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At least 739 records · Page 41Linked to original sources
PubMed1999

Intracellular compartmentalization of DNA fragments in cultured airway epithelial cells mediated by cationic lipids.

PURPOSE: The amount and intracellular distribution of DNA fragments (491-bp) was characterized after transfection in vitro with a commercially available cationic lipid. Localization of fragment to the nucleus, its subcellular distribution, and integrity within the cells was determined for various times after transfection. METHODS: Cystic fibrosis (CF) airway epithelial cells were transfected with 32P and FITC labeled single-stranded (ss) or double-stranded (ds) DNA fragments complexed with Lipofectamine at various charge ratios. RESULTS: A 511 (+/-) charge ratio was found to be the optimal ratio for transfection of both ss-and dsDNA. After a 5 h exposure, 7.51 +/- 0.89% of the radioactivity was associated with the nuclear fraction whereas only 1.07 +/- 0.23%, was found in the nuclear fraction when dsDNA was used. The nuclear radioactivity detected after a 24 h exposure was only 1/3 of that after 5 h. Analysis of fragment stability in the cytosolic and nuclear fractions showed the presence of intact fragment in each subcellular compartment. No intranuclear/intracellular fragment could be detected in control experiments with naked DNA. Conclusions. The results from these experiments indicate that small fragments of DNA can be efficiently and rapidly transferred intact to the cell nucleus using cationic lipids and that ssDNA fragments are more effective than dsDNA fragments for nuclear delivery.

Bronchi↗
PubMedDate not supplied

High molecular weight DNA fragmentation assay: a sensitive method for monitoring in vivo response to antileukemic chemotherapy.

A large number of antineoplastic agents exert their cytotoxic effects through induction of apoptosis. However, despite the availability of an increasing number of methods to identify apoptosis, attempts to detect apoptosis in clinical samples are rarely successful. Here we report the reliability of the high molecular weight (HMW) DNA fragmentation assay in monitoring the in vivo response of a patient diagnosed with B-CLL to fludarabine (F-ara-A) infusion monotherapy by tracing 50 Kb DNA fragments in his peripheral blood at different time-points before and after infusion. The HMW DNA fragmentation assay proved qualitative and quantitative sensitivity in following the progression of apoptosis in the patient's circulating population of leukemic blasts while, under the same circumstances, conventional cytometric parameters of apoptosis remained negative.

Aged↗
PubMed1997

Apoptotic DNA fragmentation, and its in vitro prevention by nicotinamide, in lymphocytes from HIV-1-seropositive patients and in HIV-1-infected MT-4 cells.

Apoptosis seems to play an important role in the decline of CD4+ T-cells in patients infected with HIV-1. Moreover, extensive interest in apoptosis comes from the observation that it correlates both with the progression and the severity of HIV-1 infection. A cross-sectional study was made to evaluate whether such correlation may also extend to the early phases of ex vivo apoptosis, after 20 h of culture. DNA fragmentation, a parameter associated with apoptosis, was evaluated with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end labelling (TUNEL) technique, which preferentially labels apoptosis in comparison to necrosis. The results obtained indicate that a negative correlation exists between the proportion of lymphocytes exhibiting DNA strand breaks and the absolute number of CD4+ T-cells per microliter. DNA fragmentation was significantly higher in patients with AIDS or advanced HIV-1 infection as compared to asymptomatic patients or seronegative individuals. No significant difference was found in relation to antiretroviral therapy. Furthermore, the addition of nicotinamide to the cultures significantly reduced DNA fragmentation of both in vitro HIV-1-infected MT-4 cells and lymphocytes from six HIV-1-seropositive individuals. The results of this study confirm that DNA fragmentation, as an early marker of apoptosis, correlates with the severity of HIV-1 infection and suggest that nicotinamide may be involved in the modulation of HIV-1-related apoptosis.

Adult↗
PubMed2000

DNA fragmentation factor 45 deficient mice exhibit enhanced spatial learning and memory compared to wild-type control mice.

Programmed cell death or apoptosis is a highly regulated physiological process that is critical in development, particularly in the central nervous system. The DNA fragmentation factor 45 (DFF45 or ICAD) is a subunit of a heterodimeric DNase complex that is crucial for DNA fragmentation and normal apoptosis. To examine the neurobiological consequences of lacking DNA fragmentation and timely apoptosis during mouse development in vivo, we compared spatial learning behaviors in DFF45 mutant and wild-type control mice. We found that DFF45 mutant mice exhibit enhanced spatial learning and memory compared to wild-type mice. Moreover, both the granule cell density and total granule cell number in the hippocampal dentate gyrus region are higher in the DFF45 mutant brains than in the wild-type brains. We propose that the increase in granule cell number in the dentate region due to the DFF45 mutation changes the neuronal network underlying spatial learning and memory in DFF45 mutant mice.

