Peritrophic membrane role in enhancing digestive efficiency Theoretical and experimental models

The pentrophic membrane (PM) is an anatomical structure surrounding the food bolus in most insects. Rejecting the idea that PM has evolved from coating mucus to play the same protective role as it, novel functions were proposed and experimentally tested. The theoretical principles underlying the digestive enzyme recycling mechanism were described and used to develop an algorithm to calculate enzyme distributions along the midgut and to infer secretory and absorptive sites. The activity of a Spodoptera frugiperda microvillar aminopeptidase decreases by 50% if placed in the presence of midgut contents. S. frugiperda trypsin preparations placed into dialysis bags in stirred and unstirred media have activities of 210 and 160%, respectively, over the activities of samples in a test tube. The ectoperitrophic fluid (EF) present in the midgut caeca of Rhynchosciara americana may be collected. If the enzymes restricted to this fluid are assayed in the presence of PM contents (PMC) their activities decrease by at least 58%. The lack of PM caused by calcofluor feeding impairs growth due to an increase in the metabolic cost associated with the conversion of food into body mass. This probably results from an increase in digestive enzyme excretion and useless homeostatic attempt to reestablish destroyed midgut gradients. The experimental models support the view that PM enhances digestive efficiency by: (a) prevention of non-specific binding of undigested material onto cell Surface; (b) prevention of excretion by allowing enzyme recycling powered by an ectoperitrophic counterflux of fluid; (c) removal from inside PM of the oligomeric molecules that may inhibit the enzymes involved in initial digestion; (d) restriction of oligomer hydrolases to ectoperitrophic space (ECS) to avoid probable partial inhibition by non-dispersed undigested food. Finally,PM functions are discussed regarding insects feeding on any diet. (C) 2008 Elsevier Ltd. All rights reserved.

Antipyretic and anti-inflammatory properties of the ethanolic extract, dichloromethane fraction and costunolide from Magnolia ovata (Magnoliaceae)

Aim of the study: Magnolia ovata (A.St.-Hil.) Spreng (formerly Talauma ovata), known as ""pinha-do-brejo"" or ""baguacu"", is a large tree widely distributed in Brazil. Its trunk bark has been used in folk medicine against fever. However, no data have been published to support the antipyretic ethnopharmacological use. This study investigated the antipyretic and anti-inflammatory effects of the ethanolic extract (EEMO). dichloromethane fraction (DCM), and the isolated compound costunolide. Materials and methods: The antipyretic and anti-inflammatory activities were evaluated in experimental models of fever and inflammation in mice. Results: The oral administration of EEMO, DCM and costunolide inhibited carrageenan (Cg)-induced paw oedema (ID(50) 72.35 (38.64-135.46) mg/kg, 5.8 (2.41-14.04) mg/kg and 0.18 (0.12-0.27) mg/kg, respectively) and was effective in abolishing lipopolysaccharide (LPS)-induced fever (30 mg/kg, 4.5 mg/kg and 0.15 mg/kg, respectively). EEMO was also effective in reducing cell migration in the pleurisy model. Intraplantar injection of costunolide also reduced the paw oedema, myeloperoxidase and N-acetyl-glucosaminidase activity induced by Cg in mice. Conclusions: Collectively, these results show, for the first time, that extracts obtained from Magnolia ovata possess antipyretic and anti-inflammatory properties, and costunolide appears to be the compound responsible for these effects. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Metastatic melanoma positively influences pregnancy outcome in a mouse model: could a deadly tumor support embryo life?

