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PubMed2005

Effects of N-acylethanolamines and various antimitotic agents on apoptotic DNA fragmentation in conventionally normal and tumor tissue of human adrenals.

AIM: To study effects of N-acylethanolamines (NAE) and various antimitotic agents: taxol, colchicine, and cytochalasin B on the DNA fragmentation extent in conventionally normal (CNT) and tumor tissue of human adrenal cortex. METHODS: Six types of adrenal tumor tissue of 84 patients were analyzed. Slices of tissue was incubated at 37 degrees C for 3 h in the presence of NAEs and antimitotics, DNA was extracted and analysed in agarose gel. RESULTS: It was established that NAEs enhanced apoptosis in conventionally normal and tumor tissue of adrenal glands. In general, tumor tissue was more sensitive to NAEs and antimitotic compounds than conventionally normal tissue. NAEs in combination with colchicine and cytochalasin B enhanced DNA fragmentation in some types of tumor tissue and did not influence, or even reduced it in CNT. Taxol revealed selective action depending on tumor tissue type. Considerable individual differences were reported in sensitiveness of different types of tumors to the NAEs and antimitotic agents. CONCLUSIONS: Taxol and combination of NAEs with colchicine and cytochalasin B are inductors of apoptosis in the adrenal tumor cells and thus promising for further investigation.

Adrenal Gland Neoplasms↗
PubMed1997

Etoposide-induced PC12 cell death: apoptotic morphology without oligonucleosomal DNA fragmentation or dependency upon de novo protein synthesis.

Etoposide, a topoisomerase II inhibitor used in cancer therapy, has been shown to induce apoptosis in vitro in a variety of cell types. In the present study, we have characterized the effects of etoposide on undifferentiated rat pheochromocytoma PC12 cells. Etoposide killed PC12 cells in a time- and concentration-dependent manner. 20-24 h incubation with 10 micrograms/ml etoposide induced 25-50% cell death. Hoechst 33258 staining revealed apoptotic morphology in dying cells. No evidence was found of either oligonucleosomal DNA fragmentation, as shown by agarose gel electrophoresis, or endonuclease involvement, as shown by the inability of aurintricarboxylic acid to prevent cell death. Cycloheximide and actinomycin-D were unable to prevent etoposide cytotoxicity indicating that the process is not dependent upon de novo protein or mRNA synthesis. NGF (5 ng/ml) prevented etoposide-induced PC12 cell death. These results offer an example of how the morphological features of apoptosis are not necessarily associated with oligonucleosomal DNA fragmentation or with de novo macromolecule synthesis.

Analysis of Variance↗
PubMed2002

Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm.

BACKGROUND: Sperm cell death appears to be a cause of male infertility. The objective of this study was to determine the most reliable method for the evaluation of sperm quality in semen samples during sperm preparation for IVF. METHODS: Conventional analysis of semen samples was compared with several cytofluorometric methods detecting death-associated changes. Neat semen from infertile patients and sperm prepared by PureSperm gradient were studied by conventional microscopy and analysed for mitochondrial membrane potential (Delta Psi(m)), generation of reactive oxygen species, DNA fragmentation and cell viability. RESULTS: In neat semen, a positive correlation was found between the percentage of Delta Psi(m)(high) sperm cells and standard semen parameters (concentration/motility). Sperm cells depicting Delta Psi(m)(high) and cells with low DNA fragmentation displayed high fertilization rate after IVF. The only changes that could be detected in prepared sperm were changes in Delta Psi(m), with Delta Psi(m)(high) sperm positively correlated with forward motility and also with high fertilization rates after IVF. CONCLUSION: Analysis of mitochondrial membrane potential is the most sensitive test by which to determine sperm quality. These findings promise development of a test that may help to predict successful IVF.

Adult↗
PubMed1988

Single-stranded DNA fragments holding unusual conformation.

