Table of Content

30 December 2001, Volume 19 Issue 6

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论著

Cloning and Characterization of Three Novel Genes Encoding Transmembrane Proteins of Schistosoma japonicum

ZHOUDongming;YIXinyuan;ZENGXianfang;ZENGQingren;ZHOUJinchun;WANGMin;ZHANGShunke;HUANGFushen

2001, 19(6):  1-324. 

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　Objective To clone and analyze novel antigen molecules of Schistosoma japonicum (Sj), and to provide effective vaccine candidate antigens against schistosomiasis japonica. Methods Sj adult cDNA library was screened using sera of mice infected with Trichinella spiralis (Ts) and the inserts of positive clones were specifically amplified by PCR. The positive clones were sequenced and the sequence data were analyzed using Nucleotide BLAST software of NCBI and Expert Protein Analysis System of Swiss Institute of Bioinformatics. Results Nine positive clones were obtained after three rounds of immunoscreening. The size of these inserts ranged from 0.6 kb to 2.1 kb. Among five novel genes, Sj Ts1, Sj Ts3 and Sj Ts5 (GenBank accession number: AY005816, AF299080 and AY024352,respectively) encode transmembrane proteins with 83, 83 and 233 amino acids, respectively. Sj Ts1 protein predicted contains one possible transmembrance helix, one N myristoylation site, two phosphorylation sites for protein kinase C and one for tyrosine kinase, Sj Ts3 protein contains two possible transmembrance helices and one casein kinaseⅡphosphorylation site, whereas Sj Ts5 protein has five possible transmembrance helices, one N glycosylation site, one N myristoylation site, two phosphorylation sites for cAMP and cGMP dependent protein kinase and four for protein kinase C and one for casein kinase Ⅱ. Conclusion Three novel genes encoding three transmembrane proteins might be developed as new vaccine candidates against Sj infection.


Enrichment and Screening of Up-regulated Genes of the Mosquito Anopheles stephensi in Response to Malaria Parasite

XUXiaochun;QUFengyi;SONGGuanhong;XUJiannong

2001, 19(6):  2-329. 

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　Objective To isolate and identify genes related to malaria parasite infection in vector mosquito, and to explore the mechanisms. Methods Anopheles stephensi infected with Plasmodium yoelii was used as tester (T) group, while uninfected but normal blood fed as driver (D) one. Engorged female mosquitoes of two groups were collected separately at 24 hours after biting. An enriched subtractive cDNA pool was generated through the course of suppression subtractive hybridization (SSH) and selective PCR amplification. The subtracted library was screened by hybridization using T and D cDNA mixture as probes, respectively. The positive clones, which produced stronger signal when probed with T than with D, were sequenced and their sequence homologues in GenBank database were searched with BLAST by internet. Results The analysis of subtraction efficiency showed that the differentially expressed genes in T comparing to in D were enriched significantly. In dot blot screening, 24 of 58 randomly selected clones (41.4%) were shown up regulation in malaria infected mosquitoes. The BLAST search of 23 genes revealed that 12 were homologous to functionally known genes, 4 were homologous to functionally unknown entries, and 7 were novel without any relatives. Nine of the 23 genes (39.1%) also hit homologous sequences in the An. gambiae EST database generated from an immune competent cell line treated with lipopolysaccharide (LPS). Conclusion An enriched cDNA pool of the mosquito genes which up regulated responsively at the early stage of malaria parasite infection was obtained. Expression screening against the pool indicated that various biochemical processes and mechanisms might be involved in the response of mosquito to parasite infection, especially those related with the innate immune system and energy metabolism.


Observation on Hemocytes of Culex pipiens quinquefasciatus Larvae Infected by Lagenidium giganteum

MOFei;BAOHuaien

2001, 19(6):  3-332. 

