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PubMed2003

The effect of hypocapnia (PaCO2 27 mmHg) on CaM kinase IV activity, Bax/Bcl-2 protein expression and DNA fragmentation in the cerebral cortex of newborn piglets.

The present study tests the hypothesis that a PaCO(2) of 27 mmHg for 1 hr results in increased neuronal nuclear Ca(++)/calmodulin-dependent protein kinase IV (CaM kinase IV) activity, pro-apoptotic protein expression and DNA fragmentation in the cerebral cortex of newborn piglets. Hypocapnic (HC) and normocapnic newborn piglets were studied. Tissue levels of ATP and phosphocreatine (PCr) were lower in the HC group. CaM kinase IV activity and Bax protein density were higher in the HC group. Bcl-2 protein density was the same in both groups, resulting in an increased ratio of Bax/Bcl-2 in the HC group. Density of nuclear DNA fragments was greater in the HC group and varied inversely with ATP and PCr levels. We conclude that hypocapnia (PaCO(2) 27 mmHg) results in increased expression of pro-apoptotic proteins and fragmentation of nuclear DNA in newborn piglets.

Animals↗
PubMed2006

Exposure of lymphocytes to high doses of Actinobacillus actinomycetemcomitans cytolethal distending toxin induces rapid onset of apoptosis-mediated DNA fragmentation.

We have shown previously that treatment of human lymphocytes with the Actinobacillus actinomycetemcomitans cytolethal distending toxin (Cdt) results in dose-dependent G2 arrest, followed 24 h later by apoptotic cell death. Here we demonstrated that for Jurkat cells exposed to high concentrations of Cdt (>0.2 ng/ml) there was a dose-dependent increase in the level of S-phase cells and a concomitant decrease in the level of G2 cells. Fluorescence-activated cell sorter analysis demonstrated that the S-phase cells did not incorporate BrdU and likely represented cells that arrested in G2 and underwent significant DNA fragmentation. Analysis of the kinetics of the appearance of both S-phase cells and apoptotic cells supported this interpretation. Cells exposed to low doses of toxin exhibited G2 arrest at 24 h, but at 48 and 72 h there were also decreases in the level of G2 cells and concomitant increases in the levels of S, G0/G1, and sub-G0 cells; these changes were paralleled by increased numbers of apoptotic cells. Cells exposed to high doses of toxin exhibited these changes 24 to 48 h earlier. We also examined the relationship between G2 arrest, DNA fragmentation, and activation of the apoptotic cascade. We employed two inhibitors of apoptosis, overexpression of Bcl-2 and the caspase-3 inhibitor zvad. Both inhibitors blocked Cdt-induced apoptosis, Cdt-induced DNA fragmentation, and phosphorylation of the histone H2AX. However, the cells retained the ability to undergo G2 arrest in the presence of the toxin. Thus, it appears that high doses of Cdt induce rapid onset of DNA degradation resulting from activation of the apoptotic cascade.

Aggregatibacter actinomycetemcomitans↗
PubMed1995

Correlation of DNA fragment sizes within loci in the presence of non-detectable alleles.

At present most forensic databases of DNA profiling of individuals consist of DNA fragment sizes measured from Southern blot restriction fragment length polymorphism (RFLP) analysis. Statistical studies of these databases have revealed that, when fragment sizes are measured from RFLP analysis, some of the single-band patterns of individuals may actually be due to heterozygosity of alleles in which fragment size resulting from one allele remains undetected. In this work, we evaluate the effect of such allelic non-detectability on correlation of fragment sizes within individuals at a locus, and its impact on the inference of independence of fragment sizes within loci. We show that when non-detectable alleles are present in a population at a locus, positive correlations of fragment sizes are expected, which increase with the proportion of non-detectable alleles at the locus. Therefore, a non-zero positive correlation is not a proof of allelic dependence within individuals. Applications of this theory to the current forensic RFLP databases within the US show that there is virtually no evidence of significant allelic dependence within any of the loci. Therefore, the assumption that DNA fragment sizes within loci are independent is valid, and hence, the population genetic principles of computing DNA profile frequencies by multiplying binned frequencies of fragment sizes are most likely to be appropriate for forensic applications of DNA typing data.

Alleles↗
PubMedDate not supplied

Adding Zn2+ induces DNA fragmentation and cell condensation in cultured human Chang liver cells.

