Desenvolvimento de pastilha potencialmente probiótica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influência da espessura do substrato dental sobre a eficácia clareadora e citotoxicidade de diferentes protocolos de clareamento

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação do potencial citotóxico, genotóxico e mutagênico da melitina em cultura de células

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Avaliação do potencial citotóxico, genotóxico e mutagênico da melitina em cultura de células

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

alpha-6 Integrin Expression in Bovine Spermatogonial Cells Purified by Discontinuous Percoll Density Gradient

The study of spermatogonial stem cells (SSCs) provides a model to better understand adult stem cell biology. Besides the biomedical potential to perform studies of infertility in many species, SSCs hold a promising application at animal transgenesis. Because stem cells are thought to be associated with basement membranes, expression of alpha-6 integrin has been investigated as a marker of type A spermatogonial cells, which are considered SSCs because of their undifferentiated status and self-renewal ability. In this manner, the aim of this study was to isolate type A SSCs from adult bulls by a two-step enzymatic procedure followed by a discontinuous Percoll density gradient purification and verify the expression of alpha-6 integrin by flow cytometry and real-time RT-PCR before and after Percoll purification. Spermatogonial cells were successfully obtained using the two-step enzymatic digestion. An average of 1 x 10(5) viable cells per gram of testis was isolated. However, the discontinuous Percoll did not purify isolated cells regarding alpha-6 integrin expression. Flow cytometry analysis demonstrated no differences in the alpha-6 integrin expression between cell samples before and after Percoll purification (p = 0.5636). The same was observed in the real-time PCR analysis (p > 0.05). In addition to alpha-6 integrin, the expression of GFR alpha-1 and PGP9.5, known bovine SSCs markers, was detected in all samples studied. Considering that Percoll can reduce cell viability, it is possible to conclude that Percoll density gradient is not suitable to purify bovine SSC, according to alpha-6 integrin expression.

Neurotoxicity of Anhydroecgonine Methyl Ester, a Crack Cocaine Pyrolysis Product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaineAEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaineAEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Characterization and in vitro evaluation of bacterial cellulose membranes functionalized with osteogenic growth peptide for bone tissue engineering

The aim of this study was to characterize the physicochemical properties of bacterial cellulose (BC) membranes functionalized with osteogenic growth peptide (OGP) and its C-terminal pentapeptide OGP[10-14], and to evaluate in vitro osteoinductive potential in early osteogenesis, besides, to evaluate cytotoxic, genotoxic and/or mutagenic effects. Peptide incorporation into the BC membranes did not change the morphology of BC nanofibers and BC crystallinity pattern. The characterization was complemented by Raman scattering, swelling ratio and mechanical tests. In vitro assays demonstrated no cytotoxic, genotoxic or mutagenic effects for any of the studied BC membranes. Culture with osteogenic cells revealed no difference in cell morphology among all the membranes tested. Cell viability/proliferation, total protein content, alkaline phosphatase activity and mineralization assays indicated that BC-OGP membranes enabled the highest development of the osteoblastic phenotype in vitro. In conclusion, the negative results of cytotoxicity, genotoxicity and mutagenicity indicated that all the membranes can be employed for medical supplies, mainly in bone tissue engineering/regeneration, due to their osteoinductive properties.

Evaluation of Distinct Freezing Methods and Cryoprotectants for Human Amniotic Fluid Stem Cells Cryopreservation

Amniotic fluid (AF) was described as a potential source of mesenchymal stem cells (MSCs) for biomedicine purposes. Therefore, evaluation of alternative cryoprotectants and freezing protocols capable to maintain the viability and stemness of these cells after cooling is still needed. AF stem cells (AFSCs) were tested for different freezing methods and cryoprotectants. Cell viability, gene expression, surface markers, and plasticity were evaluated after thawing. AFSCs expressed undifferentiated genes Oct4 and Nanog; presented typical markers (CD29, CD44, CD90, and CD105) and were able to differentiate into mesenchymal lineages. All tested cryoprotectants preserved the features of AFSCs however, variations in cell viability were observed. In this concern, dimethyl sulfoxide (Me2SO) showed the best results. The freezing protocols tested did not promote significant changes in the AFSCs viability. Time programmed and nonprogrammed freezing methods could be used for successful AFSCs cryopreservation for 6 months. Although tested cryoprotectants maintained undifferentiated gene expression, typical markers, and plasticity of AFSCs, only Me2SO and glycerol presented workable viability ratios.

Ursolic acid directly promotes protein accretion in myotubes but does not affect myoblast proliferation

Ursolic acid (UA) has been recently proposed as a potential candidate for the treatment of muscle wasting conditions because of its protein sparring/anabolic effects. Despite this finding, it is unknown whether this response is the consequence of a direct effect on the muscle fibre or if it is mediated by neural or other systemic factors. In the present study, we sought to determine if UA has direct effects in skeletal muscle cells, whether it can increase myoblast proliferation and whether UA can become myotoxic at higher doses. Our results demonstrate that UA directly promoted protein accretion in cultured myotubes but did not modulate myoblast proliferation. At higher doses, UA compromised cell viability in both myoblasts and myotubes. We conclude that the anabolic properties of UA seen in vivo and in vitro are likely a direct effect on the muscle cell, but at higher doses, the benefits decline in favour of a myotoxic outcome. Copyright (C) 2012 John Wiley & Sons, Ltd.

