[The immunological responses induced by Mycobacterium tuberculosis heat shock protein 16.3 and its synthetic peptide in mice].

OBJECTIVE: To evaluate the immune responses and resistance against Mycobacterium tuberculosis (MTB) infection in the mice induced by HSP16.3 of MTB and its synthetic peptide. METHODS: BALB/c mice were immunized subcutaneously 3 times at 2 week interval at the base of tail. The doses of HSP16.3 protein and synthetic peptide were both 50 microg each time. A single dose of BCG (5 x 10(6) CFU/mouse) was used to immunize the mice. The concentrations of specific antibodies in serum obtained at 0, 2, 4, 6, 8 weeks after the first immunization and the titer of serum obtained at 8th week, were analyzed by enzyme linked immunosorbent assay (ELISA). Four weeks after the final immunization, 8 mice from each group were sacrificed and single-cell suspensions of splenocytes were prepared, some of which were used for lymphocyte proliferation by MTT colorimetry with HSP16.3 stimulation, and the remaining cells were used for IFN-gamma level assay by sandwich ELISA. The remaining mice in each group were challenged intravenously with 10(5) colony forming units (CFU) of MTB H(37)Rv and were sacrificed 4 weeks after infection, and the number of bacteria in the spleens and lungs were determined by plating serial dilutions of homogenized tissue on Middlebrook 7H10 agar. The statistical significance of differences among means was assessed by an LSD-t test. RESULTS: The level of specific antibody to HSP16.3 protein and the peptide increased rapidly in the former 4 weeks and moderately in the later weeks. The average antibody-specific titers of 3 experiment groups (HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA) were higher than the BCG group. The indexes of spleen lymphocyte proliferation (SI) of the 3 experiment groups (3.13 +/- 0.18, 3.21 +/- 0.21 and 2.40 +/- 0.15) were significantly higher than the BCG group (1.67 +/- 0.12) and the saline group (1.04 +/- 0.09) respectively. The SI of HSP16.3 protein + DDA + MPL group (3.13 +/- 0.18) and synthetic peptide + DDA + MPL group (3.21 +/- 0.21) were higher than the synthetic peptide + IFA group (2.40 +/- 0.15). The IFN-gamma levels induced among the 3 experiment groups [(182 +/- 6), (194 +/- 9) and (179 +/- 8) mg/L] were lower than the BCG group [(275 +/- 10) mg/L], but higher than the saline group [(71 +/- 3) mg/L]. The IFN-gamma level induced among the 3 experiment groups did not show any marked difference. Although the protection induced by HSP16.3 protein + DDA + MPL, synthetic peptide + DDA + MPL and synthetic peptide + IFA all showed resistance against MTB H(37)Rv infection in the spleens or lungs (the bacterial logarithmic loads of spleen: 6.74 +/- 0.14, 6.60 +/- 0.13 and 6.81 +/- 0.28; the bacterial logarithmic loads of lung: 5.81 +/- 0.21, 5.74 +/- 0.27 and 6.65 +/- 0.32), none of them was better than the conventional BCG (the bacterial logarithmic loads of spleen and lung: 5.95 +/- 0.17 and 5.62 +/- 0.23). CONCLUSIONS: Both HSP16.3 and its synthetic peptide can be considered as TB vaccine candidates or effective components in TB vaccines.

Effect of electromagnetic pulse exposure on permeability of blood-testicle barrier in mice.

OBJECTIVE: To study the effect of electromagnetic pulse (EMP) exposure on the permeability of blood-testicle barrier (BTB) in mice. METHODS: Adult male BALB/c mice were exposed to EMP at 200 kV/m for 200 pulses with 2 seconds interval. The mice were injected with 2% Evans Blue solution through caudal vein at different time points after exposure, and the permeability of BTB was monitored using a fluorescence microscope. The testis sample for the transmission electron microscopy was prepared at 2 h after EMP exposure. The permeability of BTB in mice was observed by using Evans Blue tracer and lanthanum nitrate tracer. RESULTS: After exposure, cloudy Evans Blue was found in the testicle convoluted seminiferous tubule of mice. Lanthanum nitrate was observed not only between testicle spermatogonia near seminiferous tubule wall and sertoli cells, but also between sertoli cells and primary spermatocyte or secondary spermatocyte. In contrast, lanthanum nitrate in control group was only found in the testicle sertoli cells between seminiferous tubule and near seminiferous tubule wall. CONCLUSION: EMP exposure could increase the permeability of BTB in the mice.

