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ABSTRACT: DnaJ, cooperating with DnaK and GrpE, promotes the folding of unfolded hydrophobic polypeptides, dissociates protein complexes and translocates protein across membranes. Additionally, DnaJ from Streptococcus pneumoniae (SpDnaJ) is involved in the infectious disease process and is being developed as a potential vaccine to prevent bacterial infection. Here the expression, purification, crystallization and preliminary crystallographic analysis of SpDnaJ are reported. The crystals belong to space groups I222 or I212121 and the diffraction resolution is 3.0 Å with unit-cell parameters a = 47.68, b = 104.45, c = 234.57 Å. The crystal most likely contains one molecule in the asymmetric unit, with a VM value of 3.24 Å(3) Da(-1) and a solvent content of 62.1%.
Acta Crystallographica Section F Structural Biology and Crystallization Communications 03/2013; 69(Pt 3):267-9. · 0.51 Impact Factor
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ABSTRACT: DCs are essential for host immune response to pathogens. Pneumococcal diseases still remain to be a major global-health issue, and HSP100/ClpP is a ubiquitously present virulence determinant for Streptococcus pneumoniae. Here, we show that ClpP expression facilitates the uptake and phagocytosis of pneumococci by human DCs, and it could increase apoptosis of DCs infected with pneumococci. Furthermore, pneumococcal ClpP is required for optimal production of inflammatory cytokines and chemokines and an efficient activation of adaptive immune response in DCs. Complementary, purified rClpP protein recognizes TLR4 and functionally activates human DCs by augmenting the expression of surface molecules and the production of inflammatory cytokines and chemokines dependent on MAPKs and NF-κB signaling pathways. Besides, ClpP-treated DCs induce T cell proliferation and contribute to Th1 immune response. This study describes a novel role of ClpP in the interaction of DCs with pneumococci that could provide new insight for the progression of pneumococcal diseases and has important implications for designing pneumococcal protein vaccines.
Journal of leukocyte biology 02/2013; · 4.99 Impact Factor
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ABSTRACT: IL-27 is involved in inflammatory reactions. CXCL10 is an important chemokine contributing to airway inflammatory diseases. In this study, we investigated whether IL-27 modulated the synthesis of CXCL10 in primary human lung fibroblasts (HLF). HLF were activated by IL-27 alone, or in combination with other cytokines. CXCL10 synthesis was measured by real-time PCR and ELISA. Examination of transcriptional regulation was performed via transient transfection of promoter constructs, whereas mRNA stability was assessed by actinomycin D chase and real-time PCR. The underlying signaling pathways were studied by Western blot and intracellular staining using flow cytometry. Our results demonstrated that IL-27 induced and synergized with TNF-α to upregulate CXCL10 mRNA and protein levels in a steroid-insensitive manner. This synergistic CXCL10 production was dependent on transcriptional regulation of CXCL10 gene promoter activity and the enhanced stability of CXCL10 mRNA by IL-27 and TNF-α, and this synergism was regulated by the activation of p38 MAPK and PI3K-Akt dominantly and in a little part via NF-kB. Interestingly, IL-27 promoted basal and enhanced TNF-α-induced phosphorylation of p38 MAPK and Akt, but not IkBα. Besides, the enhanced CXCL10 mRNA stability occurred via a p38 MAPK-dependent pathway. Finally, clinical analysis showed that IL-27 was detected in BAL of patients with asthma, COPD, and PTB, and the increased IL-27 levels was correlated with the increased CXCL10 levels in patients with COPD and PTB. Our findings suggest that IL-27 has the potential to amplify airway inflammation via the induction of CXCL10 from HLF in combination with TNF-α.
American Journal of Respiratory Cell and Molecular Biology 01/2013; · 5.13 Impact Factor
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Wen Zhong,
Wenchun Xu,
Hong Wang,
Yuanshuai Huang,
Ju Cao,
Yi Gong,
Xiuyu Xu,
Xun Min,
Yanqing Zhang,
Jie Dong, Yibing Yin,
Xuemei Zhang
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ABSTRACT: Streptococcus pneumoniae resides on the mucosal surface of the upper respiratory tract and is ready to spread and trigger clinical diseases. Hence the vaccine that can eliminate the nasopharyngeal colonization was thought to be an ideal protective strategy against pneumococcal invasive diseases. Caseinolytic protease X (ClpX), a pneumococcal caseinolytic protease ATPase subunit, was shown to be a non-transmembrane protein by bioinformatics analysis. Consistent with the in silico prediction, the secretory expression of ClpX, instead of surface exposure, was further confirmed by flow cytometry and Western blot. Furthermore, ClpX was highly conserved in nine different serotypes of S. pneumoniae at both gene and protein concentrations. In addition, the anti-ClpX IgG antibody levels in human serum samples were much higher in healthy children, compared with pediatric patients, and displayed an age-related increase. Finally, ClpX protein antigen was introduced to BALB/c mice through a mucosal route, and protection against nasopharyngeal colonization and lethal infection caused by different S. pneumoniae serotypes was successfully elicited. Our findings suggest that ClpX is a potential candidate antigen that could be incorporated in pneumococcal protein vaccines.
