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 505 records · Page 28Linked to original sources
PubMed2003

Hypoxia-induced caspase-3 activation and DNA fragmentation in cortical neurons of newborn piglets: role of nitric oxide.

Hypoxia results in generation of nitric oxide (NO) free radicals, activation of caspase-3, and genomic DNA fragmentation. The present study tests the hypothesis that hypoxia-induced caspase-3 activation and DNA fragmentation are nitric oxide mediated. Studies were conducted in newborn piglets, divided into normoxic (n = 5), hypoxic (n = 5), and hypoxic-7-NINA (n = 6). Hypoxic-7-NINA group received the neuronal nitric oxide synthase inhibitor, 7-Nitroindazole (7-NINA). Caspase-3 activity was determined spectrofluorometrically using enzyme-specific substrates. Sections from the neocortex were stained with an antiserum recognizing active caspase-3. Purified DNA was separated by gel electrophoresis. Administration of 7-NINA resulted in decreased immunoreactivity of caspase-3 (mean LI: 20.2%) as compared to the untreated hypoxia group (mean LI: 57.5%) (P < 0.05). 7-NINA attenuated caspase-3 enzymatic activity as well in comparison to the untreated hypoxia group (P < 0.05). Furthermore, multiple low molecular weight bands corresponding to DNA fragments were present in the hypoxic but not in the normoxic or hypoxic-7-NINA groups. Inhibition of nNOS abates the hypoxia-induced increase in active caspase-3 immunoreactivity, as well as enzymatic activity in cortical neurons, and DNA fragmentation in brain homogenates. We conclude that the coordinate increase of capase-3 activity and fragmentation of nuclear DNA in the hypoxic newborn piglet brain are NO mediated.

Adenosine Triphosphate↗
PubMed1999

Relationship between dead cells and DNA fragmentation in bovine embryos produced in vitro and stored at 4 degrees C.

DNA fragmentation and its relationship with dead cells were examined in bovine blastocysts produced in vitro and stored at 4 degrees C for 1-5 days. Survival and development to the hatching and hatched blastocyst stage decreased with increasing storage time. Both were significantly lower at 72 hr than at 48 hr. None of the embryos stored for 120 hr developed to the hatching or hatched blastocyst stage. The proportion of dead cells per embryo increased progressively as the time of storage increased, until 69% of embryonic cells were dead after 120 hr of storage. There was no significant difference between the proportions of DNA fragmentation per embryo stored for 0 and 24 hr (12% vs 16%). However, the proportion of DNA fragmentation in embryos stored for longer than 48 hr was significantly greater than that in embryos stored for less than 24 hr. There were no significant differences among those stored for longer than 48 hr (28-33%). These results suggest that the reduced developmental competence of bovine embryos stored at 4 degrees C is characterized by necrotic change rather than apoptotic change.

Animals↗
PubMed1997

Changes in high molecular weight DNA fragmentation following human blood exposure to styrene-7,8-oxide.

Styrene-7,8-oxide (S7,8O) is the major in vivo metabolite of styrene and is a genotoxic agent potentially carcinogenic to humans. It is known to cause DNA strand breaks and adducts. We studied high molecular weight DNA fragmentation in white blood cells following incubation of blood with S7,8O from individuals with no previous exposure to this compound. To better understand the effects of S7,8O, we also examined blood exposed to hydrogen peroxide (H2O2) a typical oxidant that is linked to oxidative stress. All individuals in this study showed a variable reduction in high molecular weight DNA fragments determined by laser densitometry compared to untreated controls for both S7,8O and H2O2 treated samples. This decrease was independent of the concentration and length of exposure of blood to S7,8O and H2O2. An increase in low molecular weight DNA fragments from samples treated with S7,8O and H2O2, compared to untreated samples was also noted. Similarities in the reduction of HMW-DNA fragments after S7,8O and H2O2 exposure suggested a similar mechanism of HMW-DNA damage. It was surmised that S7,8O exposure in blood may induce high molecular weight DNA fragmentation due to oxidative stress.

Carcinogens↗
PubMed1991

DNA helicase and duplex DNA fragment unwinding activities of polyoma and simian virus 40 large T antigen display similarities and differences.

