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At least 253 records · Page 14Linked to original sources
PubMed2002

CpG motifs of bacterial DNA exacerbate colitis of dextran sulfate sodium-treated mice.

Inflammatory bowel disease (IBD) is characterized by a dysregulated intestinal immune response with elevated levels of the Th1 cytokines TNF, IL-12 and IFN-gamma. The luminal flora has been implicated as a major factor contributing to the initiation and perpetuation of chronic intestinal inflammation by as yet unknown mechanisms. Bacterial DNA contains unmethylated cytosine-guanosine dinucleotides (CpG) which strongly activate Th1-mediated immune responses. To test whether these CpG-motifs contribute to intestinal inflammation we treated mice with dextran-sulfate-sodium (DSS)-induced acute or chronic colitis for 5 days with CpG-containing oligodeoxynucleotides (CpG-ODN). Colonic inflammation was assessed by histological scoring. Colonic cytokine RNA was quantified by reverse transcription-PCR and cytokine secretion from mesenterial lymph node cells by ELISA. In chronic colitis, CpG-ODN treatment severely aggravated inflammation by 50%. Colonic expression of IFN-gamma and TNF was elevated (200- and 150-fold, respectively) and IFN-gamma and IL-12 secretion from lymph node cells was increased 5,000- and 8-fold, respectively, compared to GpG-ODN-treated controls. Similar effects were obtained in acute colitis. In conclusion, CpG-motifs of bacterial DNA have proinflammatory activity by strengthening the Th1 arm of immunity in DSS-induced colitis, and might therefore play a significant role in the initiation and perpetuation of inflammation in IBD.

Acute Disease↗
PubMed2004

Detection of bacterial DNA in painful degenerated spinal discs in patients without signs of clinical infection.

A local inflammatory and potentially painful response, of which the ultimate cause is unknown, has been described in nervous tissues in contact with degenerated disc material in patients with low back and leg pain. With the rationale that a possible cause of such inflammation could be bacterial infection, we utilized PCR (polymerase chain reaction) amplification of the 16S rRNA (ribosomal RNA) gene followed by gene sequencing, to investigate whether bacterial DNA might be detected in the degenerative discs of 10 patients operated for disc herniation or post-discectomy syndrome. One patient with disc hernia harbored DNA homologous to Bacillus cereus, and in one patient suffering from post-discectomy syndrome, Citrobacter braaki/freundii DNA was detected. The finding demonstrates that 16S rRNA PCR can be a useful tool in search of bacterial DNA in degenerated discs, which in turn may be indicative of low-grade infection, manifesting itself only as pain rather than as clinical infection.

Adult↗
PubMed1995

Amplification of bacterial DNA using highly conserved sequences: automated analysis and potential for molecular triage of sepsis.

OBJECTIVE: The clinical diagnosis of sepsis remains difficult, particularly in the young child, and would be improved by a rapid and reliable method for identification of bacteria in blood and other body fluids. Polymerase chain reaction (PCR) amplification of highly conserved DNA sequences found in all bacteria would permit fast and sensitive determination of the presence of bacteria in clinical specimens. METHODOLOGY: A primer pair for highly conserved regions of bacterial DNA encoding 16S ribosomal RNA (rDNA) was utilized for PCR amplification. PCR products were analyzed by gel electrophoresis, and, after modification of the primers, by an automated 96-well plate reader. RESULTS: rDNA was amplified from 12 different species of bacteria, including Gram-negative and -positive organisms. No signal was observed when total human DNA was used as template. Colorimetric analysis of amplified sequences using a 96-well format was also successful. CONCLUSIONS: We conclude that a single primer pair designed to anneal to a highly conserved region of bacterial DNA can amplify DNA specimens from a variety of different bacteria, while not amplifying human DNA. Such a molecular genetics approach can be fully automated with existing robotic technology. Because of speed, sensitivity, and cost, molecular triage of patients with signs and symptoms of possible bacterial infection will decrease morbidity and mortality among those with unrecognized bacteremia who are managed as outpatients, and will dramatically reduce hospital expenses from individuals who are admitted and are not bacteremic.

Autoanalysis↗
PubMed1999

Exacerbation of viral and autoimmune animal models for multiple sclerosis by bacterial DNA.