[Expression in E.coli and bioactivity assay of Micrococcus luteus resuscitation promoting factor domain and its mutants].

OBJECTIVE: To express Micrococcus luteus resuscitation promoting factor (Rpf) domain and its mutants in prokaryotic cells, and to investigate their bioactivity. METHODS: The gene of Rpf domain and its mutants (E54K, E54A) were amplified by polymerase chain reaction (PCR) from the genome of Micrococcus luteus and cloned into pMD18-T vector. After sequenced, the Rpf domain and its mutant gene were subcloned into expression vector PGEX-4T-1, and transfected into E. coli DH5alpha. The expressed product was purified by affinity chromatography using GST Fusion Protein Purification bead. The aim proteins were identified by SDS-PAGE analysis and by Western blot with monoclonal antibodies against Rpf domain (mAb). The bioactivity of the proteins was analyzed by stimulating the resuscitation of Mycobacterium smegmatis. RESULTS: The sequences of the PCR products were identical to those of the Rpf domain and its mutant gene in GenBank. The relative molecular mass identified by SDS-PAGE analysis was consistent with that had been reported, which was also confirmed by Western blot analysis that there were specific bindings at 32 000 with Rpf domain mAb. The purified GST-Rpf domain could stimulate resuscitation of Mycobacterium smegmatis. Replacements E54A and especially E54K resulted in inhibition of Rpf resuscitation activity. CONCLUSIONS: Rpf domain and two kinds of its mutant protein were obtained, and its effects on the resuscitation of dormant Mycobacterium smegmatis were clarified.

Short hairpin RNA targeting survivin inhibits growth and angiogenesis of glioma U251 cells.

Survivin is a novel tumor-associated gene, its overexpression mostly associates with carcinogenesis and development. Nevertheless, the precise role of survivin in initiation and progression of gliomas is still not completely clear. We constructed here three short hairpin RNA (shRNA) targeting survivin plasmid vectors and introduced them into glioma U251 cells. The three shRNAs were efficiently and specifically able to knockdown the survivin expression in transiently transfected U251 cells. The stable transfectants expressing the shRNA having the strongest inhibitory effect against survivin exhibited decreased cell growth, increased spontaneous apoptosis, mitotic catastrophe and cell cycle arrest. Furthermore, in nude mice xenografts, the stable transfectants presented decreased de novo glioma formation and reduced development of angiogenesis. Results from this study indicate that survivin plays an important role in malignant proliferation, antiapoptosis and angiogenesis of gliomas, which may become an attractive target for gene therapy of gliomas, while RNA interference (RNAi) mediated by shRNA may become a new promising strategy for cancer gene therapy.

Biphasic effect of androgens on prostate cancer cells and its correlation with androgen receptor coactivator dopa decarboxylase.

The aim of this study was to explore the mechanism underlying the dual effect of androgen on prostate cancer cells and further explore its correlation with dopa decarboxylase (DDC), an androgen receptor (AR) coactivator and a traditional neuroendocrine differentiation (NED) marker. Cell proliferation and cycling after treatment with synthetic nonmetabolizable androgen R1881 was determined by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) method and flow cytometry. Differential gene expression was analyzed by cDNA microarrays. DDC expression during the dual effect of R1881 was further explored with microarray, quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blot, and enzyme activity assays. Proliferation of LNCaP cells was inhibited by 1 nM R1881 but stimulated by 0.1 nM R1881. Compared with the untreated cells, 320 (2.26%; 170 up-regulated, 150 down-regulated) and 4608 (32.65%; 2046 up-regulated, 2562 down-regulated) genes were found to be expressed differentially in the 1 nM and 0.1 nM R1881-treated cells, respectively. The results were partially confirmed by RT-PCR and Western blot. The DDC gene was down-regulated in the 1 nM R1881-treated cells and up-regulated in 0.1 nM R1881- and 30 nM hydroxyflutamide-treated cells. The enzymatic activity of DDC in the latter 2 groups was also strengthened. Meanwhile, the NED markers CgA and synaptophysin were not affected by these AR activators. R1881 had a dose-dependent biphasic effect on LNCaP cell proliferation. AR coactivator DDC was respectively down- and up-regulated in high and low concentrations of R1881. DDC up-regulation by exogenous AR activators is not accompanied by up-regulation of definitive NED markers.

