Tityus serrulatus venom and toxins Ts1, Ts2 and Ts6 induce macrophage activation and production of immune mediators

Scorpion envenomation induces a systemic immune response, and neurotoxins of venom act on specific ion channels, modulating neurotransmitter release or activity. However, little is known about the immunomodulatory effects of crude venom from scorpion Tityus serrulatus (TsV) or its toxins (Ts1, Ts2 and Ts6) in combination with lipopolysaccharide (LPS). To investigate the immunomodulatory effects of TsV and its toxins (Ts1, Ts2 and Ts6), J774.1 cells were stimulated with different concentrations (25, 50 and 100 mu g/mL) of venom or toxins pre-stimulated or not with LPS (0.5 mu g/mL). Macrophage cytotoxicity was assessed, and nitric oxide (NO) and cytokine production were analyzed utilizing the culture supernatants. TsV and its toxins did not produce cytotoxic effects. Depending on the concentrations used, TsV, Ts1 and Ts6 stimulated the production of NO, interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha in J774.1 cells, which were enhanced under LPS co-stimulation. However, LPS + Ts2 inhibited NO, IL-6 and TNF-alpha production, and Ts2 alone stimulated the production of IL-10, suggesting an anti-inflammatory activity for this toxin. Our findings are important for the basic understanding of the mechanisms involved in macrophage activation following envenomation: additionally, these findings may contribute to the discovery of new therapeutic compounds to treat immune-mediated diseases. (C) 2011 Elsevier Ltd. All rights reserved.

Isolation and functional characterization of proinflammatory acidic phospholipase A(2) from Bothrops leucurus snake venom

In the present study, an acidic PLA(2), designated BI-PLA(2), was isolated from Bothrops leucurus snake venom through two chromatographic steps: ion-exchange on CM-Sepharose and hydrophobic chromatography on Phenyl-Sepharose. Bl-PLA(2) was homogeneous on SDS-PAGE and when submitted to 2D electrophoresis the molecular mass was 15,000 Da and pl was 5.4. Its N-terminal sequence revealed a high homology with other Asp49 acidic PLA(2)s from snake venoms. Its specific activity was 159.9 U/mg and the indirect hemolytic activity was also higher than that of the crude venom. Bl-PLA(2) induced low myotoxic and edema activities as compared to those of the crude venom. Moreover, the enzyme was able to induce increments in IL-12p40, TNF-alpha, IL-1 beta and IL-6 levels and no variation of IL-8 and IL-10 in human PBMC stimulated in vitro, suggesting that Bl-PLA2 induces proinflammatory cytokine production by human mononuclear cells. Bothrops leucurus venom is still not extensively explored and knowledge of its components will contribute for a better understanding of its action mechanism. (C) 2011 Elsevier Inc. All rights reserved.

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.

Comparative effects of lantadene A and its reduced metabolite on mitochondrial bioenergetics

Lantana (Lantana camara Linn.) is a noxious weed to which certain medicinal properties have been attributed, but its ingestion has been reported to be highly toxic to animals and humans, especially in the liver. The main hepatotoxin in lantana leaves is believed to be the pentacyclic triterpenoid lantadene A (LA), but the precise mechanism by which it induces hepatotoxicity has not yet been established. This work addressed the action of LA and its reduced derivative (RLA) on mitochondrial bioenergetics. At the concentration range tested (5-25 mu M), RLA stimulated state-4 respiration, inhibited state-3 respiration, circumvented oligomycin-inhibited state-3 respiration, dissipated membrane potential and depleted ATP in a concentration-dependent manner. However. LA did not stimulate state-4 respiration, nor did it affect the other mitochondrial parameters to the extent of its reduced derivative. The lantadenes didn`t inhibit the CCCP-uncoupled respiration but increased the ATPase activity of intact coupled mitochondria. The ATPase activity of intact uncoupled or disrupted mitochondria was not affected by the compounds. We propose, therefore, that RLA acts as a mitochondrial uncoupler of oxidative phosphorylation, a property that arises from the biotransformation (reduction) of LA, and LA acts in other mitochondrial membrane components rather than the ATP synthase affecting the mitochondrial bioenergetics. Such effects may account for the well-documented hepatoxicity of lantana. (C) 2010 Elsevier Ltd. All rights reserved.

