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At least 973 records · Page 54Linked to original sources
PubMed1994

Cytotoxic lymphocytes require granzyme B for the rapid induction of DNA fragmentation and apoptosis in allogeneic target cells.

We have generated H-2b mice with a homozygous null mutation in the granzyme (gzm) B gene. Gzm B is a neutral serine protease with Aspase activity that is found only in the granules of activated cytolytic T cells, natural killer cells, and lymphokine-activated killer cells. Gzm B-/- mice develop normally and have normal hematopoiesis and lymphopoiesis. In vitro, cytotoxic T lymphocytes (CTL) derived from gzm B-/- animals are able to induce 51Cr release from allotarget cells, but with reduced efficiency. However, gzm B-/- CTL have a profound defect in their ability to induce rapid DNA fragmentation and apoptosis in allogeneic target cells. This defect is kinetic since DNA fragmentation is partially compensated and 51Cr release is completely rescued with long incubation times. We conclude that gzm B serves a critical and nonredundant role for the rapid induction of target cell DNA fragmentation and apoptosis by alloreactive cytotoxic T lymphocytes.

Animals↗
PubMed1986

Increased ionic strength: effects on DNA fragmentation and poly(ADP-ribose) polymerase activity in HeLa nuclei.

Poly(ADP-ribose) polymerase activity was measured in a crude nuclear fraction isolated from HeLa cells. It was found that the addition of ammonium sulfate or other salts to the standard incubation medium inhibited the formation of poly(ADP-ribose). Through the use of alkaline sucrose density gradients it was also noted that this same increase in ionic strength inhibited the in vitro breakdown of the HeLa DNA. Additional experiments with alkaline sucrose density gradients and deoxyribonuclease I showed that the in vitro activity of poly(ADP-ribose) polymerase is largely dependent upon DNA fragmentation but that DNA fragmentation at least in vitro is not dependent upon the formation of poly(ADP-ribose). These observations imply that this nuclear enzyme is not extremely sensitive to changes in the ionic strength of the reaction media but is affected indirectly, supposedly through changes in the endonuclease activity of the HeLa nuclei. If this proves to be true, then the addition of salt to the incubation medium for poly(ADP-ribose) polymerase could prove to be a valuable tool for the study of ADP-ribosylation reactions.

Ammonium Sulfate↗
PubMed1998

DNA fragmentation induced by a cytoplasmic extract from irradiated cells.

Apoptosis is a mode of cell death characterized by distinct morphological features and DNA fragmentation. The program that leads to apoptosis has been considered to be predominantly extranuclear, and a signal transduction pathway to the nucleus exists during apoptosis, while characteristic events occur in the nucleus. As for radiation-induced apoptosis, the signal transduction pathway remains unclear, especially the sites where the primary effect of radiation occurs. In this study, we demonstrate that a cytoplasmic extract prepared from irradiated cells has the ability to cause DNA fragmentation and that caspase-3 is activated in this extract. Normal nuclei of HeLa S3 cells were added to a cytoplasmic extract made from HL60 cells which had been irradiated with 30 Gy of 137Cs gamma rays and were incubated. Agarose gel electrophoresis of the added nuclei showed a characteristic DNA laddering pattern. This reaction was blocked by a caspase-3 inhibitor but not by an ICE inhibitor. These observations suggest that a signal transduction pathway from an unknown target of gamma radiation may exist upstream of caspase-3 during radiation-induced apoptosis.

Caspase 3↗
PubMed2001

DNA fragmentation in mammalian cells exposed to various light ions.

