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PubMed2006

DNA fragmentation induced in human fibroblasts by accelerated (56)fe ions of differing energies.

DNA fragmentation was studied in the fragment size range 0.023-5.7 Mbp after irradiation of human fibroblasts with iron-ion beams of four different energies, i.e., 200 MeV/nucleon, 500 MeV/nucleon, 1 GeV/nucleon and 5 GeV/nucleon, with gamma rays used as the reference radiation. The double-strand break (DSB) yield (and thus the RBE for DNA DSB induction) of the four iron-ion beams, which have LETs ranging from 135 to 442 keV/mum, does not vary greatly as a function of LET. As a consequence, the variation of the cross section for DSB induction mainly reflects the variation in LET. However, when the fragmentation spectra were analyzed with a simple theoretical tool that we recently introduced, the results showed that spatially correlated DSBs, which are absent after gamma irradiation, increased markedly with LET for the iron-ion beams. This occurred because iron ions produce DNA fragments smaller than 0.75 Mbp with a higher probability than gamma rays (a probability that increases with LET). These sizes include those expected from fragmentation of the chromatin loops with Mbp dimensions. This result does not exclude a correlation at distances smaller than the lower size analyzed here, i.e. 23 kbp. Moreover, the DSB correlation is dependent on dose, decreasing when dose increases; this can be explained with the argument that at increasing dose there is an increasing fraction of fragments produced by DSBs caused by separate, uncorrelated tracks.

Cell Line↗

PubMed2001

Effect of moderate hypocapnic ventilation on nuclear DNA fragmentation and energy metabolism in the cerebral cortex of newborn piglets.

Previous studies have shown that severe hypocapnic ventilation [arterial carbon dioxide partial pressure (PaCO(2)) 7-10 mm Hg] in newborn animals results in decreased cerebral blood flow and decreased tissue oxidative metabolism. The present study tests the hypothesis that moderate hypocapnic ventilation (PaCO(2) 20 mm Hg) will result in decreased cerebral oxidative metabolism and nuclear DNA fragmentation in the cerebral cortex of normoxemic newborn piglets. Studies were performed in 10 anesthetized newborn piglets. The animals were ventilated for 1 h to achieve a PaCO(2) of 20 mm Hg in the hypocapnic (H) group (n = 5) and a PaCO(2) of 40 mm Hg in the normocapnic, control (C) group (n = 5). Tissue oxidative metabolism, reflecting tissue oxygenation, was documented biochemically by measuring tissue ATP and phosphocreatine (PCr) levels. Cerebral cortical nuclei were purified, nuclear DNA was isolated, and DNA content was determined. DNA samples were separated, stained, and compared with a standard DNA ladder. Tissue PCr levels were significantly lower in the H group than the C group (2.32 +/- 0.66 versus 3.73 +/- 0.32 micromol/g brain, p < 0.05), but ATP levels were preserved. Unlike C samples, H samples displayed a smear pattern of small molecular weight fragments between 100 and 12,000 bp. The density of DNA fragments was eight times higher in the H group than the C group, and DNA fragmentation varied inversely with levels of PCr (r = 0.93). These data demonstrate that moderate hypocapnia of 1 h duration results in decreased oxidative metabolism that is associated with DNA fragmentation in the cerebral cortex of newborn piglets. We speculate that hypocapnia-induced hypoxia results in increased intranuclear Ca(2+) flux, which causes protease and endonuclease activation, DNA fragmentation, and periventricular leukomalacia in newborn infants.

Adenosine Triphosphate↗

PubMed1997

Guanine tetraplex formation by short DNA fragments containing runs of guanine and cytosine.

Using CD spectroscopy, guanine tetraplex formation was studied with short DNA fragments in which cytosine residues were systematically added to runs of guanine either at the 5' or 3' ends. Potassium cations induced the G-tetraplex more easily with fragments having the guanine run at the 5' end, which is just an opposite tendency to what was reported for (G+T) oligonucleotides. However, the present (G+C) fragments simultaneously adopted other conformers that complicated the analysis. We demonstrate that repeated freezing/thawing, performed at low ionic strength, is a suitable method to exclusively stabilize the tetraplex in the (G+C) DNA fragments. In contrast to KCl, the repeated freeze/thaw cycles better stabilized the tetraplex with fragments having the guanine run on the 3' end. The tendency of guanine blocks to generate the tetraplex destabilized the d(G5).d(C5) duplex whose strands dissociated, giving rise to a stable tetraplex of (dG5) and single-stranded (dC5). In contrast to d(G3C3) and d(G5C5), repeated freezing/thawing induced the tetraplex even with the self-complementary d(C3G3) or d(C5G5); hence the latter oligonucleotides preferred the tetraplex to the apparently very stable duplex. The tetraplexes only included guanine blocks while the 5' end cytosines interfered neither with the tetraplex formation nor the tetraplex structure.