Zinc (Zn) is a trace element in human cells and regarded as an essential nutrient with established deficiency states affecting multiple organs in the body. However, it has been reported that Zn uptake is associated with some serious harmful effects, such as inhibition of DNA synthesis and enhanced toxicity from reactive oxygen species. We have previously shown that in vivo administration of Zn2+ in C57/6J mice induces weight loss and massive hair loss where the normal course hair becomes replaced by fine vello hair, simulating the side effects from cancer chemotherapy where oxidative free radical damage is implicated in association with DNA fragmentation and programmed cell death (PCD). Here, in vitro flow cytometric studies on human Chang liver showed Zn2+ causing cell condensation with DNA fragmentation that occurred in a dose-dependent manner, an effect replicated by micrococcal nuclease digestion. Specific terminal deoxynucleotidyl transferase-(TdT) mediated labeling of 3'-OH ends of DNA nicks corroborated the flow cytometric profiles of propidium iodide-DNA binding where degradation of both 2 and 4 N genomic DNA resulted in a solitary 1N peak presentation. DNA degradation concomitant with cell condensation is seen as an established hallmark of PCD. We further showed that Zn2+ could enhance the generation of hydroxyl free radicals (OH.) by the transition metal vanadium. Glutathione, the cell's main reducing agent, underwent corresponding reduction. The results suggested that Zn supplementation could induce features resembling PCD.

Apoptosis↗
PubMed2000

Subarachnoid hemolysate produces DNA fragmentation in a pattern similar to apoptosis in mouse brain.

Stroke and traumatic brain/spinal cord injuries are often associated with hemorrhage. Despite the relative frequency of hemorrhage in the central nervous system (CNS), little is known about what role blood and hemoglobin (Hb) play in mediating cellular injury. Since Hb and hemolysate have been associated with generation of oxidative stress and cell injury, we examined whether apoptosis was present after cortical exposure to subarachnoid hemolysate. Subarachnoid hemorrhage (SAH) was induced in CD-1 mice (n=25) by injection of 50 microl of autologous hemolysate over the right parietal cortex. Saline-injected mice (n=13) were used as controls. Subjects were sacrificed at 24 h. Transcardiac perfusion fixation was performed on a subgroup of hemolysate- (n=15) and saline-injected (n=9) animals. Sections were stained for DNA fragmentation using the terminal deoxyuridine nick end-labeling (TUNEL) method and also immunostained for the hemeoxygenase-1 (HO-1) protein to assess blood distribution. In the remaining animals (n=6 SAH, n=4 saline), DNA was extracted and precipitated from 40 mg of tissue and subjected to electrophoresis on a 1.5% agarose gel. DNA fragmentation was evident on TUNEL staining in 10/15 subjects injected with hemolysate as compared to 0/9 subjects injected with saline (p<0.01, Fisher exact test). TUNEL-positive cells were most abundant closest to the site of cortical SAH, as evidenced by HO-1 immunoreactivity. TUNEL-positive cells were also seen remotely in the hippocampus and basal forebrain. The presence of apoptosis was suggested by DNA laddering on electrophoresis in the hemolysate-injected subjects (4/6 animals). No laddering was evident in saline-injected subjects (n=4). These results provide evidence that the presence of subarachnoid blood products is associated with DNA fragmentation and apoptotic cell death.

Animals↗
PubMed2004

[Screening of hypermethylated DNA fragments in tumor tissue derived from patients with lung cancer].

The investigations on the role of DNA methylation in carcinogenesis have been mainly focused on promoter hypermethylation of tumor suppressor genes. As a number of genes associated with cancer development may be influenced by DNA methylation, identification of these genes is of great importance for understanding the epigenetic alteration in carcinogenesis. In this study, hypermethylated regions of genomic DNA from Chinese lung cancer patients were identified by a modified methylation-sensitive arbitrarily primed PCR (MS-AP-PCR). Eight hypermethylated DNA fragments (HMDF) were separated from a PCR product region between 300 and 500bp in size. After cloning, sequencing and searching with Blast and NewCpGseek programs,the result showed that all of them were typical CpG island sequences, four fragments had 99% approximately 100% homology to regions on human chromosome 2, 7, 9 and 10, respectively,but only one revealed to be known gene. Neural Network Promoter Prediction, TSSG and TSSW programs were run to analyze possible functions of the rest 7 fragments, of which 4 were identified as candidate promoter regions, indicating that they might belong to new genes. The hypermethylated DNA fragments identified in this study might be specific epigenetic alterations in the Chinese lung cancer.