Theiler's murine encephalomyelitis virus (TMEV) infection and relapsing-remitting experimental allergic encephalomyelitis (R-EAE) have been used to investigate the viral and autoimmune etiology of multiple sclerosis (MS), a possible Th1-type mediated disease. DNA immunization is a novel vaccination strategy in which few harmful effects have been reported. Bacterial DNA and oligodeoxynucleotides, which contain CpG motifs, have been reported to enhance immunostimulation. Our objectives were two-fold: first, to ascertain whether plasmid DNA, pCMV, which is widely used as a vector in DNA immunization studies, could exert immunostimulation in vitro; and second, to test if pCMV injection could modulate animal models for MS in vivo. We demonstrated that this bacterially derived DNA could induce interleukin (IL)-12, interferon (IFN)gamma, (Th1-promoting cytokines), and IL-6 production as well as activate NK cells. Following pCMV injections, SJL/J mice were infected with TMEV or challenged with encephalitogenic myelin proteolipid protein (PLP) peptides. pCMV injection exacerbated TMEV-induced demyelinating disease in a dose-dependent manner. Exacerbation of the disease did not correlate with the number of TMEV-antigen positive cells but did with an increase in anti-TMEV antibody. pCMV injection also enhanced R-EAE with increased IFNgamma and IL-6 responses. These results caution the use of DNA vaccination in MS patients and other possible Th1-mediated diseases.

Animals↗
PubMed1989

Direct genomic sequencing of bacterial DNA: the pyruvate kinase I gene of Escherichia coli.

The genomic sequencing procedure is applied to the direct sequencing of uncharacterized regions of bacterial DNA by a "multiplex walking" approach. Samples of bulk Escherichia coli DNA are cut with various restriction enzymes, subjected to chemical sequencing degradations, run in a sequencing gel, and transferred to nylon membranes. When a labeled oligomer is hybridized to a membrane, a sequence ladder appears wherever the probe lies near a restriction cut. New probes, based on sequence that lies beyond other restriction sites, are then synthesized, and the membranes are reprobed to reveal new sequence. Repeated cycles of oligomer probe synthesis and subsequent reprobing permit rapid sequence walking along the genome. This oligomer walking technique was used to sequence the pyruvate kinase (EC 2.7.1.40) gene in E. coli without resorting to cloning or to library construction. The sequenced region was amplified by the polymerase chain reaction and subsequently transcribed and translated using both in vivo and in vitro systems, and the resultant gene product characterized to show that the gene encodes the type I isoform of pyruvate kinase.

Base Sequence↗
PubMed2006

Assessing the potential of bacterial DNA profiling for forensic soil comparisons.

A pilot study was undertaken to evaluate DNA profiling of the bacterial community in soil as an alternative to geological methods for forensic soil comparisons. Soil samples from three different ecosystems were compared, and the variation within and between ecologically different sites was determined by using terminal restriction fragment (TRF) analysis of 16S ribosomal DNA. Comparison of TRF profiles revealed that samples from within a specific ecosystem (e.g., a field) showed a significantly higher similarity to each other than to those from another ecosystem (e.g., a forest). In addition, some profile features were unique to specific ecosystems. These features may allow the determination of characteristic profiles that will facilitate identification of ecologically different sites, so that a given sample collected from a suspect could be identified as originating from, for example, a field, rather than a forest. The implications of these preliminary findings for forensic investigations are discussed.

DNA Restriction Enzymes↗
PubMed1982

Molecular fate of heterologous bacterial DNA in competent Bacillus subtilis. II. Unstable association of heterologous DNA with the recipient chromosome.

In CsCl density gradients of lysates from competent Bacillus subtilis cells, which had been exposed to heterologous bacterial DNA, very little donor-recipient complex (DRC) formation could be detected. The present study demonstrates that photocrosslinking of such lysates by irradiation with long-wave UV light in the presence of 4,5',8-trimethylpsoralen results in a dramatic increase in the amount of heterologous DRC. This phenomenon may be interpreted as the stabilization of a pre-existing weak association between entered heterologous donor DNA and one recipient strand in unpaired regions of the chromosome. When a recombination-deficient mutant is used, the amount of stabilizable heterologous DRC is reduced to the same extent as the specific transforming activity of homologous DNA. Although the amount of stabilizable complex is related to the degree of homology between donor and recipient DNA, this relation is not a quantitative one. Probably the association is caused by very short regions of base pairing between the donor and recipient moieties in the complex. Heating of a lysate at 70 degrees prior to photocrosslinking prevents stabilization, apparently because the regions of base pairing are rapidly melted out. The results described in this paper can be best interpreted as the fixation of a process in which entered donor DNA in competent cells tries to find homologous stretches in the recipient chromosome.

