通讯机构：
[Wu, YM ] ;Univ S China, Pathogenic Biol Inst, Hengyang 421001, Peoples R China.
摘要：
This study was designed to investigate the molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression and apoptosis in human monocytic cell line THP-1 stimulated by lipoproteins ( LPs) prepared from Mycoplasma genitalium. Cultured cells were stimulated with M. genitalium LP to analyze the production of proinflammatory cytokines and expression of their mRNA by ELISA and RT-PCR, respectively. Cell apoptosis was also detected by Annexin V-FITC-propidium iodide ( PI) staining and acridine orange ( AO)-ethidium bromide ( EB) staining. The DNA-binding activity of nuclear factor-kappa B ( NF-kappa B) was assessed by electrophoretic mobility shift assay ( EMSA). Results showed that LP stimulated THP-1 cells to produce tumor necrosis factor-alpha ( TNF-alpha), interleukin-1 beta ( IL-1 beta), and IL-6 in a dose-dependent manner. The mRNA levels were also upregulated in response to LP stimulation. LPs were also found to increase the DNA-binding activity of NF-kappa B, a possible mechanism for the induction of cytokine mRNA expression and the cell apoptosis. These effects were abrogated by PDTC, an inhibitor of NF-kappa B. Our results indicate that M. genitalium-derived LP may be an important etiological factor of certain diseases due to the ability of LP to produce proinflammatory cytokines and induction of apoptosis, which is probably mediated through the activation of NF-kappa B. Copyright (C) 2008 YimouWu et al.
作者机构：
[Chen Lin; Yu Min-jun; Wan Yan-ping; Zhu Cui-ming; Cao Qing-xiang; Liu An-yuan; Chan Xi] Univ S China, Inst Pathogen Biol, Hengyang 421001, Peoples R China.
通讯机构：
[Wan Yan-ping] U;Univ S China, Inst Pathogen Biol, Hengyang 421001, Peoples R China.
关键词：
human cytomegalovirus ( HCMV);IE1;macrophages;IL-1 beta;TNF-alpha;apoptosis
摘要：
Objective: To investigate the effect of human cytomegalovirus (HCMV) IE1 protein on the secretory activity and apoptosis of macrophages. Methods: The eukaryotic expression vector pEGFP-C1/IE1 was used to transfect THP-1-macrophages. 48 h after transfection, the expression and localization of GFP or GFP-IE1 was observed under fluorescent microscope. The levels of IL-1β and TNF-α in the culture media were examined by ELISA, and the mRNA expression of them was analyzed by RT-PCR. Cell undergoing apoptosis were determined by flow cytometry using the propidium iodide (PI) staining method. The data were analyzed by SPSS 13.0. Results: As observed under fluorescent microscope, the expressions of GFP-IE1 and GFP by plasmid pEGFP-C1/IE1 or pEGFP-C1 in THP-1-macrophages could be found in nuclei or whole cells. Conclusion: As demonstrated by RT-PCR and ELISA, mRNA and protein expressions of IL-1β and TNF-α and promotes apoptosis in THP-1-macrophages.
作者机构：
[Wang X.; Zhu C.; Cai H.; Wan Y.] Institute of Pathogenic Biology, University of South China, Hengyang 421001, China;School of Public Health, University of South China, Hengyang 421001, China;[Hengling Cai] Institute of Pathogenic Biology, University of South China, Hengyang 421001, China, School of Public Health, University of South China, Hengyang 421001, China
通讯机构：
[Aitao He] I;Institute of Pathogenic Biology, University of South China, Hengyang, China<&wdkj&>School of Public Health, University of South China, Hengyang, China
摘要：
Mycoplasma penetrans was shown to be involved in alteration of several eukaryotical cells functions and a causative agent in urogenital infectious diseases. Lipid-associated membrane proteins (LAMPs) may be responsible for the pathogenicity of some mycoplamas. In this study, we investigated whether M. penetrans LAMPs have pathogenic potential by inducing apoptosis in mouse macrophages. As analyzed by annexin-V – fluorescein isothiocyanate staining, significant early- and late-stage apoptosis was induced in M. penetrans LAMPs-challenged mouse macrophages. And agarose gel electrophoresis of the DNA of M. penetrans LAMPs-challenged cells revealed a ladder-like pattern of migration of DNA indicative of apoptosis. The possible molecular mechanisms responsible for the induction of apoptosis were also investigated by characterizing the activation of nuclear transcription factor κB (NFκB). NFκB was activated and translocated into the nucleus in mouse macrophages stimulated by M. penetrans LAMPs. The activation of NFκB and M. penetrans LAMPs-induced apoptosis in mouse macrophages was partially inhibited by the NFκB-specific inhibitor pyrrolidine dithiocarbamate. Thus, this study demonstrates that M. penetrans LAMPs may be an important etiological factor owing to their ability to induce apoptosis in mouse macrophages, which is probably mediated through the activation of NFκB.
作者机构：
[Wu Yi-Mou; Chen Chun-Lian; Wan Yan-Ping; Zhu Cui-Ming; Liu An-Yuan; Yin Wei-Guo; Wang Jing] Univ S China, Inst Pathogen Biol, Hengyang 421001, Peoples R China.;[Chen Chun-Lian] Guilin Med Coll, Affiliated Hosp, Guilin 541001, Peoples R China.
