Human Placenta DNA Research Articles

Proof of principle: Physiological transfer of small numbers of bacteria from mother to fetus in late-gestation pregnant sheep.

Fetal development is thought to proceed in a sterile environment. Recent reports of the presence of bacterial DNA in human placenta, the transfer of live bacteria from mother to fetus after hypoxia in the pregnant sheep, and the presence of bacteria in the meconium of newborn infants have suggested that the fetus might be exposed to bacteria in utero. The present experiments were designed to test the hypothesis that small numbers of bacteria introduced into the maternal bloodstream (too few to induce fever or changes in maternal food consumption), can be found in the fetus days later. We injected 100 colony forming units of green-, red- and far red- fluorescent protein (GFP, RFP, FRFP) expressing S. aureus into late-gestation pregnant sheep intravenously. Five to 7 days later, the animals were euthanized and tissues collected for analysis of GFP. The inoculations did not cause any fever or other measurable behavioral response in the ewes, but did result in the appearance of GFP DNA, and protein in various tissues within the fetuses. Immunohistochemical analysis reveals GFP protein-containing bacteria that appear to be mostly contained within other cells. We were unable to recover any live GFP-expressing bacteria from the fetal tissues. We conclude that S. aureus, and perhaps other bacteria, gain access to the fetus, although it is not clear from these experiments that they survive in the fetus. It is possible that these low inocula and their progeny were effectively cleared by the fetal immune system.

Discovery of Bacteria in Meconium of Normal, Uncatheterized, and Uninfected Late‐Gestation Fetal Sheep

Fetal development is thought to proceed in a sterile environment. Recent reports of the presence of bacterial DNA in human placenta, the transfer of live bacteria from mother to fetus after hypoxia in the pregnant sheep, and the presence of bacteria in the meconium of newborn infants have suggested that the fetus might be exposed to bacteria in utero. We performed the present studies to build on our discovery of bacterial rRNA in the meconium of normal fetal sheep. Specifically, the present experiments were designed to test the hypothesis that intact bacteria could be visualized histologically in meconium from normal, uncatheterized, and uninfected fetal sheep. Pregnant ewes, uncatheterized and free of clinical disease, were euthanized in late gestation. At necropsy, meconium was isolated from fetal colon using RNAse and DNAse‐free instruments and snap‐frozen, then stored at −80C until analyzed. Approximately 150 mg of meconium was homogenized and both RNA and DNA extracted in Trizol. RNA was isolated, converted to cDNA using random hexamers, then subjected to endpoint PCR using primers 341F and 806R that amplify a 465 bp region of bacterial 16S rRNA. All assays were performed with both positive and negative controls. In meconium, 7/7 fetuses (143 days gestation; term=147 days) harbored bacterial RNA. Smears of meconium were stained using Gram stain or hematoxylin stain. Gram stain revealed Gram positive and Gram negative organisms with morphologies that are consistent with the genus/species of bacteria that we have previously identified in meconium using 16S rRNA gene sequencing in DNA isolated from meconium. Hematoxylin staining confirmed the morphology of bacteria observed in the Gram‐stained samples. Bacteria in meconium appear in clusters that are consistent with live, replicating, organisms. The present results support previous work from this laboratory demonstrating that bacteria gain access to the normal fetal sheep. These results are consistent with our observations that bacteria are present in low numbers in amniotic fluid. It is possible that amniotic fluid containing bacteria is swallowed by the fetus, that bacteria survive the transit through the immature fetal gastrointestinal tract, and that the bacterial population expands in the nutrient‐rich environment of the fetal colon.Support or Funding InformationThis work was supported by HD033053 and AI120195.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

DNA Barcoding Coupled with High Resolution Melting Analysis Enables Rapid and Accurate Distinction of Aspergillus species.

