The effect of S100A6 on nuclear translocation of CacyBP/SIP in colon cancer cells.

BACKGROUND: Calcyclin Binding Protein/(Siah-1 interacting protein) (CacyBP/SIP) acts as an oncogene in colorectal cancer. The nuclear accumulation of CacyBP/SIP has been linked to the proliferation of cancer cells. It has been reported that intracellular Ca2+ induces the nuclear translocation of CacyBP/SIP. However, the molecular mechanism of CacyBP/SIP nuclear translocation has yet to be elucidated. The purpose of this study was to test whether the Ca2+-dependent binding partner S100 protein is involved in CacyBP/SIP nuclear translocation in colon cancer SW480 cells. METHODS: The subcellular localization of endogenous CacyBP/SIP was observed following the stimulation of ionomycin or BAPTA/AM by immunofluorescence staining in SW480 cells. S100A6 small interfering RNAs (siRNA) were transfected into SW480 cells. Immunoprecipitation assays detected whether S100 protein is relevant to the nuclear translocation of CacyBP/SIP in response to changes in [Ca2+]i. RESULTS: We observed that endogenous CacyBP/SIP is translocated from the cytosol to the nucleus following the elevation of [Ca2+]i by ionomycin in SW480 cells. Co-immunoprecipitation experiments showed that the interaction between S100A6 and CacyBP/SIP was increased simultaneously with elevated Ca2+. Knockdown of S100A6 abolished the Ca2+ effect on the subcellular translocation of CacyBP/SIP. CONCLUSION: Thus, we demonstrated that S100A6 is required for the Ca2+-dependent nuclear translocation of CacyBP/SIP in colon cancer SW480 cells.

Infection of mouse bone marrow-derived immature dendritic cells with classical swine fever virus C-strain promotes cells maturation and lymphocyte proliferation.

In this study, the interactions of classical swine fever virus (CSFV) C-strain and the virulent GSLZ strain with mouse bone marrow-derived immature dendritic cells (BM-imDCs) were investigated for the first time. Both the C-strain and the virulent GSLZ strain could effectively infect and replicate in mouse BM-imDCs. C-strain-infected BM-imDCs showed a greatly enhanced degree of maturation, and could effectively promote the expansion and proliferation of allogeneic naive T cells. The C-strain induced a stronger Th1 response. Infection with the virulent GSLZ strain had no obvious influence on cell maturation or lymphocyte proliferation, and failed to induce any obvious immune response. The results of this study provided initial information for research of the immunologic mechanisms of CSFV using mouse DCs as the model cells.

Phylogenetic relationships of the glycoprotein gene of bovine ephemeral fever virus isolated from mainland China, Taiwan, Japan, Turkey, Israel and Australia.

BACKGROUND: The glycoprotein (G) gene sequences of bovine ephemeral fever virus (BEFV) strains derived from mainland China have not been compared with those of the isolates from other countries or areas. Therefore, the G genes of four BEFV isolates obtained from mainland China were amplified and sequenced. A phylogenetic tree was constructed in order to compare and analyze the genetic relationships of the BEFV isolates derived from mainland China and different countries and areas. RESULTS: The complete BEFV G gene was successfully amplified and sequenced from four isolates that originated from mainland China. A total of fifty-one BEFV strains were analyzed based on the G gene sequence and were found to be highly conserved. A phylogenetic tree showed that the isolates were grouped into three distinct lineages depending on their source of origin. The antigenic sites of G1, G2 and G3 are conserved among the isolates, except for several substitutions in a few strains. CONCLUSIONS: The phylogenetic relationships of the BEFV isolates that originated from mainland China, Taiwan, Japan, Turkey, Israel and Australia were closely related to their source of origin, while the antigenic sites G1, G2 and G3 are conserved among the BEFV isolates used in this work.

Discovery and characterization of gene cassettes-containing integrons in clinical strains of Riemerella anatipestifer.

Forty-eight prevalent strains of Riemerella anatipestifer (RA) isolated in China were tested for susceptibility to eighteen antibiotics and investigated for the frequencies and characteristics of integrons and gene cassettes. All isolates were resistant to between three and ten antimicrobial drugs. Forty-seven isolates contained class 1 integron (97.92%), and 15 of the 47 isolates contained class 2 integron (31.25%). Class 3 integron was not detected in the strains analysed. Three different cassette arrays (aadA1, aadA5 and aacA4-aadA1) of class 1 integron and one gene cassette (sat2-aadA1) of class 2 integron were discovered. Three out of the four cassette arrays were novel, with the exception of aadA5. The location of integrons was confirmed by transforming extracted plasmids into an integron-negative strain of Escherichia coli (E. coli) BL21 (DE3). Class 1 integrons were always discovered in plasmids, while class 2 integrons could be located on plasmids or in the chromosome. This is the first description of class 2 integrons, three novel cassette arrays and the location of integrons in RA species.