Elucidation of how effects of densely ionizing radiation at cellular level are linked to DNA damage is fundamental for a better understanding of the mechanisms leading to genomic damage (especially chromosome aberrations) and developing biophysical models to predict space radiation effects. We have investigated the DNA fragmentation patterns induced in Chinese hamster V79 cells by 31 keV/micrometer protons, 123 keV/micrometer helium-4 ions and gamma rays in the size range 0.023-5.7 Mbp, using calibrated Pulsed Field Gel Electrophoresis (PFGE). The frequency distributions of fragments induced by the charged particles were shifted towards smaller sizes with respect to that induced by comparable doses of gamma rays. The DSB yields, evaluated from the fragments induced in the size range studied, were higher for protons and helium ions than for gamma rays by a factor of about 1.9 and 1.2, respectively. However, these ratios do not adequately reflect the RBE observed on the same cells for inactivation and mutation induced by these beams. This is a further indication for the lack of correlation between the effects exerted at cellular level and the initial yield of DSB. The dependence on radiation quality of the fragmentation pattern suggests that it may have a role in damage repairability. We have analyzed these patterns with a "random breakage" model generalized in order to consider the initial non-random distribution of the DNA molecules. Our results suggest that a random breakage mechanism can describe with a reasonable approximation the DNA fragmentation induced by gamma rays, while the approximation is not so good for light ions, likely due to the interplay between ion tracks and chromatin organization at the loop level.

Animals↗
PubMed2002

Immunohistochemical and biochemical assessment of caspase-3 activation and DNA fragmentation following transient focal ischemia in the rat.

In the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, deltaC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co-localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was confined to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post-reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a confirmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase-specific cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our findings suggest a possible association between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury.

Animals↗
PubMed2003

Chronic cerebral hypoperfusion induces white matter lesions and loss of oligodendroglia with DNA fragmentation in the rat.

Cerebrovascular white matter lesions represent an age-related neurodegenerative condition that appears as a hyperintense signal on magnetic resonance images. These lesions are frequently observed in aging, hypertension and cerebrovascular disease, and are responsible for cognitive decline and gait disorders in the elderly population. In humans, cerebrovascular white matter lesions are accompanied by apoptosis of oligodendroglia, and have been thought to be caused by chronic cerebral ischemia. In the present study, we tested whether chronic cerebral hypoperfusion induces white matter lesions and apoptosis of oligodendroglia in the rat. Doppler flow meter analysis revealed an immediate reduction of cerebral blood flow ranging from 30% to 40% of that before operation; this remained at 52-64% between 7 and 30 days after operation. Transferrin-immunoreactive oligodendroglia decreased in number and the myelin became degenerated in the medial corpus callosum at 7 days and thereafter. Using the TUNEL method, the number of cells showing DNA fragmentation increased three- to eightfold between 3 and 30 days post-surgery compared to sham-operated animals. Double labeling with TUNEL and immunohistochemistry for markers of either astroglia or oligodendroglia showed that DNA fragmentation occurred in both of these glia. Messenger RNA for caspase-3 increased approximately twofold versus the sham-operated rats between 1 and 30 days post-surgery. Immunohistochemistry revealed up-regulation of caspase-3 in the oligodendroglia of the white matter, and also in the astroglia and neurons of the gray matter. Molecules involved in apoptotic signaling such as TNF-alpha and Bax were also up-regulated in glial cells. These results indicate that chronic cerebral hypoperfusion induces white matter degeneration in association with DNA fragmentation in oligodendroglia.

Animals↗
PubMed1996

[The physical mapping of human chromosomes. II. The production of unique chromosome-specific DNA fragments using the polymerase chain reaction with oligonucleotide primers to conservative regions of Alu repeats].

Unique DNA fragments localised between Alu-repeats have been produced by PCR. The reaction was carried out with oligonucleotide primers to conservative regions of Alu-repeats. The DNA fragments from different pulls, individual clones, chromosome-specific clonotecs derived from phage lambda, cosmids and individual human chromosomes served as matrixes. The possibilities are discussed of Alu-primer applying in production of exceptional physical features of DNA molecules, suitable for constructing clone couple groups and for direct physical mapping on the DNA of isolated chromosomes, missing the stage of cloning.

Base Sequence↗
PubMedDate not supplied

[The pattern of DNA fragmentation in pediatric neuromuscular disorders].

