C-terminal processing of the teneurin proteins: Independent actions of a teneurin C-terminal associated peptide in hippocampal cells

Many neuropsychiatric conditions have a common set of neurological substrates associated with the integration of sensorimotor processing. The teneurins are a recently described family of proteins that play a significant role in visual and auditory development. Encoded on the terminal exon of the teneurin genes is a family of bioactive peptides, termed teneurin C-terminal associated peptides (TCAP), which regulate mood-disorder associated behaviors. Thus, the teneurin-TCAP system could represent a novel neurological system underlying the origins of a number of complex neuropsychiatric conditions. However, it is not known if TCAP-1 exerts its effects as part of a direct teneurin function, whereby TCAP represents a functional region of the larger teneurin protein, or if it has an independent role, either as a splice variant or post-translational proteolytic cleavage product of teneurin. In this study, we show that TCAP-1 can be transcribed as a smaller mRNA transcript. After translation, further processing yields a smaller 15. kDa protein containing the TCAP-1 region. In the mouse hippocampus, immunoreactive (ir) TCAP-1 is exclusively localized to the pyramidal layers of the CA1, CA2 and CA3 regions. Although the localization of TCAP and teneurin in hippocampal regions is similar, they are distinct within the cell as most ir-teneurin is found at the plasma membrane, whereas ir-TCAP-1 is predominantly found in the cytosol. Moreover, in mouse embryonic hippocampal cell culture, FITC-labeled TCAP-1 binds to the plasma membrane and is taken up into the cytosol via dynamin-dependent caveolae-mediated endocytosis. Our data provides novel evidence that TCAP-1 is structurally and functionally distinct from the larger teneurins. © 2012.

HDAC inhibition decreases XIST expression on female IVP bovine blastocysts

During initial development, both X chromosomes are active in females, and one of them must be silenced at the appropriate time in order to dosage compensate their gene expression levels to male counterparts. Silencing involves epigenetic mechanisms, including histone deacetylation. Major X chromosome inactivation (XCI) in bovine occurs between hatching and implantation, although in vitro culture conditions might disrupt the silencing process, increasing or decreasing X-linked gene expression. In this study, we aimed to address the roles of histone deacetylase inhibition by trichostatin A (TSA) on female preimplantation development.We tested the hypothesis that by enhancing histone acetylation, TSA would increase the percentage of embryos achieving 16-cell stage, reducing percentage of embryos blocked at 8-cell stage, and interfere with XCI in IVF embryos. We noticed that after TSA treatment, acetylation levels in individual blastomeres of 8-16 cell embryos were increased twofold on treated embryos, and the samewas detected for blastocysts. Changes among blastomere levels within the same embryo were diminished on TSA group, as low-acetylated blastomeres were no longer detected. The percentage of embryos that reached the 5th cleavage cycle 118 h after IVF, analyzed by Hoechst staining, remained unaltered after TSA treatment. Then, we assessed XIST and G6PD expression in individual female bovine blastocysts by quantitative real-time PCR. Even though G6PD expression remained unaltered after TSA exposure, XIST expression was eightfold decreased, and we also detected a major decrease in the percentage of blastocysts expressing detectable XIST levels after TSA treatment. Based on these results, we conclude that HDAC is involved on XCI process in bovine embryos, and its inhibition might delay X chromosome silencing and attenuate aberrant XIST expression described for IVF embryos. © 2013 Society for Reproduction and Fertility.

Antiprotozoal activity of quinonemethide triterpenes from Maytenus ilicifolia (Celastraceae)

The present study describes the leishmanicidal and trypanocidal activities of two quinonemethide triterpenes, maytenin (1) and pristimerin (2), isolated from Maytenus ilicifolia root barks (Celastraceae). The compounds were effective against the Trypanosomatidae Leishmania amazonensis and Leishmania chagasi and Trypanosoma cruzi, etiologic agents of leishmaniasis and Chagas' disease, respectively. The quinonemethide triterpenes 1 and 2 exhibited a marked in vitro leishmanicidal activity against promastigotes and amastigotes with 50% inhibitory concentration (IC50) values of less than 0.88 nM. Both compounds showed IC50 lower than 0.3 nM against Trypanosoma cruzi epimastigotes. The selectivity indexes (SI) based on BALB/c macrophages for L. amazonensis and L. chagasi were 243.65 and 46.61 for (1) and 193.63 and 23.85 for (2) indicating that both compounds presented high selectivity for Leishmania sp. The data here presented suggests that these compounds should be considered in the development of new and more potent drugs for the treatment of leishmaniasis and Chagas' disease. © 2013 by the authors.

