Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “DNA Fragmentation”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,243 records · Page 69Linked to original sources
PubMed2003

Lens epithelial cells express CD95 and CD95 ligand treatment induces cell death and DNA fragmentation in vitro.

PURPOSE: Despite advances in intraocular lens design and material, posterior capsule opacification remains one of the major problems in modern cataract surgery. Therefore, the use of antiproliferative agents has been advocated. CD95 ligand (CD95L, Fas, Apo-1) is a death ligand that triggers apoptosis in susceptible target cells. Apoptosis allows for the safe disposal of cells without damaging the surrounding tissue. The goal of this study was to characterize and evaluate CD95L-induced cell death in cultured lens epithelial cells (LEC). METHODS: Expression of CD95 in untreated porcine LEC was investigated by flow cytometry. Cell death after CD95L or CD95 agonistic antibody treatment was assessed by crystal violet assay and DNA fragmentation was measured by comet assay. RESULTS: The presence of CD95 was observed in LEC. CD95L treatment resulted in a time--and concentration-dependent killing of LEC, which was synergistically enhanced by the addition of cyclohexamide. CD95L treatment induced DNA fragmentation. CONCLUSIONS: The present study confirms the use of apoptosis-inducing CD95L in the inhibition of LEC proliferation. Further studies are needed before clinical application of CD95L to inhibit posterior capsule opacification will be feasible.

Animals↗
PubMed2000

Vascular ultrastructure and DNA fragmentation in swine infected with Shiga toxin-producing Escherichia coli.

Shiga toxins (Stx) produced by Escherichia coli cause systemic vascular damage that manifests as edema disease in swine and hemolytic uremic syndrome in humans. In vitro, Stx inhibit protein synthesis and, depending on circumstances, induce necrosis, apoptosis, or both. The mechanism of in vivo Stx-mediated vascular damage is not known. The ability of Stx to cause apoptosis of vasculature in vivo was studied in pigs with edema disease that was produced by oral inoculation with Stx-producing E. coli. Arterioles of ileum and brain were evaluated by terminal dUTP nick-end labeling (TUNEL) assay for DNA fragmentation in myocytes (10 infected pigs, 5 control pigs) and by transmission electron microscopy for ultrastructural changes characteristic of apoptosis (17 infected pigs, 8 control pigs). In comparison with controls, increased numbers of TUNEL-positive arterioles were detected in 6/10 (60%) subclinically affected pigs 14-15 days after inoculation. Ultrastructurally, lesions in myocytes consisted of lysis (necrosis), with cytoplasmic debris and nuclear fragments contained between intact basement membranes. Endothelial cell changes ranged from acute swelling to necrosis and detachment from basement membrane. Subclinically affected pigs (n = 14) tended to have changes predominantly in myocytes, whereas pigs with clinical illness (n = 3) more commonly had changes in endothelial cells. The arteriolar lesions and clinical signs of edema disease are attributed to the effects of Stx on vasculature. Therefore, our findings suggest that the Stx-induced arteriolar lesions seen in this study were primarily necrotic, not apoptotic. We suspect that necrosis was the principal cause of the DNA fragmentation detected.

Animals↗
PubMed2003

Spatial resolution of phospholipid scramblase 1 (PLSCR1), caspase-3 activation and DNA-fragmentation in the human hippocampus after cerebral ischemia.

Reports on non-neural cells have shown that enhanced activity of the Ca(2+)-dependent/ATP-independent phospholipid scramblase (PLSCR1) is, at least in part, responsible for surface exposure of phosphatidylserine and the collapse of plasma membrane asymmetry in injured or apoptotic cells. To shed some light on mechanisms with a potential to lead to apoptotic death of human neurones following ischemic/hypoxic injury, we examined the immunoreactivity of hippocampal neurones for PLSCR1, caspase-3, cytochrome c and DNA-fragmentation in 22 individuals with clinically symptomatic cerebral ischemia after cardiac arrest or severe hypotension. WE FOUND: (1) significant differences in the percentage of PLSCR1-immunoreactive neurones between controls and short survivors; statistically strong differences between the frequency of immunoreactive neurones among the subfields studied with lowest levels in the CA3; preferential distribution of immunoreactive neurones in controls within the regio entorhinalis, subfield CA1, and hilum. Additionally, these areas exhibited staining of fibre bundles which probably correspond to perforant path, alvear path and collateral's of Schaffer, (2) caspase-3 was upregulated in a region-specific manner with marked activation in the selectively vulnerable hippocampal areas, (3) cytochrome c was redistributed, (4) DNA-fragmentation represented by scattered TUNEL-positive cells increased predominantly during the first 3 days after ischemia, and particularly in the regions of greatest susceptibility to hypoxic injury. This study presents the first evidence that PLSCR1, and probably remodelling of plasma membrane phospholipids (PL), plays a role in ischemic injury in the human hippocampus.

