Caracterização funcional da BaltMTx, uma miotoxina isolada da peçonha de Bothrops alternatus

CHAPTER II: Snake venoms are a complex mixture of organic and inorganic compounds, proteins and peptides such as aminotransferases, acetylcholinesterase, hyaluronidases, L-amino acid oxidase, phospholipase A2, metalloproteases, serine proteases, lectins, disintegrins, and others. Phospholipase A2 directly or indirectly influence the pathophysiological effect on envenomation, as well as their participation in the digestion of the prey. They have several other activities such as hemolytic indirect action, cardiotoxicity, aggregating of platelets, anticoagulant, edema, myotoxic and inflammatory activities. In this work, we describe the functional characterization of BaltMTx, a PLA2 from Bothrops alternatus that inhibits platelet aggregation and present bactericidal effect. The purification of BaltMTx was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, followed by hydrophobic chromatography on Phenyl–Sepharose and affinity chromatography on HiTrap™ Heparin HP). The protein was purified to homogeneity as judged by its migration profile in SDS–PAGE stained with coomassie blue, and showed a molecular mass of about 15 kDa under reducing conditions and approximately 25 kDa in non-reducing conditions. BaltMTx showed a rather specific inhibitory effect on platelet aggregation induced by epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by collagen or adenosine diphosphate. BaltMTx also showed antibacterial activity against Staphylococcus aureus and Escherichia coli. High concentrations of BatlMTx stimulated the proliferation of Leishmania (Leishmania) infantum and Leishmania (Viania) braziliensis. BaltMTx induced production of inflammatory mediators such as IL-10, IL-12, TNF-α and NO. BaltMTx could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders as well as bactericidal agent.Coordenação de Aperfeiçoamento de Pessoal de Nível SuperiorDissertação (Mestrado)CAPÍTULO II: Peçonhas de serpentes são uma mistura complexa de compostos orgânicos e inorgânicos, proteínas e peptídeos, tais como as aminotransferases, acetilcolinesterases, hialuronidases, L-aminoácido-oxidases, fosfolipases A2, metaloproteases, serinoproteases, lectinas, desintegrinas, entre outros. As fosfolipases A2 influenciam direta ou indiretamente no efeito fisiopatológico no envenenamento, pois além de sua participação na digestão das presas, elas apresentam várias outras atividades como ação hemolítica indireta, neurotoxicidade, cardiotoxicidade, ação agregadora de plaquetas, atividades anticoagulante, edematogênica, miotóxica e inflamatória. Neste trabalho, descrevemos a caracterização funcional de BaltMTx, uma PLA2 de Bothrops alternatus que inibe a agregação plaquetária e apresenta efeito bactericida. A purificação de BaltMTx foi realizada por uma combinação de três passos cromatográficos (cromatografia de troca iônica em DEAE-Sephacel, seguido de cromatografia de interação hidrofóbica em Phenyl-Sepharose, e cromatografia de afinidade em Heparina HiTrap™ HP). A proteína foi purificada até à homogeneidade, tal como avaliado pelo seu perfil de migração em SDS-PAGE, corado com azul de Coomassie, que mostrou uma massa molecular de cerca de 15 kDa sob condições redutoras e cerca de 25 kDa em condições não redutoras. BaltMTx mostrou um efeito inibidor específico sobre a agregação plaquetária induzida por epinefrina em plasma humano rico em plaquetas, de uma maneira dose dependente. Todavia, apresentou pouco ou nenhum efeito sobre a agregação plaquetária induzida por colágeno e adenosinadifosfato. BaltMTx, também, mostrou atividade antibacteriana contra Staphylococcus aureus e Escherichia coli. Altas concentrações de BatlMTx estimulou a proliferação de Leishmania (Leishmania) infantum e Leishmania (Viania) braziliensis. BaltMTx induziu a produção de mediadores inflamatórios tais como IL-10, IL-12, TNF-α e NO. BaltMTx apresenta grande interesse médico, tanto como uma nova ferramenta para o desenvolvimento de novos agentes terapêuticos para a prevenção e tratamento de distúrbios trombóticos, quanto como agente bactericida

14-weeks combined exercise epigenetically modulated 118 genes of menopausal women with prediabetes