Experimental Biology and Medicine 06/2012; 237(6):694-702. · 2.64 Impact Factor
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ABSTRACT: An electrochemical immunosensor for detection of neuron specific enolase (NSE) was designed by immobilizing NSE covalently functionalized single-walled carbon nanotubes (NSE-SWNTs) on a glassy carbon electrode. The NSE-SWNTs not only enhanced electrochemical signal but also presented abundant antigen domains for competitive immunological recognition to anti-NSE primary antibody and then gold nanoprobes labeled with alkaline phosphatase conjugated secondary antibody (AP-anti-IgG/AuNPs). The AP-anti-IgG/AuNPs exhibited highly catalytic activity toward enzyme substrate and significantly amplified the amperometric signal for target molecule detection. Based on the dual signal amplification of SWNTs and gold nanoprobe, the immunosensor could response down to 0.033 ng mL(-1) NSE with a linear range from 0.1 ng mL(-1) to 2 μg mL(-1), and showed acceptable precision and reproducibility. The designed immunosensor was amenable to direct quantification of target protein with a wide range of concentration in complex clinical serum specimens. The assay results were in a good agreement with the reference values. The proposed electrochemical immunosensor provided a pragmatic platform for convenient detection of tumor markers in clinical diagnosis.
Talanta 05/2012; 93:433-8. · 3.79 Impact Factor
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ABSTRACT: Protein-based vaccines are considered to be the next-generation of pneumococcal vaccines. Here we evaluated the protection elicited by immunization with recombinant glutamyl tRNA synthetase (Gts), polyamine transport protein D (PotD) and sortase A (SrtA) antigens in preclinical mouse models. In mucosal immunization studies, intranasal immunization with either Gts, PotD or SrtA could significantly reduce pneumococcal nasopharyngeal and lung colonization and significantly increase mice survival times following invasive pneumococcal challenge, and combinations of these antigens could enhance this protection. In systemic immunization studies, intraperitoneal immunization with multiple protein antigens also provided better protection against pneumococcal sepsis caused by different pneumococcal strains. Finally, passive immunization studies showed an additive effect by using multiple anti-sera when compared to single anti-sera. Therefore, a multicomponent protein-based pneumococcal vaccine composed of Gts, PotD or SrtA could confer protection against pneumococcal colonization as well as invasive infections in terms of efficacy of protection and serotype coverage.
Vaccine 03/2012; 30(24):3624-33. · 3.77 Impact Factor
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ABSTRACT: Pneumolysin (Ply) is an essential virulence factor of S. pneumoniae, which can induce apoptosis in a variety of host cells to facilitate infection of pathogenic bacteria by as yet unclear mechanisms. To confirm the apoptosis-inducing properties of pneumolysin in endothelial cells, human umbilical vein endothelial cells (HUVECs) were exposed to pneumolysin. The proliferation of HUVECs was inhibited by pneumolysin in a dose- and time-dependent manner. Flow cytometry analysis and ultrastructural changes of the cells indicated the apoptotic response. Exposure to pneumolysin significantly increased the number of apoptotic cells and the activities of caspases-3 and -8. This change was associated with activation of p38 mitogen-activated protein kinase (MAPK) and suppression of extracellular signaling regulation kinase (ERK)1/2. Pre-exposure to the p38 MAPK inhibitor SB-203850 prevented human endothelial cells from apoptosis induced by pneumolysin. In conclusion, these findings demonstrate that pneumolysin induces apoptosis in endothelial cells and the involvement of p38 MAPK activation and ERK1/2 deactivation.