INTRODUCTION: An apoptotic mode of cell death has been identified in the developing skeletal muscle of invertebrates. It has been also involved in the pathogenesis of muscle degeneration in the dystrophin-deficient mdx mouse. This has raised an interest on the possible role of apoptosis in human neuromuscular disorders, but the existing studies have offered conflicting results. OBJECTIVE: To analyze the presence of apoptosis in several pediatric neuromuscular disorders. PATIENTS AND METHODS: Identification of cells with apoptotic morphology, detection of DNA fragmentation in situ by means of the TUNEL assay and study of the pattern of DNA fragmentation by agarose gel electrophoresis and Southern blot hybridization in muscle biopsies of 29 patients and three normal controls. RESULTS: No morphological features of apoptosis were found in any of the samples examined. The TUNEL method did not label the myonuclei from any of the biopsies, though some mastocytes stained positively. Agarose gel electrophoresis of extracted DNA and Southern blot hybridization with a genomic DNA probe did not show oligonucleosomal fragmentation. However, smearing of DNA was observed in samples from three patients affected with Duchenne's muscular dystrophy, one with Becker's muscular dystrophy and one with infantile spinal muscular atrophy. CONCLUSIONS: The present results do not favor the hypothesis of persistent apoptosis in some of the pediatric neuromuscular disorders, although they do not exclude that some sort of programmed cell death occurs either excessively or anachronically at other stages of the pathogenesis of these diseases.

Adolescent↗
PubMed1990

A simple method for isolation of DNA fragments associated with the nuclear lamina in vivo.

We describe a simple method for the purification of DNA fragments associated with the nuclear lamina in vivo. Ehrlich ascite tumor cells are first u.v.-irradiated to crosslink DNA to proteins. The nuclear lamina is then isolated and purified by low-speed centrifugation through a cushion of 40% sucrose. The material sedimenting through the created density barrier represents nuclear lamina of a very high purity, free from any DNA fragments except those which were in a crosslinking distance to it in vivo.

Animals↗
PubMed1996

Apoptosis in renal cell carcinoma: detection by in situ end-labeling of fragmented DNA and correlation with other prognostic factors.

Because stage and grade of renal cell carcinoma (RCC) sometimes fail to predict the patient's outcome, additional prognostic predictors are needed. Apoptosis is a process of programmed cell death seen in both normal and neoplastic tissues, which has been shown to have prognostic significance in some tumor types. Forty-seven RCCs were studied for size, grade, stage, apoptosis, and proliferation. Fragmented DNA, a hallmark of apoptosis, was detected in situ by a process in which the fragmented DNA was labeled with a biotinylated nucleotide, which, in turn, was detected by an avidin-biotin-peroxidase labeling system. The proliferating tumor cells were detected by immunostaining with the MIB-1 antibody. The apoptotic index and proliferation index of each RCC were expressed as the number of tumor cells undergoing apoptosis and proliferation per 1,000 tumor cells. For grade I to IV RCCs, the median proliferative index was 13, 41, 119, and 143; the median apoptotic index was 8, 12, 39, and 73. For stage I to IV RCCs, the median proliferative index was 21, 34, 70, and 144; the median apoptotic index was 8, 9, 20, and 69. There was a statistically significant correlation of tumor grade, stage, and size with both proliferative index and apoptotic index. There was a statistically significant correlation between proliferation index and apoptotic index. In conclusion, apoptosis can be easily and reliably recognized by the in situ end labeling of fragmented DNA and may help predict the outcome of RCC.

Antibodies, Monoclonal↗
PubMed1989

Infectivity of vesicles prepared from chilo iridescent virus inner membrane: evidence for recombination between associated DNA fragments.

