Mechanism of action of a nitroimidazole-thiadiazole derivate upon Trypanosoma cruzi tissue culture amastigotes

Megazol (CL 64,855) a very effective drug in experimental infections by Trypanosoma cruzi, and also in in vitro assays with vertebrate forms of the parasite, had its parasite, had its activity upon macromolecule biosynthesis tested using tissue culture-derived amastigote forms. Megazol presented a drastic inhibition of [3H]-uridine incorporation, suggesting a selective activity upon protein synthesis. Comparing the three drugs, megazol was more potent than nifurtimox and benznidazole in inhibiting protein an DNA synthesis. Megazol showed a 91% of inhibition of [3H]-leucine incorporation whereas nifurtimox and benznidazole, 0% and 2%, respectively. These latter two drugs inhibited the incorporation of all the precursors tested at similar levels, but the concentration of benznidazole was always three times higher, suggesting different mechanisms of action or, more probably, a greater efficiency of the 5-nitrofuran derivate in relation to the 2-nitroimidazole. So, wes conclude that the mode of action of megazol is different from the ones of nifurtimox and benznidazole and that its primary effect is associated with an impairment of protein synthesis.

Direct Visualization Of The Action Of Triton X-100 On Giant Vesicles Of Erythrocyte Membrane Lipids.

The raft hypothesis proposes that microdomains enriched in sphingolipids, cholesterol, and specific proteins are transiently formed to accomplish important cellular tasks. Equivocally, detergent-resistant membranes were initially assumed to be identical to membrane rafts, because of similarities between their compositions. In fact, the impact of detergents in membrane organization is still controversial. Here, we use phase contrast and fluorescence microscopy to observe giant unilamellar vesicles (GUVs) made of erythrocyte membrane lipids (erythro-GUVs) when exposed to the detergent Triton X-100 (TX-100). We clearly show that TX-100 has a restructuring action on biomembranes. Contact with TX-100 readily induces domain formation on the previously homogeneous membrane of erythro-GUVs at physiological and room temperatures. The shape and dynamics of the formed domains point to liquid-ordered/liquid-disordered (Lo/Ld) phase separation, typically found in raft-like ternary lipid mixtures. The Ld domains are then separated from the original vesicle and completely solubilized by TX-100. The insoluble vesicle left, in the Lo phase, represents around 2/3 of the original vesicle surface at room temperature and decreases to almost 1/2 at physiological temperature. This chain of events could be entirely reproduced with biomimetic GUVs of a simple ternary lipid mixture, 2:1:2 POPC/SM/chol (phosphatidylcholine/sphyngomyelin/cholesterol), showing that this behavior will arise because of fundamental physicochemical properties of simple lipid mixtures. This work provides direct visualization of TX-100-induced domain formation followed by selective (Ld phase) solubilization in a model system with a complex biological lipid composition.

Presynaptic Neuromuscular Action Of A Methanolic Extract From The Venom Of Rhinella Schneideri Toad.

Rhinella schneideri, previously known as Bufo paracnemis, is a common toad in many regions of Brazil. Its venom exerts important cardiovascular effects on humans and other animals. Although this toad venom has been the subject of intense investigations, little is known about its neuromuscular activity. The neurotoxicity of a methanolic extract of R. schneideri venom was tested on mouse phrenic nerve-diaphragm (PND) preparations mounted for conventional twitch tension recording - in response to indirect stimulation - and for electrophysiological measurements. Venom extract (50 μg/mL) increased the muscle twitch tension in PND preparations but did not significantly alter the resting membrane potential values. Electrophysiological evaluations showed that the extract (50 μg/mL) significantly augmented the frequency of miniature end-plate potential (from 38 ± 3.5 to 88 ± 15 after 60 minutes; n = 5; p < 0.05) and quantal content (from 128 ± 13 to 272 ± 34 after five minutes; n = 5; p < 0.05). Pretreatment with ouabain (1 μg/mL) for five minutes prevented the increase in quantal content (117 ± 18 and 154 ± 33 after five and 60 minutes, respectively). These results indicate that the methanolic extract of R. schneideri venom acts primarily presynaptically to enhance neurotransmitter release in mouse phrenic-diaphragm preparations.

Action Of Essential Oils From Brazilian Native And Exotic Medicinal Species On Oral Biofilms.