We have characterized the biochemical activities of purified polyoma (Py) large T antigen (T Ag) that was capable of mediating the replication of a plasmid containing the Py origin (ori(+) DNA) in mouse cell extracts. We report here that like the T Ag encoded by simian virus 40 (SV40), Py T Ag has DNA helicase and double-stranded DNA fragment unwinding activities. Py T Ag displaced DNA fragments greater than 1,600 nucleotides which were annealed to complementary sequences in single-stranded M13 by translocating in the 3' to 5' direction. Both helicase and double-stranded DNA fragment unwinding reactions were completely dependent upon NTP hydrolysis, displaying a strong preference for ATP and dATP. At low T Ag concentrations, significantly more Py ori(+) DNA fragment was unwound compared with a fragment lacking the replication origin. However, at higher ratios of Py T Ag to DNA, equivalent to those used in replication reactions, unwinding of both ori-containing and -lacking fragments was equally efficient. This is in contrast to SV40 T Ag which exhibited a more stringent requirement for SV40 origin sequences under similar conditions. Furthermore, some of the nucleotides that supported the helicase and unwinding activities of Py T Ag were different from those for the same SV40 T Ag reactions. We have also observed that in contrast to the very poor replication of linear SV40 ori(+) DNA by SV40 T Ag in human cell extracts, linear Py ori(+) DNA was replicated efficiently in mouse cell extracts by Py T Ag. However, despite the fact that linear Py ori(+), SV40 ori(+), and ori(-) DNA fragments could be unwound with comparable efficiency by Py T Ag, only fragments containing the Py replication origin were replicated in vitro. These results suggest that the initiation of DNA synthesis at the Py origin of replication requires features in addition to unwinding of the template.

Antigens, Polyomavirus Transforming↗
PubMed1994

A differential cloning procedure of complex genomic DNA fragments.

We have developed a differential cloning procedure designed for cloning of anonymous restriction DNA fragments whose molecular sizes differ between two genomic DNA preparations from higher organisms. The procedure, which was extensively revised from the original one, consists of several steps as summarized below. (i) Digestion of two DNA preparations (target and reference DNA) with the same restriction enzyme (4 base cutter). (ii) Biotinylation of target DNA and conversion of reference DNA to nonamplifiable form by terminal dephosphorylation. (iii) Electrophoresis of the two DNA preparations through a synthetic gel with a large excess of reference DNA as a competitor. (iv) In-gel alkaline dissociation of DNA, followed by reassociation (in-gel competitive reassociation). (v) Elution of DNA from the gel and PCR after adapter ligation and adsorption of DNA onto streptavidin-coated matrix. By repeating these steps, we attained substantial enrichment (approximately 10,000-fold) of DNA fragments which were originally present at one copy or less per complex mammalian genome. The details of the procedure and its unique characteristics in cloning of altered genomic DNA fragments, particularly from mammalian genome, are discussed.

Adenine Phosphoribosyltransferase↗
PubMed1991

Anti-CD3-induced cell death in T cell hybridomas: mitochondrial failure and DNA fragmentation are distinct events.

Triggering of the T cell receptor of T cell hybridomas leads to interleukin (IL) 2 secretion, inhibition of spontaneous growth, degradation of genomic DNA and cell death. We have investigated the relationship between the ability of mitochondria to convert 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), DNA fragmentation and growth arrest in hybridomas stimulated with anti-CD3/T cell receptor antibodies. We describe a variant T hybridoma whose mitochondrial function remains unaffected upon stimulation with anti-CD3 antibody, although it does undergo DNA fragmentation. By contrast, treatment of another anti-CD3-stimulated T hybridoma with endonuclease inhibitor completely inhibits the DNA fragmentation response but not mitochondrial failure induced by anti-CD3 antibody. Thus, we have been able to dissociate anti-CD3-induced mitochondrial failure and DNA fragmentation, suggesting that they are separate events. Although both undoubtedly contribute to cell death induced by activation the primary cause of death may be mitochondrial failure rather than DNA fragmentation.

Animals↗
PubMed1991

[Enzymatic ligation of DNA fragments containing phosphoamide modification of the internucleotide bonds].

