Objective To reconstitute a transactivation system in yeast (yeast model) for screening the pesticides acting on ecdysone metabolism route and eventually influencing the process of ecdysis. Methods The fragment of 5 times repeated EcRE from Drosophila melanogaster was synthesized and the HSP27 promoter from D. melanogaster genome was amplified with PCR. The two sequences were connected and followed by a reporting gene——green fluorescence protein(GFP) gene. The EcRE-HSP27 promoter-GFP fragment was inserted into the expression plasmid pPIC3.5 and integrated into the yeast chromosome to construct yeast A. EcR and USP coding sequences of Aedes albopictus were synthesized, and these two fragments were inserted into Pichia pastoris expression plasmid pGAPZ as two respective reading frames. The two reading frames were integrated into Pichia pastoris chromosome in another recombinant site(pGAPZ and pPIC3.5k share different recombinant sites while being integrated into Pichia pastoris yeast chromosome). EcR and USP were constituted and expressed in the yeast. This recombinant yeast was called yeast B. The model yeast was thus constructed. A known ecdysone agonist-tebufenozide was used to test the yeast model. The effect of tebufenozide on the model yeast was observed under fluorescent microscope. Semi-quantitative RT-PCR was used to test the transcrip-tion level of GFP in the tebufenozide affected yeast and the control. Results In the model yeast, the intracellular expressed EcR and USP constituted EcR/USP heterodimer interacting with EcRE, the expression of GFP was activated, and green fluorescence was observed in model yeast under fluorescent microscope. Tebufenozide affected model yeast showed less fluorescence in comparison to the control model yeast, indicating that the transcription of GFP was suppressed by tubufenozide. Yeast housekeeping gene Actin-1 was used as inner control, semi-quantitative RT-PCR was operated and the result was scanned. The ratio of the brightness of GFP to Actin-1 was calculated automatically, and that of tubufenozide added yeast and the control yeast was 0.614 and 1.134 respectively. This result showed a low transcription level of GFP in tebufenozide affected model yeast, comparing to that of the control. Conclusion The ecdysone-related transacting system in yeast has been constructed, and the model yeast can be used to screen the ecdysone agonists which can act on the ecdysone metabolic route.

To prepare a large amount of pure alive Schistosoma japonicum eggs, rabbit was infected with 2000 cercariae and its liver was taken aseptically 38-45 days after infection and homogenized. The homogenate was screened through different sieves(60, 120, 200, 300, 360 meshes per inch respectively), and washed with 1.2% NaCl. The eggs and leftover were then digested with 0.25% trypsin for 2 hours, sieved over 360 meshes per inch and washed with RPMI 1640 medium. The collected eggs reached to (95.1?6.4)% of live eggs, with a high efficiency.

Objective To express and purify the Schistosoma japonicum adenylate kinase(AK) protein of which the cDNA sequence was subcloned into the pET32a(+) soluble expression plasmid, and to evaluate its immunoreactivity. MethodsThe recombinant plasmid was transformed into {E.coli} BL21, and induced with IPTG for expression. The bacterial lysis was conducted by ultrasonication and the supernatant was analyzed by SDS_PAGE after boiling and centrifugation. The target protein was purified with the Ni_NTA agarose. The proteins on the gel were transferred to the PVDF film and then the immunoreactivity was tested by Western blotting using anti_6_His antibody, sera from rabbits 6 weeks after infected with cercariae or UV_attenuated cercariae. The purified protein was coated for ELISA to test the cercariae infected rabbit serum and the normal rabbit serum. Results The molecular weight of the target fusion protein was {Mr 40 000} after being induced with IPTG. The fused protein showed a single band when reacted with anti_6_His antibody, the cercariae infected rabbit serum and attenuated cercariae infected rabbit serum. Conclusion The AK protein is expressed as a fused protein with thioredoxin and its molecular weight is about {Mr 40 000}. This protein has a positive immunoreactivity with sera of rabbits infected with cercariae and UV_attenuated cercariae.