Aged↗
PubMed2002

Evidence of oncotic cell death and DNA fragmentation in human hypertrophic chondrocytes in chondro-osteophyte.

OBJECTIVE: To investigate the population and morphology of in situ terminal deoxynucleotidyle transferase (TdT)-mediated dUTP nick end-labeling (TUNEL) stain positive non-apoptotic chondrocytes in hypertrophic zone of human chondro-osteophytes. MATERIALS AND METHODS: Chondro-osteophytes from osteoarthritic patients were obtained at joint replacement surgery. Apoptosis was verified by light microscopic examination of Safranin O stained sections and TUNEL stain. TUNEL staining was also performed on hydrophilic resin embedded semi-thin and ultra-thin sections combined with the treatment with streptavidin-gold conjugates, observed by light microscopy with silver enhancement technique (TUNEL-LM with SE) and transmission electron microscopy (TUNEL-TEM) respectively for the simultaneous evaluation of cellular structure and DNA fragmentation. RESULTS: In paraffin embedded sections (N=18), 31.5+/-6.1% of cells in the hypertrophic zone were TUNEL positive, but only 3.8+/-1.2% cells in this zone showed apoptotic appearances with cell shrinkage and nuclear condensation. Both in TUNEL-TEM and TUNEL-LM with SE, gold particles, which indicate DNA fragmentation, were observed within the nucleus of morphologically apoptotic chondrocytes, as well as of disintegrated, swollen chondrocytes. CONCLUSIONS: In human chondro-osteophytes, hypertrophic chondrocytes might die by oncotic cell death with DNA fragmentation, as well as apoptosis.

Adult↗
PubMed1998

Caspase-3 is required for DNA fragmentation and morphological changes associated with apoptosis.

Interleukin 1beta-converting enzyme-like proteases (caspases) are crucial components of cell death pathways. Among the caspases identified, caspase-3 stands out because it is commonly activated by numerous death signals and cleaves a variety of important cellular proteins. Studies in caspase-3 knock-out mice have shown that this protease is essential for brain development. To investigate the requirement for caspase-3 in apoptosis, we took advantage of the MCF-7 breast carcinoma cell line, which we show here has lost caspase-3 owing to a 47-base pair deletion within exon 3 of the CASP-3 gene. This deletion results in the skipping of exon 3 during pre-mRNA splicing, thereby abrogating translation of the CASP-3 mRNA. Although MCF-7 cells were still sensitive to tumor necrosis factor (TNF)- or staurosporine-induced apoptosis, no DNA fragmentation was observed. In addition, MCF-7 cells undergoing cell death did not display some of the distinct morphological features typical of apoptotic cells such as shrinkage and blebbing. Introduction of the CASP-3 gene into MCF-7 cells resulted in DNA fragmentation and cellular blebbing following TNF treatment. These results indicate that although caspase-3 is not essential for TNF- or staurosporine-induced apoptosis, it is required for DNA fragmentation and some of the typical morphological changes of cells undergoing apoptosis.

Animals↗
PubMed1984

Analysis of 5'-ends of short DNA fragments excreted by phytohemagglutinin stimulated lymphocytes.

Human peripheral blood lymphocytes were stimulated with phytohemagglutinin and the excreted DNA was isolated from the medium after four days of incubation of cells. The excreted DNA was labeled at the 5'-end with [gamma-32P]ATP and polynucleotide kinase. Analysis of the end-labeled material revealed a size distribution with a chain length of 6 - 60 nucleotides. These short DNA fragments did not contain ribo-nucleotides at their 5'-termini. P1 nuclease digestion did not release specific deoxyribonucleoside monophosphates from the 5'-end of the excreted DNA fragments. These results point to the non-specific degradation of DNA excreted by stimulated lymphocytes.

DNA↗
PubMed2000

Caspases that are activated during generation of nuclear polyglutamine aggregates are necessary for DNA fragmentation but not sufficient for cell death.

