RESUMO
Snake bites are a severe problem in the countryside of Brazil and are usually attributed to snakes of the genera Bothrops, Crotalus, and Lachesis. Snake venom can release ectoenzymes and nucleotidases that modulate the purinergic system. In addition to serum therapy against snake poisoning, medicinal plants with anti-infammatory activities, such as Tabebuia aurea, is empirically applied in accidents that occur in difcult-to-access areas. This study aimed was to verify the presence and activity of nucleotidases in the crude venom of Bothrops mattogrossensis (BmtV) in vitro and characterize the modulation of purinergic components, myeloid diferentiation, and infammatory/oxidative stress markers by BmtV in vivo and in vitro. Moreover, our study assessed the inhibitory activities of specioside, an iridoid isolated from Tabebuia aurea, against the efects of BmtV. Proteomic analysis of venom content and nucleotidase activity confrm the presence of ectonucleotidase-like enzymes in BmtV. In in vivo experiments, BmtV altered purinergic component expression (P2X7 receptor, CD39 and CD73), increased neutrophil numbers in peripheral blood, and elevated oxidative stress/infammatory parameters such as lipid peroxidation and myeloperoxidase activity. BmtV also decreased viability and increased spreading index and phagocytic activity on macrophages. Specioside inhibited nucleotidase activity, restored neutrophil numbers, and mediate the oxidative/infammatory efects produced by BmtV. We highlight the efects produced by BmtV in purinergic system components, myeloid diferentiation, and infammatory/oxidative stress parameters, while specioside reduced the main BmtV-dependent efects.
RESUMO
Increased collagen-derived advanced glycation end-products (AGEs) are consistently related to painful diseases, including osteoarthritis, diabetic neuropathy, and neurodegenerative disorders. We have recently developed a model combining a two-dimensional glycated extracellular matrix (ECM-GC) and primary dorsal root ganglion (DRG) that mimicked a pro-nociceptive microenvironment. However, culturing primary cells is still a challenge for large-scale screening studies. Here, we characterized a new model using ECM-GC as a stimulus for human sensory-like neurons differentiated from SH-SY5Y cell lines to screen for analgesic compounds. First, we confirmed that the differentiation process induces the expression of neuron markers (MAP2, RBFOX3 (NeuN), and TUBB3 (ß-III tubulin), as well as sensory neuron markers critical for pain sensation (TRPV1, SCN9A (Nav1.7), SCN10A (Nav1.8), and SCN11A (Nav1.9). Next, we showed that ECM-GC increased c-Fos expression in human sensory-like neurons, which is suggestive of neuronal activation. In addition, ECM-GC upregulated the expression of critical genes involved in pain, including SCN9A and TACR1. Of interest, ECM-GC induced substance P release, a neuropeptide widely involved in neuroinflammation and pain. Finally, morphine, the prototype opiate, decreased ECM-GC-induced substance P release. Together, our results suggest that we established a functional model that can be useful as a platform for screening candidates for the management of painful conditions.
Assuntos
Analgésicos/análise , Analgésicos/farmacologia , Colágeno/farmacologia , Avaliação Pré-Clínica de Medicamentos , Modelos Biológicos , Células Receptoras Sensoriais/citologia , Animais , Antígenos de Neoplasias/metabolismo , Biomarcadores/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Matriz Extracelular/metabolismo , Galectina 3/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Glicosilação/efeitos dos fármacos , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/genética , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Receptores da Neurocinina-1/genética , Receptores da Neurocinina-1/metabolismo , Células Receptoras Sensoriais/efeitos dos fármacos , Células Receptoras Sensoriais/metabolismo , Substância P/metabolismo , beta-Endorfina/metabolismoRESUMO
BACKGROUND: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. RESULTS: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. CONCLUSIONS: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. METHODS: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.
RESUMO
Background: Ependymoma (EPN), the third most common pediatric brain tumor, is a central nervous system (CNS) malignancy originating from the walls of the ventricular system. Surgical resection followed by radiation therapy has been the primary treatment for most pediatric intracranial EPNs. Despite numerous studies into the prognostic value of histological classification, the extent of surgical resection and adjuvant radiotherapy, there have been relatively few studies into the molecular and cellular biology of EPNs. Results: We elucidated the ultrastructure of the cultured EPN cells and characterized their profile of immunophenotypic pluripotency markers (CD133, CD90, SSEA-3, CXCR4). We established an experimental EPN model by the intracerebroventricular infusion of EPN cells labeled with multimodal iron oxide nanoparticles (MION), thereby generating a tumor and providing a clinically relevant animal model. MRI analysis was shown to be a valuable tool when combined with effective MION labeling techniques to accompany EPN growth. Conclusions: We demonstrated that GFAP/CD133+CD90+/CD44+ EPN cells maintained key histopathological and growth characteristics of the original patient tumor. The characterization of EPN cells and the experimental model could facilitate biological studies and preclinical drug screening for pediatric EPNs. Methods: In this work, we established notoriously challenging primary cell culture of anaplastic EPNs (WHO grade III) localized in the posterior fossa (PF), using EPNs obtained from 1 to 10-year-old patients (n = 07), and then characterized their immunophenotype and ultrastructure to finally develop a xenograft model.
