Biocompatible hybrids based on nanographene oxide covalently linked to glycolporphyrins: synthesis, characterization and biological evaluation

The major limitation in the development of hybrids based on graphene oxide (GO) and porphyrins is their dispersibility and stability in aqueous systems due to the hydrophobic character induced by porphyrins. Most of the previous approaches reported the direct functionalization of GO with polyethylene glycol (PEG) chains followed by the self-assembly of porphyrins by π-π interactions. Here, new hybrids were prepared using porphyrins previously functionalized with different number/types of glycol branches to be covalently attached through esterification to the carboxyl groups of GO sheets of nanometric dimensions. The number of the glycol chains and its relative position in the porphyrin core showed to be fundamental to improve the hybrids dispersion and stability in aqueous solutions. The best performing hybrids were characterized by transmission electron microscopy, X-ray photoelectron spectroscopy, Fourier transform infrared, UV-Vis absorption and fluorescence spectroscopy. The in vitro biocompatibility assessment of these hybrids was conducted using human Saos-2 cells. Their effects on cell proliferation and viability, the generation of reactive oxygen species as well as the cell morphology after cell uptake were analysed. The results demonstrate the biocompatibility of these hybrid nanomaterials with human Saos-2 cells, which is very promising for future application in biomedicine namely in cancer therapy.publishe

The inhibitory effect of aluminium on the (Na+/K+)ATPase activity of rat brain cortex synaptosomes

Partilhar no CESAMThe effect of AlCl3 on the (Na+ /K+)ATPase activity of freeze-thawed synaptosomes, isolated from rat brain cortex, has been studied. The AlCl3 action on the enzyme hydrolytic activity was examined using in vitro and in vivo approaches. Following exposure to AlCl3 using both in vitro (synaptosomes incubated in the presence of AlCl3 for 5 min) and in vivo (synaptosomes isolated from rats that received 0.03 g AlCl3/day for 4 months) approaches, the (Na+/K+)ATPase activity was inhibited in a concentration-dependent way. The maximal inhibitory effect (similar to60%) was observed in the presence of a AlCl3 concentration >75 muM and at non-limiting ATP concentrations. Conversely, AlCl3 did not inhibit the enzyme activity when UTP was used as substrate instead of ATP. Analysis of the substrate dependence of membrane-bound (Na+/K+)ATPase by a computer simulation model suggests that the AlCl3-induced inhibitory effect is characterised by a reduction of the rate-limiting step velocity of the reaction cycle. Moreover, it seems that aluminium can induce impairment of the interprotomeric interaction within the oligomeric ensemble of membrane-bound (Na+/K+)ATPase. In fact, this effect was accompanied by a slight, but significant, decrease of readily accessible SH groups, which are involved in the maintenance of the membrane-bound (Na+/K+)ATPase oligomeric structure. In conclusion, during exposure to aluminium, reduction of the activation of membrane-bound (Na+/K+)ATPase by high ATP concentrations occurs, which results in a partial inhibition of the enzyme. (C) 2003 Elsevier Inc. All rights reserved.FCTPOCTI/BSE/46721/2002BD/21343/9

Aluminum accumulation and membrane fluidity alteration in synaptosomes isolated from rat brain cortex following aluminum ingestion: effect of cholesterol

Partilhar com CESAMIn the present work, we studied the effect of cholesterol/phospholipid (CH/PL) molar ratio on aluminum accumulation and aluminum-induced alteration of membrane fluidity in rat brain cortex synaptosomes. We observed that sub-acute (daily supply of 1.00 g of AlCl3 during 10 days) and chronic (daily supply of 0.03 g of AlCl3 during 4 months) exposure to dietary aluminum leads to a synaptosomal aluminum enrichment of 45 and 59%, respectively. During chronic exposure to AlCl3, the enhancement of aluminum content was prevented by administration of colestipol (0.31 g/day), which decreased the synaptosomal membrane CH/PL molar ratio (nmol/nmol) from 1.2 to 0.4. Fluorescence anisotropy analysis, using 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-(4-(trimethylamino)phenyl)-6-phenylhexa-1,3,5-triene (TMA-DPH), showed that after treatment with colestipol a decrease in membrane order occurs at the level of hydrophilic lipid-water surface and deeper hydrophobic region of the synaptosomal membrane. When the rats were exposed to aluminum, it was observed a significant enhancement of membrane fluidity, which was more pronounced at the level of the membrane hydrophilic regions. Meanwhile, when chronic exposure to dietary AlCl3 was accompanied by treatment with colestipol, the aluminum-induced decrease in membrane order was negligible when compared to TMA-DPH and DPH anisotropy values measured upon colestipol treatment. In contrast, in vitro incubation of synaptosomes (isolated from control rats) with AlCl3 induced a concentration-dependent rigidification of this more hydrophilic membrane region. The opposite action of aluminum on synaptosomal membrane fluidity, during in vivo and in vitro experiments, appears to be explained by alteration of synaptosomal CH/PL molar ratio, since a significant reduction (similar to80%) of this parameter occurs during in vivo exposure to aluminum. In conclusion, during in vivo exposure to aluminum, fluidification of hydrophilic regions and reduction of CH/PL molar ratio of presynaptic membranes accompany the accumulation of this cation, which appear to restrict aluminum retention in brain cortex nerve terminals. (C) 2002 Elsevier Science Ireland Ltd. and the Japan Neuroscience Society. All rights reserved.FCT - grants from PRAXIS XX