Base Sequence↗

PubMed1989

N.m.r. and c.d. studies of the DNA fragments d(TATATATA) and d(TATATA) in solution.

DNA fragments d(TATATATA) and d(TATATA) were studied in low-salt aqueous solutions and found to coexist in more than one conformer. 1H-n.m.r. demonstrates that single-stranded and double-stranded states are involved in the conformational coexistence. Circular dichroism spectroscopy indicates a global B-DNA stacking of bases in the fragments. 31P-n.m.r. resonances of the TpA and ApT phosphodiester bonds are substantially separated in the spectra of both d(TATATATA) and d(TATATA) duplexes to suggest an alternating architecture of their backbones. In fact, the oligonucleotide duplexes are much more alternating than the corresponding polynucleotide under the same solution conditions. The alternating character of the d(TATATATA) double helix is further enhanced in molar caesium fluoride solutions. The oligonucleotide isomerization into X-DNA is, however, accompanied by gel formation, which makes high resolution n.m.r. measurements impossible.

Base Sequence↗

PubMed1996

Cooperativity in the binding of echinomycin to DNA fragments containing closely spaced CpG sites.

Quantitative footprinting has been used to investigate cooperative binding of the antitumor antibiotic echinomycin to DNA fragments containing closely spaced CpG steps. The sequences of the designed DNA fragments contained two pairs of strong echinomycin binding sites: a pair of ACGT sites together with an ACGT site and a TCGA site, either directly adjacent or separated by two or four A.T base pairs. The results demonstrate that the binding of echinomycin to the sequences ACGTACGT and TCGAACGT is highly cooperative. The extent of cooperativity depends on the nature of the sequences clamped by the antibiotic and diminishes as the distance between the binding sites is increased. Various methods of extracting the information necessary to establish cooperativity have been compared. Beyond the specific interest in echinomycin-DNA interaction, the present quantitative footprinting study provides a model that may be generally applicable for designing investigations into cooperativity in drug-DNA recognition.

Antibiotics, Antineoplastic↗

PubMed1993

Microplate capture hybridization of amplified parvovirus B19 DNA fragment labelled with digoxigenin.

A capture hybridization technique in microplate has been developed for the identification of polymerase chain reaction (PCR) amplified B19 DNA fragment in clinical specimens. The amplified 104 bp B19 DNA fragment, located in the gene coding for structural proteins, was directly labelled during the amplification reaction by incorporation of digoxigenin-labelled dUTP. The amplified product was then captured by a probe immobilized on microplate wells. The capture hybridization reaction was visualized as an enzyme-linked immunosorbent assay using anti-digoxigenin Fab fragment labelled with peroxidase. Thirty-five serum samples were tested by our capture hybridization assay and the results were in accordance with the results obtained by Southern blot analysis of PCR amplified product. Our microplate capture hybridization assay showed a high sensitivity and reproducibility and appears to be a practical and reliable test for routine screening of B19 parvovirus DNA in clinical specimens.

Base Sequence↗

PubMed2006

Viability and DNA fragmentation in differently sorted boar spermatozoa.

Sperm cell defense against DNA damage relies on two factors: the tight packaging of chromatin, based on condensation and substitution of histones with protamines, and the antioxidant agents present in seminal plasma. These defenses are extremely important as mature sperm is unable to repair DNA damage and even if a successful fertilization occurs, embryo undergoes apoptosis at the time of genomic activation. Sex-sorting exposes spermatozoa to stress sources such as high pressure, laser beam and electrical charge. The aim of this work was to determine how sorting procedures affect viability and DNA integrity in boar spermatozoa, by using the newly developed Sperm-Sus-Halomax. Four sperm populations were considered: CONTROL (no treatment), REAL (sex-sorted semen), BULK (semen sorted without sex separation) and NO LASER (semen only exposed to the high pressure, but including also cells normally discarded from sex-sorting). A significantly (P=0.019) lower viability in NO LASER (64.71%) than in CONTROL (78.6%) and REAL (80.5%) groups was found; this was accompanied by a significantly (P=0.001) higher DNA fragmentation index (DFI) in NO LASER group (6.86%) respect to CONTROL (3.30%) and REAL (3.42%) groups. BULK group did not show any difference in viability or DFI as compared to the other groups. In conclusion, we may believe that sex-sorting procedure as a whole does not affect either viability or DFI and that shear mechanical forces are a relevant source of DNA damage for sorted semen.