Loop-mediated isothermal amplification assay targeting the omp25 gene for rapid detection of Brucella spp.

A novel loop-mediated isothermal amplification (LAMP) assay was established to detect Brucella species DNA in milk and blood samples of animals and humans. This LAMP assay based on the sequence of highly repetitive omp25 gene was able to detect 9fg/µl Brucella spp. DNA with high sensitivity, which was 10 times higher than the nested PCR. The LAMP was evaluated for its specificity using 19 strains of six Brucella species and 28 related non-Brucella micro-organism strains as controls. The target 19 Brucella strains were all amplified, and no cross-reaction was found with all the non-Brucella micro-organism strains. Both nested PCR and LAMP assays were then used to detect Brucella spp. DNA in 78 milk samples and 113 blood samples from animals and 11 blood samples from humans, and the established LAMP assay yielded 99.0% concordance rate with the nested PCR. The LAMP assay should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of Brucellosis.

Loop-mediated isothermal amplification assay targeting the ompA gene for rapid detection of Riemerella anatipestifer.

A novel loop-mediated isothermal amplification (LAMP) assay was developed and evaluated for the detection of Riemerella anatipestifer (RA) infection. The LAMP assay exhibited a higher sensitivity than conventional polymerase chain reaction (PCR) and microbial isolation. The specificity of the assay was determined by restriction enzyme digestion of the LAMP products and detection of Escherichia coli, Salmonella enterica and Pasteurella multocida. The LAMP assay was able to detect RA effectively in samples of the reference strains, isolated strains and infected duck brains. This assay is a useful tool for the diagnosis of RA infection in the clinical setting.

A reverse-transcription, loop-mediated isothermal amplification assay for detection of bovine ephemeral fever virus in the blood of infected cattle.

A novel reverse-transcription, loop-mediated isothermal amplification (RT-LAMP) assay for the detection of bovine ephemeral fever virus (BEFV) was developed and evaluated in this study. The RT-LAMP assay exhibited higher sensitivity when compared with conventional reverse-transcription polymerase chain reaction (RT-PCR) and virus isolation methods. The specificity of the assay was determined by digestion of the RT-LAMP products with restriction enzyme and detection of BEFV serogroup rabies virus (RV). Using RT-LAMP, RT-PCR and virus isolation methods, 36 blood samples were tested and the results indicated that RT-LAMP could detect early infection with BEFV. The RT-LAMP method is useful for the diagnosis of BEFV infection in blood samples.

An indirect ELISA of classical swine fever virus based on quadruple antigenic epitope peptide expressed in E.coli.

In this study, a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV) E2 glycoprotein was expressed in E. coli to a obtain target protein. This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs. The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve (ROC) analysis based on 30 negative sera and 80 positive samples. The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination (IHA) test. The inter-assay and intra-assay coefficients of variation (CVs) for 16 sera were both ≤6.8%. No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus (BVDV) antibodies was observed.

Immunization trials with an avian chlamydial MOMP gene recombinant adenovirus.

Chicks were inoculated with a live vector vaccine of avian chlamydial MOMP gene recombinant adenovirus to evaluate efficacy, safety and viability of the vaccine. Five batches of the recombinant adenovirus vaccines, which were prepared using the 22nd generation avian chlamydial MOMP gene recombinant adenovirus cultured in HEK293 cells, were used to vaccinate 7 days-old chicks negative for chlamydial antibody. The recombinant adenovirus vaccine was shown to be both safe and effective in inducing specific immunity in vaccinated chicks.

Serological detection of bovine ephemeral fever virus using an indirect ELISA based on antigenic site G1 expressed in Pichia pastoris.

