The mpn668 gene of Mycoplasma pneumoniae encodes a novel organic hydroperoxide resistance protein.

Mycoplasma pneumoniae (M. pneumoniae), as an obligate parasite, has evolved a protective strategy for coping with oxidative challenges caused by M. pneumoniae itself as well as the host immune system. However, to date, few antioxidant enzymes have been identified in mycoplasmas. In this report, we identified a protein encoded by the mpn668 gene from M. pneumoniae with a putative function as an organic hydroperoxide reductase (Ohr). The results indicated that the recombinant 140 amino acid protein, designated rMPN668, displayed hydroperoxidase activity towards both organic (tert-butyl hydroperoxide) and inorganic (hydrogen peroxide) hydroperoxides in the presence of a reducing agent such as dithiothreitol. Moreover, the expression of mpn668 in M. pneumoniae is upregulated in response to oxidative stress. Additionally, homology modeling of MPN668 and a molecular dynamics simulation suggest that both Cys55 and Cys119 form part of the active site of the protein. Mutants in which Cys55 or Cys119 were replaced with a serine lack antioxidant activity, indicating that MPN668 is a Cys-based peroxidase, consistent with it representing a new member of the Ohr family.

Hypothetical protein Cpn0423 triggers NOD2 activation and contributes to Chlamydia pneumoniae-mediated inflammation.

BACKGROUND: Chlamydia pneumoniae (C. pneumoniae) is pathogenic to humans, by causing pulmonary inflammation or bronchitis in both adolescents and young adults. However, the molecular signals linking C. pneumoniae components to inflammation remain elusive. This study was to investigate the effect of Chlamydia-specific Cpn0423 of C. pneumoniae on C. pneumoniae-mediated inflammation. RESULTS: Cpn0423 was detected outside of C. pneumoniae inclusions, which induced production of several cytokines including macrophage inflammatory protein-2 (MIP-2) and interleukins (ILs). Production of the Cpn0423-induced cytokines was markedly reduced in cells pretreated with NOD2-siRNA, but not with negative control oligonucleotides. Mice treated with Cpn0423 through intranasal administration exhibited pulmonary inflammation as evidenced by infiltration of inflammatory cells, increased inflammatory scores in the lung histology, recruitment of neutrophils and increased cytokines levels in the BALF. CONCLUSION: Cpn0423 could be sensed by NOD2, which was identified as an essential element in a pathway contributing to the development of C. pneumoniae -mediated inflammation.

Death domain associated protein (Daxx), a multi-functional protein.

Death domain associated protein (Daxx), a multi-functional protein, plays an important role in transcriptional regulation, cell apoptosis, carcinogenesis, anti-virus infection and so on. However, its regulatory mechanisms for both cell survival and apoptosis remain largely obscure. Our review of recent studies shows that Daxx has many interesting functional dualities and can provide a reference for further research on Daxx.

Prenatal exposure to PFOS caused mitochondia-mediated apoptosis in heart of weaned rat.

Perfluorooctanyl sulfonate (PFOS), a cardiac toxicity compound, has been widely detected in the environment and in organisms. However, the toxic mechanism is not clear. Our previous study indicated that prenatal PFOS exposure led to swollen mitochondrial with vacuolar structure and loss of cristae in offsping's heart. The purpose of this study was to investigate the effect of PFOS on the apoptosis in developing heart and mitochondria-mediated apoptosis pathway. Pregnant Sprague-Dawley (SD) rats were exposed to PFOS at doses of 0.1, 0.6, and 2.0 mg/kg-d and 0.05% Tween 80 as control by gavage from gestation day 2 (GD 2) to GD 21. Apoptosis, as well as expression of apoptosis related genes associated with mitochondrial-mediated apoptosis pathway, including p53, bcl-2, bax, cytochrome c, caspase-9, and caspase-3 were analyzed in heart tissues from weaned (postnatal day 21, PND 21) offspring. The results showed that prenatal PFOS exposure resulted in apoptosis in the offspring's heart. The mRNA and protein expression levels of p53, bax, cytochrome c, caspase-9, and caspase-3 in the offspring's heart were enhanced in various PFOS-treated groups, meanwhile, the bcl-2 expression levels were decreased. Our results indicated that prenatal PFOS exposure induced the apoptosis of weaned offspring rat heart tissue via mitochondria-mediated apoptotic pathway.

