Molecular characterization of a calcium-binding protein SjCa8 from <Emphasis Type="Italic">Schistosoma japonicum</Emphasis>

A full-length cDNA encoding a cercarial stage-specifically expressed 8-kDa calcium-binding protein (SjCa8) was isolated from Schistosoma japonicum cercarial cDNA library using microarray screen. The putative gene coding for SjCa8 is of 371 bp with an open reading frame of 69 amino acid (aa). The deduced aa sequence showed 83% identity with the Schistosoma mansoni 8-kDa CaBP, 47% identity with Clonorchis sinensis calcium-binding protein, and 38% identity with Fasciola hepatica putative calcium-binding protein. Also, it shares more than 30% identity with the calmodulin of many different species, while the most significant similarity between them lies around the two calcium-binding loop regions. There are two potential sites for phosphorylation and one potential site for N-myristoylation in the sequence. The SjCa8 has also been predicted to contain a single pair of EF-hand Ca(2+)-binding domain. The recombinant SjCa8 (rSjCa8) protein expressed and purified from E. coli has been demonstrated to possess the calcium-binding activity. Immune serum from UV-attenuated S. japonicum cercariae-immunized rabbit detected rSjCa8 by Western blot assay, while the sera from S. japonicum naturally infected rabbit and normal rabbit could not. These findings may contribute to the development of an effective vaccine against schistosomiasis.     

Immunological cross-reactivity analysis on recombinant histamine-releasing factors from <Emphasis Type="Italic">Schistosoma japonicum</Emphasis>, <Emphasis Type="Italic">Clonorchis sinensis</Emphasis>, and Wistar rat

Studies have demonstrated a declined incidence of allergic disorders in the population with helminthic infection. Though several hypotheses have been proposed to explain how helminthic infection protected people against allergies, the underlying mechanisms remain poorly understood. A human histamine-releasing factor (HRF) has been proved to be closely related to the development of allergic disorders and the homologues are ubiquitously expressed in all eukaryotic organisms including parasites. To study the role of this HRF in the relationship between parasitic infection and allergic diseases with experimental model of rats, the cDNA of the homologues of the human HRF from Wistar rat, Schistosoma japonicum, and Clonorchis sinensis containing a coding region of 519, 510, and 510 bp, respectively, were cloned. In addition, the cross-reactivity between recombinant rat HRF (rRHRF) and recombinant S. japonicum HRF (rSjHRF) as well as that between rRHRF and recombinant C. sinensis HRF (rCsHRF) was identified with ELISA and Western blotting. Based on their detected cross-reactivities, a hypothesis was put forward that the anti-parasitic HRFs antibodies could inhibit the effects of host HRF and those of parasitic HRFs and thus decreased the host sensitivities to allergens.     

Histochemical and enzyme-histochemical studies of <Emphasis Type="Italic">Oncomelania hupensis</Emphasis> (Gredler, 1881), the intermediate host of <Emphasis Type="Italic">Schistosoma japonicum</Emphasis>

The distribution of glycogen, DNA and histone, and localization of activity of ten enzymes such as glucose-6-phosphatase (G-6-Pase), cytochrome oxidase (CCO), lactate dehydrogenase (LDH), 5′-nucleotidase (5′-NT), succinate dehydrogenase (SDH), glucose-6-phosphate dehydrogenase (G-6-PDH), alkaline phosphatase (ALP), acid phosphatase (ACP), Mg²⁺-adenosine triphosphatase (Mg²⁺-ATPase), and cholinesterase (CHE) in Oncomelania hupensis (Gredler, 1881) snails, the intermediate host of Schistosoma japonicum, was surveyed using the histochemical and enzyme-histochemical techniques. The results showed that the glycogen with high activity was widely distributed in snails. DNA showed its strongest reaction in testis. The histone was distributed in sites of the radula, parenchyma of penis and ovary. LDH and SDH were widely distributed in snails and all were abundant. Sites of the strongest enzyme activities of G-6-PDH were evident in the reproductive system together with central ganglia. Only the ovary showed a strong enzyme activity of G-6-Pase. Most tissues and organs contained CCO, and the activity of this enzyme was very strong. 5′-NT showed a strong enzyme activity in the ovary and testis. Mg²⁺-ATPase was localized in sites of the liver, stomach and reproductive gland. A strong enzyme activity of ALP appeared in the digestive system. ACP showed a low activity in snails. The central ganglia, nervous stem, liver, branchial duct, epithelia of the head and foot regions showed a very strong enzyme activity of CHE. The findings could provide a theoretical basis for development of highly effective molluscicides with low toxicity to other biota, as well as means for novel snail control strategies.     