We describe a high-resolution melting (HRM) analysis method that is rapid, reproducible, and able to identify reference strains and further 40 clinical isolates of Aspergillus fumigatus (14), A. lentulus (3), A. terreus (7), A. flavus (8), A. niger (2), A. welwitschiae (4), and A. tubingensis (2). Asp1 and Asp2 primer sets were designed to amplify partial sequences of the Aspergillus benA (beta-tubulin) genes in a closed-, single-tube system. Human placenta DNA, further Aspergillus (3), Candida (9), Fusarium (6), and Scedosporium (2) nucleic acids from type strains and clinical isolates were also included in this study to evaluate cross reactivity with other relevant pathogens causing invasive fungal infections. The barcoding capacity of this method proved to be 100% providing distinctive binomial scores; 14, 34, 36, 35, 25, 15, 26 when tested among species, while the within-species distinction capacity of the assay proved to be 0% based on the aligned thermodynamic profiles of the Asp1, Asp2 melting clusters allowing accurate species delimitation of all tested clinical isolates. The identification limit of this HRM assay was also estimated on Aspergillus reference gDNA panels where it proved to be 10-102 genomic equivalents (GE) except the A. fumigatus panel where it was 103 only. Furthermore, misidentification was not detected with human genomic DNA or with Candida, Fusarium, and Scedosporium strains. Our DNA barcoding assay introduced here provides results within a few hours, and it may possess further diagnostic utility when analyzing standard cultures supporting adequate therapeutic decisions.

Simultaneous Quantification of Three Lipid Peroxidation-Derived Etheno Adducts in Human DNA by Stable Isotope Dilution Nanoflow Liquid Chromatography Nanospray Ionization Tandem Mass Spectrometry

Etheno DNA adducts are promutagenic DNA lesions derived from exogenous industrial chemicals, as well as endogenous sources including lipid peroxidation. Furthermore, levels of etheno adducts in tissue DNA are elevated in cancer-prone tissues. In this study, we have developed a highly sensitive and specific stable isotope dilution nanoflow LC-nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS) assay for simultaneous detection and accurate quantification of 1,N(6)-etheno-2'-deoxyadenosine (epsilondAdo), 3,N(4)-etheno-2'-deoxycytidine (epsilondCyt), and 1,N(2)-etheno-2'-deoxyguanosine (1,N(2)-epsilondGuo) in tissue DNA. Typically, [(13)C(1),(15)N(2)]epsilondAdo, [(15)N(3])epsilondCyd, and [(13)C(1),(15)N(2)]1,N(2)-epsilondGuo were added to calf thymus, human placenta, or human leukocyte DNA as internal standards, and the mixture was subjected to enzyme hydrolysis to form the nucleosides. The etheno adducts in DNA hydrolysate were enriched by a reversed phase solid-phase extraction column before analysis by nanoLC-NSI/MS/MS under the highly selective reaction monitoring (H-SRM) mode. This nanoLC-NSI/MS/MS assay achieved attomole-level sensitivity with the detection limit of 0.73, 160, and 34 amol for the respective standard epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo injected on-column, while the quantification limit for the entire assay was 0.18, 4.0, and 3.4 fmol, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in human placental DNA were 28.2, 44.1, and 8.5 adducts in 10(8) normal nucleosides, respectively. The levels of epsilondAdo, epsilondCyd, and 1,N(2)-epsilondGuo in 11 human leukocyte DNA samples were 16.2 +/- 5.2, 11.1 +/- 5.8, and 8.6 +/- 9.1 (mean +/- S.D.) in 10(8) normal nucleotides, respectively, starting from 30 mug of DNA or 1-1.5 mL of blood, and all the relative standard deviations were within 10%. An aliquot equivalent to 6 mug of DNA hydrolysate was used for analysis by this nanoLC-NSI/MS/MS. Thus, this highly sensitive and specific nanoLC-NSI/MS/MS method is suitable for accurate quantification of the three lipid peroxidation-derived etheno DNA adducts as noninvasive biomarkers in clinical studies for cancer risk assessment and for evaluation of preventive agents.