DNA fragments with the point amidophosphate (cyclohexylamido- or morpholido-) modification in the sugar-phosphate backbone were synthesized and separated into individual diastereoisomer. The isomers were separated by the reversed-phase HPLC (RPC), and chirality at phosphorus was assigned by a stereochemical correlation scheme using phosphorothioate standards. The RPC-retention time values for Rp-isomers were found to be lower than for Sp-analogues. Amidophosphate DNA fragments were used as P- and OH-components in the T4 DNA-ligation. The enzyme does not ligate amidated fragments with modified internucleotide linkage near 5'- or 3'-end, independently of the amidophosphate chirality. When an unmodified phosphodiester linkage separates the amidophosphate group from 3'-end in O-component, the ligation occurs only with Sp-isomer, whereas Rp-analogue does not give the ligation product. In the P-component of the ligation, configuration of the modified linkage separated from 5'-phosphate by an unmodified linkage does not affect the result of the enzymatic reaction: both Sp-and Rp-stereomers do take part in the ligation. As a result of the ligation of the modified fragments on unmodified templates a set of 31-mers was obtained. They contain FokI and EcoRI recognition sites with the cleavage points of both endonucleases coinciding and being amidated. Upon treatment of duplex DNA consisted of unmodified and amidated strands with these endonucleases Sp-configuration did not hinder the cleavage of the unmodified strand, whereas Rp-configuration inhibited the EcoRI and did not affect the FokI cleavage.

Base Sequence↗
PubMed1991

Ca2+/Mg(2+)-dependent nuclease: tissue distribution, relationship to inter-nucleosomal DNA fragmentation and inhibition by Zn2+.

Ca2+/Mg(2+)-dependent endonuclease has been implicated in the extensive internucleosomal DNA fragmentation that accompanies apoptosis (gene-directed cell death). We present further evidence that this enzyme is involved in apoptosis. Ca2+/Mg2+ nuclease activity was increased about 6-fold during colchicine-induced apoptosis in human chronic lymphocytic leukaemia cells. The increase in activity coincided with onset of DNA fragmentation. Spleen, liver, kidney and thymus expressed high levels of this enzyme while lung, brain, heart and testis contained little activity. Cells from tissues with high Ca2+/Mg2+ nuclease activity underwent rapid DNA fragmentation in response to a Ca2+ flux. Physiological concentrations of Zn2+ known to inhibit both apoptosis and DNA fragmentation also inhibited Ca2+/Mg2+ nuclease activity.

Animals↗
PubMed2006

Hypoxia-induced Bax and Bcl-2 protein expression, caspase-9 activation, DNA fragmentation, and lipid peroxidation in mitochondria of the cerebral cortex of newborn piglets: the role of nitric oxide.

