Showing papers by "Chihiro Sugimoto published in 1992"

Antigenic differences between Japanese Theileria sergenti and other benign Theileria species of cattle from Australia (T. buffeli) and Britain (T. orientalis).

Abstract: Serological comparisons among piroplasm antigens of the benignTheileria species of cattle from Japan, Australia and Britain, which are frequently referred to asT. sergenti, T. buffeli andT. orientalis, were carried out. The results obtained from comparative enzyme-linked immunosorbent assay (ELISA) using sera from infected cattle suggest thatT. sergenti could be differentiated from bothT. buffeli andT. orientalis by their serological dissimilarities. Western blotting combined with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) revealed that both the 33-kDa piroplasm protein ofT. sergenti and the similar 34-kDa protein ofT. buffeli andT. orientalis corresponded to immunodominant antigens against cattle. The other 32-kDa proteins ofT. buffeli andT. orientalis also represented immunodominant antigens. Cross-reactivities of the 32-and 34-kDa proteins were observed betweenT. buffeli andT. orientalis, whereas the 33-kDa protein ofT. sergenti could be differentiated from the similar 34-kDa proteins ofT. buffeli/orientalis. The present study suggests thatT. sergenti should be separated fromT. buffeli andT. orientalis on the basis of their serological dissimilarities.

Cloning and characterization of the casein kinase II alpha subunit gene from the lymphocyte-transforming intracellular protozoan parasite Theileria parva.

Abstract: Theileria parva is an obligate intracellular protozoan parasite which is the causative agent of East Coast fever, an acute, leukemia-like disease of cattle. The intralymphocytic stage of the parasite induces blastogenesis and clonal expansion of quiescent bovid lymphocytes. Experiments in our laboratory have shown a marked increase of casein kinase II- (CK II-) like activity in T. parva-transformed lymphocytes. We have also detected CK II activity in purified T. parva schizonts. To explore the significance of this increase, we used a Drosophila melanogaster CK II alpha cDNA probe [Saxena et al. (1987) Mol. Cell Biol. 7, 3409-3417] to isolate a T. parva genomic clone encoding a CK II catalytic subunit. The clone contains a 1.3-kb open reading frame coding for a predicted protein of 420 amino acids (M(r) 50,200). Northern blot analysis revealed a single transcript of 1.65 kb. The deduced T. parva CK II catalytic subunit sequence shows, over 321 residues comprising the C-terminus of the molecule, extensive identity with CK II alpha and alpha' sequences from both vertebrate and invertebrate organisms. The T. parva CK II subunit amino acid sequence displays 68% identity with the Drosophila alpha subunit and 67% with the Caenorhabditis elegans alpha subunit but only 58% and 56% sequence identity with the Saccharomyces cerevisiae alpha and alpha' subunits, respectively. Comparison of the T. parva sequence with higher eukaryotic alpha and alpha' sequences reveals that it is most identical with the alpha subunit. A unique component of the T. parva CK II alpha subunit is a 99 amino acid sequence at the N-terminus, which contains a sequence motif with features characteristic of signal peptides.

Molecular cloning and immunological analysis of immunodominant piroplasm surface proteins of Theileria sergenti and T. buffeli.

Abstract: cDNA libraries of Theileria sergenti and T. buffeli piroplasms were constructed in lambda gt11 and screened with rabbit anti-piroplasm sera. A major antigen of T. sergenti (33 kDa) and that of T. buffeli (34 kDa) was identified from the recombinant phages by using recombinant antigen-selected monospecific antibodies. The reactivities of the cloned proteins with rabbit antisera, infected calf sera and mouse monoclonal antibody suggested that the 33 and 34 kDa proteins expressed species-common and species-specific epitopes. The DNA probes from these recombinant clones showed species-specific hybridizations in Southern blotting with genomic DNA from piroplasms. These results indicate that the Japanese T. sergenti can be distinguishable from the Australian T. buffeli with regard to a polymorphism of the major immunodominant proteins of piroplasm.

Analysis of Theileria parva immunodominant schizont surface antigen by two-dimensional polyacrylamide gel electrophoresis and immunoblotting.

Abstract: Merozoites of Eimeria acervulina, Eimeria maxima, Eimeria necatrix, and Eimeria tenella were compared by gel electrophoresis, western-blotting with chicken antiserum, indirect fluorescent antibody reactions, and antiserum neutralization. Merozoites from the 4 species had dissimilar patterns of proteins and antigens in soluble and membrane fractions. Coomassie blue staining of SDS-PAGE gels revealed 16-22 protein bands depending on the species of merozoite but only 3 bands per species in the membrane fractions. Homologous and heterologous antisera recognized 5-12 soluble fraction bands and 3-7 membrane fraction bands on immunoperoxidase-stained western blots, depending on the species. When antisera from infected chickens were used in an indirect fluorescent antibody reaction, the merozoites of E. tenella and E. necatrix had a strong reaction with homologous and heterologous antisera. Merozoites of E. acervulina and E. maxima reacted with homologous antisera but had a weak or no reaction with heterologous antisera. Chicken antiserum against E. tenella had no effect on the viability of E. tenella merozoites when they were inoculated into chicken embryos.

Preliminary biochemical characterization of 'veil' structure purified from Theileria sergenti-, T. buffeli- and T. orientalis-infected bovine erythrocytes.

Abstract: A structure called ‘veil’ in Theileria sergenti-, T. buffeli - or T. orientalis -infected bovine erythrocytes was purified from erythrocyte lysates by Percoll density-gradient centrifugation. On electron microscopical examination, the veils consisted of electron-dense material showing a periodicity of striations and were not surrounded by membranes. Analyses of veil proteins by one- and two-dimensional polyacrylamide gel electrophoresis revealed that the veils contain haemoglobin and several other basic proteins of molecular weight ranging from 15·5 to 46 kDa. By comparing the protein patterns of the veil with those of purified piroplasms and uninfected bovine erythrocytes, these basic proteins appeared to be of parasite origin. It would appear likely that the veils formed by intra-erythrocytic precipitation of haemoglobin and proteins excreted by the parasites. Differences in veil constituents were found between T. sergenti, T. buffeli and T. orientalis .

Detection and characterization of Theileria sergenti proteinases.

Abstract: The lysate of Theileria sergenti piroplasms was tested for proteinases using sodium dodecyl sulfate-polyacrylamide gel electrophoresis in which substrate was included in gel matrix. Six proteinases of molecular weight 330, 125, 98, 94, 67 and 58 kilodalton (kDa) were detected. From the results of the Triton X-114 phase partition, 330, 125 and 58 kDa proteinases were partitioned into aqueous phase, which indicated that they were not associated with parasite membranes. All these three enzymes were classified into metalloproteinase family because of their sensitivities to metal-ion chelating compounds, ethylenediaminetetraacetic acid (EDTA) and 1,10-phenanthroline. On the other hand, 98 and 94 kDa proteinases were membrane-associated metalloproteinases which were preferentially inhibited by 1,10-phenanthroline. Another metalloproteinase of 67 kDa which was inhibited by EDTA and 1,10-phenanthroline was not associated with parasite membranes. Proteinases of 98 and 94 kDa degraded heat-denatured hemoglobin.