Simultaneous Quantification of 1,N2-Propano-2′-deoxyguanosine Adducts Derived from Acrolein and Crotonaldehyde in Human Placenta and Leukocytes by Isotope Dilution Nanoflow LC Nanospray Ionization Tandem Mass Spectrometry

Humans are exposed to acrolein and crotonaldehyde due to environmental pollution and endogenous lipid peroxidation. These aldehydes react with the 2'-deoxyguanosine moiety of DNA, forming the exocyclic 1,N2-propano-2'-deoxyguanosine adducts AdG and CdG. These adducts are mutagenic lesions, and they play an important role in cancer and neurodegenerative diseases. In this study, a highly sensitive and quantitative assay was developed for simultaneous detection and quantification of AdG and CdG isomers in human placenta and leukocyte DNA by isotope dilution nanoflow LC with nanospray ionization tandem mass spectrometry (nanoLC-NSI/MS/MS). The on-column detection limits (S/N > or = 3) of AdG and CdG were 15 and 8.9 amol, respectively. The quantification limits of AdG and CdG for the entire assay were 619 and 297 amol, respectively, corresponding to 9.8 and 4.7 adducts in 10(9) normal nucleotides, respectively, starting with 20 microg of DNA. Different enzyme hydrolysis methods were compared, and the optimal hydrolysis conditions were employed for the assay. Levels of AdG and CdG in human placental DNA (20 microg) were 108 and 26 in 10(8) normal nucleotides, respectively, with the respective relative standard deviation (RSD) of 2.6% and 3.1% (n = 3). Levels of AdG and CdG in 9 human leukocyte DNA samples were 78 +/- 23 (mean +/- SD) and 6.2 +/- 3.8 (mean +/- SD) in 10(8) normal nucleotides, respectively, starting from 30 microg of DNA. Using this assay, only 4-6 microg of DNA sample was subjected to this nanoLC-NSI/MS/MS system for analysis. Only 1-1.5 mL of blood is needed for measuring AdG and CdG levels in leukocyte DNA. Thus, it is clinically feasible using this highly sensitive assay to investigate the potential of using these adducts as noninvasive biomarkers for DNA damage resulting from acrolein and crotonaldehyde and to study their roles in cancer development and prevention.

Human interferon regulatory factor-1 gene and its promoter sequences revealed by population-based complete gene sequencing

Interferon regulatory factor-1 (IRF-1) plays important roles in host immunity, cell proliferation and apoptosis. The current GenBank sequence for human IRF-1 (accession number: L05072) was derived from a human placenta DNA library and reported in 1992. In one recent population-based sequence study, we observed consistent discrepancies between our IRF-1 sequence data and GenBank reference sequences suggesting that, current IRF-1 reference sequence was not representative for all populations. By complete gene sequencing, we obtained a representative full-length IRF-1 sequence from a single subject. Compared to submission L05072, our population-based data contains: 35 nucleotide additions, 8 nucleotide removals and another 12 nucleotide replacements. A single nucleotide difference was observed in the IRF-1 promoter sequence compared to GenBank sequence (X53095). These changes were confirmed in 350 Kenyans and 28 non-African donors. The accuracy of a reference sequence is crucial for downstream genetic and functional studies and this study provides more complete and accurate data on the sequence of the human IRF-1 gene and its immediate promoter region.

Effect of Glycation on the Structure and Dynamics of DNA: A Critical Spectroscopic Approach

Glycated DNA is considered to be a pathogenic factor for diabetes mellitus. Here we present a novel and preliminary study on normal and glycated (with fructose and glucose-6-phosphate as reducing sugars) human placenta DNA using agarose gel electrophoresis and photon correlation spectroscopy. The former is used to find structural alterations, while the latter is exploited to observe differences in the dynamics between normal (i.e., pure) and glycated DNA molecules. For scattering angles up to 90 degrees , we obtained a quasi-single-exponential relaxation process for the pure DNA, whereas at higher scattering angles the relaxation of pure DNA becomes broader with a stretching parameter beta approximately 0.6 at 130 degrees. Interestingly, for both the glycated DNAs stretched relaxation profiles and higher relaxation rates (Omega) are observed for all scattering angles. Moreover, a separate and very fast relaxation (e.g., relaxation time tau approximately 2 micros at 90 degrees ) can be noticed for both the glycated DNAs at all the studied scattering angles. Thus, the dramatic changes in the relaxation parameters (Omega, tau, and beta) of the glycated DNA show at the molecular level, for the first time, that the structure and dynamics of DNA are strongly affected by glycation. Implications of the results are discussed.

