Induction of Crown-gall: partial homology between tumor-cell DNA, bacterial DNA and the G+C--rich DNA of stressed normal cells.
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Binding Sites↗
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Results for “DNA, Bacterial”
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Presence of bacterial DNA and bacterial peptidoglycans in joints of patients with rheumatoid arthritis and other arthritides.
OBJECTIVE: The continuous presence of bacteria or their degraded antigens in the synovium may be involved in the pathogenesis of rheumatoid arthritis (RA). The aim of this study was to determine the presence of bacterial nucleic acids and bacterial cell wall constituents in the joints of patients with RA and other forms of arthritis. METHODS: Joint samples were obtained from patients with RA (n = 26), septic arthritis (n = 2), inflammatory osteoarthritis (n = 5), and gout (n = 6), and joint trauma (n = 1). Universal 16S-ribosomal RNA primers were used to detect the presence of bacterial DNA in these samples, using stringent regimens for sample collection and molecular microbiologic analysis. Automated sequencing and comparative data analysis were performed to identify the species. The presence of bacterial peptidoglycan-polysaccharide complexes in synovial tissue was detected by immunohistologic analysis with a specific antibody. RESULTS: The bacterial species cultured from the synovium could be identified in both of the patients with septic arthritis. DNA amplicons were also detected in the synovial fluid and/or tissue samples from 5 patients with RA and 2 patients with crystal-induced arthritis; these originated from multiple bacterial species. Staining for peptidoglycan-polysaccharide complexes was positive in the synovial tissue of both patients with septic arthritis, 16 with RA, 4 with inflammatory osteoarthritis, 4 with crystal-induced arthropathy, and 1 with joint trauma. The staining was mainly found in cells in the synovial sublining, including macrophages. CONCLUSION: The results indicate that bacterial DNA and bacterial cell wall constituents are retained in the joints of some patients with arthritis, where they might enhance synovial inflammation.
Adult↗
Universal DNA primers amplify bacterial DNA from human fetal membranes and link Fusobacterium nucleatum with prolonged preterm membrane rupture.
A large number of bacterial species have been identified in fetal membranes after preterm labour (PTL) associated with intrauterine infection by microbiological culture. In this study, we have investigated a molecular and bioinformatic approach to organism identification which surmounts the need for specific and diverse microbiological culture conditions required by conventional methods. Samples of fetal membranes were taken from 37 preterm infants, and 6 normal term controls delivered by caesarean section, in which bacteria had been detected by in situ hybridization of 16S ribosomal RNA using a generic probe. Degenerate primers were designed to amplify bacterial 16S ribosomal DNA by PCR and used to amplify bacterial DNA from human fetal membranes. Amplicons were cloned, sequenced and bacteria were identified bioinformatically by comparison of sequences with known bacterial DNA genomes. In situ hybridization using an organism specific probe was then used to confirm the presence of the commonest identified organism in tissue samples. Bacterial DNA amplified from 15/43 samples, all from preterm deliveries, and the bioinformatic approach identified organisms in all cases. Multiple bacteria were identified including Mycoplasma hominis, Pasturella multocida, Pseudomonas PH1, Escherichia coli and Prevotella bivia. The commonest organism Fusobacterium nucleatum was found in 9/15 (60%) of samples. Ten of the 12 samples obtained after prolonged membrane rupture were positive for bacterial DNA, and 7 of these (70%) contained DNA from F. nucleatum. Bacteria from fetal membranes may be identified by molecular and bioinformatic methods. Further work is warranted to investigate the apparent linkage between F. nucleatum, fetal membrane rupture and preterm delivery.
Bacterial Typing Techniques↗
[Influence of cultivation conditions on bacterial DNA content and bacterial membrane permeability].
The study included measurement of intracellular and extracellular DNA content and evaluation of bacterial membrane condition during plankton growth and in isolated communities. This was performed at different stages of bacterial development and after heating at the temperature of heat shock system activation. The content of intracellular bacterial DNA in the community decreased after 72 hours of cultivation growth more prominently than during plankton growth. Change of intracellular bacterial DNA content in the community overtook the increase of membrane permeability, which may indicate the onset of apoptosis. Heating at 420' decreased the permeability of bacterial membranes in the community, but did not affect this parameter in the bacteria during plankton growth. Extracellular DNA was found in the matrix of the communities after 24 hours of growth. Appearance of this DNA after 48-74 hours of cultivation may be associated with apoptotic process.
Bacteria↗
The irreversible binding of azacytosine-containing DNA fragments to bacterial DNA(cytosine-5)methyltransferases.
