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At least 595 records · Page 33Linked to original sources
PubMed2003

Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW, HTK, and Celsior solutions.

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4 degrees C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not hanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.

Adenosine↗
PubMed1999

A rapid and efficient method for preparation of genomic DNA suitable for analysis of both high and low molecular weight DNA fragmentation during neuronal apoptosis.

In this paper, we describe a fast (within 1 h) and efficient method for the preparation of intact genomic DNA suitable for the analysis of DNA integrity in neuronal cells undergoing apoptosis. The procedure involves the embedding of apoptotic cells into low-melting point agarose, in situ cell lysis and purification of DNA samples followed by resolution of fragmented DNA by agarose gel electrophoresis. The method has no restrictions on the number of simultaneous DNA preparations, allows detection of both high and low molecular weight DNA fragments, and is suitable for the preparation of DNA samples from cultured cells, fresh or frozen tissues, as well as from purified nuclei. This procedure is also applicable for the preparation of intact genomic DNA for the megabase-scale analysis.

Animals↗
PubMed2001

DNA fragmentation, an indicator of apoptosis, in cultured black tiger shrimp Penaeus monodon infected with white spot syndrome virus (WSSV).

Fifty black tiger shrimp Penaeus monodon from commercial cultivation ponds in Malaysia were examined by Tdt-mediated dUTP nick-end labeling (TUNEL) fluorescence assay and agarose gel electrophoresis of DNA extracts for evidence of DNA fragmentation as an indicator of apoptosis. From these specimens, 30 were grossly normal and 20 showed gross signs of white spot syndrome virus (WSSV) infection. Of the 30 grossly normal shrimp, 5 specimens were found to be positive for WSSV infection by normal histology and by nested polymerase chain reaction (PCR) analysis. All of the specimens showing gross signs of WSSV infection were positive for WSSV by normal histology, while 5 were positive by nested PCR only (indicating light infections) and 15 were positive by 1-step PCR (indicating heavy infections). Typical histological signs of WSSV infection included nuclear hypertrophy, chromatin condensation and margination. None of the 25 grossly normal shrimp negative for WSSV by 1-step PCR showed any signs of DNA fragmentation by TUNEL assay or agarose gel electrophoresis of DNA extracts. The 10 specimens that gave PCR-positive results for WSSV by nested PCR only (i.e., 5 grossly normal shrimp and 5 grossly positive for WSSV) gave mean counts of 16 +/- 8% TUNEL-positive cells, while the 25 specimens PCR positive by 1-step PCR gave mean counts of 40 +/- 7% TUNEL-positive cells. Thus, the number of TUNEL positive cells present in tissues increased with increasing severity of infection, as determined by gross signs of white spots on the cuticle, the number of intranuclear inclusions in histological sections, and results from single and nested PCR assays. DNA extracts of PCR-positive specimens tested by agarose gel electrophoresis showed indications of DNA fragmentation either as smears or as 200 bp ladders. Given that DNA fragmentation is generally considered to be a hallmark of apoptosis, the results suggested that apoptosis might be implicated in shrimp death caused by WSSV.

Animals↗
PubMed2006

Sperm DNA fragmentation in boars is delayed or abolished by using sperm extenders.

The semen quality of seven young adult boars was assessed for percentages of sperm motility, normal acrosomes, abnormal sperm, cells positive to sHOST (short Hipoosmotic Swelling Test), HPNA cells (sHOST Positive with Normal Acrosome cells) and the percentage of sperm heads, which exhibited DNA fragmentation using the Sperm Chromatin Dispersion test (SCD). These parameters were analysed in sperm samples both undiluted and diluted using a commercial extender and stored at 15 degrees C for 21 days. Results showed that semen quality decreases faster in the undiluted semen samples from day 0 to day 7 compared to diluted semen samples that remained with a high quality up to day 11. The undiluted semen exhibited a low DNA fragmentation index (DFI) during the first days and then a significant increase from day 7 up to day 21. This increase in the DFI coincided with the lowest levels of the other semen quality parameters. On the contrary, the samples diluted in the commercial extender showed very low levels of DNA fragmentation in all boars during the preservation period. When the evolution of DNA fragmentation was analysed in the undiluted samples, differences were found among boars. These differences were not shown in the samples diluted in the extender where the basal DFI remained stable during the 21 days. The main conclusion of this study was that some sperm extenders delay or partially prevent sperm DNA fragmentation.

