Morphological and enzymatical observations in Oncomelania hupensis after molluscicide treatment: implication for future molluscicide development.

A preparation of niclosamide named 50 % wettable powder of niclosamide ethanolamine salt (WPN), the only chemical molluscicide available in China, has been widely used for Oncomelania hupensis control over the past 20 years, but its molluscicidal mechanism has not been elucidated yet. Recently, a derivative of niclosamide, the salt of quinoid-2',5-dichloro-4'-nitro-salicylanilide (Liu Dai Shui Yang An, LDS), has been proven to have equivalent molluscicidal effects as WPN but with lower cost and significantly lower toxicity to fish than WPN. In our previous study, gene expression profiling of O. hupensis showed significantly effects after these two molluscicides had been applied. This study was designed to use morphological and enzymological analyses to further elucidate the mechanism by which these molluscicides cause snail death. After WPN or LDS treatment, the number of mitochondria of O. hupensis was reduced and their cristae appeared unclear, heterochromatin gathered to be polarized, ribosome numbers of the rough endoplasmic reticulums (rERs) decreased, myofilaments in muscle cells became disordered and loose, and cytoplasm in some liver cells was concentrated. Damage of cell structures and organelles suggested inhibited movement ability and effects on liver and energy metabolism following treatment. In parallel, activities of enzymes related with carbohydrate metabolism were inhibited except lactate dehydrogenase (LDH) increased in muscle tissue, and activities of enzymes related with stress response increased followed by decreasing to lower levels than those of the H2O-treated group. This shift of carbohydrate metabolism patterns led to insufficient energy supply and lactic acid accumulation, and variations of nitric oxide synthase (NOS), alanine aminotransferase (ALT), and superoxide dismutase (SOD) during process of molluscicide treatment suggested a stress response of snail to the molluscicides at early stages and later fatal damage in liver and nervous system. In general, effects of WPN and LDS were similar although LDS-treated snails showed more serious damage in the liver and a stronger inhibition of enzymes related with aerobic respiration and stress response. This was consistent with the transcriptome profile obtained previously. However, considering enzyme activities at post-transcriptional and protein levels, comprehensive identification and annotation of potential enzyme-related genes and regulation pattern would be necessary to provide great benefit for understanding of potential mechanism of these molluscicides and even for future molluscicide development.

[The Effect of Lipopolysaccharide-Induced Mouse B Cell Activation on Schistosoma japonicum Development].

OBJECTIVE: To investigate the effect of lipopolysaccharide (LPS)-induced B cell activation on the development of Schistosoma japonicum. METHODS: Eighteen BALB/c nude mice deficient in T cells and 23 BALB/c SCID mice deficient in T and B cells were used in this study. Each was infected with 30 ± 1 S. japonicum cercariae. The nude (n=9, NL group) and SCID (n=12, SL group) mice then received 2-3 (every two weeks) intraperitoneal injections with LPS (100 µg/mL, 0.2 mL for each mouse). The remaining nude(n=9, N group) and SCID (n=11, S group) mice received PBS injection as control. The mice were sacrificed on days 28 and 36 after infection (n=4/5, 4/5, 5/6, 6/6 for N, NL, S and SL groups, respectively), and adult worms were collected by hepatic portal vein perfusion. The collecting rate of the adult worms was calculated, the body-length measured, and pairs of worms recorded. The liver tissue was collected and digested with 5% KOH, and the number of eggs per gram of liver tissue was calculated. The levels of TGF-ß, IFN-γ and IL-10 in peripheral blood were evaluated. Spleen cell suspension was prepared for detecting the proportion of regulatory B cells (Bregs) in splenic lymphocytes. RESULTS: On day 28 after infection, the body-lengths of male worms in NL and N groups were (7.65±2.85) mm and (5.28±1.64) mm (P<0.01), and those of female worms were (9.64±1.99) mm and (7.49±1.63) mm (P<0.01), respectively. On day 36 after infection, the number of eggs per gram of liver tissue was significantly higher in the NL group than in the N group (1 088±297 vs 715±404, P<0.05), and significantly lower in the SL group than in the S group (217±33 vs 573±160, P<0.01). The proportions of CD(hi)CD5(+)CD19(+) Bregs in N group on days 28 and 36 after infection were (12.73±0.96)% and (37.15±3.04)% (P<0.05), respectively, with no significant difference with that of NL group. The serum levels of TGF-ß and IFN-y on day 28 after infection were significantly different between N and NL groups (TGF-ß, 101.75±46.72 vs 260.90±45.34 pg/mL; IFN-y, 7.91±1.62 vs 14.11±3.72 pg/mL, both P<0.01). Similarly, significant difference was found for the plasma level of IL-10 on day 36 after infection between the S and N groups (41.85±3.14 vs 66.25±4.16 pg/mL, P<0.01), and between the SL and NL groups (44.48±3.87 vs 72.22±17.76 pg/mL, P<0.01), but not between the LPS groups and the control groups. CONCLUSION: LPS can induce the release of cytokines (e.g. TGF-ß) from B cells of mice infected with S. japonium, to facilitate the early development of adult female and male worms.