An improved synthesis for the (Z)-14-methyl-9-pentadecenoic acid and its topoisomerase I inhibitory activity

An improved synthesis for the (Z)-14-methyl-9-pentadecenoic acid was developed based on the appropriate use of (trimethylsilyl)acetylene as the key reagent in the synthesis. The reported synthesis started with commercially available 8-bromo-1-octanol and furnished the desired acid in seven steps and in a 16% overall yield, a significant improvement over the previous reported synthesis for this fatty acid. The synthesis reported herein afforded sufficient amounts to study the acid topoisomerase I inhibitory potential and it was found that the title acid inhibits the human placenta DNA topoisomerase I enzyme at concentrations of 500 μM.

STR - Analysis of maternal and foetal DNA in human placenta: a preliminary study

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Multiplexed PCR genotyping of HPVs from plantaris verrucae

Background Plantaris verrucae are a common diagnosis in childhood, consume a significant amount of health-care resources, have many painful treatment options and many recurrences. Objectives The objective of this study was to design and test a single site-anchored, multiplexed and expandable PCR assay for common types of cutaneous HPVs. Study design Common forward and unique reverse primers were selected from the E2 open reading frames of five cutaneous HPV genotypes. These were analyzed for sensitivity and selectivity using pHPV plasmids and several control DNAs in an optimized multiplexed assay. This standardized assay was used to analyze human verruca plantaris tissue for genome type and to evaluate the effect of a commonly used treatment protocol. Results A sensitive, multiplexed PCR assay for human cutaneous HPV genotypes 1a, 2a and 4 was developed. Specific-unique primers and a consensus anchor primer were selected within the HPV E2 region to produce amplicons varying by greater than 100 bp. In analytical sensitivity studies, fewer than 100 genome copies of HPV1a and 2a were detected, and fewer than 1000 copies of HPV4 were detected. The multiplexed assay did not amplify regions of human placenta, calf thymus, CaSki or SiHa DNA and E. coli, pBR322 or non-HPV virus DNAs. In combination with a forensic DNA extraction procedure, the multiplexed HPV assay detected and identified HPV types in 23 of 51 (45%) deep plantaris verrucae. Two patients were found with two different genotypes in single deep plantaris verruca. Detection of the HPV genome was followed as a function of tissue ablation and Mediplast treatment in one patient. In healing tissue, the genome content was reduced but had not totally disappeared. Conclusions The multiplexed HPV assay can be used to determine genotype prevalence that may correlate with treatment efficacy.

Exploring the antioxidant property of bioflavonoid quercetin in preventing DNA glycation: A calorimetric and spectroscopic study

Reducing sugars for example glucose, fructose, etc., and their phosphate derivatives non-enzymatically glycate biological macromolecules (e.g., proteins, DNA and lipids) and is related to the production of free radicals. Here we present a novel study, using differential scanning calorimetry (DSC) along with UV/Vis absorption and photon correlation spectroscopy (PCS), on normal and glycated human placenta DNA and have explored the antioxidant property of the naturally occurring polyhydroxy flavone quercetin (3,3′,4′,5,7-pentahydroxyflavone) in preventing the glycation. The decrease in the absorption intensity of DNA in presence of sugars clearly indicates the existence of sugar molecules between the two bases of a base pair in the duplex DNA molecule. Variations were perceptible in the PCS relaxation profiles of normal and glycated DNA. The melting temperature of placenta DNA was decreased when glycated suggesting a decrease in the structural stability of the double-stranded glycated DNA. Our DSC and PCS data showed, for the first time, that the dramatic changes in the structural properties of glycated DNA can be prevented to a significant extent by adding quercetin. This study provides valuable insights regarding the structure, function, and dynamics of normal and glycated DNA molecules, underlying the manifestation of free radical mediated diseases, and their prevention using therapeutically active naturally occurring flavonoid quercetin.