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　Objective To explore the possible defensive immunity of Culex pipiens quinquefasciatus larvae after being infected by Lagenidium giganteum . Methods To count and analyze morphologically hemocytes of the mosquito larvae with Giemsa's stain and by both phase contrast microscope and ordinary microscope. Results ① Following the prolonged duration of infection, the number of hemocytes featured as increase, intense increase and gradual decrease. ② 48 and 72 h after infection the proportion of plasmatocytes and granulocytes increased significantly, but that of prohemocytes showed a relatively slow increase, while 96 and 120 h after infection the proportion of spherulocytes and oenocytoids increased gradually and that of plasmatocyte and granulocytes showed a slow decrease. ③ After L. giganteum infection, vacuolation and morpholysis were found in plasmatocytes. Conclusion The changes of hemocytes in amount,classification and morphology after infection revealed that Lagenidium giganteum can induce some defensive immune reactions in the larvae of Culex pipiens quinquefasciatus possibly due to a release of antigens or toxic substances by the fungus.


Inhibition of in vitro Translation of Esterase mRNA of Dipterex-Resistant Mosquito (Culex pipiens pallens) by Antisense Nucleic Acids

ZHUHuaimin;ZOUYading

2001, 19(6):  4-335. 

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　Objective To examine the inhibitory effect of antisense nucleic acid on the in vitro translation of esterase mRNA from dipterex resistant Culex pipiens pallens . Methods 18 mer nucleic acid was synthesized and complementary to the translation initiation site of mRNA of dipterex resistant mosquitoes. The ODNs were annealed to the corresponding mRNA molecules and they were added to rabbit reticulocyte cell free system. The translation products were analyzed by SDS PAGE. After fixing, the gel was exposed to X ray film by autoradiography for analysis of protein synthesis. Results Six μmol/L of ODNs elicited a 50% reduction in specific protein expression , and 20 μmol/L of ODNs inhibited the expression of esterase by 80%. The SDS PAGE showed that the band of reduced amounts of 65 kDa protein for resistant mosquito was almost the same as that for sensitive sample. Conclusion Antisense oligonucleic acids to the esterase mRNA of dipterex resistant mosquito could effectively inhibit its in vitro translation.


Protective Immunity Induced by 23 kDa Membrane Protein DNA Vaccine of Schistosoma japonicum Chinese Strain in Mice

RENJiangong;ZHUYinchang;D.A.Harn;YUChuanxin;YINXuren;SIJin;HEWei;XUMing;HUAWanquan;XUYongliang

2001, 19(6):  5-339. 

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　Objective To develop 23 kDa membrane protein DNA vaccine of Schistosoma japonicum Chinese strain and test its protective efficacy in infected C57BL/6 mice. Methods The full length cDNA encoding SjC23 amplified from pUC19 SjC23 subcloned into pcDNA3.1. 48 female mice were divided into three groups: A, B and C. Group A (control group) was each immunized im with 100 μg of pcDNA3.1; group B (SjC23 group) was each immunized im with 100 μg of pcDNA3.1 SjC23; group C (SjC23+IL 12) was each immunized im with a mixture of 100 μg of pcDNA3.1 SjC23, 100 μg of pcDNA3.1 p35 and 100 μg of pcDNA p40, followed by two boosts of the same DNA once every two weeks. All the mice were challenged with 45 cercariae at week 8, killed and perfused for worms at week 14. The expression of SjC23 and p35, p40 in muscle tissue was determined by immuno histochemical method. By the culture of spleen cells, the production of IL 2,IL 4,IL 10 and IFN γ after the stimulation of rSjC23 HD was determined two weeks before and after challenge. Anti SjC23 antibodies were tested by Western blotting. Results SjC23 and p35, p40 were all expressed on the membrane and in the plasma of muscle cells of the infected mice. Significant increase of IL 2 and IFN γ in SjC23 and SjC23+IL 12 groups was observed before and after challenge. Western blotting showed that after the third immunization (before challenge) 8 out of 10 sera from SjC23 group and 9 out of 10 sera from SjC23+IL 12 group were positive. The worm reduction rate in SjC23 group and SjC23 +IL 12 group was 26.9% and 35.4%, respectively; the number of eggs in liver tissue was reduced by 22.2% and 28.4%, respectively. Conclusion pcDNA3.1 SjC23 DNA vaccine could induce partial protection against Schistosoma japonicum in C57BL/6 mice.


Environmental Investigation in Areas where Anopheles anthropophagus Distributed in Hubei Province

HUANGGuangquan;LIHanfan;ZHANGHuaxun;LIUJingyuan;ZHANGJibin;CHENGuoying;YUPinghong;MINGGuizhen

2001, 19(6):  6-343. 