In the chemical synthesis of DNA, we found that the single-stranded DNA (ssDNA) fragments containing the sequence GCGAAAGC showed higher mobilities than the fragments without this sequence on a denaturing polyacrylamide gel electrophoresis. Physical structure of these DNA fragments was studied by enzyme digestion and optical analysis. The abnormal mobilities on electrophoresis seem to depend on an unusual conformation.

Base Sequence↗
PubMed1996

Intracellular metabolism of Ara-C and resulting DNA fragmentation and apoptosis of human AML HL-60 cells possessing disparate levels of Bcl-2 protein.

We examined the effects of high intracellular levels of Bcl-2 on the metabolism and DNA incorporation of high-dose Ara-C (HIDAC) as well as on Ara-C-induced DNA strand breaks and apoptosis of human AML HL-60 cells. HL-60/Bcl-2 and HL-60/neo cells were created by retrovirally transfecting the human AML HL-60 cells with the pZip-bcl-2 and pZip-neo plasmids, respectively. As compared to HL-60/neo, HL-60/Bcl-2 cells contained significantly higher (approximately 10-fold) p26Bcl-2, but equivalent levels of Bax and undetectable levels of Bcl-xL. HIDAC (10 or 100 microM for 4 h) produced the kilobase size and internucleosomal DNA fragmentation associated with apoptosis in HL-60/neo but not in HL-60/Bcl-2 cells. Significantly greater loss of survival (by MTT assay) and flowcytometric and morphologically recognizable apoptosis were observed in HL-60/neo cells. HIDAC did not affect Bcl-2 levels in either cell type. The intracellular accumulation of Ara-CTP relative to dCTP, Ara-C DNA incorporation and Ara-C-induced early DNA damage in the form of strand breaks (detected by alkaline elution assay) were not significantly different between HL-60/Bcl-2 and HL-60/neo cells. In addition, HIDAC treatment caused similar DNA synthesis inhibition in the two cell types. These results indicate that high intracellular levels of Bcl-2 operate distally to inhibit the final apototic cell death pathway by preventing the conversion of HIDAC-induced early DNA damage into lethal DNA fragmentation associated with apoptosis.

Antimetabolites, Antineoplastic↗
PubMed1987

Identification of 28 DNA fragments that detect RFLPs in 13 distinct physical regions of the short arm of chromosome 5.

A series of 175 lambda phage carrying human inserts isolated from a library that is specific for the short arm of human chromosome 5 (5p) have been regionally mapped on 5p using a deletion mapping panel of 16 human-hamster cell hybrids, each of which contains a chromosome 5 with a different deletion in the short arm. Seventy-five single copy DNA fragments were screened with 12 restriction enzymes for their ability to detect restriction fragment length polymorphisms (RFLPs). Twenty-eight of these DNA fragments, which are located in 13 distinct physical regions of 5p, were found to detect RFLPs. These DNA markers make it possible to construct a linkage map that will span the entire length of 5p and will allow the relationship between genetic and physical distance for this region of the genome to be examined at a high level of resolution.

Chromosome Mapping↗
PubMed1993

A ribosomal DNA fragment of Listeria monocytogenes and its use as a genus-specific probe in an aqueous-phase hybridization assay.

cDNAs were prepared from the total RNA of Listeria monocytogenes ATCC 19118 and used as probes to screen a genomic library of the same strain. Four clones were identified which contained ribosomal DNA fragments. Recombinant DNA from one of them was fractionated and differentially hybridized with the cDNA probes to RNA of L. monocytogenes and Kurthia zopfii. The resulting hybridization pattern revealed an HpaII fragment of 0.8 kb that was specific for the L. monocytogenes strain. The nucleotide sequence of this fragment showed 159 bases of the 3' end of the 16S rRNA gene, 243 bases of the spacer region, and 382 bases of the 5' end of the 23S rRNA gene. In dot blot hybridization assays, the 32P-labeled 784-bp fragment was specific only for Listeria species. Dot blot assays revealed that the 32P-labeled fragment can easily detect > or = 10 pg of total nucleic acids from pure cultures of L. monocytogenes, which corresponds to approximately 300 bacteria. This fragment was also used as a probe in an assay named the heteroduplex nucleic acid (HNA) enzyme-linked immunosorbent assay. In this system, the biotinylated DNA probe is hybridized in the aqueous phase with target RNA molecules and then specific HNAs are captured by HNA-specific antibodies. Captured HNA molecules are revealed with an enzyme conjugate of streptavidin. In a preliminary HNA enzyme-linked immunosorbent assay, the 784-bp fragment maintained its specificity for Listeria spp. and could detect 5 x 10(2) cells in artificially contaminated meat homogenate.

