Potencial de produção de gás metano e dióxido de carbono in vitro dos ingredientes utilizados em dietas para ovinos

This study aimed to evaluate the potential of CH4 and CO2 in vitro production of soybean hulls, sunflower meal, corn, citrus pulp and corn silage. Four rumen-cannulated sheep were fed diets containing the evaluated ingredients at 40:60 forage:concentrate ratio. The gases produced by samples incubation were measured by injection into a gas chromatograph equipped with flame ionization detector. The experimental design was completely randomized with repeated measures, with three replicates for each evaluated food at four different periods. Under the experimental conditions, we verified different potential gas production among the ingredients. The citrus pulp meal was the ingredient with the greatest potential for CO2production. Corn silage and soybean hulls showed the greatest potential while citrus pulp and sunflower meal showed the least potential for CH4 production, when expressed in mL/g of degraded dry matter; therefore, they can be considered, among the evaluated ingredients, those with the lowest environmental impact.

Sources of omega-6 fatty acids do not alter the rumen degradation and transit of fibre from dairy cow diets

The objective of this study was to evaluate the behaviour of fibre in the digestive tract on the basis of the passage kinetics of forage and concentrate particles in cows fed different omega-6 fatty-acid sources. The scientific hypothesis of this study was that omega-6 fatty acids do not interfere with the digestion of fibre in the diets of dairy cows. Five primiparous lactating Holstein cows were used in the experiment. The experimental diets were: control (C), ground soyabean (GS), cottonseed (CS), soyabean oil (SO), calcium salts of fatty acids (CSFA). The global mean estimates for the parameters of passage rate (gamma) were 0.038 and 0.055 h(-1) for forage and concentrate, respectively. The only significant effect with respect to the passage rate was a high negative correlation between the concentrate passage rate and dry matter intake. There was less undegradable neutral detergent fibre (NDF) in treatments without added lipid. Dietary supplementation with lipid sources does not alter the kinetic parameters of roughage and concentrate particle passage or in vitro NDF degradation. Sources of omega-6 fatty acids do not alter the rumen degradation and transit of fibre.

Stabilization of dentin matrix after cross-linking treatments, in vitro

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Differential Expression of IGF Family Members in Heat-Stressed Embryos Produced In Vitro from OPU-Derived Oocytes of Nelore (Bos indicus) and Holstein (Bos taurus) Cows

Contents The IGF system is related to embryo quality. We aim to determine the effect of the heat stress on the mRNA expression of IGF1 and IGF2, IGFR1 and IGFR2, IGFBP2 and IGFBP4, and PAPPA in in vitro production (IVP) blastocysts from Nelore and Holstein after ovum pick up (OPU) to better understand the differences between these breeds.Oocytes from four Nelore and seven Holstein were collected in six OPU sessions. Following in vitro maturation and fertilization using six Nelore or Holstein sires, embryos were divided into control (cultured at 39 degrees C) and heat stress (HS; exposed to 41 degrees C for 9h). Blastocysts were submitted to RNA extraction. The IGF1 expression was higher in blastocysts under HS in both breeds, and the expression of IGFBP2 and IGFBP4 was higher in Holstein blastocysts under HS. The high PAPPA expression and the low expression of IGFBP2 and IGFBP4 are associated with a more efficient degradation of IGFBPs, which results in greater IGF bioavailability in Nelore blastocysts and may contribute to the superior HS tolerance in Nelore, when compared to Holstein.

In vitro effect of caffeic acid phenethyl ester on matrix metalloproteinases (MMP-1 and MMP-9) and their inhibitor (TIMP-1) in lipopolysaccharide-activated human monocytes

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Membrane lipid profile monitored by mass spectrometry detected differences between fresh and vitrified in vitro-produced bovine embryos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

In vitro Metabolism of Grandisin, a Lignan with Anti-chagasic Activity

Tetrahydrofuran lignans represent a well-known group of phenolic compounds capable of acting as antiparasitic agents. In the search for new medicines for the treatment of Chagas disease, one promising compound is grandisin which has shown significant activity on trypomastigote forms of Trypanosoma cruzi. In this work, the in vitro metabolism of grandisin was studied in the pig cecum model and by biomimetic phase I reactions, aiming at an ensuing a preclinical pharmacokinetic investigation. Although grandisin exhibited no metabolization by the pig microbiota, one putative metabolite was formed in a biomimetic model using Jacobsen catalyst. The putative metabolite was tested against T. cruzi revealing loss of activity in comparison to grandisin.