Essential oils (EO) obtained from twenty medicinal and aromatic plants were evaluated for their antimicrobial activity against the oral pathogens Candida albicans, Fusobacterium nucleatum, Porphyromonas gingivalis, Streptococcus sanguis and Streptococcus mitis. The antimicrobial activity of the EO was evaluates by microdilution method determining Minimal Inhibitory Concentration. Chemical analysis of the oils compounds was performed by Gas chromatography-mass spectrometry (CG-MS). The most active EO were also investigated as to their actions on the biolfilm formation. The most of the essential oils (EO) presented moderate to strong antimicrobial activity against the oral pathogens (MIC--Minimal Inhibitory Concentrations values between 0.007 and 1.00 mg/mL). The essential oil from Coriandrum sativum inhibited all oral species with MIC values from 0.007 to 0.250 mg/mL, and MBC/MFC (Minimal Bactericidal/Fungicidal Concentrations) from 0.015 to 0.500 mg/mL. On the other hand the essential oil of C. articulatus inhibited 63.96% of S. sanguis biofilm formation. Through Scanning Eletronic Microscopy (SEM) images no changes were observed in cell morphology, despite a decrease in biofilm formation and changes on biofilm structure. Chemical analysis by Gas Chromatography-Mass Spectrometry (GC-MS) of the C. sativum essential oil revealed major compounds derivatives from alcohols and aldehydes, while Cyperus articulatus and Aloysia gratissima (EOs) presented mono and sesquiterpenes. In conclusion, the crude oil from C. articulatus exhibited the best results of antimicrobial activity e ability to control biofilm formation. The chemical analysis showed the presence of terpenes and monoterpenes such as a-pinene, a-bulnesene and copaene. The reduction of biofilms formation was confirmed from SEM images. The results of this research shows a great potential from the plants studied as new antimicrobial sources.

Interference with Hemozoin Formation Represents an Important Mechanism of Schistosomicidal Action of Antimalarial Quinoline Methanols

Background: The parasitic trematode Schistosoma mansoni is one of the major causative agents of human schistosomiasis, which afflicts 200 million people worldwide. Praziquantel remains the main drug used for schistosomiasis treatment, and reliance on the single therapy has been prompting the search for new therapeutic compounds against this disease. Our group has demonstrated that heme crystallization into hemozoin (Hz) within the S. mansoni gut is a major heme detoxification route with lipid droplets involved in this process and acting as a potential chemotherapeutical target. In the present work, we investigated the effects of three antimalarial compounds, quinine (QN), quinidine (QND) and quinacrine (QCR) in a murine schistosomiasis model by using a combination of biochemical, cell biology and molecular biology approaches. Methodology/Principal Findings: Treatment of S. mansoni-infected female Swiss mice with daily intraperitoneal injections of QN, and QND (75 mg/kg/day) from the 11(th) to 17(th) day after infection caused significant decreases in worm burden (39%-61%) and egg production (42%-98%). Hz formation was significantly inhibited (40%-65%) in female worms recovered from QN- and QND-treated mice and correlated with reduction in the female worm burden. We also observed that QN treatment promoted remarkable ultrastructural changes in male and female worms, particularly in the gut epithelium and reduced the granulomatous reaction to parasite eggs trapped in the liver. Microarray gene expression analysis indicated that QN treatment increased the expression of transcripts related to musculature, protein synthesis and repair mechanisms. Conclusions: The overall significant reduction in several disease burden parameters by the antimalarial quinoline methanols indicates that interference with Hz formation in S. mansoni represents an important mechanism of schistosomicidal action of these compounds and points out the heme crystallization process as a valid chemotherapeutic target to treat schistosomiasis.

Inhibitory action of toxic compounds present in lignocellulosic hydrolysates on xylose to xylitol bioconversion by Candida guilliermondii

The inhibitory action of acetic acid, ferulic acid, and syringaldehyde on metabolism of Candida guilliermondii yeast during xylose to xylitol bioconversion was evaluated. Assays were performed in buffered and nonbuffered semidefined medium containing xylose as main sugar (80.0 g/l), supplemented or not with acetic acid (0.8-2.6 g/l), ferulic acid (0.2-0.6 g/l), and/or syringaldehyde (0.3-0.8 g/l), according to a 2(3) full factorial design. Since only individual effects of the variables were observed, assays were performed in a next step in semidefined medium containing different concentrations of each toxic compound individually, for better understanding of their maximum concentration that can be present in the fermentation medium without affecting yeast metabolism. It was concluded that acetic acid, ferulic acid, and syringaldehyde are compounds that may affect Candida guilliermondii metabolism (mainly cell growth) during bioconversion of xylose to xylitol. Such results are of interest and reveal that complete removal of toxic compounds from the fermentation medium is not necessary to obtain efficient conversion of xylose to xylitol by Candida guilliermondii. Fermentation in buffered medium was also considered as an alternative to overcome the inhibition caused by these toxic compounds, mainly by acetic acid.

