EVALUATION OF IN VITRO AND IN VIVO EFFECTS OF SEMIPURIFIED PROTEINASE INHIBITORS FROM THEOBROMA SEEDS ON MIDGUT PROTEASE ACTIVITY OF LEPIDOPTERAN PEST INSECTS

We have characterized in vitro and in vivo effects of trypsin inhibitors from Theobroma seeds on the activity of trypsin- and chymotrypsin-like proteins from Lepidopteran pest insects. The action of semipurified trypsin inhibitors from Theobroma was evaluated by the inhibition of bovine trypsin and chymotrypsin activities determined by the hydrolysis of N-Benzoyl-DL-Arginine-p-Nitroanilide (BAPA) and N-Succinyl-Ala-Ala-Pho-Phe p-Nitroanilide (S-(Ala)2ProPhe-pNA). Proteinase inhibitor activities from Theobroma cacao and T. obovatum seeds were the most effective in inhibiting trypsin-like proteins, whereas those from T. obovatum and T. sylvestre were the most efficient against chymotrypsin-like proteins. All larvae midgut extracts showed trypsin-like proteolytic activities, and the putative trypsin inhibitors from Theobroma seeds significantly inhibited purified bovine trypsin. With respect to the influence of Theobroma trypsin inhibitors on intact insects, the inclusion of T. cacao extracts in artificial diets of velvet bean caterpillars (Anticarsia gemmatalis) and sugarcane borer (Diatraea saccharalis) produced a significant increase in the percentage of adult deformation, which is directly related to both the survival rate of the insects and oviposition.

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.

Effects of flavomycin on ruminal fermentation, In situ degradability and In vivo digestibility in bovine fed sugarcane diets

Problem statement: The aim of the present study was to characterize and differentiate the effects of addition of flavomycin or monensin on ruminal fermentation and degradability as well as on total digestibility in bovine. Approach: Twelve non-pregnant and non-lactating cows (736 kg of BW) were randomly assigned to three treatments: control, flavomycin (20 mg animal-1 day-1) and monensin (300 mg animal-1 day-1). The trial lasted 21 days. The last 10 days were used for external marker administration (15 g of chromic oxide animal-1 day-1). The last 5 days of the trial were used for feces collection and evaluation of corn grain, soybean meal or sugarcane ruminal degradability and the 21st day was used for ruminal fluid sampling. Results: Monensin increased 27.2%, on average, propionate molar proportion at 0, 4, 6, 8, 10 and 12 h after feeding, compared to control and flavomycin groups. When compared to control, flavomycin reduced the degradation rate of soybean meal CP in 31.0%, decreasing the effective degradability when passage rates of 5 and 8% h-1 were used. Dry matter intake, pH, total Short Chain Fatty Acids (tSCFA) or ammoniacal Nitrogen (NH3-N) concentration were not influenced by the addition of either antibiotics. Effective degradability of sugarcane NDF was not influenced by the use of either antibiotic; neither were the TDN nor the digestibility of DM, CP, EE, NFE, ADF, NDF, GE or starch of the diet. Conclusion/Recommendations: In the present study, it was possible to show the beneficial effects of monensin but not of flavomycin, on rumen fermentation

Dose-dependent effects of angiotensin-(1-7) on the NHE3 exchanger and [Ca(2+)](i) in in vivo proximal tubules

The acute direct action of angiotensin-(1-7) [ANG-(1-7)] on bicarbonate reabsorption (JHCO(3)(-)) was evaluated by stationary microperfusions on in vivo middle proximal tubules in rats using H ion-sensitive microelectrodes. The control JHCO(3)(-) is 2.82 ± 0.078 nmol·cm(-2)·s(-1) (50). ANG-(1-7) (10(-12) or 10(-9) M) in luminally perfused tubules decreases JHCO(3)(-) (36 or 60%, respectively), but ANG-(1-7) (10(-6) M) increases it (80%). A779 increases JHCO(3)(-) (30%) and prevents both the inhibitory and the stimulatory effects of ANG-(1-7) on it. S3226 decreases JHCO(3)(-) (45%) and changes the stimulatory effect of ANG-(1-7) to an inhibitory effect (30%) but does not affect the inhibitory effect of ANG-(1-7). Our results indicate that in the basal condition endogenous ANG-(1-7) inhibits JHCO(3)(-) and that the biphasic dose-dependent effect of ANG-(1-7) on JHCO(3)(-) is mediated by the Mas receptors via the Na(+)/H(+) exchanger 3 (NHE3). The control value of intracellular Ca(2+) concentration ([Ca(2+)](i)), as monitored using fura-2 AM, is 101 ± 2 nM (6), and ANG-(1-7) (10(-12), 10(-9), or 10(-6)M) transiently (3 min) increases it (by 151, 102, or 52%, respectively). A779 increases the [Ca(2+)](i) (25%) but impairs the stimulatory effect of all doses of ANG-(1-7) on it. The use of BAPTA or thapsigargin suggests a correlation between the ANG-(1-7) dose-dependent effects on [Ca(2+)](i) and JHCO(3)(-). Therefore, the interaction of the opposing dose-dependent effects of ANG II and ANG-(1-7) on [Ca(2+)](i) and JHCO(3)(-) may represent an physiological regulatory mechanism of extracellular volume and/or pH changes. However, whether [Ca(2+)](i) modification is an important direct mechanism for NHE3 activation by these peptides or is a side effect of other signaling pathways will require additional studies.

