IBA application for rooting of Eucalyptus benthamii Maiden and Cambage x Eucalyptus dunnii Maiden minicuttings

IBA application for rooting of Eucalyptus benthamii Maiden and Cambage x Eucalyptus dunnii Maiden minicuttings. Eucalyptus has great importance in the forestry sector and many advances in the area of improvement have been achieved with the advent of biotechnological techniques. However, some promising genotypes still do not have multiplication protocols with cloning techniques, such as minicutting. The study aimed to evaluate IBA concentrations for survival, rooting and vegetative vigor of E. benthamii x E. dunnii minicuttings, and determine the maximum technical efficiency dose. Ministumps H12, H19 and H20 clones were cultivated in a clonal minigarden under a semi-hydroponic system. For rooting, the rninicutting basal portion was plunged in hydro-alcoholic solutions, whose concentrations were: 0; 2,000; 4,000; 6,000 and 8,000 mg L(-1) of IBA. The experiment was conducted in a completely randomized design, with the factors consisting of three clones and five IBA concentrations, with five replications, containing 10 minicuttings per replication. IBA positively influenced the minicutting rooting processes, with differentiated behavior between the clones, with 30.32 to 55.45% rooting variation. The positives increments occurred until the highest IBA concentration for H12 and H19 clones. However, the range between 4,000 and 6,000 mg L(-1) of IBA treatments promoted the best rooting results for the H20 clone.

Allele-Specific Expression of the MAOA Gene and X Chromosome Inactivation in In Vitro Produced Bovine Embryos

During embryogenesis, one of the two X chromosomes is inactivated in embryos. The production of embryos in vitro may affect epigenetic mechanisms that could alter the expression of genes related to embryo development and X chromosome inactivation (XCI). The aim of this study was to understand XCI during in vitro, pre-implantation bovine embryo development by characterizing the allele-specific expression pattern of the X chromosome-linked gene, monoamine oxidase A (MAOA). Two pools of ten embryos, comprised of the 4-, 8- to 16-cell, morula, blastocyst, and expanded blastocyst stages, were collected. Total RNA from embryos was isolated, and the RT-PCR-RFLP technique was used to observe expression of the MAOA gene. The DNA amplicons were also sequenced using the dideoxy sequencing method. MAOA mRNA was detected, and allele-specific expression was identified in each pool of embryos. We showed the presence of both the maternal and paternal alleles in the 4-, 8-to 16-cell, blastocyst and expanded blastocyst embryos, but only the maternal allele was present in the morula stage. Therefore, we can affirm that the paternal X chromosome is totally inactivated at the morula stage and reactivated at the blastocyst stage. To our knowledge, this is the first report of allele-specific expression of an X-linked gene that is subject to XCI in in vitro bovine embryos from the 4-cell to expanded blastocyst stages. We have established a pattern of XCI in our in vitro embryo production system that can be useful as a marker to assist the development of new protocols for in vitro embryo production. Mol. Reprod. Dev. MoL Reprod. Dev. 77: 615-621, 2010. (C) 2010 Wiley-Liss, Inc.

A New Approach to Ammonium Sulphate Feeding for Fed-Batch Arthrospira (Spirulina) platensis Cultivation in Tubular Photobioreactor

Arthrospira platensis was cultivated in tubular photobioreactor using different photosynthetic photon flux densities (PPFD) and protocols of (NH(4))(2)SO(4) fed-hatch supply. Results were evaluated by variance analysis selecting maximum cell concentration (X(m)), cell productivity (P(x)), nitrogen-to-cell conversion factor (Y(X/N)) and biomass, protein and lipid contents as responses. At PPFD of 120 and 240 mu mol-photons/m(2) s, a parabolic profile of (NH(4))(2)SO(4) addition aiming at producing biomass with 7% nitrogen content ensured X(m) values (14.1 and 12.2 g/L, respectively) comparable to those obtained with NaNO(3). At PPFD of 240 mu mol-photons/m(2) s, P(x) (1.69 g/Ld) was 36% higher, although the photosynthetic efficiency (3.0%) was less than one-half that at PPFD of 120 mu mol-photons/m(2) s. Biomass was shown to be constituted by about 35% proteins and 10% lipids, without any dependence on PPFD or kind of nitrogen source. These results highlight the possible use of (NH(4))(2)SO(4) as alternative, cheap nitrogen source for A. platensis cultivation in tubular photobioreactors. (C) 2010 American Institute of Chemical Engineers Biotechnol. Prog., 26: 1271-1277, 2010

