Effects of zoledronic acid on odontoblast-like cells

The aim of the study was to evaluate the effects of a highly potent bisphosphonate, zoledronic acid (ZOL), on cultured odontoblast-like cells MDPC-23. The cells (1.5 × 104 cells/cm2) were seeded for 48 h in wells of 24-well dished. Then, the plain culture medium (DMEM) was replaced by fresh medium without fetal bovine serum. After 24 h, ZOL (1 or 5 μM) was added to the medium and maintained in contact with the cells for 24 h. After this period, the succinic dehydrogenase (SDH) enzyme production (cell viability-MTT assay), total protein (TP) production, alkaline phosphatase (ALP) activity, and gene expression (qPCR) of collagen type I (Col-I) and ALP were evaluated. Cell morphology was assessed by SEM. Five μM ZOL caused a significant decrease in SDH production. Both ZOL concentrations caused a dose-dependent significant decrease in TP production and ALP activity. ZOL also produced discret morphological alterations in the MDPC-23 cells. Regarding gene expression, 1 μM ZOL caused a significant increase in Col-I expression. Although 5 μM ZOL did not affect Col-I expression, it caused a significant alteration in ALP expression (ANOVA and Tukey's test, p < 0.05). ZOL presented a dose-dependent cytotoxic effect on the odontoblast-like cells, suggesting that under clinical conditions the release of this drug from dentin could cause damage to the pulpo-dentin complex. © 2012 Elsevier Ltd.

Effects of Ginkgo biloba on chemically-induced mammary tumors in rats receiving tamoxifen

Background: Ginkgo biloba extract (GbE) is used extensively by breast cancer patients undergoing treatment with Tamoxifen (TAM). Thus, the present study investigated the effects of GbE in female Sprague-Dawley (SD) rats bearing chemically-induced mammary tumors and receiving TAM.Methods: Animals bearing mammary tumors (≥1 cm in diameter) were divided into four groups: TAM [10 mg/kg, intragastrically (i.g.)], TAM plus GbE [50 and 100 mg/kg, intraperitoneally (i.p.)] or an untreated control group. After 4 weeks, the therapeutic efficacy of the different treatments was evaluated by measuring the tumor volume (cm3) and the proportions of each tumor that were alive, necrotic or degenerative (mm2). In addition, labeling indexes (LI%) were calculated for cell proliferation (PCNA LI%) and apoptosis (cleaved caspase-3 LI%), expression of estrogen receptor-alpha (ER-α) and p63 biomarkers.Results: Overall, the tumor volume and the PCNA LI% within live tumor areas were reduced by 83% and 99%, respectively, in all TAM-treated groups when compared to the untreated control group. GbE treatment (100 mg/kg) reduced the proportions of live (24.8%) and necrotic areas (2.9%) (p = 0.046 and p = 0.038, respectively) and significantly increased the proportion of degenerative areas (72.9%) (p = 0.004) in mammary tumors when compared to the group treated only with TAM. The expression of ER-α, p63 and cleaved caspase-3 in live tumor tissues was not modified by GbE treatment.Conclusions: Co-treatment with 100 mg/kg GbE presented a slightly beneficial effect on the therapeutic efficacy of TAM in female SD rats bearing mammary tumors. © 2013 Dias et al.; licensee BioMed Central Ltd.

Osteogenesis-inducing calcium phosphate nanoparticle precursors applied to titanium surfaces

This study investigated the effects of the morphology and physicochemical properties of calcium phosphate (CaP) nanoparticles on osteogenesis. Two types of CaP nanoparticles were compared, namely amorphous calcium phosphate (ACP) nano-spheres (diameter: 9-13 nm) and poorly crystalline apatite (PCA) nano-needles (30-50 nm x 2-4 nm) that closely resemble bone apatite. CaP particles were spin-coated onto titanium discs and implants; they were evaluated in cultured mouse calvarial osteoblasts, as well as after implantation in rabbit femurs. A significant dependence of CaP coatings was observed in osteoblast-related gene expression (Runx2, Col1a1 and Spp1). Specifically, the PCA group presented an up-regulation of the osteospecific genes, while the ACP group suppressed the Runx2 and Col1a1 expression when compared to blank titanium substrates. Both the ACP and PCA groups presented a more than three-fold increase of calcium deposition, as suggested by Alizarin red staining. The removal torque results implied a slight tendency in favour of the PCA group. Different forms of CaP nanostructures presented different biologic differences; the obtained information can be used to optimize surface coatings on biomaterials. © 2013 IOP Publishing Ltd.

