RESUMO
A series of 10 1,4-bis(3-aminopropyl)piperazine compounds was found to display antiplasmodial activity with 50% growth inhibition between 30 and 250 nM, on three Plasmodium falciparum strains differently sensitive to chloroquine. By affinity chromatography using one of these compounds, a 52-kDa protein was isolated from P. falciparum, microsequenced and cloned. It corresponded to a single copy gene encoding a 453 amino acid protein displaying the typical features of protein disulfide isomerases, a thiol metabolizing enzyme belonging to the thiol: disulfide oxidoreductase superfamily, which was not previously described in malarial species.
Assuntos
Antiprotozoários , Plasmodium falciparum/enzimologia , Isomerases de Dissulfetos de Proteínas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Cromatografia de Afinidade , Cromatografia em Gel , Clonagem Molecular , Colômbia , Humanos , Dados de Sequência Molecular , Peso Molecular , Plasmodium falciparum/genética , Plasmodium falciparum/isolamento & purificação , Isomerases de Dissulfetos de Proteínas/isolamento & purificação , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Tanzânia , TailândiaRESUMO
Hybrid molecules were constructed with either polyclonal antibodies against Trypanosoma cruzi antigens or monoclonal antibody against Trypanosoma brucei brucei low-density lipoprotein (LDL)-receptor conjugated with chlorambucil. Physical-chemical analysis of the hybrid molecule showed four chlorambucil coupling sites in each IgG and a binding constant in the order of 10(4). Maintenance of IgG integrity was indicated by its circular dichroism pattern. Biologic activity of the hybrid molecule was shown by its inhibitory effect on the mobility and proliferation of the parasite. An IgG-chlorambucil conjugate, produced with monoclonal antibody anti-T. b. brucei LDL-receptor, led to the immobilization of the T. cruzi forms, albeit at a much lesser level than that obtained with a mouse polyclonal anti-T. cruzi IgG linked to the drug. Targeting experimental T. cruzi infection with a specific IgG-chlorambucil conjugate resulted in consistent reduction of parasitemia and mortality, thus showing its potential usefulness in controlling the acute form of the disease.
Assuntos
Doença de Chagas/terapia , Clorambucila/uso terapêutico , Imunoconjugados/uso terapêutico , Imunoglobulina G/uso terapêutico , Animais , Membrana Celular/metabolismo , Doença de Chagas/parasitologia , Doença de Chagas/patologia , Imunoconjugados/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Movimento/fisiologia , Parasitemia/terapia , Coelhos , Análise de Sobrevida , Trypanosoma cruzi/crescimento & desenvolvimento , Trypanosoma cruzi/metabolismo , Trypanosoma cruzi/fisiologiaRESUMO
Specific interactions between parasites and extracellular matrix components are an important mechanism in the dissemination of Chagas' disease. Binding of the extracellular matrix proteins to Trypanosoma cruzi receptors has been described as a significant step in this phenomenon. In this study, a specific proteinase activity was identified in cell-free extracts of amastigote, trypomastigote and epimastigote forms of T. cruzi using the collagenase fluorogenic substrate N-Suc-Gly-Pro-Leu-Gly-Pro-7-amido-4-methylcoumarin. Isolation of this activity was achieved by a four-step FPLC procedure. Optimal enzyme activity was found to occur at pH 8.0 and was associated with a single T. cruzi 80 kDa protein (Tc 80 proteinase) on SDS/PAGE under reducing conditions. An internal peptide sequence of Tc 80 proteinase was obtained (AGDNYTPPE), and no similarity was found to previously described proteinases of T. cruzi. This enzyme activity is strongly inhibited by HgCl2, tosyl-lysylchloromethane ('TLCK') p-chloromercuribenzoate and benzyloxycarbonyl-Phe-Ala-diazomethane. The purified enzyme was able to hydrolyse purified human [14C]collagen types I and IV at neutral pH, but not 14C-labelled BSA, rat laminin, rabbit IgG or small proteins such as insulin or cytochrome c. In addition, Tc 80 proteinase activity was found to be secreted by T. cruzi forms infective to mammalian cells. Furthermore we demonstrated that purified Tc 80 proteinase mediates native collagen type I hydrolysis in rat mesentery. This feature is compared with that of Clostridium histolyticum collagenase. These findings suggest that Tc 80 proteinase may facilitate T. cruzi host-cell infection by degrading the collagens of the extracellular matrix and could represent a good target for Chagas' disease chemotherapy.
