RESUMO
Terminalia actinophylla has been used for anti-diarrheic and haemostatic purposes in Brazil. The fly spot data obtained after exposure of marker-heterozygous Drosophila melanogaster larvae to T. actinophylla ethanolic extract (TAE) in the standard (ST) and high bioactivation (HB) crosses revealed that TAE did not induce any statistically significant increment in any spot categories. Differences between the two crosses are related to cytochrome P450 (CYPs) levels. In this sense, our data pointed out the absence of TAE-direct and indirect mutagenic and recombinagenic action in the Somatic Mutation and Recombination Test (SMART). When the anti-genotoxicity of TAE was analyzed, neither mitomycin C (MMC) nor ethylmethanesulfonate (EMS) genotoxicity was modified by the post-exposure to TAE, which suggests that TAE has no effect on the mechanisms involved in the processing of the lesions induced by both genotoxins. In the mwh/flr(3) genotype, co-treatment with TAE may lead to a significant protection against the genotoxicity of MMC and a weak but significant effect in the toxic genetic action of EMS. The overall findings suggested that the favorable modulations by TAE could be, at least in part, due to its antioxidative potential.
Assuntos
Antimutagênicos/farmacologia , Drosophila melanogaster/efeitos dos fármacos , Testes de Mutagenicidade/métodos , Extratos Vegetais/farmacologia , Terminalia/química , Animais , Brasil , Cruzamentos Genéticos , Sistema Enzimático do Citocromo P-450/genética , Drosophila melanogaster/genética , Etanol , Feminino , Larva/efeitos dos fármacos , Larva/genética , Masculino , Mitomicina/toxicidade , Asas de Animais/efeitos dos fármacosRESUMO
A keratinolytic Xanthomonas maltophilia strain (POA-1), cultured on feather meal broth, using keratin as its sole source of carbon and nitrogen, secretes several extracellular peptidases. The major serine peptidase was purified to homogeneity by a five-step procedure. Its purity was evaluated by capillary zone electrophoresis. This enzyme has a molecular mass of 36 kDa, an optimum pH of 9.0, and an optimum temperature of 60 degrees C. The inhibitory profile using protease inhibitors shows that this enzyme is a serine endopeptidase. Besides keratin, the enzyme is active upon the substrates azokeratin, azocasein, and the following fluorogenic peptide substrates: Abz-Leu-Gly-Met-Ile-Ser-Leu-Met-Lys-Arg-Pro-Gln-EDDnp, Abz-Lys-Leu-Cys(SBzl)-Gly-Pro-Lys-Gln-EDDnp, and Abz-Lys-Pro-Cys(SBzl)-Phe-Ser-Lys-Gln-EDDnp.
Assuntos
Plumas/metabolismo , Queratinas/metabolismo , Serina Endopeptidases/isolamento & purificação , Serina Endopeptidases/metabolismo , Stenotrophomonas maltophilia/enzimologia , Animais , Cromatografia em Agarose , Peptídeo Hidrolases , Inibidores de Proteases , Stenotrophomonas maltophilia/metabolismoRESUMO
We describe a nested polymerase chain reaction (PCR) for the detection of Babesia equi in equine infected erythrocytes using oligonucleotides designed on the published sequence of a B. equi merozoite antigen gene (ema-1). A 102bp DNA fragment is specifically amplified from B. equi but not from Babesia caballi, Babesia bovis or Babesia bigemina DNA. In a mock infection we were able to detect down to six infected cells in 10(8) equine erythrocytes or to detect the parasite in blood with an equivalent parasitemia of 0.000006%. Furthermore, gene polymorphism was found by performing a PCR-RFLP (PCR combined with restriction fragment length polymorphism) on both the 102bp and the entire ema-1 gene DNA amplified from two B. equi isolates, Florida (USA) and Pelotas (Southern Brazil) isolates. The polymorphism was confirmed by sequencing the entire ema-1 gene from the B. equi isolate Pelotas. Our results demonstrate that the ema-1 based nested PCR is a valuable technique for routine detection of B. equi in chronically infected horses. It may be used for epidemiological and phylogenetic studies of the parasite as well as monitoring B. equi infected horses in chemotherapeutic trials.
Assuntos
Antígenos de Protozoários , Babesia/isolamento & purificação , Babesiose/veterinária , DNA de Protozoário/análise , Doenças dos Cavalos/diagnóstico , Sequência de Aminoácidos , Animais , Babesia/genética , Babesiose/diagnóstico , Sequência de Bases , Doença Crônica , Eritrócitos/parasitologia , Amplificação de Genes , Cavalos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase/veterinária , Polimorfismo de Fragmento de Restrição , Proteínas de Protozoários/genética , Mapeamento por Restrição/veterinária , Sensibilidade e Especificidade , Análise de Sequência de DNA/veterináriaRESUMO
A cysteine proteinase gene homologous to cathepsins L genes was isolated from a B. microplus cDNA library. The precursor protein deduced from the nucleotide sequence contains 332 amino acid residues consisting of a signal sequence (pre-region), a pro-region and a mature proteinase. The DNA fragment coding for the proenzyme was cloned and expressed using the E. coli expression vector pMAL-p. The recombinant protein (MBP+PROCP) once activated is able to hydrolyze synthetic substrates as well as protein substrates like hemoglobin, vitellin and gelatin. Its optimal enzymatic activity on both fluorogenic and protein substrates was found to occur at an acidic pH. Expression of the proteinase gene was tested by RT-PCR with tick larvae RNA. Detection of amplified sequences indicates that the gene is expressed at this stage of the tick life cycle and the molecule is therefore potentially a target for chemotherapy or an immunogen in a vaccine.
Assuntos
Catepsinas/genética , Clonagem Molecular , Cisteína Endopeptidases/genética , Endopeptidases , Carrapatos/genética , Animais , Sequência de Bases , Catepsina L , Cisteína Endopeptidases/biossíntese , Cisteína Endopeptidases/metabolismo , DNA Complementar/genética , Precursores Enzimáticos/genética , Escherichia coli/genética , Amplificação de Genes , Vetores Genéticos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
DNA from seven isolates of the cattle tick Boophilus microplus was analyzed by restriction fragment length polymorphism (RFLP). Three different cDNA clones, named P-9, P-25 and CP-12, isolated from a B. microplus cDNA library, were used as DNA probes. DNA sequences of P-9 have high similarity to ribosomal genes, whereas P-25 does not show significant homology with known sequences within databases. CP-12 is a cDNA clone encoding a cysteine endopeptidase gene. A limited degree of polymorphism was detected with P-9 and P-25, while CP-12 showed a different pattern of bands for each tick isolate. These findings suggest the existence of a complex genotypic diversity of the tick B. microplus population in endemic regions.