Adult↗
PubMed1999

Necrosis of schwann cells during tellurium-induced primary demyelination: DNA fragmentation, reorganization of splicing machinery, and formation of intranuclear rods of actin.

We present a cytological, immunocytochemical, and biochemical study of the cell death of mature myelinating Schwann cells (SCs) in the primary demyelinating neuropathy induced by tellurium (Te). Weaned rats were fed a diet containing 1.1% elemental Te. The animals were killed daily within the first week of Te diet. After 4 to 6 days of Te treatment some SCs underwent degeneration and necrosis. By electron microscopy analysis, degenerating SCs showed chromatin condensation, detachment from the nuclear envelope of condensed chromatin clumps, aggregation of interchromatin granule clusters, formation of intranuclear bundles of microfilaments, and cytoplasmic vesiculation. By confocal laser fluorescence microscopy, chromatin regions were stained with the TUNEL method for in situ labeling of DNA fragmentation and exhibited a progressive reduction of histone signal. In addition, splicing small nuclear ribonucleoprotein (snRNP) factors were redistributed in a few large nuclear domains and bright foci of intranuclear actin were observed. DNA electrophoresis revealed a smear pattern of DNA fragmentation in sciatic nerve samples from Te-treated animals. Upon Te treatment, no degradation of the caspase substrates poly (ADP-ribose) polymerase and lamin B was detected by Western blots or immunocytochemistry, respectively. The peculiar structural rearrangement of the transcription and splicing machinery as well as the vesicular degeneration of the cytoplasm in degenerating SCs support an autophagic cell death of the necrotic type. Unlike the apoptosis of pre-remyelinating SCs (11), this caspase independent cell death of necrotic type involves mature pre-demyelinating SCs and eliminates SCs injured by the neurotoxic effect of Te.

Actins↗
PubMed1999

Isolation of differentially expressed genes by cloning transcriptionally active DNA fragments.

During studies of erythroid cell growth and differentiation induced by erythropoietin (Epo), we developed a method that allows the identification and isolation of genes based upon their transcriptional activity. Transcriptionally active genomic DNA fragments from Epo-treated cells and control cells are purified from inactive chromatin using mercury affinity chromatography, based on the mechanism that the thiol groups of histone H3 on transcriptionally active chromatin are exposed to the solvent and therefore are easily accessible. Using the purified genomic DNA fragments from the two populations of cells, a subtractive hybridization strategy is used to isolate and clone genes that are differentially expressed in the absence or in the presence of Epo.

Animals↗
PubMed2003

A striking correlation between lethal activity and apoptotic DNA fragmentation of liver in response of D-galactosamine-sensitized mice to a non-lethal amount of lipopolysaccharide.

AIM: To observe whether challenge of bacterial lipopolysaccharide (LPS) with D-galactosamine (D-GalN) in mice will result in apoptotic characteristic of vital organs. METHODS: The experimental group of mice was challenged directly with bacterial LPS (0.05 microg) in the presence of D-GalN (20 mg), and the control group of mice was challenged either with bacterial LPS or with D-GalN alone. The organs including brain, kidney, heart, spleen, lung, and liver were removed at an indicated time point under ether anesthesia, and immediately homogenized, from which DNA was extracted. The DNA obtained from these organs was analyzed by agarose gel electrophoresis to determine whether the DNA laddering phenomenon existed. The amount of plasma LDH and GOT was detected in mice challenged with bacterial LPS in the presence of D-GalN, and either bacterial LPS or D-GalN alone. RESULTS: Apoptotic DNA fragmentation was initially seen at 4 h after challenge only in the livers of mice challenged with bacterial LPS and D-GalN, all mice in this group challenged with bacterial LPS and D-GalN died at 7 h after challenge; in contrast, the animals in the control group were all alive and the DNA was integral. CONCLUSION: The liver is the only specific target organ where apoptotic DNA fragmentation was present in mice treated with D-GalN and challenged with bacterial LPS and the liver impairment might be the critical cause of the lethality of mice elicited by bacterial LPS.