The incidence of melanoma is increasing worldwide. It is one of the leading cancers in pregnancy and the most common malignancy to metastasize to placenta and fetus. There are no publications about experimental models of melanoma and pregnancy. We propose a new experimental murine model to study the effects of melanoma on pregnancy and its metastatic process. We tested several doses of melanoma cells until we arrived at the optimal dose, which produced tumor growth and allowed animal survival to the end of pregnancy. Two control groups were used: control (C) and stress control (SC) and three different routes of inoculation: intravenous (IV), intraperitoneal (IP) and subcutaneous (SC). All the fetuses and placentas were examined macroscopically and microscopically. The results suggest that melanoma is a risk factor for intrauterine growth restriction but does not affect placental weight. When inoculated by the SC route, the tumor grew only in the site of implantation. The IP route produced peritoneal tumoral growth and also ovarian and uterine metastases in 60% of the cases. The IV route produced pulmonary tumors. No placental or fetal metastases were obtained, regardless of the inoculation route. The injection of melanoma cells by any route did not increase the rate of fetal resorptions. Surprisingly, animals in the IV groups had no resorptions and a significantly higher number of fetuses. This finding may indicate that tumoral factors released in the host organism to favor tumor survival may also have a pro-gestational action and consequently improve the reproductive performance of these animals.

Effects of voluntary running on spatial memory and mature brain-derived neurotrophic factor expression in mice hippocampus after status epilepticus

Voluntary physical activity improves memory and learning ability in rodents, whereas status epilepticus has been associated with memory impairment. Physical activity and seizures have been associated with enhanced hippocampal expression of BDNF, indicating that this protein may have a dual role in epilepsy. The influence of voluntary physical activity on memory and BDNF expression has been poorly studied in experimental models of epilepsy. In this paper, we have investigated the effect of voluntary physical activity on memory and BDNF expression in mice with pilocarpine-incluced epilepsy. Male Swiss mice were assigned to four experimental groups: pilocarpine sedentary (PS), pilocarpine runners (PRs), saline sedentary (SS) and saline runners (SRs). Two days after pilocarpine-induced status epilepticus, the affected mice (PR) and their running controls (SR) were housed with access to a running wheel for 28 days. After that, the spatial memory and the expression of the precursor and mature forms of hippocampal BDNF were assessed. PR mice performed better than PS mice in the water maze test. In addition, PR mice had a higher amount of mature BDNF (14 kDa) relative to the total BDNF (14 kDa + 28 kDa + 32 kDa forms) content when compared with PS mice. These results show that voluntary physical activity improved the spatial memory and increased the hippocampal content of mature BDNF of mice with pilocarpine-induced status epilepticus. (C) 2009 Elsevier B.V. All rights reserved.

BnP1, a novel P-I metalloproteinase from Bothrops neuwiedi venom: Biological effects benchmarking relatively to jararhagin, a P-IIISVMP

Snake venom metalloproteinases (SVMPs) have been extensively studied and their effects associated with the local bleeding observed in human accidents by viper snakes. Representatives of P-I and P-III classes of SVMPs similarly hydrolyze extracellular matrix proteins or coagulation factors while only P-III SVMPs induce significant hemorrhage in experimental models. In this work, the effects of P-I and P-III SVMPs on plasma proteins and cultures of muscle and endothelial cells were compared in order to enlighten the mechanisms involved in venom-induced hemorrhage. To reach this comparison, BnP1 was isolated from B. neuwiedi venom and used as a weakly hemorrhagic P-I SVMPs and jararhagin was used as a model of potently hemorrhagic P-III SVMP. BnP1 was isolated by size exclusion and anion-exchange chromatographies, showing apparent molecular mass of approximately 24kDa and sequence similarity with other members of SVMPs, which allowed its classification as a group P-I SVMP. The comparison of local effects induced by SVMPs showed that BnP1 was devoid of significant myotoxic and hemorrhagic activities and jararhagin presented only hemorrhagic activity. BnP1 and jararhagin were able to hydrolyze fibrinogen and fibrin, although the latter displayed higher activity in both systems. Using HUVEC primary cultures, we observed that BnP1 induced cell detachment and a decrease in the number of viable endothelial cells in levels comparable to those observed by treatment with jararhagin. Moreover, both BnP1 and jararhagin induced apoptosis in HUVECs while only a small increase in LDH supernatant levels was observed after treatment with jararhagin, suggesting that the major mechanism involved in endothelial cell death is apoptosis. Jararhagin and BnP1 induced little effects on C2C12 muscle cell cultures, characterized by a partial detachment 24h after treatment and a mild necrotic effect as evidenced by a small increase in the supernatants LDH levels. Taken together, our data show that P-I and P-III SVMPs presented comparable effects except for the hemorrhagic activity, suggesting that hydrolysis of coagulation factors or damage to endothelial cells are not sufficient for induction of local bleeding. (C) 2007 Elsevier Ltd. All rights reserved.

