[Show abstract][Hide abstract] ABSTRACT: 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 DPPC 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 FCCP-uncoupled respirations are inhibited by both fullerenes when glutamate/malate or succinate was used as substrates. 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 per 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 ADP 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: In the search for new leishmanicidal agents, Thymus capitellatus Hoffmanns. & Link (family Lamiaceae) volatile extract and its major compounds, 1,8-cineole and borneol, were tested against Leishmania infantum, L. tropica and L. major. Plant volatile extract (essential oil) was analysed by GC and GC-MS and the activity of essential oil on Leishmania promastigotes viability was assessed using tetrazolium-dye colorimetric method (MTT). The MTT test was also used to assess the cytotoxicity of essential oil on macrophages and bovine aortic endothelial cells. Effects on parasites were also analyzed by flow cytometry in order to assess mitochondrial transmembrane electrochemical gradient (JC-1), analyze phosphatidylserine externalization (annexin V–FITC, propidium iodide) and evaluate cell cycle (DNase-free, RNase, PI). Morphological and ultrastructural studies were performed by light, scanning and transmission electron microscopy. T. capitellatus volatile extract exhibited anti-parasite activity on Leishmania species, with IC50 values ranging from 35 to 62 μg/ml. However, major compounds 1,8-cineole and borneol did not showed biological activity suggesting that these monoterpenes are not responsible for the antileishmanial activity of T. capitellatus essential oil. Appearance of aberrant-shaped cells, mitochondrial swelling and autophagosomal structures were some of the ultrastructural alterations exhibited among treated promastigote cells. T. capitellatus promoted leishmanicidal effect by triggering a programmed cell death as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. The volatile extract did not induced cytotoxic effects on mammalian cells. Taken together, these results suggest that T. capitellatus may represent a valuable source for therapeutic control of leishmaniasis in humans and animals.
[Show abstract][Hide abstract] ABSTRACT: In order to contribute for the search of new drugs for leishmaniasis, we study the susceptibility of Leishmania infantum, Leishmania tropica and Leishmania major to Cymbopogon citratus essential oil and major compounds, mrycene and citral. C. citratus and citral were the most active inhibiting L. infantum, L. tropica and L. major growth at IC(50) concentrations ranging from 25 to 52 μg/ml and from 34 to 42 μg/ml, respectively. L. infantum promastigotes exposed to essential oil and citral underwent considerable ultrastructural alterations, namely mitochondrial and kinetoplast swelling, autophagosomal structures, disruption of nuclear membrane and nuclear chromatin condensation. C. citratus essential oil and citral promoted the leishmanicidal effect by triggering a programmed cell death. In fact, the leishmanicidal activity was mediated via apoptosis as evidenced by externalization of phosphatidylserine, loss of mitochondrial membrane potential, and cell-cycle arrest at the G(0)/G(1) phase. Taken together, ours findings lead us to propose that citral was responsible for anti-Leishmania activity of the C. citratus and both may represent a valuable source for therapeutic control of leishmaniasis.
[Show abstract][Hide abstract] ABSTRACT: To better understand the taxonomy of Pulicaria, the pollen wall architecture of the six Iberian species were investigated using light (LM) and scanning electron microscopy
(SEM). The exine structure of Pulicaria odora was also investigated using transmission electron microscopy (TEM). Statistical analysis was performed to distinguish taxonomically
significant morphometric information from all the measured parameters of pollen grains. It was found that the exine sculpture
characters, with special importance paid to the spines, were the most useful of all characters to define Pulicaria pollen types and separate the species. Three pollen types distinguishable through the spines morphology and the inter-spinular
sculpture are described: P. microcephala pollen type (incl. P. microcephala), P. vulgaris pollen type (incl. P. vulgaris), and P. dysenterica pollen type (incl. P. dysenterica, P. odora, P. paludosa and P. sicula). A dichotomous key to these Pulicaria pollen types is proposed. The distribution of P. dysenterica, P. odora, P. paludosa and P. sicula in more than one leaf node in the classification tree reveals that the pollen grains of these species are difficult to segregate.
Therefore, the construction of a satisfactory dichotomous key to the P. dysenterica pollen type species is not feasible. Yet, the different spines apex morphology between P. microcephala and P. paludosa and the existence of significant differences in five of the eight studied quantitative pollen characters of these two taxa,
supports the opinion that the Berlengas Islands endemic P. microcephala should be accepted as a separate species. In addition, the differences among the spines morphology of P. vulgaris, P. microcephala, and the other four Iberian (and European) species, strengthen the conclusion that the section Pulicaria is non-monophyletic.