DNA containing 5-azacytosine is an irreversible inhibitor of DNA(cytosine-5)methyltransferase. This paper describes the binding of DNA methyltransferase to 32P-labeled fragments of DNA containing 5-azacytosine. The complexes were identified by gel electrophoresis. The EcoRII methyltransferase specified by the R15 plasmid was purified from Escherichia coli B(R15). This enzyme methylates the second C in the sequence CCAGG and has a molecular mass of 60,000 Da. Specific binding of enzyme to DNA fragments could be detected if either excess unlabeled DNA or 0.8% sodium dodecyl sulfate was added to the reaction mixture prior to electrophoresis. Binding was dependent upon the presence of both the CCAGG sequence and azacytosine in the DNA fragment. S-Adenosylmethionine stimulated the formation of the complex. The complex was stable to 6 M urea but could be digested with pronase. These DNA fragments could be used to detect the presence of several different methyltransferases in crude extracts of E. coli. No DNA protein complexes could be detected in E. coli B extracts, a strain that contains no DNA(cytosine-5)methyltransferases. The chromosomally determined methylase with the same specificity as the purified EcoRII methylase could be detected in crude extracts of E. coli K12 strains. The MspI methylase cloned in E. coli HB101 could also be detected in crude extracts. These enzymes are the only proteins that bind azacytosine-containing DNA in crude extracts of E. coli.
Cytosine↗
Stimulation of DNA synthesis in bacterial DNA--membrane complexes after low doses of ionizing radiation.
DNA--membrane complexes from three strains of E. coli were irradiated and changes in the rates of DNA synthesis were observed. Doses from 1--10 krad to complexes from W3110 and pol A1 strains gave up to a 100 per cent increase in DNA synthesis; under the same conditions, no change was observed in Bs-1. The degree of stimulation did not depend on the presence of oxygen during irradiation, and a post-irradiation incubation was necessary to achieve activation. The properties of all three complexes were similar when unirradiated. Irradiation of intact organisms under conditions which produced marked, oxygen-dependent inhibition of the Bs-1 complex had no significant effect on those from W3110 and Pol A1. Enhanced DNA synthesis is concluded to be due wholly to repair of preexisting DNA. It is further postulated that DNA synthesis in untreated complexes (E. coli B's, W3110 and Pol A1) is mainly of the repair-type and does not necessarily take place at the site of DNA--membrane attachment.
Animals↗
[Detection of bacterial DNA using the polymerase chain reaction (PCR)].
Enzymatic amplification of DNA using the polymerase chain reaction (PCR) is a very sensitive and rapid way of detecting specific DNA sequences. Bacterial DNA can be detected in a wide variety of samples provided at least partial sequence information is available. For a great number of bacteria PCR detection methods have been published. Most important for the pathologist are mycobacteriae (M. tuberculosis, avium, etc.). Borellia burgdorferi, Listeria monozytogenes and chlamydiae (Ce. trachomatis, C. psittaci). Fresh or fixed paraffin embedded tissues, exfoliated cells, whole blood, serum, sputum, urine, ascites or pleural fluid etc. can be analyzed. The time needed to perform the analysis varies between 5 hours and 2 days mostly depending on the DNA extraction method. Several potential pitfalls have to be avoided. The most common problem is contamination of reagents with target DNA. Amplification of DNA from biological samples may be prevented by enzyme inhibitors (salts, proteins). This problem can at least partially be avoided by changing the DNA purification method. Several additional problems may arise if bacterial DNA has to be amplified. Bacterial walls may have to be disrupted using heat or detergent for accessibility of target DNA. Positive results have to be judged carefully. Unlike the situation in retroviral infections with the virus sometimes present in the absence of disease, in the majority of bacterial infections the presence of bacteria signals manifest disease. A possible exception may be the finding of mycobacterial DNA in sarcoidosis patients who can be treated with steroids without provoking tuberculosis. PCR is especially useful in situations where rapid results are necessary or only fixed tissue is available.(ABSTRACT TRUNCATED AT 250 WORDS)
Bacteria↗
Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo.
The loss of transgene expression has been a major obstacle to the development of nonviral vectors for the treatment of human diseases. We previously demonstrated that bacterial DNA linked to a mammalian expression cassette resulted in transcriptional silencing of the transgene in vivo. To confirm these studies and develop a means to produce a robust DNA vector that is not silenced in vivo, we developed a phage phiC31 integrase-mediated intramolecular recombination technology to prepare minicircle vector DNA devoid of the bacterial backbone and then compared the transgene expression profile of the minicircle with different molecular forms of plasmid DNAs in mice. We demonstrate that minicircular DNAs devoid of bacterial sequences expressed 45- and 560-fold more serum human factor IX and alpha1-antitrypsin, respectively, compared to standard plasmid DNAs transfected into mouse liver. Our data suggest that minicircles are capable of expressing high and persistent levels of therapeutic products in vivo and have a great potential to serve as episomal vectors for the treatment of a wide variety of diseases.
