[show abstract][hide abstract] ABSTRACT: It is not known whether precursors of methotrexate, such as 2, 4-diamino-6-hydroxymethyl-pteridine (DAP) and 2, 4-diamino-N10-methyl-pteroic acid (DAMPA), could target the dihydrofolate reductase-thymidylate synthase (DHFR-TS) enzyme of Babesia and inhibit the parasite growth. Therefore, we have determined whether DAP and DAMPA as well as other chemically related compounds like pteroic acid (PA) and N10-Triflouropteroic acid (N10TFPA) could target the DHFR-TS enzyme of B. gibsoni and inhibit its growth. DAMPA was a more-potent inhibitor of the B. gibsoni growth in vitro (50% inhibition concentration [IC50] = 2.4 ± 0.20 μM) [mean ± standard error of the mean] than DAP (IC50 = 78 ± 15 μM). Moreover, DAMPA potently inhibited enzymatic activity of recombinant DHFR-TS of B. gibsoni (IC50 = 2.6 ± 0.15 μM) than DAP (IC50 > 100 μM). In contrast, PA and N10-TFPA did not inhibit the activity of the recombinant enzyme and growth of B. gibsoni. The inhibition of the recombinant enzyme activity by DAMPA mirrored with inhibition of the parasite growth indicating that the purified recombinant enzyme could be used for preliminary screening of some antifolate precursors. Therefore, both DAP and DAMPA inhibit growth of B. gibsoni by targeting the DHFR-TS enzyme of the parasite.
J. Protozool. Res. 20, 70-81 (2010). 01/2010; 20:70-81.
[show abstract][hide abstract] ABSTRACT: We cloned and expressed a novel gene encoding a 32-kDa merozoite protein of Babesia gibsoni (BgP32). The length of nucleotide sequence of the cDNA was 1464 bp with an open reading frame of 969 bp. The truncated recombinant BgP32 (rBgP32) without a signal peptide and C-terminal hydrophobic sequence was expressed in Escherichia coli as a soluble glutathione-S-transferase (GST) fusion protein. Western blotting demonstrated that the native protein was 32-kDa, consistent with molecular weight of the predicted mature polypeptide. Enzyme-linked immunosorbent assay (ELISA) using rBgP32 detected specific antibodies from 8 days to 541 days post-infection in the sequential sera from a dog experimentally infected with B. gibsoni. Moreover, the antigen did not cross-react with B. canis subspecies and closely related protozoan parasites, indicating that rBgP32 is a specific diagnostic antigen. Analysis of 47 sera taken from dogs with anaemic signs revealed that rBgP32 detected a higher proportion of B. gibsoni seropositive samples (77%) than its previously identified rBgP50 (68%) homologue. These results indicate that the BgP32 is a novel immunodominant antigen of B. gibsoni, and rBgP32 might be useful for diagnosis of B. gibsoni infection.
[show abstract][hide abstract] ABSTRACT: Apoptosis has been found to help in the defence against pathogens. Infection with the obligate intracellular parasite Toxoplasma gondii is known to trigger host-cell apoptosis. When using a T. gondii-infected macrophage cell line, J774A.1, treatment with IFN-gamma significantly enhanced apoptosis in noninfected bystander cells while parasitized cells became relatively resistant. Infection and IFN-gamma treatment activated the expression of inducible nitric oxide synthase (iNOS), and the production of nitric oxide (NO) and treatment of cells with an iNOS inhibitor, N(G)-monomethlyl-L-arginine acetate (L-NMMA) reduced the apoptosis frequency. However, the reversal was only partial suggesting that not only NO, but also other, as of yet, unknown factors are induced. Finally, we studied the effect in vivo by infecting mice with either a virulent or an avirulent strain. Challenge with the virulent strain lead to a higher parasite burden, induced host-cell apoptosis in peritoneal cells, and produced higher levels of IFN-gamma and NO. Moreover, treatment of mice with a NO synthase inhibitor, aminoguanidine, partially inhibited the host-cell apoptosis induced by the parasite infection. Altogether, our findings indicate that apoptosis in bystander host cells is due to the secretion of NO and other soluble factors released by parasite-infected cells.