Rapid detection of rifampin-resistant clinical isolates of Mycobacterium tuberculosis by reverse dot blot hybridization.

OBJECTIVE: A PCR-reverse dot blot hybridization (RDBH) assay was developed for rapid detection of rpoB gene mutations in 'hot mutation region' of Mycobacterium tuberculosis (M. tuberculosis). METHODS: 12 oligonucleotide probes based on the wild-type and mutant genotype rpoB sequences of M. tuberculosis were designed to screen the most frequent wild-type and mutant genotypes for diagnosing RIF resistance. 300 M. tuberculosis clinical isolates were detected by RDBH, conventional drug-susceptibility testing (DST) and DNA sequencing to evaluate the RDBH assay. RESULTS: The sensitivity and specificity of the RDBH assay were 91.2% (165/181) and 98.3% (117/119), respectively, as compared to DST. When compared with DNA sequencing, the accuracy, positive predictive value (PPV) and negative predictive value (NPV) of the RDBH assay were 97.7% (293/300), 98.2% (164/167), and 97.0% (129/133), respectively. Furthermore, the results indicated that the most common mutations were in codons 531 (48.6%), 526 (25.4%), 516 (8.8%), and 511 (6.6%), and the combinative mutation rate was 15 (8.3%). One and two strains of insertion and deletion were found among all strains, respectively. CONCLUSION: Our findings demonstrate that the RDBH assay is a rapid, simple and sensitive method for diagnosing RIF-resistant tuberculosis.

Development of ELISAs for the detection of urogenital chlamydia trachomatis infection targeting the pORF5 protein.

OBJECTIVE: To prepare antibodies against pORF5 plasmid protein of Chlamydia trachomatis and develop double-antibody sandwich enzyme-linked immunosorbent assays (DAS-ELISAs) for the detection of genital C. trachomatis infections. METHODS: The pORF5 protein was expressed in Escherichia coli and used to immunize BALB/c mice and New Zealand rabbits to produce monoclonal antibodies (mAbs) and polyclonal antibody (pAb) for DAS-ELISAs. Clinical samples from 186 urogenital infection patients (groups I) and 62 healthy donors (groups II) were detected in parallel by the DAS-ELISAs developed in this study and by IDEIA PCE commercial ELISA. RESULTS: Two hybridoma cell lines, named 2H4 and 4E6, stably secreting specific mAbs against pORF5 were obtained. The mAb 2H4 was recognized by 32 (17.20%, positive recognition rate) and 25 (13.44%), mAb 2H4 by 0 (0%) and 2 (3.22%) samples from groups I and II, respectively. The sensitivities of mAbs 2H4 and 4E6 were 92.11% and 77.78% and the specificities were 100% and 96.88%, respectively in relation to the IDEIA PCE commercial ELISA. The sensitivities of detection for the DAS-ELISAs were 10 ng/mL (based on 2H4) and 18 ng/mL (based on 4E6). CONCLUSION: Two DAS-ELISAs were developed in this study that provided a feasible and effective assay that could be considered alternative tools for the serodiagnosis of C. trachomatis infection.

A study of the technique of western blot for diagnosis of lyme disease caused by Borrelia afzelii in China.

OBJECTIVE: To study the technique of Western blot for the diagnosis of Lyme disease caused by Borrelia afzelii in China and to establish the standard criteria by operational procedure. METHODS: FP1, which is the representative strain of B. afzelii in China, was analyzed by SDS-PAGE, electro transfer and immunoblotting assays. The molecular weights of the protein bands of FP1 were analyzed by Gel-Pro analysis software. In a study using 451 serum samples (159 patients with Lyme disease and 292 controls), all observed bands were recorded. The accuracy of the WB as a diagnostic test was established by using the ROC curve and Youden index. RESULTS: Criteria for a positive diagnosis of Lyme disease were established as at least one band of P83/100, P58, P39, OspB, OspA, P30, P28, OspC, P17, and P14 in the IgG test and at least one band of P83/100, P58, P39, OspA, P30, P28, OspC, P17, and P41 in the IgM test. For IgG criteria, the sensitivity, specificity and Youden index were 69.8%, 98.3%, and 0.681, respectively; for IgM criteria, the sensitivity, specificity and Youden index were 47%, 94.2%, and 0.412, respectively. CONCLUSION: Establishment of WB criteria for B. afzelii is important in validating the diagnostic assays for Lyme disease in China.