Assuntos
Colágeno/metabolismo , Endopeptidases/metabolismo , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Cromatografia por Troca Iônica , Colágeno/química , Endopeptidases/química , Endopeptidases/isolamento & purificação , Humanos , Cinética , Laminina/metabolismo , Peso Molecular , Fragmentos de Peptídeos/química , Inibidores de Proteases/farmacologia , Coelhos , Ratos , Especificidade por SubstratoRESUMO
Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.
Assuntos
Endopeptidases/metabolismo , Plasmodium falciparum/enzimologia , Animais , Eritrócitos/parasitologia , Genes de Protozoários/genética , Plasmodium falciparum/genética , Coelhos , Espectrina/administração & dosagemRESUMO
Numerous proteinase activities have been shown to be essential for the survival of Plasmodium falciparum. One approach to antimalarial chemotherapy, would be to block specifically one or several of these activities, by using compounds structurally analogous to the substrates of these proteinases. Such a strategy requires a detailed knowledge of the active site of the proteinase, in order to identify the best substrate for the proteinase. Aiming at developing such a strategy, two proteinases previously identified in our laboratory, were chosen for further characterization of their molecular structure and properties: the merozoite proteinase for erythrocytic invasion (MPEI), involved in the erythrocyte invasion by the merozoites, and the Pf37 proteinase, which hydrolyses human spectrin in vitro.
Assuntos
Animais , Coelhos , Endopeptidases , Plasmodium falciparum , Eritrócitos , Genes de Protozoários , Plasmodium falciparum , EspectrinaRESUMO
A new alkaline proteinase activity was identified in cell-free extracts of Trypanosoma cruzi epimastigotes on the basis of its ability to hydrolyze the fluorogenic substrate N-Z-Gly-Gly-Arg-AMC. The optimal activity was at pH 8.0. After a three step-chromatography procedure using two anionic columns (DEAE-Sepharose and Mono Q) and a chromatofocusing column (Mono P), the proteolytic activity was associated with a single 120 kDa protein and was called Tc 120 proteinase. The molecular mass of the proteinase was confirmed by direct visualization of the proteolytic activity using a fluorometric assay on SDS-PAGE. The Tc 120 proteinase which also cleaves N-Z-Arg-AMC, N-Z-Phe-Arg-AMC and N-glutaryl-Gly-Arg-AMC substrates, is a cysteine-type proteinase with an unusual low sensitivity to E-64.
Assuntos
Cisteína Endopeptidases/isolamento & purificação , Trypanosoma cruzi/enzimologia , Sequência de Aminoácidos , Animais , Cisteína Endopeptidases/química , Cisteína Endopeptidases/metabolismo , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Peso Molecular , Peptídeos/metabolismo , Inibidores de Proteases/farmacologia , Proteínas de Protozoários/química , Proteínas de Protozoários/isolamento & purificação , Proteínas de Protozoários/metabolismo , Especificidade por SubstratoRESUMO
A vaccine strategy against Babesia divergens bovine babesiosis was successfully developed after perfecting of an efficient in vitro culture. Crude supernatants and purified fractions were able to induce a vaccine protection in gerbils against B. divergens infection. More, supernatants induced an effective vaccine protection in cattle. The role of B. divergens exoantigens of 17, 37, 46, 70 and 90 kDa in the development of the immune response was clearly demonstrated in gerbils, cattle, and man.
Assuntos
Babesia/imunologia , Babesiose/prevenção & controle , Doenças dos Bovinos/prevenção & controle , Vacinas Protozoárias , Animais , Antígenos de Protozoários/imunologia , Babesiose/imunologia , Bovinos , Doenças dos Bovinos/imunologia , Gerbillinae , Parasitologia/métodos , VacinaçãoAssuntos
Babesia/crescimento & desenvolvimento , Meios de Cultura Livres de Soro/farmacologia , Lipoproteínas HDL/farmacologia , Parasitologia/métodos , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Babesia/efeitos dos fármacos , Babesia/metabolismo , Eritrócitos/parasitologia , Humanos , Metabolismo dos Lipídeos , Lipoproteínas LDL/farmacologia , Lipoproteínas VLDL/farmacologia , Fosfatidilcolinas/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismoRESUMO
A vaccine strategy against Babesia divergens bovine babesiosis was sucessfully developed after perfecting of an efficient in vitro culture. Crude supernatants and purified fractions were able to induce a vaccine protection in gerbils against B. divergens infection. More, supernatants induced an effective vaccine protection in cattle. The role of B. divergens exoantigens of 17, 37, 46, 70 and 90 kDa in the development of the immune response was cleary demonstrated in gerbils, cattle, and man.