Anti-inflammatory mechanisms of the annexin A1 protein and its mimetic peptide Ac2-26 in models of ocular inflammation in vivo and in vitro

Annexin A1 (AnxA1) is a protein that displays potent anti-inflammatory properties, but its expression in eye tissue and its role in ocular inflammatory diseases have not been well studied. We investigated the mechanism of action and potential uses of AnxA1 and its mimetic peptide (Ac2-26) in the endotoxin-induced uveitis (EIU) rodent model and in human ARPE-19 cells activated by LPS. In rats, analysis of untreated EIU after 24 and 48 h or EIU treated with topical applications or with a single s.c. injection of Ac2-26 revealed the anti-inflammatory actions of Ac2-26 on leukocyte infiltration and on the release of inflammatory mediators; the systemic administration of Boc2, a formylated peptide receptor (fpr) antagonist, abrogated the peptide's protective effects. Moreover, AnxA1-/- mice exhibited exacerbated EIU compared with wild-type animals. Immunohistochemical studies of ocular tissue showed a specific AnxA1 posttranslational modification in EIU and indicated that the fpr2 receptor mediated the anti-inflammatory actions of AnxA1. In vitro studies confirmed the roles of AnxA1 and fpr2 and the protective effects of Ac2-26 on the release of chemical mediators in ARPE-19 cells. Molecular analysis of NF-κB translocation and IL-6, IL-8, and cyclooxygenase-2 gene expression indicated that the protective effects of AnxA1 occur independently of the NF-κB signaling pathway and possibly in a posttranscriptional manner. Together, our data highlight the role of AnxA1 in ocular inflammation, especially uveitis, and suggest the use of AnxA1 or its mimetic peptide Ac2-26 as a therapeutic approach. Copyright © 2013 by The American Association of Immunologists, Inc.

Análise da ocorrência de transposição em regiões reguladoras dos genes da família Cyp em espécies de Drosophila

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

Caracterização genética e análise de polimorfismos da região promotora do gene da enzima esteraoil-coenzima a dessaturase em báfalas leiteiras

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

Efeito da infecção pelo vírus da febre amarela no mecanismo de splicing celular

Pós-graduação em Microbiologia - IBILCE

Genomic regions harboring insecticide resistance-associated Cyp genes are enriched by transposable element fragments carrying putative transcription factor binding sites in two sibling Drosophila species

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

A protein kinase screen of Neurospora crassa mutant strains reveals that the SNF1 protein kinase promotes glycogen synthase phosphorylation

Glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. The activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. To identify the protein kinases affecting glycogen metabolism in Neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. We identified multiple kinases that have already been described as controlling glycogen metabolism in different organisms, such as NcSNF1, NcPHO85, NcGSK3, NcPKA, PSK2 homologue and NcATG1. In addition, many hypothetical kinases have been implicated in the control of glycogen metabolism. Two kinases, NcIME-2 and NcNIMA, already functionally characterized but with no functions related to glycogen metabolism regulation, were also identified. Among the kinases identified, it is important to mention the role of NcSNF1. We showed in the present study that this kinase was implicated in glycogen synthase phosphorylation, as demonstrated by the higher levels of glycogen accumulated during growth, along with a higher glycogen synthase (GSN) ±glucose 6-phosphate activity ratio and a lesser set of phosphorylated GSN isoforms in strain Ncsnf1KO, when compared with the wild-type strain. The results led us to conclude that, in N. crassa, this kinase promotes phosphorylation of glycogen synthase either directly or indirectly, which is the opposite of what is described for Saccharomyces cerevisiae. The kinases also play a role in gene expression regulation, in that gdn, the gene encoding the debranching enzyme, was down-regulated by the proteins identified in the screen. Some kinases affected growth and development, suggesting a connection linking glycogen metabolism with cell growth and development.