<Emphasis Type="Italic">Penelope</Emphasis>-like retrotransposons in the genome of the Asian blood fluke <Emphasis Type="Italic">Schistosoma japonicum</Emphasis> (Trematoda: Schistosomatidae)

Mobile genetic elements (in particular, retrotransposons) make up a significant part of eukaryotic genomes and are one of the main sources of genomic variability and instability. More than 60 years have passed already since their discovery, but, nevertheless, certain classes of them, such as Penelope-like elements (PLEs), remain poorly studied. In this work, we present the results of in silico search for PLEs in the genome assembly of the Asian schistosome S. japonicum (one of the most epidemiologically important human parasites). We present a new evaluation of their representation in the S. japonicum genome and showed that their high heterogeneity apparently reflect several transposition events. It was demonstrated that PLEs in the schistosome genome are represented by two groups of canonical copies differing by amino-acid sequences of RT and EN domains. Data on the representation, structural peculiarities, possible functional load, and evolution of Penelope-like retroelements of the parasite are discussed.     

Cloning,expression, and characterization of salivary apyrase from <Emphasis Type="Italic">Aedes albopictus</Emphasis>

Apyrases (ATP diphosphohydrolase) hydrolyze the phosphodiester bonds of nucleoside tri- and diphosphates to orthophosphate and mononucleodides. They can inhibit platelet activation by depletion of adenosine diphosphate. In the current study, the Escherichia coli expression vector pET-19b equipped with an N-terminal histidine tag was applied to express the apyrase of Aedes albopictus. The gene-coding mature apyrase protein was amplified by RT-PCR and cloned into pET-19b. Soluble apyrase protein with high purity was successfully obtained by utilization of the suitable renaturation approach and Ni-NTA purification column. Four monoclonal antibodies to apyrase from A. albopictus were produced in male BALB/c mice immunized with the renatured apyrase. Using immunofluorescence assay and immunoblotting analysis, recombinant apyrase showed fine consistency with native apyrase. From kinetic analysis, it had a K _m of 11.6 μM and V _max of 1.02 nM/S/μg protein for adenosine triphosphate. Adenosine diphosphate-induced platelet aggregation was inhibited by approximately 6% when 0.4 μM recombinant apyrase was added and by about 9.5% when the concentration of recombinant apyrase was 0.8 μM. The effect on platelet aggregation was dose dependent. In conclusion, the apyrase of A. albopictus was cloned and expressed highly in the E. coli expression system. Recombinant apyrase protein showed biological activity, and anti-apyrase monoclonal antibody was also prepared.     

Cysteine protease inhibitor of <Emphasis Type="Italic">Schistosoma japonicum</Emphasis> - A parasite-derived negative immunoregulatory factor

Studies have shown that cysteine protease inhibitors from some parasites have immunosuppressive effects on the host. We previously have cloned a novel cysteine protease inhibitor from Schistosoma japonicum and purified its recombinant version (protein named rSj-C). Its possible inhibitory effect on the host immune response has not been described.This study shows that rSj-C inhibits lysosomal cysteine protease of murine dendritic cells (DCs). After DCs were incubated with rSj-C and then with soluble adult worm antigen (AWA) of S. japonicum, the mean fluorescence intensity of MHC class II antigens on the surface of DCs decreased significantly by flow cytometry. These results indirectly proved that rSj-C can suppress exogenous-antigen presentation by DCs. The flow cytometric assay revealed that in comparison with control groups, the proportion of CD4⁺CD25⁺Foxp3⁺ T cells among CD4⁺CD25⁺ T cells of Schistosom-infected mice increased significantly 8 weeks after the infected mice were injected with rSj-C (p ? 0.05). Additionally, the expression levels of cytokines IL-4 and TGF-β produced by T cells increased significantly as compared with these levels in the normal group (p ? 0.05). These results clearly show that the cysteine protease inhibitor from S. japonicum is a new parasite-derived immunosuppressive factor.     