Effect of cytotoxic T-lymphocyte-associated protein 4 on CD4(+)CD25(+) regulatory T cells in murine schistosomiasis japonica.

In a previous study we demonstrated that CD4(+)CD25(+) regulatory T cells (Tregs) contributed to the escape of Schistosoma japonicum (S. japonicum) from the host's immune responses. In this paper, we studied the effect of cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on CD4(+)CD25(+) Tregs in murine Schistosomiasis japonica and its corresponding role in the immune evasion of S. japonicum in mice. The results showed substantial reductions of worm burden and egg production in worm groups treated with anti-CD25 or anti-CTLA-4 monoclonal antibodies (mAb) compared to an infected but untreated control. The reduction effect was even enhanced in an experimental group co-treated with both mAbs. Compared to the control group, the percentage of CD4(+)CD25(+) Tregs was very much lower in the anti-CD25 mAb group as determined by FACS analyses and higher in the anti-CTLA-4 mAb group. ELISA analyses showed that both the anti-CTLA-4 mAb and the co-treated groups had higher levels of cytokines compared to the control group as well as larger egg granuloma sizes as determined by microscopical analyses of liver sections of infected mice. These results suggest that treatment with an anti-CTLA-4 mAb allows the host to clear S. japonicum, but at the cost of elevated pathological damage. The latter indicated a role of CTLA-4 in granuloma formation. Moreover, CD4(+)CD25(+) Tregs and CTLA-4 may exert synergistic effects during immune evasion processes by enhancing Th1-type immune response.

Cloning and characterization of a bone morphogenetic protein homologue of Schistosoma japonicum.

Bone morphogenetic proteins (BMPs) are known to play an important role in the regulation of cell proliferation, survival, differentiation and apoptosis in many vertebrates and invertebrates through the TGF-ß signaling pathway. Although the TGF-ß signaling pathway exists in schistosomes, BMP homologue, a ligand of TGF-ß in Schistosoma japonicum, has not yet been identified. In this study, a BMP homologue of S. japonicum was cloned and characterized. The full length SjBMP cDNA is 3,020 bp and encodes 928 amino acids, which include a TGF-ß superfamily conserved domain at the C-terminus. BLAST analysis showed that, SjBMP has 68%, 51% and 43% homology with BMP from Schistosoma mansoni, Schmidtea mediterranea and Dugesia japonica at the amino acid level, respectively. According to data from real-time PCR, SjBMP was expressed in lung-stage schistosomula, 21-day liver-stage schistosomula, 50-day adult worms (the male and female), and eggs. The PCR data also indicated that, there was a ≈ 27- and ≈ 37-fold increase of SjBMP transcripts in the lung-stage schistosomula and eggs, respectively, and that there was relatively more SjBMP transcript in the adult male worm than in the adult female, in which the hepatic schistosomula was set as the calibrator for calculation. In situ hybridization based on FITC-labeled specific antisense oligonucleotide probes showed that SjBMP mRNA localized to the ovary of female worms and the integument and epithelium of female and male worms. After treatment with double-stranded RNA (dsRNA) at a concentration of 8 × 10(-2) µg/ml, which was added to the culture medium every other day for a week, the level of SjBMP mRNA in the cultured adult mixed-sex S. japonicum decreased at a range of ≈ 25-98% within 7 days compared with the level of SjBMP mRNA in the blank control group. On the 2nd day, the number of eggs produced per pair of worms decreased 28.7%, and the percent of normal eggs also decreased (12.7% vs. 4.3%) in the SjBMP dsRNA-treated group when compared with the eggs laid by the blank control group. No difference was detected between the two groups on the 7th day of treatment, because the eggs of the untreated worms were also mostly abnormal, similar to the eggs laid by the treated group. In addition, no significant difference in the morphological structure of the adult worms was observed. Thus, the preliminary in vitro experiment indicated that SjBMP may be involved in the oviposition behavior of S. japonicum, and further studies based on the recombinant virus vector-induced steady knockdown of SjBMP or in vivo experiments are required for more in-depth investigation.

