Vacuole-mitochondrial cross-talk during apoptosis in yeast: a model for understanding lysosome-mitochondria-mediated apoptosis in mammals.

The yeast apoptosis field emerged with the finding that key components of the apoptotic machinery are conserved in these simple eukaryotes. Thus it became possible to exploit these genetically tractable organisms to improve our understanding of the intricate mechanisms of cell death in higher eukaryotes and of severe human diseases associated with apoptosis dysfunctions. Early on, it was recognized that a mitochondria-mediated apoptotic pathway showing similarities to the mammalian intrinsic pathway was conserved in yeast. Recently, lysosomes have also emerged as central players in mammalian apoptosis. Following LMP (lysosomal membrane permeabilization), lysosomal proteases such as cathepsins B, D and L are released into the cytosol and can trigger a mitochondrial apoptotic cascade. CatD (cathepsin D) can also have anti-apoptotic effects in some cellular types and specific contexts. Nonetheless, the mechanisms underlying LMP and the specific role of cathepsins after their release into the cytosol remain poorly understood. We have recently shown that yeast vacuoles, membrane-bound acidic organelles, which share many similarities to plant vacuoles and mammalian lysosomes, are also involved in the regulation of apoptosis and that the vacuolar protease Pep4p, orthologue of the human CatD, is released from the vacuole into the cytosol in response to acetic acid. Here, we discuss how the conservation of cell-death regulation mechanisms in yeast by the lysosome-like organelle and mitochondria may provide new insights into the understanding of the complex interplay between the mitochondria and lysosome-mediated signalling routes during mammalian apoptosis.

Antioxidant activity of synthetic diarylamines: a mitochondrial and cellular approach.

We investigated the antioxidant properties of two synthetic diarylamines, MJQ1 and MJQ2. For one of them (MJQ1) the synthesis procedure is herein described. The compounds showed maximal protection of ADP/Fe(2+) induced mitochondrial lipid peroxidation for 50nM (MJQ1) and 60muM (MJQ2) concentrations. Both compounds were also effective in the prevention of mitochondrial DeltaPsi collapse. The effective antioxidant dose of MJQ1 in mitochondria (50nM) also proved to protect lipid peroxidation in PC12 cells and the effect seems not to be related with the compound's iron chelating ability. The modified structure of MJQ1 clearly resulted in an improvement of its antioxidant and toxic profile, evaluated in mitochondria and whole cells. This study demonstrates a high potential of these diarylamines, as radical scavengers, whose chemical structures can be manipulated if a specific target is well characterized.

Phenolic Imidazole Derivatives with Dual Antioxidant/Antifungal Activity: Synthesis and Structure-Activity Relationship.

BACKGROUND: Previous publications show that the addition of a phenolic antioxidant to an antifungal agent, considerably enhances the antifungal activity. OBJECTIVE: Synthesis of novel compounds combining phenolic units with linear or cyclic nitrogencontaining organic molecules with antioxidant/antifungal activity using methodologies previously developed in the group. METHODS: Several N- [1,2-dicyano-2- (arylidenamino) vinyl]-O-alkylformamidoximes 3 were synthesized and cyclized to 4,5-dicyano-N- (N´-alcoxyformimidoyl)-2-arylimidazoles 4 upon reflux in DMF, in the presence of manganese dioxide or to 6-cyano-8-arylpurines 5 when the reagent was refluxed in acetonitrile with an excess of triethylamine. These compounds were tested for their antioxidant activity by cyclic voltammetry, DPPH radical (DPPH•) assay and deoxyribose degradation assay. The minimum inhibitory concentration (MIC) of all compounds was evaluated against two yeast species, Saccharomyces cerevisiae and Candida albicans, and against bacteria Bacillus subtilis (Gram-positive) and Escherichia coli (Gram negative). Their cytotoxicity was evaluated in fibroblasts. RESULTS: Among the synthetised compounds, five presented higher antioxidant activity than reference antioxidant Trolox and from these compounds, four presented antifungal activity without toxic effects in fibroblasts and bacteria. CONCLUSION: Four novel compounds presented dual antioxidant/antifungal activity at concentrations that are not toxic to bacteria and fibroblasts. The active molecules can be used as an inspiration for further studies in this area.

Protective role of new nitrogen compounds on ROS/RNS-mediated damage to PC12 cells.

Reactive oxygen (ROS) and nitrogen (RNS) species are known to be involved in many degenerative diseases. This study reports four new nitrogen compounds from organic synthesis, identified as FMA4, FMA7, FMA762 and FMA796, which differ mainly by the number of hydroxyl groups within their phenolic unit. Their potential role as antioxidants was evaluated in PC12 cells by assessing their protection against oxidative and nitrosative insults. The four compounds, and particularly FMA762 and FMA796, were able to protect cells against lipid peroxidation and intracellular ROS/RNS formation to a great extent. Their protective effects were likely mediated by their free radicals scavenging ability, as they appeared to be involved neither in the induction of natural antioxidant enzymes like GSH-PX and SOD, nor in the inhibition of NOS. Nevertheless, these results suggest a promising potential for these compounds as ROS/RNS scavengers in pathologies where oxidative/nitrosative stress are involved.