MicroRNAs targeting the immunomodulatory HLA-G gene: a new survey searching for microRNAs with potential to regulate HLA-G

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

Immunolocalization of myosin Va in the developing nervous system of embryonic chicks

Myosins are molecular motors associated with the actin cytoskeleton that participate in the mechanisms of cellular motility. During the development of the nervous system, migration of nerve cells to specific sites, extension of growth cones, and axonal transport are dramatic manifestations of cellular motility. We demonstrate, via immunoblots, the expression of myosin Va during early stages of embryonic development in chicks, extending from the blastocyst period to the beginning of the fetal period. The expression of myosin Va in specific regions and cellular structures of the nervous system during these early stages was determined by immunocytochemistry using a polyclonal antibody. Whole mounts of chick embryos at 24-30-h stages showed intense immunoreactivity of the neural tube in formation along its full extent. Cross-sections at these stages of development showed strong labeling in neuroepithelial cells at the basal and apical regions of the neural tube wall. Embryos at more advanced periods of development (48h and 72 h) showed distinctive immunolabeling of neuroepithelial cells, neuroblasts and their cytoplasmic extensions in the mantle layer of the stratified neural tube wall, and neuroblasts and their cytoplasmic extensions in the internal wall of the optic cup, as well as a striking labeling of cells in the apparent nuclei of cranial nerves and budding fibers. These immunolocalization studies indicate temporal and site-specific expression of myosin Va during chick embryo development, suggesting that myosin Va expression is related to recruitment for specific cellular tasks.

Adição de ácido linolênico e L-carnitina na maturação oocitária: efeitos sobre o metabolismo celular, potencial de desenvolvimento e criotolerância de embriões bovinos produzidos in vitro

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

Funktionelle Rolle von SNARE-Proteinen bei der Myelinisierung

Die Hauptfunktion der Oligodendrozyten im zentralen Nervensystem ist die Myelinisierung. Dabei umwickelt die Zelle mit Ausläufern ihrer Plasmamembran mehrmals die Axone. In den Phasen der aktiven Myelinisierung produziert ein Oligodendrozyt eine Fläche von 5000-50000 µm2 Myelin pro Tag, wobei große Mengen der Myelinkomponenten über vesikulären Transport zur Zelloberfläche transportiert werden müssen. Die Fusion der Vesikel mit ihrer Zielmembran wird duch SNARE-Proteine (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor proteins) kontrolliert, die durch spezifische Interaktionen zwischen R- (Vesikel) und Q- (Zielmembran) SNAREs auch zur Spezifität der Fusion beitragen. Um die SNAREs den oligodendroglialen Transportrouten zuzuordnen, wurde deren Expression, Regulation und subzelluläre Lokalisation in primären Oligodendrozyten, in Oli-neu Zellen und im Myelin untersucht. Die Plasmamembran-Q-SNAREs Syntaxin 3, Syntaxin 4 und SNAP23, sowie das endosomale R-SNAREs VAMP3 zeigten eine zunehmende Expression im Verlauf der Oligodendrozytenreifung, die Expression von SNAP29 hingegen verminderte sich. Zudem akkumulierten die SNARE-Proteine Syntaxin 2, Syntaxin 3, Syntaxin 4 und VAMP7 im adulten Myelin, was für ihre Beteiligung an der Myelinisierung spricht. Co-Immunpräzipitationen ergaben u.a. Interaktionen zwischen den SNARE-Proteinen VAMP3 (R), Syntaxin 4 (Qa) und SNAP23 (Qbc). Anhand der beschriebenen Analyse konnten die SNARE-Proteine den oligodendroglialen Transportwegen zugeordnet werden. Immunzytochemische Analysen zeigten, dass das Hauptmyelinprotein PLP mit dem R-SNARE des Recycling Endosoms VAMP3, und mit dem spät endosomal, lysosomalen SNARE VAMP7 kolokalisiert. Um deren Rolle im PLP-Transport zu untersuchen, wurden verschiedene VAMP3- bzw. VAMP7-Silencing-Experimente durchgeführt. In beiden Fällen führte dies zu einer reduzierten Menge an PLP an der Zelloberfläche. Die Ergebnisse lassen somit auf zwei unabhängige Transportwege für PLP zur Plasmamembran von Oligodendrozyten schließen. Der VAMP7-abhängige PLP-Transport wurde zusätzlich in vivo in AP3-defizienten Mäusen, welche VAMP7 fehlsortieren, überprüft. Die Gehirne der mutanten Mäuse enthielten weniger Myelin, das isolierte Myelin enthielt weniger PLP und CNP. VAMP7 scheint also bei der Myelinisierung die Fusion von PLP- und CNP-enthaltenden Vesikeln mit der Myelinmembran zu vermitteln.