In vivo and in vitro antioxidant activity and hepatoprotective properties of polyphenols from Halimeda opuntia (Linnaeus) Lamouroux

Antioxidant activity and hepatoprotective properties of the aqueous extract and tetrahydrofuran-extracted phenolic fractions of Halimeda opuntia (Linnaeus) Lamouroux were investigated in rats with chemically induced liver injury. Total polyphenols were determined by using the Folin-Ciocalteau reagent. Liver damage was induced by CCl4 and assessed by a histological technique. Reverse transcription/polymerase chain reaction (RT/PCR) analysis showed increased superoxide dismutase (SOD) and catalase (CAT) gene expression and activities in the group treated with free phenolic acid (FPA) fractions of H. opuntia, suggesting inducing effects on both enzymes. In addition, rats treated with FPA fractions displayed lower liver thiobarbituric acid reactive substance (TBARS) levels than those observed for rats in the CCl4-treated group. These data suggest that the phenolic fractions from H. opuntia may protect the liver against oxidative stress-inducing effects of chemicals by modulating its antioxidant enzymes and oxidative status.

(+)alpha-Tocopheryl succinate inhibits the mitochondrial respiratory chain complex I and is as effective as arsenic trioxide or ATRA against acute promyelocytic leukemia in vivo

The vitamin E derivative (+)alpha-tocopheryl succinate (alpha-TOS) exerts pro-apoptotic effects in a wide range of tumors and is well tolerated by normal tissues. Previous studies point to a mitochondrial involvement in the action mechanism; however, the early steps have not been fully elucidated. In a model of acute promyelocytic leukemia (APL) derived from hCG-PML-RAR alpha transgenic mice, we demonstrated that alpha-TOS is as effective as arsenic trioxide or all-trans retinoic acid, the current gold standards of therapy. We also demonstrated that alpha-TOS induces an early dissipation of the mitochondrial membrane potential in APL cells and studies with isolated mitochondria revealed that this action may result from the inhibition of mitochondrial respiratory chain complex I. Moreover, alpha-TOS promoted accumulation of reactive oxygen species hours before mitochondrial cytochrome c release and caspases activation. Therefore, an in vivo antileukemic action and a novel mitochondrial target were revealed for alpha-TOS, as well as mitochondrial respiratory complex I was highlighted as potential target for anticancer therapy. Leukemia (2012) 26, 451-460; doi:10.1038/leu.2011.216; published online 26 August 2011

In vitro and in vivo evaluation of the biocompatibility of a calcium phosphate/poly(lactic-co-glycolic acid) composite

This study assess the effects of bioceramic and poly(lactic-co-glycolic acid) composite (BCP/PLGA) on the viability of cultured macrophages and human dental pulp fibroblasts, and we sought to elucidate the temporal profile of the reaction of pulp capping with a composite of bioceramic of calcium phosphate and biodegradable polymer in the progression of delayed dentine bridge after (30 and 60 days) in vivo. Histological evaluation of inflammatory infiltrate and dentin bridge formation were performed after 30 and 60 days. There was similar progressive fibroblast growth in all groups and the macrophages showed viability. The in vivo study showed that of the three experimental groups: BCP/PLGA composite, BCP and calcium hydroxide (Ca(OH)(2)) dentin bridging was the most prevalent (90 %) in the BCP/PLGA composite after 30 days, mild to moderate inflammatory response was present throughout the pulp after 30 days. After 60 days was observed dentine bridging in 60 % and necrosis in 40 %, in both groups. The results indicate that understanding BCP/PLGA composite is biocompatible and by the best tissue response as compared to calcium hydroxide in direct pulp capping may be important in the mechanism of delayed dentine bridge after 30 and 60 days.