Objective To analyze the difference among antigens of Angiostrongylus cantonensis in different developmental stages and identify dominant diagnostic antigen for angiostrongyliasis. Methods Antigens of A.cantonensis in different developmental stages were analyzed by SDS-PAGE and immunoblot. Results The protein bands of all developmental stages were similar on SDS-PAGE. The Mr 40 000 , 50 000 , 66 000 and 80 000 antigens reacted not only with the sera of rats infected by A.cantonensis but also with the sera of normal rats. The Mr 104 000 antigen could be discerned by sera of rats infected with A.cantonensis for 2 weeks. The Mr 32 000 antigen could be recognized by sera of rats 2 weeks after infection, and the reaction became stronger with the infection continued. Conclusion The Mr 40 000 , 50 000 , 66 000 and 80 000 antigens might result in the unspecific reaction in the immunodiagnosis of angiostrongyliasis using the crude antigen of A.cantonensis. The Mr 104 000 of larva, Mr 33 000 of adult females and Mr 32 000 of the worms might be used as candidate antigens in early diagnosis and epidemiological survey of angiostrongyliasis.

Objective To screen the Schistosoma japonicum female specific expressing genes. Methods{S.japonicum} adult worms were collected from the rabbits’ vein after six-week infection by affusing method. The adult worms were stabilized by RNA-later liquid, the male and female worms were carefully separated with nipper. The high quality total RNA was extracted and mRNA was obtained after purification. Double stranded cDNAs were synthesized after reverse transcription. Female subtractive (female as tester, male as driver) and male subtractive (male as tester, female as driver) cDNA libraries were constructed. The differentially expressed genes were further screened by dot-blot hybridization. The clones were selected and sequenced, which showed apparently higher signals when hybridizing with the female subtracting male probes, than those signals when hybridizing with the male subtracting female probes. The homology of these sequences was searched with BLAST program. The semi-quantitative PCR was applied to test the differential gene expression in female and male adult worms. Result Female subtracting male and male subtracting female cDNA libraries were constructed with SSH technique. After dot-blot hybridization, 50 clones were tested to be the potential female differentially expressed genes and were sequenced. 42 expressing sequence tags (ESTs) were received. After bioinformatics analysis, 17 fragments (about 40^5%) showed high identity with the S.japonicum egg-shell protein genes, 17 sequences(about 40^5%) were highly homologous to unknown S.japoniucm genes and partly homologous to female specific 800 protein. 8 fragments (about 19^0%) showed high identity with other S.japonicum unknown genes. The fragments in clones of 577, 579, 668, 695, 720, and 708 were tested by RT-PCR to be the differentially expressed genes in female adult worms using S.japonicum actin gene as the internal standard. These fragments were highly homologous to S.japonicum egg shell protein gene AY222885, AY222895, AB017097, AF519182, M32281, and S.japonicum unknown gene AY813556 respectively. Conclusion SSH is essential to screen the differentially expressed genes of {S.japonicum} female worms. A number of female specific genes have been found by this method.

We detected the seroprevalence of Toxoplasma gondii (T.gondii) in the wild birds in northeast China.The wild bird's blood was collected from the cutaneous ulnar vein and the serum was isolated and used for detection of anti T.gondii antibody by modified agglutination test (MAT).Results showed that totally 179 birds' serum samples were collected.Twenty serum samples (11.17％) were positive with T.gondii antibody,which belonged to 9 orders,17 families and 31 species.The seroprevalence against T.gondii was about 5.26％ (1/19) in Columbiformes,9.09％ (9/99) in Passeriformes,14.29％ (3/21) in Falconiformes,15.00％ (5/22) in Piciformes,16.67％ (1/6) in Coraciiformes,and 25.00％ (1/4) in Anseriformes.Based on their feeding behavior,the seroprevalence was 12.00％ (3/25) in carnivorous wild birds,10.60％ (15/141) in omnivorous wild birds,and 15.38％ (2/13) in the wild birds feeding on aquatic animals or plants.These wild birds also can be sorted as migratory and sedentary (non-migratory) according to their migration habits,and the serum positivity was 11.67％ (14/120),and 10.71％ (6/59) respectively.The seroprevalence against Toxoplasma gondii in wild birds in northeast China is about 11.17％,which indicates a common infection of Toxoplasma gondii in wild birds.