Animals↗
PubMed2001

Nuclear accumulation of exogenous DNA fragments in viable cells mediated by FGF-2 and DNA release upon cellular injury.

We and others have previously shown that basic fibroblast growth factor (FGF-2 or bFGF) can be used as a targeting molecule to help carry plasmid DNA into cells when the growth factor molecule is physically coupled to the DNA molecule being delivered. Herein we report our observations on the FGF-mediated uptake of exogenous labeled DNA into cultured cells in a manner that is representative of that which may occur under physiological conditions at sites of wounded tissue. Cellular debris at such sites contains nucleic acid fragments released from dead cells, as well as growth factors such as FGF-2 that function early in the wound repair process. Using a cell culture model designed to mimic the local environment of a wound with respect to the presence of soluble FGF-2 and DNA fragments, we have shown that FGF-2 is able to direct the cellular uptake and nuclear localization of fragments of exogenous DNA via the FGF receptor into intact and healthy cells. Furthermore, we can monitor and quantitate this type of FGF-mediated DNA delivery by using indirect immunofluorescence of bromodeoxyuridine-labeled exogenous DNA. Our results suggest that this type of FGF-mediated DNA fragment uptake could allow for the transduction of viable nearest neighbor cells at sites of injury in vivo. Such a phenomenon may lead to mutational aberrations in the recipient cells and enhance the probability of wound carcinogenesis.

Active Transport, Cell Nucleus↗
PubMed1993

Rapid sizing of individual fluorescently stained DNA fragments by flow cytometry.

Large, fluorescently stained restriction fragments of lambda phage DNA are sized by passing individual fragments through a focused continuous wave laser beam in an ultrasensitive flow cytometer at a rate of 60 fragments per second. The size of the fluorescence burst emitted by each stained DNA fragment, as it passes through the laser beam, is measured in one millisecond. One hundred sixty four seconds of fluorescence burst data allow linear sizing of DNA with an accuracy of better than two percent over a range of 10 to 50 kbp. This corresponds to analyzing less than 1 pg of DNA. Sizing of DNA fragments by this approach is much faster, requires much less DNA, and can potentially analyze large fragments with better resolution and accuracy than with gel-based electrophoresis.

Bacteriophage lambda↗
PubMed1998

Induction of apoptotic DNA fragmentation by nonsteroidal anti-inflammatory drugs in cultured rat gastric mucosal cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) have been shown to cause apoptosis in several cell lines including transformed chicken embryo fibroblasts and human colon cancer cells. We herein report the apoptotic effect of NSAIDs in a non-transformed cell line derived from the rat gastric mucosa, RGMI (rat gastric mucosa cell first). 1-[p-Chlorobenzoyl]-5-methoxy-2-methylindole-3-acetic acid (indomethacin) and sodium 2-(2,6-dichloroanilino)phenylacetate (sodium diclofenac), potent and non-selective inhibitors of cyclooxygenase, were found to induce DNA fragmentation in RGM1 cells in a time- and concentration-dependent manner. The expression of mRNA for cyclooxygenase-2 was hardly detected in the intact cells but was clearly enhanced when the cells were incubated with the two NSAIDs. In contrast, the expression of mRNA for cyclooxygenase-1 was constitutive and was never affected by NSAIDs. The effect of [3,4-di(4-methoxyphenyl)-5-isoxazolyl] acetic acid (mofezolac), a potent and highly preferential inhibitor of cyclooxygenase-1, and N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulphonamide (NS-398), a selective inhibitor of cyclooxygenase-2, on DNA fragmentation and cyclooxygenase-2 mRNA expression was weak compared to the effect of indomethacin or sodium diclofenac. The DNA fragmentation induced by sodium diclofenac was hardly affected by the exogenous addition of 16,16-dimethyl prostaglandin E2 but was inhibited by caspase inhibitors such as Ac-YVAD-CHO and Ac-DEVD-CHO. The present data provide the first evidence that NSAIDs, such as indomethacin and sodium diclofenac, cause apoptotic DNA fragmentation in cultured gastric mucosal cells, and also indicate the involvement of caspases rather than the inhibition of cellular prostaglandin synthesis in the apoptotic process.

16,16-Dimethylprostaglandin E2↗
PubMed2001

Antimonial-mediated DNA fragmentation in Leishmania infantum amastigotes.