Animals↗

PubMed1996

Cleavage of CPP32 by granzyme B represents a critical role for granzyme B in the induction of target cell DNA fragmentation.

Cytotoxic T lymphocytes (CTLs) are able to recognize and destroy target cells bearing foreign antigen using one of two distinct mechanisms: granule- or Fas-mediated cytotoxicity. The exact mechanisms involved in the induction of apoptotic cell death remain elusive; however, it seems likely that a family of cysteine proteases related to interleukin-1beta converting enzyme are involved. One family member, CPP32, has been identified as an intracellular substrate for granzyme B, a CTL-specific serine protease responsible for the early induction of target cell DNA fragmentation. Here we use cytolytic cells from granzyme B-deficient mice to confirm that cleavage and activation of CPP32 represents a nonredundant role for granzyme B and that this activation plays a role in the induction of DNA fragmentation in target cells, a signature event for apoptotic cell death. A peptide inhibitor of CPP32-like proteases confirmed the function of these enzymes in fragmentation. 51Cr release was not suppressed under these conditions, suggesting that granzyme B cleavage of CPP32 is primarily involved in the induction of DNA fragmentation and not membrane damage during CTL-induced apoptosis.

Animals↗

PubMed1989

Some features of DNA fragments associated in vivo with the nuclear lamina.

Ehrlich Ascites Tumour cells were irradiated with UV-light to crosslink DNA to proteins in vivo. The DNA fragments associated with the nuclear lamina were purified and characterized. The results of the Cot analysis and the hybridization experiments suggest that the DNA fragments attached to the nuclear lamina although containing the entire complexity of genomic DNA are enriched in some highly repeated sequences.

Animals↗

PubMed1981

Isolation of DNA fragments containing replicating growing forks from both E. coli and B. subtilis.

By the use of a restriction enzyme digestion of gently lysed E. coli or B. subtilis cells, it is possible to isolate a minute fraction of the total DNA that has an unusually high sedimentation coefficient. Upon inspection of this DNA in the electron microscope, branched DNA fragments are observed. Single branched DNA fragments were analyzed by restriction enzyme and partial denaturation mapping techniques. The fragments appear to have the properties of growing forks excised from in vivo replicating intermediates. In B. subtilis, the minute fraction of DNA was also investigated by transformation assays and found to be greatly enriched for a marker near the origin and slightly enriched for a terminus marker.

Bacillus subtilis↗

PubMed1999

Detection of duck hepatitis B virus DNA fragments using on-column intercalating dye labeling with capillary electrophoresis-laser-induced fluorescence.

A rapid on-column DNA labeling technique is used to detect viral restriction DNA fragments by capillary electrophoresis-laser induced fluorescence detection. Intercalating dyes such as POPO3 or ethidium homodimer-2 are incorporated into the detection buffer. The cationic dyes migrate into the capillary during electrophoresis and bind to the oppositely migrating DNA fragments. A post-column sheath-flow fluorescence detector is used in the experiment. Excellent labeling efficiency is achieved at minimal background fluorescence by diluting the dyes to between 1 x 10(-7) M and 5 x 10(-7) M in a buffer with low ionic strength relative to the running buffer within the capillary. This dilute sheath-flow buffer allows stacking of dye molecules inside the capillary when an electric field is applied. Calibration curves using a series of DNA size markers (between 72 and 1353 base pairs) were linear over an order of magnitude in DNA concentration. Sensitivity also increased linearly with fragment length, and detection limits ranged from 4 x 10(-14) M to 5 x 10(-13) M for the size-standards. Analysis of cloned viral DNA using duck hepatitis B virus demonstrated a concentration detection limit of 3.9 x 10(-16) M. Last, the technique produced very high separation efficiency, 14 x 10(6) theoretical plates which is greater than 47 x 10(6) plates m-1, for the duck hepatitis B viral genome.