Acrosome↗
PubMed1998

Large DNA fragment sizing using native acrylamide gels on an automated DNA sequencer and GENESCAN software.

We have investigated the potential of the PE Applied Biosystems Model 373 Automated DNA Sequencer and GENESCAN software to size minisatellite alleles ranging in size from 230 bp to 2.5 kbp. We report on the use of a native (non-denaturing) acrylamide gel system and fluorescent dUTP labeling of PCR products. The observed variability in size calling ranged from +/- 0.4-bp standard deviation (SD) at the lower end of the size range to +/- 37.5-bp SD for the largest allele. Both within-gel and between-gel variability in sizing increased with larger alleles, in particular when sizes exceeded 2 kbp. Size-calling differences were observed dependent on the method used to fluorescently label the PCR products and with the fluorescent dye type and concentration used in incorporation. The benefits and limitations of the current GENESCAN software in sizing large DNA fragments are also discussed.

Alleles↗
PubMed1982

Cloning of DNA fragments from the left end of the adenovirus type 12 genome: transformation by cloned early region 1.

The human adenovirus serotype 12 (Ad-12) EcoRI-C DNA fragment (0 to 16.5 map units) was cloned in the plasmid vector pAT153. This cloned Ad-12 EcoRI-C DNA fragment was subcloned generating recombinant plasmids which contained the Ad-12 SalI-C fragment (0 to 10.3 map units), the Ad-12 HindIII-G fragment (0 to 6 X 8 map units) and the Ad-12 AccI-H fragment (0 to 4 X 7 map units). Thus, we constructed recombinant plasmids which contain Ad-12 DNA sequences which represent all or part of the virus transforming gene region. The capacity of the cloned Ad-12 EcoRI-C DNA fragment to transform rat cells in vitro was assessed using the focus assay on primary cultures of rat cells. The specific transforming activity of this recombinant plasmid was in the same range as that found for intact Ad-12 DNA. Transformed foci which were induced by the cloned Ad-12 EcoRI-C DNA fragment were established as cell lines and the presence of Ad-12 DNA in these lines was demonstrated using the Southern blotting technique.

Adenoviruses, Human↗
PubMed2002

Nicotinamide and ketamine reduce infarct volume and DNA fragmentation in rats after brain ischemia and reperfusion.

The possible ability of nicotinamide and ketamine to decrease infarction volume and DNA fragmentation was investigated in a middle cerebral artery occlusion rat model. DNA fragmentation was measured with an enzyme linked immunoassay. Control infarct volume was 223.8 +/- 10.6 mm(3). Ketamine alone did not alter infarct volume, 233.2 +/- 61.8 mm(3). Nicotinamide alone did not alter infarct volume, 235.2 +/- 62.8 mm(3). The combination of ketamine and nicotinamide decreased infarct volume to 83.8 +/- 35.2 mm(3). Ketamine produced hypothermia. Nicotinamide and ketamine decreased brain swelling and DNA fragmentation in the cerebral cortex, striatum and hippocampus in rats perfused for 6 or 24 h. Ketamine may synergize the actions of nicotinamide and partially prevent brain damage from ischemia and reperfusion.

Animals↗
PubMed2002

Nitric oxide-mediated expression of Bax protein and DNA fragmentation during hypoxia in neuronal nuclei from newborn piglets.