International Journal of Molecular Medicine 03/2012; 29(6):1025-30. · 1.98 Impact Factor
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ABSTRACT: Previous reports suggest that ClpP proteolytic activity is important not only for cell physiology but also for regulation of virulence properties of Streptococcus pneumoniae (S. pneumoniae). In order to get a more comprehensive picture of the role of ClpP protease on protein expression in S. pneumoniae D39 and how it relates to physiology and virulence, a clpP mutant strain was constructed in S. pneumoniae D39, and global proteome expression was studied by 2-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. We report here that clpP deletion affects the expression of proteins which are involved in the general stress response, nucleotide metabolism, energy metabolism, and proteins metabolism. These provide clues for understanding the role of ClpP in the physiology and pathogenesis of pneumococcus.
Current Microbiology 12/2011; 64(3):294-9. · 1.82 Impact Factor
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ABSTRACT: Although mast cells have been found in increased numbers in bronchial epithelium in asthma patients, the pathogenic role of the interaction of mast cells with bronchial epithelial cells in the development of local inflammation in asthma is not well understood. In this study, primary human bronchial epithelial cells and a human mast cell line (HMC-1) were cultured either together or separately in the presence or absence of various signaling molecule inhibitors or dexamethasone. Cytokine IL-6, and chemokines including CXCL1 and CXCL8 in cell culture supernatant were assayed by enzyme-linked immunosorbent assay (ELISA), and the activity of mitogen-activated protein kinases (MAPKs), or nuclear factor-κB (NF-κB) in co-culture system was analyzed by ELISA. Co-culture of bronchial epithelial cells and mast cells induced a significant elevation of IL-6, CXCL1 and CXCL8 in bronchial epithelial cells, and both IL-17A and IL-17F could further enhance the release of these inflammatory mediators from co-culture. The induction of IL-6, CXCL1 and CXCL8 upon the interaction of bronchial epithelial cells with mast cells was mediated by MAPKs and NF-κB signaling pathways. These data indicate that the interaction of mast cells with bronchial epithelial cells may represent a crucial mechanism of regulating local inflammatory response in allergic asthma.
Cytokine 12/2011; 56(3):823-31. · 3.02 Impact Factor
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Hua Zhou,
Miao Luo,
Xuefei Cai,
Jian Tang,
Siqiang Niu,
Wenlu Zhang,
Yuan Hu, Yibing Yin,
Ailong Huang,
Dacheng Wang,
Deqiang Wang
Proteins Structure Function and Bioinformatics 07/2011; 79(7):2346-51. · 3.39 Impact Factor
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ABSTRACT: A simple scanometric strategy was developed for ultrasensitive assay of matrix metalloproteinases based on their discriminatory and proteolytic activity by integrating a newly designed peptide-gold nanoparticle probe, a nitrilotriacetic acid modified chip and silver signal amplification.
Chemical Communications 03/2011; 47(10):2877-9. · 6.17 Impact Factor
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ABSTRACT: Zinc metalloprotease B (ZmpB) is present in all isolated pneumococcal strains and contributes to the pathogenesis of pneumococcal infection. In this study, recombinant ZmpB was cloned and expressed in Escherichia coli. The expression of ZmpB by different pneumococcal strains was detectable by Western blotting with antisera raised to recombinant ZmpB. Flow cytometry analysis demonstrated that anti-ZmpB polyclonal antibodies could bind to the cell surface of the pneumococcal strains analyzed. Both recombinant ZmpB protein and anti-ZmpB polyclonal antibodies significantly inhibited the adhesion of Streptococcus pneumoniae D39 to A549 cells. In mouse models, mucosal immunization with recombinant ZmpB could significantly reduce pneumococcal lung colonization caused by S. pneumoniae serotypes 19F and 14 and significantly increase mice survival times following invasive pneumococcal challenge with different pneumococcal strains, including serotypes 2, 3, 6B, and 14. Furthermore, intraperitoneal immunization with recombinant ZmpB in combination with the recombinant pneumolysin mutant (DeltaA146 Ply) and heat shock protein 40 (DnaJ) could enhance the protection against pneumococcal infection compared to protection provided by single-protein antigens. Passive immunization with hyperimmune antisera against these three antigens also demonstrated that the combination of three hyperimmune antisera could provide better protection than single antisera. Taken together, our results suggest that ZmpB is a good candidate pneumococcal vaccine antigen.