Autogenous bone combined with anorganic bovine bone for maxillary sinus augmentation: Analysis of the osteogenic potential of cells derived from the donor and the grafted sites

Objectives: This study aimed to comparatively evaluate the in vitro osteogenic potential of cells obtained from the mandibular ramus (MR, autogenous bone donor site) and from the maxillary sinus (MS) bone grafted with a mixture of anorganic bovine bone (ABB) and MR prior to titanium implant placement (MS, grafted implant site). Material and methods: Cells were obtained from three patients subjected to MS floor augmentation with a 1: 1 mixture of ABB (GenOx Inorg®) and MR. At the time of the sinus lift procedure and after 8 months, prior to implant placement, bone fragments were taken from MR and MS, respectively, and subjected to trypsin-collagenase digestion for primary cell culturing. Subcultured cells were grown under osteogenic condition for up to 21 days and assayed for proliferation/viability, osteoblast marker mRNA levels, alkaline phosphatase (ALP) activity and calcium content/Alizarin red staining. ALP activity was also determined in primary explant cultures exposed to GenOx Inorg® (1: 1 with MR) for 7 days. Data were compared using either the Mann-Whitney U-test or the Kruskal-Wallis test. Results: MS cultures exhibited a significantly lower osteogenic potential compared with MR cultures, with a progressive increase in cell proliferation together with a decrease in osteoblast markers, reduced ALP activity and calcium content. Exposure of MR-derived primary cultures to GenOx Inorg® inhibited ALP activity. Conclusion: These results suggest that the use of GenOx Inorg® in combination with MR fragments for MS floor augmentation inhibits the osteoblast cell differentiation at the implant site in the long term. © 2013 John Wiley & Sons A/S.

Fibronectin induces MMP2 expression in human prostate cancer cells

High-grade prostate cancers express high levels of matrix metalloproteinases (MMPs), major enzymes involved in tumor invasion and metastasis. However, the tumor cell lines commonly employed for prostate cancer research express only small amounts of MMPs when cultivated as monolayer cultures, in common culture media. The present study was conducted to ascertain whether culture conditions that include fibronectin can alter MMP2 and MMP9 expression by the human prostatic epithelial cell lines RWPE-1, LNCaP and PC-3. These cells were individually seeded at 2×104cells/cm2, cultivated until they reached 80% confluence, and then exposed for 4h to fibronectin, after which the conditioned medium was analyzed by gelatin zymography. Untreated cells were given common medium. Only RWPE-1 cells express detectable amounts of MMP9 when cultivated in common medium, whereas the addition of fibronectin induced high expression levels of pro and active forms of MMP2 in all tested cell lines. Our findings demonstrate that normal and tumor prostate cell lines express MMP2 activity when in contact with extracellular matrix components or blood plasma proteins such as fibronectin. Future studies of transcriptomes and proteomes in prostate cancer research using these cell lines should not neglect these important conclusions. © 2012 Elsevier Inc..

VEGF-C expression in oral cancer by neurotransmitter-induced activation of beta-adrenergic receptors

The aim of this study was to investigate the expression of vascular endothelial growth factor type C (VEGF-C) in oral squamous cell carcinoma (OSCC) cell lines through norepinephrine-induced activation of beta-adrenergic receptors. Human OSCC cell lines (SCC-9 and SCC-25) expressing beta-adrenergic receptors were stimulated with different concentrations of norepinephrine (0.1, 1, and 10 μM) and 1 μM of propranolol, and analyzed after 1, 6, and 24 h. VEGF-C gene expression and VEGF-C production in the cell supernatant were evaluated by real-time PCR and by ELISA, respectively. The results showed that beta-adrenergic receptor stimulation by different concentrations of norepinephrine or blocking by propranolol did not markedly alter VEGF-C expression by SCC-9 and SCC-25 cells. VEGF-C protein levels produced by oral malignant cell lines after stimulation with different norepinephrine concentrations or blocking with propranolol was statistically similar (p > 0.05) to those of the control group (nonstimulated OSCC cell lines). Our findings suggest that stimulation of beta-adrenergic receptors by means of norepinephrine does not seem to modulate the VEGF-C expression in OSCC cell lines. These findings reinforce the need for further studies in order to understand the responsiveness of oral cancer to beta-adrenergic receptor stimulation or blockage, especially with regard to VEGF-C production. © 2012 International Society of Oncology and BioMarkers (ISOBM).

