RESUMO
Bauninia forficata is trivially known as cow paw, and popularly used in Brazil for treatment of diabetes mellitus. Denominated baupain a cysteine proteinase was purified from B. forficata leaves. In this study, we investigated the baupain effect on aggregation of isolated human platelets in vitro and the results show that baupain hinders thrombin - but not ADP- and collagen- induced platelet aggregation. With synthetic quenched-fluorescent peptides, the kinetics of the cleavage site of human proteinase-activated receptor 1 / 2 / 3 and 4 [PAR-1 / 2 / 3 and 4] by baupain was determined. In conclusion, similar to bromelain and papain, baupain hinders human platelets aggregation, probably through an unspecific cleavage in the Phe-Leu bond of PAR1.
Assuntos
Cisteína Proteases/química , Cisteína Proteases/metabolismo , Fibrinolíticos/química , Folhas de Planta/enzimologia , Agregação Plaquetária/efeitos dos fármacos , Contagem de Células , Cisteína Proteases/farmacologia , Dipeptídeos/química , Fibrinolíticos/metabolismo , Fibrinolíticos/farmacologia , Corantes Fluorescentes , Humanos , Cinética , Receptores Ativados por Proteinase/química , Receptores Ativados por Proteinase/metabolismo , Trombina/metabolismoRESUMO
Immunization with peptide P10, derived from gp43, and chemotherapy were used together in an attempt to improve treatment of paracoccidioidomycosis and prevent relapses. The combined treatment showed an additive protective effect when administered at 48 h or 30 days after intratracheal challenge. Its use is recommended to improve regular chemotherapy and reduce the duration of treatment.
Assuntos
Antifúngicos/uso terapêutico , Proteínas Fúngicas/imunologia , Paracoccidioides/imunologia , Paracoccidioides/patogenicidade , Paracoccidioidomicose/tratamento farmacológico , Paracoccidioidomicose/terapia , Peptídeos/imunologia , Adjuvantes Imunológicos/uso terapêutico , Animais , Terapia Combinada , Proteínas Fúngicas/administração & dosagem , Vacinas Fúngicas/administração & dosagem , Vacinas Fúngicas/imunologia , Humanos , Imunização , Intubação Intratraqueal , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Paracoccidioides/isolamento & purificação , Peptídeos/administração & dosagem , VirulênciaRESUMO
Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambdaexc = 320 nm and lambdaem = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway
Assuntos
Animais , Apoptose , Caspases , Músculo Esquelético , Deficiência de Vitamina E , Galinhas , Fragmentação do DNA , Ativação Enzimática , Músculo EsqueléticoRESUMO
Apoptosis and necrosis are two distinct forms of cell death that can occur in response to different agents and stress conditions. In order to verify if the oxidative stress induced by dietary selenium and vitamin E deficiencies can lead muscle cells to apoptosis, one-day-old chicks were reared using diets differing in their vitamin E (0 or 10 IU/kg) and selenium (0 or 0.15 ppm) supplementation. Chick skeletal muscle tissue was obtained from 28-day-old animals and used to verify apoptosis occurrence based on caspase activity detection and DNA fragmentation. Antioxidant deficiency significantly increased caspase-like activity assessed by the hydrolysis of fluorogenic peptide substrates (Abz-peptidyl-EDDnp) at lambda exc = 320 nm and lambda em = 420 nm. Proteolytic activation was not accompanied by typical internucleosomal DNA fragmentation detected by field inversion gel electrophoresis. Although the general caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp(O-Me) fluoromethyl ketone (Z-VAD-fmk) (0 to 80 muM) did not block caspase-like activity when preincubated for 30 min with muscle homogenates, the hydrolyzed substrates presented the same cleavage profile in HPLC (at the aspartic acid residue) when incubated with the purified recombinant enzyme caspase-3. These data indicate that oxidative stress causes caspase-like activation in muscle cells and suggest that cell death associated with exudative diathesis (dietary deficiency of selenium and vitamin E) can follow the apoptotic pathway.
Assuntos
Apoptose , Caspases/metabolismo , Músculo Esquelético/citologia , Selênio/deficiência , Deficiência de Vitamina E/enzimologia , Animais , Apoptose/genética , Inibidores de Caspase , Galinhas , Fragmentação do DNA , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Músculo Esquelético/enzimologiaRESUMO
Trypsin inhibitors were purified from a saline extract of Bauhinia bauhinioides seeds by ion-exchange column chromatography on DEAE-Sephadex, gel filtration on Superose 12 column, Mono Q ion-exchange chromatography or, alternatively, by affinity chromatography on trypsin-Sepharose. Both B. bauhinioides isolated inhibitors, BbTI-I and BbTI-II, inhibit trypsin being the dissociation constant 0.6 and 0.36 nM, respectively. BbTI-II only inhibits porcine pancreatic kallikrein hydrolysis of H-Pro-Phe-Arg-AMC (Ki 2.0 nM); the bradykinin-containing sequence LGMISLMKRPPGFSPFRSSRI-NH2 and the two kininogen related flanking quenched substrates Abz-MISLMKRP-EDDnp (Ki 2.0 nM) and Abz-FRSSRQ-EDDnp (Ki 2.5 nM).
Assuntos
Fabaceae/química , Cininogênios/antagonistas & inibidores , Cininogênios/metabolismo , Proteínas de Plantas/química , Plantas Medicinais , Inibidores de Serina Proteinase/química , Calicreínas Teciduais/antagonistas & inibidores , Sequência de Aminoácidos , Hidrólise/efeitos dos fármacos , Dados de Sequência Molecular , Proteínas de Plantas/isolamento & purificação , Sementes/química , Homologia de Sequência de Aminoácidos , Inibidores de Serina Proteinase/isolamento & purificação , Especificidade por SubstratoRESUMO
We report an improved procedure for the synthesis of fully protected aminoacyl 7-amino-4-methylcoumarin amide (MCA) employing the phosphorous oxychloride anhydride method. Seven Boc-X-MCA [where X = Arg(NG Tos), Cys(S-Bzl), Thr(O-Bzl), Ser(O-Bzl), Phe, Leu and Gly] and Z-Tyr(O-Me) were synthesized using this procedure, with yields ranging from 50% to 75%. These aminoacyl-MCA derivatives were employed for the synthesis of epsilon-NH2-caproyl-Leu-X-MCA, a fluorescent peptide series, which were assayed as papain substrates. All of them were completely hydrolyzed by papain, indicating that all of the Boc-X-MCA derivatives obtained were practically free of racemization. Since epsilon-NH2-Caproyl-Leu-(S-Bzl)Cys-MCA is very susceptible to hydrolysis by papain, quite resistant to hydrolysis by chymotrypsin and not hydrolyzed by trypsin, it is recommended for assays of thiol-proteinases in which specificity is required.