The new national integrated strategy emphasizing infection sources control for schistosomiasis control in China has made remarkable achievements.

Schistosomiasis japonica remains one important public health concern that cause great loss of humans' health and social-economic development in the Peoples' Republic of China. At the end of 1990s and the beginning of 2000s, there were still about 0.8 million patients and nearly 85 million people living in the epidemic areas around China. We undertook full analysis of the epidemiological data of schistosomiasis taken from the report of schistosomiasis status in People's Republic of China from 1999 to 2010 for effectiveness assessment of China's new strategy for schistosomiasis control nationwide after its implementation since the beginning the 21st century. The schistosomiasis-endemic uncontrolled counties or towns decreased in number from 1,149 in 2002 to 643 in 2010 at a rate of 44%. The number of schistosomiasis patients decreased from nearly 800,000 to less than 326,000 in 2010 at a decrease rate of more than 50%. The number of acute schistosomiasis patients also decreased significantly, and only 43 cases were reported in 2010. The infection rates of cattle in the endemic uncontrolled provinces decreased greatly though the number of cattle and the actual snail habitat areas remained large with no obvious decline. The schistosome infection rates of human and cattle both decreased significantly by more than 64% and 75%. However, most of the uncontrolled schistosomiasis-endemic areas, schistosomiasis patients, and acute cases are generally located in the four provinces (Hunan, Hubei, Jiangxi, and Anhui) of the lake regions in the middle and lower reach of the Yangtze River, and the egg-positive rates in diagnosed human in endemic Hunan and Hubei remained higher than 10%. Therefore, the new strategy of schistosomiasis control via integrated measures emphasizing infection source control is scientific and successful around China, though it is essential to explore an effective and sustainable strategy for schistosomiasis control in the tough lake and marshland regions of China. The four provinces (Hunan, Hubei, Jiangxi, and Anhui) of the lake regions in China are the main battlefield of China's schistosomiasis control in the present and future.

Identification of differentially expressed genes in Oncomelania hupensis chronically infected with Schistosoma japonicum.

Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum. The schistosome-snail interaction is biomedically important. To identify differentially expressed transcripts in O. hupensis chronically infected with S. japonicum, suppression subtractive hybridization (SSH) was used to construct a cDNA library in each direction for transcripts that are more abundantly enriched in head-foot part of the infected O. hupensis and for those that are more abundantly enriched in the uninfected, as head-foot part contains hemocytes and hemolymph which are associated with the snail internal defense system. After differential screening, 39 transcripts were identified, including nine and 30 transcripts enriched in infected and uninfected snails, respectively. Some of the transcripts have similar homology to available sequences in current databases, including transposase, caveolin-like protein, pancreatic trypsin inhibitor-like protein, prosaposin, glutathione s-transferase (GST), and several hypothetical proteins, while most of the transcripts do not match with any sequences in available databases. The identified transcripts were involved functionally in cell growth, metabolism, signal transduction, and immune responses. Two forward library transcripts and 11 reverse library transcripts were selected for real-time PCR, and 10 of them were confirmed to be consistent with the SSH results. It is intriguing to continue functional studies for some genes such as pancreatic trypsin inhibitor; a hypothetical protein (HS576367) related to calcium ion binding; GST; and several unknown proteins (HS576353 and HS576355). These identified differentially expressed genes may be key targets for understanding the molecular mechanism of co-existence during which the snail is unable to rid itself of the schistosome in chronic infection stage.

What is the role of health education in the integrated strategy to control transmission of Schistosoma japonicum in China?

