Highly up-regulated CXCR3 expression on eosinophils in mice infected with Schistosoma japonicum.
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CXCR3, predominately expressed on memory/activated T cells, is a receptor for both interferon-gamma inducible protein-10/CXC ligand 10 (CXCL10) and monokine induced by interferon-gamma/CXCL9. We reported here that CXCR3 was highly up-regulated on infiltrating eosinophils in Schistosoma japonicum egg-induced granuloma in the mouse liver. It was also highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum. The phenomena were demonstrated at protein and mRNA levels using immunohisto- and immunocytochemistry evaluation of biopsy, flow cytometry and real-time quantitative reverse transcriptase-polymerase chain reaction technique, and verified by Northern blotting and chemotaxis assay in vitro. We also found that CCR3 expression on the infiltrating and peritoneal exudate cells was significantly decreased, CXCR4 expression was unchanged during the 42-day period of infection. We screened mRNA expression levels of the all known chemokine receptors in purified peritoneal exudate eosinophils and liver granuloma dominated by eosinophils. CXCR3 was highly and functionally up-regulated on peritoneal exudate eosinophils in mice infected with S. japonicum, meanwhile CCR3 was significantly and functionally down-regulated in these cells. The findings could lead to a better understanding of the chemokine receptor expression pattern of eosinophils at inflamed tissue sites caused by parasites. These could be also crucial for establishing a therapeutic strategy for eosinophilic inflammation via intervention in chemokine actions. (+info)
Isolation and characterization of different C-terminal fragments of dystrophin expressed in Escherichia coli.
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Dystrophin, the protein product of the Duchenne muscular dystrophy gene, is thought to belong to a family of membrane cytoskeletal proteins. Based on its deduced amino-acid sequence, it is postulated to have several distinct structural domains; an N-terminal region; a central, rod-shaped, domain; and a C-terminal domain [Koenig, Monaco & Kunkel (1988) Cell 53, 219-228]. The C-terminal domain is further divided into two regions; the first has some sequence similarity to slime mould alpha-actinin, and is rich in cysteine residues; this is followed by the C-terminal amino-acid sequence that is unique to dystrophin. Dystrophin is very difficult to purify in quantities sufficient for detailed studies of the structure/function relationships within the molecule. Therefore, in this study, we have expressed selected fragments of the C-terminal region of dystrophin, as fusion proteins, in Escherichia coli. Importantly, we describe the first successful purification, from E. coli lysates, of large quantities of fragments of dystrophin in a soluble form. The first fragment, termed CT-1, encodes the C-terminal 201 amino acids of the protein; the second, termed CT-2, spans the cysteine-rich region of the C-terminal domain. These fusion proteins were identified by their mobility in SDS/PAGE, by their interaction with appropriate affinity columns and by their reactivity with anti-dystrophin antibodies. The fragment CT-2, which spans a region containing putative EF-hand-like sequences, was found to bind Ca2+ in 45Ca2+ overlay experiments. In addition, we have discovered that the fragment CT-1, but not fragment CT-2, interacts specifically with the E. coli DnaK gene product [analogue of heat shock protein 70 (hsp70)]. This interaction is disrupted, in vitro, by the addition of ATP. Our results indicate that the two C-terminal fragments of dystrophin have differing biophysical properties, indicating that they may play distinct roles in the function of the protein. (+info)
Factors influencing the transmission of Schistosoma japonicum in the mountains of Sichuan Province of China.
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Twenty villages in the Anning River Valley of southwestern Sichuan China were surveyed for Schistosoma japonicum infections in humans and domestic animals. Also surveyed were human water contact patterns, snail populations, cercarial risk in irrigation systems, and agricultural land use. Few animals were infected, while village prevalence of infection in humans ranged from 3% to 68% and average village eggs per gram of stool ranged from 0 to 110. Except for occupation and education, individual characteristics were not strong determinants of infection intensity within a village. Differences in human infection intensity between these villages are strongly associated with crop type, with low-intensity villages principally growing rice, in contrast to villages devoting more land to vegetables and tobacco. Cercarial risk in village irrigation systems is associated with snail density and human infection intensity through the use of manure-based fertilizer. Some of the agricultural and environmental factors associated with infection risk can be quantified using remote sensing technology. (+info)
Silica sol-gel amperometric immunosensor for Schistosoma japonicum antibody assay.
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An amperometric immunosensor was constructed by dispersing graphite, schistosoma-japonicum antigen (SjAg) and silica sol-gel at low temperature. The performance characteristics of the prepared immunosensor were examined in the buffered solution of o-aminophenol (o-AP) used as a substrate. It exhibited excellent physical and electrochemical stability with a renewable external surface. A competitive binding assay was employed to determine schistosoma-japonicum antibody (SjAb) with the aid of horseradish peroxidase labeled SjAb (HRP-SjAb). The experimental parameters for SjAb assay were optimized, including the amount of labeled SjAb in incubation solution, incubation time, temperature and the pH of solution. The use of o-AP substrate and amperometric detection at -250 mV (vs. SCE) results in a determination limit of 0.32 microg/ml and a linear range extending up to 0.18 microg/ml. The results of SjAb assay in serum samples demonstrate the feasibility of using the proposed immunosensor for clinical analysis. (+info)
Implications of the ligandin binding site on the binding of non-substrate ligands to Schistosoma japonicum-glutathione transferase.