Differences in the expression pattern of HCN isoforms among mammalian tissues: sources and implications

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels play a critical role in a broad range of cell types, but the expression of the various HCN isoforms is still poorly understood. In the present study we have compared the expression of HCN isoforms in rat excitable and non-excitable tissues at both the mRNA and protein levels. Real-time PCR and Western blot analysis revealed distinct expression patterns of the four HCN isoforms in brain, heart, pituitary and kidney, with inconsistent mRNA-protein expression correlation. The HCN2 was the most abundant mRNA transcript (95.6, 78.0 and 59.0 % in kidney heart and pituitary, respectively) except in the brain (42.0 %) whereas HCN4 was the most abundant protein isoform. Our results suggest that HCN channels are mostly produced by the HCN4 isoform in heart, which contrasts with the sharp differences in the isoform stoichiometry in pituitary (15 HCN4:2 HCN2:1 HCN1:1 HCN3), kidney (24 HCN4:2 HCN3:1 HCN2:1 HCN1) and brain (3 HCN4:2 HCN2:1 HCN1:1 HCN3). Moreover, deviations of the electrophoretic molecular weight (MW) of the HCN isoforms relative to the theoretical MW were observed, suggesting that N-glycosylation and enzymatic proteolysis influences HCN channel surface expression. We hypothesize that selective cleavage of HCN channels by membrane bound metalloendopeptidases could account for the multiplicity of properties of native HCN channels in different tissues

Ecophysiological effects of mercury bioaccumulation and biochemical stress in the deep-water mesopredator Etmopterus spinax (Elasmobranchii; Etmopteridae)

Mercury (Hg) is a non-essential metal that can have toxic effects on the fitness of organisms and tends to bioaccumulate with age and to biomagnify in higher trophic levels. Few studies have assessed oxidative stress and neurotoxicity in deep-water sharks. This study evaluated early ontogenetic changes and physiological effects (antioxidant defences, oxidative damage, aerobic metabolism and neurotransmission functions) of Hg accumulation in the white muscle and brain tissues of the velvet belly lantern shark Etmopterus spinax from the southern Iberian coast (NE Atlantic). Results suggested that the low mercury concentrations observed may induce acute effects in E. spinax before they reach sexual maturity. We found different Hg concentrations in E. spinax: [Hg] males > [Hg] females; [Hg] muscle > [Hg] brain. Females appeared to have higher redox capability translated into higher activities and levels of antioxidant defences than males. However, higher levels of oxidative damage were also observed in females. Whilst the mechanisms underlying these effects remain unknown, these results suggest differences in mercury accumulation between tissues and sex, and potentially deleterious effects on oxidative stress status and neurophysiology of E. spinax, potentially impairing swimming performance and reproduction, which could subsequently impact on the health of both individuals and population.info:eu-repo/semantics/publishedVersio

Automated high-throughput screening of carbon nanotube-based bio-nanocomposites for bone cement applications

In this work we demonstrate the potential of using an automated cell viability analyzer for developing high-throughput screening of orthopedic bioactive materials. We used a biomaterial of carbon nanotubes (CNTs)-based composite integrated with hydroxyapatite/ polymethyl methacrylate (HA/PMMA) with controlled physical and chemical properties to evaluate the usefulness of morphometric analysis in conjunction with trypan blue dye exclusion assays in MG63 cell cultures. The MG63 cell line, derived from human bone osteo - sarcoma, is often used as a model for studying osteoblast-like cellular response to bioactive materials for orthopedic surgery. The viability analyzer, Vi-CELLTM XR, Beckman Coulter, was used with trypan blue dye exclusion method in cell suspensions obtained after trypsinization along with determining the distribution plots of cell diameter and circularity, which are critical cellular characteristics. In addition, the activity of alkaline phosphatase (ALP), a typical representation of osteogenic activity of osteoblasts, was also measured spectrophotometrically using p-nitrophenol phosphate as the substrate. Comparative analysis of the frequency histogram of average cell diameter and circularity allowed for the analyses of significant alterations in cell morphology not only over time in control cultures (spherical vs. a flat morphology) but also with respect to PMMA and HA nanocomposites. After cell exposure to HA/PMMA/CNTs, a shift toward loss of cell circularity was observed. The appearances of more differentiated morphologic features were well correlated with the increase of secreted ALP activity. In conclusion, the evaluation of material-induced changes of cell morphology could represent a valuable prescreening test for bioactive properties.International Iberian Laboratory of Nanotechnology - NANO/NMed- AT/0115/2007FCT - Program Compromise with Science (to M.K.S. and P.M.)SFRH/BPD/14677/2003 (to V.S.S.