In 2009, Wang et al.'s field trial published in the New England Journal of Medicine, reported that a comprehensive strategy aiming to reduce the roles of humans and cattle as sources of Schistosoma japonicum infection in snails was implemented and proved effective and promising in dramatically reducing the percentage of infected humans and snails, which had been extended to other endemic provinces in China afterwards. This implies that the integrated schistosomiasis-control strategies of interventions including political will, financial support and residents' participation to control human and bovine sources of S. japonicum infection in snails may direct to successfully interrupt the parasitic transmission and to ultimately eliminate schistosomiasis. Confusingly, however, the role of health education, which is a critical part of the integrated strategy and should play an active role in schistosomiasis control, was not reflected. We wish the authors to provide the readers a better and clearer statement of the role of health education as part of the integrated control strategy and so we write this comment.

Artemisinin: the gifts from traditional Chinese medicine not only for malaria control but also for schistosomiasis control.

The 2011 Lasker~DeBakey Clinical Medical Research Award honors the Chinese scientist Tu Youyou who discovered artemisinin and its utility for treating malaria. A therapy based on artemisinin has saved millions of lives across the globe, especially in the developing world. Meanwhile, artemisinin and its derivatives, especially for artemether and artesunate, are showing promising preventive efficacies as high as 65-97% administrated with multiple doses at 6 mg/kg body weight by 1- or 2-week intervals for preventing schistosomiasis japonica during epidemic seasons used in China for more than one decade. So, we would like to say, to our excitement, artemisinin and its derivatives are the gifts from traditional Chinese medicine not only for malaria control but also for schistosomiasis control.

The sensitivity of artesunate against Schistosoma japonicum decreased after 10 years of use in China?

Artesuante (AS) is a good chemoprophylactic drug for preventing against schistosome infection, but Hua et al. recently reported that the sensitivity of AS against Schistosoma japonicum decreased after 10 years of use in China. There are at least three problems, to our knowledge, making the finding suspicious or inconclusive in that report. In consideration of it as the first report about the emergence of potential artemisinin derivative-resistant S. japonicum to date and the possible severe influences of the emergence of AS-resistant S. japonicum on the future choice of chemoprophylactic drugs for preventing against S. japonicum infections in China, we write this comment and call for some more rigorous and convincing trials to confirm this finding further.

Effects of protein extract from head-foot tissue of Oncomelania hupensis on the growth and gene expression of mother sporocysts of Schistosoma japonicum.

Oncomelania hupensis is the intermediate host of Schistosoma japonicum. In the present study, we investigated the effects of protein extracts from head-foot or gland tissue of O. hupensis on mother sporocysts of S. japonicum cultured in vitro. In the presence of head-foot protein extract of snails from the native province Hunan, in-vitro-transformed mother sporocysts presented not only a longer survival time and stronger motility, but also a bigger size than parasites cultured with protein extracts of glands of the same snail or head-foot tissue of a non-native snail from the Hubei province. Using suppression subtractive hybridization, two subtractive libraries were constructed on the basis of RNA of sporocysts cultured with or without native snail head-foot protein extract. A number of 31 transcripts were found to be up-regulated. Sequence analyses revealed that they represented genes involved among others in metabolic process, electron transport chain, response to chemical stimulus, and oxidation-reduction processes. Opposite to that 20 down-regulated transcripts were among others related to pseudouridine synthesis, RNA processing, and ribosome biogenesis. The differential expression of three of these transcripts, encoding cytochrome c oxidase subunit 2 (Cox2), NADH-ubiquinone oxidoreductase (ND1), and dyskeratosis congenita 1 protein (DKC1), were confirmed by real-time PCR. The promoted development and the differential gene expression of cultured sporocysts under the influence of head-foot protein extract of native O. hupensis implied not only its ability to improve in vitro culture conditions for intramolluscan stages, it may also represent a priming result with respect to the identification and characterization of factors involved in the parasite-host interplay between S. japonicum and O. hupensis.

Studies on the establishment of a co-culture system of lung stage Schistosoma japonicum with host cells.