Bacillus↗
PubMed2004

Bacterial DNA and RNA induce rat cardiac myocyte contraction depression in vitro.

Sepsis and septic shock, the systemic immunologic and pathophysiologic response to overwhelming infection, are associated with perturbation of a variety of metabolic cell pathways and with multiple organ failure (MOF) including cardiac depression. This depression has been attributed to the effect of several circulating and locally produced proinflammatory mediators. Recent data suggest that bacterial nucleic acids can produce profound systemic inflammatory responses characterized by circulatory shock in intact animals. In this study, bacterial DNA and RNA derived from pathogenic clinical S. aureus and E. coli isolates are shown to induce early concentration-dependent depression of maximum extent and peak velocity of contraction of electrically paced neonatal rat ventricular myocytes in culture. Significant but more modest depression was generated by a nonpathogenic E. coli isolate. Pretreatment with a DNase or RNase abrogated this effect. Further, synthetic, double-stranded RNA (dsRNA) also induced concentration-dependent depression of myocyte contraction, with the effect also being prevented by pretreatment with RNase. These data suggest that bacterial DNA and RNA may contribute to myocardial depression during bacterial sepsis and septic shock.

Animals↗
PubMed1987

Electron probe X-ray microanalysis of bacterial DNA.

X-ray microanalysis provides a potentially useful technique for the detection, quantitation and statistical analysis of cation levels in microsamples of freshly-isolated bacterial DNA. The dilution of the sample deposited on the electron microscope grid is important both for the visualisation of discrete macromolecules and also for the microanalytical detection of a full range of divalent cations. Approximately 50 detectable cations (30 monovalent, 20 divalent) were present per 100 phosphorus atoms, which is equivalent to one detectable cation per 2 nucleotides. Significant correlations in the occurrence of K, Ca, Ni and Zn with continuum and also with phosphorus suggests that these cations are distributed homogeneously in the sample, while Mn, Fe and Cu showed no such correlations. The occurrence of K in the different spectral analyses did not show any significant correlation with any divalent cations, whereas significant correlations did occur between divalent cations. This suggest that fundamental differences occur between monovalent and divalent cations in their association with the DNA molecule.

Calcium↗
PubMed1995

CpG motifs in bacterial DNA trigger direct B-cell activation.

Unmethylated CpG dinucleotides are more frequent in the genomes of bacteria and viruses than of vertebrates. We report here that bacterial DNA and synthetic oligodeoxynucleotides containing unmethylated CpG dinucleotides induce murine B cells to proliferate and secrete immunoglobulin in vitro and in vivo. This activation is enhanced by simultaneous signals delivered through the antigen receptor. Optimal B-cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5' purines and two 3' pyrimidines. Oligodeoxynucleotides containing this CpG motif induce more than 95% of all spleen B cells to enter the cell cycle. These data suggest a possible evolutionary link between immune defence based on the recognition of microbial DNA and the phenomenon of 'CpG suppression' in vertebrates. The potent immune activation by CpG oligonucleotides has implications for the design and interpretation of studies using 'antisense' oligonucleotides and points to possible new applications as adjuvants.

Animals↗
PubMed1991

In vivo effects of porphyrins on bacterial DNA.

The DNA damage in intact Staphylococcus aureus and E. coli cells induced by photosensitized deuteroporphyrin or hemin is described. Treatment of S. aureus cultures with hemin or photosensitized deuteroporphyrin (Dp) caused time-dependent changes in the plasmidial DNA profiles. The major observation was the disappearance of the plasmid supercoiled fraction. The chromosomal DNA was also affected by hemin and by photosensitized Dp, since its degradation products were detected after exposing the bacterial cells to the porphyrin drugs. Photosensitization of E. coli cells, pretreated with Dp and polymyxin B nonapeptide (PMBNP), also resulted in plasmidial damage. No such damage occurred when E. coli cultures were treated with hemin and PMBNP. The above results can be tightly correlated with the antimicrobial action of porphyrins. Their damage to the bacterial DNA seems to reflect one of the in vivo effects of these porphyrins.