An indirect ELISA for the serological detection of bovine ephemeral fever virus (BEFV) infection in cattle is described in which a glycosylated protein of approximately 25 kDa (including the G1 antigenic site of the virus glycoprotein) expressed in Pichia pastoris GS115 was used as the coating antigen. The optimal concentration of coated antigen was 0.3 microg/well at a serum dilution of 1:40. The optimal positive threshold value of the assay was 1.88, as derived from receiver operating characteristic curve analysis. The test had 100% sensitivity and 96.7% specificity when compared with a micro-neutralisation test using 336 positive and 180 negative sera to BEFV, respectively. The inter-assay and intra-assay coefficients of variation for 15 sera were both <5.8% and there was no evidence of cross-reactivity between the tested coating antigen and antibodies to related rabies virus. The ELISA is an inexpensive and rapid serological detection method that would be suitable for screening for BEFV infection on a large scale.

Development and application of G1-ELISA for detection of antibodies against bovine ephemeral fever virus.

The gene encoding antigenic site G(1) of bovine ephemeral fever virus (BEFV) was highly expressed in the host cell Escherichia coli. An indirect G(1)-ELISA with the recombinant protein as the coating antigen was established to detect antibodies against BEFV. The result revealed that the optimal concentration of the coated antigen was 0.5 microg/well and the dilution of serum was 1:20. It was optimal that sera with P/N >or= 2.2 were considered positive, P/N

[Expression, purification and antigenic characterization of the Epitope-G1 gene of bovine ephemeral fever virus in Escherichia coli].

The epitope-G1 gene, cloned from the pMD-G plasmid including G protein gene of bovine ephemeral fever virus (BEFV), was subcloned into expression vector pGEX-4T-1 to construct pGEX-G1 recombinant plasmid successfully. The pGEX-G1 was transformed into E. coli BL21(DE3) to be induced with IPTG. The optimal expression conditions for G1 gene were obtained, which included reaction temperature 16 degrees C, induction time 18h and IPTG concentration 0.1 mmol/L. The soluble target protein was purified with Glutathione Sepharose TM(4B) and the purity reached 80%. The inclusion body washed with 2% deoxycholic acid sodium salt and dissolved with 0.5% N-lauroyl sarcosine sodium was recovered by the way of dialysis, then the protein was purified with Glutathione Sepharose TM(4B) and its purity was above 85%. The protein purified had nicer reaction activity by analysis of Western blot. The target protein was used as coating antigen to detect the sera against BEFV by an indirect ELISA. The 12 positive sera to BEFV were detected and the average of OD490 was 1.813 +/- 0.231, while the average of OD490 from 12 negative sera was 0.359 +/- 0.032, and the distinction was very remarkable (P < 0.01). All the rabbits inoculated with the target protein had produced high titer of antibodies, which indicated that the target protein had immunological activity. The average of OD49, detecting the 8 positive sera to rabies virus (RV) with the target protein purified was 0.324 +/- 0.031 which closed the datum obtained from the negative sera to BEFV, and it showed no cross-reaction between the sera to RV and BEFV. All the results above indicated that the target protein expressed had nicer biological activity and specificity, so the protein could be used as coating antigen to develop ELISA Kit for diagnosing BEF.

Construction and immunogenicity of recombinant adenovirus expressing the major outer membrane protein (MOMP) of Chlamydophila psittaci in chicks.

Avian chlamydiosis is caused by Chlamydophila psittaci. The major outer membrane protein (MOMP) encoded by the outer membrane protein 1 (omp1) gene is an excellent candidate for genetic engineering of a vaccine against avian chlamydiosis. In this study, the MOMP gene was amplified by PCR and cloned into the transfer vector pShuttle-CMV. The recombinant plasmid was obtained by recombination between the plasmid pShuttle-CMV-MOMP and skeleton vector pAdEasy-1 in Escherichia coli strain BJ5183. The titer of recombinant adenovirus containing the MOMP gene (rAd-MOMP) of C. psittaci was 3.4x10(10)TCID(50)/ml in human embryonic kidney 293 (HEK293) monolayer cells. The expression of the MOMP in HEK293 cells infected with rAd-MOMP was confirmed by an indirect immunofluorescence assay. Specific pathogen free (SPF) chicks were inoculated with 10(6), 10(8), and 10(10)TCID(50) of rAd-MOMP/chick. Inoculated chicks generated antibodies against MOMP of C. psittaci, which were detected by an indirect hemagglutination test (IHA). The vaccinated chicks were challenged with a virulent Chinese field isolate. Nine out of 10 chicks in the vaccinated group were protected, while birds in the wild-type adenovirus control group and the PBS control group all showed clinical signs after challenge. The results indicate that the recombinant adenovirus containing the MOMP gene of C. psittaci might be a candidate vaccine against avian chlamydiosis.