Cercarial shedding from <Emphasis Type="Italic">Galba</Emphasis><Emphasis Type="Italic">truncatula</Emphasis> infected with <Emphasis Type="Italic">Fasciola</Emphasis><Emphasis Type="Italic">gigantica</Emphasis> of distinct geographic origins

Galba truncatula snails were experimentally infected with either of two different isolates of Fasciola gigantica, originating from Egypt or China, to determine the influence of these isolates on the characteristics of snail infections. The survival rates of G. truncatula on day 30 post-exposure were 90.0% and 60.2% in the Egyptian and Chinese groups, respectively. The frequency of cercaria-shedding snails within the Egyptian group was 79.8%, whereas in the Chinese group it was 22.4%. The parasite origin had a significant effect on the durations of the prepatent and patent periods. The mean number of cercariae shed from the Egyptian group was significantly greater than that shed from the Chinese group (a mean of 275.5 per cercaria-shedding snail compared with 29.0). These results could be explained by the fact that G. truncatula might be a natural intermediate host for F. gigantica in Egypt, and the greater adaptability of the Egyptian miracidia of F. gigantica to unusual snail hosts. These results demonstrate the influence of the geographic origin of the parasite on the success of trematodes infecting snails.     

Ultrastructural alterations of juvenile <Emphasis Type="Italic">Schistosoma japonicum</Emphasis> harbored in mice following mefloquine administration

The aim of the present study was to assess the ultrastructural alterations of juvenile Schistosoma japonicum induced by mefloquine. Mice infected with 14-day-old S. japonicum were treated orally with mefloquine at a single dose of 400 mg/kg. Between 8 h and 7 days after treatment, groups of two mice were sacrificed, and schistosomula were recovered for transmission electron microscopic observations. Ultrastructural damage was seen in the tegument, subtegumental musculature, parenchymal tissues, and gut epithelial cell. It was already prominent 8 h after drug administration and increased in severity rapidly to reach a peak 3 days post-treatment. Tegumental alterations were characterized by emergence of irregular and elongated cytoplasmic processes, which further fused together accompanied by indistinction of matrix and roughness of external plasma membrane. Meanwhile, in the subtegument, damage to the syncytium, swelling, and lysis of muscle bundles and parenchymal tissues were universal, which further aggravated the lesion on the tegument, followed by collapse or disintegration of damaged tegument to form numerous fragment or debris of cytoplasmic process detached from the worm surface. Severe damage to the gut epithelial cell was also observed 8 h post-mefloquine treatment, which included focal lysis of cytoplasm accompanied by formation of vacuoles and degeneration of mitochondria, emergence of enlarged and contracted nucleus with indistinct or focal disrupted nuclear membrane, and decrease in microvilli. All these alterations further increased in severity and reached the peak 3 days post-treatment. The findings of our study indicate that mefloquine exhibits a fast and potent ability to cause extensive ultrastructural damage to juvenile S. japonicum, which correlates with its high efficacy against juvenile schistosomes.     

Cysteine proteinases from promastigotes of <Emphasis Type="Italic">Leishmania</Emphasis> (<Emphasis Type="Italic">Viannia</Emphasis>) <Emphasis Type="Italic">braziliensis</Emphasis>