Comparison of the Salmonella/Microsome Microsuspension Assay with the new Microplate Fluctuation Protocol for Testing the Mutagenicity of Environmental Samples

The objective of this study was to compare the responses of the Salmonella/microsome microsuspension assay with the new microplate fluctuation protocol (MPF) for the evaluation of the mutagenic activity of environmental samples. Organic extracts of total particulate atmospheric air samples, surface waters, and effluents were tested in dose-response experiments. The assays were performed with strain TA98 in the absence and presence of S9 mix. Both protocols produced similar results, despite the fact that the maximum score of the MPF is limited to 48 wells, whereas in the regular plate assay it is possible to count up to 1,500 colonies using an automatic counter. Similar sensitivities based on the lowest dose that resulted in a positive response were obtained for both assays. The MPF procedure is less laborious (e.g., all-liquid format, use of multi-channel pipettors) and allows for automation of the pipetting and dispensing steps, thus, reducing time of the analysis which is particularly important in environmental quality monitoring programs or in effect-directed analysis. The results show that the MPF procedure is a promising tool to test environmental samples for mutagenic activity. Environ. Mol. Mutagen. 51:31-38, 2010. (C) 2009 Wiley-Liss, Inc.

Application of hydrophobic resin adhesives to acid-etched dentin with an alternative wet bonding technique

Hydrophilic dentin adhesives are prone to water sorption that adversely affects the durability of resin-dentin bonds. This study examined the feasibility of bonding to dentin with hydrophobic resins via the adaptation of electron microscopy tissue processing techniques. Hydrophobic primers were prepared by diluting 2,2-bis[4(2-hydroxy-3-methacryloyloxy-propyloxy)-phenyl] propane/triethyleneglycol dimethacrylate resins with known ethanol concentrations. They were applied to acid-etched moist dentin using an ethanol wet bonding technique that involved: (1) stepwise replacement of water with a series of increasing ethanol concentrations to prevent the demineralized collagen matrix from collapsing; (2) stepwise replacement of the ethanol with different concentrations of hydrophobic primers and subsequently with neat hydrophobic resin. Using the ethanol wet bonding technique, the experimental primer versions with 40, 50, and 75% resin exhibited tensile strengths which were not significantly different from commercially available hydrophilic three-step adhesives that were bonded with water wet bonding technique. The concept of ethanol wet bonding may be explained in terms of solubility parameter theory. This technique is sensitive to water contamination, as depicted by the lower tensile strength results from partial dehydration protocols. The technique has to be further improved by incorporating elements of dentin permeability reduction to avoid water from dentinal tubules contaminating water-free resin blends during bonding. (c) 2007 Wiley Periodicals, Inc. J Biomed Mater Res 84A: 19-29, 2008.

Evaluation of physico-chemical and phytochemical characteristics of different tinctures of barbatimao (Stryphnodendron barbatiman)

As a consequence of the large distribution and use of medicinal plants, the industries are producing products based on plant species in various pharmaceutical forms, which have been commercialized in pharmacies and natural products homes. However, there is no guarantee for the vast majority of these products, as to their effctiveness, safety, and quality, which may cause risks to the health of consumers. There it is important the establishment of standardized protocols of quality control for phytotherapeutic products. Tinctures of barbatimao are available in the Brazilian market proceeding from diverse manufacturers. With the purpose to evaluate the difference between the quality of tinctures of barbatimao proceeding from four manufactures, a comparative study of ph ysico-chenfical andphylocheinical characteristics was carried out. For physico-chemical analysis, the pH, density, dry residue and tannins content were evaluated. The phytochemical analysis was made using thin layer chromatography. The differences observed in physico-chemical and phytochemical characteristics had evidenced the lack of standardization in the production of these tinctures.

Evaluation of antiulcer activity of the main phenolic acids found in Brazilian Green Propolis