Infection and immunity 02/2011; 79(2):867-78. · 4.21 Impact Factor
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Yali Cui,
Xuemei Zhang,
Yi Gong,
Siqiang Niu,
Nanlin Yin,
Run Yao,
Wenchun Xu,
Dairong Li,
Hong Wang,
Yujuan He,
Ju Cao, Yibing Yin
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ABSTRACT: Increasing mortality, morbidity and economic costs have been paid to pneumococcal diseases every year. Currently, vaccination is the most promising strategy to reduce the occurrence of pneumococcal infection. In this study, we investigated the protective efficacy of immunization with recombinant DnaJ (hsp40) protein against infections of different serotypes of Streptococcus pneumoniae. We demonstrated that mucosal immunization with DnaJ antigen could induce both systemic and mucosal antibodies for DnaJ and stimulate the release of high levels of IL-10, IFN-γ and IL-17A. Moreover, this mucosal vaccination could reduce nasal or lung colonization of pneumococcus and elicit protection against different serotypes of invasive pneumococcal infections. As well, we found that intraperitoneal immunization with DnaJ could also protect against invasive infections caused by different serotypes of pneumococcus, and passive immunization with antibodies specific for DnaJ confirmed that this protection was antibody-mediated. Our results therefore support the potential of DnaJ as a conserved pneumococcal protein vaccine.
Vaccine 02/2011; 29(9):1736-44. · 3.77 Impact Factor
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ABSTRACT: RNase E functions as the rate-limiting enzyme in the global mRNA metabolism as well as in the maturation of functional RNAs. The endoribonuclease, binding to the PNPase trimer, the RhlB monomer, and the enolase dimer, assembles into an RNA degradosome necessary for effective RNA metabolism. The RNase E processing is found to be negatively regulated by the protein modulator RraA which appears to work by interacting with the non-catalytic region of the endoribonuclease and significantly reduce the interaction between RNase E and PNPase, RhlB and enolase of the RNA degradosome. Here we report the crystal structure of RraA from P. aeruginosa to a resolution of 2.0 Å. The overall architecture of RraA is very similar to other known RraAs, which are highly structurally conserved. Gel filtration and dynamic light scattering experiments suggest that the protein regulator is arranged as a hexamer, consistent with the crystal packing of "a dimer of trimer" arrangement. Structure and sequence conservation analysis suggests that the hexamer RraA contains six putative charged protein-protein interaction sites which may serve as binding sites for RNase E.
The Protein Journal 11/2010; 29(8):583-90. · 1.04 Impact Factor
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ABSTRACT: Surface-exposed pneumococcal virulence proteins pneumococcal surface protein A (PspA) and pneumococcal surface protein C (PspC) play important roles in the pathogenesis of invasive pneumococcal diseases. Human neutrophils are principle antimicrobial effector cells of the innate and adaptive immune systems. In this study, we investigated the effects of PspA and PspC on the up-regulation of chemokine CXCL8 in human neutrophils, and characterized the underlying intracellular signaling pathways. Both PspA and PspC were found to induce the release of newly synthesized CXCL8. Synergistic effect was observed in the combined treatment of PspA and PspC on the release of CXCL8. Products from PspA-deficient or PspC-deficient mutant pneumococcus that did not express PspA or PspC induced significantly less release of CXCL8 than wild type pneumococcus. Both PspA and PspC could activate p38 MAPK and NF-κB pathways in neutrophils, while inhibition of NF-κB and p38 MAPK could suppress the release of CXCL8 from neutrophils induced by PspA and PspC. Together, our results demonstrated that the induction of CXCL8 in human neutrophils activated by PspA and PspC was regulated by p38 MAPK and NF-κB pathways.
Microbes and Infection 11/2010; 12(12-13):1051-60. · 3.10 Impact Factor
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ABSTRACT: Our previous studies have demonstrated that bone morphogenetic protein 9 (BMP-9) is one of the most efficacious BMPs to induce osteoblast differentiation of mesenchymal stem cells (MSCs). However, the molecular mechanism underlying the BMP-9-induced osteogenic differentiation of MSCs remains to be fully elucidated. In this study, dominant negative (DN) type II TGF-β receptors were constructed and introduced into C3H10T1/2 stem cells, then in vitro and in vivo assays were carried out to analyze and identify the type II TGF-β receptors required for BMP-9-induced osteogenesis. We found that three DN type II TGF-β receptors, DN-BMPRII, DN-ActRII, and DN-ActRIIB, diminished BMP-9-induced alkaline phosphatase (ALP) activity, led to a decrease in BMP-9-induced Smad binding element (SBE)-controled reporter activity, reduced BMP-9-induced expressions of Smad6 and Smad7, and decreased BMP-9-induced mineralization in vitro and ectopic bone formation in vivo, finally resulted in decreased bone masses and immature osteogenesis. These findings strongly suggested that three wild-type II TGF-β receptors, BMPRII, ActRII and ActRIIB, may play a functional role in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells. However, C3H10T1/2 stem cells can express BMPRII and ActRII, but not ActRIIB. Using RNA interference (RNAi), we found that luciferase reporter activity and ALP activity induced by BMP-9 were accordingly inhibited along with the knockdown of BMPRII and ActRII. Taken together, our results demonstrated that BMPRII and ActRII are the functional type II TGF-β receptors in BMP-9-induced osteogenic differentiation of C3H10T1/2 cells.