DNA Damage↗
PubMed2005

CpG motifs of bacterial DNA essentially contribute to the perpetuation of chronic intestinal inflammation.

BACKGROUND & AIMS: Recently, we demonstrated a proinflammatory effect of cytosin-guanosin dinucleotide (CpG)-oligodeoxynucleotide (ODN) treatment in established dextran sulphate sodium (DSS)-induced colitis. Here, we investigated whether DNA derived from luminal bacteria plays a role in the perpetuation of chronic intestinal inflammation. METHODS: Toll-like receptor (TLR9)-deficient and wild-type (wt) control mice were used for the induction of chronic DSS colitis. Moreover, mice with established chronic colitis using different experimental models were treated with adenoviral ODN (AV-ODN) known to block CpG effects. Colonic inflammation was scored and cytokine production was quantified both in colonic tissue and draining mesenteral lymph node cells (MLC). RESULTS: Eight weeks after induction of chronic DSS colitis in TLR9-deficient mice, intestinal inflammation was significantly lower (-68%), and proinflammatory cytokine production was drastically reduced. Treatment of wt mice with chronic DSS-induced colitis with AV-ODN resulted in a significant amelioration of disease with a reduced histologic score (-43%) and reduced cytokine production of MLC (interleukin [IL]-6: -68%; interferon [IFN]-gamma: -48%) and RNA expression of the T helper (Th)1-specific transcription factor T-bet (-62%) in colonic tissue. Qualitatively, the same results were obtained in the severe combined immunodeficiency disease (SCID) transfer model of colitis and in spontaneous colitis in IL-10-deficient mice. CONCLUSIONS: Bacterial DNA derived from luminal bacteria contributes significantly to the perpetuation of chronic intestinal inflammation. Inhibition of the immune-stimulating properties of bacterial DNA using AV-ODN may offer a novel and specific tool for the treatment of inflammatory bowel disease.

Animals↗
PubMed1977

Transcription of bacterial DNA by isolated plant nuclei.

Plant nuclei prepared from protoplasts can be used as a cell-free system for testing their template activity of procaryotic DNA for plant polymerases. We were able to demonstrate that plant polymerases of Petunia hybrida are capable of transcribing linear bacterial DNA, whereas supercoiled DNA could not be used as a template.

Cell Nucleus↗
PubMed1993

An assay for the detection of bacterial DNA gyrase inhibitors.

In summary, we have developed a sensitive detection system for inhibitors of bacterial DNA gyrase. The use of B. subtilis as the host organism confers the advantage that it is sensitive to both gyrase subunit A and B inhibitors, whereas E. coli is relatively insensitive to B subunit inhibitors in vivo. Using this assay, we identified a new DNA gyrase inhibitor with a novel structure.

Anti-Bacterial Agents↗
PubMed1990

Taq polymerase contains bacterial DNA of unknown origin.

The polymerase chain reaction (PCR) was carried out with the highly conserved E. coli ribosomal RNA gene sequences 1376-1395 and 1521-1540. Using these primers and reaction conditions specified by the manufacturer(s), a 165 bp fragment was synthesized using Taq polymerase from three different sources in the absence of any added template. Restriction enzyme analysis suggests the source of this bacterial DNA is neither E. coli nor Thermus aquaticus. A variety of different methods to eliminate it such as treatment with DNase, restriction enzyme digestion, and CsCl2 density gradient centrifugation were unsuccessful. Since the bacteria in which the Taq polymerase is produced are not the source of the DNA, some step(s) in the purification or reagents added to the enzyme must be involved. Thus it is likely other biological products are similarly contaminated. Although the problem is easily dealt with by running a no-template control and choosing other primers if a problem exists, it is important to recognize the potential for a false-positive result.

Base Sequence↗
PubMed2005

A simple and economic preservation method for genomic bacterial DNA from clinically significant pathogens.

Bacterial culture was allowed to dry to completeness on Columbia agar base with defibrinated horse blood. Following 6 months storage at room temperature, microbial DNA was extracted and successfully amplified by PCR. This storage technique has the advantage over other methods of not requiring (i) a DNA extraction protocol prior to storage and (ii) refrigeration and/or freezing. This technique maybe useful in the transportation of bacterial genomic DNA in nonviable cells as well as reliable method for the storage of DNA in underdeveloped countries.

Base Sequence↗