Effect of cytofectins on the immune response of murine macrophages to mammalian DNA

DNA, depending on base sequence, can induce a wide range of immune responses. While bacterial DNA is stimulatory, mammalian DNA is inactive alone and can, moreover, inhibit the response to bacterial DNA. To determine whether the mode of cell entry affects the immune properties of mammalian DNA, we have investigated the effects of the cytofectin agents Fugene 6 (Roche Diagnostics Corp., Indianapolis, IN), Lipofectin and Lipofectamine (Life Technologies, Grand Island, NY) on the responses of murine macrophages to DNA from calf thymus and human placenta. Whereas calf thymus and human placenta DNA alone failed to stimulate J774 or RAW264.7 cell lines or bone marrow-derived macrophages, these DNAs in complexes with cytofectin agents stimulated macrophages to produce nitric oxide but not interleukin 12. Both single-stranded and double-stranded DNAs were active in the presence of cytofectins. Macrophage activation by the DNA-cytofectin complexes was reduced by chloroquine, suggesting a role of endosomal acidification in activation. As shown by flow cytometry and confocal microscopy, the cytofectins caused an increase in the uptake of DNA into cells. Our findings indicate that macrophages vary in their response to DNA depending on uptake pathway, suggesting that activation by DNA reflects not only sequence but also context or intracellular location.

Cytotoxicity and DNA topoisomerase inhibitory activity of benz[f]indole-4,9-dione analogs.

A series of benz[f]indole-4,9-diones, based on the antitumor activity of 1,4-naphthoquinone, were synthesized and evaluated for their cytotoxic activity in cultured human cancer cell lines A549 (lung cancer), Col2 (colon cancer), and SNU-638 (stomach cancer), and also for the inhibition of human DNA topoisomerases I and II activity in vitro. Several compounds including 2-amino-3-ethoxycarbonyl-N-methyl-benz[f]indole-4,9-dione showed a potential cytotoxic activity judged by IC50<20.0 microg/ml in the panel of cancer cell lines. Especially, 2-hydroxy-3-ethoxycarbonyl-N-(3,4-dimethylphenyl)-benz[f]indole-4,9-dione had potential selective cytotoxicity against lung cancer cells (IC50=0.4 microg/ml)) compared to colon (IC50>20.0 microg/ml) and stomach (IC50>20.0 microg/ml) cancer cells. To further investigate the cytotoxic mechanism, the effects of test compounds on DNA topoisomerase I and II activities were used. In a topoisomerase I-mediated relaxation assay using human placenta DNA topoisomerase I and supercoiled pHOTI plasmid DNA, 2-amino-3-ethoxycarbonyl-N-(4-fluorophenyl)-benz[f]indole-4,9-dione had the most potent inhibitory activity among the compounds tested. However, most of the compounds showed only weak inhibition of the DNA topoisomerase II-mediated KDNA (Kinetoplast DNA) decatenation assay, except for 2-amino-3-ethoxycarbonyl-N-(4-methylphenyl)-benz[f]indole-4,9-dione and 2-amino-3-ethoxycarbonyl-N-(2-bromoehtyl)-benz[f]indole-4,9-dione with a moderate inhibitory activity. These results suggest that several active compounds had relatively selective inhibitory activity against toposiomearse I compared to toposiomerase II. No obvious correlation was observed between the cytotoxicity of the individual compound and the inhibitory activity of DNA relaxation and decatenation by topoisomerase I and II, respectively, in vitro.