Development and evaluation of a MAb-based ELISA for detection of Chlamydophila pneumoniae infection with variable domain 2 and 3 of the major outer membrane protein.

OBJECTIVE: This paper aims to develop a monoclonal antibodies (MAbs)- based ELISA for detecting Chlamydophila pneumoniae (C. pneumoniae) antigens in humans with the variable domains (VD) 2 and 3 of the major outer membrane protein (MOMPVD2-VD3) and to assess its sensitivity and specificity by comparing with a widely used MAb that is able to recognize the elementary bodies of C. pneumoniae. METHODS: MOMPVD2-VD3 were overexpressed in Escherichia coli and purified by affinity chromatography. Mice were immunized with the recombinant antigen, and hybridomas secreting MAbs were screened. Three stable hybridomas clones were selected and named 5D6, 7G3, and 8C9. The MAbs-based ELISA was scrutinized for species-specific recognition with a number of human throat swab samples from Group I (156 patients with typical respiratory illness clinically confirmed before) and Group II (57 healthy donors). RESULTS: In Group I, 55 positive cases were detected by anti-EB MAb-based ELISA, 51 cases were positive by MAbs 5D6-based ELISA, and 33 and 38 cases were positive by MAb 8C9 and 7G3-based ELISA respectively. Of the 57 samples from Group II "healthy donors", 5 were positive and 52 were negative with both anti-EB and 5D6-based tests, while 2 and 3 positive cases were identified by the other two MAb-based ELISAs respectively. CONCLUSION: The novel MOMPVD2-VD3 MAb-based assay may have higher specificity than the anti-EB MAb, which may possibly be used as an alternative tool for the diagnosis of C. pneumoniae infection.

In vitro activities of erythromycin, tetracycline and levofloxacin alone and in dual combinations against ureaplasma spp.

PURPOSE: It was the aim of our study to evaluate the in vitro activities of tetracycline (TET), erythromycin (ERY) and levofloxacin (LVX) alone and in dual combinations against ureaplasmas. METHODS: The minimum inhibitory concentrations (MICs) of 51 ureaplasmal strains were determined by microdilution assay. RESULTS: TET was the most active when the antibiotics were used alone. The combinations resulted in significantly decreased MICs for every agent compared with the use of single antibiotics (p < 0.05, respectively), except for ERY in the ERY-LVX pair (p > 0.05), and decreased the MICs more significantly in the strains with an MIC ≥4 mg/l compared with MIC <4 mg/l, except for the TET-ERY pair. The ERY-LVX pair increased ERY MICs significantly in the MIC <4 mg/l group (p < 0.05). The combinations resulted in more beneficial MICs in strains where both agents had an MIC ≥4 mg/l compared with those where either had an MIC ≥4 mg/l, as well as in strains where either agent had an MIC <4 mg/l compared with those where both had an MIC <4 mg/l. CONCLUSIONS: Drugs in dual combinations always give more beneficial MICs against ureaplasmas than one agent alone. Combinational benefits prefer strains with a higher initial MIC.

HPV Prevalence and Genotype Distribution Among Women From Hengyang District of Hunan Province, China.