In vivo hydroquinone exposure impairs MCP-1 secretion and monocyte recruitment into the inflamed lung

Alveolar macrophages (AMs) are important cells in the resolution of the inflammatory process and they come into direct contact with inhaled pollutants. Hydroquinone (HQ) is an environmental pollutant and a component of cigarette smoke that causes immunosuppressive effects. In the present work, we showed that mice exposed to low levels of aerosolized HQ (25 ppm; 1 h/day/5 days) presented impaired mononuclear cell migration to the lipopolysaccharide (LPS)-inflamed lung. This may have been due to reduced monocyte chemoattractant protein-1 (MCP-1) secretion into bronchoalveolar lavage fluid (BALF), and it was not related to alterations to mononuclear cell mobilization into the blood or adhesion molecules expression on mononuclear cell membranes. Corroborating the actions of HQ on MCP-1 secretion, reduced MCP-1 concentrations were also found in the supernatant of ex vivo AM and tracheal tissue collected from HQ-exposed mice. A direct action of HQ on MCP-1 secretion, resulting from impaired gene synthesis, was verified by in vitro incubation of naive AMs or tracheal tissue with HQ. The role of reduced levels of MCP-1 in the BALF on monocyte migration was analysed in the human monocytic lineage THP-1 in in vitro chemotaxis assays, which showed that the reduced concentrations of MCP-1 found in the BALF or cell supernatants from HQ-exposed mice impaired cell migration. Considering the fact that MCP-1 presents a broad spectrum of actions on pathophysiological conditions and that resident mononuclear cells are involved in lung tissue homeostasis and in immune host defence, the mechanism of HQ toxicity presented herein might be relevant to the genesis of infectious lung diseases in smokers and in inhabitants of polluted areas. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Toxicity of photodynamic therapy with LED associated to Photogem (R): An in vivo study

The aims of this study were to evaluate the effects of PhotogemA (R)-mediated photosensitization on rat palatal mucosa and the biodistribution of the photosensitizer in this tissue. A solution of PhotogemA (R) (500 or 1000 mg/l) was applied to the palatal mucosa for 30 min and the exposure time to blue LED (460 nm) was 20 min (144 J/cm(2)). At 0, 1, 3, and 7 days, palatal mucosa was photographed for macroscopic analysis. After killing, the palate was removed for microscopic analysis. Thermal mapping evaluated temperature change in the tissue during irradiation. All experimental groups revealed intact mucosa in the macroscopic analysis. Tissue alterations were observed microscopically for only four out of 80 animals subjected to PDT. Fluorescence emitted by PhotogemA (R) was identified and was limited to the epithelial layer. A temperature increase from 35 to 41A degrees C was recorded. PhotogemA (R)- mediated PDT was not toxic to the rat palatal mucosa.

In vivo hydroquinone exposure causes tracheal hyperresponsiveness due to TNF secretion by epithelial cells

Hydroquinone (HQ) is the main oxidative substance in cigarette smoke and a toxic product of benzene biotransformation. Although the respiratory tract is an inlet pathway of HQ exposure, its effect on airway muscle responsiveness has not been assessed. We thus investigated the effects of low dose in vivo HQ-exposure on tracheal responsiveness to a muscarinic receptor agonist. Male Swiss mice were exposed to aerosolised 5% ethanol/saline solution (HQ vehicle; control) or 0.04 ppm HQ (1 h/day for 5 days) and tracheal rings were collected 1 h after the last exposure. HQ exposure caused tracheal hyper-responsiveness to methacholine (MCh), which was abolished by mechanical removal of the epithelium. This hyperresponsiveness was not dependent on neutrophil infiltration, but on tumour necrosis factor (TNF) secretion by epithelial cells. This conclusion was based on the following data: (1) trachea from HQ-exposed mice presented a higher amount of TNF, which was abrogated following removal of the epithelium; (2) the trachea hyperresponsiveness and TNF levels were attenuated by in vivo chlorpromazine (CPZ) treatment, an inhibitor of TNF synthesis. The involvement of HQ-induced TNF secretion in trachea mast cell degranulation was also demonstrated by the partial reversion of tracheal hyperresponsiveness in sodium cromoglicate-treated animals, and the in vivo HQ-exposure-induced degranulation of trachea connective tissue and mucosal mast cells, which was reversed by CPZ treatment. Our data show that in vivo HQ exposure indirectly exacerbates the parasympathetic-induced contraction of airway smooth muscle cells, mediated by TNF secreted by tracheal epithelial cells, clearly showing the link between environmental HQ exposure and the reactivity of airways. (C) 2012 Elsevier Ireland Ltd. All rights reserved.