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation. Here, we synthesized diapocynin and studied its effect on inhibition of gp91(phox) RNA expression. We found that diapocynin strongly inhibited the expression of gp91(phox)mRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-alpha production, diapocynin had a much more pronounced effect, on both TNF-alpha and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91(phox) mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched. (C) 2010 Elsevier Inc. All rights reserved.

INSULIN REGULATES CYTOKINES AND INTERCELLULAR ADHESION MOLECULE-1 GENE EXPRESSION THROUGH NUCLEAR FACTOR-kappa B ACTIVATION IN LPS-INDUCED ACUTE LUNG INJURY IN RATS

Diabetic patients have increased susceptibility to infection, which may be related to impaired inflammatory response observed in experimental models of diabetes, and restored by insulin treatment. The goal of this study was to investigate whether insulin regulates transcription of cytokines and intercellular adhesion molecule 1 (ICAM-1) via nuclear factor-kappa B (NF-kappa B) signaling pathway in Escherichia coli LIPS-induced lung inflammation. Diabetic male Wistar rats (alloxan, 42 mg/kg, iv., 10 days) and controls were instilled intratracheally with saline containing LPS (750 mu g/0.4 mL) or saline only. Some diabetic rats were given neutral protamine Hagedorn insulin (4 IU, s.c.) 2 h before LIPS. Analyses performed 6 h after LPS included: (a) lung and mesenteric lymph node IL-1 beta, TNF-alpha, IL-10, and ICAM-1 messenger RNA (mRNA) were quantified by real-time reverse transcriptase-polymerase chain reaction; (b) number of neutrophils in the bronchoalveolar lavage (BAL) fluid, and concentrations of IL-1 beta, TNF-alpha, and IL-10 in the BAL were determined by the enzyme-linked immunosorbent assay; and (c) activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were quantified by Western blot analysis. Relative to controls, diabetic rats exhibited a reduction in lung and mesenteric lymph node IL-1 beta (40%), TNF-alpha (similar to 30%), and IL-10 (similar to 40%) mRNA levels and reduced concentrations of IL-1 beta (52%), TNF-alpha (62%), IL-10 (43%), and neutrophil counts (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were almost suppressed in diabetic rats. Treatment of diabetic rats with insulin completely restored mRNA and protein levels of these cytokines and potentiated lung ICAM-1 mRNA levels (30%) and number of neutrophils (72%) in the BAL. Activation of NF-kappa B p65 subunit and phosphorylation of I-kappa B alpha were partially restored by insulin treatment. In conclusion, data presented suggest that insulin regulates transcription of proinflammatory (IL-1 beta, TNF-alpha) and anti-inflammatory (IL-10) cytokines, and expression of ICAM-1 via the NF-kappa B signaling pathway.

CD4(+) CD25(+) Foxp3(+) Regulatory T Cells, Dendritic Cells, and Circulating Cytokines in Uncomplicated Malaria: Do Different Parasite Species Elicit Similar Host Responses?

Clearing blood-stage malaria parasites without inducing major host pathology requires a finely tuned balance between pro- and anti-inflammatory responses. The interplay between regulatory T (Treg) cells and dendritic cells (DCs) is one of the key determinants of this balance. Although experimental models have revealed various patterns of Treg cell expansion, DC maturation, and cytokine production according to the infecting malaria parasite species, no studies have compared all of these parameters in human infections with Plasmodium falciparum and P. vivax in the same setting of endemicity. Here we show that during uncomplicated acute malaria, both species induced a significant expansion of CD4(+) CD25(+) Foxp3(+) Treg cells expressing the key immunomodulatory molecule CTLA-4 and a significant increase in the proportion of DCs that were plasmacytoid (CD123(+)), with a decrease in the myeloid/plasmacytoid DC ratio. These changes were proportional to parasite loads but correlated neither with the intensity of clinical symptoms nor with circulating cytokine levels. One-third of P. vivax-infected patients, but no P. falciparum-infected subjects, showed impaired maturation of circulating DCs, with low surface expression of CD86. Although vivax malaria patients overall had a less inflammatory cytokine response, with a higher interleukin-10 (IL-10)/tumor necrosis factor alpha (TNF-alpha) ratio, this finding did not translate to milder clinical manifestations than those of falciparum malaria patients. We discuss the potential implications of these findings for species-specific pathogenesis and longlasting protective immunity to malaria.