The basic treatment of leishmaniasis consists in the administration of pentavalent antimonials. The mechanisms that contribute to pentavalent antimonial toxicity against the intracellular stage of the parasite (i.e., amastigote) are still unknown. In this study, the combined use of several techniques including DNA fragmentation assay and in situ and cytofluorometry terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling methods and YOPRO-1 staining allowed us to demonstrate that potassium antimonyl tartrate, an Sb(III)-containing drug, was able to induce cell death associated with DNA fragmentation in axenic amastigotes of Leishmania infantum at low concentrations (10 microg/ml). This observation was in close correlation with the toxicity of Sb(III) species against axenic amastigotes (50% inhibitory concentration of 4.75 microg/ml). Despite some similarities to apoptosis, nuclease activation was not a consequence of caspase-1, caspase-3, calpain, cysteine protease, or proteasome activation. Altogether, our results demonstrate that the antileishmanial toxicity of Sb(III) antimonials is associated with parasite oligonucleosomal DNA fragmentation, indicative of the occurrence of late events in the overall process of apoptosis. The elucidation of the biochemical pathways leading to cell death could allow the isolation of new therapeutic targets.

Animals↗
PubMed2003

DNA fragmentation, apoptosis and cell cycle arrest induced by zearalenone in cultured DOK, Vero and Caco-2 cells: prevention by Vitamin E.

Zearalenone (ZEN) is a non-steroidal oestrogenic mycotoxin produced by several Fusarium species growing on cereals. ZEN and its metabolites bind to human oestrogen receptors and hence display oestrogenic and anabolic properties. Several lines of investigation suggest that ZEN may be genotoxic in vivo. ZEN damages DNA in Bacillus subtilis recombination tests, and it induces sister chromatid exchange and chromosomal aberration in CHO cells. ZEN also induces DNA-adduct formation in mouse tissues and SOS repair process in lysogenic bacteria. In the present study, ZEN genotoxicity has been confirmed in three cell-lines, Vero, Caco-2 and DOK at concentrations of 10, 20 and 40 microM. Under these conditions, ZEN induces concentration-dependent DNA fragmentation resulting in DNA laddering patterns on agarose gel electrophoresis. This observation is consistent with apoptosis, which was confirmed by observations of formation of apoptotic bodies. Moreover, ZEN induces cell cycle arrest in the three cell-lines characterised by an increase of the number of cells in the G2/M phase of the cell cycle. Vitamin E (25 microM) added simultaneously with ZEN partially reduces DNA fragmentation and apoptotic body formation after 24h incubation. Vitamin E may act by maintaining prolonged cell cycle arrest during which time DNA repair takes place.

Animals↗
PubMed2000

Lack of association between smoking and DNA fragmentation in the spermatozoa of normal men.

Male factor infertility patients can have anomalies in their sperm nuclei, displaying high levels of loosely packaged chromatin and damaged DNA. The primary objectives of this study were to compare the extent of DNA fragmentation in the spermatozoa of healthy light and heavy smokers versus non-smokers, and to investigate its correlation with concentrations of the smoking markers cotinine and cadmium. A secondary objective was to compare the concentrations of blood cadmium and serum cotinine with corresponding concentrations in seminal plasma. Ninety-seven healthy male volunteers were divided into three groups: non-smokers, light and heavy smokers. There was no difference between the three groups with respect to age, number of ejaculations per week, serum testosterone concentration, and parameters of semen analysis. The percentages of DNA fragmentation in spermatozoa were not statistically different in the heavy smokers (12.11%), light smokers (11.66%) and non-smokers (20.41%). Serum and seminal plasma concentrations of cotinine were significantly higher in heavy smokers compared with the other groups (P < 0.0001). Median values for blood cadmium concentration were higher in heavy smokers (4.50 microg/l) than in light smokers (0.20 microg/l) and non-smokers (0.20 microg/l) (P < 0.001). Cadmium concentration in seminal plasma was significantly higher in heavy smokers (0.20 microg/l) than in light smokers (0.10 microg/l) and non-smokers (0. 10 microg/l) (P < 0.05). In summary, our results indicate no association between smoking and DNA fragmentation in the spermatozoa of healthy men.

Adult↗
PubMed2003

Guanylyl cyclase inhibitors NS2028 and ODQ and protein kinase G (PKG) inhibitor KT5823 trigger apoptotic DNA fragmentation in immortalized uterine epithelial cells: anti-apoptotic effects of basal cGMP/PKG.