Most cervical cancers were closely associated with human papillomavirus (HPV) infections. Therefore, understanding the ecological diversity of HPV prevalence and genotype distribution among various populations in different geographical regions was essential for optimizing HPV vaccination and maximizing the vaccination effects. A total of 12,053 patient data from the three-level hospitals in Hengyang city were retrospectively analyzed. In this study, the HPV prevalence was 10.16% overall, and the multiple-type infection rate was 1.83%. The HR-HPV infection rate was 8.52%. The top six HPV genotypes were as follows in descending order: HPV16, HPV58, HPV52, HPV39, HPV51, and HPV53. The HPV prevalence in the group above 60 years old was the most, and their HR-HPV infection rate corresponded to the most too. The infection rates of HPV and HR-HPV among outpatients were both lower than those among the hospitalized-patients, respectively. Among the hospitalized-patients, the infection rates of HPV and HR-HPV among the 50-60 years group were the most in both. The HR-HPV ratio-in-positive among HPV-positive patients with the histopathologic examination was higher than that among those patients without. Among 52 HPV-positive patients with cervical squamous carcinoma, the ratio-in-positive of HPV16 was 61.54%. This study demonstrated that the HPV prevalence varied with age among women from Hengyang district of Hunan province in China and showed that HPV16, HPV58, HPV52, HPV39, HPV51, and HPV53 genotypes were more popularly distributed in this region, which could provide the experimental basis for Chinese public health measures on cervical cancer prevention.

Mycoplasma lipoproteins and Toll-like receptors.

Mycoplasmas, the smallest free-living, self-replicating bacteria with diameters of 200 to 800 nm, have been reported to be associated with human diseases. It is well known that the mycoplasma lipoprotein/peptide is able to modulate the host immune system, whose N-terminal structure is an important factor in inducing immunity and distinguishing Toll-like receptors (TLRs). However, there is still no clear elucidation about the pathogenic mechanism of mycoplasma lipoprotein/peptide and the signaling pathway. Some researchers have focused on understanding the structures of these proteins and the relationships between their structure and biological function. This review provides an update on the research in this field.

[Application of the recombinant protein MOMP(VD2-VD3) from Chlamydia pneumoniae in sero diagnosis].

To express the recombinant protein MOMP(VD2-VD3) of Chlamydia pneumoniae, and research on the immunocompetence of the MOMP(VD2-VD3) to support serodiagnosis,PCR and gene recombinant technique was used to clone the targeted DNA fragment from a strain AR-39. The recombinant plasmid was induced in E. coli BL21 after having constructed the prokaryotic expression system, then the immunocompetence of the expression product was analyzed by Western blot and indirected ELISA which is based on the animal experimentation. A group of control sera and 126 sera from patients with coronary heart disease were examined by using ELISAs based on the recombinant protein (MOMP(VD2-VD3), and then the results were evaluated comparing with a commercial ELISAs kit. The results of the Western blot and indirected ELISA showed ompA(VD2-VD3) gene inserted in pET30a could express a recombinant protein with the molecular weight of 24kDa in BL21 and specifically reacted with the antibodies against the MOMP. Specific humoral response was elicited after immune the BALB/c mouse with protein and the specific antibody titer was more than 1:20480. Using a panel of control sera, the participation of the recombinant antigen, the sensitivity and the specificity of the indirected ELISAs were 100% respectively. Comparisons between two methods in detecting 126 sero samples, the concordance of two tests was 96.3%. The results reported here show that the recombinant protein with excellent immunocompetence could benefit the research on the serodiagnosis to Chlamydia pneumoniae.

[Mycoplasma genitalium lipid-associated membrane proteins induce human monocytic cell express proinflammatory cytokines and apoptosis by activating nuclear factor kappaB].

Designed to investigate the potential pathogenicity of Mycoplasma genitalium (M. genitalium) and its molecular mechanisms responsible for the induction of proinflammatory cytokines gene expression in human monocytic cells (THP-1) stimulated by lipid-associated membrane proteins (LAMPs) prepared from M. genitalium. THP-1 cells were stimulated with LAMPs to analyze the production of proinflammatory cytokines and the expression of mRNA was detected by RT-PCR. Cell apoptosis was detected in THP-1 cells by Annexin V-propidium iodide staining. The activity of transcriptional factors, NF-kappaB, was examined in THP-1 cells treated with LAMPs by EMSA. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, on the production of proinflammatory cytokines, the expression of mRNA and apoptosis were also examined in THP-1 cells treated with LAMPs. M. genitalium LAMPs stimulate THP-1 cells to produce TNF-alpha, IL-1beta and IL-6 in dose- and time-dependent manner. The mRNA levels and cell apoptosis are also downregulated in response to LAMPs stimulation and inhibited by PDTC treatment. M. genitalium LAMPs are found to trigger NF-kappaB activation, a possible mechanism for the induction of mRNA expression and the cell apoptosis. This study demonstrated that M. genitalium may be an important etiological factor of certain disease due to the ability of LAMPs to stimulated the expression of mRNA and apoptosis, which is probably mediated through the activation of NF-kappaB.