The present study tests the hypothesis that nitric oxide mediates the hypoxia-induced increase in expression of Bax and in DNA fragmentation in the cerebral cortex of newborn piglets, and that administration of N-nitro-L-arginine (NNLA), a nitric oxide synthase inhibitor, will prevent a change in hypoxia-induced expression of apoptotic genes and DNA damage. Piglets were assigned to normoxic, hypoxic, or NNLA-pretreated hypoxic groups. Cerebral tissue hypoxia was documented biochemically by measuring ATP and phosphocreatine (PCr) levels. Cerebral cortical neuronal nuclei were isolated and nuclear proteins were separated electrophoretically and probed with specific antibodies against Bcl-2 or Bax proteins. Neuronal nuclear DNA from normoxic, hypoxic, and NNLA-pretreated hypoxic animals was isolated, separated by electrophoresis on 1% agarose gel and stained with ethidium bromide. Cerebral hypoxia resulted in an increase in nuclear membrane Bax protein levels from 121.33+/-47.7 optical density (OD)xmm(2) in normoxic to 273.67+/-67.3 ODxmm(2) in hypoxic group (P<0.05 vs. normoxic), but levels in NNLA-pretreated hypoxic group were 155.78+/-48.3 ODxmm(2) (P<0.05 vs. hypoxic, P=NS vs. normoxic). Similarly, cerebral hypoxia resulted in the density of DNA fragments increasing from 1530.3+/-309.8 OD/mm(2) in the normoxic group to 5383.3+/-775 OD/mm(2) in the hypoxic group (P<0.05), while levels in NNLA-pretreated hypoxic group were 3574.0+/-952 OD/mm(2) (P<0.05 compared to hypoxic and normoxic groups). The data show that NNLA-pretreatment prevents the hypoxia-induced increase in Bax expression and DNA fragmentation demonstrating that the hypoxia-induced Bax gene expression and the DNA fragmentation are NO-mediated.

Animals↗
PubMed2004

A novel inhibitor that protects apoptotic DNA fragmentation catalyzed by DNase gamma.

The internucleosomal cleavage of genomic DNA is the biochemical hallmark of apoptosis. DNase gamma, a Ca(2+)/Mg(2+)-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases. We identified here 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel and potent DNase gamma inhibitor using stable HeLa S3 transfectants of DNase gamma (HeLa-gamma cells). DR396 inhibited apoptotic DNA fragmentation in HeLa-gamma cells induced by staurosporine (STS) and in rat splenocytes exposed to gamma-ray irradiation in a dose-dependent manner. This compound potently and selectively inhibited DNase gamma activity with an IC(50) value of 3.2 microM. DR396 did not delay the apoptotic processes as judged by the morphological changes and the cleavage of a death substrate, poly(ADP-ribose) polymerase (PARP). Furthermore, the compound did not prevent apoptotic DNA fragmentation in Jurkat cells induced by anti-Fas antibody (Ab), which is catalyzed by caspase-activated DNase (CAD). These findings clearly indicate that DR396 exerts chemical knockdown effect of DNase gamma on cells, suggesting that the compound could be an attractive tool for understanding of the physiological significance of DNase gamma.

Animals↗
PubMed1996

Role of adhesion molecules in natural killer cell-induced DNA fragmentation of cytomegalovirus-infected fibroblasts.

We investigated the roles of CD11a, CD11b, CD18, and CD54 adhesion molecules in non-adherent peripheral blood mononuclear cells (NPBMC)-induced DNA fragmentation of cytomegalovirus (CMV)-infected cells. DNA fragmentation in the supernatant from CMV-infected cells and NPBMC was assayed, and cytotoxicity against CMV-infected cells was calculated. Treatment of NPBMC with monoclonal antibodies to CD11a, CD11b, CD18, and CD54 significantly reduced cytotoxicity against CMV-infected cells. A combination of anti-CD11a, anti-CD11b, and anti-CD18 antibodies further inhibited cytotoxicity against CMV-infected cells. Cytotoxicity against CMV-infected cells treated with anti-CD54 antibody was also significantly inhibited. Binding of effector cells to target cells was not affected by treatment of NPBMC or CMV-infected cells with antiadhesion molecule antibodies. These results indicate that LFA-1 (CD11a/CD18), Mac-1 (CD11b/CD18), and ICAM-1 (CD54) adhesion molecules are involved in natural killer (NK) cell-induced DNA fragmentation in CMV-infected cells.