Biochemical and Functional Characterization of a Metalloprotease from the Thermophilic Fungus Thermoascus aurantiacus

Protease production was carried out in solid state fermentation. The enzyme was purified through precipitation with ethanol at 72% followed by chromatographies in columns of Sephadex G75 and Sephacryl S100. It was purified 80-fold and exhibited recovery of total activity of 0.4%. SDS-PAGE analysis indicated an estimated molecular mass of 24.5 kDa and the N-terminal sequence of the first 22 residues was APYSGYQCSMQLCLTCALMNCA. Purified protease was only inhibited by EDTA (96.7%) and stimulated by Fe(2+) revealing to be a metalloprotease activated by iron. Optimum pH was 5.5, optimum temperature was 75 degrees C, and it was thermostable at 65 degrees C for 1 h maintaining more than 70% of original activity. Through enzyme kinetic studies, protease better hydrolyzed casein than azocasein. The screening of fluorescence resonance energy transfer (FRET) peptide series derived from Abz-KLXSSKQ-EDDnp revealed that the enzyme exhibited preference for Arg in P(1) (k(cat)/K(m) = 30.1 mM(-1) s(-1)).

Relaxation evoked by extracellular Ca(2+) in rat aorta is nerve-independent and involves sarcoplasmic reticulum and L-type Ca(2+) channell

The perivascular nerve network expresses a Ca(2+) receptor that is activated by high extracellular Ca(2+) concentrations and causes vasorelaxation in resistance arteries. We have verified the influence of perivascular nerve fibers on the Ca(2+)-induced relaxation in aortic rings. To test our hypothesis, either pre-contracted aortas isolated from rats after sensory denervation with capsaicin or aortic rings acutely denervated with phenol were stimulated to relax with increasing extracellular Ca(2+) concentration. We also studied the role of the endothelium on the Ca(2+)-induced relaxation, and we verified the participation of endothelial/nonendothelial nitric oxide and cyclooxygenise-arachidonic acid metabolites. Additionally, the role of the sarcoplasmic reticulum, K(+) channels and L-type Ca(2+) channels on the Ca(2+)-induced relaxation were evaluated. We have observed that the Ca(2+)-induced relaxation is completely nerve independent, and it is potentiated by endothelial nitric oxide (NO). In endothelium-denuded aortic rings, indomethacin and AH6809 (PGF(2 alpha) receptor antagonist) enhance the relaxing response to Ca(2+). This relaxation is inhibited by thapsigargin and verapamil, while was not altered by tetraethylammonium. In conclusion, we have shown that perivascular nervous fibers do not participate in the Ca(2+)-induced relaxation, which is potentiated by endothelial NO. In endothelium-denuded preparations, indomethacin and AH6809 enhance the relaxation induced by Ca(2+). The relaxing response to Call was impaired by verapamil and thapsigargin, revealing the importance of L-type Ca(2+) channels and sarcoplasmic reticulum in this response. (c) 2008 Elsevier Inc. All rights reserved.

The Aspergillus nidulans nucA(EndoG) Homologue Is Not Involved in Cell Death

Upon apoptosis induction, translocation of mammalian mitochondrial endonuclease G (EndoG) to the nucleus coincides with large-scale DNA fragmentation. Here, we describe for the first time a homologue of EndoG in filamentous fungi by investigating if the Aspergillus nidulans homologue of the EndoG gene, named nucA(EndoG), is being activated during farnesol-induced cell death. Our results suggest that NucA is not involved in cell death, but it plays a role in the DNA-damaging response in A. nidulans.