Background: Pre-diabetes precedes Diabetes Mellitus (DM) disease and is a critical period for hyperglycemia treatment, especially for menopausal women, considering all metabolic alterations due to hormonal changes. Recently, the literature has demonstrated the role of physical exercise in epigenetic reprogramming to modulate the gene expression patterns of metabolic conditions, such as hyperglycemia, and prevent DM development. In the present study, we hypothesized that physical exercise training could modify the epigenetic patterns of women with poor glycemic control. Methods: 48 post-menopause women aged 60.3 ± 4.5 years were divided according to their fasting blood glucose levels into two groups: Prediabetes Group, PG (n=24), and Normal Glucose Group, NGG (n=24). All participants performed 14 weeks of physical exercise three times a week. The Infinium Methylation EPIC BeadChip measured the participants’ Different Methylated Regions (DMRs). Results: Before the intervention, the PG group had 12 DMRs compared to NGG. After the intervention, five DMRs remained different. Interestingly, when comparing the PG group before and after training, 118 DMRs were found. The enrichment analysis revealed that the genes were related to different biological functions such as energy metabolism, cell differentiation, and tumor suppression. Conclusion: Physical exercise is a relevant alternative in treating hyperglycemia and preventing DM in post-menopause women with poor glycemic control

Associação entre a ooforectomia bilateral precoce e o desenvolvimento do parkinsonismo e Doença de Parkinson em mulheres na pré-menopausa

O parkinsonismo é um distúrbio do sistema nervoso de maior incidência masculina do que feminina, visto que, por mecanismos fisiológicos, o estrogênio possui efeitos neuroprotetores, com funções como aumento da dopamina, um neurotransmissor essencial para o controle das funções motoras. Além disso, previne a formação dos corpúsculos de Lewy e da agregação da α-sinucleína, responsáveis pela progressão da Doença de Parkinson. Por isso, a doença se apresenta diferentemente nas mulheres. A remoção cirúrgica de ambos os ovários em mulheres na pré-menopausa para a prevenção do câncer de ovário parece favorecer o surgimento da doença, tendo em vista a perda da produção do hormônio protetor. Assim, o objetivo do estudo é analisar a associação entre a ooforectomia bilateral precoce e o desenvolvimento de parkinsonismo e Doença de Parkinson em mulheres na pré-menopausa. Trata-se de uma revisão bibliográfica sistemática, do tipo quantitativa, que utilizou as plataformas do PubMed, SciELO e Cochrane Library como bases de dados para seleção dos artigos, todos na língua inglesa. Foram utilizadas literaturas publicadas com recorte temporal de 2017 a 2022. De acordo com as literaturas analisadas, a ooforectomia bilateral precoce em mulheres na pré-menopausa aumenta o risco do desenvolvimento de parkinsonismo. Desse modo, a diminuição dos procedimentos cirúrgicos profiláticos para câncer de ovário nas pacientes com risco médio de malignidade reduziria o risco dessa condição

Evaluation of functional activities of Bhalternin, identification and synthesis of the platelet aggregation inhibitor peptide region

Platelets plays important roles in primary hemostasis, which is responsible for hemostatic plug formation in the presence of vascular injury. The processes exerted by platelets consist of platelet adhesion, activation and aggregation, and imbalances in these mechanisms cause vascular occlusion, with consequent occurrence of thrombotic disease. This is considered a serious public health problem due to its high incidence and high mortality rate, and the available treatments have several limitations, such as increased risk of bleeding and unresponsiveness by patients. The search for new components that act on platelet function is extremely important for the diagnosis and treatment of these diseases, and snake venoms present substances capable of interfering with platelet function. There are several classes of ophidic proteins that interact with platelet receptors, and the peptides present in venom have stood out due to their lower complexity and lower production cost. The aim of this study was to evaluate the interference of bhalternin, a serinoprotease isolated from Bothrops alternatus venom, on platelet aggregation. As a result, it was found that the protein, after denatured, was able to inhibit platelet aggregation by interacting with the P2Y12 ADP receptor. In addition, the peptide region responsible for this activity has been identified and designated BhaltPIP, which has been synthesized and is in patenting process. The effect of bhalternin on tumor cells was also evaluated, and was noted that the protein interferes with prostate tumor cell growth without acting on the growth of normal cell. Thus, it is concluded that bhalternin has functions on both tumor cells and platelet aggregation, and the BhaltPIP peptide derived from this protein has therapeutic potential for treatment and diagnosis of hemostatic disorders.CENAPAD - Centro Nacional de Processamento de Alto Desempenho em São PauloTese (Doutorado)As plaquetas exercem importantes funções na hemostasia primária, a qual é responsável pela formação do tampão hemostático na presença de uma injúria vascular. Os processos exercidos pelas plaquetas consistem em adesão, ativação e agregação plaquetária, e desequilíbrios nesses mecanismos provocam oclusão vascular, com consequente ocorrência de doença trombótica. Esta é considerada grave problema de saúde pública, devido a sua alta incidência e elevada taxa de mortalidade, e os tratamentos disponíveis possuem várias limitações, como o aumento de risco de sangramento e a não responsividade pelos pacientes. A pesquisa de novos componentes que atuem na função plaquetária é de extrema importância para o diagnóstico e tratamento destas doenças, e as peçonhas de serpentes possuem em sua composição substâncias capazes de interferirem na função das plaquetas. São várias as classes de proteínas ofídicas que interagem com receptores plaquetários, entretanto os peptídeos presentes na peçonha têm ganhado destaque devido a sua menor complexidade e menor custo de produção. O objetivo deste trabalho foi avaliar a interferência da bhalternina, uma serinoprotease isolada da peçonha de Bothrops alternatus, na agregação plaquetária. Foi constatado que a proteína, após ser desnaturada, foi capaz de inibir a agregação plaquetária ao interagir com o receptor de ADP P2Y12. Ademais, a região peptídica responsável por essa atividade foi identificada e denominada BhaltPIP, a qual foi sintetizada e está em processo de patenteamento. Também foi avaliado o efeito da bhalternina em células tumorais in vitro, onde se notou que a proteína interfere no crescimento de célula tumoral de próstata sem atuar sobre o crescimento de células normais. Assim, conclui-se que a bhalternina apresenta funções tanto em células tumorais como na agregação plaquetária, sendo que o peptídeo BhaltPIP, derivado desta proteína, possui potencial terapêutico para tratamento e diagnóstico de distúrbios hemostáticos.2021-08-1