Effects of Aspergillus spp. exogenous fibrolytic enzymes on in vitro fermentation of tropical forages

BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen micro-organisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages. Copyright (c) 2012 Society of Chemical Industry

Degradation of [D-Leu]-Microcystin-LR by solar heterogeneous photocatalysis (TiO2)

The present study investigates the use of solar heterogeneous photocatalyis (TiO2) for the destruction of [D-Leu]-Microcystin-LR, powerful toxin of widespread occurrence within cyanobacteria blooms. We extracted [D-Leu]-Microcystin-LR from a culture of Microcystis spp. and used a flat plate glass reactor coated with TiO2 (Degussa, P25) for the degradation studies. The irradiance was measured during the experiments with the aid of a spectroradiometer. After the degradation experiments, toxin concentrations were determined by HPLC and mineralization by TOC analyses. Acute and chronic toxicities were, quantified using mice and phosphatase inhibition in vitro assays, respectively. According to the performed experiments, 150 min were necessary to reduce the toxin concentration to the WHO's guideline for drinking water (from 10 to 1 mu g L-1) and to mineralize 90% of the initial carbon content. Another important finding is that solar heterogeneous photocatalysis was a destructive process indeed, not only for the toxin, but also for the other extract components and degradation products generated. Moreover, toxicity tests using mice have shown that the acute effect caused by the initial sample was removed. However, tests using the phosphatase enzyme indicated that it may be formed products capable of inducing chronic effects on mammals. The performed experiments indicate the feasibility of using solar heterogeneous photocatalysis for treating contaminated water with [D-Leu]-Microcystin-LR, not only due to its destruction, but also to the significant removal of organic matter and acute toxicity that can be achieved. (C) 2012 Elsevier Ltd. All rights reserved.

Degradation of resin-dentin bonds of etch-and-rinse adhesive system to primary and permanent teeth

The aim of this in vitro study was to compare the degradation of resin-dentin bonds of an etch-and-rinse adhesive system to primary and permanent teeth. Flat superficial coronal dentin surfaces from 5 primary second molars and 5 permanent third molars were etched with phosphoric acid and bonded with an adhesive system (Adper Single Bond 2, 3M ESPE). Blocks of resin composite (Z250, 3M ESPE) were built up and the teeth sectioned to produce bonded sticks with a 0.8 mm(2) cross-sectional area. The sticks of each tooth were randomly divided and assigned to be subjected to microtensile testing immediately (24 h) or after aging by water storage (6 months). Data were analyzed by two-way repeated measures ANOVA and Tukey post hoc test (alpha = 0.05). Failure mode was evaluated using a stereomicroscope (400x). Microtensile values significantly decreased after the 6 months aging, independent of the dentin substrate. In 24 h, the values obtained to primary dentin were lower compared with permanent dentin. This difference was not maintained after aging. Adhesive/mixed failure was predominant in all experimental groups. In conclusion, degradation of resin-dentin bonds of the etch-and-rinse adhesive system occurred after 6 months of water storage; however, the reduction in bond strength values was higher for permanent teeth.

Influence of caffeine on the survival of leaf-cutting ants Atta sexdens rubropilosa and in vitro growth of their mutualistic fungus

BACKGROUND: Leaf-cutting ants collect plant fresh material for the cultivation of their mutualistic fungus. Atta sexdens rubropilosa Forel (Hymenoptera: Formicidae) cause great economic losses through their foraging activity, mainly in agriculture. The main control method is the application of granulated toxic baits incorporated with an active ingredient (AI). The present goal is to evaluate the effect of caffeine on in vitro growth of the mutualistic fungus and on the survival of the leaf-cutting ants, aiming to verify the potential toxicity of this secondary metabolite over these organisms. RESULTS: Three distinct patterns of fungal growth correlated with caffeine concentration were observed: (1) no effect (0.01% caffeine); (2) intermediate growth reduction (0.05% caffeine); (3) drastic growth reduction (0.10 and 0.50% caffeine). The highest caffeine concentration causes fungus death in the first week. As for insect survival, caffeine does not seem to exert any effect. The treatments with diet containing caffeine showed similar values of M50, irrespective of caffeine concentration. CONCLUSION: As caffeine was shown to reduce growth of the mutualistic fungus of Atta sexdens rubropilosa, but with no conclusive effect on insect survival, a hypothetical explanation for the selection of different Coffea species by this leaf-cutting ant species might be associated with caffeine toxicity to the fungus. Copyright (C) 2011 Society of Chemical Industry

Impact of Protease Inhibitors on Dentin Matrix Degradation by Collagenase

This proof-of-concept study assessed whether the reduction of the degradation of the demineralized organic matrix (DOM) by pre-treatment with protease inhibitors (PI) is effective against dentin matrix loss. Bovine dentin slices were demineralized with 0.87 M citric acid, pH 2.3, for 36 hrs. In sequence, specimens were treated or not (UT, untreated) for 1 min with gels containing epigallocatechin 3-gallate (EGCG, 400 A mu M), chlorhexidine (CHX, 0.012%), FeSO4 (1 mM), NaF (1.23%), or no active compound (P, placebo). Specimens were then stored in artificial saliva (5 days, 37 degrees C) with the addition of collagenase (Clostridium histolyticum, 100 U/mL). We analyzed collagen degradation by assaying hydroxyproline (HYP) in the incubation solutions (n = 5) and evaluated the dentin matrix loss by profilometry (n = 12). Data were analyzed by ANOVA and Tukey's test (p < 0.05). Treatment with gels containing EGCG, CHX, or FeSO4 led to significantly lower HYP concentrations in solution and dentin matrix loss when compared with the other treatments. These results strongly suggest that the preventive effects of the PI tested against dentin erosion are due to their ability to reduce the degradation of the DOM.