The role of seasonality on the inhibitory effect of Brazilian green propolis on the oxidative metabolism of neutrophils

The reactive oxygen species (ROS) produced by neutrophils are involved in the pathogenesis of several diseases, for which the intake of antioxidants could benefit patients either as a prophylactic or therapeutic treatment. Propolis is among the known antioxidants, and its chemical composition may vary under the influence of seasonality, which may interfere in its biological properties. This work evaluates the role of seasonality on the production of some important compounds of propolis samples produced monthly from November 2001 through October 2002 as well as the effect of these samples on the oxidative metabolism of stimulated neutrophils, by using both luminol and lucigenin to produce chemiluminescence (CLlum and CLluc, respectively). The cytotoxicity of the most active extracts to neutrophils was also investigated. The inhibitory effect of the propolis samples varied significantly during the studied period for both assays (3.4 +/- 1.1 to 16.0 +/- 1.1 mu g/mL for CLlum and 6.2 +/- 2.0 to 30.0 +/- 5.0 mu g/mL for CLluc), which was also observed in the quantitative profile of the main analyzed compounds (aromadendrin-4`-methyl ether, artepillin C, and baccharin). This effect started to become more prominent during the fall and, among all the studied extracts, the one obtained in May displayed the highest inhibitory effect on CL production (3.4 +/- 1.1 mu g/mL for alum and 6.2 +/- 2.0 mu g/mL for CLluc). The HPLC qualitative profiles of the extracts of propolis samples were quite similar, but there was a huge variation in terms of quantitative profile. It seems that aromadendrin-4`-methyl ether and baccharin play an essential role in the antioxidant activity, while artepillin C is not very important for this effect. The extracts presenting the highest antioxidant activity were produced in May, June, and August, and they did not display cytotoxicity at 25 mu g/mL; quercetin, used as control, was not toxic to neutrophils at 8.5 mu g/mL (C) 2010 Elsevier B.V. All rights reserved.

Role of phospholipase C and protein kinase C in Aspergillus nidulans during growth on pectin or glucose: Effects on germination and duplication cycle

The effects of PLC and Pkc inhibitors on Aspergillus nidulans depend on the carbon source. PLC inhibitors Spm and C48/80 delayed the first nuclear division in cultures growing on glucose, but stimulated it in media supplemented with pectin. Less intense were these effects on the mutant transformed with PLC-A gene rupture (AP27). Neomycin also delayed the germination in cultures growing on glucose or pectin; however, on glucose, the nuclear division was inhibited whereas in pectin it was stimulated. These effects were minor in AP27. The effects of Ro-31-8425 and BIM (both Pkc inhibitors) were also opposite for cultures growing on glucose or pectin. On glucose cultures of both strains BIM delayed germination and the first nuclear division, whereas on pectin both parameters were stimulated. Opposite effects were also detected when the cultures were growing on glucose or pectin in the presence of Ro-31-8425.

Budlein A from Viguiera robusta inhibits leukocyte-endothelial cell interactions, adhesion molecule expression and inflammatory mediators release

Budlein A has been reported to exert some analgesic and anti-inflammatory properties. In this study, we have evaluated its effect on LPS-induced leukocyte recruitment in vivo and the mechanisms involved in its anti-inflammatory activity. In vivo, intravital videomicroscopy was used to determine the effects of budlein A on LPS-induced leukocyte-endothelial cell interactions in the murine cremasteric microcirculation. In vitro, the effects of budlein A on LPS-induced cytokine, chemokine and nitrites release, T-cell proliferative response as well as cell adhesion molecule expression (CAM) were evaluated. In vivo, intraperitoneal administration of budlein A (2.6 mM/kg) caused a significant reduction of LPS-induced leukocyte rolling flux, adhesion and emigration by 84, 92 and 96% respectively. In vitro, T-cell proliferative response was also affected by budlein A. When murine J774 macrophages were incubated with the sesquiterpene lactone, LPS-induced IL-1 beta, tumor necrosis factor-alpha (TNF-alpha) and keratinocyte-derived chemokine (KC) release were concentration-dependently inhibited. In human umbilical vein endothelial cells (HUVECs), budlein A also reduced the production of TNF-alpha, monocyte chemoattractant protein-1 (MCP-1), IL-8, nitrites and CAM expression elicited by LPS. Budlein A is a potent inhibitor of LPS-induced leukocyte accumulation in vivo. This effect appears to be mediated through inhibition of cytokine and chemokine release and down-regulation of CAM expression. Thus, it has potential therapeutic interest for the control of leukocyte recruitment that occurs in different inflammatory disorders. (C) 2009 Elsevier GrnbH. All rights reserved.