通讯机构：
[Wan Yan-Ping] U;Univ S China, Inst Pathogen Biol, Hengyang 421001, Peoples R China.
关键词：
HBV;X protein;Macrophages;TNF-α;IL-1β
摘要：
Objective: To provide the experimental basis for further studying the molecular transformation mechanism of Hepatitis B virus (HBV) X protein (HBx) on hepatocellular carcinoma. Methods: Reconstructed plasmid pcDNA3.1(+)-HBx was transfected into THP-1 macrophages. Expression of HBx was assayed in macrophages lysate by Western-blotting, and TNF-α and IL-1β contents were detected respectively by ELISA. All the data were analyzed by SPSS13.0. Results: In THP-1 macrophages, the pcDNA3.1(+)-HBx plasmid expressed HBx with a molecular weight of about 17 KDa demonstrated by Western-blotting. The secreted TNF-α and IL-1β from macrophages were determined by ELISA, the results from analysis of all groups showed as following: control group was different from LPS group and pcDNA3.1(+) group (P<0.01), and so was pcDNA3.1(+)-HBx group; but there was no obvious difference between pcDNA3.1(+) group and LPS group (P>0.05), all of which indicated that transient overexpression of HBx enhanced LPS-induced production of TNF-α and IL-1β by macrophages. Conclusion: Transient overexpression of HBx up-regulates LPS-induced TNF-α and IL-1β secretion of macrophages.
摘要：
Background. This study was designed to investigate the potential pathogenicity of Mycoplasma penetrans (M. penetrans) and its molecular mechanisms responsible for the induction of iNOS gene expression in mouse macrophages stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. penetrans. Methods. Mouse macrophages were stimulated with M. penetrans LAMPs to assay the production of nitric oxide (NO). The expression of inducible nitric oxide synthase (iNOS) was detected by RT-PCR and Western blotting. The activity of nuclear factor κB (NF-κB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-κB, on the production of nitric oxide and the expression of iNOS were also assessed in mouse macrophages treated with M. penetrans LAMPs by indirect immunofluorescence and Western blotting. Results. M. penetrans LAMPs stimulated mouse macrophages to produce nitric oxide in a dose- and time-dependent manner. The mRNA and protein levels of iNOS were also upregulated in response to LAMP stimulation and inhibited by PDTC treatment. M. penetrans LAMPs were found to trigger NF-κB activation, a possible mechanism for the induction of iNOS expression. Conclusion. This study demonstrated that M. penetrans may be an important etiological factor of certain diseases due to the ability of M. penetrans LAMPs to stimulate the expression of iNOS, which is probably mediated through the activation of NF-κB.
摘要：
Background. Daxx has been identified as a nuclear protein that involves in apoptosis and transcriptional repression. Daxx co-localizes with the promyelocytic leukemia (PML) protein and regulates transcription. Human Daxx (hDaxx) is a protein that functions as a transcriptional regulation through its interaction with some DNA-associated proteins. The aim of this study was to explore the transcriptional regulatory effect of hDaxx interacting with adenovirus (Ad) 12 E1B (Ad12E1B) 55-kDa oncoprotein. Methods. The co-localization of hDaxx-Ad12E1B or hDaxx-PML protein in the nucleus was observed under a confocal microscope. Interaction of hDaxx and Ad12E1B was analyzed by yeast two-hybrid assay. Direct binding of hDaxx and Ad12E1B was analyzed using coimmunoprecipitation and Western blot in vivo and in vitro. The activity of a luciferase reporter gene, which was regulated by an hDaxx modulated thymidine kinase (TK) promoter, was detected in an automat luminometer. Results. Ad12E1B, which co-localized with hDaxx in the nuclei of G401-CC3 cells, disrupted the co-localization of hDaxx and PML in the PML oncogenic domains (PODs). hDaxx bound directly to Ad12E1B in vivo and in vitro. hDaxx interacted with Ad12E1B along its full length. Ad12E1B enhanced transcriptional repression activity of hDaxx. Conclusion. Ad12E1B disrupts the co-localization of hDaxx with PML in PODs and enhances transcriptional repression activity of hDaxx.
摘要：
Objective: To construct a recombinant plasmid containing the outer membrane protein 2 (Omp2) gene of Chlamydia trachomatis and express Omp2 in E.coli. Methods: The omp2 gene of C. trachomatis serovar D was cloned into pQE30 vector following PCR amplification from genomic DNA. E. coli M15 transformants were induced to express the fusion protein by IPTG and the product was identified by SDS-PAGE and Western blot. Results: Confirmed by enzyme cleavage analysis and DNA sequencing, a correct recombinant plasmid pQE30/omp2 was constructed. The fusion protein from the transformants was approximately 60 kDa in size in SDS-PAGE analysis, which could specially react with anti-6 × His mouse monoclonal IgG antibodies. Conclusion: We successfully expressed Omp2 in E. coil M15, providing an efficient and simple system for assaying the immunological properties of Omp2.
