RESUMO
A proteína Kint3-4 (PKint3-4), codificadora da angiostatina, é reconhecida por sua potencialidade antiangiogênica. O presente estudo teve como objetivo avaliar o efeito da proteína Kint3-4 no crescimento do tumor sólido de Ehrlich. Para isso, foram analisados a curva de desenvolvimento tumoral, o índice apoptótico e a dosagem de hemoglobina, a fim de se avaliar a angiogênese, em 20 camundongos Swiss fêmeas, inoculadas com o tumor sólido de Ehrlich em seus coxins plantares. Os resultados demonstraram a participação de PKint3-4 na indução à apoptose de células neoplásicas, na diminuição da concentração de hemoglobina e, principalmente, na diminuição do desenvolvimento tumoral. Sugere-se que a ação antitumoral, determinada pela sequência proteica utilizada, possa estar associada ao papel antiangiogênico da angiostatina, que indiretamente aumentaria o índice apoptótico das células neoplásicas, e/ou a uma ação direta da proteína Kint3-4 sobre essas células, estimulando-as a sofrerem apoptose.(AU)
Assuntos
Animais , Feminino , Camundongos , Camundongos/fisiologia , Antígenos de Neoplasias/administração & dosagem , Proteínas , Angiostatinas , Hemoglobinas/análise , Células Tumorais CultivadasRESUMO
A proteína Kint3-4 (PKint3-4), codificadora da angiostatina, é reconhecida por sua potencialidade antiangiogênica. O presente estudo teve como objetivo avaliar o efeito da proteína Kint3-4 no crescimento do tumor sólido de Ehrlich. Para isso, foram analisados a curva de desenvolvimento tumoral, o índice apoptótico e a dosagem de hemoglobina, a fim de se avaliar a angiogênese, em 20 camundongos Swiss fêmeas, inoculadas com o tumor sólido de Ehrlich em seus coxins plantares. Os resultados demonstraram a participação de PKint3-4 na indução à apoptose de células neoplásicas, na diminuição da concentração de hemoglobina e, principalmente, na diminuição do desenvolvimento tumoral. Sugere-se que a ação antitumoral, determinada pela sequência proteica utilizada, possa estar associada ao papel antiangiogênico da angiostatina, que indiretamente aumentaria o índice apoptótico das células neoplásicas, e/ou a uma ação direta da proteína Kint3-4 sobre essas células, estimulando-as a sofrerem apoptose.
RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: The leaves of Hancornia speciosa Gomes are popularly used in Brazil to treat diabetes and hypertension. Cardiovascular diseases are the main cause of death worldwide and their incidences are increasing in Brazilian population. The present study aimed to investigate the hypotensive effect and the mechanism of action of Hancornia speciosa Gomes. METHODS: A fraction of the ethanolic extract of leaves from Hancornia speciosa (SFH) was obtained and standardized by its content on rutin, bornesitol and quinic acid. Systolic blood pressure (SBP) of normotensive mice was measured by tail plethysmography. SFH was given orally and SBP was monitored for 5h. Angiotensin-converting enzyme (ACE) inhibitor activity of SFH (1mg/kg) or captopril (10mg/kg) was measured by colorimetric methods. Serum nitrite levels were measured by spectrophotometry. RESULTS: SFH induced a dose-dependent hypotensive effect in normotensive mice. The serum activity of ACE and the level of angiotensin II were significantly reduced by SFH and by captopril. Administration of SFH induced a significant increase on plasmatic level of nitrites and the systemic inhibition of nitric oxide synthase by L-NAME (20mg/kg) reduced the hypotensive effect of SFH. CONCLUSIONS: The present work demonstrated that Hancornia speciosa has a potent hypotensive effect in normotensive mice. The inhibition of ACE leading to reduction on angiotensin II and increase on NO levels might account for the hypotensive effect. These results support the use of Hancornia speciosa by traditional medicine as antihypertensive.