Cytotoxicity evaluation of Curcuma zedoaria (Christm.) Roscoe fluid extract used in oral hygiene products

Objective. This in vitro study evaluated the cytotoxic effects of the Curcuma zedoaria (Christm.) Roscoe (popular name: zedoary) fluid extract, as used in preparations for oral hygiene, mostly for anti-septic purposes. Materials and methods. The cell viability and cell growth were assessed by Trypan blue dye exclusion assay using the LMF cell line derived from oral mucosa. Cell viability (short-term assay) was measured 0, 6, 12 and 24 h after contact with the fluid extract. Cell growth (long-term assay) was analyzed in 1, 3, 5 and 7 days. The experimental groups were those testing the fluid extract obtained from the zedoary rhizome and the extractor liquid (ethanol 70 degrees GL) in the concentrations of 0.01-0.0001% v/v. Fresh DMEM were used in the control cultures. Results. Short-term assay-all studied cultures maintained stable cell viability; Long-term assay-there was progressive cell growth in all studied cultures. Conclusion. According to the results, the zedoary fluid extract presents low cytotoxicity and probably can be used in the oral hygiene products.

Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondria] apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax alpha protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells, and mitochondria dysfunction might be involved in the deleterious effects after long-term GC treatment.

Dietary carotenoid lutein protects against DNA damage and alterations of the redox status induced by cisplatin in human derived HepG2 cells

Several epidemiological and experimental studies has been reported that lutein (LT) presents antioxidant properties. Aim of the present study was to investigate the protective effects of LT against oxidative stress and DNA damage induced by cisplatin (cDDP) in a human derived liver cell line (HepG2). Cell viability and DNA-damage was monitored by MU and comet assays. Moreover, different biochemical parameters related to redox status (glutathione, cytochrome-c and intracellular ROS) were also evaluated. A clear DNA-damage was seen with cDDP (1.0 mu M) treatment. In combination with the carotenoid, reduction of DNA damage was observed after pre- and simultaneous treatment of the cells, but not when the carotenoid was added to the cells after the exposure to cDDP. Exposure of the cells to cDDP also caused significant changes of all biochemical parameters and in co-treatment of the cells with LT, the carotenoid reverted these alterations. The results indicate that cDDP induces pronounced oxidative stress in HepG2 cells that is related to DNA damage and that the supplementation with the antioxidant LT may protect these adverse effects caused by the exposure of the cells to platinum compound, which can be a good predict for chemoprevention. (C) 2011 Elsevier Ltd. All rights reserved.

Response of Saccharomyces cerevisiae to Cadmium and Nickel Stress: The Use of the Sugar Cane Vinasse as a Potential Mitigator

Most of the metals released from industrial activity, among them are cadmium (Cd) and nickel (Ni), inhibit the productivity of cultures and affect microbial metabolism. In this context, the aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Cd and Ni on cell growth, viability, budding rate and trehalose content of Saccharomyces cerevisiae, likely because of adsorption and chelating action. For this purpose, the yeast was grown batch-wise in YED medium supplemented with selected amounts of vinasse and Cd or Ni. The negative effects of Cd and Ni on S. cerevisiae growth and the mitigating one of sugar cane vinasse were quantified by an exponential model. Without vinasse, the addition of increasing levels of Cd and Ni reduced the specific growth rate, whereas in its presence no reduction was observed. Consistently with the well-proved toxicity of both metals, cell viability and budding rate progressively decreased with increasing their concentration, but in the presence of vinasse the situation was remarkably improved. The trehalose content of S. cerevisiae cells followed the same qualitative behavior as cell viability, even though the negative effect of both metals on this parameter was stronger. These results demonstrate the ability of sugar cane vinasse to mitigate the toxic effects of Cd and Ni.

C-Phycocyanin protects SH-SY5Y cells from oxidative injury, rat retina from transient ischemia and rat brain mitochondria from Ca2+/phosphate-induced impairment

Oxidative stress and mitochondrial impairment are essential in the ischemic stroke cascade and eventually lead to tissue injury. C-Phycocyanin (C-PC) has previously been shown to have strong antioxidant and neuroprotective actions. In the present study, we assessed the effects of C-PC on oxidative injury induced by tert-butylhydroperoxide (t-BOOH) in SH-SY5Y neuronal cells, on transient ischemia in rat retinas, and in the calcium/phosphate-induced impairment of isolated rat brain mitochondria (RBM). In SH-SY5Y cells, t-BOOH induced a significant reduction of cell viability as assessed by an MTT assay, and the reduction was effectively prevented by treatment with C-PC in the low micromolar concentration range. Transient ischemia in rat retinas was induced by increasing the intraocular pressure to 120 mmHg for 45 min, which was followed by 15 min of reperfusion. This event resulted in a cell density reduction to lower than 50% in the inner nuclear layer (INL), which was significantly prevented by the intraocular pre-treatment with C-PC for 15 min. In the RBM exposed to 3 mM phosphate and/or 100 mu M Ca2+, C-PC prevented in the low micromolar concentration range, the mitochondrial permeability transition as assessed by mitochondrial swelling, the membrane potential dissipation, the increase of reactive oxygen species levels and the release of the pro-apoptotic cytochrome c. In addition, C-PC displayed a strong inhibitory effect against an electrochemically-generated Fenton reaction. Therefore, C-PC is a potential neuroprotective agent against ischemic stroke, resulting in reduced neuronal oxidative injury and the protection of mitochondria from impairment. (C) 2012 Elsevier Inc. All rights reserved.