Treatment of CIV particles with octylglucoside at high ionic strength leads to the solubilization of the inner viral membrane. Incubation of permissive cells (Cf124 cells) with vesicles obtained after dialysis of the detergent shows that this fraction is infectious. This infectivity, which is very low, could only be detected after two serial passages on permissive cells. This phenomenon is, however, reproducible. Isopycnic centrifugation analysis shows that some DNA cosediments with the vesicles. Extraction and purification of this DNA confirm the presence of a large DNA fragment of about 50.10(6) Da. Digestion with restriction endonucleases demonstrated that this DNA did not correspond to a particular fragment but to a population of DNA fragments of homogeneous size arising from various regions of the viral genome. Purified viral DNA was not infectious, the presence of DNA in the vesicles could not account therefore for their infectivity. Experiments of non-genetic reactivation of purified CIV DNA by UV-irradiated virus suggest that one (or several) structural component(s) of CIV particles must be involved in the first stages of the viral replication cycle. In addition, transfection of cells with large overlapping DNA fragments could generate infectious particles when the cells were superinfected with UV-irradiated virus. It can be supposed that the vesicle suspensions, which probably contain the reactivating factor, are composed of a population of vesicles which are all different in their DNA content. Infectivity of such suspensions would be the consequence of a recombination between large overlapping DNA fragments.

Aedes↗
PubMed2000

Differences in nuclear DNA fragmentation and mitochondrial integrity of semen and prepared human spermatozoa.

Sperm DNA integrity is essential for accurate transmission of genetic material to offspring. Fragmentation of genomic DNA is an initial hallmark of apoptosis (programmed cell death). The aim of this study was to determine sperm nuclear DNA integrity and mitochondrial function, to quantify possible apoptosis and to investigate any relationship between these parameters. Semen samples (n = 25) were prepared by discontinuous Percoll density centrifugation (95.0:47.5). DNA integrity was determined using a modified alkaline single cell gel electrophoresis (Comet) assay. DNA fragmentation, possibly indicative of apoptosis, was detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL). Mitochondrial transmembrane potential was determined using the mitochondrial probe 5,5',6,6'-tetrachloro-1,1', 3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The DNA integrity of prepared spermatozoa was significantly greater than that of semen (P < 0.005). Further, the percentage of spermatozoa with fragmented DNA and the degree of fragmentation within these cells in prepared spermatozoa is significantly less than in semen (P < 0.005). There is a significant correlation between DNA damage quantified using the Comet assay and DNA fragmentation determined using TUNEL (R = 0.562, P < 0.01). The percentage of spermatozoa with dysfunctional, possibly apoptotic, mitochondria was significantly lower in prepared spermatozoa than in neat semen samples (P < 0.001). There was a negative correlation between the percentage of spermatozoa with dysfunctional mitochondria and the percentage of progressively motile spermatozoa (R = -0.67, P < 0.01).

Adult↗
PubMed1987

A possible role for K+ channels in tumor cell injury. Membrane vesicle shedding and nuclear DNA fragmentation.

Mastocytoma P815 tumor cells subjected to low temperature (O degrees C/l h) and shifted to 22 degrees or 37 degrees C undergo morphological, physiological and biochemical changes which are analogous to those induced by immune effector cells, i.e., changes in cell-surface morphology and membrane permeability, elevated O2 consumption rates and nuclear DNA fragmentation [18-21]. Utilizing this low-temperature shift method for the induction of cell injury, we investigated the possible role of K+ channels in this process. Results show that the two classical K+ channel blockers, tetraethylammonium (TEA) and 4-aminopyridine (4-AP), inhibited the low temperature-induced cell-surface membrane vesicle shedding as well as the nuclear DNA-fragmentation process. These results indicate that K+ channel function is required for tumor-cell injury as manifested by nuclear DNA fragmentation and cell-surface membrane vesicle (MV) shedding.

4-Aminopyridine↗
PubMed1999

Effects of exogenous nitric oxide and hyperoxia on lung fibroblast viability and DNA fragmentation.