Adult↗
PubMed1998

Suppression of the ptsH mutation in Escherichia coli and Salmonella typhimurium by a DNA fragment from Lactobacillus casei.

A DNA fragment from Lactobacillus casei that restores growth to Escherichia coli and Salmonella typhimurium ptsH mutants on glucose and other substrates of the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) has been isolated. These mutants lack the HPr protein, a general component of the PTS. Sequencing of the cloned fragment revealed the absence of ptsH homologues. Instead, the complementation ability was located in a 120-bp fragment that contained a sequence homologue to the binding site of the Cra regulator from enteric bacteria. Experiments indicated that the reversion of the ptsH phenotype was due to a titration of the Cra protein, which allowed the constitutive expression of the fructose operon.

Bacterial Proteins↗
PubMed1991

Evolution and sequence analysis of a human Y-chromosomal DNA fragment.

A Y-chromosomal DNA fragment has been isolated from a human Y-Charon 21A recombinant library. Evolutionary analysis of 1F5 indicates that the size and sequence of this fragment have been conserved in higher primates. Deletion mapping and in situ hybridization analysis have localized 1F5 to the middle euchromatic portion of the long arm of the human Y chromosome at Yq11.2. Sequence analysis revealed the presence of an atypical Alu element and two regions rich in polypyrimidine-polypurine residues.

Base Sequence↗
PubMed1994

Genetic variation detected by quantitative analysis of end-labeled genomic DNA fragments.

The continuing efforts to evaluate specific human populations for altered germinal mutation rates would profit from more efficient and more specific approaches than those of the past. To this end, we have explored the potential usefulness of two-dimensional electrophoresis of DNA fragments obtained from restriction-enzyme-digested genomic DNA. This permits the analysis, on a single preparation, of approximately 2000 DNA fragments varying in size from 1.0 to 5.0 kb in the first dimension and from 0.3 to 2.0 kb in the second dimension. To enter into a genetic analysis, these fragments must exhibit positional and quantitative stability. With respect to the latter, if spots that are the product of two homologous DNA fragments are to be distinguished with the requisite accuracy from spots that are the product of only one fragment, the coefficient of variation of spot intensity should be approximately < or = 0.12. At present, 482 of the spots in our preparations meet these standards. In an examination of preparations based on three Japanese mother/father/child trios, 43 of these 482 spots were found to exhibit variation that segregated within families according to Mendelian principles. We have established the feasibility of cloning a variant fragment from such gels and establishing its nucleotide sequence. This technology should be highly efficient in monitoring for mutations resulting in loss/gain/rearrangement events in DNA fragments distributed throughout the genome.

Base Sequence↗
PubMed1985

Chromosomol DNA fragments from mouse cells exposed to an intercalating agent contain a 175-kdalton terminal polypeptide.

A 175 kdalton (kDa) polypeptide is bound covalently to the chromosomal DNA fragments from mouse cells exposed to the intercalating agent 4'-[(9-acridinyl)-amino]methansulphon-m-anisidide. Electron microscopy shows a terminal protein on the DNA fragments, whose 5'-termini are blocked. Since the relative molecular mass of topoisomerase II polypeptide chains is also about 175 kDa and topoisomerase II inhibitors prevent intercalator-induced DNA fragmentation, we propose that the polypeptide bound covalently to the 5'-terminus of the DNA fragments is a polypeptide derived from frequently integrated topoisomerase II operating to normalize torsional stress resulting from intercalation.

