Le VEGF induit la synthèse du PAF par l’entremise de l’activation du VEGFR-2 : identification des phosphotyrosines impliquées

Notre laboratoire a démontré que la capacité proinflammatoire du vascular endothelial growth factor (VEGF-A165) implique la synthèse endothéliale du facteur d’activation plaquettaire (PAF) via l’activation du récepteur tyrosine kinase homodimérique VEGFR-2/R-2. La synthèse du PAF requiert l’activation de la p38 MAPK et p42/44 MAPK qui activent la phospholipase A2 secrétée de type V (sPLA2-V). Nous avons découvert que la synthèse aigue de prostacycline (PGI2) induite par le VEGF-A165 requiert l’activation des récepteurs hétérodimériques VEGFR-1/R-2. L’activation sélective des récepteurs du VEGF peut donc agir comme balance dans la synthèse de facteurs pro-(PAF) et anti-(PGI2) inflammatoire. Cependant, les tyrosines impliquées dans la transphosphorylation de VEGFR-2/R-2 menant à la synthèse du PAF sont inconnues. Par mutagenèse dirigée, nous avons effectué des transfections transitoires de cellules endothéliales avec des plasmides codant pour le VEGFR-2 dont les tyrosines ciblées ont été remplacées de façon séquentielle par une phénylalanine. Un vecteur vide pcDNA a été utilisé comme contrôle négatif. La stimulation des cellules endothéliales de l’aorte bovine (BAEC) transfectées avec le VEGF-A165 (1nM) pendant 15 minutes augmente la synthèse du PAF de 300%, laquelle était similaire dans les BAEC non transfectées. Dans les BAEC transfectées avec les vecteurs pcDNA codant pour les mutations Y801F, Y1059F, Y1175F et Y1214F, nous avons observé une réduction de 54, 73, 68, et 57% respectivement de la synthèse du PAF induite par le VEGF par rapport au pcDNA témoin. Nos résultats apportent un nouvel aperçu sur le mécanisme par lequel le VEGF induit la synthèse du PAF qui est connu pour sa contribution dans l’activité pro-inflammatoire du VEGF.

Embryonic and Uterine Characteristics of Diapause

L’implantation retardée ou diapause embryonnaire décrit l'arrêt ou le retardement pendant l'embryogenèse. Chez le vison, la diapause est corrélée avec une sécrétion pituitaire insuffisante de la prolactine, ayant pour résultat la différentiation incomplète du corpus luteum et réduction de la progestérone. Des études antérieures suggèrent que le blastocyste de vison en diapause demeure dans un état de quiescence ou se développe lentement. Pour élucider ceci, la réplication de l'ADN a été étudiée. Les résultats démontrent synthèse de l’ADN et prolifération cellulaire dans les embryons au stade de morula, avant la diapause et dans les blastocystes après la réactivation. La réplication de l'ADN a été également détectée dans des blastocystes en diapause et en diapause prolongée. L'implantation est considérée comme une interaction bidirectionnelle entre le blastocyste et l'utérus. Il a été montré que les prostaglandines sont importantes pour la vascularisation de l’utérus au moment de l’implantation et peuvent réactiver l'utérus des visons après la diapause. La concentration protéinique et la localisation de la phospholipase citosolique A2 (CPLA2) et de la cyclooxygenase 2 (COX2) ont été étudiées dans l'utérus de vison. L’expression de la CPLA2 et COX2 étaient sur-régulées au moment de l'implantation. Il est connu que la prolactine active les corpus luteum des visons. L'idée de un lien entre la prolactine et la voie de signalisation des prostaglandines a été testée en mesurant les récepteurs de prolactine. Les résultats montrent une augmentation de l’expression des récepteurs de prolactine à l'implantation suggérant que la prolactine pourrait activer la voie de prostaglandine à l'utérus par son propre récepteur. La conclusion, les embryons pendant la diapause ne sont pas arrêtées complètement et les protéines liées à la voie de prostaglandine sont implique dans la réactivation de l'utérus.