Aim of the study: In a previous study, our group described the gastric protective effect of the hydroalcoholic extract of Brazilian green propolis. The main compounds found in Brazilian green propolis include phenolic acids, such as: caffeic, ferulic, p-coumaric and cinnamic acids. This study was therefore carried out to evaluate the antiulcerogenic property of the main phenolic acids found in Brazilian Green Propolis. Material and methods: The anti-ulcer assays were performed using the following protocols: nonsteroidal-antinflammatory drug (NSAID)-induced ulcer, ethanol-induced ulcer, and stress-induced ulcer. The effects of the phenolic acids on gastric content volume, pH and total acidity, using the pylorus ligated model, were also evaluated. Results: It was observed that treatment using doses of 50 and 250 mg/kg of caffeic, ferulic, p-coumaric and cinnamic acids and positive controls (omeprazol or cimetidine) significantly diminished the lesion index, the total area of the lesion and the percentage of lesion in comparison with the negative control groups. In addition, the percentage of ulcer inhibition was significantly higher in the groups treated with the different phenolic acids, cimetidine or omeprazol, in all the protocols used, compared with the negative control groups. In the model to determine gastric secretion, using ligated pylorus, treatment with phenolic acids and cimetidine reduced the volume of gastricjuice and total acidity and significantly increased the gastric pH (p < 0.05), compared with the control group, with the exception of the group treated with 50 mg/kg of p-coumaric acid, in which no significant difference was observed, compared with the control. In relation to the acute toxicity, none sign of toxicity was observed when phenolic acids, used in this study, were administered for rats in dose of 2000 mg/kg. Conclusions: In conclusion, the results of this study show that caffeic, ferulic, p-coumaric and cinnamic acids display antiulcer activity. (c) 2008 Elsevier Ireland Ltd. All rights reserved.

In vitro studies of cutaneous retention of magnetic nanoemulsion loaded with zinc phthalocyanine for synergic use in skin cancer treatment

In this study was developed a new nano drug delivery system (NDDS) based on association of biodegradable surfactants with biocompatible magnetic fluid of maguemita citrate derivative. This formulation consists in a magnetic emulsion with nanostructured colloidal particles. Preliminary in vitro experiments showed that the formulation presents a great potential for synergic application in the topical release of photosensitizer drug (PS) and excellent target tissue properties in the photodynamic therapy (PDT) combined with hyperthermia (HPT) protocols. The physical chemistry characterization and in vitro assays were carried out by Zn(II) Phtalocyanine (ZnPc) photosensitizer incorporated into NDDS in the absence and the presence of magnetic fluid, showed good results and high biocompatibility. In vitro experiments were accomplished by tape-stripping protocols for quanti. cation of drug association with different skin tissue layers. This technique is a classical method for analyses of drug release in stratum corneum and epidermis+ dermis skin layers. The NDDS formulations were applied directly in pig skin (tissue model) fixed in the cell`s Franz device with receptor medium container with a PBS/EtOH 20% solution (10mM, pH 7.4) at 37 degrees C. After 12 h of topical administration stratum corneum was removed from fifty tapes and the ZnPc retained was evaluated by solvent extraction in dimetil-sulphoxide under ultrasonic bath. These results indicated that magnetic nanoemulsion (MNE) increase the drug release on the deeper skin layers when compared with classical formulation in the absence of magnetic particles. This could be related with the increase of biocompatibility of NDDS due to the great affinity for the polar extracelullar matrix in the skin and also for the increase in the drug partition inside of corneocites wall. (C) 2008 Elsevier B.V. All rights reserved.

Photophysical studies and in vitro skin permeation/retention of Foscan (R)/nanoemulsion (NE) applicable to photodynamic therapy skin cancer treatment.

In this work we evaluated the photophysical and in vitro properties of Foscan (R), a second-generation photosensitizer drug (PS) widely used in systemic clinical protocols for cancer therapy based on Photodynamic Therapy (PDT). We employed biodegradable nanoemulsions (NE) as a colloidal vehicle of the oil/water (o/w) type focusing in topical administration of Foscan (R) and other photosensitizer drugs. This formulation was obtained and stabilized by the methodology described by Tabosa do Egito et al.,(30) based on the mixture of two phases: an aqueous solution and an organic medium consisting of nonionic surfactants and oil. The photodynamic potential of the drug incorporated into the NE was studied by steady-state and time-resolved spectroscopic techniques. We also analyzed the in vitro biological behavior carried out in mimetic biological environment protocols based on the animal model. After topical application in a skin animal model, we evaluated the Foscan (R)/NE diffusion flux into the skin layers (stratum corneum and epidermis + dermis) by classical procedures using Franz Diffusion cells. Our results showed that the photophysical properties of PS were maintained after its incorporation into the NE when compared with homogeneous organic medium. The in vitro assays enabled the determination of an adequate profile for the interaction of this system in the different skin layers, with an ideal time lag of 6 h after topical administration in the skin model. The Foscan (R) diffusion flux (J) was increased when this PS was incorporated into the NE, if compared with its flux in physiological medium. These parameters demonstrated that the NE can be potentially applied as a drug delivery system (DDS) for Foscan (R) in both in vitro and in vivo assays, as well as in future clinical applications involving topical skin cancer PDT.