RESUMO
The strategic position of factor Xa (FXa) in blood coagulation makes it a compelling target for the development of new anticoagulants. Blood-sucking animals have in their salivary glands mixtures of anticoagulants, which could be used for designing novel antithrombotic compounds. Herein, we describe Vizottin, the first FXa inhibitor from the salivary complex of the leech Haementeria vizottoi . Vizottin was purified by gel filtration and reverse-phase chromatography, and shown to have anticoagulant effects in human plasma, prolonging the recalcification time in a dose-dependent manner (IC50 40 nM). Vizottin induced blood incoagulability in FX-deficient plasma, whereas in normal and reconstituted plasma, Vizottin doubled the prothrombin time at 160 nM. This peptide competitively inhibited human FXa (K(i) 2 nM) like FXa inhibitors from other leeches, albeit via a distinct mechanism of action. At high concentrations, vizottin inhibited the amidolytic activity of factor VIIa/tissue factor (IC50 96.4 nM). Vizottin inhibited FXa in the prothrombinase complex and Gla-domainless FXa. Moreover, vizottin did not interfere with FX activation induced by RVV-X, a known enzyme that requires the Gla-domain of FX for activation. Competition experiments in the presence of FXa and GGACK-FXa (active site blocked) demonstrated that the inhibition of FXa by vizottin is through binding to the active site rather than an exosite. This novel inhibitor appears to exert its inhibitory effects through direct binding to the active site of FXa in a time-dependent manner, but not involving a tight-binding model. In this context, vizottin is a promising model for designing novel anticoagulants for the treatment of thrombotic diseases.
Assuntos
Anticoagulantes/farmacologia , Inibidores do Fator Xa , Sanguessugas/química , Clorometilcetonas de Aminoácidos/farmacologia , Animais , Anticoagulantes/isolamento & purificação , Coagulação Sanguínea/efeitos dos fármacos , Testes de Coagulação Sanguínea , Domínio Catalítico/efeitos dos fármacos , Cromatografia em Gel , Cromatografia de Fase Reversa , Compostos Cromogênicos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Ativação Enzimática/efeitos dos fármacos , Fator VIIa/antagonistas & inibidores , Fibrinolíticos/isolamento & purificação , Fibrinolíticos/farmacologia , Humanos , Lipoproteínas/farmacologia , Ligação Proteica/efeitos dos fármacos , Glândulas Salivares/química , Proteínas e Peptídeos Salivares/farmacologiaRESUMO
A novel acidic Asp49 phospholipase A(2) was isolated from Bothrops erythromelas (jararaca malha-de-cascavel) snake venom by four chromatographic steps. BE-I-PLA2 present a molecular weight of 13,649.57 Da as estimated by mass spectrometry. N-terminal and four internal peptides were sequenced, covering around one-third of the complete toxin sequence. The complete BE-I-PLA2 cDNA was cloned from a B. erythromelas venom-gland cDNA library. The cDNA sequence possesses 457 bp and encodes a protein with significant sequence similarity to many other phospholipase A(2) from snake venoms. When tested in platelet rich plasma, the enzyme showed a potent inhibitory effect on aggregation induced by arachidonic acid and collagen, but not ADP. On the other hand, BE-I-PLA2 did not modify aggregation in washed platelet. Furthermore, no action of BE-I-PLA2 on the principal platelets receptors was observed. Chemical modification with p-bromophenacyl bromide abolished the enzymatic activity of BE-I-PLA2, but its anti-platelet activity was only partially inhibited. In human umbilical-cord veins endothelial cells, BE-I-PLA2 was neither apoptotic nor proliferative but stimulated endothelial cells to release prostaglandin I(2), suggesting an increase of its potential anti-platelet activity in vivo. Further studies are required in order to determine the exact mechanism of action of BE-I-PLA2 in the inhibition of platelet aggregation.
Assuntos
Bothrops/genética , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/isolamento & purificação , Células Endoteliais/metabolismo , Epoprostenol/metabolismo , Fosfolipases A/genética , Fosfolipases A/isolamento & purificação , Sequência de Aminoácidos , Animais , Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Clonagem Molecular , Venenos de Crotalídeos/química , Venenos de Crotalídeos/metabolismo , Venenos de Crotalídeos/farmacologia , DNA Complementar/química , DNA Complementar/genética , Relação Dose-Resposta a Droga , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Epoprostenol/biossíntese , Feminino , Fosfolipases A2 do Grupo II , Fosfolipases A2 do Grupo IV , Humanos , Masculino , Dados de Sequência Molecular , Fosfolipases A/metabolismo , Fosfolipases A/farmacologia , Fosfolipases A2 , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/farmacologia , Proteínas de Répteis , Análise de Sequência de DNA , Análise de Sequência de Proteína , Homologia de Sequência de AminoácidosRESUMO
Using a cDNA library made from Lonomia obliqua caterpillar bristles, we identified a transcript with a 603 bp open reading frame. The deduced protein corresponds to Lopap, a prothrombin activator previously isolated by our group from the bristles of this species. The mature protein is composed by 185 amino acids and shares similarity with members of the lipocalin family. The cDNA encoding the mature form was amplified by PCR, subcloned into pAE vector and used to transform Escherichia coli BL21(DE3) cells. As for the native Lopap, the recombinant fusion protein shows enzymatic activity, promotes prothrombin hydrolysis, generates fragments similar to prethrombin-2 and fragment 1.2 as intermediates, and generates thrombin as the final product. In addition, structural bioinformatics studies indicated several interesting molecular features, including the residues that could be responsible for Lopap's serine protease-like activity and the role of calcium binding in this context. Such catalytic activity has never been found in other members of the lipocalin family. This is the first report describing the recombinant production and biochemical characterization of a Lonomia obliqua lipocalin, as well as the structural features that could be responsible for its serine protease-like catalytic activity.