The present study tests the hypothesis that cerebral hypoxia results in increased ratio of Bax/Bcl-2, activation of caspase-9, lipid peroxidation, and DNA fragmentation in mitochondria of the cerebral cortex of newborn piglets and that the inhibition of nitric oxide synthase by N-nitro-L-arginine during hypoxia will prevent the events leading to mitochondrial DNA fragmentation. To test this hypothesis, six piglets, 3-5 days old, were divided into three groups: normoxic (n=5), hypoxic (n=5), and hypoxic-nitric oxide synthase (n=4). Hypoxic animals were exposed to a FiO2 of 0.6 for 60 min. Nitric oxide synthase (40 mg/kg) was infused over 60 min prior to hypoxia. Tissue hypoxia was confirmed by measuring levels of ATP and phosphocreatine. Cerebral cortical tissue mitochondria were isolated and purified using a discontinuous ficoll gradient. Mitochondrial Bax and Bcl-2 proteins were determined by Western blot. Caspase-9 activity in mitochondria was determined spectro-fluorometrically using fluorogenic substrate for caspase-9. Fluorescent compounds, an index of mitochondrial membrane lipid peroxidation, were determined spectrofluorometrically. Mitochondrial DNA was isolated and separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. ATP levels (micromol/g brain) were 4.52+/-0.34 in normoxic, 1.18+/-0.29 in hypoxic (P<0.05) and 1.00+/-0.26 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic). Phosphocreatine levels (micromol/g brain) were 3.61+/-0.33 in normoxic, 0.70+/-0.20 in hypoxic (P<0.05 vs. normoxic) and 0.57+/-0.14 in hypoxic-nitric oxide synthase animals (P<0.05 vs. normoxic, P=NS vs. hypoxic). Bax density in mitochondrial membranes was 160+/-28 in normoxic and 324+/-65 in hypoxic (P<0.001 vs. normoxic). Bcl-2 density mitochondria was 96+/-18 in normoxic and 98+/-20 in hypoxic (P=NS vs. normoxic). Mitochondrial caspase-9 activity (nmol/mg protein/h) was 1.32+/-0.23 in normoxic and 2.25+/-0.24 in hypoxic (P<0.01 vs. normoxic). Levels of fluorescent compounds (microg of quinine sulfate/g protein) were 12.48+/-4.13 in normoxic and 37.92+/-7.62 in hypoxic (P=0.003 vs. normoxic). Densities (ODxmm2) of low molecular weight DNA fragments were 143+/-38 in normoxic, 365+/-152 in hypoxic, (P<0.05 vs. normoxic) and 163+/-25 in hypoxic-nitric oxide synthase animals (P<0.05 vs. hypoxic, P=NS vs. normoxic). The data demonstrate that hypoxia results in increased mitochondrial proapoptotic protein Bax, increased mitochondrial caspase-9 activity, increased mitochondrial lipid peroxidation, and increased fragmentation of DNA in mitochondria of the cerebral cortex of newborn piglets. The administration of a nitric oxide synthase inhibitor, nitric oxide synthase, prior to hypoxia prevented fragmentation of mitochondrial DNA, indicating that the hypoxia-induced mitochondrial DNA fragmentation is NO-mediated. We propose that NO free radicals generated during hypoxia lead to NO-mediated altered expression of Bax leading to increased ratio of pro-apoptotic/anti-apoptotic protein resulting in modification of mitochondrial membrane, and subsequently Ca2+-influx and fragmentation of mitochondrial DNA.

Adenosine Triphosphate↗
PubMed1997

Major DNA fragmentation is a late event in apoptosis.

Apoptosis, the terminal morphological and biochemical events of programmed cell death, is characterized by specific changes in cell surface and nuclear morphology. In addition, DNA fragmentation in an internucleosomal pattern is detectable in mass cultures of apoptotic cells. However, DNA fragmentation and nuclear morphological changes may not necessarily be associated events. In this study, we examined OVCAR-3 and KB human carcinoma cells using time-lapse video phase-contrast microscopy to characterize the surface and nuclear morphological features of apoptosis in response to treatment with either taxol or ricin. The surface morphological features of apoptosis were the same in both cell types and with both drugs. Using an in situ nick-translation histochemical assay, these single cells were also examined for DNA strand breaks during apoptosis. Surface morphological changes demonstrated discrete stages of cell rounding, surface blebbing, followed by cessation of movement and the extension of thin surface microspikes, followed much later by surface blistering and cell lysis. Nuclear features examined by DAPI cytochemistry demonstrated apoptotic nuclear condensation very early in this sequence, usually at the time of initial surface blebbing. The nick-translation assay, however, demonstrated DNA strand breaks at a much later time, only after the formation of separated apoptotic bodies or after final cell lysis. This study points out the differences between surface and nuclear morphological changes in apoptosis, and the large temporal separation between nuclear morphological changes and major DNA fragmentation detectable by this in situ technique. This result suggests caution in using in situ nick-translation as a direct correlate of internucleosomal DNA fragmentation in apoptosis.

Apoptosis↗
PubMed1993

Detection of DNA fragmentation in apoptosis: application of in situ nick translation to cell culture systems and tissue sections.

