EPR spin trapping evaluation of ROS production in human fibroblasts exposed to cerium oxide nanoparticles: Evidence for NADPH oxidase and mitochondrial stimulation

To better understand the antioxidant (enzyme mimetic, free radical scavenger) versus oxidant and cytotoxic properties of the industrially used cerium oxide nanoparticles (nano-CeO(2)), we investigated their effects on reactive oxygen species formation and changes in the antioxidant pool of human dermal and murine 3T3 fibroblasts at doses relevant to chronic inhalation or contact with skin. Electron paramagnetic resonance (EPR) spin trapping with the nitrone DEPMPO showed that pretreatment of the cells with the nanoparticles dose-dependently triggered the release in the culture medium of superoxide dismutase- and catalase-inhibitable DEPMPO/hydroxyl radical adducts (DEPMPO-OH) and ascorbyl radical, a marker of ascorbate depletion. This DEPMPO-OH formation occurred 2 to 24h following removal of the particles from the medium and paralleled with an increase of cell lipid peroxidation. These effects of internalized nano-CeO(2) on spin adduct formation were then investigated at the cellular level by using specific NADPH oxidase inhibitors, transfection techniques and a mitochondria-targeted antioxidant. When micromolar doses of nano-CeO(2) were used, weak DEPMPO-OH levels but no loss of cell viability were observed, suggesting that cell signaling mechanisms through protein synthesis and membrane NADPH oxidase activation occurred. Incubation of the cells with higher millimolar doses provoked a 25-60-fold higher DEPMPO-OH formation together with a decrease in cell viability, early apoptosis induction and antioxidant depletion. These cytotoxic effects could be due to activation of both the mitochondrial source and Nox2 and Nox4 dependent NADPH oxidase complex. Regarding possible mechanisms of nano-CeO(2)-induced free radical formation in cells, in vitro EPR and spectrophotometric studies suggest that, contrary to Fe(2+) ions, the Ce(3+) redox state at the surface of the particles is probably not an efficient catalyst of hydroxyl radical formation by a Fenton-like reaction in vivo.     

Density of insect‐pollinated grassland plants decreases with increasing surrounding land‐use intensity

Pollinator declines have raised concerns about the persistence of plant species that depend on insect pollination, in particular by bees, for their reproduction. The impact of pollinator declines remains unknown for species‐rich plant communities found in temperate seminatural grasslands. We investigated effects of land‐use intensity in the surrounding landscape on the distribution of plant traits related to insect pollination in 239 European seminatural grasslands. Increasing arable land use in the surrounding landscape consistently reduced the density of plants depending on bee and insect pollination. Similarly, the relative abundance of bee‐pollination‐dependent plants increased with higher proportions of non‐arable agricultural land (e.g. permanent grassland). This was paralleled by an overall increase in bee abundance and diversity. By isolating the impact of the surrounding landscape from effects of local habitat quality, we show for the first time that grassland plants dependent on insect pollination are particularly susceptible to increasing land‐use intensity in the landscape.     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

Male-Specific Lethal Mutations of DROSOPHILA MELANOGASTER

A total of 7,416 ethyl methanesulfonate (EMS)-treated second chromosomes and 6,212 EMS-treated third chromosomes were screened for sex-specific lethals. Four new recessive male-specific lethal mutations were recovered. When in homozygous condition, each of these mutations kills males during the late larval or early pupal stages, but has no detectable effect in females. One mutant, mle^ts, is a temperature sensitive allele of maleless, mle (Fukunaga, Tanaka and Oishi 1975), while the other three mutants identify two new loci: male-specific lethal-1 (msl-1) (two alleles) at map position 2-53.3 and male-specific lethal-2 (msl-2) at 2-9.0.——The male-specific lethality associated with these mutants is not related to the sex per se of the mutant flies, since sex-transforming genes fail to interact with these mutations. Moreover, the presence or absence of a Y chromosome in males or females has no influence on the male-specific lethal action of these mutations. Finally, no single region of the X chromosome, when present as a duplication, is sufficient to rescue males from the lethal effects of msl-1 or msl-2. These results suggest that the number of complete X chromosomes determines whether a fly homozygous for a male-specific lethal mutation lives or dies.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.     

