Interaction of fullerene nanoparticles with biomembranes: from the partition in lipid membranes to effects on mitochondrial bioenergetics.

Partition and localization of C60 and its derivative C60(OH)18-22 in lipid membranes and their impact on mitochondrial activity were studied, attempting to correlate those events with fullerene characteristics (size, surface chemistry, and surface charge). Fluorescence quenching studies suggested that C60(OH)18-22 preferentially populated the outer regions of the bilayer, whereas C60 preferred to localize in deeper regions of the bilayer. Partition coefficient values indicated that C60 exhibited higher affinity for dipalmitoylphosphatidylcholine and mitochondrial membranes than C60(OH)18-22. Both fullerenes affected the mitochondrial function, but the inhibitory effects promoted by C60 were more pronounced than those induced by C60(OH)18-22 (up to 20 nmol/mg of mitochondrial protein). State 3 and p-trifluoromethoxyphenylhydrazone-uncoupled respirations are inhibited by both fullerenes when glutamate/malate or succinate was used as substrate. Phosphorylation system and electron transport chain of mitochondria are affected by both fullerenes, but only C60 increased the inner mitochondrial membrane permeability to protons, suggesting perturbations in the structure and dynamics of that membrane. At concentrations of C60(OH)18-22 above 20 nmol/mg of mitochondrial protein, the activity of FoF1-ATP synthase was also decreased. The evaluation of transmembrane potential showed that the mitochondria phosphorylation cycle decreased upon adenosine diphosphate addition with increasing fullerenes concentration and the time of the repolarization phase increased as a function of C60(OH)18-22 concentration. Our results suggest that the balance between hydrophilicity and hydrophobicity resulting from the surface chemistry of fullerene nanoparticles, rather than the cluster size or the surface charge acquired by fullerenes in water, influences their membrane interactions and consequently their effects on mitochondrial bioenergetics.

Studies on the toxicity of an aqueous suspension of C60 nanoparticles using a bacterium (gen. Bacillus) and an aquatic plant (Lemna gibba) as in vitro model systems.

The increasing use of C60 nanoparticles and the diversity of their applications in industry and medicine has led to their production in a large scale. C60 release into wastewaters and the possible accumulation in the environment has raised concerns about their ecotoxicological impact. In the present study, an aqueous suspension of C60 nanoparticles was prepared and its potential toxicity studied in laboratory, using a bacterium (Bacillus stearothermophilus) and an aquatic plant (Lemna gibba) as model systems. C60 nanoparticles inhibited the growth of L. gibba, in contrast to that of the bacterium. Consistently, the ultrastructure and respiratory activity of bacterial cells were not affected by C60, but the contents of chlorophylls a and b and chloroplast oxygen production decreased considerably in L. gibba. Altogether, our results suggest that C60 aqueous dispersions must be viewed as an environmental pollutant, potentially endangering the equilibrium of aquatic ecosystems.

Anti-Giardia activity of phenolic-rich essential oils: effects of Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris, and Lippia graveolens on trophozoites growth, viability, adherence, and ultrastructure.

The present work evaluates the anti-Giardia activity of phenolic-rich essential oils obtained from Thymbra capitata, Origanum virens, Thymus zygis subsp. sylvestris chemotype thymol, and Lippia graveolens aromatic plants. The effects were evaluated on parasite growth, cell viability adherence, and morphology. The tested essential oils inhibited the growth of Giardia lamblia. T. capitata essential oil is the most active followed by O. virens, T. zygis subsp. sylvestris, and L. graveolens oils. The tested essential oils at IC50 (71-257) microg/ml inhibited parasite adherence (p < 0.001) since the first hour of incubation and were able to kill almost 50% of the parasites population in a time-dependent manner. The main ultrastructural alterations promoted by essential oils were deformations in typical trophozoite appearance, often roundly shape, irregular dorsal and ventral surface, presence of membrane blebs, electrodense precipitates in cytoplasm and nuclei, and internalization of flagella and ventral disc. Our data suggest that essential oils induced cell death probably by processes associated to the loss of osmoregulation caused by plasmatic membrane alterations. Experiments revealed that the essential oils did not present cytotoxic effects in mammalian cells. In conclusion, T. capitata, O. virens, T. zygis subsp. sylvestris chemotype thymol, and L. graveolens essential oils have antigiardial activity in vitro and seem to have potential for the treatment of the parasitic disease caused by the protozoan G. lamblia.

Interaction of lipid bodies with other cell organelles in the maturing pollen of Magnolia x soulangeana (Magnoliaceae).