Since DNA fragmentation is a key feature of programmed cell death (PCD) and also occurs in certain stages of necrosis, we have adapted the methodology of in situ nick-translation (ISNT) to detect DNA fragmentation on a single-cell level. We first established the technique for cell preparations. Apoptosis was induced by gamma-irradiation on freshly isolated rat thymocytes. After fixation procedures, ISNT was performed by overnight incubation either with fluorescein-12-dUTP or with digoxigenin-labeled 11-dUTP and DNA polymerase I. The enzymatic incorporation of labeled nucleotides at sites of DNA fragmentation was detected by flow cytometry either directly or indirectly with fluorescein-conjugated anti-digoxigenin. The quantitative results demonstrated close correlation with morphological essays for apoptosis, DNA gel electrophoresis, and ISNT. Proliferating cells determined by bromodeoxyuridine immunofluorescence were not labeled by ISNT. Immunocytochemistry for cell surface antigens in combination with ISNT allowed the identification of specific cell types undergoing PCD. Furthermore, the simultaneous application of photolabeling techniques with ethidium monoazide and ISNT led to the identification of DNA fragmentation in cells with still intact membranes. Extending ISNT to tissue sections of paraformaldehyde-fixed, paraffin-embedded material reliably revealed labeling of cells with typical morphological features of apoptosis. However, this technique was not useful in detecting early stages of necrotic cell death.

Animals↗
PubMed2005

Lethal effect and apoptotic DNA fragmentation in response of D-GalN-treated mice to bacterial LPS can be suppressed by pre-exposure to minute amount of bacterial LPS: dual role of TNF receptor 1.

AIM: To investigate whether induction of tolerance of mice to lipopolysaccharide (LPS) was able to inhibit apoptotic reaction in terms of characteristic DNA fragmentation and protect mice from lethal effect. METHODS: Experimental groups of mice were pretreated with non-lethal amount of LPS (0.05 microg). Both control and experimental groups simultaneously were challenged with LPS plus D-GalN for 6-7 h. The evaluations of both DNA fragmentations from the livers and the protection efficacy against lethality to mice through induction of tolerance to LPS were conducted. RESULTS: In the naive mice challenge with LPS plus D-GalN resulted in complete death in 24 h, whereas a characteristic apoptotic DNA fragmentation was exclusively seen in the livers of mice receiving LPS in combination with D-GalN. The mortality in the affected mice was closely correlated to the onset of DNA fragmentation. By contrast, in the mice pre-exposed to LPS, both lethal effect and apoptotic DNA fragmentation were suppressed when challenged with LPS/D-GalN. In addition to LPS, the induction of mouse tolerance to TNF also enabled mice to cross-react against death and apoptotic DNA fragmentation when challenged with TNF and/or LPS in the presence of D-GalN. Moreover, this protection effect by LPS could last up to 24 h. TNFR1 rather than TNFR2 played a dual role in signaling pathway of either induction of tolerance to LPS for the protection of mice from mortality or inducing morbidity leading to the death of mice. CONCLUSION: The mortality of D-GalN-treated mice in response to LPS was exceedingly correlated to the onset of apoptosis in the liver, which can be effectively suppressed by brief exposure of mice to a minute amount of LPS. The induced tolerance status was mediated not only by LPS but also by TNF. The developed tolerance to either LPS or TNF can be reciprocally cross-reacted between LPS and TNF challenges, whereas the signaling of induction of tolerance and promotion of apoptosis was through TNFR1, rather than TNFR2.

Animals↗
PubMed1995

DNA fragmentation of human infarcted myocardial cells demonstrated by the nick end labeling method and DNA agarose gel electrophoresis.

Myocardial tissue taken from 19 autopsy cases of myocardial infarction were examined both by the nick and labeling method (NELM) and by DNA agarose gel electrophoresis in order to demonstrate the localization of cells with fragmented DNA and to confirm the internucleosomal cleavage of DNA biochemically. The nuclei corresponding to those with the histological features of acute myocardial infarction in hematoxylin and eosin (H&E)-stained sections were stained strongly positive with the nick end labeling method. Myocardial cells corresponding to those with nick end labeling method-stained nuclei, on the other hand, had mostly pyknotic and karyolytic nuclei and some unremarkable nuclei, even nuclear ghosts, and showed degenerated cytoplasm, including contraction band necrosis in H&E-stained preparations. The agarose gel electrophoresis of DNA extracted from the corresponding areas mentioned above showed the ladder pattern of internucleosomal cleavage characteristic of apoptosis. The present study revealed that infarcted myocardial cells with nuclear outlines, even nuclear ghosts, showed a distinct DNA fragmentation with the ladder pattern of internucleosomal cleavage. It is concluded from this study that the damaged myocardial cells of acute myocardial infarction represent a coagulation necrosis having the biochemical nature of apoptosis.