Mutational Events in the Triplo- and Haplo-Lethal Region (83de) of the DROSOPHILA MELANOGASTER Genome

The Drosophila melanogaster genome contains a single region (at 83DE on the polytene chromosome map) for which both heterozygous deficiency and heterozygous duplication are inviable. Seven EMS-induced mutations have been recovered that are viable in combination with a duplication of this region. Two classes of mutations are reported: (1) Mutations that allow survival of flies with either a duplication or a normal third chromosome. These mutations retain Ki, a closely linked marker on the mutagenized chromosome. They fail to complement, and one has been mapped to the vicinity of 83DE. (2) Mutations that allow survival only in heterozygous combination with a duplication and have lost the Ki marker. These mutations represent new deletions of the dose-sensitive information. The possible structural organization of the 83DE region is discussed in light of these two classes of mutations.     

Analysis of the blocking activity of charybdotoxin homologs and iodinated derivatives against Ca2+-Activated K+ channels

Summary Two charybdotoxin peptides were purified from venom of the Israeli scorpion,Leiurus quinquestriatus hebraeus. Microsequencing of the most abundant toxin, ChTX-Lq1, revealed identity with the 37-residue peptide previously sequenced by Gimenez-Gallego et al. [Gimenez-Gallego, G., et al.,Proc. Natl. Acad. Sci. USA 85:3329–3333 (1988)]. Sequence data on the minor peptide, ChTX-Lq2, showed substantial homology to ChTX-Lq1 with differences observed at eight positions. These two charybdotoxin sequences, along with that of noxiustoxin, define a distinct family of scorpion peptide toxins with activity against K⁺ channels. Both charybdotoxin homologs inhibited Ca²⁺-dependent K⁺ efflux from human erythrocytes with similar potency,K _0.5-40nm. In planar bilayer assays of single K(Ca) channels from rat muscle, ChTX-Lq1 and ChTX-Lq2 blocked with intrinsicK _d's of 1.3 and 43nm, respectively, in the presence of 50mm external KCl. A new application of dwell-time histogram analysis of single-channel blocking events was used to characterize the kinetic homogeneity of toxin samples and the blocking kinetics of ChTX derivatives. The lower blocking affinity of ChTX-Lq2 was the combined result of a faster dissociation rate and a slower association rate as compared to ChTX-Lq1. The blocking activity of two mono-iodinated derivatives of ChTX-Lq1 was also analyzed. Blocked dwell-time histograms of the iodinated peptides were characterized by predominately brief (0.2–2 sec) blocking events in comparison to the native toxin (20 sec). Histogram analysis revealed that mono-iodination of ChTX-Lq1 impairs blocking activity by adverse effects on both dissociation and association rate constants. Frequency density histograms of single channel blocking events provide a sensitive assay of toxin purity suitable for quantitating structure-activity relationships of charybdotoxin derivatives.     

Choline and betaine as inducer agents of Pseudomonas aeruginosa phospholipase C activity in high phosphate medium

In Pseudomonas aeruginosa, choline or betaine employed as the sole carbon and nitrogen source in a high phosphate medium induced a phospholipase C and an acid phosphatase activity but not an alkaline phosphatase activity. The P. aeruginosa strain utilized in this work does not possess a constitutive phospholipase C, since under culture conditions identical to those utilized by other authors (J. Bacteriol. 93, 670-674 (1967) and J. Bacteriol. 150, 730-738 (1982), our phospholipase C proved to be an inorganic phosphate-repressible enzyme. These findings enable us to conclude that although the phosphate control for the synthesis of phospholipase C may exist, it is expressed only under certain favorable culture conditions.