Muscarinic and Nicotinic Modulation of Thalamo-Prefrontal Cortex Synaptic Pasticity In Vivo

The mediodorsal nucleus of the thalamus (MD) is a rich source of afferents to the medial prefrontal cortex (mPFC). Dysfunctions in the thalamo-prefrontal connections can impair networks implicated in working memory, some of which are affected in Alzheimer disease and schizophrenia. Considering the importance of the cholinergic system to cortical functioning, our study aimed to investigate the effects of global cholinergic activation of the brain on MD-mPFC synaptic plasticity by measuring the dynamics of long-term potentiation (LTP) and depression (LTD) in vivo. Therefore, rats received intraventricular injections either of the muscarinic agonist pilocarpine (PILO; 40 nmol/mu L), the nicotinic agonist nicotine (NIC; 320 nmol/mu L), or vehicle. The injections were administered prior to either thalamic high-frequency (HFS) or low-frequency stimulation (LFS). Test pulses were applied to MD for 30 min during baseline and 240 min after HFS or LFS, while field postsynaptic potentials were recorded in the mPFC. The transient oscillatory effects of PILO and NIC were monitored through recording of thalamic and cortical local field potentials. Our results show that HFS did not affect mPFC responses in vehicle-injected rats, but induced a delayed-onset LTP with distinct effects when applied following PILO or NIC. Conversely, LFS induced a stable LTD in control subjects, but was unable to induce LTD when applied after PILO or NIC. Taken together, our findings show distinct modulatory effects of each cholinergic brain activation on MD-mPFC plasticity following HFS and LFS. The LTP-inducing action and long-lasting suppression of cortical LTD induced by PILO and NIC might implicate differential modulation of thalamo-prefrontal functions under low and high input drive.

Application of tetra-isopalmitoyl ascorbic acid in cosmetic formulations: Stability studies and in vivo efficacy

Liposoluble vitamin C derivatives, such as tetra-isopalmitoyl ascorbic acid (IPAA), are often used in dermocosmetic products due to their higher stability than vitamin C free form as well as its proposed effects in skin; however, there are no studies analyzing IPAA stability or its in vivo effects when present in dermocosmetic formulations. Thus, this study aimed to evaluate chemical stability and pre-clinical and clinical efficacy of dermocosmetic formulations containing IPAA in skin hydration and microrelief. Chemical stability of the formulations added with 1% IPAA was evaluated by heat stress during 35 days by HPLC. For pre-clinical evaluation, experimental formulations were topically applied on hairless skin mice during 5 days and animal skins were analyzed by non-invasive biophysic techniques (water content of stratum corneum, TEWL, viscoelasticity, and microrelief) and by histopathological studies. For clinical efficacy tests, the formulations were topically applied to the forearm and face of human volunteers, and 3 h and 15 days after applications, the skins were evaluated by the same non-invasive techniques mentioned before. Results showed that formulations containing IPAA had medium stability and had pronounced moisturizing effects on stratum corneum and on viable epidermis. These formulations also improved skin microrelief especially in relation to skin smoothness and roughness. (C) 2012 Elsevier B.V. All rights reserved.

Bixin and norbixin protect against DNA-damage and alterations of redox status induced by methylmercury exposure in vivo

Populations in the Amazon are exposed to organic mercury via consumption of contaminated foods. These ethnic groups consume a specific plant seed annatto which contains certain carotenoids. The aim of this study was to find out if these compounds (bixin, BIX and norbixin, NOR), protect against DNA-damage caused by the metal. Therefore, rats were treated orally with methylmercury (MeHg) and with the carotenoids under conditions that are relevant to humans. The animals were treated either with MeHg (30 mu g/kg/bw/day), BIX (0.110 mg/kg/bw/day), NOR (0.011.0 mg/kg/bw/day) or combinations of the metal compound and the carotenoids consecutively for 45 days. Subsequently, the glutathione levels (GSH) and the activity of catalase were determined, and DNA-damage was measured in hepatocytes and leukocytes using single cell gel electrophoresis assays. Treatment with the metal alone caused a decrease in the GSH levels (35%) and induced DNA damage, which resulted in increased DNA migration after electrophoresis in liver and blood cells, whereas no effects were seen with the carotenoids alone. When BIX or NOR were given in combination with organic mercury, the intermediate and the highest concentrations of the carotenoids (1.0 and 10.0 mg/kg/bw/day BIX and 0.1 and 1.0 mg/kg/bw/day NOR) protected against DNA-damage. Furthermore, we found with both carotenoids, a moderate increase in the GSH levels in both metal-treated and untreated animals, while the activities of catalase remained unchanged. Our results indicate that consumption of BIX and NOR may protect humans against the adverse health effects caused by exposure to organic mercury. Environ. Mol. Mutagen., 2012. (c) 2012 Wiley Periodicals, Inc.