Truncated polypeptides containing expanded polyglutamine (polyQ) stretches tend to form cytoplasmic or nuclear aggregates in cultured cells, leading to cell death. Although it has been shown recently that caspase-8 coaggregates with polyQ and is activated during polyQ-mediated cell death, little is known of the location and timing of caspase-8 activation by nuclear polyQ aggregates. Also, the relationship between nuclear polyQ aggregate-mediated cell death and activation of other caspases is unclear. In P19 embryonal carcinoma (EC) cells, which can be made to differentiate into neuronal cells, polyQ72 repeats preferentially aggregate in the nucleus. Nuclear aggregates of polyQ72 induced P19 EC cell death, with a high frequency of cells exhibiting morphology characteristic of apoptosis (i.e., roundness, cell shrinkage, chromatin condensation) and DNA fragmentation. In the present study, we used antisera that specifically recognized the active forms of caspase-8, -3, and -9 but not their proforms, and showed that only caspase-8 and -3 were activated during the generation of polyQ72 aggregates in P19 EC cell nuclei. Furthermore, we showed that the caspase inhibitor z-VAD-fmk inhibited DNA fragmentation, but only partially inhibited the appearance of apoptotic morphology. Thus, caspase activation, including caspase-8 and -3, is necessary for polyQ-mediated DNA fragmentation but not sufficient for polyQ-mediated cell death in P19 EC cells.

Apoptosis↗
PubMedDate not supplied

Endonuclease activity and induction of DNA fragmentation in human myelogenous leukemic cell lines.

When four human myelogenous leukemic cell lines (HL-60, ML-1, U-937, THP-1) were exposed to either ascorbic acid, hydrogen peroxide, etoposide, tumor necrosis factor, hyperthermia or UV irradiation, their growth inhibition and oligonucleosome-size DNA fragmentation were induced. Non-myelogenous leukemic cell lines (MOLT-4, K-562) were similarly sensitive to ascorbic acid and hydrogen peroxide, but relatively resistant to etoposide, TNF, hyperthermia and UV irradiation. Furthermore, these treatments except for UV irradiation, did not induce any apparent DNA fragmentation in MOLT-4 and K-562 cells. An autodigestion experiment revealed that all of these six cell lines contained divalent cation-independent endonuclease activity as a major endonuclease. The ability of this endonuclease to produce oligonucleosome-size DNA fragmentation was stimulated at acidic, but not at neutral pH. Since this enzyme activity was not detected in the lysosomal enzyme-free nuclei, prepared from all six cell lines, the cytoplasmic localization of this enzyme was suggested. The results suggest that the endonuclease activity might be differently regulated between myelogenous and non-myelogenous leukemic cell lines.

Apoptosis↗
PubMed1978

A cloned Drosophila DNA fragment which codes for a 4 S RNA species.

A collection of random Drosophila melanogaster DNA fragments cloned individually in Escherichia coli was screened for the presence of sequences complementary to the 4 S, 5 S and 5.8 S RNA species produced in the D. melanogaster Kc tissue culture line. Four D. melanogaster DNA fragments were found which possessed sequences complementary to the 4 S RNA species but not complementary to the 5 S or 5.8 S RNA. One such cloned fragment (6.81 kilobase in length) was characterized further. It hybridizes in situ to region 22A-C of the left arm of chromosome 2 and does not contain repetitive sequences detectable by renaturation (cot) analysis. This same region was reported earlier by Steffensen and Wimber (Genetics (1971) 69, 163--178) to hybridize in situ to bulk tRNA extracted from D. melanogaster.

Animals↗
PubMed2002

Direct cloning of the unknown flanking DNA fragments from a large insert without restriction mapping.

A simple technique for direct cloning of the target DNA fragments from a large insert according to its adjacent known sequence is described here. In this new subcloning method, a large DNA insert is digested and ligated with a linearized plasmid vector to construct a subclone library that is subjected to screening. The bacterial clones in this library are individually picked, grown in a 96-well plate, and then pooled across the rows or columns. Target clones are obtained from the ordered separate pools by PCR-screening with a set of primers, one specific for the adjacent known sequence and the other serving as "anchor primer" specific for the vector sequence. This direct subcloning procedure was efficiently demonstrated by cloning a specific DNA region from a large insert within 2 days without mapping the starting DNA or isolating the digested DNA fragment.

Cloning, Molecular↗
PubMed1994

Reiterated DNA fragments in defective genomes of Autographa californica nuclear polyhedrosis virus are competent for AcMNPV-dependent DNA replication.

