chironomus sancticaroli and cladoceran were the most sensible species. The aqueous extract of H. coronarium rhizomes was toxic on tested conditions, suggesting that the rhizome compounds may interfere on aquatic organisms and in the dynamic of trophic webs of aquatic ecosystems on invaded areas.