Antiulcerogenic Activity of the Essential Oil of Baccharis dracunculifolia on Different Experimental Models in Rats

Baccharis dracunculifolia DC (Asteraceae), a native plant from Brazil, commonly known as `Alecrim-do-campo` is widely used in folk medicine to treat inflammation, hepatic disorders and stomach ulcers, and it is the most important botanical source of Southeastern Brazilian propolis, known as green propolis. Its essential oil is composed of non-oxygenated and oxygenated terpenes. In this work, the effects of the essential oil obtained from the aerial parts of R dracunculifolia on gastric ulcers were evaluated. The antiulcer assays were undertaken using the following protocols in rats: nonsteroidal antiinflammatory drug (NSAID)-induced ulcer, ethanol-induced ulcer, stress-induced ulcer, and determination of gastric secretion using ligated pylorus. The treatment in the doses of 50, 250 and 500 mg/kg of R dracunculifolia essential oil significantly diminished the lesion index, the total lesion area and the percentage of lesions in comparison with both positive and negative control groups. With regard to the model of gastric secretion a reduction of gastric juice volume and total acidity was observed, as well as an increase in the gastric pH. No sign of toxicity was observed in the acute toxicity study. Considering the results, it is suggested that the essential oil of B. dracunculifolia could probably be a good therapeutic agent for the development of new phytotherapeutic medicine for the treatment of gastric ulcer. Copyright (C) 2009 John Wiley & Sons, Ltd.

Evaluation of Antigenotoxic Effects of Plant Flavonoids Quercetin and Rutin on HepG2 Cells

The flavonoid quercetin and its derivative rutin were investigated for genotoxicity/antigenotoxicity activity in human hepatoma HepG2 cells using the comet assay. The extract cytotoxicity was evaluated using the trypan blue exclusion dye method with quercetin and rutin concentrations ranging from 0.1 to 200.0 mu g/mL of culture medium. Three minor non-cytotoxic concentrations were chosen to evaluate the genotoxicity and antigenotoxicity of the flavonoids (0.1, 1.0 and 5.0 mu g/mL) through comet assay. The cultures were treated with three different concentrations of rutin or quercetin (genotoxicity) or their association with Aflatoxin B1 (AFB1), methyl methanesulfonate (MMS) or doxorubicin (DXR) (antigenotoxicity test) in three protocols: pretreatment, simultaneous treatment and post-treatment. The cell cultures were also treated with 1% DMSO (control group), AFB1, MMS and DXR (positive-control). Statistical analyses were performed using ANOVA and Dunnett`s test (p <= 0.05). Quercetin at concentrations higher than 10.0 mu g/mL or rutin higher than 50.0 mu g/mL exhibited a cytotoxic effect on the cells, showing that quercetin is more cytotoxic than rutin. Furthermore, neither compound was able to induce genotoxicity in the concentrations evaluated. On the other hand, both flavonoids reduced DNA damage induced by AFB1, MMS and DXR in all treatment protocols. Copyright (C) 2011 John Wiley & Sons, Ltd.

Suppressive action of melatonin on the TH-2 immune response in rats infected with Trypanosoma cruzi

Control of the acute phase of Trypanosoma cruzi infection is critically dependent on cytokine-mediated macrophage activation to intracellular killing, natural killer (NK) cells, CD4(+) T cells, CD8(+) T cells and B cells. Cell-mediated immunity in T. cruzi infection is also modulated by cytokines, but in addition to parasite-specific responses, autoimmunity can be also triggered. Importantly, cytokines may also play a role in the cell-mediated immunity of infected subjects. Here we studied the role of cytokines in the regulation of innate and adaptive immunity during the acute phase of T. cruzi infection in Wistar rats. Melatonin is an effective regulator of the immune system. Macrophages and T lymphocytes, which have melatonin receptors, are target cells for the immunomodulatory function of melatonin. In this paper melatonin was orally given via two protocols: prior to and concomitant with infection. Both treatments were highly effective against T. cruzi with enhanced action for the concomitant treatment. The data suggest an up-regulation of the TH-1 immune response as all analyzed parameters, interleukin (IL)-4, IL-10, transforming growth factor-beta 1 and splenocyte proliferation, displayed reduced levels as compared with the untreated counterparts. However, the direct effects of melatonin on immune cells have not been fully investigated during T. cruzi infection. We conclude that in light of the current results, melatonin exerted important therapeutic benefits through its immune regulatory effects.