Matrix metalloproteinase-21, the human orthologue for XMMP, is expressed during fetal development and in cancer

We have characterized a novel human matrix metalloproteinase (MMP-21) from human placenta DNA complementary to RNA (cDNA). The 569 amino acid translation of the cDNA includes all the typical features of an MMP family member, namely a signal sequence, a prodomain with a PRCGVPD motif, a zinc-binding catalytic domain with an HEIGHVLGL sequence, and a hemopexin-like domain flanked by two cysteine residues. Furthermore, MMP-21 has a furin activation sequence, but no transmembrane sequence nor a cytoplasmic domain. As in Xenopus laevis and Cynops pyrrhogaster there is an additional insertion of approximately 30 amino acids between the prodomain and the catalytic domain, which is poorly conserved between the species and is in human MMP-21 especially proline rich. The MMP-21 gene has seven exons and is located in chromosome 10. This new MMP is the human orthologue for XMMP and CyMMP expressed during gastrulation of X. laevis and C. pyrrhogaster, respectively. A 2.5 kb messenger RNA was observed in fetal liver by Northern analysis. By reverse transcription-polymerase chain reaction, MMP-21 is expressed in various human fetal and adult tissues as well as in cancer cell lines. MMP-21 protein can also be detected in malignancies such as ovarian and colon carcinomas by immunohistochemical staining. Our findings suggest that MMP-21 functions in embryogenesis and tumor progression.

Detection of cytomegalovirus DNA in human placenta.

Although human cytomegalovirus (CMV) is one of the most common causes of viral intrauterine and perinatal infection, its distribution in the placenta is poorly understood. The purpose of this study was to determine the frequency of CMV DNA positivity in placentas, to demonstrate the localization of the viral genome, and to identify the clinical features related to placental CMV. A total of 254 placentas from 231 mothers were investigated, and the maternal serum CMV immunoglobulin antibodies were measured. Specimens from both the placental parenchyma and the placental membrane close to the ruptured site in each placenta were examined for the presence of CMV DNA using dot blot hybridization after PCR amplification. None of 57 placentas from seronegative mothers was positive for CMV DNA. Of 197 placentas from seropositive mothers, 60 (30.5%) had CMV DNA in either the parenchyma or the membrane by dot blot analysis. In situ hybridization was carried out on these 60 placentas, and the localization of the viral genome was established in 19; CMV DNA was localized mostly to the villi, including the mesenchyme and trophoblasts, extravillous trophoblasts, and decidual cells. The mean gestational age at delivery was significantly later in the CMV DNA-positive placentas than in the negative placentas (36.9 +/- 5.1 vs 34.7 +/- 6.2 weeks, P = 0.0059). CMV DNA was detected in only 6 of 33 placentas delivered in the second trimester, and all six were associated with either severe maternal nephritis or severe chorioamnionitis. These results suggest that the CMV genome is common in placentas at later gestational ages and in those of earlier gestational ages with certain maternal complications.

The determination of DNA based on light-scattering of a complex formed with histone

The interaction of histone with nucleic acids was characterized by light-scattering measurement using a common spectrofluorometer. Thereby, a sensitive and convenient method for the determination of nucleic acids was established. At pH 4.5–6.5, the interaction of histone with nucleic acids resulted in considerable light-scattering , and four characteristic peaks at 298, 450, 503, and 551 nm were observed. The light-scattering was applied to the determination of nucleic acids. The experiments indicated that, under optimal conditions, a linear relationship was obtained between the light-scattering intensity (I LS) and the concentration of nucleic acids. The linear ranges were 0.02–2.0 μg ml −1 for fish sperm DNA (fsDNA), 0.05–1.5 μg ml −1 for calf thymus DNA (ctDNA), 0.05–2.5 μg ml −1 for Herring testis DNA (HtDNA), and 0.05–1.5 μg ml −1 for human placenta DNA (hpDNA). The detection limits were 2.0 ng for fish sperm DNA, 2.0 ng for calf thymus DNA, 5.0 ng for Herring testis DNA, and 3.0 ng for human placenta DNA. The nucleic acids in yeast cell extraction were determined by simple vortex extraction. The results were satisfactory, and the recovery rates were in the range of 88–108%.

Quantification of etheno-DNA adducts using liquid chromatography, on-line sample processing, and electrospray tandem mass spectrometry.