In vitro and in vivo investigation of the biological and mechanical behaviour of resin-modified glass-ionomer cement containing chlorhexidine

Objectives: To evaluate: (1) the in vitro antibacterial, cytotoxic and mechanical properties of a resin-modified glass ionomer cement (RMGIC) containing different concentrations of chlorhexidine (CHX) and (2) the in vivo microbiologic action of the best concentration of CHX associated with the RMGIC applied on remaining dentine after indirect pulp treatment (IPT). Methods: For the in vitro studies, RMGIC was associated with 0.2, 0.5, 1.25 and 2.5% CHX. Microbiologic evaluation consisted of an agar diffusion test on cariogenic bacteria for 24 h. Odontoblast-like cell metabolism and morphology analyses measured the cytotoxic effects of the RMGIC groups after 24 h. The same groups were submitted to compressive and diametral tensile strength. The in vivo treatment consisted of IPT using an RMGIC associated with the best CHX concentration. Clinical and microbiologic evaluations were performed before and after 3 months. Results: The use of 1.25% CHX significantly improved the antibacterial effects of the evaluated RMGIC, without causing any detrimental effects to the odontoblast-like cells and on the mechanical properties. This RMGIC and CHX combination completely eliminated mutans streptococci after 3 months of IPT. Conclusion: The RMGIC and 1.25% CHX mixture showed great biological and mechanical behaviour and could be a good treatment against caries progression. Clinical significance: The association of CHX with a liner RMGIC opens a new perspective for arresting residual caries after IPT. © 2012 Elsevier Ltd. All rights reserved.

Chemoprotective activity of the isoflavones, genistein and daidzein on mutagenicity induced by direct and indirect mutagens in cultured HTC cells

Isoflavones are phenolic compounds widely distributed in plants and found in a high percentage in soybeans. They have important biological properties and are regarded as potential chemopreventive agents. The aim of this study was to verify the preventive effect of two soy isoflavones (genistein and daidzein) by a micronucleus assay, analysis of GST activity, and real-time RT-PCR analysis of GSTa2 gene expression. Mutagens of direct (doxorubicin) and indirect (2-aminoanthracene) DNA damage were used. Hepatoma cells (HTC) were treated with genistein or daidzein for 26 h at noncytotoxic concentrations; 10 μM when alone, and 0.1, 1.0 and 10 μM when combined with genotoxic agents. The micronucleus test demonstrated that both isoflavones alone had no genotoxic effect. Genistein showed antimutagenic effects at 10 μM with both direct and indirect DNA damage agents. On phase II enzyme regulation, the current study indicated an increase in total cytoplasmic GST activity in response to genistein and daidzein at 10 μM supplementation. However, the mRNA levels of GSTa2 isozymes were not differentially modulated by genistein or daidzein. The results point to an in vitro antimutagenic activity of genistein against direct and indirect DNA damage-induced mutagenicity. © 2012 Springer Science+Business Media B.V.

Comparative analysis of Enterococcus faecalis biofilm formation on different substrates

Introduction: The aim of this study was to compare Enterococcus faecalis biofilm formation on different substrates. Methods: Cell culture plates containing growth medium and E. faecalis (ATCC 29212) were used to grow biofilm on bovine dentin, gutta-percha, hydroxyapatite, or bovine bone. Substrates were incubated at 37°C for 14 or 21 days, and the medium was changed every 48 hours. After the growth induction periods, specimens (n = 5 per group and per induction period) were stained by using Live/Dead, and the images were analyzed under a confocal microscope. The total biovolume (μm3), live bacteria biovolume (μm3), and substrate coverage (%) were quantified by using the BioImage-L software. Results obtained were analyzed by nonparametric tests (P =.05). Results: Biofilm formation was observed in all groups. Gutta-percha had the lowest total biovolume at 14 days (P <.05) and hydroxyapatite the highest at 21 days (P <.05). No significant difference was observed in green biovolume at 14 days. At 21 days, however, hydroxyapatite had the highest volume (P <.05). The percentages of coverage were similar among all substrates at 21 days (P >.05), but at 14 days, bovine bone presented the highest coverage (P <.05). Conclusions: E. faecalis was capable of forming biofilm on all substrates during both growth periods; hydroxyapatite presented the highest rates of biofilm formation. The type of substrate influenced the biofilm characteristics, according to the parameters evaluated. © 2013 American Association of Endodontists.

Use of different carbon sources in cultivation of baker's yeast for production of glycerol-3-phosphate dehydrogenase

The physiological state of yeast cells changes during culture growth as a consequence of environmental changes (nutrient limitations, pH and metabolic products). Cultures that grow exponentially are heterogeneous cell populations made up of cells regulated by different metabolic and/or genetic control systems. The strain of baker's yeast selected by plating commercial compressed yeast was used for the production of glycerol-3- phosphate dehydrogenase. Glycerol-3-phosphate dehydrogenase (GPD) has been widely used in the enzyme assays with diverse compounds of industrial interest, such as glycerol or glycerol phosphate, as well as a number of important bioanalytical applications. Each cell state determines the level of key enzymes (genetic control), fluxes through metabolic pathways (metabolic control), cell morphology and size. The present study was carried out to determine the effects of environmental conditions and carbon source on GPD production from baker's yeast. Glucose, glycerol, galactose and ethanol were used as carbon sources. Glycerol and ethanol assimilations required agitation, which was dependent on the medium volume in the fermentation flask for the greatest accumulation of intracellular GPD. Enzyme synthesis was also affected by the initial pH of the medium and inoculum size. The fermentation time required for a high level of enzyme formation decreased with the inoculum size. The greatest amount of enzyme (0.45 U/ml) was obtained with an initial pH of 4.5 in the medium containing ethanol or glycerol. The final pH was maintained in YP-ethanol, but in the YP-glycerol the final pH increased to 6.9 during growth.