Expression and immunogenicity of recombinant Mycobacterium bovis Bacillus Calmette-Guérin strains secreting the antigen ESAT-6 from Mycobacterium tuberculosis in mice.

BACKGROUND: Tuberculosis remains the leading cause of human death. Currently, Bacillus Calmette-Guérin (BCG) is the only available vaccine against tuberculosis but its efficacy is highly variable. Thus, developing new tuberculosis vaccines becomes an urgent task. In this study, we evaluated in BALB/c mice the humoral and cellular immune responses of recombinant BCG expressing the antigen ESAT-6 from Mycobacterium tuberculosis. METHODS: Escherichia coli-BCG shuttle plasmid named pDE22-esat-6 was constructed by inserting the BamHI/EcoRI digested esat-6 gene PCR product into the similarly digested parental plasmid pDE22. BCG cells were transformed with pDE22-esat-6, which was named recombinant BCG (rBCG). BALB/c mice were immunized subcutaneously on the back with 100 microl normal saline containing 10(6) CFU of BCG or rBCG. They were sacrificed after 4 weeks to detect their humoral and cellular responses. RESULTS: There was no any significant differences in the growth characteristics between the conventional BCG and rBCG. In immunized mice, the IgG antibody titres of rBCG group were as high as 1:8000, which was significantly higher than that in BCG group (1:1400, P < 0.05). The elicited IFN-gamma level of rBCG group was (1993 +/- 106) pg/ml, which was also significantly higher than that in BCG group ((1463 +/- 105) pg/ml, P < 0.05). The splenocyte proliferation index of rBCG group reached 4.34 +/- 0.31, which was higher than that of BCG group (3.79 +/- 0.24, P < 0.05). CONCLUSION: rBCG secreted expressing antigen ESAT-6 stimulated stronger humoral and cellular immune responses than BCG did, and, therefore may be the better vaccine against mycobacterium tuberculosis.

[Inhibition of growth and angiogenesis of U251 cell xenograft in vivo by short hairpin RNA targeting survivin gene].

OBJECTIVE: To observe the impact of specific short hairpin RNA (shRNA) targeting survivin gene on tumorigenesis and angiogenesis of human brain glioblastoma U251 cells in vivo of nude mice. METHODS: U251 cells, U251-SR cells transfected stably with shRNA eukaryotic expression vector pWH1-SR targeting survivin gene, and U251-P cells transfected stably with blank pWH1 vector, were inoculated respectively into subcutaneous tissue in flank of 15 nude mice (each group 5 mice), and the tumor growth status was observed and measured. Protein expressions of survivin, proliferating cell nuclear antigen (PCNA) and factor VIII related antigen (F VIII RAg) were investigated by immunohistochemistry SABC method, apoptotic cells were screened by TUNEL method, furthermore proliferative index (PI), apoptotic index (AI) and microvessel density (MVD) were measured respectively in each group of tumor specimens. RESULTS: Comparing with those in U251 and U251-P groups, in U251-SR group, the tumorigenesis time delayed, tumor grew slowly, both tumor volume and tumor weight decreased significantly (P < 0.01 for both); Survivin protein expression was down-regulated markedly; PI and MVD decreased significantly, whereas AI increased remarkably (P < 0.01 for all). CONCLUSIONS: The specific shRNA targeting survivin gene can inhibit significantly tumorigenesis and angiogenesis of U251 cells in vivo.

Change of the cell cycle after flutamide treatment in prostate cancer cells and its molecular mechanism.