Crotalphine induces potent antinociception in neuropathic pain by acting at peripheral opioid receptors

Neuropathic pain is an important clinical problem and it is usually resistant to the current therapy. We have recently characterized a novel analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in an experimental model of neuropathic pain induced in rats by chronic constriction, of sciatic nerve. The effect of the peptide was compared to that induced by the crude venom, which confirmed that crotalphine is responsible for the antinociceptive effect of the crotalid venom on neuropathic pain. For characterization of neuropathic pain, the presence of hyperalgesia, allodynia and spontaneous pain was assessed at different times after nerve constriction. These phenomena were detected 24 h after surgery and persisted at least for 14 days. The pharmacological treatments were performed on day 14 after surgery. Crotalphine (0.2-5 mu g/kg) and the crude venom (400-1600 mu g/kg) administered p.o. inhibited hyperalgesia, allodynia and spontaneous pain induced by nerve constriction. The antinociceptive effect of the peptide and crude venom was long lasting, since it was detected up to 3 days after treatment. Intraplantar injection of naloxone (1 mu g/paw) blocked the antinociceptive effect, indicating the involvement of opioid receptors in this phenomenon. Gabapentin (200 mg/kg, p.o.), and morphine (5 mg/kg, s.c.), used as positive controls, blocked hyperalgesia and partially inhibited allodynia induced by nerve constriction. These data indicate that crotalphine induces a potent and long lasting opioid antinociceptive effect in neuropathic pain that surpasses that observed with standard analgesic drugs. (C) 2008 Elsevier B.V. All rights reserved.

Effects of Low Molecular Weight Sulfated Galactan Fragments From Botryocladia Occidentalis on the Pharmacological and Enzymatic Activity of Spla2 From Crotalus Durissus Cascavella

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Evaluation of anti-inflammatory activity of derivatives from aerial parts of Baccharis uncinella

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The extract of the jellyfish Phyllorhiza punctata promotes neurotoxic effects

Phyllorhiza punctata (P. punctata) is a jellyfish native to the southwestern Pacific. Herewith we present the biochemical and pharmacological characterization of an extract of the tentacles of P. punctata. The tentacles were subjected to three freezethaw cycles, homogenized, ultrafiltered, precipitated, centrifuged and lyophilized to obtain a crude extract (PHY-N). Paralytic shellfish poisoning compounds such as saxitoxin, gonyautoxin-4, tetrodotoxin and brevetoxin-2, as well as several secretory phospholipase A2 were identified. PHY-N was tested on autonomic and somatic neuromuscular preparations. In mouse vas deferens, PHY-N induced phasic contractions that reached a peak of 234 +/- 34.7% of control twitch height, which were blocked with either 100 mu m of phentolamine or 1m m of lidocaine. In mouse corpora cavernosa, PHY-N evoked a relaxation response, which was blocked with either L-NG-Nitroarginine methyl ester (0.5 m m) or 1m m of lidocaine. PHY-N (1, 3 and 10 mu g ml(-1)) induced an increase in tonus of the biventercervicis neuromuscular preparation that was blocked with pre-treatment of galamine (10 mu m). Administration of 6 mg kg(-1) PHY-N intramuscularly produced death in broilers by spastic paralysis. In conclusion, PHY-N induces nerve depolarization and nonspecifically increases neurotransmitter release. Copyright (C) 2011 John Wiley & Sons, Ltd.

Antagonism of myotoxic and paralyzing activities of bothropstoxin-I by surarnin

Polyanionic substances are known to inhibit the myotoxic effects of some crotalide snake venoms. Bothropstoxin-I (BthTX-I), a basic Lys49 phospholipase (PLA(2)) homologue from Bothrops jararacussu venom, besides inducing muscle damage, also promotes the blockade of both directly and indirectly evoked contractions in mouse neuromuscular preparation. In this work, we evaluated the ability of suramin, a polysulfonated naphtylurea derivative, to antagonize the myotoxic and the paralyzing activities of BthTX-I on mice neuromuscular junction in vitro. Myotoxicity was assessed by light and electronic microscopic analysis of extensor digitorum longus (EDL) muscles; paralyzing activity was evaluated through the recording of both directly and indirectly evoked contractions of phrenic-diaphragm (PD) preparations. BthTX-I (1 muM) alone, or pre-incubated with suramin (10 muM) at 37degreesC for 15 min was added to the preparations for 120 min. BthTX-I induced histological alterations typical of myonecrosis in 14.6 +/- 1.0% of EDL muscle fibers. In addition, BthTX-I blocked 50% of both directly and indirectly evoked contractions in PD preparations in 72.1 +/- 9.1 and 21.1 +/- 2.0 min, respectively. Pre-incubation with suramin abolished both the muscle-damaging and muscle-paralyzing activities of BthTX-I. Since suramin is a polyanionic substance, we suggested that its effects result from the formation of inactive acid-base complexes with BthTX-I. (C) 2003 Elsevier Ltd. All rights reserved.