A New Platelet-Aggregation-Inhibiting Factor Isolated from Bothrops moojeni Snake Venom

This work reports the purification and functional characterization of BmooPAi, a platelet-aggregation-inhibiting factor from Bothrops moojeni snake venom. The toxin was purified by a combination of three chromatographic steps (ion-exchange on DEAE-Sephacel, molecular exclusion on Sephadex G-75, and affinity chromatography on HiTrap™ Heparin HP). BmooPAi was found to be a single-chain protein with an apparent molecular mass of 32 kDa on 14% SDS-PAGE, under reducing conditions. Sequencing of BmooPAi by Edman degradation revealed the amino acid sequence LGPDIVPPNELLEVM. The toxin was devoid of proteolytic, haemorrhagic, defibrinating, or coagulant activities and induced no significant oedema or hyperalgesia. BmooPAi showed a rather specific inhibitory effect on ristocetin-induced platelet aggregation in human platelet-rich plasma, whereas it had little or no effect on platelet aggregation induced by collagen and adenosine diphosphate. The results presented in this work suggest that BmooPAi is a toxin comprised of disintegrin-like and cysteine-rich domains, originating from autolysis/proteolysis of PIII SVMPs from B. moojeni snake venom. This toxin may be of medical interest because it is a platelet aggregation inhibitor, which could potentially be developed as a novel therapeutic agent to prevent and/or treat patients with thrombotic disorders

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

Abstract Background Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO3 2− groups, present in BaltDC, form hydrogen bonds with the PO2 − groups present in the non-lipid portion of the membrane platelets. Conclusions BaltDC may be of medical interest since it was able to inhibit platelet aggregation

BaltDC: purification, characterization and infrared spectroscopy of an antiplatelet DC protein isolated from Bothrops alternatus snake venom

<div><p>Abstract Background: Snake venoms are a complex mixture of proteins, organic and inorganic compounds. Some of these proteins, enzymatic or non-enzymatic ones, are able to interact with platelet receptors, causing hemostatic disorders. The possible therapeutic potential of toxins with antiplatelet properties may arouse interest in the pharmacological areas. The present study aimed to purify and characterize an antiplatelet DC protein from Bothrops alternatus snake venom. Methods: The protein, called BaltDC (DC protein from B. alternatus snake venom), was purified by a combination of ion-exchange chromatography on DEAE-Sephacel column and gel filtration on Sephadex G-75. The molecular mass was estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The amino acid sequence of the N-terminal region was carried out by Edman degradation method. Platelet aggregation assays were performed in human platelet-rich plasma (PRP). Infrared (IR) spectroscopy was used in order to elucidate the interactions between BaltDC and platelet membrane. Results: BaltDC ran as a single protein band on SDS-PAGE and showed apparent molecular mass of 32 kDa under reducing or non-reducing conditions. The N-terminal region of the purified protein revealed the amino acid sequence IISPPVCGNELLEVGEECDCGTPENCQNECCDA, which showed identity with other snake venom metalloproteinases (SVMPs). BaltDC was devoid of proteolytic, hemorrhagic, defibrinating or coagulant activities, but it showed a specific inhibitory effect on platelet aggregation induced by ristocetin and epinephrine in PRP. IR analysis spectra strongly suggests that PO 3 2 − groups, present in BaltDC, form hydrogen bonds with the PO 2 − groups present in the non-lipid portion of the membrane platelets. Conclusions: BaltDC may be of medical interest since it was able to inhibit platelet aggregation.</p></div