Leishmania (Viannia) braziliensis is the major causative agent of American tegumentary leishmaniasis, a disease that has a wide geographical distribution and is a severe public health problem. The cysteine proteinase B (CPB) from Leishmania spp. represents an important virulence factor. In this study, we characterized and localized cysteine proteinases in L. (V.) braziliensis promastigotes. By a combination of triton X-114 extraction, concanavalin A-affinity, and ion exchange chromatographies, we obtained an enriched fraction of hydrophobic proteins rich in mannose residues. This fraction contained two proteinases of 63 and 43 kDa, which were recognized by a CPB antiserum, and were partially sensitive to E-64 in enzymatic assays with the peptide Glu-Phe-Leu. In confocal microscopy, the CPB homologues localized in the peripheral region of the parasite. This data together with direct agglutination and flow cytometry assays suggest a surface localization of the CPB homologues. The incubation of intact promastigotes with phospholipase C reduced the number of CPB-positive cells, while anti-cross-reacting determinant and anti-CPB antisera recognized two polypeptides (63 and 43 kDa) derived from phospholipase C treatment, suggesting that some CPB isoforms may be glycosylphosphatidylinositol-anchored. Collectively, our results suggest the presence of CPB homologues in L. braziliensis surface and highlight the need for further studies on L. braziliensis cysteine proteinases, which require enrichment methods for enzymatic detection.     

Effects of <Emphasis Type="Italic">Schistosoma mansoni</Emphasis> and <Emphasis Type="Italic">Schistosoma haematobium</Emphasis> infections on calcium content in their intermediate hosts

Flame atomic absorption spectrometry was performed to determine the alteration of calcium concentration in the soft parts and shells of Biomphalaria alexandrina and Bulinus truncatus due to the infection with Schistosoma mansoni and S. haematobium, respectively. The results showed significant lowering in the calcium content of the shells of cercariae shedding B. alexandrina and B. truncatus relative to the calcium content in the shells of uninfected ones. In contrast, the calcium content in the soft parts of cercariae shedding snails was higher than in the soft parts of uninfected snails; the differences were statistically significant. Generally, calcium content was significantly higher in the shells than in the soft parts of the snails, regardless infected or uninfected. The results obtained and the hypothesis of hypercalcification in shells of infected snails were discussed.     

Redescription of <Emphasis Type="Italic">Lemuricola</Emphasis> (<Emphasis Type="Italic">Madoxyuris</Emphasis>) <Emphasis Type="Italic">bauchoti</Emphasis> (Nematoda,Oxyuridae) from <Emphasis Type="Italic">Lemur catta</Emphasis> in Madagascar

Lemuricola (Madoxyuris) bauchoti Chabaud, Brygoo et Petter, 1965 is redescribed from material collected from the ring-tailed lemur, Lemur catta, from the Beza Mahafaly Special Reserve in Madagascar using the scanning electron microscope. This is a new host record and the first oxyurid reported from the ring-tailed lemur. Previously, records of each species of the subgenus Madoxyuris have been restricted to a single host species, but the close relationship between these nematodes and their Strepsirrhini hosts will only be proven when additional records fill in the gaps in their distribution.     

Repeat induced point mutation in two asexual fungi, <Emphasis Type="Italic">Aspergillus niger</Emphasis> and <Emphasis Type="Italic">Penicillium </Emphasis><Emphasis Type="Italic">chrysogenum</Emphasis>

Repeat induced point mutation (RIP) is a gene silencing mechanism present in fungal genomes. During RIP, duplicated sequences are efficiently and irreversibly mutated by transitions from C:G to T:A. For the first time, we have identified traces of RIP in transposable elements of Aspergillus niger and Penicillium chrysogenum, two biotechnologically relevant fungi. We found that RIP in P. chrysogenum has affected a large set of sequences, which also contain other mutations. On the other hand, RIP in A. niger is limited to only few sequences, but literally all mutations are RIP-like. Surprisingly, RIP occurred only in transposon sequences that have disrupted open reading frames in A. niger, a phenomenon not yet reported for other fungi. In both fungal species, we identified two sequences with strong sequence similarity to Neurospora crassa RID. RID is a putative DNA methyltransferase and the only known enzyme involved in the RIP process. Our findings suggest that both A. niger and P. chrysogenum either had a sexual past or have a sexual potential. These findings have important implications for future strain development of these fungi.     