Assuntos
Inibidores da Enzima Conversora de Angiotensina/farmacologia , Anti-Hipertensivos/farmacologia , Apocynaceae , Pressão Sanguínea/efeitos dos fármacos , Óxido Nítrico/metabolismo , Extratos Vegetais/farmacologia , Administração Oral , Angiotensina II/sangue , Inibidores da Enzima Conversora de Angiotensina/administração & dosagem , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/isolamento & purificação , Animais , Anti-Hipertensivos/administração & dosagem , Anti-Hipertensivos/química , Anti-Hipertensivos/isolamento & purificação , Apocynaceae/química , Captopril/farmacologia , Etanol/química , Masculino , Camundongos , Óxido Nítrico Sintase/antagonistas & inibidores , Óxido Nítrico Sintase/metabolismo , Nitritos/sangue , Extratos Vegetais/administração & dosagem , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta , Plantas Medicinais , Pletismografia , Solventes/química , Fatores de Tempo , Regulação para CimaRESUMO
Disintegrins and disintegrins-like proteins are able to inhibit platelet aggregation and integrin-mediated cell adhesion. The aim of this study was to produce one disintegrin-like cloned from Bothrops leucurus venom gland and to characterize it regarding biological activity. The recombinant protein was purified by one step procedure involving anion-exchange chromatography (DEAE-cellulose) and presented a molecular mass of 10.4 kDa. The purified protein was able to inhibit platelet aggregation induced by collagen (IC50 = 0.65 µM) and to inhibit growth of Ehrlich tumor implanted in mice by more than 50% after 7 days administration of 10 µg/day. No effects were observed upon adenosine 5'-diphosphate (ADP)-and arachidonic acid (AA)-induced platelet aggregation. The recombinant protein was recognized by an antibody specific for jararhagin one metalloproteinase isolated from Bothrops jararaca venom, and therefore it was named leucurogin. Anti-angiogenesis effect of leucurogin was evaluated by the sponge implant model. After 7 days administration leucurogin inhibited, in a dose dependent way, the vascularization process in the sponge. Leucurogin represents a new biotechnological tool to understand biological processes where disintegrins-like are involved and may help to characterize integrins that can be involved in development and progression of malignant cells.
Assuntos
Bothrops/metabolismo , Carcinoma de Ehrlich/tratamento farmacológico , Desintegrinas/farmacologia , Proteínas Recombinantes/farmacologia , Sequência de Aminoácidos , Inibidores da Angiogênese/genética , Inibidores da Angiogênese/isolamento & purificação , Inibidores da Angiogênese/metabolismo , Inibidores da Angiogênese/farmacologia , Animais , Bothrops/genética , Clonagem Molecular , Venenos de Crotalídeos , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/isolamento & purificação , Masculino , Metaloendopeptidases , Camundongos , Dados de Sequência Molecular , Neovascularização Fisiológica/efeitos dos fármacos , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/isolamento & purificação , Inibidores da Agregação Plaquetária/farmacologia , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Veneno de Bothrops jararacaRESUMO
AIM: The present study was undertaken to determine the effects of type 2 diabetes (T2D) on plasma kallikrein activity (PKA) and postexercise hypotension (PEH). METHODS: Ten T2D patients (age: 53.6±1.3 years; body mass index: 30.6±1.0kg/m(2); resting blood glucose: 157.8±40.2mgdL(-1)) and 10 non-diabetic (ND) volunteers (age: 47.5±1.0 years; body mass index: 28.3±0.9kg/m(2); resting blood glucose: 91.2±10.5mgdL(-1)) underwent two experimental sessions, consisting of 20min of rest plus 20min of exercise (EXE) at an intensity corresponding to 90% of their lactate threshold (90LT) and a non-exercise control (CON) session. Blood pressure (BP; Microlife BP 3AC1-1 monitor) and PKA were measured during rest and every 15min for 135min of the postexercise recovery period (RP). RESULTS: During the RP, the ND individuals presented with PEH at 30, 45 and 120min (P<0.05) while, in the T2D patients, PEH was not observed at any time. PKA increased at 15min postexercise in the ND (P<0.05), but not in the T2D patients. CONCLUSION: T2D individuals have a lower PKA response to exercise, which probably suppresses its hypotensive effect, thus reinforcing the possible role of PKA on PEH.
