RESUMO
Paraquat has been shown to be a highly toxic compound for humans and animals, and many cases of acute poisoning and death have been reported over the past few decades. The present study was undertaken to evaluate comprehensively herbicides (Paraquat) and some plant extracts to biochemical aspects of Lymnaea natalensis snails. It was found that the exposure of L. natalensis to Paraquat and plant extracts led to a significant reduction in the infectivity of Fasciola gigantica miracidia to the snail. The glucose level in hemolymph of exposed snails was elevated, while the glycogen showed a decrease in soft tissues when compared with the control group. In addition, the activity level of some enzymes representing glycolytic enzymes as hexokinase (HK), pyruvate kinase (PK), phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glucose phosphate isomerase (GPI) in snail's tissues were reduced in response to the treatment. It was concluded that the pollution of the aquatic environment by herbicide would adversely affect the metabolism of the L. natalensis snails. Snails treated with Agave attenuate, Ammi visnaga, and Canna iridiflora plant had less toxic effect compared to snails treated with Paraquat.
Assuntos
Herbicidas/toxicidade , Lymnaea/efeitos dos fármacos , Paraquat/toxicidade , Extratos Vegetais/toxicidade , Animais , Fasciola/crescimento & desenvolvimento , Glucose-6-Fosfato Isomerase/metabolismo , Hexoquinase/metabolismo , L-Lactato Desidrogenase/metabolismo , Dose Letal Mediana , Lymnaea/metabolismo , Lymnaea/parasitologia , Fosfofrutoquinases/metabolismo , Compostos Fitoquímicos/toxicidade , Piruvato Quinase/metabolismoRESUMO
A full-length complementary DNA (cDNA) encoding Cu/Zn-superoxide dismutase was isolated from Fasciola gigantica that on nucleotide sequencing showed a close homology (98.9%) with Cu/Zn-superoxide dismutase (SOD) of the temperate liver fluke, F. hepatica. Expression of the gene was found in all the three developmental stages of the parasite viz. adult, newly excysted juvenile and metacercaria at transcriptional level by reverse transcription-polymerase chain reaction (RT-PCR) and at the protein level by Western blotting. F. gigantica Cu/Zn-SOD cDNA was cloned and expressed in Escherichia coli. Enzyme activity of the recombinant protein was determined by nitroblue tetrazolium (NBT)-polyacrylamide gel electrophoresis (PAGE) and this activity was inactivated by hydrogen peroxide but not by sodium azide, indicating that the recombinant protein is Cu/Zn-SOD. The enzyme activity was relatively stable at a broad pH range of pH 4.0-10.0. Native Cu/Zn-superoxide dismutase protein was detected in the somatic extract and excretory-secretory products of the adult F. gigantica by Western blotting. NBT-PAGE showed a single Cu/Zn-SOD present in the somatic extract while three SODs are released ex vivo by the adult parasite. The recombinant superoxide dismutase did not react with the serum from buffaloes infected with F. gigantica. The role of this enzyme in defense by the parasite against the host reactive oxygen species is discussed.
Assuntos
DNA Complementar/isolamento & purificação , Fasciola/enzimologia , Regulação Enzimológica da Expressão Gênica , Superóxido Dismutase/isolamento & purificação , Matadouros , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Búfalos/parasitologia , DNA Complementar/química , DNA de Helmintos/química , DNA de Helmintos/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Fasciola/genética , Fasciola/crescimento & desenvolvimento , Fasciola hepatica/enzimologia , Fasciola hepatica/genética , Fasciolíase/parasitologia , Fasciolíase/veterinária , Concentração de Íons de Hidrogênio , Indicadores e Reagentes , Estágios do Ciclo de Vida/genética , Nitroazul de Tetrazólio , RNA de Helmintos/genética , RNA de Helmintos/isolamento & purificação , Coelhos , Proteínas Recombinantes/química , Análise de Sequência de DNA , Superóxido Dismutase/química , Superóxido Dismutase/genéticaRESUMO
A full-length cDNA encoding the Fasciola gigantica calcium-binding protein 1 (FgCaBP1) was cloned from an adult stage cDNA expression library in an immunoscreen using rabbit immune serum against the parasite's excretion/secretion antigens. The deduced amino acid sequence showed 96.3% identity to Fh22CBP of Fasciola hepatica. During development in the mammalian host FgCaBP1 RNA was detected in metacercariae, juveniles and adults and was exclusively localized to the tegumental cell bodies. Immune serum of a rabbit infected with F. gigantica detected recombinant FgCaBP1 starting from the sixth week of infection. Immune sera of mice infected with Schistosoma mansoni and Schistosoma mekongi cross-reacted with recombinant FgCaBP1 in immunoblots. Recombinant FgCaBP1 showed calcium and magnesium-binding activity by a mobility shift during non-denaturing PAGE in the presence of Ca2+ or Mg2+, respectively. A polyclonal mouse anti-rFgCaBP1 antiserum detected the native protein as a major component of the parasite's tegumental antigens in immunoblots and as a strictly tegumental antigen in tissue cross-sections of adult and juvenile parasites. Comparative sequence analysis of homologs from Fasciola and Schistosoma present in the GenBank database revealed sequence signatures specific to these trematode proteins and thereby indicates their origin from a single ancestor. FgCaBP1 contains two adjacent, N-terminal located EF-hands and a C-terminal located domain similar to dynein light chain type 1. Independent structure predictions of the two domains suggest that they will fold according to the already determined structures of the EF-hand motif and the dynein light chain type 1 proteins.