Interactions between mycoplasma lipid-associated membrane proteins and the host cells.

Mycoplamas are a group of wall-less prokaryotes widely distributed in nature, some of which are pathogenic for humans and animals. There are many lipoproteins anchored on the outer face of the plasma membrane, called lipid-associated membrane proteins (LAMPs). LAMPs are highly antigenic and could undergo phase and size variation, and are recognized by the innate immune system through Toll-like receptors (TLR) 2 and 6. LAMPs can modulate the immune system, and could induce immune cells apoptosis or death. In addition, they may associate with malignant transformation of host cells and are also considered to be cofactors in the progression of AIDS.

[Expression of inducible nitric oxide synthase was regulated by activating nuclear factor kappaB in mouse macrophages stimulated with ureaplasma urealyticum lipid-associated membrane proteins].

The aim was to investigate the molecular mechanisms responsible for the inducible nitric oxide synthase (iNOS) gene expression stimulated by lipid associated membrane proteins (LAMPs) of Ureaplasma urealyticum (Uu). Mouse macrophages were stimulated by Ureaplasma urealyticum LAMPs to analyze the production of nitric oxide (NO) and the expression of iNOS detected by RT-PCR and Western blot. The activation of NF-kappaB was examined in mouse macrophages treated with LAMPs by indirect immunofluorescence (IFA), immunocytochemistry and Western blot. The effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB and of cycloheximide (CHX), a protein synthase inhibitor, on the expression of iNOS and on the activation of NF-kappaB were also investigated in mouse macrophages treated with LAMPs. Results showed Ureaplasma urealyticum LAMPs stimulated mouse macrophages to express iNOS and thus produce NO in dose- and time-dependent manners by activating nuclear factor kappaB. The activation of NF-kappaB and the expression of iNOS were inhibited by LAMPs combination with PDTC or CHX. In conclusion, these findings suggested Ureaplasma urealyticum may be an important pathogenic factor due to the ability of LAMPs to stimulate the expression of iNOS, which is probably medicated by the activation of NF-kappaB.

[DNA sequence polymorphism of Chlamydia trachomatis omp1 gene].

A method for detection and genotyping of genital Chlamydia trachomatis infections based on omp1 gene amplification and sequencing was developed, and the character of omp1 gene of Chlamydia trachomatis was analysed. Urethral or endocervical specimens were collected from 323 patients attending STD clinics in Hengyang, Shanghai, Guangzhou and Jiangmen from November, 2003 to May, 2004. DNA was extracted by usual method, and an approximately 980bp fragment from the major outer membrane protein (omp1) gene of Chlamydia trachomatis was amplified by nested polymerase chain reaction (nPCR). The PCR products were purified by DNA agarose gel purification system and the sequence of the omp1 gene was determined by using an ABI PRISM 3700 Genetic Analyser, and genotyping was performed by BLAST similarity search. Multiple alignment was performed with CLUSTAL package (CLUSTAL X), and a phylogenetic tree was constructed by Mega3 software to illustrate the evolutionary relationships between clinical isolates and reference strains of C. trachomatis obtained from GenBank. All variable sequences were submitted to GenBank by Banklt programe. The overall prevalence of urogenital chlamydial infection was 29.7% (96 of 323). All the 96 C. trachomatis-positive cases were sequenced, and 10 genotypes and 28 genetic variants were detected. The most prevalent genotype was E(34.4%), followed by J(25.0%), D(12.5%), F(8.3%), G(7.3%), H(3.1%), Ba(3.1%), K(3.1%), Da (2.1% ), 1 (1.1%). The distribution of C. trachomatis genotypes in the four cities in sourth China was similar to other countries in the world. The omp1 gene was highly conserved for genotype E and F, but appeared slightly less conserved for other genotypes, where the sequences displayed one or several nucleotide substitutions relatived to the corresponding reference sequence. And a similar recombination was found between genotypes Ba and D in CD1. Phylogenetic tree showed that Chlamydia trachomatis genotypes were mainly divided into three clusters, according to previous grouping in the B, F-G, and C complexes. Clusters F-G and C were characterized by small genetic distances within each cluster, but clusters B displayed larger genetic distances. And the clinical isolates were highly related to the reference strains. It is concluded that the isolated Chlamydia trachomatis strains exhibit remarkable omp1 DNA sequence polymorphism, which can encourage for vaccine design and infection control.