Animals↗
Inflammatogenic properties of bacterial DNA following cutaneous exposure.
Bacterial DNA and oligodeoxynucleotides containing cytosine-phosphate-guanosine sequences and thereby mimicking prokaryotic DNA, have recently been shown to exert potent immunostimulatory properties. As skin normally harbors bacteria, and as the bacterial content and the levels of bacterial degradation products increase during skin infection, we analyzed the potential inflammatogenic role of bacterial DNA and oligodeoxynucleotides in a mouse model of cutaneous inflammation. Bacterial DNA from Staphylococcus aureus was injected intradermally into mice and its inflammatogenic properties were compared with synthetic phosphodiester and phosphorothioate cytosine-phosphate-guanosine- or GpC-containing oligodeoxynucleotides. A peak inflammatory infiltrate in the skin was seen already 2 d after injection with either bacterial DNA or the phosphodiester cytosine-phosphate-guanosine-oligodeoxynucleotides. In contrast, nuclease-resistant phosphorothioate cytosine-phosphate-guanosine-induced dermatitis peaked 7 d after intradermal injection. The inflammatory infiltrates consisted mainly of macrophages, and depletion of this cell population resulted in a significant (p=0.0001) decrease in the severity of inflammation, which suggests that macrophages play a central part in inflammatory responses in the skin following exposure to cytosine-phosphate-guanosine-containing oligodeoxynucleotides. A significant decrease in local inflammatory infiltrate was also seen in mice with deficiencies in neutrophil or lymphocyte populations, which indicates that these cell populations may also be involved in mediating inflammatory signals after the injection of immunostimulatory DNA sequences. In summary, our results suggest that bacterial DNA is an important virulence determinant and inflammatory stimulus during skin infections.
Administration, Topical↗
DNA-DNA hybridization studies between bacterial DNA, crown gall tumor cell DNA and the normal cell DNA.
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DNA↗
An African swine fever virus gene with similarity to bacterial DNA binding proteins, bacterial integration host factors, and the Bacillus phage SPO1 transcription factor, TF1.
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Amino Acid Sequence↗
Effect of sex on the induction of anti-DNA antibodies in normal mice immunized with bacterial DNA.
Immunization of normal mice with bacterial DNA elicits a significant IgG anti-DNA response and has been explored as a model of systemic lupus erythematosus. To determine whether this induced response is influenced by sex, we have measured anti-DNA levels in normal male and female BALB/c mice immunized with single stranded DNA from E. coli as complexes with methylated bovine serum albumin (mBSA) in adjuvant. By ELISA assays, anti-DNA levels of immunized females were approximately 16-fold higher than those of immunized males; levels of antibodies to the mBSA carrier were similar, however. The antibodies from females and males showed a similar degree of cross-reactivity when assayed using other natural and synthetic DNA antigens, including mammalian DNA. These findings suggest the potentiation of anti-DNA production in females by antigen-specific mechanisms and provide further evidence that immunization with bacterial DNA replicates features of autoantibody production in SLE.
Animals↗
Intracellular cytokine expression in peritoneal monocyte/macrophages obtained from patients with cirrhosis and presence of bacterial DNA.
BACKGROUND: The detection of bacterial DNA in serum and ascitic fluid from patients with cirrhosis and ascites is interpreted as molecular evidence of intestinal bacterial translocation and considered sufficient to activate the cellular immune response. In vitro studies on ascitic fluid culture have shown a close relationship between the synthesis of several cytokines and nitric oxide and the presence of bacterial DNA. Since different cell types give rise to cytokines, flow cytometry becomes a powerful tool to discriminate between populations involved in a bacterial challenge. OBJECTIVE: To study the pre-activation status of macrophage/monocyte population ex vivo according to the presence of bacterial DNA. PATIENTS: Patients with cirrhosis and culture-negative, non-neutrocytic ascites, with or without the presence of bacterial DNA in blood and ascitic fluid were studied. METHODS: Flow cytometry analysis of intracellular cytokine expression in monocyte/macrophages from ascitic fluid was performed in basal conditions and after 12 h of cell stimulation adding lypopolysaccharide. RESULTS: Monocyte/macrophages from patients with bacterial DNA showed a significantly higher production of interleukin-6 and tumor necrosis factor alpha in basal conditions than that in cells from patients without the presence of bacterial DNA. The addition of lipopolysaccharide produced a non-significant increment in the expression of these cytokines in patients with the presence of bacterial DNA, while this increment became significant in the other group of patients. CONCLUSIONS: Bacterial translocation in patients with cirrhosis and ascites increases the basal intracellular cytokine expression, reducing its functional reserve capability.
Aged↗
Template-free primer-independent DNA synthesis by bacterial DNA polymerases I using the DnaB protein from Escherichia coli.