[Show abstract][Hide abstract] ABSTRACT: The present work evaluates the anti-Giardia activity of Syzygium aromaticum and its major compound eugenol. The effects were evaluated on parasite growth, adherence, viability and ultrastructure. S. aromaticum essential oil (IC(50)=134 μg/ml) and eugenol (IC(50)=101 μg/ml) inhibited the growth of G. lamblia. The essential oil inhibited trophozoites adherence since the first hour of incubation and was able to kill almost 50% of the parasites population in a time dependent manner. The eugenol inhibited G. lamblia trophozoites adherence since the third hour and not induce cell lyses. The main morphological alterations were modifications on the cell shape, presence of precipitates in the cytoplasm, autophagic vesicles, internalization of flagella and ventral disc, membrane blebs, and intracellular and nuclear clearing. Taken together, our findings lead us to propose that eugenol was responsible for the anti-giardial activity of the S. aromaticum essential oil and both have potential for use as therapeutic agents against giardiasis.
[Show abstract][Hide abstract] ABSTRACT: To investigate the role of the plasmalemmic cord in the pollen grains of members of the Magnoliaceae, anthers of Magnolia × soulangeana Soul.-Bod. were incubated for 1, 2.5, and 5 h in lead and lanthanum salt solutions. Ultrastructural analysis revealed differences in the labelling of the pollen grains with the time of exposure to the heavy metal salt, which is likely related to metal tolerance mechanisms operating in the pollen grains. Within 1 h of treatment, both tracers produced a fine precipitate lining the entire vegetative cell (VC) plasmalemma (including the plasmalemmic cord) as well as the generative cell (GC) plasmalemma. Heavy deposits were not found inside both the VC and the GC. At later times, the amount of heavy deposits increased on the pollen wall surface and, particularly, in the two outer intine layers. Also, heavy deposits were found in membrane-bound cell components of the VC and the GC, but never in the cytosol. In the VC, the cell components more frequently labelled were the protein storage vacuoles. In both pollen cells, multivesicular bodies, dictyosome cisternae, and small vesicles were also labelled. We hypothesize that the latter organelles participate in the metal ions accumulation (end of the trip), together with the protein vacuoles, or in the transport of metal ions to the vacuoles following the endocytotic uptake of these ions. The presence of both coated and uncoated pits and vesicles in the treated and the untreated pollen reinforces the latter hypothesis. Another significant result was that the tracers, after diffusing through the microchannels in the exine and crossing the intine layers, moved through the plasmalemmic cord to reach the GC periplasm. We suggest that the plasmalemmic cord may provide a system of communication for the flux of solutes from the anther loculus to both pollen cells and (or) from the VC to the GC.Key words: pollen grain, ultrastructure, metal uptake, metal tolerance, lead nitrate, lanthanum nitrate.
Canadian Journal of Botany 02/2011; 82(12):1758-1767. DOI:10.1139/b04-133 · 1.40 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lavanders (Lavandula spp.) are aromatic plants with high industrial and commercial value, used in food industry, perfumery and pharmaceutical preparations. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Lavandula pedunculata and to evaluate the potential of these plants for essential oil production with industrial application.In vitro cultures were established on media with different concentrations of benzyladenine. Trichome morphology was examined by SEM and their secretion analyzed by histochemical tests. Essential oils were isolated by hydrodistillation and analyzed by GC and GC-MS. The essential oils and trichomes of in vitro plants were compared with those of field-growing plants.Best propagation rates were achieved with 0.25mg/l BA. Rooting occurred without treatment with auxins thus preventing callus growth. Trichomes and essential oils of the in vitro propagated plants were similar to those of the parent plants. Two chemotypes were characterized: 1,8-cineole/camphor type and fenchone type.Since trichomes and essential oils of L. pedunculata micropropagated plantlets are identical to those produced by field-growing plants, in vitro cultures can be used for essential oil production without affecting natural resources. Moreover, the quality of the essential oils is assured and this protocol can be further applied to the propagation of selected chemotypes for industrial purposes.
[Show abstract][Hide abstract] ABSTRACT: 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.