Acta Biochimica et Biophysica Sinica 10/2010; 42(10):699-708. · 1.38 Impact Factor
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ABSTRACT: A pragmatic and simple strategy was developed for ultrasensitive detection of protein with good specificity and low matrix effect, which combined aptamer-initiated rolling circle amplification with a Au nanoparticle probe and convenient scanometric readout.
Chemical Communications 09/2010; 46(36):6720-2. · 6.17 Impact Factor
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Zhihua Tao,
Mo Shen,
Yanbo Zheng,
Xiaolu Mao,
Zhanguo Chen, Yibing Yin,
Kaiyuan Yu,
Zhiliang Weng,
Hui Xie,
Chengdi Li,
Xiuling Wu,
Yuanping Hu,
Xiaohua Zhang,
Ouchen Wang,
Qitong Song,
Zhixian Yu
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ABSTRACT: Prostate cancer (PCa) is the second most common cancer in men, and its incidence is still increasing. PCA3 is the most prostate cancer specific biomarker. Here we confirmed that both exon 3 and exon 4 are in the prostate-specific region of the PCA3 gene, and established the methodology of real-time fluorescent quantitative RT-PCR (FQ-RT-PCR) detecting PCA3 mRNA with primer spanning exons 1 and 3, and evaluated its clinical utility in a Chinese population. What disclosed that PCA3 mRNA is prostate cancer specific and shows increased expression in prostate cancer. It could be a reliable molecular marker in prostate cancer diagnosis. Exon 3-based real-time FQ-RT-PCR may prove useful in prostate cancer diagnosis, given that the associated primer would span only exons 1 and 3, relative to other models spanning exons 1 to 4. A shorter amplicon would not only enhance the efficiency of real-time FQ-RT-PCR, but may also simplify the quantification of PCA3 mRNA.
Experimental and Molecular Pathology 08/2010; 89(1):58-62. · 2.42 Impact Factor
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ABSTRACT: To investigate the effect of putative lipoate-protein ligase (LPL) on the virulence of Streptococcus pneumoniae.
lpl gene deficient strain was constructed by LFH-PCR and identified by PCR and sequencing. The cell adherence assay and mice challenge assay were used to observe the differences between wild strain and the mutant in the pathopoiesis of Streptococcus pneumoniae.
Mice virulence experiments showed that the median lethal time of wide type and the lpl mutant are both 12 h, no statistics difference. The ability of adherence of the mutant was greater than the wild strain (P < 0.01); The capsule stain in-vivo showed that the wild strain and the mutant both had the capsule.
lpl gene inhibits the adherence to host, but no affect on the ability to infection mice by
ACTA MICROBIOLOGICA SINICA 06/2010; 50(6):774-9.
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ABSTRACT: To screen in vivo genes of Streptococcus pneumoniae controlled by comE gene.
The comE-deficient strain was constructed by using insertion inactivation and identified by PCR and sequencing. The BALB/c mouse was used as test animal and injected with D39 wild type and D39 comE-deficient strain via intraperitoneal injection. The mice blood was obtained about 24 hours after the injection through posterior orbital venous plexus approach. Then the bacteria induced in vivo were collected from the blood and their RNA were extracted to measure the mRNA expression levels of each in vivo-induced gene by RT-PCR.
The differences of the expressions of 8 in vivo-induced genes in D39 and D39 comE-deficient strain were statistically significant (P < 0.05) and in which spd_0300, spd_0414, spd_ 0622, spd_1663, spd_1719, spd_0235, spd_0873 were up-regulated by transformation and meanwhile spd_1672 was down-regulated.
In vivo-induced genes spd_0300, spd_0414, spd_0622, spd_1663, spd_1719, spd 0235, spd_0873, spd_1672 regulated by transformation were screened out and they may participate in the processes such as growth regulation, temperature reception, carbohydrate metabolism, and lipoids metabolism. The bacterial transformation may enhance the virulence via regulating some in vivo-induced genes expression.
ACTA MICROBIOLOGICA SINICA 03/2010; 50(3):310-5.