Aged↗
PubMed2004

Sequence analysis of amplified DNA fragments containing the region encoding the putative lipase substrate-binding domain and genotyping of Aeromonas hydrophila.

DNA fragments were amplified by PCR from all tested strains of Aeromonas hydrophila, A. caviae, and A. sobria with primers designed based on sequence alignment of all lipase, phospholipase C, and phospholipase A1 genes and the cytotonic enterotoxin gene, all of which have been reported to have the consensus region of the putative lipase substrate-binding domain. All strains showed lipase activity, and all amplified DNA fragments contained a nucleotide sequence corresponding to the substrate-binding domain. Thirty-five distinct nucleotide sequence patterns and 15 distinct deduced amino acid sequence patterns were found in the amplified DNA fragments from 59 A. hydrophila strains. The deduced amino acid sequences of the amplified DNA fragments from A. caviae and A. sobria strains had distinctive amino acids, suggesting a species-specific sequence in each organism. Furthermore, the amino acid sequence patterns appear to differ between clinical and environmental isolates among A. hydrophila strains. Some strains whose nucleotide sequences were identical to one another in the amplified region showed an identical DNA fingerprinting pattern by repetitive extragenic palindromic sequence-PCR genotyping. These results suggest that A. hydrophila, and also A. caviae and A. sobria strains, have a gene encoding a protein with lipase activity. Homologs of the gene appear to be widely distributed in Aeromonas strains, probably associating with the evolutionary genetic difference between clinical and environmental isolates of A. hydrophila. Additionally, the distinctive nucleotide sequences of the genes could be attributed to the genotype of each strain, suggesting that their analysis may be helpful in elucidating the genetic heterogeneity of Aeromonas.

Aeromonas↗
PubMed1997

Glutamate metabotropic receptor agonist 1S,3R-ACPD induces internucleosomal DNA fragmentation and cell death in rat striatum.

Glutamate metabotropic receptor mediated mechanisms have been implicated in both neuroprotection and neurotoxicity. To characterize these mechanisms further in vivo, the effects of an intrastriatally injected metabotropic receptor agonist, trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (1S,3R-ACPD), were studied alone and together with N-methyl-D-aspartate (NMDA) or kainic acid (KA) receptor agonists on DNA fragmentation and nerve cell death. 1S,3R-ACPD induced internucleosomal DNA fragmentation of striatal cells in a dose-dependent manner. TUNEL and propidium iodide staining showed DNA fragmentation and profound nuclear condensation around the injection site. Fragmented nuclei were occasionally seen under light microscopy. Internucleosomal DNA fragmentation induced by 1S,3R-ACPD was attenuated by the protein synthesis inhibitor cycloheximide as well as by the non-selective and selective metabotropic receptor antagonists L-(+)-2-amino-3-phosphonopionic acid (L-AP3), (RS)-aminoindan-1,5-dicarboxylic acid and (RS)-alpha-methylserine-o-phosphate monophenyl ester, respectively. The 1S,3R-ACPD (100-900 nmol) induced death of striatal neurons was suggested by the reduction in NMDA and D1 dopamine receptors by up to 13% (P < 0.05) and 20% (P < 0.05) as well as by the decline in GAD67 mRNA (25%, P < 0.01) and proenkephalin mRNA levels (35%, P < 0.01). Interestingly, 1S,3R-ACPD attenuated internucleosomal DNA fragmentation induced by NMDA, but potentiated that induced by KA. These results suggest that metabotropic receptor stimulation leads to the death of striatal neurons by a mechanism having the biochemical stigmata of apoptosis. Moreover, metabotropic receptor stimulation evidently exerts opposite effects on pre- or postsynaptic mechanisms contributing to the NMDA and KA-induced apoptotic-like death of these neurons.

Alanine↗
PubMedDate not supplied

Cryopreservation-Thawing of fractionated human spermatozoa is associated with membrane phosphatidylserine externalization and not DNA fragmentation.