Inhibitory activity of limonene against Leishmania parasites in vitro and in vivo

Limonene is a monoterpene that has antitumoral, antibiotic and antiprotozoal activity. In this study we demonstrate the activity of limonene against Leishmania species in vitro and in vivo. Limonene killed Leishmania amazonensis promastigotes and amastigotes with 50% inhibitory concentrations of 252.0 +/- 49.0 and 147.0 +/- 46.0 mu M, respectively. Limonene was also effective against Leishmania major, Leishmania braziliensis and Leishmania chagasi promastigotes. The treatment of L. amazonensis-infected macrophages with 300 mu M limonene resulted in 78% reduction in infection rates. L. amazonensis-infected mice treated topically or intrarectally with limonene had significant reduction of lesion sizes. A significant decrease in the parasite load was shown in the lesions treated topically with limonene by histopathological examination. The intrarectal treatment was highly effective in decreasing the parasite burden, healing established lesions and suppressing the dissemination of ulcers. Limonene presents low toxicity in humans and has been shown to be effective as an agent for enhancing the percutaneous permeation of drugs. Our results suggest that limonene should be tested in different experimental models of infection by Leishmania. (C) 2009 Elsevier Masson SAS. All rights reserved.

Tamoxifen as a potential antileishmanial agent: efficacy in the treatment of Leishmania braziliensis and Leishmania chagasi infections

The aim of this study was to evaluate the efficacy of tamoxifen in vivo in experimental models of cutaneous (CL) and visceral leishmaniasis (VL) caused by Leishmania braziliensis and Leishmania chagasi, respectively. Drug activity was assessed against intracellular amastigotes by treating infected macrophage cultures and evaluating the number of infected cells. In vivo efficacy of tamoxifen was tested in L. braziliensis-infected BALB/c mice and in L. chagasi-infected hamsters. Treatment with 20 mg/kg/day tamoxifen was administered for 15 days by the intraperitoneal route. Efficacy was evaluated through measurements of lesion size, parasite burden at the lesion site or liver and spleen and survival rate. Tamoxifen killed L. braziliensis and L. chagasi intracellular amastigotes with 50% inhibitory concentrations (IC(50)) of 1.9 +/- 0.2 and 2.4 +/- 0.3 mu M, respectively. Treatment of L. braziliensis-infected mice with tamoxifen resulted in significant reductions in lesion size and 99% decrease in parasite burden, compared with mock-treated controls. L. chagasi-infected hamsters treated with tamoxifen showed significant reductions in liver parasite load expressed as Leishman-Donovan units and 95% to 98% reduction in spleen parasite burden. All animals treated with tamoxifen survived while 100% of the mock-treated animals had died by 11 weeks after the interruption of treatment. Tamoxifen is effective in the treatment of CL and VL in rodent models.

Mechanisms underlying the long-term regulation of NHE3 by parathyroid hormone

The activity of the Na(+)/H(+) exchanger NHE3 is regulated by a number of factors including parathyroid hormone (PTH). In the current study, we used a renal epithelial cell line, the opossum kidney (OKP) cell, to elucidate the mechanisms underlying the long-term effects of PTH on NHE3 transport activity and expression. We observed that NHE3 activity was reduced 6 h after addition of PTH, and this reduction persisted almost unaltered after 24 h. The decrease in activity was associated with diminished NHE3 cell surface expression at 6, 16, and 24 h after PTH addition, total cellular NHE3 protein at 16 and 24 h, and NHE3 mRNA abundance at 24 h. The lower levels of NHE3 mRNA were associated to a small, but significant, decrease in mRNA stability. Additionally, by analyzing the rat NHE3 gene promoter activity in OKP cells, we verified that the regulatory region spanning the segment -152 to +55 was mildly reduced under the influence of PTH. This effect was completely abolished by the presence of the PKA inhibitor KT 5720. In conclusion, long-term exposure to PTH results in reduction of NHE3 mRNA levels due to a PKA-dependent inhibitory effect on the NHE3 promoter and a small reduction of mRNA half-life, and decrease in the total amount of protein which is preceded by endocytosis of the apical surface NHE3. The decreased NHE3 expression is likely to be responsible for the reduction of sodium, bicarbonate, and fluid reabsorption in the proximal tubule consistently perceived in experimental models of PTH disorders.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.