Assuntos
Diabetes Mellitus Tipo 2/sangue , Exercício Físico/fisiologia , Hipotensão/etiologia , Calicreínas/sangue , Pressão Sanguínea/fisiologia , Diabetes Mellitus Tipo 2/complicações , Feminino , Humanos , Cinética , Masculino , Pessoa de Meia-IdadeRESUMO
Endothelium-dependent vasorelaxation in large vessels is mainly attributed to Nomega-nitro-L-arginine methyl ester (L-NAME)-sensitive endothelial nitric oxide (NO) synthase (eNOS)-derived NO production. Endothelium-derived hyperpolarizing factor (EDHF) is the component of endothelium-dependent relaxations that resists full blockade of NO synthases (NOS) and cyclooxygenases. H2O2 has been proposed as an EDHF in resistance vessels. In this work we propose that in mice aorta neuronal (n)NOS-derived H2O2 accounts for a large proportion of endothelium-dependent ACh-induced relaxation. In mice aorta rings, ACh-induced relaxation was inhibited by L-NAME and Nomega-nitro-L-arginine (L-NNA), two nonselective inhibitors of NOS, and attenuated by selective inhibition of nNOS with L-ArgNO2-L-Dbu-NH2 2TFA (L-ArgNO2-L-Dbu) and 1-(2-trifluoromethylphehyl)imidazole (TRIM). The relaxation induced by ACh was associated with enhanced H2O2 production in endothelial cells that was prevented by the addition of L-NAME, L-NNA, L-ArgNO2-L-Dbu, TRIM, and removal of the endothelium. The addition of catalase, an enzyme that degrades H2O2, reduced ACh-dependent relaxation and abolished ACh-induced H2O2 production. RT-PCR experiments showed the presence of mRNA for eNOS and nNOS but not inducible NOS in mice aorta. The constitutive expression of nNOS was confirmed by Western blot analysis in endothelium-containing vessels but not in endothelium-denuded vessels. Immunohistochemistry data confirmed the localization of nNOS in the vascular endothelium. Antisense knockdown of nNOS decreased both ACh-dependent relaxation and ACh-induced H2O2 production. Antisense knockdown of eNOS decreased ACh-induced relaxation but not H2O2 production. Residual relaxation in eNOS knockdown mouse aorta was further inhibited by the selective inhibition of nNOS with L-ArgNO2-L-Dbu. In conclusion, these results show that nNOS is constitutively expressed in the endothelium of mouse aorta and that nNOS-derived H2O2 is a major endothelium-dependent relaxing factor. Hence, in the mouse aorta, the effects of nonselective NOS inhibitors cannot be solely ascribed to NO release and action without considering the coparticipation of H2O2 in mediating vasodilatation.
Assuntos
Aorta Torácica/enzimologia , Fatores Biológicos/metabolismo , Endotélio Vascular/enzimologia , Fatores Relaxantes Dependentes do Endotélio/metabolismo , Peróxido de Hidrogênio/metabolismo , Óxido Nítrico Sintase Tipo I/metabolismo , Vasodilatação , Acetilcolina/farmacologia , Animais , Aorta Torácica/efeitos dos fármacos , Catalase/metabolismo , Relação Dose-Resposta a Droga , Endotélio Vascular/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hidrazinas/farmacologia , Imidazóis/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico/metabolismo , Doadores de Óxido Nítrico/farmacologia , Óxido Nítrico Sintase Tipo I/antagonistas & inibidores , Óxido Nítrico Sintase Tipo I/genética , Óxido Nítrico Sintase Tipo III/metabolismo , Nitroarginina/farmacologia , Nitroprussiato/farmacologia , Oligonucleotídeos Antissenso/metabolismo , RNA Mensageiro/metabolismo , Vasodilatação/efeitos dos fármacos , Vasodilatadores/farmacologiaRESUMO
Two proteins with phospholipase A(2) (PLA(2)) activity were purified to homogeneity from Bothrops leucurus (white-tailed-jararaca) snake venom through three chromatographic steps: Conventional gel filtration on Sephacryl S-200, ion-exchange on Q-Sepharose and reverse phase on Vydac C4 HPLC column. The molecular mass for both enzymes was estimated to be approximately 14 kDa by SDS-PAGE. The N-terminal sequences (48 residues) show that one enzyme presents lysine at position 48 and the other an aspartic acid in this position, and therefore they were designated blK-PLA(2) and blD-PLA(2) respectively. blK-PLA(2) presented negligible levels of PLA(2) activity as compared to that of blD-PLA(2). The PLA(2) activity of both enzymes is Ca(2+)-dependent. blD-PLA(2) did not have any effect upon platelet aggregation induced by arachidonic acid, ADP or collagen, but strongly inhibits coagulation and is able to stimulate Ehrlich tumor growth but not angiogenesis.