The objective of these studies was to evaluate the effect of cryopreservation-thawing of human spermatozoa on DNA fragmentation and membrane integrity. This was a prospective, controlled cohort study, performed at a university-based infertility center. Ejaculates were examined from 5 donors and 16 men undergoing infertility evaluation. Purified sperm populations were prepared by gradient centrifugation, cryopreserved using a manual method and TEST-yolk buffer and glycerol (TYB-G), followed by quick-thaw. Annexin V binding was used for assessing membrane translocation of phosphatidylserine, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) was utilized for the evaluation of DNA fragmentation. The results were as follows: the percentage of live cells with intact membranes (annexin V-, live) was significantly reduced after cryopreservation-thawing. On the other hand, the percentages of live cells with phosphatidylserine translocation (annexin V-, live) and of necrotic (dead) cells increased significantly after thawing. TUNEL revealed percentages of cells with DNA fragmentation in the prefreeze and postthaw samples that were not significantly different. In a further attempt to examine differences in response to various cryoprotection protocols, experiments were carried out using no cryoprotection, glycerol alone, or TYB-G. Samples frozen with TYB-G demonstrated significantly higher percentages of live cells without phosphatidylserine translocation than the other conditions. We concluded that cryopreservation-thawing of human sperm from patients and donors was associated with membrane change, as revealed by membrane translocation of phosphatidylserine, while having no major impact on DNA fragmentation.

Annexin A5↗
PubMed1992

Expectation maximization algorithm for identifying protein-binding sites with variable lengths from unaligned DNA fragments.

An Expectation Maximization algorithm for identification of DNA binding sites is presented. The approach predicts the location of binding regions while allowing variable length spacers within the sites. In addition to predicting the most likely spacer length for a set of DNA fragments, the method identifies individual sites that differ in spacer size. No alignment of DNA sequences is necessary. The method is illustrated by application to 231 Escherichia coli DNA fragments known to contain promoters with variable spacings between their consensus regions. Maximum-likelihood tests of the differences between the spacing classes indicate that the consensus regions of the spacing classes are not distinct. Further tests suggest that several positions within the spacing region may contribute to promoter specificity.

Algorithms↗
PubMed2005

Metalloporphyrin-based superoxide dismutase mimic attenuates the nuclear translocation of apoptosis-inducing factor and the subsequent DNA fragmentation after permanent focal cerebral ischemia in mice.

BACKGROUND AND PURPOSE: Recently, apoptosis- inducing factor (AIF), a mitochondrial proapoptotic protein, and its nuclear translocation have been reported in caspase-independent neuronal apoptosis. In this study, we investigated the contribution of reactive oxygen species (ROS) to the nuclear translocation of AIF and the subsequent DNA fragmentation after permanent focal cerebral ischemia (pFCI) using manganese tetrakis (4-benzoic acid) porphyrin (MnTBAP), which mimics mitochondrial superoxide dismutase. METHOD: Adult male ICR mice were subjected to pFCI by intraluminal suture blockade of the middle cerebral artery. Immunohistochemistry and Western blot analysis were performed. Large-scale DNA fragmentation was evaluated by pulse field gel electrophoresis, and apoptotic cell death was quantified. MnTBAP was injected into the ventricle to determine whether the removal of ROS contributes to AIF translocation and the subsequent DNA fragmentation. RESULTS: Western blot analysis showed that the nuclear translocation of AIF occurred as early as 2 hours after pFCI. AIF translocation was not blocked by a pan-caspase inhibitor. MnTBAP-treated mice had attenuated AIF translocation and blocked large-scale DNA fragmentation. Caspase-3 activity was similarly inhibited between the pan-caspase inhibitor- and MnTBAP-treated mice, but the amount of apoptosis-associated DNA fragmentation in the MnTBAP-treated mice was less than in the pan-caspase inhibitor-treated mice (P<0.001). CONCLUSIONS: These results suggest that the MnTBAP, a mitochondrial O2- scavenger, may attenuate the caspase-independent nuclear translocation of AIF after pFCI and subsequent apoptosis-associated DNA fragmentation.

Animals↗