The effect of starch and starch-bioactive glass composite microparticles on the adhesion and expression of the osteoblastic phenotype of a bone cell line.

There is a clear need for the development of microparticles that can be used simultaneously as carriers of stem/progenitor cells and as release systems for bioactive agents, such as growth factors or differentiation agents. In addition, when thinking on bone-tissue-engineering applications, it would be very useful if these microparticles are biodegradable and could be made to be bioactive. Microparticles with all those characteristics could be cultured together with adherent cells in appropriate bioreactors to form in vitro constructs that can then be used in tissue-engineering therapies. In this work, we have characterized the response of MC3T3-E1 pre-osteoblast cells to starch-based microparticles. We evaluated the adhesion, proliferation, expression of osteoblastic markers and mineralization of cells cultured at their surface. The results clearly show that MC3T3-E1 pre-osteoblast cells adhere to the surface of both polymeric and composite starch-based microparticles and express the typical osteoblastic marker genes. Furthermore, the cells were found to mineralize the extracellular matrix (ECM) during the culture period. The obtained results indicate that starch-based microparticles, known already to be biodegradable, bioactive and able to be used as carriers for controlled release applications, can simultaneously be used as carriers for cells. Consequently, they can be used as templates for forming hybrid constructs aiming to be applied in bone-tissue-engineering applications.

Starch-based microparticles as vehicles for the delivery of active platelet-derived growth factor.

In a previous work, we described the use of starch-based microparticles as vehicles for the controlled release of corticosteroids. The goal of the present work is to evaluate the potential of these microparticles to incorporate and release platelet-derived growth factor (PDGF). The loading efficiency and release profile were evaluated, and PDGF was incorporated into and released from the matrix of starch-based microparticles. The release profile shows rapid release of PDGF in the first 24 h, after which there was a slow but constant release for up to 8 weeks. The maintenance of the PDGF biological activity after incorporation and release was evaluated by its mitogenic effect over osteoblastic cells, and it was shown to be comparable to that of PDGF supplemented to the culture medium. This proves that the incorporation and release did not affect the biological activity of the growth factor (GF). The results clearly demonstrate that starch-based microparticles are suitable vehicles for the incorporation and release of GFs. When combined with previous results, these materials also suggest their ability to enhance the regenerating potential of tissue engineering hybrid constructs.

Oxidative stress protection by newly synthesized nitrogen compounds with pharmacological potential.

In this study we used new nitrogen compounds obtained by organic synthesis whose structure predicted an antioxidant potential and then an eventual development as molecules of pharmacological interest in diseases involving oxidative stress. The compounds, identified as FMA4, FMA5, FMA7 and FMA8 differ in the presence of hydroxyl groups located in the C-3 and/or C-4 position of a phenolic unit, which is possibly responsible for their free radicals' buffering capacity. Data from the DPPH discoloration method confirm the high antiradical efficiency of the compounds. The results obtained with cellular models (L929 and PC12) show that they are not toxic and really protect from membrane lipid peroxidation induced by the ascorbate-iron oxidant pair. The level of protection correlates with the drug's lipophilic profile and is sometimes superior to trolox and equivalent to that observed for alpha-tocopherol. The compounds FMA4 and FMA7 present also a high protection from cell death evaluated in the presence of a staurosporine apoptotic stimulus. That protection results in a significant reduction of caspase-3 activity induced by staurosporine which by its turn seems to result from a protection observed in the membrane receptor pathway (caspase-8) together with a protection observed in the mitochondrial pathway (caspase-9). Taken together the results obtained with the new compounds, with linear chains, open up perspectives for their use as therapeutical agents, namely as antioxidants and protectors of apoptotic pathways. On the other hand the slight pro-oxidant profile obtained with the cyclic structures suggests a different therapeutic potential that is under current investigation.

Bone tissue engineering: state of the art and future trends.

Although several major progresses have been introduced in the field of bone regenerative medicine during the years, current therapies, such as bone grafts, still have many limitations. Moreover, and in spite of the fact that material science technology has resulted in clear improvements in the field of bone substitution medicine, no adequate bone substitute has been developed and hence large bone defects/injuries still represent a major challenge for orthopaedic and reconstructive surgeons. It is in this context that TE has been emerging as a valid approach to the current therapies for bone regeneration/substitution. In contrast to classic biomaterial approach, TE is based on the understanding of tissue formation and regeneration, and aims to induce new functional tissues, rather than just to implant new spare parts. The present review pretends to give an exhaustive overview on all components needed for making bone tissue engineering a successful therapy. It begins by giving the reader a brief background on bone biology, followed by an exhaustive description of all the relevant components on bone TE, going from materials to scaffolds and from cells to tissue engineering strategies, that will lead to "engineered" bone. Scaffolds processed by using a methodology based on extrusion with blowing agents.