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　Objective To investigate the natural and socio economical environment in the areas where Anopheles anthropophagus distributed in Hubei Province. Methods 5% of the villages in distribution area of An. anthropophagus were randomly selected for the investigation which included vegetation, soil, water, temperature, farming, resting places of the mosquito. Results The vegetation of An. anthropophagus area consisted of forest, bush,sod and crops. The area was densely covered with river, ditches and ponds, the water pH being 6.1～7.7. The soil texture was either yellow, yellow brown, grey or black, contained 2.72% organic material. There were one or two seasons of rice plantation in the area and the amount of insecticide used was 0.828 kg/mu in the rice field. The annual average temperature was 16.9 ℃ and the humidity was 76.9%. The breed period of An. anthropophagus was from June to September. There are two peaks of mosquito population in area with two seasons of rice plantation, and one peak in one rice season. The composition of An. anthropophagus resting in households, cowsheds and pigpens was 80.9%,12.2% and 6.0%, respectively. Conclusion The distribution of An. anthropophagus was in point, flaky or belt form in low hill, hillock, shallow hilly plain. These areas were full of vegetation and source of water, the principal crop was single cropping of rice, and soil texture is yellow or yellow brown. The major resting place of the mosquito was human dwellings.


The Effect of Nitric Oxide Donor on the DNA Content in Toxoplasma gondii Tachyzoite

PENGBiwen;HUJianshi;ZHAORong;JIANGMingsen;LINJianyin

2001, 19(6):  7-347. 

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　Objective To determine the role of sodium nitroprusside (SNP) in regulating DNA synthesis of Toxoplasma gondii tachyzoites. Methods Hypodiploid peak of tachyzoite DNA induced by SNP was assessed according to DNA fragmentation. The effect of SNP on appearance of hypodiploid peak and the effect of Ca 2+ on the growth of tachyzoites were evaluated. The intracellular Ca 2+ chelator (BAPTA/AM), antagonist of Ca 2+ channel(verapamil) and the extracellular Ca 2+ chelator (EGTA) were used. The change of DNA content was measured by flow cytometry. Results SNP inhibited DNA synthesis of tachyzoites in a dose and time dependent pattern. The antiproliferative effect of SNP on tachyzoites was inhibited by verapamil, EGTA and BAPTA/AM. The inhibition of the growth of tachyzoites by SNP was associated with increased subploid peak through a Ca 2+ dependent mechanism. Conclusion SNP induced a hypodiploid peak in tachyzoites by altering the Ca 2+ concentration in the plasma of tachyzoite, resulting in damages of the parasite.


Climbing-upward Inhibition of Oncomelania hupensis by Niclosamide Combination

ZHUDan;XIEFaxian;BAOZiping;ZHUDabei;NIChuanhua

2001, 19(6):  8-350. 

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　Objective To develop a combination of molluscicide/insecticide with a good molluscicidal effect, especially an inhibitory effect on climbing upward of Oncomelania hupensis . Methods Experiments on molluscicidal and snail climbing inhibition effect of the combination of niclosamide and shachongding [(dimethylamine) trithacyclohexane hydrochloride] were conducted by immersion and spraying in laboratory. Results The combination of niclosamide (0.2 mg/L) and shachongding (0.1 mg/L) showed an inhibition of 92.67% of snails to climb up. There was no significant difference in molluscicidal effect between the combination (LC 90 0.198 mg/L) and niclosamide alone (LC 90 0.207 mg/L). Conclusion The combination showed an effective inhibition on the climbing upward of snails in water so as to improve the molluscicidal effect and reduce the cost of mollusciciding.


Effect of Acryl Thiourea on Liver Pathologic Changes in Mice Infected with Schistosoma japonicum

HELi;JIANGMingsen;CAIGuobin;YANGMengxiang;YIXinyuan;ZENGXianfang

2001, 19(6):  9-353. 

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　Objective To observe the effect of acryl thiourea, an inhibitor of phenol oxidase, on pathological changes in the liver of mice infected with Schistosoma japonicum . Methods From day 22 to day 42 postinfection with cercariae, the mice of the acryl thiourea group were each injected i.p. with acryl thiourea at a dose of 300 mg/kg every other day. The mice were killed on the 42nd day postinfection to observe the pathological changes in the liver. Results Compared to the infected control group, the liver tissue of the acryl thiourea group showed scattered foci of inflammatory cell infiltration, the mean diameter and area of the foci were significantly reduced ( P <0.01), and there were no eggs in the center of the foci except for some granules. Conclusion After i.p. injections of acryl thiourea, no typical egg granuloma was found in the liver of infected mice. This was possibly due to the inhibition of schistosome phenol oxidase activity and so the female adult schistosomes could not produce normal eggs.