Etheno-DNA adducts are promutagenic lesions present in normal animal and human tissues that are believed to be important in the etiology of cancer related to diet and lifestyle. A method has been developed for the quantification of trace levels of etheno-DNA adducts using on-line sample preparation coupled with liquid chromatography and electrospray tandem mass spectrometry. The use of automated solid-phase extraction and stable labeled internal standards permitted the robust determination of ethenodeoxyadenosine contained in crude DNA hydrolysates from untreated rodent and human tissues at levels on the order of one adduct in 10(8) normal nucleotides from 100 microg of DNA. Inherent analyte response and matrix interference made sensitivity for simultaneous determination of ethenodeoxycytidine approximately 5-fold lower. The method was applied to the analysis of liver DNA from untreated and urethane-treated B6C3F1 mice, untreated rat liver, human placenta, and several commercial DNA preparations. Some sources of potential artifactual formation of etheno-DNA adducts were investigated.

Inhibition of murine macrophage IL-12 production by natural and synthetic DNA.

DNA is a complex macromolecule whose immunological properties vary with sequence and structure. To determine whether DNA can inhibit immune responses, the effects of mammalian DNA and synthetic phosphodiester (Po) and phosphorothioate (Ps) oligonucleotides (ODNs) on IL-12 production were tested using murine macrophages. With bacterial DNA as a stimulant, calf thymus DNA and human placenta DNA blocked IL-12 production by splenic and bone marrow macrophages. A (dG)(30) Po ODN and all single-base Ps 30 mer ODNs were also effective inhibitors. The Ps ODNs also blocked IL-12 production induced by lipopolysaccharide (LPS) and a stimulatory Ps ODN. With the J774 cell line, single-base Ps ODNs inhibited IL-12 production induced by bacterial DNA, LPS, and a stimulatory Ps ODN. Together, these results indicate that DNA has inhibitory properties, suggesting that mammalian DNA could limit immune activation during inflammation and counteract the effects of bacterial DNA.

Anti-vaccinia virus effect of M13 bacteriophage DNA

Single-stranded DNA derived from M13 phage was evaluated for antiviral activity in mice infected with vaccinia virus. M13 DNA at a dose as low as 16.7 mg/kg was effective in reducing the number of tail lesions caused by vaccinia virus by more than 90%. A single administration of M13 DNA 1 day before infection was sufficient to reduce significantly the number of tail lesions caused by vaccinia virus. Denatured eukaryotic nucleic acids such as calf thymus DNA and human placenta DNA were not effective. A mixture of nucleotides prepared according to the nucleotides composition of M13 DNA was also ineffective. Within 4 h after the administration of M13 DNA, the serum interferon (IFN, predominantly type β) titer rose from undetectable levels to as much as approximately 700 IU/ml. IFN was detectable for up to 12 h after the administration of M13 DNA. IFN titers as high as 1050 IU/ml were detected in vitro when M13 DNA was added to spleen cultures. We conclude that at least part of the antiviral activity of M13 DNA can be explained on the basis of IFN induction.

Different effects of histone H1 on de novo DNA methylation in vitro depend on both the DNA base composition and the DNA methyltransferase.

We have characterized the inhibition exerted by histone H1 on the activity of human placenta DNA (cytosine-5-)-methyltransferase. Our experiments demonstrate that the extent of inhibition depends on the DNA base composition, AT-rich substrates being more severely affected than GC-rich substrates and CpG-rich islands. With bacterial SssI methylase, the effect is completely reversed since its activity on AT-rich substrates undergoes a 4-5-fold stimulation upon the addition of H1. Poly(L-lysine) mimicks H1 effects, suggesting an essential role of lysine residues in both the inhibitory and stimulatory effects of H1. By comparison of the different behaviors of the two enzymes, the inhibitory effect over the eukaryotic enzyme might be accounted for by hypothesizing a competition between minor groove-binding motifs (SPKK-like) present in placenta methylase as well as in histone H1.