AIM: To explore the effect of androgen receptor (AR) on the expression of the cell cycle-related genes, such as CDKN1A and BTG1, in prostate cancer cell line LNCaP. METHODS: After AR antagonist flutamide treatment and confirmation of its effect by phase contrast microscope and flow cytometry, the differential expression of the cell cycle-related genes was analyzed by a cDNA microarray. The flutamide treated cells were set as the experimental group and the LNCaP cells as the control. We labeled cDNA probes of the experimental group and control group with Cy5 and Cy3 dyes, respectively, through reverse transcription. Then we hybridized the cDNA probes with cDNA microarrays, which contained 8 126 unique human cDNA sequences and the chip was scanned to get the fluorescent values of Cy5 and Cy3 on each spot. After primary analysis, reverse transcription polymerase chain reaction (RT-PCR) tests were carried out to confirm the results of the chips. RESULTS: After AR antagonist flutamide treatment, three hundred and twenty-six genes (3.93%) expressed differentially, 97 down-regulated and 219 up-regulated. Among them, eight up-regulated genes might be cell cycle-related, namely CDC10, NRAS, BTG1, Wee1, CLK3, DKFZP564A122, CDKN1A and BTG2. The CDKN1A and BTG1 gene mRNA expression was confirmed to be higher in the experimental group by RT-PCR, while p53 mRNA expression had no significant changes. CONCLUSION: Flutamide treatment might up-regulate CDKN1A and BTG1 expression in prostate cancer cells. The protein expressions of CDKN1A and BTG1 play an important role in inhibiting the proliferation of cancer cells. CDKN1A has a great impact on the cell cycle of prostate cancer cells and may play a role in the cancer cells in a p53-independent pathway. The prostate cancer cells might affect the cell cycle-related genes by activating AR and thus break the cell cycle control.

[Screening and construction of recombinant BCG strains expressing the Ag85B-ESAT6 fusion protein].

OBJECTIVE: To screen and construct recombinant BCG strains which express the Ag85B-ESAT6 fusion protein. METHODS: The heat shock protein 60 (Hsp60) and the alpha-ss signal peptide encoding sequence were amplified by PCR from Mycobacterium tuberculosis H(37)Rv and cloned into E. coli/Mycobacteria shuttle vector-pOLYG. The resulting expression vector was named pDE22, and then ag85b and esat6 genes were cloned into pDE22 at different sites. The resulting recombinant plasmids Ag85B-ESAT6 and ESAT6-Ag85 were electroporated into BCG. Positive clones were screened by hygromycin resistance and confirmed by PCR. Recombinant BCG culture supernatants were collected and analyzed by SDS-PAGE and Western blot. RESULTS: Two recombinant BCG strains were obtained, which secreted the 37,000 fusion protein in their culture supernatant, which was confirmed by Western blot with specific immune serum against Ag85B and ESAT6. CONCLUSIONS: Recombinant BCG strains expressing Ag85B and ESAT6 fusion proteins of Mycobacterium tuberculosis were constructed. They may serve as new vaccine candidates for preventing tuberculosis.

[Immunity characteristics induced by a genetic vaccine expressing Ag85B-ESAT6 fusion protein].