A Strong Protective Action of Guttiferone-A, a Naturally Occurring Prenylated Benzophenone, Against Iron-Induced Neuronal Cell Damage

Guttiferone-A (GA) is a natural occurring polyisoprenylated benzophenone with several reported pharmacological actions. We have assessed the protective action of GA on iron-induced neuronal cell damage by employing the PC12 cell line and primary culture of rat cortical neurons (PCRCN). A strong protection by GA, assessed by the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carbox-anilide (XTT) assay, was revealed, with IC(50) values <1 mu M. GA also inhibited Fe(3+)-ascorbate reduction, iron-induced oxidative degradation of 2-deoxiribose, and iron-induced lipid peroxidation in rat brain homogenate, as well as stimulated oxygen consumption by Fe(2+) autoxidation. Absorption spectra and cyclic voltammograms of GA Fe(2+)/Fe(3+) complexes suggest the formation of a transient charge transfer complex between Fe(2+) and GA, accelerating Fe(2+) oxidation. The more stable Fe(3+) complex with GA would be unable to participate in Fenton-Haber Weiss-type reactions and the propagation phase of lipid peroxidation. The results show a potential of GA against neuronal diseases associated with iron-induced oxidative stress.

Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3 beta-ol in the isolated rat aorta

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3 beta-ol (PA-3 beta-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3 beta-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca(2+)]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3 beta-ol (10, 50 and 100 mu mol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3 beta-ol also reduced CaCl(2)-induced contraction in Ca(2+)-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 mu mol/l). PA-3 beta-ol (1-300 mu mol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca(2+) mobilization study showed that PA-3 beta-ol (100 mu mol/l) and verapamil (1 mu mol/l) inhibited the increase in Ca(2+)-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 mu mol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 mu mol/l) and 1H-[1,2,4] Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ 1 mu mol/l) produced a rightward displacement of the PA-3 beta-ol concentration-response curves. On the other hand, 7-nitroindazole (100 mu mol/l), 1400 W (1 mu mol/l), indomethacin (10 mu mol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3 beta-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3 beta-ol involve extracellular Ca(2+) influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway. (C) 2009 Elsevier B.V. All rights reserved.

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.

Mode of action of back-line application of spinosad, a non-systemic parasiticide, on sheep

Spinosad, applied as a jetting solution or dip is an efficacious, non-systemic treatment for the control of Bovicola ovis in sheep. This paper describes the effect of back-line treatment width and group housing of animals on the efficacy of spinosad for the control of lice. A 0.4 mg/kg liveweight dose was found to be the suboptimal dose of spinosad for the control of body lice in a dose titration study and was used to investigate application and housing effects in a second study. Lousy Merino sheep were treated with either a narrow 3-cm application of spinosad or with a wider 25-cm swathe. After treatment they were either kept alone or in groups of 6 sheep per pen. Lice were counted at day 0 and every 14 days to 70 days after treatment before estimation of the percentage of lice control and analysis of treatment effects. A much higher percentage of lice control was achieved with 0.4 mg/kg in the second study than in the first, possibly because of differences in formulation used. The wider application width gave significantly higher (P < 0.05) control of lice than the narrow application when sheep were either housed alone or in groups up to day 42 post-treatment. Greater control of lice was seen in group-housed sheep compared with sheep housed individually (P < 0.05) up to day 70. Using broader application widths combined with holding the animals together after treatment with pour-on formulations may optimise the delivery and efficacy of ectoparasiticides for livestock.

Action of aromatase inhibitor for treatment of uterine leiomyoma in perimenopausal patients

Objective: To assess the effect of the aromatase inhibitor on patients with leiomyoma in the reproductive stage regarding reduction of uterine volume and control of symptoms. Design: Clinical study. Setting: Academic clinical practice. Patient(s): Twenty patients, over 35 years of age, with symptomatic uterine leiomyoma. Intervention(s): Anastrozol, 1 mg/day for 12 weeks. Main Outcome Measure(s): Measurement of uterine volume, assessment of symptoms related to uterine leiomyoma, serum assay of follicle stimulating hormone (FSH), and estradiol. Results: Average reduction of uterine volume of 9.32%, attaining up to 32%, and reduction of symptoms of uterine leiomyoma (menstrual volume, duration of menstruation, and dysmenorrhea). No significant change in serum levels of FSH and estradiol during use of the medication were observed. Conclusion(S): Anastrozol proved to be effective in reducing the volume of the uterus-leiomyoma structure, leading to the control of symptoms connected with the disorder without changes in serum FSH and estradiol. (Fertil Steril (R) 2009;91:240-3. (c) 2009 by American Society for Reproductive Medicine.)