Parasitology Research 03/2010; 106(5):1205-15. DOI:10.1007/s00436-010-1800-7 · 2.10 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To better understand relationships within the Asteroideae, the pollen morphology and exine structure of 10 genera and 15 species
of Gnaphaliinae were investigated using light, scanning electron, and transmission electron microscopy. All taxa have a Gnaphalioid
pattern of exine with an evidently rough foot layer. The tectal complex consists of three main layers that differ in morphology
and thickness: a tectum, a median columellar layer, and an internal interlaced sub-columellar layer. The apertural system
consists of an ectoaperture, a mesoaperture, and an endoaperture, which intersect, respectively, the tectal complex, the foot
layer and the upper part of the endexine, and the inner part of the endexine. On the basis of pollen characteristics, especially
those of the spines, all the species examined may be gathered into two groups, one including Helichrysum foetidum, H. italicum, Plecostachys serpyllifolia, and Pseudognaphalium luteo-album and the other including the other 11 species of Gnaphaliinae now investigated. In addition, because Gnaphalieae has been
proposed as sister group to several tribes and clades of Asteroideae, a comparison was made between exine pattern in the Gnaphalieae
and that in its putatively related tribes. On the basis of previous phylogenetic studies and our present pollen data we suggest
that either Astereae, Astereae-Anthemideae clade, or Heliantheae s.l.-Anthemideae clade are the best candidates for the sister
taxon/sister group of Gnaphalieae.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Dendritic cells (DC) are major players in initiating immune responses by activating T-lymphocytes. They act as sentinels in peripheral tissues, continuously scavenging for antigens in their immediate surroundings. Their involvement in T-cell responses consists of a linear progression of events starting with capture of antigens in peripheral tissues, such as the skin, followed by migration to draining lymphoid organs and presentation of antigen-derived peptides to induce T-cell priming. In the last years, a number of DC lines have been generated and the electron microscopy has been used to define their characteristics, thus contributing to their formal validation. A cell line with features of early DC precursors was previously established from fetal mouse skin (FSDC), but its ultrastructural organization was not fully investigated. Here we report such investigation using light (LM), scanning electron (SEM) and transmission electron microscopy (TEM). The goal is to use FSDC as a model system to evaluate the safety and effectiveness of selected plant extracts, fractions and isolated polyphenols against inflammatory processes. Our preliminary results with these cells showed anti-inflammatory activity for some of the extracts we are currently investigating.
Microscopy and Microanalysis 07/2009; 15(S3). DOI:10.1017/S1431927609990638 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lavandula species are aromatic plants that produce and accumulate essential oils in different types of glandular trichomes. The oils protect the aerial parts of the plant against herbivores and pathogens, and are of great economic value for fragrance, pharmaceutical, food and flavour industries. In Portugal there are five spontaneous species of Lavandula which are included in three different sections: Section Lavandula (L. latifolia), Section Pterostoechas (L. multifida) and Section Stoechas (L. luisieri, L. pedunculata, L. viridis). Our preliminary observations showed that these species have a high diversity of trichomes. This, associated with the fact that trichome morphology is often referred as an useful characteristic for systematic purposes, led us to investigate leaf trichome morphology in the five Portuguese species, using scanning electron microscopy (SEM).
Microscopy and Microanalysis 07/2009; 15(S3). DOI:10.1017/S143192760999064X · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Pulicaria is the third largest genus of the tribe Inuleae. It comprises ca. 80 species of shrubs, shrublets and herbs with a European, North African and Asian distribution. All of the 5 European species are represented in the Iberian Peninsula. The genus has taxonomic problems, as recent cladistic analysis showed that, like the other two great genera of Inuleae (Inula and Blumea), Pulicaria is a paraphyletic taxon. In fact, the species investigated so far were placed in two distinct clades. Moreover, the species status of Pulicaria microcephala, a Portuguese endemism of Berlengas Islands, is still controversial. As already demonstrated by several authors, the palynological data are of great importance to understand the evolution, taxonomy and ecology of the Asteraceae. However, in spite of a few relevant contributions, the palynology of Pulicaria is still insufficiently known. In the present work we investigated the palynology of the Iberian species of Pulicaria using light (LM) and scanning electron microscopy (SEM). The exine structure of Pulicaria paludosa was also investigated using transmission electron microscopy (TEM).
Microscopy and Microanalysis 07/2009; 15(S3). DOI:10.1017/S1431927609990626 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The selection of native Lavandula species and their economic exploitation have increased in the last few years. Micropropagation techniques have been used as an alternative for vegetative propagation allowing the multiplication of selected genotypes and chemotypes. Our previous studies showed that the essential oils of Lavandula pedunculata have an important antifungal activity against dermatophyte strains. Therefore, a new line of investigation concerning the in vitro culture of this species is justified. In the present study we compare the morphology of the leaf trichomes and the chemical composition of their essential oils in both field-growing and in vitro propagated plants.
Microscopy and Microanalysis 09/2008; 14. DOI:10.1017/S143192760808971X · 1.88 Impact Factor