[Cloning and expression of outer membrane protein gene Gpd from Treponema pallidum and preliminary studies on its immunogenicity in rabbits].

To construct the recombinant plasmid of Eukaryotic expression containing Gpd gene from Treponema Pallidum and study its immunogenicity in New Zealand White rabbits. Gpd gene was amplified from the genomic DNA of T. pallidum and cloned into appropriate site of pcDNA3. 1 ( + ) vector. After verified that the Gpd antigen gene could be expressed in HeLa cells by Western blot and immunocytochemistry, recombinant plasmids pcDNA3.1 ( + )-Gpd, control plasmid pcDNA3. 1 ( + ) or PBS buffer were administered in three groups of New Zeal and White rabbits. Booster immunizations were employed at 2-week interval for three times. ELISA was used for the quantitative detection of the specific antibody in the sera of rabbits. The proliferation response of spleen cells was detected by MTT assay. The results of the Western blot and immunocytochemistry showed that Gpd gene constructed in pcDNA3.1 ( + ) vector could express a fusion protein with a calculated molecular mass of 41kD in HeLa cells and react with positive blood serum from syphilis patients. The significant specific antibody IgG titers were observed and the highest titer was 1:1024 in rabbits after three times with pcDNA3.1 ( + )-Gpd. The proliferation response of spleen cells were significantly higher than that of rabbits injected with pcDNA3.1 ( + ) (p < 0.05). All above results establish a solid basis for future studying the biological activities of Gpd and benefit the development of the Syphilis DNA vaccine.

The type III secretion system (T3SS) of Chlamydophila psittaci is involved in the host inflammatory response by activating the JNK/ERK signaling pathway.

Chlamydophila psittaci (C. psittaci) is a human zoonotic pathogen, which could result in severe respiratory disease. In the present study, we investigated the role and mechanism of the type III secretion system (T3SS) of C. psittaci in regulating the inflammatory response in host cells. C. psittaci-infected THP-1 cells were incubated with the specific T3SS inhibitor INP0007, inhibitors of ERK, p38, or JNK, and the levels of inflammatory cytokines were analyzed using Q-PCR and ELISA. The levels of ERK, p38, and JNK phosphorylation were analyzed by Western blot. Our results verified that INP0007 inhibited chlamydial growth in vitro, but the coaddition of exogenous iron completely reversed the growth deficit. INP0007 inhibited the growth of C. psittaci and decreased the levels of IL-8, IL-6, TNF-α, and IL-1ß. Exogenous iron restored the chlamydial growth but not the production of inflammatory cytokines. These results demonstrated that the expression of inflammatory cytokines during infection was associated with the T3SS which reduced by incubation with ERK and JNK inhibitors, but not with p38 inhibitor. We concluded that the T3SS elicited inflammatory responses by activating the JNK or ERK signaling pathways in the infection of C. psittaci.

Transcriptional repression of hDaxx enhanced by adenovirus 12 E1B 55-kDa oncoprotein interacting with hDaxx.

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.

Activation of nuclear factor kappaB and induction of inducible nitric oxide synthase by lipid-associated membrane proteins isolated from Mycoplasma penetrans.

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 kappaB (NF-kappaB) and the effects of pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, 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-kappaB 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-kappaB.