The cGMP/protein kinase G (PKG) signalling pathway, at basal levels, has anti-apoptotic/pro-survival effects in certain neural cells. The present study determined apoptosis-regulating effects of basal cGMP/PKG in an immortalized uterine epithelial cell line, HRE-H9 cells, using two soluble guanylyl cyclase (sGC) inhibitors, NS2028 and ODQ, and a PKG inhibitor, KT5823. A new quantitative, ultrasensitive technique using capillary electrophoresis with laser-induced fluorescent detector (CE-LIF), recently developed in our laboratory, was used to quantify levels of apoptotic DNA fragmentation. NS2028 and ODQ increased apoptotic DNA fragmentation by 3.5- and 9-fold respectively, suggesting that lowering basal cGMP levels causes spontaneous apoptosis. 8-Br-cGMP, a cell-permeable cGMP analogue that directly activates PKG, reduced ODQ-induced apoptosis by 81%, indicating that replacement of lowered cGMP with a direct PKG activator prevents apoptosis. Western blot analysis, using PKG type I (PKG-I)-specific antibody, indicated that HRE-H9 cells express PKG-I at moderate levels. Inhibiting basal PKG activity with KT5823 increased apoptotic DNA fragmentation by 9.8-fold. Overall, the data show that inhibitors of basal sGC and PKG activities in immortalized uterine epithelial cells cause apoptosis, suggesting that normal basal levels of cGMP and PKG activity may be necessary to prevent a spontaneous development of apoptosis in these cells.

Animals↗
PubMed2007

FragMatch--a program for the analysis of DNA fragment data.

FragMatch is a user-friendly Java-supported program that automates the identification of taxa present in mixed samples by comparing community DNA fragment data against a database of reference patterns for known species. The program has a user-friendly Windows interface and was primarily designed for the analysis of fragment data derived from terminal restriction fragment length polymorphism analysis of ectomycorrhizal fungal communities, but may be adapted for other applications such as microsatellite analyses. The program uses a simple algorithm to check for the presence of reference fragments within sample files that can be directly imported, and the results appear in a clear summary table that also details the parameters that were used for the analysis. This program is significantly more flexible than earlier programs designed for matching RFLP patterns as it allows default or user-defined parameters to be used in the analysis and has an unlimited database size in terms of both the number of reference species/individuals and the number of diagnostic fragments per database entry. Although the program has been developed with mycorrhizal fungi in mind, it can be used to analyse any DNA fragment data regardless of biological origin. FragMatch, along with a full description and users guide, is freely available to download from the Aberdeen Mycorrhiza Group web page (http://www.aberdeenmycorrhizas.com).

Algorithms↗
PubMed1990

Separation of DNA fragments by high-resolution ion-exchange chromatography on a nonporous QA column.

A nonporous QA column (a strong anion exchanger) was used for HPLC of DNA fragments. This column was successfully employed to separate small (ca. 10 bp) and intermediate size (ca. 10 kbp) DNA fragments from each other. The column also separated double-stranded DNA from its single-stranded form, and circular DNA molecules from linear ones. The entire separation process was completed within 60 min. The recovery of DNA fragments in each run was above 95%. High resolution was obtained both at an analytical level (microgram scale) and at a preparative level (100 micrograms scale). In view of time efficiency, recovery, and resolution, the nonporous QA column is superior to other porous ion-exchange columns and expected to be a very useful tool in molecular biological studies.

Anions↗
PubMed2005

Nucleotide exchange and excision technology (NExT) DNA shuffling: a robust method for DNA fragmentation and directed evolution.

DNA shuffling is widely used for optimizing complex properties contained within DNA and proteins. Demonstrated here is the amplification of a gene library by PCR using uridine triphosphate (dUTP) as a fragmentation defining exchange nucleotide with thymidine, together with the three other nucleotides. The incorporated uracil bases were excised using uracil-DNA-glycosylase and the DNA backbone subsequently cleaved with piperidine. These end-point reactions required no adjustments. Polyacrylamide urea gels demonstrated adjustable fragmentation size over a wide range. The oligonucleotide pool was reassembled by internal primer extension to full length with a proofreading polymerase to improve yield over Taq. We present a computer program that accurately predicts the fragmentation pattern and yields all possible fragment sequences with their respective likelihood of occurrence, taking the guesswork out of the fragmentation. The technique has been demonstrated by shuffling chloramphenicol acetyltransferase gene libraries. A 33% dUTP PCR resulted in shuffled clones with an average parental fragment size of 86 bases even without employment of a fragment size separation, and revealed a low mutation rate (0.1%). NExT DNA fragmentation is rational, easily executed and reproducible, making it superior to other techniques. Additionally, NExT could feasibly be applied to several other nucleotide analogs.