Role of hypoxia and growth and differentiation factor-5 on differentiation of human mesenchymal stem cells towards intervertebral nucleus pulposus-like cells

There is evidence that mesenchymal stem cells (MSCs) can differentiate towards an intervertebral disc (IVD)-like phenotype. We compared the standard chondrogenic protocol using transforming growth factor beta-1 (TGFß) to the effects of hypoxia, growth and differentiation factor-5 (GDF5), and coculture with bovine nucleus pulposus cells (bNPC). The efficacy of molecules recently discovered as possible nucleus pulposus (NP) markers to differentiate between chondrogenic and IVD-like differentiation was evaluated. MSCs were isolated from human bone marrow and encapsulated in alginate beads. Beads were cultured in DMEM (control) supplemented with TGFß or GDF5 or under indirect coculture with bNPC. All groups were incubated at low (2 %) or normal (20 %) oxygen tension for 28 days. Hypoxia increased aggrecan and collagen II gene expression in all groups. The hypoxic GDF5 and TGFß groups demonstrated most increased aggrecan and collagen II mRNA levels and glycosaminoglycan accumulation. Collagen I and X were most up-regulated in the TGFß groups. From the NP markers, cytokeratin-19 was expressed to highest extent in the hypoxic GDF5 groups; lowest expression was observed in the TGFß group. Levels of forkhead box F1 were down-regulated by TGFß and up-regulated by coculture with bNPC. Carbonic anhydrase 12 was also down-regulated in the TGFß group and showed highest expression in the GDF5 group cocultured with bNPC under hypoxia. Trends in gene expression regulation were confirmed on the protein level using immunohistochemistry. We conclude that hypoxia and GDF5 may be suitable for directing MSCs towards the IVD-like phenotype.

Ethanol Production from Hardboard Manufacturing Process Wastewater: Methods for Improving Product Yields from Hemicellulose Hydrolysates

Waste effluents from the forest products industry are sources of lignocellulosic biomass that can be converted to ethanol by yeast after pretreatment. However, the challenge of improving ethanol yields from a mixed pentose and hexose fermentation of a potentially inhibitory hydrolysate still remains. Hardboard manufacturing process wastewater (HPW) was evaluated at a potential feedstream for lignocellulosic ethanol production by native xylose-fermenting yeast. After screening of xylose-fermenting yeasts, Scheffersomyces stipitis CBS 6054 was selected as the ideal organism for conversion of the HPW hydrolysate material. The individual and synergistic effects of inhibitory compounds present in the hydrolysate were evaluated using response surface methodology. It was concluded that organic acids have an additive negative effect on fermentations. Fermentation conditions were also optimized in terms of aeration and pH. Methods for improving productivity and achieving higher ethanol yields were investigated. Adaptation to the conditions present in the hydrolysate through repeated cell sub-culturing was used. The objectives of this present study were to adapt S. stipitis CBS6054 to a dilute-acid pretreated lignocellulosic containing waste stream; compare the physiological, metabolic, and proteomic profiles of the adapted strain to its parent; quantify changes in protein expression/regulation, metabolite abundance, and enzyme activity; and determine the biochemical and molecular mechanism of adaptation. The adapted culture showed improvement in both substrate utilization and ethanol yields compared to the unadapted parent strain. The adapted strain also represented a growth phenotype compared to its unadapted parent based on its physiological and proteomic profiles. Several potential targets that could be responsible for strain improvement were identified. These targets could have implications for metabolic engineering of strains for improved ethanol production from lignocellulosic feedstocks. Although this work focuses specifically on the conversion of HPW to ethanol, the methods developed can be used for any feedstock/product systems that employ a microbial conversion step. The benefit of this research is that the organisms will the optimized for a company's specific system.