A vaccine against S. pyogenes: Design and experimental immune response

Streptococcus pyogenes causes severe invasive infections: the post-streptococcal sequelae of acute rheumatic fever (RF) and rheumatic heart disease (RHD), acute glomerulonephritis, and uncomplicated pharyngitis and pyoderma. Efforts to produce a vaccine against S. pyogenes began several decades ago, and different models have been proposed. Here, we describe the methodology used in the development of a new vaccine model, consisting of both T and B protective epitopes constructed as synthetic peptides and recombinant proteins. Two adjuvants were tested in an experimental inbred mouse model: a classical Freund`s adjuvant and a new adjuvant (AFCo1) that induces mucosal immune responses and is obtained by calcium precipitation of a proteoliposome derived from the outer membrane of Neisseria meningitides B. The StreptInCor vaccine epitope co-administrated with AFCo1 adjuvant induced mucosal (IgA) and systemic (IgG) antibodies as preferential Th1-mediated immune responses. No autoimmune reactions were observed, suggesting that the vaccine epitope is safe. (c) 2009 Elsevier Inc. All rights reserved.

Continuum, Discrete, and Explicit Solvation Models for Describing the Low-Lying Absorption Spectrum of the Pterin Acid in Aqueous Environment

The absorption spectrum of the acid form of pterin in water was investigated theoretically. Different procedures using continuum, discrete, and explicit models were used to include the solvation effect on the absorption spectrum, characterized by two bands. The discrete and explicit models used Monte Carlo simulation to generate the liquid structure and time-dependent density functional theory (B3LYP/6-31G+(d)) to obtain the excitation energies. The discrete model failed to give the correct qualitative effect on the second absorption band. The continuum model, in turn, has given a correct qualitative picture and a semiquantitative description. The explicit use of 29 solvent molecules, forming a hydration shell of 6 angstrom, embedded in the electrostatic field of the remaining solvent molecules, gives absorption transitions at 3.67 and 4.59 eV in excellent agreement with the S(0)-S(1) and S(0)-S(2) absorption bands at of 3.66 and 4.59 eV, respectively, that characterize the experimental spectrum of pterin in water environment. (C) 2010 Wiley Periodicals, Inc. Int J Quantum Chem 110: 2371-2377, 2010

The specificity of frutalin lectin using biomembrane models

Frutalin is a homotetrameric alpha-D-galactose (D-Gal)-binding lectin that activates natural killer cells in vitro and promotes leukocyte migration in vivo. Because lectins are potent lymphocyte stimulators, understanding the interactions that occur between them and cell surfaces can help to the action mechanisms involved in this process. In this paper, we present a detailed investigation of the interactions of frutalin with phospho- and glycolipids using Langmuir monolayers as biomembrane models. The results confirm the specificity of frutalin for D-Gal attached to a biomembrane. Adsorption of frutalin was more efficient for the galactose polar head lipids, in contrast to the one for sulfated galactose, in which a lag time is observed, indicating a rearrangement of the monolayer to incorporate the protein. Regarding ganglioside GM1 monolayers, lower quantities of the protein were adsorbed, probably due to the farther apart position of D-galactose from the interface. Binary mixtures containing galactocerebroside revealed small domains formed at high lipid packing in the presence of frutalin, suggesting that lectin induces the clusterization and the forming of domains in vitro, which may be a form of receptor internalization. This is the first experimental evidence of such lectin effect, and it may be useful to understand the mechanism of action of lectins at the molecular level. (C) 2010 Elsevier B.V. All rights reserved.