Functional polymorphisms in <Emphasis Type="Italic">XRCC</Emphasis>-<Emphasis Type="Italic">1</Emphasis> and <Emphasis Type="Italic">APE</Emphasis>-<Emphasis Type="Italic">1</Emphasis> contribute to increased apoptosis and risk of ulcerative colitis

Objective  

The present study was designed to investigate the role of X-ray cross-complementing group 1 (XRCC1) and apurinic/apyrimidinic endonuclease 1 (APE1) polymorphisms in apoptosis and the risk of ulcerative colitis (UC).     

Association analysis of <Emphasis Type="Italic">SLC22A4</Emphasis>, <Emphasis Type="Italic">SLC22A5</Emphasis> and <Emphasis Type="Italic">DLG5</Emphasis> in Japanese patients with Crohn disease

Crohn disease (CD) is an inflammatory bowel disease characterized by chronic transmural, segmental, and typically granulomatous inflammation of the gut. Recently, two novel candidate gene loci associated with CD, SLC22A4 and SLC22A5 on chromosome 5 known as IBD5 and DLG5 on chromosome 10, were identified through association analysis of Caucasian CD patients. We validated these candidate genes in Japanese patients with CD and found a weak but possible association with both SLC22A4 (P=0.028) and DLG5 (P=0.023). However, the reported genetic variants that were indicated to be causative in the Caucasian population were completely absent in or were not associated with Japanese CD patients. These findings imply significant differences in genetic background with CD susceptibility among different ethnic groups and further indicate some difficulty of population-based studies.     

Heterologous expression and characterization of <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> vacuolar carboxypeptidase Y in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

To investigate the intracellular transport mechanism of the vacuolar carboxypeptidase of Schizosaccharomyces pombe (SpCPY), SpCPY was expressed in Saccharomyces cerevisiae and its biosynthesis and sorting were examined. When Sac. cerevisiae prc1Delta, devoid of intrinsic (Sc) CPY activity, was transformed with a plasmid carrying the Sch. pombe cpy1(+) gene, CPY activity was restored. Pulse-chase experiments revealed that SpCPY is initially synthesized in a pro-precursor form and then converted to a heterodimer, the mature form, in Sac. cerevisiae cells. SpCPY was not processed into intermediate or mature forms in pep4 mutant cells, indicating that SpCPY was proteolytically cleaved in a PEP4-dependent manner in Sac. cerevisiae. Several vps mutants, which are defective in vacuolar protein-sorting, exhibited a defect in the maturation of SpCPY. Moreover, the maturation of SpCPY was severely inhibited in a vps10 strain, although the pro- segment of SpCPY does not contain a QRPL-like sequence, which is the putative targeting signal of ScCPY. When SpCPY was expressed in a wild-type strain, more than 90% of ScCPY was normally sorted to the vacuole, indicating that SpCPY does not compete with ScCPY for vacuolar sorting. In contrast, expression of SpCPY resulted in a missorting of a ScCPY-invertase fusion protein to the cell surface. These results suggested that there are two different binding sites for SpCPY and ScCPY on Vps10p and that the binding of SpCPY to Vps10p interferes with the binding of a ScCPY-invertase fusion protein.     

Identification and characterization of a novel <Emphasis Type="Italic">Tritimovirus</Emphasis> species isolated from wild <Emphasis Type="Italic">Trisetum flavescens</Emphasis> L., family <Emphasis Type="Italic">Poaceae</Emphasis>

Yellow oat-grass plants (Trisetum flavescens L.) with mild mosaic and pronounced dwarfing symptoms were observed at different locations in the Czech Republic. Electron microscope observations of symptomatic plants revealed the presence of filamentous particles and inclusion bodies characteristic of the family Potyviridae. The virus was readily mechanically transmitted to its original host plus a narrow host range of monocot species. Serological assays of infected plant extracts using antiserum specific to the closest species in the family Potyviridae were negative. The 3′ end of the viral genome was cloned, sequenced and compared to sequences of species in the family Potyviridae. The virus is more closely related to viruses in the genus Tritimovirus than to other genera within the Potyviridae. Based on phylogenetic analyses of the coat protein cistron and flanking genomic regions, we propose this is a distinct viral species of the genus Tritimovirus, tentatively named Yellow oat-grass mosaic virus (YOgMV).