Identification of archaeal proteins that affect the exosome function in vitro

Abstract Background The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

Potential antiproteolytic effects of L-leucine: observations of in vitro and in vivo studies

The purpose of present review is to describe the effect of leucine supplementation on skeletal muscle proteolysis suppression in both in vivo and in vitro studies. Most studies, using in vitro methodology, incubated skeletal muscles with leucine with different doses and the results suggests that there is a dose-dependent effect. The same responses can be observed in in vivo studies. Importantly, the leucine effects on skeletal muscle protein synthesis are not always connected to the inhibition of skeletal muscle proteolysis. As a matter of fact, high doses of leucine incubation can promote suppression of muscle proteolysis without additional effects on protein synthesis, and low leucine doses improve skeletal muscle protein ynthesis but have no effect on skeletal muscle proteolysis. These research findings may have an important clinical relevancy, because muscle loss in atrophic states would be reversed by specific leucine supplementation doses. Additionally, it has been clearly demonstrated that leucine administration suppresses skeletal muscle proteolysis in various catabolic states. Thus, if protein metabolism changes during different atrophic conditions, it is not surprising that the leucine dose-effect relationship must also change, according to atrophy or pathological state and catabolism magnitude. In conclusion, leucine has a potential role on attenuate skeletal muscle proteolysis. Future studies will help to sharpen the leucine efficacy on skeletal muscle protein degradation during several atrophic states.

Neuroprotective activity of guanosine in an in vitro model of Alzheimer's disease

The β-Amyloid (βA) peptide is the major component of senile plaques that are one of the hallmarks of Alzheimer’s Disease (AD). It is well recognized that Aβ exists in multiple assembly states, such as soluble oligomers or insoluble fibrils, which affect neuronal viability and may contribute to disease progression. In particular, common βA-neurotoxic mechanisms are Ca2+ dyshomeostasis, reactive oxygen species (ROS) formation, altered signaling, mitochondrial dysfunction and neuronal death such as necrosis and apoptosis. Recent study shows that the ubiquitin-proteasome pathway play a crucial role in the degradation of short-lived and regulatory proteins that are important in a variety of basic and pathological cellular processes including apoptosis. Guanosine (Guo) is a purine nucleoside present extracellularly in brain that shows a spectrum of biological activities, both under physiological and pathological conditions. Recently it has become recognized that both neurons and glia also release guanine-based purines. However, the role of Guo in AD is still not well established. In this study, we investigated the machanism basis of neuroprotective effects of GUO against Aβ peptide-induced toxicity in neuronal (SH-SY5Y), in terms of mitochondrial dysfunction and translocation of phosphatidylserine (PS), a marker of apoptosis, using MTT and Annexin-V assay, respectively. In particular, treatment of SH-SY5Y cells with GUO (12,5-75 μM) in presence of monomeric βA25-35 (neurotoxic core of Aβ), oligomeric and fibrillar βA1-42 peptides showed a strong dose-dependent inhibitory effects on βA-induced toxic events. The maximum inhibition of mitochondrial function loss and PS translocation was observed with 75 μM of Guo. Subsequently, to investigate whether neuroprotection of Guo can be ascribed to its ability to modulate proteasome activity levels, we used lactacystin, a specific inhibitor of proteasome. We found that the antiapoptotic effects of Guo were completely abolished by lactacystin. To rule out the possibility that this effects resulted from an increase in proteasome activity by Guo, the chymotrypsin-like activity was assessed employing the fluorogenic substrate Z-LLL-AMC. The treatment of SH-SY5Y with Guo (75 μM for 0-6 h) induced a strong increase, in a time-dependent manner, of proteasome activity. In parallel, no increase of ubiquitinated protein levels was observed at similar experimental conditions adopted. We then evaluated an involvement of anti and pro-apoptotic proteins such as Bcl-2, Bad and Bax by western blot analysis. Interestingly, Bax levels decreased after 2 h treatment of SH-SY5Y with Guo. Taken together, these results demonstrate that Guo neuroprotective effects against βA-induced apoptosis are mediated, at least partly, via proteasome activation. In particular, these findings suggest a novel neuroprotective pathway mediated by Guo, which involves a rapid degradation of pro-apoptotic proteins by the proteasome. In conclusion, the present data, raise the possibility that Guo could be used as an agent for the treatment of AD.