Prevalence and characterization of Enterococcus spp. isolated from Brazilian foods

Enterococci can be used in the food industry as starter or probiotic cultures. However, enterococci are also implicated in severe multi-resistant nosocomial infections. In this study, the prevalence of enterococci in selected Brazilian foodstuffs (raw and pasteurized milk, meat products, cheeses and vegetables) was evaluated. Phenotypic and PCR protocols were used for species identification. Tests for production of gelatinase, haemolysin, bacteriocin and bile salt hydrolysis were done with all enterococci isolates, whereas molecular determination of virulence markers (genes esp, gel, ace, as, efaA, hyl and cylA) and antibiotic resistance was checked only for Enterococcus faecium and Enterococcus faecalis isolates. The antibiotic-resistant isolates were assayed for biofilm formation and adhesion to mammalian cells. From the 120 food samples analyzed, 52.5% were positive for enterococci, meat and cheese being the most contaminated. E. faecium was the predominant species, followed by E. faecalis, E. casseliflavus and Enterococcus gallinarum. Phenotypic tests indicated that 67.7% of isolates hydrolyzed bile salts, 15.2% produced bacteriocin, 12.0% were beta-hemolytic and 18.2% produced gelatinase. Antibiotic resistance (gentamicin, tetracycline and erythromycin) and genes encoding for virulence traits were more frequent in E. faecalis than in E. faecium. Three E. faecium isolates were resistant to vancomycin. Among antibiotic-resistant isolates, 72.4% of E. faecalis were able to form biofilm and 13.8% to adhere to Caco-2 cells. Antibiotic-resistant E. faecalis and E. faecium isolates were grouped by RAPD-PCR and a scattered distribution was noted, indicating that resistance was not related to a particular clone. The spread of virulence/resistance traits in isolates of the two species and different RAPD-types suggest the pathogenic potential of both species. By contrast, the recovery of bacteriocinogenic E. faecium isolates with no virulence traits suggests their potential for biotechnological applications. In conclusion, our results showed that enterococci from Brazilian foods present important dualist aspects for food safety. (C) 2008 Elsevier Ltd. All rights reserved.

Anti-eosinophilic effect of Lafoensia pacari in toxocariasis

We previously reported the anti-inflammatory activity of Lafoensia pacari extract in Toxocara canis infection, a model of systemic IL-5-dependent eosinophil migration. In the present study, we describe the kinetics of the anti-inflammatory activity of L. pacari extract and compare it with dexamethasone. T canis-infected mice were submitted to different treatment protocols and the cells present in bronchoalveolar space and peritoneal cavity were collected at the end of each treatment period. The results showed that L. pacari extract effectively inhibited eosinophil migration only when the treatment was initiated before the peak of eosinophil migration (1st to 18th; 12th to 18th and 12th to 24th day post-infection). When eosinophil migration was established, administration of L. pacari extract had no effect on it (treatment 18th to 24th day post-infection). Dexamethasone was effective in inhibiting eosinophil migration in all periods studied. We suggest that L pacari extract can potentially be a natural alternative treatment of eosinophilic diseases. (c) 2007 Published by Elsevier GmbH.

Evaluation of the genotoxic and anti-genotoxic activities of Silybin in human hepatoma cells (HepG2)

Silybin (SB), a constituent of the medicinal plant Silybum marianum, is reported to be a potent hepatoprotective agent, but little is currently known regarding its genotoxicity, mutagenicity and potential chemopreventive properties. In this study, we evaluated the ability of SB to induce DNA migration and micronuclei (MN) formation in human hepatoma cells (HepG2). Also, possible preventive effects of SB on MN formation induced by three different mutagens, bleomycin (BLEO), benzo[a] pyrene (B[alpha] P) and aflatoxin B(1) (AFB(1)), were studied. To clarify the possible mechanism of SB antimutagenicity, three treatment protocols were applied: pretreatment, in which SB was added before the application of the mutagens; simultaneous treatment, in which SB was added during treatment and post-treatment, in which SB was added after the application of the mutagens. At concentrations up to 100 mu M, SB was non-genotoxic, while at a concentration of 200 mu M, SB induced DNA migration, generated oxidized DNA bases, reduced cell viability, decreased the replicative index of the cells and induced oxidative stress. It is noteworthy that SB was able to reduce the genotoxic effect induced by B[alpha] P, BLEO and AFB1 in pretreatment and simultaneous treatments but had no significant effect on DNA damage induction in post-treatment. Taken together, our findings indicate that SB presents anti-genotoxic activity in vitro, which suggests potential use as a chemopreventive agent.