BhSGAMP-1, a Gene That Encodes an Antimicrobial Peptide, Is Developmentally Regulated by the Direct Action of 20-OH Ecdysone in the Salivary Gland of Bradysia hygida (Diptera, Sciaridae)

Recently we have shown that BhSGAMP-1 is a developmentally regulated reiterated gene that encodes an antimicrobial peptide (AMP) and is expressed exclusively in the salivary glands, at the end of the larval stage. We show, for the first time, that a gene for an AMP is directly activated by 20-OH ecdysone. This control probably involves the participation of short-lived repressor(s). We also found that the promoter of BhSGAMP-1 is not equipped with elements that respond to infection, provoked by the injection of microorganisms, in the salivary glands or in the fat body. We produced polyclonal antibodies against the synthetic peptide and found that the BhSGAMP-1 peptide is secreted in the saliva. The BhSGAMP-1 gene was also activated during the third larval molt. These facts confirm our hypothesis that this preventive system of defense was selected to produce an environment free of harmful microorganisms in the insect`s immediate vicinity, during molts. genesis 47:847-857, 2009. (C) 2009 Wiley-Liss, Inc.

Mechanisms of Action of Eicosapentaenoic Acid in Bladder Cancer Cells In Vitro: Alterations in Mitochondrial Metabolism, Reactive Oxygen Species Generation and Apoptosis Induction

Purpose: Eicosapentaenoic acid has been tested in bladder cancer as a synergistic cytotoxic agent in the form of meglumine-eicosapentaenoic acid, although its mechanism of action is poorly understood in this cancer. The current study analyzed the mechanisms by which eicosapentaenoic acid alters T24/83 human bladder cancer metabolism in vitro. Materials and Methods: T24/83 human bladder cancer cells were exposed to eicosapentaenoic acid for 6 to 24 hours in vitro and incorporation profiles were determined. Effects on membrane phospholipid incorporation, energy metabolism, mitochondrial activity, cell proliferation and apoptosis were analyzed Reactive oxygen species and lipid peroxide production were also determined. Results: Eicosapentaenoic acid was readily incorporated into membrane phospholipids with a considerable amount present in mitochondrial cardiolipin. Energy metabolism was significantly altered by eicosapentaenoic acid, accompanied by decreased mitochondrial membrane potential, and increased lipid peroxide and reactive oxygen species generation. Subsequently caspase-3 activation and apoptosis were detected in eicosapentaenoic acid exposed cells, leading to decreased cell numbers. Conclusions: These findings confirm that eicosapentaenoic acid is a potent cytotoxic agent in bladder cancer cells and provide important insight into the mechanisms by which eicosapentaenoic acid causes these changes. The changes in membrane composition that can occur with eicosapentaenoic acid likely contribute to the enhanced drug cytotoxicity reported previously in meglumine-eicosapentaenoic acid/epirubicin/mitomycin studies. Dietary manipulation of the cardiolipin fatty acid composition may provide an additional method for stimulating cell death in bladder cancer. In vivo studies using intravesical and dietary manipulation of fatty acid metabolism in bladder cancer merit further attention.

Antibothropic action of Casearia sylvestris Sw. (flacourtiaceae) extracts

Casearia sylvestris Sw., popularly known in Brazil as `guacatonga`, has been used as antitumor, antiseptic, antiulcer, local anaesthetic and healer in folk medicine. Snakebite envenomation by Bothrops jararacussu (Bjssu) constitutes a relevant public health hazard capable of inducing serious local damage in victims. This study examined the pharmacological action of apolar and polar C sylvestris leaf extracts in reverting the neuromuscular blockade and myonecrosis, which is induced by Bjssu venom and its major toxin bothropstoxin-I on the mouse phrenic nerve-diaphragm preparations. The polar methanol extract (ME) was by far the most efficacious. ME not only prevented myonecrosis and abolished the blockade, but also increased ACh release. Such facilitation in neuromuscular transmission was observed with ME alone, but was accentuated in preparations incubated with ME plus venom or toxin. This established synergy opens an interesting point of investigation because the venom or toxin in contact with ME changes from a blocking to a facilitating effect. It is suggested that rutin, known to have potent antioxidant properties, and one of the components present in the ME, could have a role in the observed effects. Since commercial rutin did not reproduce the ME effects, it is likely that a rutin-containing phytocomplex is neutralizing the bothropic envenoming effects. Copyright (C) 2008 John Wiley & Sons, Ltd.