Calibration↗

PubMed1997

Progressive increase of apoptosis in prostatic intraepithelial neoplasia and carcinoma: comparison between in situ end-labeling of fragmented DNA and detection by routine hematoxylin-eosin staining.

OBJECTIVE: Apoptosis has attracted significant attention in the study of tumors during recent years. The first goal of this study was to evaluate the number of apoptotic cells and bodies in benign glands, in high-grade prostatic intraepithelial neoplasia, and in malignant prostatic glands. The second objective was to compare the effectiveness of in situ end-labeling of fragmented DNA (ISEL) with the use of routine hematoxylin-eosin (H&E) stains in the assessment of apoptosis rates. METHODS: The percentage of apoptosis was measured with ISEL and H&E stains in sections from 16 prostatectomies performed for previously untreated peripheral prostatic adenocarcinomas. RESULTS: Both methods showed progressive increase of the rates of apoptosis from benign glands (0.34% to 0.38%), to high-grade prostatic intraepithelial neoplasia (1.44% to 1.39%), to carcinoma (2.69% to 2.75%). The increase in apoptosis rate in prostatic intraepithelial neoplasia and carcinomas is one more indication of the continuum in the pathogenetic process leading to invasive prostatic carcinoma. Student's t test revealed no statistically significant difference in the percentage of apoptosis rendered by ISEL and H&E staining. CONCLUSIONS: From a practical point of view, evaluation of apoptosis with H&E stains can be readily performed using routine clinical material. The procedure is inexpensive, and it gives good tissue morphology. However, quantitative measurements may be time-consuming and observer-dependent. The apoptotic bodies are clearly identifiable with ISEL, making quantitation easy and even amenable to automated counting methods. Disadvantages of ISEL are significantly higher costs and poor tissue morphology. We conclude that accurate evaluation of apoptosis may be performed reliably with both routine H&E staining and the ISEL method. The decision to choose one method over the other depends on the economic resources available and the amount of material to be evaluated.

Adenocarcinoma↗

PubMed1999

Nuclear DNA fragmentation in Creutzfeldt-Jakob disease: does a mere positive in situ nuclear end-labeling indicate apoptosis?

The method of in situ end-labeling of nuclear DNA fragmentation was used in the study of ten patients (two biopsies, eight autopsies) with sporadic Creutzfeldt-Jakob disease (CJD). All the patients had the typical morphological lesions including neuron loss, spongiform change and astrocytosis. Four of them also showed prion protein (PrP) deposits in the cerebral cortex, and two of them kuru-like plaques in the cerebellum. A few cells with DNA breaks were found in the two biopsy cases; one of them, suffering from a panencephalopathic form of the disease, showed positive nuclei not only in the cerebral cortex but also in the subcortical white matter. Variable numbers of positive nuclei were observed in the gray and white matter in the eight autopsy cases, in which, although the distribution of positive cells roughly correlated with the distribution of neuron loss, no clear relationship was found as regards the distribution and degree of cell labeling and the degree of neuron loss. Furthermore, large numbers of positive cells were concentrated in a particular area, whereas a few cells were seen in a neighboring equally affected area. Positive glial cells in the plexiform layer of the CA1 area of the hippocampus, and in the frontal white matter were frequently encountered. Staining of the cytoplasm in a minority of cells was interpreted as the result of nuclear DNA leakage. None of the stained cells had the typical morphology of apoptosis; most particularly, peripheral chromatin condensation and formation of apoptotic bodies were not seen in any case. PrP deposits did not result in an increase of nuclear DNA breaks either within the area or in adjacent regions. Although positive cells were also observed in autopsy cases of controls which were processed in the same way, positive labeling as a whole was higher in CJD than in age-matched controls. These results show that brain nuclear DNA is vulnerable in CJD, and suggest that increased DNA vulnerability has a role in cell death and neuron loss. Since nuclear shrinkage and positive nuclear staining with the method of in situ end-labeling of nuclear DNA fragmentation are not exclusive to apoptosis, further information is needed to categorize cell death in CJD as apoptosis. Necrosis or other forms of cell death, as well as increased DNA vulnerability to agonal changes of the individuals, and to postmortem delay in the fixation of the tissues, may account for additional positive staining in cases examined at autopsy.

Aged↗

PubMed1982

[Polymorphism of HLA genes: I. Demonstration of a close correlation between DNA fragments determined by BglI restriction enzyme and HLA class I antigens].