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The binding interactions between dimeric glutathione transferase from Schistosoma japonicum (Sj26GST) and bromosulfophthalein (BS) or 8-anilino-1-naphthalene sulfonate (ANS) were characterised by fluorescence spectroscopy and isothermal titration calorimetry (ITC). Both ligands inhibit the enzymatic activity of Sj26GST in a non-competitive form. A stoichiometry of 1 molecule of ligand per mole of dimeric enzyme was obtained for the binding of these ligands. The affinity of BS is higher (K(d)=3.2 microM) than that for ANS (K(d)=195 microM). The thermodynamic parameters obtained by calorimetric titrations are pH-independent in the range of 5.5 to 7.5. The interaction process is enthalpically driven at all the studied temperatures. This enthalpic contribution is larger for the ANS anion than for BS. The strongly favourable enthalpic contribution for the binding of ANS to Sj26GST is compensated by a negative entropy change, due to enthalpy-entropy compensation. DeltaG degrees remains almost invariant over the temperature range studied. The free energy change for the binding of BS to Sj26GST is also favoured by entropic contributions at temperatures below 32 degrees C, thus indicating a strong hydrophobic interaction. Heat capacity change obtained for BS (DeltaC(p) degrees =(-580.3+/-54.2) cal x K(-1) mol(-1)) is twofold larger (in absolute value) than for ANS (DeltaC(p) degrees =(-294.8+/-15.8) cal x K(-1) mol(-1)). Taking together the thermodynamic parameters obtained for these inhibitors, it can be argued that the possible hydrophobic interactions in the binding of these inhibitors to L-site must be accompanied by other interactions whose contribution is enthalpic. Therefore, the non-substrate binding site (designed as ligandin) on Sj26GST may not be fully hydrophobic. (+info)
Antigen-specific antibody isotype patterns to schistosoma japonicum recombinant and native antigens in a defined population in Leyte, the Philippines.
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We describe antibody isotype patterns resulting from Schistosoma japonicum infection among 155 individuals 5-76 years old from a community in Leyte, The Philippines. Their exposure, infection, and reinfection status had been observed in a previously described water contact study used to categorically classify them as putative resistant or susceptible individuals. Antigens tested for specific antibody isotype responses were soluble worm antigen preparation (SWAP) and soluble egg antigen (SEA) and a panel of recombinant molecules. The study was aimed primarily at evaluating antigen-specific antibody responses and their potential in inducing protection among putative resistant individuals. Specific antibody responses suggestive of an involvement in protection were an IgE response to SWAP among females less than 20 years of age (5-19) and IgA responses to SWAP in the younger (5-19 years) age groups. Compatible with other studies on human schistosomes, IgM reactivities to SWAP and SEA in the 5-19-year-old age group predicted susceptibility as did IgG4 responses to recombinant paramyosin. (+info)
Structure and bioactivity of neuropeptide F from the human parasites Schistosoma mansoni and Schistosoma japonicum.
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The blood flukes Schistosoma mansoni and Schistosoma japonicum inflict immense suffering as agents of human schistosomiasis. Previous investigations have found the nervous systems of these worms contain abundant immunoreactivity to antisera targeting invertebrate neuropeptide Fs (NPFs) as well as structurally similar neuropeptides of the mammalian neuropeptide Y (NPY) family. Here, cDNAs encoding NPF in these worms were identified, and the mature neuropeptides from the two species differed by only a single amino acid. Both neuropeptides feature the characteristics common among NPFs; they are 36 amino acids long with a carboxyl-terminal Gly-Arg-X-Arg-Phe-amide and Tyr residues at positions 10 and 17 from the carboxyl terminus. Synthetic S. mansoni NPF potently inhibits the forskolin-stimulated accumulation of cAMP in worm homogenates, with significant effects at 10(-11) m. This is the first demonstration of an endogenous inhibition of cAMP by an NPF, and because this is the predominant pathway associated with vertebrate NPY family peptides, it demonstrates a conservation of downstream signaling pathways used by NPFs and NPY peptides. (+info)
Cloning and expression in Escherichia coli of a new gene of Schistosoma japonicum encoding casein kinase II beta subunit.
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BACKGROUND: Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase II beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E. coli). METHODS: The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3'RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E. coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. RESULTS: A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase II beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E. coli JM109. The recombinant protein could be recognized by the S. japonicum infected rabbit serum. CONCLUSION: The full-length cDNA sequences encoding S. japonicum casein kinase II beta subunit were firstly sequenced, cloned, and expressed in E. coli. (+info)