The pollen grain maturation in Magnolia x soulangeana was studied ultrastructurally and cytochemically using both the light and transmission electron microscope. Emphasis was given on the storage lipid bodies of the vegetative cell (VC) and their interaction with other cell organelles. Stereological analysis of electron micrographs was performed to evaluate the variation in volume density (V(V)), surface density, and surface-to-volume ratio (S/V) of various cell organelles during pollen maturation. The size and numerical density of the lipid bodies, and their frequency of association with other cell organelles, were also determined. It was noted that during pollen ontogeny and maturation, the lipid bodies changed their pattern of distribution in the VC cytoplasm, which may be a good marker for the succeeding stages of pollen development. Also, the size, osmiophily, and V(V) of the lipid bodies were progressively reduced during pollen maturation whereas the S/V was significantly increased. This seems to indicate that the lipid bodies are mobilized in part during this period of pollen maturation. In particular, the intermediate and mature pollen showed a high percentage of lipid bodies establishing a physical contact with either glyoxysomes, either protein storage vacuoles, or small vesicles presumably originated from dictyosomes. This physical contact was found in both the chemically fixed and rapid freeze-fixed pollen indicating that it is neither artifactual nor casual. On the basis of this intimate association with other cell organelles and the morphometric analysis performed, we suggest that the mobilization of lipid bodies is likely mediated not only by glyoxysomes but also by other catabolic pathways involving the interaction of lipid bodies with either protein storage vacuoles or small Golgi vesicles.

Chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav.

The chemical composition and antifungal activity of the essential oils of Lavandula pedunculata (Miller) Cav., harvested in North and Central Portugal, were investigated. The essential oils were isolated by hydrodistillation and analyzed by GC and GC/MS. The minimal-inhibitory concentration (MIC) and the minimal-lethal concentration (MLC) of the essential oils and of their major constituents were used to evaluate the antifungal activity against different strains of fungi involved in candidosis, dematophytosis, and aspergillosis. The oils were characterized by a high percentage of oxygenated monoterpenes, the main compounds being 1,8-cineole (2.4-55.5%), fenchone (1.3-59.7%), and camphor (3.6-48.0%). Statistical analysis differentiated the essential oils into two main types, one characterized by the predominance of fenchone and the other one by the predominance of 1,8-cineole. Within the 1,8-cineole chemotype, two subgroups were well-defined taking into account the percentages of camphor. A significant antifungal activity of the oils was found against dermatophyte strains. The essential oil with the highest content of camphor was the most active with MIC and MLC values ranging from 0.32-0.64 microl/ml.

Differential roles of PI3-Kinase, MAPKs and NF-kappaB on the manipulation of dendritic cell T(h)1/T(h)2 cytokine/chemokine polarizing profile.

Dendritic cells (DC) are professional antigen-presenting cells with a unique capacity to initiate and modulate immune responses by their ability to prime naïve T-cells. Upon stimuli, DC experience several morphologic, phenotypic and functional changes in a process referred to as maturation. This process is crucial to the biological functions of DC since their maturation status confer them the ability to polarize distinct T-cell subsets. In this work we explored the relevance of PI3-Kinase, Mitogen-Activated Protein Kinases (MAPKs) and NF-kappaB on cytokines/chemokines and co-stimulatory molecules expression. As experimental model, we used a fetal skin-derived dendritic cell line (FSDC) induced to mature by treatment with lipopolysaccharide (LPS). Morphology and ultrastructure were analyzed by confocal and electron microscopies, respectively. Levels of phosphorylated proteins were evaluated by Western blot, production of cytokines/chemokines was analyzed by protein arrays and the expression of surface molecules was evaluated by flow cytometry. The effect of specific inhibitors of the studied signaling pathways on the transcription of cytokines/chemokines and co-stimulatory molecules was accessed by Quantitative Real-Time RT-PCR. The results showed that LPS induces significant morphological and ultrastructural changes in FSDC. Western blot analysis revealed that LPS challenge promotes an early and transient activation of NF-kappaB, ERK1/2, p38 MAPK, along with a more sustained PI3 kinase/AKT activation. The co-stimulatory CD40, CD80, CD86 and antigen-presenting MHC class I and II molecules were increased and among secreted molecules, interleukin IL-6, CCL5, G-CSF, CCL2, CXCL2 were strongly up-regulated. Using a pharmacological approach we observed that LPS-induced increase of these molecules was differentially regulated by the distinct signaling pathways. Moreover, the polarizing T(h)2 cytokines/chemokines induced by LPS in FSDC were found to be positively regulated by NF-kappaB and ERK and negatively modulated by p38 MAPK. Altogether these results suggest that the use of pharmacological inhibitors to manipulate DC maturation, namely the polarizing T(h)1/T(h)2 cytokine/chemokine profile, may be useful in the development of more specific immunotherapeutic protocols.

Non-selective toxicological effects of the insect juvenile hormone analogue methoprene. A membrane biophysical approach.

The Gram-positive bacterium, Bacillus stearothermophilus, was used as a model organism to identify the non-selective toxic effects of the currently used insecticide methoprene (isopropyl(2E,4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate). A significant decrease of the yield of bacterial cultures and a premature appearance of ultrastructural abnormalities in cells cultured in the presence of the insecticide were taken as indicators of cytotoxicity. A putative correlation of this cytotoxicity with methoprene-induced perturbations on membrane lipid organization was investigated, using differential scanning calorimetry and the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its propionic acid derivative (DPH-PA). The membrane physical effects depended on the lipid bilayer composition and packing. The most striking effect was a progressive broadening and shifting to lower temperatures, with increasing methoprene concentrations, of the main transition phase of the dimyristoyl- or dipalmitoylphosphatidylcholine bilayers and of the lateral phase separation of liposomes reconstituted with the lipid extracts of B. stearothermophilus.