Due to their role in eliciting protective Th1 cell-mediated immune responses in definitive hosts lung stage schistosomula are in the focus of intensive research. In vitro culture approaches in the past exhibited significant differences in gene expression profiles between lung stage schistosomula isolated from hosts and those cultured conventionally. Therefore, new approaches to culture schistosomula are of broad interest. In the present study, co-culture systems of schistosomula of Schistosoma japonicum and different vertebrate host cells were tested. Among these, human hepatic venous endothelial cells (ED25) turned out to be very suitable and interesting feeder cells. Compared with controls cultured in vitro or co-cultured with other cells, schistosomula co-cultured with ED25 cells shared more similarities in morphology and tegumental structures with schistosomula directly obtained from infected mice as microscopically determined. According to results from a suppression subtractive hybridization approach to compare transcriptional differences of co-cultured and host group or control group parasites, four candidate transcripts encoding cathepsin L precursor, heat shock protein 70, glyceraldehyde 3-phosphate dehydrogenase, and programmed cell death protein 10 were shown to be differently expressed among the three groups by real-time PCR. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis finally confirmed not only congruent protein patterns but also interesting differences among the compared schistosomula groups. The co-culture system between schistosomula of S. japonicum and ED25 cells established in the present study improved existing cultivation attempts. Although some differences to host-derived schistosomula were still observed, co-culture with ED25 cells positively influenced parasite morphology and gene expression in a more host-like manner.

[An easy way to purify the inclusion body protein with high purity from prokaryotic expression cells].

To clone partial ORF of SjBMP and to construct the recombinant SjBMP-pET-28a(+) plasmids, and then to transform them into the competent cells E. coli BL21 (DE3), finally a positive clone was used to be induced by IPTG. The bacterial aggregates with target protein expressed as inclusion bodies were purified by the methods of Ni(2+)-NTA affinity purification under denaturation condition and SDS-PAGE gel extraction. The purified protein was used to immune rabbits and make antiserum against the SjBMP, and the antiserum were then used to identify the rSjBMP by Western blotting. The target protein obtained by Ni(2+)-NTA Agarose affinity purification was not pure with unspecific proteins, but the protein further purified by SDS-PAGE gel extraction and the dialysis bag horizontal electrophoresis was quite pure, and the recovery rate was more than 11.0%. Meanwhile, Western blotting was used to identify the recombinant SjBMP protein by antiserum, only a specific single strip appeared, which suggested the protein purified by this method kept its antigenicity, and could be used for common immunological studies. Therefore, the SDS-PAGE gel extraction combining with electroosmosis and dialysis recycling are good and easy to purify the inclusion body proteins.

Conservation and variation in mitochondrial genomes of gastropods Oncomelania hupensis and Tricula hortensis, intermediate host snails of Schistosoma in China.

The complete mitochondrial genomes of intermediate host snails for Schistosoma in China were sequenced, including the sub-species Oncomelania hupensis hupensis in two types, and O. hupensis robertsoni, intermediate hosts for S. japonicum, and Tricula hortensis, the intermediate host of S. sinensium. Four genomes have completely the same gene order as in other caenogastropods, containing 13 protein-coding genes and 22 transfer RNAs. The gene size, start codon and termination codon are mostly the same for all protein-coding genes. However, pairwise sequence alignments revealed quite different degrees of variation. The ribbed-shelled O. hupensis hupensis and the smooth-shelled but with varix O. hupensis hupensis had a lower level of genetic distance (3.1% for protein-coding genes), but the coden usages differed obviously in the mitochondrial genomes of these two types of snails, implying that their genetic difference may be larger than previously recognized. The mean genetic distance between O. hupensis hupensis and O. hupensis robertsoni was 12% for protein-coding genes, indicating a higher degree of genetic difference. In consideration of the difference in morphology and distribution, we considered that O. hupensis hupensis and O. hupensis robertsoni can be considered as separate species. The ribbed-shelled O. hupensishupensis and smooth-shelled O. hupensis robertsoni were phylogenetically clustered together within a same clade, which was then clustered with T. hortensis, confirming their close relationship. However, species or sub-species in the Oncomelania from southeastern Asian countries should be included in future study in order to resolve the phylogenetic relationship and origination of all snails in the genus.

[Research progress on the antioxidant enzyme family in medical trematode].

Reactive oxygen species (ROS), generated in the metabolism process of aerobic organisms, can induce oxidative damages in the body. These organisms are all equipped with an excellent defense system to protect themselves and antioxidant enzymes play an important role in the system. Parasitic trematodes have to eliminate ROS not only from themselves but also from the immune system of their hosts. To better understand the structures and specialties of the antioxidant enzymes in trematodes is conducive to the study on reproductive physiology of trematode and on drug and vaccine development. This paper summarizes the research progress on the family of antioxidant enzymes in trematodes including glutathione peroxidase (GPx), superoxide dismutase (SOD) and peroxiredoxin (PRx) in the past decades.