Chloramphenicol O-Acetyltransferase↗
PubMed2006

Identification of a novel single-stranded DNA fragment associated with human hepatitis.

By performing nonspecific polymerase chain reaction followed by elimination of chromosome-derived sequences, foreign DNA fragments were obtained from the serum of a patient with non-A-E hepatitis. One of the sequences, named NV-F, contained a partial open reading frame and was detected in 17 (24.6%) of 69 patients with non-A-E hepatitis, including 1 with fulminant hepatitis (vs. in 5 [2.8%] of 180 healthy individuals). A peptide was synthesized accordingly, to detect serum anti-NV-F antibody, which was found in 49 (75.4%) of 65 patients positive for NV-F. This DNA fragment was sensitive to S1 nuclease digestion. Cesium chloride gradient analysis revealed that the NV-F-associated particles had buoyant densities of 1.33-1.39 and 1.22-1.25 g/mL. Immunofluorescence analysis revealed that the novel antigen was present in the hepatocytes of patients infected with NV-F. In conclusion, we have identified a novel single-stranded DNA fragment derived from a virus-like agent associated with human hepatitis.

Adult↗
PubMed1998

Monochloramine induced DNA fragmentation in gastric cell line MKN45.

Monochloramine (NH2Cl) is known to be one of the virulence factors in Helicobacter pylori-associated gastric mucosal injury. The present study was designed to examine NH2Cl-evoked DNA fragmentation in the gastric epithelial cell line MKN45. NH2Cl was produced by mixing NH3 with sodium hypochlorite (NaClO). MKN45 cells were exposed to NH2Cl, NH3, or NaClO in Hanks' balanced salt solution. DNA cleavage was evaluated quantitatively by photometeric enzyme immunoassay for the in vitro determination of cytoplasmic mono- and oligonucleosomes. Damage to the plasma membrane was assessed by measuring the activity of lactate dehydrogenase in the supernatants. Separately, DNA ladder formation was performed to confirm the incidence of DNA fragmentation. NH2Cl (0.001-0.01 mM) significantly increased the cytoplasmic mono- and oligonucleosomes, suggesting the incidence of DNA cleavage. The DNA ladder was clearly evoked by NH2Cl. NH2Cl induced a DNA fragmentation, one of the important aspects in apoptosis, in the gastric cell line MKN45.

Adenocarcinoma↗
PubMed1998

Dependence of the ligation efficiency of large DNA fragments isolated from agarose gels on the purification method.

The isolation of large DNA fragments (> 5 kb) from agarose gels can be problematic. The DNA yield is often low, and the purification may be insufficient for subsequent reactions such as sequencing or ligation. Here we have compared a number of commonly used methods and commercial kits for DNA recovery. Large DNA fragments (12 and 14 kb, respectively) were isolated from agarose gel and purified, the recovery yield was tested, and a well-defined amount of purified DNA was used in a standard ligation reaction. We observed dramatic differences in the efficiency of this ligation reaction depending on which recovery method had been used. However, the respective ligation efficiencies of the different methods were not related to their recovery yields.

Animals↗
PubMed2005

Early nuclear translocation of endonuclease G and subsequent DNA fragmentation after transient focal cerebral ischemia in mice.

We investigated whether the endonuclease G (endoG) translocated from mitochondria to nucleus after transient focal cerebral ischemia (tFCI), thereby contributed to subsequent DNA fragmentation. Adult male mice were subjected to 60min of focal cerebral ischemia by intraluminal suture blockade of the middle cerebral artery. Western blot analysis for endoG was performed at various time points of tFCI. Nuclear endoG was detected as early as 4h after tFCI in the ischemic brain, and correspondingly mitochondrial endoG showed a significant reduction at 4h after reperfusion (p<0.01). Immunohistochemistry of endoG confirmed that the nuclear translocation of endoG was detected as early as 4h after tFCI in the middle cerebral artery (MCA) territory of the ischemic brain. Double immunofluorescent staining with endoG and AIF showed that endoG was predominantly colocalized with AIF at 24h after tFCI. Double staining with endoG immunohistochemistry and TdT-mediated dUTP-biotin nick end labeling showed a spatial relationship between endoG expression and DNA fragmentation at 24h after tFCI. These data suggest that the early nuclear translocation of endoG occurs and could induce DNA fragmentation in the ischemic brain after tFCI.

Active Transport, Cell Nucleus↗