Nitrogen compounds prevent h9c2 myoblast oxidative stress-induced mitochondrial dysfunction and cell death.

Oxidative stress has been connected to various forms of cardiovascular diseases. Previously, we have been investigating the potential of new nitrogen-containing synthetic compounds using a neuronal cell model and different oxidative stress conditions in order to elucidate their potential to ameliorate neurodegenerative diseases. Here, we intended to extend these initial studies and investigate the protective role of four of those new synthetic compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative damage induced to H9c2 cardiomyoblasts by tert-butylhydroperoxide (t-BHP). The data indicates that FMA762 and FMA796 decrease t-BHP-induced cell death, as measured by both sulforhodamine B assay and nuclear chromatin condensation evaluation, at non-toxic concentrations. In addition, the two mentioned compounds inhibit intracellular signalling mechanisms leading to apoptotic cell death, namely those mediated by mitochondria, which was confirmed by their ability to overcome t-BHP-induced morphological changes in the mitochondrial network, loss of mitochondrial membrane potential, increased expression of the pro-apoptotic proteins p53, Bax and AIF and activation of caspases-3 and -9. Importantly, our results indicate that the compounds' ROS scavenging ability plays a crucial role in the protection profile, as a significant decrease in t-BHP-induced oxidative stress occurred in their presence. Data obtained indicates that some of the test compounds may clearly prove valuable in a clinical context by diminishing cardiac injury associated to oxidative stress without any toxicity.

Novel nitrogen compounds enhance protection and repair of oxidative DNA damage in a neuronal cell model: comparison with quercetin.

Oxidative DNA damage has been described as an important type of damage that occurs in neuronal cells, with severe implications in many neurodegenerative diseases and in aging. We have previously reported the protection of four new synthetic nitrogen compounds (FMA4, FMA7, FMA762 and FMA796) against oxidative stress conditions. In this work, we studied their effects on oxidative DNA damage induced in rat pheochromocytoma (PC12) cells, using the Comet assay, and compared them with a natural antioxidant, quercetin. Among the compounds tested, FMA762 and FMA796 were the most effective in preventing tert-butylhydroperoxide (t-BHP)-induced formation of DNA strand breaks and in improving the cells' capacity to repair this kind of damage. These effects were similar to the ones of quercetin, a flavonoid with known antioxidant activity. Moreover, contrarily to quercetin, they increased the repair capacity of oxidised bases induced with the photosensitiser Ro 19-8022. This effect seems to be mediated by an increase in DNA repair enzymes activity, assessed by the in vitro BER assay, but no regulation at the expression of OGG1 and APE1 genes was detected. In addition to other properties previously found for the nitrogen compounds, they now prove their effectiveness against oxidative stress-induced DNA damage in the neuronal cell model used.

Oxidative DNA damage protection and repair by polyphenolic compounds in PC12 cells.

Biological systems are frequently exposed to excessive reactive oxygen species, causing a disturbance in the cells natural antioxidant defence systems and resulting in damage to all biomolecules, including nucleic acids. In fact, oxidative DNA damage is described as the type of damage most likely to occur in neuronal cells. In this study, three polyphenolic compounds, luteolin, quercetin and rosmarinic acid, were investigated for their protective effects against oxidative DNA damage induced in PC12 cells, a neuronal cell model. Although luteolin and quercetin prevented the formation of strand breaks to a greater extent than rosmarinic acid, this last one presented the highest capacity to repair strand breaks formation. In addition, rosmarinic acid was the only compound tested that increased the repair of oxidized nucleotidic bases induced with the photosensitizer compound [R]-1-[(10-chloro-4-oxo-3-phenyl-4H-benzo[a]quinolizin-1-yl) carbonyl]-2-pyrrolidine-methanol (Ro 19-8022). The activity of repair enzymes was indicated by the in vitro base excision repair assay, using a cell-free extract obtained from cells previously treated with the compounds to incise DNA. The protective effect of rosmarinic acid was further confirmed by the increased expression of OGG1 repair gene, observed through real time RT-PCR. The data obtained is indicative that rosmarinic acid seems to act on the intracellular mechanisms responsible for DNA repair, rather than by a direct effect on reactive oxygen species scavenging, as deducted from the effects observed for luteolin and quercetin. Therefore, these results suggest the importance of these polyphenols, and in particular rosmarinic acid, as protectors of oxidative stress-induced DNA damage that commonly occurs in several pathological conditions, such as neurodegenerative diseases.