Animals↗
PubMed1986

Nucleosome phasing on a DNA fragment from the replication origin of simian virus 40 and rephasing upon cruciform formation of the DNA.

Nucleosomes were reconstituted in vitro from a fragment of DNA spanning the simian virus 40 minimal replication origin. The fragment contains a 27-base-pair palindrome (perfect inverted repeat). DNA molecules with stable cruciform structures were generated by heteroduplexing this DNA fragment with mutants altered within the palindromic sequence (C. Nobile and R. G. Martin, Int. Virol., in press). Analyses of the structural features of the reconstituted nucleosomes by the DNase I footprint technique revealed two alternative DNA-histone arrangements, each one accurately phased with respect to the uniquely labeled DNA ends. As linear double-stranded DNA, a unique core particle was formed in which the histones strongly protected the regions to both sides of the palindrome. The cruciform structure seemed to be unable to associate with core histones and, therefore, an alternative phasing of the histone octamer along the DNA resulted. Thus, nucleosome positioning along a specific DNA sequence appears to be influenced in vitro by the secondary structure (linear or cruciform) of the 27-base-pair palindrome. The formation of cruciform structures in vivo, if they occur, might therefore represent a molecular mechanism by which nucleosomes are phased.

Animals↗
PubMed1982

Isolation of DNA fragment containing phoS gene of Escherichia coli K-12.

The DNA fragment containing the phoS gene, a regulatory gene for alkaline phosphatase, has been isolated from Escherichia coli K-12 chromosomal DNA by cutting off the DNA with Hind III restriction enzyme and by cloning the gene with plasmid vector pTP 4 which was constructed in this study. The isolated fragment was of about 12.3 kbp and seemed to contain the phoT, glmS, and bgl genes. The 12.3 kbp Hind III fragment was subjected to restriction enzymes EcoR I, BamH I, Sal I, and Pst I, and was found to possess two EcoR I, no BamH I, a Sal I, and four Pst I sites. Partial deletion using these restriction enzymes suggested that the about 6 kbp Hind III-Pst I fragment contained the phoS and phoT genes. Further analysis with other restriction enzymes revealed that the 6 kbp Hind III-Pst I fragment contained a BstE II, two Mlu I and four Hpa I sites. The deletion of these restriction sites using single-strand-specific nuclease S1 suggested that the BstE II and one of Mlu I sites were in the phoT gene, and the BstE II and two Mlu I sites were not in the phoS gene.

Culture Media↗
PubMed2006

Unusually strong positive cooperativity in binding of peptides to latent membrane protein-1 DNA fragments of the Epstein-Barr viral gene.

The DNA-binding preferences of two oligopeptide amides, (His-Pro-Arg-Lys)(3)NH(2) (HR-12) and (Ser-Pro-Arg-Lys)(3)NH(2) (SP-12), have been examined by quantitative DNase I footprinting studies. Two different DNA fragments were investigated: a pair of 5'-(32)P-labeled duplexes from pBR322 with one or other of the complementary strands labeled and a corresponding pair of 5'-(32)P-labeled duplexes representing fragments of the latent membrane protein (LMP-1) gene from a pathogenic Epstein-Barr virus variant derived from nasopharyngeal carcinoma. The major objective was to examine molecular recognition and cooperative features associated with sequence-selective binding of synthetic peptides to the LMP-1 fragments. At various binding sites on the pBR322 fragments, Hill coefficients (n(H)) ranging from 1.9-2.2 were observed; these results indicate modest positive cooperativity between binding sites for both peptides. By contrast, unusually high values of n(H), ranging from 4.0-9.3, were observed at various binding sites on the LMP-1 fragments. Allosteric models can be constructed to interpret the observed cooperative interactions between different DNA recognition sites in the LMP-1 gene upon binding of the peptide ligands. It is noteworthy that these models feature a novel network of cooperativity interconnecting multiple DNA allosteric sites. The evidence of sequence selectivity and strong cooperativity discovered in this work may prove to be a general feature of peptide interactions with some nucleic acids.