Objective To investigate the value of apparent diffusion coefficient (ADC) of diffusion-weighted magnetic resonance imaging (DW-MRI) in distinguishing benign from malignant breast lesions. Methods ADC in 26 normal breasts, 24 malignant breast lesions, and 30 benign breast lesions confirmed by operation and pathology were calculated, respectively, and their differentiations in statistics were compared. The differentiations of different ADCs (b=1000-0, 500-0, 1000-500 s/mm2) were also compared. EPI (TR 2900 ms, TE 84 ms, thickness 5 mm) was used in order to acquire the imaging. Results There were significant differences among the ADC values of normal breast tissue, benign, and malignant lesions. The ADC of malignant lesions was lower than those of normal breast tissue and benign lesions, and the ADC of benign lesions was lower than that of normal breast tissue. There were significant differences among the ADC value of b=1000-0, 1000-500, and 500-0 s/mm2. The lower the b value, the higher the ADC. The sensitivity and specificity of ADC for the diagnosis of malignant lesion were 64% and 96.7% if the upper bound of 95% confidence interval was set as a differential level. Conclusion The differentiation of benign from malignant breast lesions by ADC is applicable, although the sensitivity is low, the specificity is high.

Objective To discuss CT,MR and~1H-MRS features of central neurocytoma(CNC).Methods Imaging findings ofneurocytomas in 7 cases confirmed by pathology were retrospectively analyzed with literature review.2 cases were examined by CT、MR and~1 H-MRS,2 cases only by MR,and 3 cases only by CT.Results All the tumors were located in the lateral ventricles.There were different degree hydrocephalus in all cases.The masses were heterogeneous appearance on CT with necrotic area and fine to course calcifications.Heterogeneous enhancement was seen in the solid portion.The tumors were isointense and hypointense on T_1WI and heterogeneous on T_2WI.Heterogeneous enhancement was also seen on MRI.The in vivo~1H-MRS showed prominent choline(Cho) and low N-acetyl aspartate(NAA) compared to the normal.Conclusion Central neurocytoma should be considered when a tumor was located at the lateral ventricles especially septum pellucidum in young patients.CT,MR and~1H-MRS are helpful in making a preoperative diagnosis.

NHL proteins, which play important roles in regulation of cell proliferation and differentiation, have been extensively studied on mammals. Here, we cloned a member of NHL protein family namely BmBrat in silkworm. The full-length cDNA sequence of BmB rat was obtained by means of the rapid amplification of cDNA ends (RACE), including 3 614 bp. The ORF is 2 580 bp long, encoding a protein with 859 amino acid residues. The molecular weight is 94.3 kDa and the isoeledtric point (pI) is 6.65. The BmBrat expression profile was detected by RT-PCR at L5D3 larval stage, and it was expressed in all tissues, including silk gland, midgut, fat body and malpighian tubule. However, it was highly expressed in ovary and head. The expression profile was also detected at different stage of embryo development, and reached a peak at the 4th and 5th days of the embryonic period. Anti-BmBrat polyclonal antibody was generated f6llowing prokaryotic expression, protein purification and mice immunization, which is highly specific and effective for recognizing BmBrat protein through Western blotting and immunofluorescence staining. Subcellular localization of BmBrat in hemocytes revealed that it was specifically expressed in cytoplasm. This study provides a foundation for further research of the biological function of BmBrat gene.
Animals ; Bombyx ; Cloning, Molecular ; DNA, Complementary ; Insect Proteins ; genetics ; metabolism ; Larva ; Mice

Objective To observe the major adverse reaction of irinotecan in combination with fluorouracil for advanced colorectal cancer,and sum up the nursing experience of acetylcholine syndrome and acute delayed diarrhea.Methods Pathologically confirmed 45 cases of patients with advanced colorectal cancer were treated with modified FOLFIRI regimen for total of 190 cycles.Results Acetylcholine syndrome rate was 28.9%,the incidence of acute delayed diarrhea was 40.0% ,more than 3 grade diarrhea was 6.7%.Conclusion The incidence of acute delayed diarrhea in Chinese patients is lower than in caucasians,and reasonable care measures can reduce the occurrence of adverse reactions,irinotecan in combination with fluorouracil is a safe and effective regimen for Chinese patients.