Calcareous corpuscles are a characteristic structure found in larval and adult stage cestodes. These corpuscles are known to contain several protein components and to possess protein-binding activity. However, the proteins bound to calcareous corpuscles in situ have not been studied. The present study was undertaken to identify the proteins on calcareous corpuscles. Calcareous corpuscles were purified from the plerocercoids (= spargana) of Spirometra erinacei, and serially dissolved using 0.1 M sulfamic acid solution. Collected supernatants were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining. The results showed that only the fraction remaining after the 19th dissolved fraction contained proteins. A total of 20 protein molecules were detected in gel, with major bands at 56, 53, 46, 40, 35, 29, 28, 24.5, 21, 19, 16, 13, 10 and 8 kDa. In particular, the proteins corresponding to the 21 and 16 kDa bands were most abundant. Our results demonstrated for the first time the protein contents of the calcareous corpuscles of spargana. Further studies on the functions of these proteins are required.
Animals ; Centrifugation ; Electrophoresis, Polyacrylamide Gel ; Helminth Proteins/analysis/*metabolism ; Molecular Weight ; Protein Binding ; Research Support, Non-U.S. Gov't ; Silver Staining ; Sparganum/isolation & purification/*metabolism ; Spirometra/*metabolism ; Sulfonic Acids

OBJECTIVETo construct the recombinant plasmid for Angiostrongylus cantonensis (AC) galectin (GAL) cDNA and analyze the immunological activity of the recombinant protein.

METHODSAcGAL cDNA was screened from the cDNA library and amplified by PCR. The amplified fragment was subcloned into the expression vector pET32a(+) and expressed in E.coli. The inclusion body was washed, degenerated, refolded by dialysis, and condensed for SDS-PAGE and Western blot analysis of the protein.

RESULTSFor the first time the full-length cDNA of AcGAL was cloned (GenBank GeneID: DQ384534). Restriction enzyme digestion indicated that the recombinant plasmid pET32a(+)-AcGAL was successfully constructed. SDS-PAGE analysis confirmed high expression of the recombinant protein AcGAL in E.coil in the form of inclusion bodies, which possessed good immunoreactivity as shown by Western blot analysis.

CONCLUSIONThe success in cloning and identification, the recombinant AcGAL may provide basis for further diagnostic study of angiostrongyliasis.

Angiostrongylus cantonensis ; genetics ; metabolism ; Animals ; Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Galectins ; genetics ; metabolism ; Helminth Proteins ; genetics ; metabolism ; Molecular Sequence Data ; Recombinant Proteins ; metabolism ; Sequence Analysis, DNA

Paramyosin is a myofibrillar protein present in helminth parasites and plays multifunctional roles in host-parasite interactions. In this study, we identified the gene encoding paramyosin of Clonorchis sinensis (CsPmy) and characterized biochemical and immunological properties of its recombinant protein. CsPmy showed a high level of sequence identity with paramyosin from other helminth parasites. Recombinant CsPmy (rCsPmy) expressed in bacteria had an approximate molecular weight of 100 kDa and bound both human collagen and complement 9. The protein was constitutively expressed in various developmental stages of the parasite. Imunofluorescence analysis revealed that CsPmy was mainly localized in the tegument, subtegumental muscles, and the muscle layer surrounding the intestine of the parasite. The rCsPmy showed high levels of positive reactions (74.6%, 56/75) against sera from patients with clonorchiasis. Immunization of experimental rats with rCsPmy evoked high levels of IgG production. These results collectively suggest that CsPmy is a multifunctional protein that not only contributes to the muscle layer structure but also to non-muscular functions in host-parasite interactions. Successful induction of host IgG production also suggests that CsPmy can be applied as a diagnostic antigen and/or vaccine candidate for clonorchiasis.
Amino Acid Sequence ; Animal Structures/chemistry ; Animals ; Antibodies, Helminth/blood ; Cloning, Molecular ; Clonorchis sinensis/chemistry/*genetics ; Collagen/metabolism ; Complement C9/metabolism ; Helminth Proteins/chemistry/*genetics/immunology/metabolism ; Immunoglobulin G/blood ; Molecular Sequence Data ; Molecular Weight ; Protein Binding ; Rats ; Rats, Sprague-Dawley ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Tropomyosin/chemistry/*genetics/immunology/metabolism