Description of DNA fragments associated to HLA class I gene is possible by using restriction enzymes which determine these fragments and specific DNA probes which permit their detection. In one family, with a child presenting a recombination between HLA-A and C, six fragments determined by the enzyme BglI were found to be polymorphic. The informative fragments segregate with HLA, either with a whole haplotype or with one of the two recombinant segments of the HLA complex. In a small sample of population they correlate with one (A11) or with a group of known cross-reactive antigens serologically defined (A3 and A11; A25 and A26. Another fragment is associated with unknown cross-reactive antigens (A2 and A29).

DNA↗

PubMed1994

Evidence of nuclear DNA fragmentation following hypoxia-ischemia in the infant rat brain, and transient forebrain ischemia in the adult gerbil.

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day. Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.

Animals↗

PubMed1992

A rapid and simple method for labeling short DNA fragments using Taq polymerase.

This report describes a new method for labeling PCR-generated short length (60-120 bp) double-stranded DNA fragments for use as hybridization probes. The method utilizes gene-specific primers identical to those for PCR generation of non-radioactive DNA fragments. Radioactive probes are synthesized by Taq DNA polymerase without using PCR. Single-stranded (sense or antisense) and double-stranded probes can be individually prepared by selection of the appropriate primers. The labeling reaction reached maximum incorporation within 30 min with mean specific activities of 1.05 x 10(9) dpm/microgram (antisense single-stranded), and 1.62 x 10(9) dpm/microgram (double-stranded) were obtained using templates 69-117 of nucleotides. This method offers a simple and rapid means of generating antisense probes for Northern blot analyses and double-stranded probes for Southern blot analyses that provide highly intense signals with low background.

Animals↗

PubMed1999

Visualizing ion relaxation in the transport of short DNA fragments.

Ion relaxation plays an important role in a wide range of phenomena involving the transport of charged biomolecules. Ion relaxation is responsible for reducing sedimentation and diffusion constants, reducing electrophoretic mobilities, increasing intrinsic viscosities, and, for biomolecules that lack a permanent electric dipole moment, provides a mechanism for orienting them in an external electric field. Recently, a numerical boundary element method was developed to solve the coupled Navier-Stokes, Poisson, and ion transport equations for a polyion modeled as a rigid body of arbitrary size, shape, and charge distribution. This method has subsequently been used to compute the electrophoretic mobilities and intrinsic viscosities of a number of model proteins and DNA fragments. The primary purpose of the present work is to examine the effect of ion relaxation on the ion density and fluid velocity fields around short DNA fragments (20 and 40 bp). Contour density as well as vector field diagrams of the various scalar and vector fields are presented and discussed at monovalent salt concentrations of 0.03 and 0.11 M. In addition, the net charge current fluxes in the vicinity of the DNA fragments at low and high salt concentrations are briefly examined and discussed.

Biophysical Phenomena↗

PubMed1994

Down-regulation of c-myc gene expression with induction of high molecular weight DNA fragments by fluorodeoxyuridine.

5-Fluoro-2'-deoxyuridine (FdUrd), a potent inhibitor of thymidylate synthase, induces extensive bulk DNA damage at drug concentrations that produce significant in vitro growth inhibition of human ileocecal carcinoma (HCT-8) cells. Constant- and pulsed-field gel electrophoresis (CFGE and PFGE), to detect size distribution of DNA double-strand breaks and repair kinetics, in parallel with northern and western blot analyses, to quantitate c-myc gene and protein expression, were utilized to analyze drug effects. At 24-hr post in vitro drug treatment, when maximum bulk DNA damage was detected, FdUrd produced a broad range of high molecular weight DNA fragments, clustering between 0.1 and 5.7 megabases in size, and resulted in a decrease in the level of c-myc transcripts and protein with no significant effect on the level of v-myc and H-ras. These effects preceded the observed cellular growth inhibition. Addition of the reduced folate leucovorin potentiated the effects induced by FdUrd, indicating that thymidylate synthase inhibition is an important initial step in drug effect followed by DNA fragmentation and suppression of c-myc expression. Changes in the integrity of the genetic materials and regulatory genes occurred prior to the observed cell growth inhibition by FdUrd, suggesting that these molecular alterations by FdUrd may be associated with subsequent FdUrd-induced cell growth inhibition.

Cell Division↗