Base Sequence↗
PubMed1981

1H NMR studies of lac-operator DNA fragments.

The hydrogen-bonded imino protons of a 14 base pair double-stranded DNA fragment comprising one half of the lac operator of E. coli were investigated by 360 MHz H NMR. From combined melting studies of this synthetic 14 b.p. fragment and its two constituent 7 b.p. fragments a nearly complete assignment for the low-field proton resonances was obtained. The experimental spectra are compared with calculated spectra and with the spectrum of a 51 b.p. DNA restriction fragment from E. coli containing the complete lac operator. Structural information on these oligonucleotides is presented. This study is a prerequisite for future 1H NMR investigations of the interaction of the lac operator with the lac repressor.

DNA, Bacterial↗
PubMed2004

[Site-directed mutagenesis and promoter functional analysis of RM07 DNA fragment from Halobacterium halobium in Escherichia coli].

The RM07 DNA fragment from Halobacterium halobium was inserted upstream of chloramphenicol acetyltransferase (cat) reporter gene in pKK232-8 in two different orientations (positively or negatively), generating the RM07-cat fusion plasmids pRM07-1 (+) and pRM07-1 (-). These two plasmids and pKK232-8 were transformed into Escherichia coli HB101 respectively, then the antibiotic resistance level and the chloramphenicol acetyltransferase protein concentration of different transformants were detected. The research results revealed that HB101/pRM07-1 (+) was resistant to chloramphenicol and could grow on the plate containing chloramphenicol, but HB101/pRM07-1 (-) and HB101/pKK232-8 were sensitive to it. Therefore, the results suggested that the positive RM07 fragment had promoter activity in Escherichia coli while the negative RM07 fragment did not. Site-directed mutagenesis of RM07 was performed by PCR mutagenesis method. The effect of specific nucleotide mutations on the chloramphenicol resistance level of different transformants was detected. The different transformants containing different nucleotide mutations were inoculated on the plates containing various concentrations of chloramphenicol and then incubated at the same time. The change of chloramphenicol resistance level reflected the change of promoter activity. By using this method, the important nucleotide responsible for the promoter function of RM07 in Escherichia coli was determined. The promoter activity of RM07 in Escherichia coli was also improved greatly by modifying the nucleotide component.

Chloramphenicol O-Acetyltransferase↗
PubMed2003

Combination and cleavage of HBV DNA fragments by triple helix-forming oligonucleotides modified with manganese porphyrin in vitro.

OBJECTIVE: To observe the ability of triple helix-forming oligonucleotides (TFOs) modified with manganese porphyrin to combine with and cleave HBV DNA fractions. METHODS: TFO were modified with manganese porphyrin and acridines, and then reacted with the (32)P labeled HBV DNA fragments at 37 degrees C in vitro (pH 7.4). Electrophoretic mobility shift assays and DNase I footprinting tests were used to show the affinity and specificity of TFO to bind to target sequences. The ability of TFO to cleave HBV DNA fragments was tested by cleavage experiments. RESULTS: TFO modified with manganese porphyrin and acridine could bind to the target sequence in a sequence-dependent manner, with a Kd value of 3.5 x 10(-7) mol/L and a relative affinity of 0.008. In the presence of potassium monopersulfate (KHSO(5)), TFO modified with manganese porphyrin and acridine could cleave the target sequence where the triplex DNA was formed. CONCLUSION: In the presence of KHSO(5), TFO modified with manganese porphyrin and acridine could bind and cleave the target HBV-DNA in a sequence-dependent manner.

DNA↗
PubMed2002

Adenovirus vector-mediated transfer of 9 kDa granulysin induces DNA fragmentation in HuD antigen-expressing small cell lung cancer murine model cells.