We previously reported on the generation of approximately 50-kb size defective genomes (DGs) which appeared to retain less than 2.2% of the standard Autographa californica nuclear polyhedrosis virus (AcMNPV) DNA between 85.0 and 87.2 MU while the rest of the virus DNA had been largely deleted (Lee and Krell, J. Virol., 66:4339-4347, 1992). To investigate these presumably repeated sequences further, we cloned and analyzed the most abundant hypermolar 1.80-kb Xhol DNA fragment as well as a minor but also supermolar 1.74-kb Xhol fragment of the DGs. These two DNA segments collectively covered 2371-bp of the standard AcMNPV DNA with a 1174-bp overlap around the Xhol site at 85.9 MU. Analysis of DGs by two-dimensional gel electrophoresis indicated that the 1.80- and 1.74-kb Xhol fragments (and most other novel Xhol fragments of the DG) were organized as tandem repeats in the DGs. We identified, in the DG population, small supercoiled DNA molecules approximately 1.0- to 8.2-kb (and possibly up to around 50 kb, the size of the major defective DNA species) in size and which contained the same DNA sequence as that of the major 1.80-kb repeat in the DGs. Furthermore, these cloned repeat sequences, represented by pLK1.80 and pLK1.74 showed AcMNPV infection-dependent autonomous replication, suggesting that an origin of DNA replication might reside within the HindIII to EcoRI segment (85.1 to 86.6 MU) of the HindIII-K fragment.

Animals↗
PubMed1992

Oxidized low density lipoproteins elicit DNA fragmentation of cultured lymphoblastoid cells.

Lymphoblastoid cell lines continuously pulsed with mildly oxidized low density lipoproteins, exhibited a significant increase of DNA fragmentation induced by oxidized LDL internalized by cells. DNA fragmentation was associated with an increasing number of morphologically characteristic apoptotic cells simultaneously with the increase of cytotoxicity indexes, and the activation of the poly(ADP-ribose) polymerase, a nuclear enzyme stimulated by DNA strand breaks. The potential involvement of these biochemical and morphological changes in atherogenesis is discussed.

B-Lymphocytes↗
PubMed1985

Characterization of DNA fragments encoding fimbriae of the uropathogenic Escherichia coli strain KS71.

Recombinant plasmids were constructed that expressed the KS71A, KS71B and KS71C fimbrial antigens of the pyelonephritogenic Escherichia coli strain KS71 (O4:K12) in E. coli HB101. The KS71C-encoding genes were located on a 6.4 kb HindIII-XhoI fragment obtained from the recombinant cosmid pKTH145 that expresses this antigen. Spontaneous KS71C-mutants were isolated that contained a 0.8 kb insert in a specific restriction fragment of KS71C-encoding recombinant plasmids. The KS71B-encoding segment was located on a 11.5 kb deletable DNA fragment of recombinant cosmid pKTH144. A DNA fragment encoding the KS71A fimbria was obtained on a 12 kb EcoRI fragment of the recombinant cosmid expressing this antigen in E. coli HB101 and closely resembled the KS71B-encoding fragment. In the recombinant cosmid, the KS71B-expressing region was flanked by homologous DNA segments. A similar stretch of DNA was found close to the KS71A-expressing DNA region.

Cloning, Molecular↗
PubMed1995

Purification and cloning of DNA fragments fractionated on agarose gels.

Purification of DNA fragments from acrylamide or agarose gels is a commonly used technique in the molecular biology laboratory. This article describes a rapid, efficient, and inexpensive method of purifying DNA fractions from an agarose gel. The purified DNA is suitable for use in a wide range of applications including ligation using DNA ligase. The procedure uses standard high-melting-temperature agarose and normal TBE electrophoresis buffer. In addition, the protocol does not involve the use of highly toxic organic solvents such as phenol.

Cloning, Molecular↗
PubMed1993

Isolation of specific DNA fragments of Mycobacterium avium and their possible use in diagnosis.

We cloned and sequenced two DNA fragments (DT1 and DT6) from Mycobacterium avium serotype 2 for use in the identification of members of the M. avium-M. intracellulare complex (MAC). Reference strains of MAC belonging to serovars 1 to 28 were examined by using these DNA fragments as probes. The study revealed that the DT6 probe hybridized with DNAs from M. avium strains (serovars 1 to 6, 8 to 11, and 21), while the DT1 probe hybridized with DNAs from serovars 2, 3, 7, 12 to 20, and 23 to 25. DT1- and DT6-derived oligonucleotides were selected for use as primers in a polymerase chain reaction test. Amplification of the DT1 and DT6 sequences may provide the basis for a rapid and reliable assay for the detection of mycobacteria belonging to MAC.

Animals↗