We have reported that a 24 kDa protein (22U homologous; As22U) of Anisakis simplex larvae could elicit several Th2-related chemokine gene expressions in the intestinal epithelial cell line which means that As22U may play a role as an allergen. In order to determine the contribution of As22U to allergic reactions, we treated mice with 6 times intra-nasal application of recombinant As22U (rAs22U). In the group challenged with rAs22U and ovalbumin (OVA), the number of eosinophils in the bronchial alveolar lavage fluid (BALF) was significantly increased, as compared to the group receiving only OVA. In addition, mice treated with rAs22U and OVA showed significantly increased airway hyperresponsiveness. Thus, severe inflammation around the airway and immune cell recruitment was observed in mice treated with rAs22U plus OVA. The levels of IL-4, IL-5, and IL-13 cytokines in the BALF increased significantly after treatment with rAs22U and OVA. Similarly, the levels of anti-OVA specific IgE and IgG1 increased in mice treated with rAs22U and OVA, compared to those treated only with OVA. The Gro-alpha (CXCL1) gene expression in mouse lung epithelial cells increased instantly after treatment with rAs22U, and allergy-specific chemokines eotaxin (CCL11) and thymus-and-activation-regulated-chemokine (CCL17) gene expressions significantly increased at 6 hr after treatment. In conclusion, rAs22U may induce airway allergic inflammation, as the result of enhanced Th2 and Th17 responses.
Administration, Intranasal ; Animals ; Anisakiasis/*immunology/parasitology ; Anisakis/*immunology/metabolism ; Bronchoalveolar Lavage Fluid ; Chemokines/metabolism ; Cytokines/analysis/*metabolism ; Eosinophils/metabolism ; Female ; Gene Expression Regulation/*immunology ; Helminth Proteins/*immunology ; Hypersensitivity/*immunology/parasitology ; Immunoglobulin E/immunology ; Immunoglobulin G/immunology ; Larva/immunology/metabolism ; Lung/metabolism ; Mice ; Mice, Inbred C57BL ; Recombinant Proteins/immunology ; Th17 Cells/metabolism ; Th2 Cells/metabolism

Many immune down-regulatory molecules have been isolated from parasites, including cystatin (cystain protease inhibitor). In a previous study, we isolated and characterized Type I cystatin (CsStefin-1) of the liver fluke, Clonorchis sinensis. To investigate whether the CsStefin-1 might be a new host immune modulator, we induced intestinal inflammation in mice by dextran sodium sulfate (DSS) and treated them with recombinant CsStefin-1 (rCsStefin-1). The disease activity index (DAI) increased in DSS only-treated mice. In contrast, the DAI value was significantly reduced in rCsStefin-1-treated mice than DSS only-treated mice. In addition, the colon length of DSS only-treated mice was shorter than that of rCsStefin-1 treated mice. The secretion levels of IFN-gamma and TNF-alpha in the spleen and mesenteric lymph nodes (MLNs) were significantly increased by DSS treatment, but the level of TNF-alpha in MLNs was significantly decreased by rCsStefin-1 treatment. IL-10 production in both spleen and MLNs was significantly increased, and IL-10+F4/80+ macrophage cells were significantly increased in the spleen and MLNs of rCsStefin-1 treated mice after DSS treatment. In conclusion, rCsStefin-1 could reduce the intestinal inflammation occurring after DSS treatment, these effects might be related with recruitment of IL-10 secreting macrophages.
Animals ; Antigens, Differentiation/analysis ; Clonorchis sinensis/*enzymology ; Colon/pathology ; Cystatins/*metabolism ; Cytokines/secretion ; Dextran Sulfate/toxicity ; Female ; Helminth Proteins/*metabolism ; Immunologic Factors/*metabolism ; Inflammation/chemically induced/*pathology ; Interleukin-10/analysis ; Intestines/*drug effects/pathology ; Lymph Nodes/immunology ; Macrophages/chemistry/*immunology ; Mice ; Mice, Inbred C57BL ; Severity of Illness Index ; Spleen/immunology

Wnt proteins together with their downstream effectors forms a set of important signal pathways. The Wnt signal pathway is important in a wide variety of development processes including cell growth, cell differentiation, cell polarity and apoptosis. Wnt4 is a key regulator of gonadal differentiation in humans and mice, playing a pivotal role in early embryogenesis. With RACE technique based on a EST identified in our lab, a novel gene including a complete open reading frame was cloned and named Sjwnt4 (GenBank accession No. DQ643829). Sequence analyses showed that SjWnt4 had a typical characteristics of Wnt family proteins, sharing 43% similarity to Dugesia japonica and 37% to human Wnt4. The ORF of Sjwnt4 contains 1311 nucleotides, encoding 436 amino acid with 49.6 kD molecular weight. Real-time PCR analysis from the worms of various stages of S. japonicum revealed that the mRNA level of Sjwnt4 is highest in the 19 days schistosomula, followed by 44 days female worms, 14 days schistosomula, 31 days adult worms and 44 days male worms, suggesting a stage-and-gender differential express. The Sjwnt4 cDNA fragment was subcloned into a modified expression vector pGEX-4T-2 and transformed into E. coli BL21 (DE3) cells, and the production of recombinant Sjwnt4 protein fused to a GST tag was analysed. In the presence of IPTG, the 76kD fusion protein was expressed in included bodies. Western-blotting revealed that the fusion protein could be recognized by the rabbit serum specific to Schistosoma japonicum adult worm antigen preparation. The study provides important basis for investigating the regulation mechanism of the Wnt signaling pathway during the development especially gonadal differentiation processes of Schistosoma japonicum.
Amino Acid Sequence ; Animals ; Blotting, Western ; Cloning, Molecular ; DNA, Complementary ; chemistry ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; Female ; Gene Expression Regulation, Developmental ; Helminth Proteins ; genetics ; immunology ; metabolism ; Immune Sera ; immunology ; Male ; Molecular Sequence Data ; Rabbits ; Recombinant Proteins ; immunology ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Schistosoma japonicum ; genetics ; growth & development ; metabolism ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid ; Sex Factors ; Signal Transduction ; genetics ; Time Factors ; Wnt Proteins ; genetics ; immunology ; metabolism