OBJECTIVE: To evaluate the humoral and cell-mediated immune responses induced by a genetic vaccine expressing the Ag85B-ESAT6 fusion protein, and to investigate its protective effect against Mycobacterium tuberculosis (MTB) challenge. METHODS: Fifty BALB/c mice were randomized into 5 groups and subjected to the following treatments respectively: immunization with normal saline, BCG, pcDNA3, A(Z)-pcDNA3-E(F) and E(Z)-pcDNA3-A(F) for 3 times at 2-week intervals. The stimulation index (SI) of the splenic lymphocytes from the immunized mice was measured by the methyl thiazolyl tetrazolium (MTT) method, and the level of secreted IFN-gamma upon antigen-specific stimulation was detected by ELISA. The immunized mice were intravenously infected with 10(5) colony forming unit (CFU) of MTB H(37)Rv. The numbers of MTB CFU in spleens were determined 4 weeks later. RESULTS: The specific antibody titers in the sera of mice immunized with plasmid A(Z)-pcDNA3-E(F) and E(Z)-pcDNA3-A(F) were 1:1,000 and 1:1,500 respectively, and the SI was 2.2 and 2.4 respectively, while the SI of the normal saline group and the plasmid pcDNA3 immunized group was only 0.9 and 1.1 respectively. The IFN-gamma concentrations in cultured supernatant of splenic lymphocytes from mice immunized with plasmid A(Z)-pcDNA3-E(F) [(5.48 +/- 0.38) ng/ml] and E(Z)-pcDNA3-A(F) [(5.76 +/- 0.51) ng/ml] were significantly higher than those of the normal saline group [(0.50 +/- 0.25) ng/ml] and the plasmid pcDNA3 immunized group [(1.20 +/- 0.33) ng/ml, P < 0.05], but were not significantly different with that of the BCG immunized group [(5.55 +/- 0.31) ng/ml]. Compared with plasmid pcDNA3 immunized group, the bacterial load (lg, CFU/g) in spleen was 6.08 +/- 0.25 which dramatically reduced in mice immunized with recombinant plasmids, but the protective efficacy of mice immunized with plasmid A(Z)-pcDNA3-E(F) (4.63 +/- 0.11) or E(Z)-pcDNA3-A(F) (4.50 +/- 0.32) was lower than that of the BCG vaccination group (4.09 +/- 0.27). CONCLUSION: The cell-mediated immune response induced by genetic vaccine expressing the Ag85B-ESAT6 fusion protein was similar to that induced by BCG immunization.

[Construction and bio-activity of the chimeric protein of BMP2-EGFP].

OBJECTIVE: To construct the recombined retroviral expression vector of BMP2/pLEGFP and investigate the bio-activity of the expressed chimeric protein. METHODS: The recombinant vector constructed by gene recombinant technology was analyzed by restriction enzyme digestion and PCR. BMP2/pLEGFP was transfected into COS-7 cells with liposome transfection reagents for transient expression. The expression of chimeric protein BMP2/EGFP was identified by fluorescent microscope and Western blotting. The bio-activity was examined by the cellular activity and animal heterotopic osteogenesis experiment. RESULTS: The recombinant plasmid proved successful by restriction enzyme digestion and PCR. The expression of the chimeric protein was shown by fluorescent microscope and Western blotting. The chimeric protein had the double bio-activities of BMP2 and EGFP identified by the cellular activity and animal heterotopic osteogenesis tests. CONCLUSION: The recombinant vector of BMP2/pLEGFP is successfully constructed by the gene recombinant technology and its chimeric protein has double bioactivities of BMP2 and EGFP.

[Mycobacterium tuberculosis secreting protein Ag85B-ESAT6 fused expression and purification].

OBJECTIVE: To investigate the fused expression of secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis, and to provide a promising preventive subunit vaccine against tuberculosis. METHODS: The gene encoding Ag85B and ESAT6 protein was amplified by PCR from genome of Mycobacterium tuberculosis H(37)Rv strain, and inserted into cloning vector P(GEM)-T-easy. After sequence analysis, and digestion by restriction endonuclease, Ag85B-ESAT6 was cloned into corresponding sites of the expression vector P(PRO) EXHT, and the recombinant plasmid was transformed into expressive strain E. coli DH5 alpha, induced with IPTG and fusion protein was purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with two fusion protein was detected by ELISA. RESULTS: The sequences of Ag85B and ESAT6 by PCR amplification were identical to those reported by GenBank. The recombinant plasmid fused expression protein of Ag85B-ESAT6 with relative molecular mass (Mr) of 37,000, which was confirmed by Western-blot analysis with specific monoclonal antibody against 6 x HismAb. The fused expression protein was insoluble. It could be purified by Ni-NTA purification system. The specific antibody titer in the sera of BALB/c mouse immunized with fusion Ag85B-ESAT6 was 1:1000 and that of mouse immunized with fusion protein ESAT6-Ag85B was 1:5000. CONCLUSIONS: Secreted protein Ag85B-ESAT6 of Mycobacterium tuberculosis was successfully fused expressed in E. Coli DH5 alpha. It may become a new type of vaccine against tuberculosis.