Phytohaemagglutinin's effect on Neospora caninum antigen production

The parasite Neospora caninum affects mainly cattle and dogs. This study aimed to evaluate the effect of phytohaemagglutinin (PHE) in antigen production of N. caninum NC-1 strain in gerbils (Meriones unguiculatus) and in vitro. 20 gerbils were used, 10 inoculated intraperitoneally with 1 × 106 tachyzoites and 10 with 1 × 106 tachyzoites plus 300 μL/mL of PHE. 16 bottles of Vero cell culture were inoculated, 8 with 1.5 × 105 tachyzoites and 8 with 1.5 × 105 tachyzoites plus 30 μL/mL of PHE. Serology of gerbils was performed on day 0 and before euthanasia. Tachyzoites present in peritoneal fluid and cell culture bottles were quantified by Neubauer chamber and by real-time PCR (qPCR). PHE has not interfered in the production of tachyzoites of N. caninum in intraperitoneal inoculated gerbils and the effect of PHE in cell culture had a negative impact, considering the qPCR technique as the gold standard.

Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Toll-like receptor 2 knockdown modulates interleukin (IL)-6 and IL-8 but not stromal derived factor-1 (SDF-1/CXCL12) in human periodontal ligament and gingival fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll-like receptors (TLRs), recognize pathogen-associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll-like receptor 2 (TLR2) and an antagonist or agonist for Toll-like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)-6, IL-8, and stromal derived factor-1 (SDF-1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA-mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL-6, IL-8, and CXCL12mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzymelinked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL-6 and IL-8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL-6 and IL-8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.

Silibinin attenuates oxidative metabolism and cytokine production by monocytes from preeclamptic women.

Silibinin is a polyphenolic plant flavonoid with anti-inflammatory properties. The present study investigated the effect of silibinin on oxidative metabolism and cytokine production - tumor necrosis factor-alpha (TNF-α), interleukin (IL)12, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-10, and transforming growth factor beta (TGF-β1) - by peripheral blood monocytes (PBM) from preeclamptic pregnant women. It is a case-controlled study involving women with preeclampsia (PE, n = 30) compared with normotensive pregnant (NT, n = 30) and with non-pregnant (NP, n = 30) women. Monocytes were obtained and cultured with or without silibinin (5 μM or 50 μM) for 18 h. Superoxide anion (O2-) and hydrogen peroxide (H2O2) release were determined by specific assays, and cytokine levels were determined by immunoenzymatic assays (ELISA). Monocytes from preeclamptic women cultured without stimulus released higher levels of O22, H2O2 and TNF-α, and lower levels of IL-10 and TGF-β1 than did monocytes from NT and NP women. Treatment in vitro with silibinin significantly inhibited spontaneous O2- and H2O2 release and TNF-α production by monocytes from preeclamptic women. The main effect of silibinin was obtained at 50 μM concentration. Thus, silibinin exerts anti-oxidative and anti-inflammatory effects on monocytes from preeclamptic pregnant women by inhibiting the in vitro endogenous release of reactive oxygen species and TNF-α production.

Fontes de carbono de baixo custo para a produção de xilanase termoestável por aspergillus niger

A strain of the flamentous fungus Aspergillus niger was isolated and shown to possess extracellular xylanolytic activity. These enzymes have biotechnological potential and can be employed in various industries. This fungus produced its highest xylanase activity in a medium made up of 0.1% CaCO3, 0.5% NaCl, 0.1% NH4Cl, 0.5% corn steep liquor and 1% carbon source, at pH 8.0. A low-cost hemicellulose residue (powdered corncob) proved to be an excellent inducer of the A. niger xylanolytic complex. Filtration of the crude culture medium with suspended kaolin was ideal for to clarify the extract and led to partial purifcation of the xylanolytic activity. The apparent molecular mass of the xylanase was about 32.3 kDa. Maximum enzyme activity occurred at pH 5.0 and 55-60oC. Apparent Km was 10.41 ± 0.282 mg/mL and Vmax was 3.32 ± 0.053 U/mg protein, with birchwood xylan as the substrate. Activation energy was 4.55 kcal/mol and half-life of the crude enzyme at 60oC was 30 minutes. Addition of 2% glucose to the culture medium supplemented with xylan repressed xylanase production, but in the presence of xylose the enzyme production was not affected.