[In vitro effect of 5-hydroxytryptamine on motility and length of Schistosoma japonicum primary sporocysts].

OBJECTIVE: To study in vitro the effect of 5-hydroxytryptamine (5-HT) on the motility and length of primary sporocysts of Schistosoma japonicum and select the optimal concentration and time of 5-HT. METHODS: Eggs of S. japonicum were harvested from livers of the infected mice 6-8 weeks after infection, which were then incubated with water. The miracidia were axenically cultured in 1/2 RPMI 1640 containing 10% calf serum and a moderate amount of antibiotics (100 U/ml penicillin G and 100 microg/ml streptomycin) for 48 hours. They became mother sporocysts and were divided into two groups. Parasites in the first group were treated by 5-HT under concentrations of 0, 0.1, 1, 10, 100 and 1000 micromol/L for 48 hours, respectively. Those parasites in the second group were treated by 5-HT of 10 micromol/L for 0.16, 6, 24 and 48 hours, respectively. The motility, length and succinic dehydrogenase (SDH) activity of the parasites were measured under Olympus microscope. RESULTS: Along with the increase of 5-HT concentration, the motility and length of the mother sporocysts all increased gradually. With the 5-HT concentration of 10 micromol/L, both of them reached a maximum value, being (65.6 +/- 1.5)% and (131.4 +/- 9.2) microm respectively. Meanwhile, along with the prolongation of treatment time, the motility and length also increased gradually. The motility reached a maximal value at 24 hours post-treatment. So did the length and SDH activity of the parasites at 48 hours post-treatment. CONCLUSION: 5-HT shows a significant effect on the motility and length of mother sporocysts of S. japonicum and its optimal concentration is 10 micromol/L under in vitro conditions.

Influence of enterococci on human sperm membrane in vitro.

AIM: To study the influence of enterococci on human sperm membrane in vitro. METHODS: Ejaculated human sperm were artificially infected with beta-hemolytic or non-beta-hemolytic enterococci at the bacteria: sperm ratio of 50:1 at 37 degrees . Sperm membrane integrity was examined after incubation for 1, 3 and 5 h by hypoosmotic swelling (HOS) test and electron microscopy. RESULTS: Sperm infected with beta-hemolytic enterococci had lower HOS scores compared with non-beta-hemolytic strains or uninfected control (P < 0.01). The HOS test scores of sperm infected with beta-hemolytic enterococci increased in the presence of phosphatidylcholine, an inhibitor of hemolysin. Non-beta-hemolytic strains showed no significant difference in swelling rate, compared with the control group (P > 0.05). It was shown by electron microscopy that beta-hemolytic enterococci caused significant rupture of human sperm membrane. CONCLUSION: Beta-hemolytic enterococci caused human sperm membrane injury, and might be mediated by the hemolysin of enterococci.

[Primary culture of the cells from Oncomelania hupensis liver].

OBJECTIVE: To develop a method for primary culture of cells from Oncomelania hupensis liver, and to observe the distribution of succinate dehydrogenase (SDH) and lactate dehydrogenase (LDH) in the cultured cells. METHODS: O. hupensis was anatomized to separate the liver. Livers were soaked in 0.2% benzalkonium bromide and washed by physiological saline containing antibiotics in turns. Cells from the liver were harvested by mechanical mulling and filtering. The isolated cells were then incubated with methods of the combination culture and standing suspension culture, respectively. The culture medium for the cells was a mixture of Medium 199 (50 ml), 0.3% lactoalbumin hydrolysate dissolved in a balanced salt solution (BBS, 30 ml), and fetal calf serum (FCS, 20 ml) containing a moderate amount of antibiotics (100 IU/ml penicillin, 100 microg/ml streptomycin and 50 microg/ml kanamycin) at pH 7.2-7.4 under the temperature of 26.5 degrees C. The cells were stained by using Giemsa and Pearson methods (for SDH and LDH respectively) to observe the shape of cultured cells and enzyme distribution in cells. The living and stained cells were microscopically observed. RESULTS: Under microscope, the attached cells incubated with method of the combination culture showed round, elliptic, triangular and irregular shapes, with more round and elliptic cells. The size was approximately (4-16) microm x (6-20) microm in average. The clustered cells with an unclear nucleus and abundant and lucid cytoplasm were smaller than diffused cells with a large, obvious nuclei and less cytoplasm. Degeneration was observed after culturing for 5-7 days. The cultured cells could be divided into two types based on the color shown after Giemsa staining. The first type cells showed blue cytoplasm and mauve nuclei while the second type cells were opposite. There were blue granules in different sizes and shade in the cytoplasm after SDH and LDH staining. It was difficult for the cells to attach the wall of the culture flask using method of the standing suspension culture. The shape of the cultured cells were almost round with unclear nuclei, and the size was about (4-6) microm x (6-8) microm in average. The cells incubated with the standing suspension method were found to be contaminated after culturing for 3 days. CONCLUSION: The combination culture method is suitable for primary culture of the cells from O. hupensis liver and the cells show activities of both SDH and LDH in cytoplasm.