Aminoacridines↗
PubMed1997

Chromosome assignment of aberrant NotI restriction DNA fragments in primary hepatocellular carcinoma.

DNA aberrations in human hepatocellular carcinoma (HCC) were studied by two-dimensional DNA electrophoresis analysis. Five intensified and 60 dwindling spots were detected recurrently in the two-dimensional profile which showed about 3000 restriction DNA fragments as distinctive spots. We assigned these aberrant spots to chromosomes, using the chromosome-assigned two-dimensional profile. Four of the five intensified, and 53 of the 60 dwindling spots were given chromosome assignments. Intensified spots were assigned to chromosomes 5, 6, 9 through 12, 16 and 18. Among the dwindling spots, the highest incidence of aberrations was found on chromosome 16, followed by 9 through 12 and chromosome 2. No aberrations were detected in chromosomes 7, 21, 22 or Y.

Carcinoma, Hepatocellular↗
PubMedDate not supplied

The effects of aging and neurodegeneration on apoptosis-associated DNA fragmentation and the benefits of nicotinamide.

In this work, the tertiary butylhydroperoxide- (t-BuOOH) treated mouse was used as a model to study the oxidative stress that is associated with various neurodegenerative diseases. DNA was found to be an early target of t-BuOOH attack. Necrosis was associated with extensive DNA fragmentation that occurred in almost all regions of the brain within 20 min following intracerebroventricular (icv) injection of 109.7 mg/kg t-BuOOH. Apoptosis was associated with high levels of DNA fragmentation that was observed at 48 h after icv administration of 21.9 mg/kg t-BuOOH. Susceptibility to DNA damage was found to be age-dependent, since 24-mo-old mice exhibited consistently higher and more pervasive DNA damage than 8 mo-old-mice. Extensive DNA damage was seen in various brain regions in patients with Alzheimer disease (AD) and with both Alzheimer and Parkinson disease (AD-PD). These results directly implicate DNA damage in neurodegeneration. The DNA fragmentation ob-served can lead to both apoptosis and necrosis, as suggested by gel electrophoresis. Nicotinamide, a precursor of NAD in the brain, was able to prevent DNA fragmentation induced by low-dose t-BuOOH, when coadministered with the toxin.

Aged↗
PubMed1997

DNA fragmentation is not the primary event in glucocorticoid-induced thymocyte death in vivo.

Thymocyte death has been recognized as one of the best models for studying apoptosis. Our recent study, however, indicated that most thymocytes die without DNA fragmentation and become terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling-positive (TUNEL+) only after being phagocytosed by macrophages. In this study, we used histological techniques using the TUNEL method, histochemistry, immunohistochemistry, and transmission electron microscopy as well as flow cytometry to examine in vivo the effect of glucocorticoid (GC), a well-known agent for inducing thymocyte apoptosis in vitro, on thymocyte death to determine whether or not DNA fragmentation was the first event of GC-induced thymocyte death. At 2 h and 4 h after GC injection, a large number of cortical thymocytes were TUNEL+. Most TUNEL+ cells were aggregated to form clusters. Double staining of the section showed that the TUNEL+ thymocytes were phagocytosed by acid phosphatase+ and Mac-2+ macrophages. An ultrastructural study indicated that a far greater number of small pyknotic thymocytes were present in the cortex of the GC-treated thymus than were observed in the control thymus, that all those pyknotic thymocytes were TUNEL-, and moreover, that at the electron microscopic level, TUNEL+ cells were all phagocytosed by macrophages. Flow cytometric analysis did not detect a single TUNEL+ thymocyte even 4 h after the GC treatment, suggesting that virtually no free dead thymocytes were present after DNA fragmentation. These results indicate that, consistent with our previous findings with normal thymocyte death and B cell death in the germinal centers, DNA fragmentation is not involved in the cell death process of the GC-induced rapid thymocyte death in vivo.

Animals↗