Inhibition of myotoxic activity of Bothrops asper myotoxin II by the anti-trypanosomal drug surarnin

Suramin, a synthetic polysulfonated compound, developed initially for the treatment of African trypanosomiasis and onchocerciasis, is currently used for the treatment of several medically relevant disorders. Suramin, heparin, and other polyanions inhibit the myotoxic activity of Lys49 phospholipase A(2) analogues both in vitro and in vivo, and are thus of potential importance as therapeutic agents in the treatment of viperid snake bites. Due to its conformational flexibility around the single bonds that link the central phenyl rings to the secondary amide backbone, the symmetrical suramin molecule binds by an induced-fit mechanism complementing the hydrophobic surfaces of the dimer and adopts a novel conformation that lacks C2 symmetry in the dimeric crystal structure of the suramin-Bothrops asper myotoxin II complex. The simultaneous binding of suramin at the surfaces of the two monomers partially restricts access to the nominal active sites and significantly changes the overall charge of the interfacial recognition face of the protein, resulting in the inhibition of myotoxicity. (c) 2005 Elsevier Ltd. All rights reserved.

Enzymatic toxins from snake venom: structural characterization and mechanism of catalysis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Snake venomics of the Siamese Russell's viper (Daboia russelli siamensis) - Relation to pharmacological activities

The venom proteome of Daboia russelli siamensis, a snake of medical importance in several Asian countries, was analysed by 2-D electrophoresis, subsequent MS/MS and enzymatic assays. The proteome comprises toxins from six protein families: serine proteinases, metalloproteinases, phospholipases A(2), L-amino acid oxidases, vascular endothelial growth factors and C-type lectin-like proteins. The venom toxin composition correlates with the clinical manifestation of the Russell's viper bite and explains pathological effects of the venom such as coagulopathy, oedema, hypotensive, necrotic and tissue damaging effects. The vast majority of toxins are potentially involved in coagulopathy and neurotoxic effects. The predominant venom components are proteinases capable of activating blood coagulation factors and promoting a rapid clotting of the blood, and neurotoxic phospholipase A(2)s. The analysis of the venom protein composition provides a catalogue of secreted toxins. The proteome of D. r. siamensis exhibits a lower level of toxin diversity than the proteomes of other viperid snakes. In comparison to the venoms of Vipera ammodytes ammodytes and Vipera ammodytes meridionalis, the venom from D. r. siamensis showed quantitative differences in the proteolytic, phospholipase A2, L-amino acid oxidase and alkaline phosphatase activities. (c) 2009 Published by Elsevier B.V.

Folk uses and pharmacological properties of Casearia sylvestris: a medicinal review

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CRYSTALLIZATION AND PRELIMINARY DIFFRACTION DATA OF 2 MYOTOXINS ISOLATED FROM THE VENOMS OF BOTHROPS-ASPER (TERCIOPELO) AND BOTHROPS-NUMMIFER (JUMPING VIPER)

Two myotoxins isolated from B. asper (myotoxin II) and B. nummifer (myotoxin I) snake venoms have been crystallized and their diffraction properties are described. These myotoxins are phospholipase A2 variants which lack enzymatic activity; B. asper myotoxin II is a lysine-49 phospholipase. Crystals were obtained at room temperature by standard hanging-drop vapour diffusion methods. Crystals diffracted to a resolution of 2.8 and 2.3 angstrom, respectively.

Role of annexin 1 gene expression in mouse craniofacial bone development

BACKGROUND: Annexin 1 is a 37-kDa protein that has complex intra- and extracellular effects. To discover whether the absence of this protein alters bone development, we monitored this event in the annexin-A1 null mice in comparison with littermate wild-type controls. METHODS: Radiographic and densitometry methods were used for the assessment of bone in annexin-A1 null mice at a gross level. We used whole-skeleton staining, histological analysis, and Western blotting techniques to monitor changes at the tissue and cellular levels. RESULTS: There were no gross differences in the appendicular skeleton between the genotypes, but an anomalous development of the skull was observed in the annexin-A1 null mice. This was characterized in the newborn annexin-A1 null animals by a delayed intramembranous ossification of the skull, incomplete fusion of the interfrontal suture and palatine bone, and the presence of an abnormal suture structure. The annexin-A1 gene was shown to be active in osteocytes during this phase and COX-2 was abundantly expressed in cartilage and bone taken from annexin-A1 null mice. CONCLUSIONS: Expression of the annexin-A1 gene is important for the normal development of the skull in mice, possibly through the regulation of osteoblast differentiation and a secondary effect on the expression of components of the cPLA2-COX-2 system. © 2007 Wiley-Liss, Inc.