[Constructing shuttle plasmid fusion expressing Ag85B-ESAT-6 on the surface of Mycobacterium].

OBJECTIVE: To construct the E. coli.-BCG (Bacille Calmette-Guerin) shuttle vector expressing Mycobacterium tuberculosis secreted protein Ag85B-ESAT-6 on the surface of Mycobacterium vaccae. METHODS: The gene fragment containing 19 000 antigen (19-ss) were amplified by polymerase chain reaction (PCR) from the Mycobacterium tuberculosis H(37)Ra. We cloned the 19ss gene into the E. coli.-BCG shuttle vector pOLYG and named the pCW, which can shuttle and express exogenous antigen gene on cell wall of Mycobacterium. Then Mycobacterium tuberculosis secret protein Ag85B and ESAT-6 gene were cloned into the vector and determined by indirect immunofluorescence. RESULTS: The sequence of 19-ss gene was identified with Genbank reported by sequencing. The constructed E. coli.-BCG shuttle vector using 19ss gene had the function of shuttle between E. coli. and Mycobacteria. By indirect immunofluorescence technique the secreted protein Ag85B-ESAT-6 can be fused and expressed on surface of Mycobacterium vaccae. CONCLUSION: The E. coli.-BCG shuttle vector is constructed successfully which could express exogenous antigen gene as a chimeric exported membrane.

Inhibition of both EGFR and IGF1R sensitized prostate cancer cells to radiation by synergistic suppression of DNA homologous recombination repair.

Reduced sensitivity of prostate cancer (PC) cells to radiation therapy poses a significant challenge in the clinic. Activation of epidermal growth factor receptor (EGFR), type 1 insulin-like growth factor receptor (IGF1R), and crosstalk between these two signaling pathways have been implicated in the development of radiation resistance in PC. This study assessed the effects of targeting both receptors on the regulation of radio-sensitivity in PC cells. Specific inhibitors of EGFR and IGF1R, Erlotinib and AG1024, as well as siRNA targeting EGFR and IGF1R, were used to radio-sensitize PC cells. Our results showed that co-inhibiting both receptors significantly dampened cellular growth and DNA damage repair, and increased radio-sensitivity in PC cells. These effects were carried out through synergistic inhibition of homologous recombination-directed DNA repair (HRR), but not via inhibition of non-homologous end joining (NHEJ). Furthermore, the compromised HRR capacity was caused by reduced phosphorylation of insulin receptor substrate 1 (IRS1) and its subsequent interaction with Rad51. The synergistic effect of the EGFR and IGF1R inhibitors was also confirmed in nude mouse xenograft assay. This is the first study testing co-inhibiting EGFR and IGF1R signaling in the context of radio-sensitivity in PC and it may provide a promising adjuvant therapeutic approach to improve the outcome of PC patients to radiation treatment.

Genistein sensitizes bladder cancer cells to HCPT treatment in vitro and in vivo via ATM/NF-κB/IKK pathway-induced apoptosis.

Bladder cancer is the most common malignant urological disease in China. Hydroxycamptothecin (HCPT) is a DNA topoisomerase I inhibitor, which has been utilized in chemotherapy for bladder cancer for nearly 40 years. Previous research has demonstrated that the isoflavone, genistein, can sensitize multiple cancer cell lines to HCPT treatment, such as prostate and cervical cancer. In this study, we investigated whether genistein could sensitize bladder cancer cell lines and bladder epithelial cell BDEC cells to HCPT treatment, and investigated the possible underlying molecular mechanisms. Genistein could significantly and dose-dependently sensitize multiple bladder cancer cell lines and BDEC cells to HCPT-induced apoptosis both in vitro and in vivo. Genistein and HCPT synergistically inhibited bladder cell growth and proliferation, and induced G2/M phase cell cycle arrest and apoptosis in TCCSUP bladder cancer cell and BDEC cell. Pretreatment with genistein sensitized BDEC and bladder cancer cell lines to HCPT-induced DNA damage by the synergistic activation of ataxia telangiectasia mutated (ATM) kinase. Genistein significantly attenuated the ability of HCPT to induce activation of the anti-apoptotic NF-κB pathway both in vitro and in vivo in a bladder cancer xenograft model, and thus counteracted the anti-apoptotic effect of the NF-κB pathway. This study indicates that genistein could act as a promising non-toxic agent to improve efficacy of HCPT bladder cancer chemotherapy.