Effects of Commonly Used Solubilizing Agents on a Model Nerve-Muscle Synapse

Solubility represents a limiting factor when testing new compounds in animal experiments, since solubilizing agents generally have pharmacological effects that can interfere with the studied substance. Vehicles are commonly used for solubilizing certain substances including apolar and polar extracts obtained from medicinal plants. In this study, fifteen vehicles were investigated on mice neuromuscular preparations. A known in vitro neuroblocker myotoxin from Bothrops jararacussu venom, bothropstoxin-I, was used as a pharmacological tool for testing the medicinal potential of apolar and polar extracts (hexane, dichloromethane, ethyl acetate and methanol) obtained from Casearia sylvestris Sw. leaves, which in turn were used for testing their solubility and concomitantly to produce no change on basal response of indirectly stimulated mouse phrenic nerve-diaphragm preparations. Taken together in vitro biological system and extracts solubility, our results showed that dimethyl sulphoxide and polyethylene glycol 400 were the better vehicles, and methanol extract solubilized on PEG 400 was the only one able to act against the paralysis induced by the myotoxin. Thus, this study points out to the relevant role that vehicles exhibit for extracting the potential pharmacological activity of plants in a given test system.

Prior and concomitant dehydroepiandrosterone treatment affects immunologic response of cultured macrophages infected with Trypanosoma cruzi in vitro?

DHEA, a steroid hormone synthesized from cholesterol by cells of the adrenal cortex, plays an essential role in enhancing the host`s resistance to different experimental infections. Receptors for this hormone can be found in distinct immune cells (especially macrophages) that are known to be the first line defense against Trypanosoma cruzi infection. These cells operate through an indirect pathway releasing nitric oxide (NO) and cytokines such TNF-alpha and IL-12 which in turn trigger an enhancement of natural killer cells and lymphocytes which finally secrete pro and anti-inflammatory cytokines. The effects of pre- and post-infection DHEA treatment on production of IL-12, TNF alpha and NO were evaluated. T. cruzi infected macrophages post treated with DHEA displayed enhanced concentrations of TNF-alpha, IL-12 and NO. Probably, the mechanisms that induced the production of cytokines by infected cells are more efficient when the immune system has been stimulated first by parasite invasion, suggesting that the protective role of DHEA is greater when administered post infection. (C) 2011 Elsevier B.V. All rights reserved.

Cytogenetic biomonitoring of inhabitants of a large uranium mineralization area: the municipalities of Monte Alegre, Prainha, and Alenquer, in the State of Para, Brazil

Uranium is a natural radioactive metallic element; its effect on the organism is cumulative, and chronic exposure to this element can induce carcinogenesis. Three cities of the Amazon region-Monte Alegre, Prainha, and Alenquer-in North Brazil, are located in one of the largest uranium mineralization areas of the world. Radon is a radioactive gas, part of uranium decay series and readily diffuses through rock. In Monte Alegre, most of the houses are built of rocks removed from the Earth`s crust in the forest, where the uranium reserves lie. The objective of the present work is to determine the presence or absence of genotoxicity and risk of carcinogenesis induced by natural exposure to uranium and radon in the populations of these three cities. The frequency of micronuclei (MN) and chromosomal aberrations (CA) showed no statistically significant differences between the control population and the three study populations (P > 0.05). MN was also analyzed using the fluorescence in situ hybridization (FISH) technique, with a centromere-specific probe. No clastogenic and/or aneugenic effects were found in the populations. Using FISH analysis, other carcinogenesis biomarkers were analyzed, but neither the presence of the IGH/BCL2 translocation nor an amplification of the MYC gene and 22q21 region was detected. Clastogenicity and DNA damage were also not found in the populations analyzed using the alkaline comet assay. The mitotic index showed no cytotoxicity in the analyzed individuals` lymphocytes. Once we do not have data concerning radiation doses from other sources, such as cosmic rays, potassium, thorium, or anthropogenic sources, it is hard to determine if uranium emissions in this geographic region where our study population lives are too low to cause significant DNA damage. Regardless, genetic analyses suggest that the radiation in our study area is not high enough to induce DNA alterations or to interfere with mitotic apparatus formation. It is also possible that damages caused by radiation doses undergo cellular repair.