Effective lung repair after acute injury requires elimination of proliferating mesenchymal and inflammatory cells without inducing an acute inflammatory response or disturbing concomitant repair of lung microvasculature. Previous studies have shown that endogenous NO regulates programmed cell death in fibroblasts and can modulate wound fibroblast synthetic function. We hypothesized that exposure of human lung fibroblasts to NO gas would decrease viability and induce apoptotic cell death. Primary cultures of normal human lung fibroblasts were exposed for 4 h to room air (RA), 80% oxygen, NO (at either 20 or 50 ppm) blended with RA, or NO blended with 80% O(2), then incubated for 24 to 72 h. Cell viability was determined by fluorescence viability/cytotoxicity assay and DNA fragmentation by TUNEL assay. Peroxynitrite formation was assessed using immunoblotting for S-nitrosotyrosine. NO plus O(2) induced significant cell death at 20 and 50 ppm NO when compared to either RA or O(2) alone at both 24 and 72 h (p < 0.05). Incubation with superoxide dismutase (SOD), catalase (CAT) or SOD + CAT significantly decreased cell death in fibroblasts treated with NO(20)/O(2) and NO(50)/O(2) compared with controls (p < 0.05). NO(20)/O(2) and NO(50)/O(2) exposure significantly increased TUNEL mean fluorescence intensity (MFI), consistent with increased DNA fragmentation, compared to RA at 24 and 72 h (p < 0.05). Antioxidants decreased MFI in cells exposed to NO(20)/O(2) (CAT and SOD + CAT) compared to controls at 24 h (p < 0.05). Western blot analysis for S-nitrosotyrosine showed increased signal intensity in fibroblasts exposed to NO at 20 and 50 ppm plus O(2) compared to RA or O(2) alone. Incubation with SOD + CAT reduced signal intensity for peroxynitrite in cells exposed to NO(20)/O(2). We conclude that NO in hyperoxic conditions induces fibroblast cell death and DNA fragmentation, which could be partially mediated by peroxynitrite synthesis.

Antioxidants↗
PubMed1997

Gene replacement with linear DNA fragments in wild-type Escherichia coli: enhancement by Chi sites.

During conjugation and transduction of Escherichia coli even numbers of recombinational exchanges are required for replacement of a gene on the circular chromosome. We studied gene replacement using a related method of gene transfer (transformation with 6.5-kb linear DNA fragments) as an experimental model for conjugation and transduction. Two properly situated Chi sites, 5' GCTGGTGG 3', stimulated gene replacement approximately 50-fold, more than the sum of the stimulation by the individual Chi sites. Gene replacement was dependent on RecA and RecB functions. Similar results were obtained with an alternative experimental model in which linear DNA fragments were generated from phage lambda by intracellular EcoRI restriction following infection. Dual Chi site-stimulation of these RecA-, RecB-dependent recombination events thus did not depend upon the mode of delivery of the linear DNA into the cells. A single DNA fragment with two Chi sites was sufficient for gene replacement. These results support a one Chi-one exchange hypothesis ("long chunk" gene replacement), stemming from studies with purified RecBCD enzyme, and argue against models in which Chi converts RecBCD enzyme to a state capable of promoting multiple exchanges on one DNA molecule. These results also provide a method for gene targeting in wild-type E. coli and suggest a method for gene targeting in other organisms.

Bacterial Proteins↗
PubMed2000

NMDA blockade attenuates caspase-3 activation and DNA fragmentation after neonatal hypoxia-ischemia.

The aim was to study the effects of an NMDA receptor antagonist on caspase-3 activation and DNA fragmentation after hypoxia-ischemia (HI) in 7-day-old rats. Animals were treated with vehicle or MK-801 (0.5 mg/kg) directly after HI and sacrificed 8, 24 or 72h later. MK-801 reduced injury (by 53%), cells positive for active caspase-3 (by 39%) and DNA fragmentation (by 79%) in the cerebral cortex. Furthermore, MK-801 significantly decreased caspase-3 activity, and Western blots revealed a tendency towards decreased proteolytic cleavage of the caspase-3 proform. The data imply that NMDA receptors are involved in the activation of apoptotic processes in the immature brain after HI.

Animals↗
PubMed1991

Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae.

DNA fragments (generated by BamHI treatment) with no homology to the yeast genome were transformed into Saccharomyces cerevisiae. When the fragments were transformed in the presence of the BamHI enzyme, they integrated into genomic BamHI sites. When the fragments were transformed in the absence of the enzyme, they integrated into genomic G-A-T-C sites. Since the G-A-T-C sequence is present at the ends of BamHI fragments, this results indicates that four base pairs of homology are sufficient for some types of mitotic recombination.

Base Sequence↗