A membrane-associated metalloprotease of Taenia solium metacestode structurally related to the FACE-1/Ste24p protease family.

The CaaX proteases are intimately involved in the post-translational modification of prenylated proteins and play a critical role in the activation/stabilization of membrane-bound or secreted molecules constituting the CAAX protein family. In this study, we have isolated a full-length cDNA putatively encoding a type I CaaX protease of the Taenia solium metacestode (TsM), which an agent causative of human neurocysticercosis. The cDNA, designated TsSte24p, comprised 1,505 bp and coded for an open reading frame of 472 amino acids with predicted Mr 54.5 kDa. This monoexonic TsSte24p gene existed as a single copy within the TsM genome and constantly expressed in the parasite from metacestode to adult stages. The TsSte24p exhibited the typical CaaX protease topology, including seven transmembrane domains and a metalloprotease segment with a zinc-binding motif. It shared a significant degree of sequence identity with the type I CaaX proteases such as Saccharomyces cerevisiae Ste24p and Caenorhabditis elegans CeFACE-1. A comparative phylogenetic analysis demonstrated that this protein family is tightly conserved across taxa, from bacteria to mammals. The bacterially expressed recombinant TsSte24p showed proteolytic activity, with an optimal pH of 7.5. The enzyme activity was significantly inhibited by EDTA. Its activity was increased in the presence of low concentrations of the Zn2+(0.001-0.01 mM); but was reversibly down-regulated at high doses (over 0.1 mM). The native TsSte24p appeared to function as a homodimer, the subunits of which were linked to each other via covalent disulfide bond. The protein was localized in the bladder wall and scolex with differential patterns of distribution. Our results indicated that TsSte24p is a zinc-dependent metalloprotease, which belongs to the FACE-1/Ste24p protease family.

[Proteomics and its application in parasitology].

Proteomics is an important high throughout method in modern life science. In this paper, the definition, background and methods used in proteomics were introduced, and the last part was focused on its application in parasitology.

[Effect of chronic Toxoplasma infection on the spatial learning and memory capability in mice].

OBJECTIVE: To investigate the effect of chronic infection of Toxoplasma gondii on the spatial learning and memory capability in mice. METHODS: Toxoplasma tachyzoites (RH strain) were reanimated at 37 degrees C after 15 days' storage at -20 degrees C, and injected intraperitoneally to mice of the experimental group each with 7.7 x 10(5). Normal saline was given to the control group, 0.5 ml per mouse. Two months later, all mice were tested in the Morris Water Maze. Smears of the mice brain homogenate and pathological sections were examined. RESULTS: (1) The density of cysts in the brain homogenate was 15/HP, and there was no evident pathological change in the hippocampus and adjacent areas of mice in the brain in the experimental mice. (2) Latency to platform, cumulative distance to the platform, total distance traveled in both experimental and control groups decreased significantly with the increase of training days (P < 0.01). The latency and cumulative distance in experimental group were significantly longer than that of the control group (P < 0.01). (3) The searching strategy of mice in the experimental group was significantly different from that of the control group. CONCLUSION: Toxoplasma tachyzoites can induce chronic infection in mice and the infection can damage at some extent the spatial learning and memory capability of mice.