Application of Dot Immunogold Filtration Assay in Antibody Detection for Cysticercosis

LIUZhiping;LIHao;ZHULanyun;LIUSen;XUEHaichou

2001, 19(6):  10-356. 

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　Objective To detect serum antibody of cysticercosis by dot immunogold filtration assay for establishing a diagnostic kit for cysticercosis patients. Methods Cyst fluid of cysticercus of Taenia solium after dialysis was used as diagnostic antigen in dot immunogold filtration assay and enzyme linked immunosorbent assay to detect the serum antibody in patients with cysticercosis. Samples to be detected included 71 sera from patients with cysticercosis, 90 sera from healthy people, 20 sera from patients with other parasitic infections or brain tumor. Results The sensitivity and specificity of dot immunogold filtration assay were 90.1%(64/71)and 95.6%(86/90), respectively. No positive reaction was recorded in cases with other diseases except one serum from a patient with brain tumor. The coincidence rate between dot immunogold filtration assay and enzyme linked immunosorbent assay was 94.4%(152/161). Conclusion Dot immunogold filtration assay showed promising result for the diagnosis of cysticercosis.


Determination of T Lymphocytes and Trace Elements in Spleen from Rats Infected with Toxoplasma gondii

GENGZhihui;FANGYanqiu;LIULi;SHIYi;LIShuhong

2001, 19(6):  11-359. 

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　Objective To determine the level of five trace elements(Fe 2+ ?Cu 2+ ?Zn 2+ ?Ca 2+ ?Mg 2+ )in the spleen and changes of T lymphocyte and its subtype variations in peripheral blood from the rats infected with Toxoplasma gondii . Methods Twenty rats were randomly and equally divided into two groups: control group and experiment group. Each rat in the experiment group received an ip injection of 2 ml normal saline containing 1.5×10 6 tachyzoites of T. gondii . On the 64th day after injection of T.gondii , the changes in T lymphocytes (TL) and their subgroups, the helper T lymphocytes (Th) and the suppressor T lymphocytes(Ts) in the peripheral blood of the rats with T.gondii were determined by the assay of the lymphocytes labeled with intercellular acid α naphthyl acetate esterase. All the rats were killed and the atomic absorption method were used for detecting the level of trace elements in the spleen tissue. Results The number of TL and Th in experiment group was significantly lower than that of control ( P <0.01, P <0.01). The ratio of Th/Ts showed a significant difference between the two groups. The level of Fe 2+ ?Cu 2+ in experiment group was significantly reduced ( P <0.01). The amount of Mg 2+ in infected rats was higher than that of the control( P <0.01). No statistical difference in the content of Zn 2+ ?Ca 2+ was found between the two groups. Conclusion T.gondii infection might cause the changes in the TL and Th in peripheral blood and the changes in trace elements in spleen of the rats.


Improvement of Amplification Method for Cryptosporidium parvum Oocysts from Mice

HUANGKehe;YANGShiguang;TANGJianxia

2001, 19(6):  12-362. 

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　Objective To establish a simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from infected C57BL/6N mice. Methods All mice in the experimental groups were immunosuppressed by given different concentrations of dexamethasone phosphate added in drinking water throughout the experiment. The recovery and purity of the oocysts obtained using different purification methods was compared. The infectivity of the oocysts obtained from the same origin but different animals and different purification methods in a bovine fallopian tube epithelial cell culture system was studied. Results 4.16×10 9 oocysts were obtained in 30 mice in the 3rd group with dexamethasone of 20 μg/ml in drinking water. No significant difference in the oocyst recovery, purity and infectivity was found between methods using saturated saline floatation and sucrose density gradient centrifugation. The infectivity of the oocysts obtained from the same origin but different animals was similar. Conclusion A simplified amplification method for obtaining a large number of purified Cryptosporidium parvum oocysts from the infected mice was established.