OBJECTIVE: Granulysin is a tumoricidal molecule secreted by cytotoxic T cells (CTL) and natural killer (NK) cells, that induces apoptotic cell death in tumour cells. It has been demonstrated that small cell lung cancer (SCLC) cell lines are susceptible to NK cells and lymphokine activated killer (LAK) cells, and HuD antigen is assumed to be a target molecule on SCLC cells for host cellular immunity. METHODOLOGY: In order to understand the mechanism of sensitivity of SCLC to cellular immunity, we evaluated granulysin-induced apoptosis using mouse adenocarcinoma Colon 26 (Colon 26/HuD) cells transfected with the 9 kDa active form of granulysin using an adenovirus vector as a murine model of SCLC cells. RESULTS: Adenovirus vector-mediated transfer of 9 kDa granulysin increased DNA fragmentation in Colon 26/HuD cells 2.5-fold and suppressed Colon 26/HuD proliferation by 21% on day 3 (P < 0.05 for each value) compared with the control adenovirus vector transfer. In contrast, adenovirus vector-mediated transfer of 9 kDa granulysin did not increase DNA fragmentation nor suppress the proliferation of Colon 26 parent cells. CONCLUSIONS: The sensitivity of HuD-expressing tumour cells to granulysin is likely to partially explain the susceptibility of SCLC to cell-mediated immunity.

Adenocarcinoma↗
PubMed2004

Clonidine increases caspase-3 mRNA level and DNA fragmentation in the developing rat brainstem.

The densities of alpha2-adrenergic receptors, labeled by 3H-clonidine or 3H-RX821002, reach a peak in the rat brainstem during the first week of its life. This enables the agonist of alpha2-adrenergic receptor clonidine, which is used as a component of anaesthetic solution in infants and children, to have specific effects in this structure of the developing brain. Clonidine was injected into the fetal brain (5 microg in 5 microl of saline) or subcutaneously to the pups (1, 10 microg in 50 microl of saline) 3 days before investigation. Clonidine increased the level of apoptotic enzyme caspase-3 mRNA expression, as measured by RT-PCR and enhanced the DNA fragmentation, as determined by gel electrophoresis, in the brainstem of the 21-day-old fetuses and 8-day-old rats. In the cortex of 8-day-old rat, the alpha2-adrenergic receptors are at a much lower level than the brainstem. Clonidine treatment had no evident effects on caspase-3 mRNA level and DNA fragmentation in the cortex of an 8-day-old rat. The data suggest that clonidine facilitates cell death in the developing brainstem. This drug effect provides a potential mechanism whereby clonidine during early life could induce long-lasting alterations in brain neurochemistry, autonomic functions and behavior.

Adrenergic alpha-Agonists↗
PubMed1998

Effects of alkyl amine carboxyboranes on L1210 DNA fragmentation and nucleic acid metabolism.

Amine-carboxyboranes with varying alkyl chain lengths were observed to be potent cytotoxic agents inhibiting the growth of a number of histological types of murine, rat, and human tumors. These agents preferentially reduced L1210 DNA synthesis with marked inhibition of the activities of regulatory enzymes of the purine pathway. Other enzyme activities which were marginally reduced were DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, t-RNA polymerase, and nucleoside kinases. Pyrimidine nucleotide pools were not reduced but DNA strand scission occurred after 24 h incubation with the agents. The amine-carboxyboranes were not DNA topoisomerase II inhibitors at 100 microM. The agents did not cause DNA protein linked breaks themselves; nevertheless, VP-16 [etoposide] induced DNA protein linked breaks were increased two fold in the presence of the agents suggesting synergistic effects. The amine-carboxyboranes decreased protein kinase C mediated phosphorylation of L1210 topoisomerase II protein, potentially decreasing its enzymatic catalytic activity. Thus, the amine-carboxyboranes did not function like VP-16 in affording cleavable products but were synergistic with VP-16 in causing DNA fragmentation. The agents were also additive with VP-16 in reducing tumor cell number, soft-agar colony growth and DNA synthesis and in producing DNA strand scission.

Animals↗
PubMed1998

Apoptosis in the human inner ear. Detection by in situ end-labeling of fragmented DNA and correlation with other markers.