Trypanosoma cruzi: Do different sylvatic strains trigger distinct immune responses?

Strains of Trypanosoma cruzi are multiclonal populations that can be classified in groups or genotypes, differing in pathogenicity, virulence, and histotropism. In this experiment the distinct behavior of two strains of T. cruzi, MORC-1 and MORC-2, was documented. Blood parasitemia, spleen proliferation, nitric oxide, histopathology of the spleen and heart were used as tools to evaluate parasite persistence. Groups of male mice were separated and divided in three groups: Control (C), Infected (IM-1) and Infected (IM-2). The peak of parasitemia occurred on 10 days post infection for both strains. LPS stimulated animals, infected MORC-2 group displayed significant higher concentrations of NO when compared to infected MORC-1 group (P < 0.05). For ConA stimulated lymphoproliferation, infected MORC-1 group displayed higher proliferation index as compared to infected MORC-2 group. An opposite behavior for IL-4 and TNF-alpha was observed according to the strain. For MORC-1 enhanced concentrations of IL-4 were present with concomitant reduced levels of TNF-alpha, while for MORC-2 enhanced concentrations of TNF-alpha and reduced levels of IL-4 were found. The histopathology of heart and spleen showed important differences in which MORC-1 displayed statistically enhanced number of amastigote in the heart and spleen as compared to MORC-2. Concluding, each strain triggered a distinct immune response with enhanced cytokine TH-1 profile for MORC-2 and TH-2 for MORC-1. (C) 2009 Elsevier Inc. All rights reserved.

Deconstructing Tick Saliva NON-PROTEIN MOLECULES WITH POTENT IMMUNOMODULATORY PROPERTIES

Dendritic cells (DCs) are powerful initiators of innate and adaptive immune responses. Ticks are blood-sucking ectoparasite arthropods that suppress host immunity by secreting immunomodulatory molecules in their saliva. Here, compounds present in Rhipicephalus sanguineus tick saliva with immunomodulatory effects on DC differentiation, cytokine production, and costimulatory molecule expression were identified. R. sanguineus tick saliva inhibited IL-12p40 and TNF-alpha while potentiating IL-10 cytokine production by bone marrow-derived DCs stimulated by Toll-like receptor-2, -4, and -9 agonists. To identify the molecules responsible for these effects, we fractionated the saliva through microcon filtration and reversed-phase HPLC and tested each fraction for DC maturation. Fractions with proven effects were analyzed by micro-HPLC tandem mass spectrometry or competition ELISA. Thus, we identified for the first time in tick saliva the purine nucleoside adenosine (concentration of similar to 110pmol/mu l) as a potent anti-inflammatory salivary inhibitor of DC cytokine production. We also found prostaglandin E(2) (PGE(2) similar to 100 nM) with comparable effects in modulating cytokine production by DCs. Both Ado and PGE(2) inhibited cytokine production by inducing cAMP-PKA signaling in DCs. Additionally, both Ado and PGE(2) were able to inhibit expression of CD40 in mature DCs. Finally, flow cytometry analysis revealed that PGE(2), but not Ado, is the differentiation inhibitor of bone marrow-derived DCs. The presence of non-protein molecules adenosine and PGE(2) in tick saliva indicates an important evolutionary mechanism used by ticks to subvert host immune cells and allow them to successfully complete their blood meal and life cycle.