Assuntos
Bothrops/metabolismo , Fosfolipases A/metabolismo , Venenos de Serpentes/enzimologia , Sequência de Aminoácidos , Animais , Anticoagulantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Cálcio/metabolismo , Carcinoma de Ehrlich/induzido quimicamente , Carcinoma de Ehrlich/enzimologia , Carcinoma de Ehrlich/patologia , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Cromatografia por Troca Iônica , Ensaio de Imunoadsorção Enzimática , Hemoglobinas/metabolismo , Humanos , Isoenzimas/química , Isoenzimas/isolamento & purificação , Isoenzimas/metabolismo , Células K562 , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Peso Molecular , Fosfolipases A/química , Fosfolipases A/isolamento & purificação , Agregação Plaquetária/efeitos dos fármacos , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Venenos de Serpentes/farmacologiaRESUMO
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein(-1) min(-1)). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.
Assuntos
Metaloendopeptidases/isolamento & purificação , Pichia/enzimologia , Cromatografia por Troca Iônica , Proteínas Ligadas por GPI , Humanos , Metaloendopeptidases/genética , Pichia/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Carboxypeptidase M (CPM) is an extracellular glycosylphosphatidyl-inositol-anchored membrane glycoprotein, which removes the C-terminal basic residues, lysine and arginine, from peptides and proteins at neutral pH. CPM plays an important role in the control of peptide hormones and growth factor activity on the cell surface. The present study was carried out to clone and express human CPM in the yeast Pichia pastoris in order to evaluate the importance of this enzyme in physiological and pathological processes. The cDNA for the enzyme was amplified from total placental RNA by RT-PCR and cloned in the vector pPIC9, which uses the methanol oxidase promoter and drives the expression of high levels of heterologous proteins in P. pastoris. The cpm gene, after cloning and transfection, was integrated into the yeast genome, which produced the active protein. The recombinant protein was secreted into the medium and the enzymatic activity was measured using the fluorescent substrate dansyl-Ala-Arg. The enzyme was purified by a two-step protocol including gel filtration and ion-exchange chromatography, resulting in a 1753-fold purified active protein (16474 RFU mg protein-1 min-1). This purification protocol permitted us to obtain 410 mg of the purified protein per liter of fermentation medium. SDS-PAGE showed that recombinant CPM migrated as a single band with a molecular mass similar to that of native placental enzyme (62 kDa), suggesting that the expression of a glycosylated protein had occurred. These results demonstrate for the first time the establishment of a method using P. pastoris to express human CPM necessary to the development of specific antibodies and antagonists, and the analysis of the involvement of this peptidase in different physiological and pathological processes.
Assuntos
Humanos , Metaloendopeptidases/isolamento & purificação , Pichia/enzimologia , Cromatografia por Troca Iônica , Metaloendopeptidases/genética , Pichia/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
In this study, a single-step duplex polymerase chain reaction procedure was developed for rapid, specific and sensitive identification of Entamoeba histolytica and for its diagnostic differentiation from E. dispar. Specific oligonucleotide primers were combined for the amplification of a cysteine proteinase 5 gene target sequence of 242 bp, present only in E. histolytica. Additionally, another oligonucleotide primer pair for both the E. histolytica and E. dispar actin gene target of 300 bp was designed to amplify only from amoebae DNA. The PCR developed was specific and efficiently identified and differentiated these parasites from each other in either cultured parasites or from stool material.