The aim of this study was to obtain baseline data on the recently described special form of single cell death, apoptosis, in normal human inner ears. For this purpose, in situ end-labeling of the fragmented DNA was applied, in conjunction with apoptosis-related markers, to detect cellular elements showing programmed cell death in decalcified and paraffin-embedded tissues. Over 20 specimens were analyzed which were obtained from autopsy cases with no history of acoustic lesions confirmed by histopathology. Based on staining results, we saw no apoptotic signs in the majority of normal adult inner ears. An apoptotic cell captured in the Reissner's membrane of the cochlea from an old patient may, however, indicate an age-related subtle cell loss with the process of apoptosis. Nevertheless, the fact that more apoptosis was not found in our cases suggests that this phenomenon does not contribute significantly to the tissue homeostasis in the adult inner ear under normal conditions. These data are in accordance with our immunohistochemical findings on the p53 nucleoprotein, and proliferating cell nuclear antigen expression since there was no staining in any of the cellular elements, including the mesenchymal cells. This reflects a stationary and stable condition of cells of the vestibular and the cochlear structures, probably to maintain their integrity and the fine sensory functions. As opposed to the above findings, during inner ear development, the epithelial cells lining the cochlear lumen, the ossifying cartilage of the temporal bone, and the mesenchymal cells show different degrees of proliferation in combination with single cell death as signs of maturation of the vestibular and the cochlear apparatus. In addition, apoptosis has been demonstrated in cells of the cochlear stria vascularis from an adult patient treated with high doses of cisplatin, vinblastine and bleomycin prior to death. Furthermore, a wide range of apoptosis could be induced experimentally in a normal ear by an external perfusion of actinomycin D (ActD), which is known to produce programmed cell death in many cell types of different origins. The potential role of cytostatic agents in the apoptotic process of the inner ear needs, however, to be confirmed in large-scale specimens from patients treated with genotoxins. The fact, however, that apoptotic cells are also seen in association with ActD indicates that the fine sensory structure of the cochlea may also be a target for certain chemotherapeutic agents when administered in high doses.

Adult↗
PubMed1998

Cytotoxicity and DNA fragmentation associated with sequential gemcitabine and 5-fluoro-2'-deoxyuridine in HT-29 colon cancer cells.

The combined cytotoxic effects of the antimetabolites gemcitabine (dFdCyd) and 5-fluoro-2'-deoxyuridine (FdUrd) were studied. Cytotoxicity, biochemical perturbations, and DNA damage seen with dFdCyd and FdUrd alone and in combination were evaluated in HT-29 human colon cancer cells. A 4-h exposure to dFdCyd followed by FdUrd for 24 h produced more than additive cytotoxicity and marked S-phase accumulation. Cells progressed through the cell cycle, however, after a 22-h drug-free interval. [3H]dFdCyd was rapidly metabolized to the 5'-triphosphate and incorporated into DNA. [3H]FdUrd was anabolized exclusively to FdUrd monophosphate, and preexposure to dFdCyd did not affect FdUrd monophosphate formation. Thymidylate synthase catalytic activity was inhibited by 48% after a 4-h exposure to 10 nM FdUrd and by 80% after exposure to the combination. Sequential 4-h exposures to 15 nM dFdCyd and 10 nM FdUrd led to greater depletion of dTTP pools (29% of control) than with either drug alone. Greater effects on nascent DNA integrity were seen with sequential dFdCyd followed by FdUrd. Although parental DNA damage was not evident immediately after exposure to 15 nM dFdCyd for 4 h followed by 10 nM FdUrd for 24 h, high molecular mass DNA fragmentation was evident 72-96 h after drug removal. Sequential dFdCyd/FdUrd was associated with prominent disturbance of the cell cycle, dTTP pool depletion, dATP/dTTP imbalance, and nascent DNA damage. Induction of double-strand parental DNA damage and cell death was delayed, consistent with postmitotic apoptosis.

Antimetabolites, Antineoplastic↗
PubMed1993

The sequence of a 17.5 kb DNA fragment on the left arm of yeast chromosome XI identifies the protein kinase gene ELM1, the DNA primase gene PRI2, a new gene encoding a putative histone and seven new open reading frames.

A 17.5 kb DNA fragment of chromosome XI, located between the genetic loci mif2 and mak11 was sequenced and analysed. Ten open reading frames were identified. Two of them are the previously sequenced genes ELM1 and PRI2, two (YKL253 and YKL256) show homologies to proteins from other organisms and one (YKL262) to yeast and mouse histone.

Amino Acid Sequence↗