Expression of Mycobacterium tuberculosis ferric uptake regulator A gene in Escherichia coli and generation of monoclonal antibodies to FurA.

Ferric uptake regulator A of Mycobacterium tuberculosis (MTB), which belongs to the Fur superfamily, is situated immediately upstream of katG encoding catalase-peroxidase, a major virulence factor that also activates the pro-drug isoniazid. The feature and role of FurA in oxidative stress contribute to research on the pathogenesis of mycobacteria. In this study, four novel mouse monoclonal antibodies were generated using the prokaryotically expressed FurA protein as immunogen. The furA gene of M. tuberculosis H37Rv was inserted into a bacterial expression vector of pRSET-A and effectively expressed in Escherichia coli BL21(DE3). The expressed fusion protein existed as soluble form in cell lysates and was purified via Ni-NTA purification system. Using the fusion protein to immunize BALB/c mice, four monoclonal antibodies (H9H6, H9E12, H10H6, and H10H8) were produced. As shown by Western blot analysis and cell fluorescence microscopy assay, the four antibodies could recognize the FurA protein, respectively. Then we assessed the effect of iron on the expression of FurA in MTB H37Rv and we concluded that iron does not affect FurA expression. These results suggest that the antibodies against FurA may provide a powerful tool for elucidating FurA biofunctions and regulation mechanism in the pathogenesis of tuberculosis.

[Effects of Mycobacterium tuberculosis Hsp16.3 protein on the autophagy function of mice macrophages].

AIM: To investisate the inhibition of Hsp-16.3 on the autophagosomes formation of macrophages. METHODS: Mouse RAW264.7 macrophages were induced by rapamycin (50 ng/µL) following infection with M.tuberculosis H37Rv strains, thereafter, co-incubated with Hsp16.3 protein (25 µg/mL). The effects of Hsp16.3 protein on the autophagosomes formation was observed with transmission electron microscope. The expression of autophagy-related genes (atg8) for macrophages was detected by Western blotting. RESULTS: It was found that rapamycin-induced autophagy of macrophages infected with M.tuberculosis H37Rv enhanced localization of mycobacteria with autophagosomes. Hsp16.3 protein inhibits autophagosome formation and affects M.tuberculosis survival inside infected macrophages. Furthermore, Hsp16.3 protein significantly increased M.tuberculosis colony forming units (CFU), and decreased the expression of microtubule-associated protein light chain-3 (LC3) expression level (P<0.05). CONCLUSION: The results showed that Hsp16.3 protein inhibits the formation of autophagosomes by regulating the expression of LC3 protein.

Mechanisms involved in the blood-testis barrier increased permeability induced by EMP.

The blood-testis barrier (BTB) plays an important role in male reproductive system. Lots of environmental stimulations can increase the permeability of BTB and then result in antisperm antibody (AsAb) generation, which is a key step in male immune infertility. Here we reported the results of male mice exposed to electromagnetic pulse (EMP) by measuring the expression of tight-junction-associated proteins (ZO-1 and Occludin), vimentin microfilaments, and transforming growth factor-beta (TGF-beta3) as well as AsAb level in serum. Male BALB/c mice were sham exposed or exposed to EMP at two different intensities (200kV/m and 400kV/m) for 200 pulses. The testes were collected at different time points after EMP exposure. Immunofluorescence histocytochemistry, western blotting, laser confocal microscopy and RT-PCR were used in this study. Compared with sham group, the expression of ZO-1 and TGF-beta3 significantly decreased accompanied with unevenly stained vimentin microfilaments and increased serum AsAb levels in EMP-exposed mice. These results suggest a potential BTB injury and immune infertility in male mice exposed to a certain intensity of EMP.