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Adenosine Triphosphatases↗
Bacterial DNA as an evolutionary conserved ligand signalling danger of infection to immune cells.
During infection, the innate limb of the immune system senses danger (pathogens) via constitutively expressed pattern-recognition receptors, and responds with activation and secretion of pro-inflammatory cytokines. Cell-wall components of gram-positive and gram-negative bacteria, such as peptidoglycan, endotoxin or lipoteichoic acid, activate via CD14, a prototypic pattern-recognition receptor for carbohydrates. This review article focuses on an alternative recognition system of the innate immune system for the recognition of bacterial DNA. Bacterial DNA differs from eukaryotic DNA in its frequency of the dinucleotides CG and its lack of methylation. These structural differences appear to be sensed by cells of the innate immune system such as antigen-presenting cells. As a consequence bacterial DNA serves as an alternate ligand to signal danger of infection. Bacterial DNA and (synthetic) oligonucleotides (ODN) derived thereof are as efficient as endotoxin in activating macrophages and dendritic cells and in triggering release of pro-inflammatory cytokines. In mice sensitized with D-galactosamine (D-GalN), high doses of bacterial DNA from either gram-positive or gram-negative pathogens induce a lethal cytokine syndrome (lethal shock). Therefore, bacterial DNA may represent a hitherto unrecognized pathophysiological entity in host-parasite interactions. Moreover, recent evidence suggests that bacterial DNA or immunostimulating ODN triggers the immunostimulation of antigen-presenting cells, and can be utilized as adjuvant to enhance immune responses of the adaptive immune system towards poorly immunogenic antigens. In fact, foreign DNA might be useful as immunotherapeutically active adjuvant to direct adaptive immune responses towards Thl-dominated immune reactions. If these findings are operative in humans, immunostimulating ODN might be used to influence Th2-dominated diseases such as allergy.
Animals↗
The bouyant behavior of viral and bacterial DNA in alkaline CsCl.
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Cesium↗
Activation of RAW264.7 macrophages by bacterial DNA and lipopolysaccharide increases cell surface DNA binding and internalization.
Bacterial DNA containing unmethylated CpG motifs is a pathogen-associated molecular pattern (PAMP) that interacts with host immune cells via a toll-like receptor (TLR) to induce immune responses. DNA binding and internalization into cells is independent of TLR expression, receptor-mediated, and required for cell activation. The objective of this study was to determine whether exposure of immune cells to bacterial DNA affects DNA binding and internalization. Treatment of RAW264.7 cells with CpG oligodeoxynucleotide (ODN) for both 18 and 42 h resulted in a significant increase in DNA binding, whereas non-CpG ODN had no effect on DNA binding. Enhanced DNA binding was non-sequence-specific, inhibited by unlabeled DNA, showed saturation, was consistent with increased cell surface DNA receptors, and resulted in enhanced internalization of DNA. Treatment with Escherichia coli DNA or lipopolysaccharide (LPS) also resulted in a significant increase in DNA binding, but treatment with interleukin-1alpha, tumor necrosis factor-alpha, or phorbol 12-myristate 13-acetate had no effect on DNA binding. Soluble factors produced in response to treatment with CpG ODN or LPS did not affect DNA binding. These studies demonstrate that one consequence of activating the host innate immune response by bacterial infection is enhanced binding and internalization of DNA. During this period of increased DNA internalization, RAW264.7 cells were hypo-responsive to continued stimulation by CpG ODN, as assessed by tumor necrosis factor-alpha activity. We speculate the biological significance of increasing DNA binding and internalization following interaction with bacterial PAMPs may provide a mechanism to limit an ongoing immune inflammatory response by enhancing clearance of bacterial DNA from the extracellular environment.
Animals↗
The fine specificity of monoclonal anti-DNA antibodies induced in normal mice by immunization with bacterial DNA.
To evaluate further bacterial DNA immunization as a model to study antigen drive in the anti-DNA response, the specificity of induced monoclonal anti-DNA antibodies was characterized. A panel of IgM and IgG monoclonal anti-DNA antibodies was produced from spleen cells of BALB/c mice immunized with single-stranded DNA from E. coli complexed to methylated bovine serum albumin in complete Freund's adjuvant. The binding of these antibodies to DNA and non-DNA antigens was tested by ELISA to assess their range of polyspecificity. These monoclonal antibodies were found to bind to nucleic acid as well as non-nucleic acid antigens, such as beta-galactosidase, cardiolipin, Ro, La and Sm. These studies demonstrate that anti-DNA antibodies from normal mice, although induced by bacterial DNA, may display a broad range of antigen recognition and thus resemble lupus anti-DNA antibodies, many of which are polyspecific, in their pattern of cross-reactivity.
Animals↗