[show abstract][hide abstract] ABSTRACT: Murine monoclonal antibodies (mAbs) against Neospora caninum tachyzoites were produced to identify the cross-reactive antigens between N. caninum and Toxoplasma gondii. Ten mAbs recognizing cross-reactive antigens of both parasites were obtained and tentatively classified into 6 different groups based on their reactivity patterns in an indirect fluorescent antibody test and Western blot analysis. Three mAbs in group 1 recognized antigens located on the surface of parasites with molecular masses ranging from 28 to 76 kDa; one mAb in group 2 recognized antigens located on interior organelles of parasites with a molecular mass of 50 kDa; one mAb in group 3 recognized antigens located on interior organelles of parasites with molecular masses of 35 kDa and 14 kDa; three mAbs in group 4 recognized antigens located on interior organelles with a molecular mass of 64 kDa; one mAb in group 5 recognized antigens located on the surface of parasites with an unknown molecular mass; one mAb in group 6 recognized antigens located on the apical end of parasites with an unknown molecular mass. The mAbs in groups 1, 2, 3, and 5 showed inhibitory effects on the growth of the two parasites in vitro in a dose-dependent manner. A cDNA expression library prepared from N. caninum tachyzoite mRNA was immunoscreened with the mAb panel. Three kinds of proteins, protein disulfide isomerase (PDI), heat-shock protein 70 (HSP70), and ribosomal protein 1 (RP1), were identified as cross-reactive antigens recognized by mAbs in groups 2, 3, and 4, respectively. Some of the proteins could be useful in developing vaccines or drugs for controlling the diseases caused by the two parasites.
[show abstract][hide abstract] ABSTRACT: Cats are pivotal in the transmission of Toxoplasma gondii. To develop a sensitive and specific serodiagnostic method for feline toxoplasmosis, surface antigen 2 (SAG2) of T. gondii was expressed in Escherichia coli and its diagnostic potential evaluated in an enzyme-linked immunosorbent assay (ELISA). The ELISA with recombinant SAG2 (rSAG2) was able to differentiate very clearly between sera from cats experimentally infected with T. gondii and sera from normal cats. Serum samples collected from domestic cats in Japan were investigated by the ELISA, and the results were compared with those of a commercially available latex agglutination test (LAT) kit. Of the 192 samples screened, 42 (21.9%) were positive by ELISA. Among the 42 ELISA-positive samples, 39 were positive by LAT. There was a significant correlation between ELISA and LAT titers. All the 150 ELISA-negative samples were negative by LAT. These results indicate that the ELISA with rSAG2 expressed in E. coli should be a useful method for detection of T. gondii infection in cats.
Journal of Parasitology 09/2002; 88(4):804-7. · 1.32 Impact Factor
[show abstract][hide abstract] ABSTRACT: A detailed comparison of the accessory cell activities was carried out among murine peritoneal cavity macrophages (PEC-Mphi), peritonea] cavity macrophages stimulated with granulocyte-macrophage colony stimulating factor (GM-CSF) plus interleukin 4 (IL-4), the most popular cytokine combination widely used to generate dendritic cells (DC) and peritoneal cavity macrophage-derived DC (PEC-DC) using a two-way mixed lymphocyte reaction (MLR). All the cell types used efficiently induced statistically significant naïve T cell proliferation at all culture time points and responder:stimulator ratios used. However, marked differences were noted in the magnitude of the proliferative responses. These variations may be attributed to the intensity of expression of MHC class II glycoproteins, as well as the actual numbers of MHC class II+ cells.
Journal of Veterinary Medical Science 01/2002; 63(12):1271-7. · 0.88 Impact Factor