Modulation of the pharmacological effects of enzymatically-active PLA 2 by BTL-2, an isolectin isolated from the Bryothamnion triquetrum red alga

Background. An interaction between lectins from marine algae and PLA 2 from rattlesnake was suggested some years ago. We, herein, studied the effects elicited by a small isolectin (BTL-2), isolated from Bryothamnion triquetrum, on the pharmacological and biological activities of a PLA 2 isolated from rattlesnake venom (Crotalus durissus cascavella), to better understand the enzymatic and pharmacological mechanisms of the PLA 2 and its complex. Results. This PLA2 consisted of 122 amino acids (approximate molecular mass of 14 kDa), its pI was estimated to be 8.3, and its amino acid sequence shared a high degree of similarity with that of other neurotoxic and enzymatically-active PLA2s. BTL-2 had a molecular mass estimated in approximately 9 kDa and was characterized as a basic protein. In addition, BTL-2 did not exhibit any enzymatic activity. The PLA2 and BTL-2 formed a stable heterodimer with a molecular mass of approximately 24-26 kDa, estimated by molecular exclusion HPLC. In the presence of BTL-2, we observed a significant increase in PLA2 activity, 23% higher than that of PLA2 alone. BTL-2 demonstrated an inhibition of 98% in the growth of the Gram-positive bacterial strain, Clavibacter michiganensis michiganensis (Cmm), but only 9.8% inhibition of the Gram-negative bacterial strain, Xanthomonas axonopodis pv passiflorae (Xap). PLA2 decreased bacterial growth by 27.3% and 98.5% for Xap and Cmm, respectively, while incubating these two proteins with PLA2-BTL-2 inhibited their growths by 36.2% for Xap and 98.5% for Cmm. PLA2 significantly induced platelet aggregation in washed platelets, whereas BTL-2 did not induce significant platelet aggregation in any assay. However, BTL-2 significantly inhibited platelet aggregation induced by PLA2. In addition, PLA 2 exhibited strong oedematogenic activity, which was decreased in the presence of BTL-2. BTL-2 alone did not induce oedema and did not decrease or abolish the oedema induced by the 48/80 compound. Conclusion. The unexpected results observed for the PLA2-BTL-2 complex strongly suggest that the pharmacological activity of this PLA2 is not solely dependent on the presence of enzymatic activity, and that other pharmacological regions may also be involved. In addition, we describe for the first time an interaction between two different molecules, which form a stable complex with significant changes in their original biological action. This opens new possibilities for understanding the function and action of crude venom, an extremely complex mixture of different molecules. © 2008 Oliveira et al; licensee BioMed Central Ltd.

Triacontyl p-coumarate: An inhibitor of snake venom metalloproteinases

Snake venom metalloproteinases (SVMPs) participate in a number of important biological, physiological and pathophysiological processes and are primarily responsible for the local tissue damage characteristic of viperid snake envenomations. The use of medicinal plant extracts as antidotes against animal venoms is an old practice, especially against snake envenomations. Such plants are sources of many pharmacologically active compounds and have been shown to antagonize the effects of some venoms and toxins. The present study explores the activity of triacontyl p-coumarate (PCT), an active compound isolated from root bark of Bombacopsis glabra vegetal extract (Bg), against harmful effects of Bothropoides pauloensis snake venom and isolated toxins (SVMPs or phospholipase A2). Before inhibition assays, Bg or PCT was incubated with venom or toxins at ratios of 1:1 and 1:5 (w/w; venom or isolated toxins/PCT) for 30 min at 37 °C. Treatment conditions were also assayed to simulate snakebite with PCT inoculated at either the same venom or toxin site. PCT neutralized fibrinogenolytic activity and plasmatic fibrinogen depletion induced by B. pauloensis venom or isolated toxin. PCT also efficiently inhibited the hemorrhagic (3MDH-minimum hemorrhagic dose injected i.d into mice) and myotoxic activities induced by Jararhagin, a metalloproteinase from B. jararaca at 1:5 ratio (toxin: inhibitor, w/w) when it was previously incubated with PCT and injected into mice or when PCT was administered after toxin injection. Docking simulations using data on a metalloproteinase (Neuwiedase) structure suggest that the binding between the protein and the inhibitor occurs mainly in the active site region causing blockade of the enzymatic reaction by displacement of catalytic water. Steric hindrance may also play a role in the mechanism since the PCT hydrophobic tail was found to interact with the loop associated with substrate anchorage. Thus, PCT may provide a alternative to complement ophidian envenomation treatments. © 2012 Elsevier Ltd. All rights reserved.