Base Sequence↗
PubMedDate not supplied

Age does not influence DNA fragmentation in the hippocampus after fatal traumatic brain injury in young and aged humans compared with controls.

Paraffin sections from the hippocampus of 12 head-injured patients (Group A, aged between 4 and 12 years n = 6 and Group B, aged between 64 and 89 years n = 6) and associated age-matched controls were stained by the terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) technique for evidence of in-situ DNA fragmentation. TUNEL+ cells were of 2 Types: I (non-apoptotic) and II (apoptotic). In addition sections stained H&E, combined Luxol Fast Blue/Cresyl Violet and by immunohistochemistry for astrocytes (GFAP) and macrophages (CD68) were used to characterize the lesions. Small numbers of Type I TUNEL+ cells were seen in all sectors of the hippocampus except CA2 of both Groups A and B. Type II TUNEL+ cells were mainly found in the white matter. They constituted less than 1% of all TUNEL+ cells. There were similar or fewer TUNEL+ cells in the corresponding areas in the controls compared with the head-injured patients. However, in the dentate fascia and the CA4 sector of the Group B cases, larger numbers of TUNEL+ cells were seen in controls than after trauma. In the grey matter most TUNEL+ cells had the morphology ofnecrosis that corresponded with foci of selective neuronal damage. Only a few TUNEL+ cells were seen in white matter. The occasional Type I TUNEL+ cells were seen in grey matter. It is concluded that the amount and distribution of DNA fragmentation in children and adults is similar and therefore at least in the hippocampus does not provide an explanation for age as an independent variable of outcome after traumatic brain injury in childhood.

Age Factors↗
PubMed1992

Capillary gel electrophoresis of single-stranded DNA fragments with UV detection.

Capillary gel electrophoresis has proven to be a powerful tool in biomedical research. We report our investigation of some of the critical parameters affecting separations of single-stranded DNA fragments as monitored by ultraviolet (UV) absorbance detection. Although not as sensitive as laser-induced fluorescence (LIF), UV absorbance detection allows one to calculate quite accurately, and inexpensively, the molarity of each separated DNA fragment and, moreover, the signal "fading" effect normally observed with LIF detection can be, in many cases, substituted for fluorescence to detect the many different single-stranded DNAs, as well as for detection of sequencing reactions.

Coliphages↗
PubMed1994

Cell nucleus and DNA fragmentation are not required for apoptosis.

Apoptosis is the predominant form of cell death and occurs under a variety of physiological and pathological conditions. Cells undergoing apoptotic cell death reveal a characteristic sequence of cytological alterations including membrane blebbing and nuclear and cytoplasmic condensation. Activation of an endonuclease which cleaves genomic DNA into internucleosomal DNA fragments is considered to be the hallmark of apoptosis. However, no clear evidence exists that DNA degradation plays a primary and causative role in apoptotic cell death. Here we show that cells enucleated with cytochalasin B still undergo apoptosis induced either by treatment with menadione, an oxidant quinone compound, or by triggering APO-1/Fas, a cell surface molecule involved in physiological cell death. Incubation of enucleated cells with the agonistic monoclonal anti-APO-1 antibody revealed the key morphological features of apoptosis. Moreover, in non-enucleated cells inhibitors of endonuclease blocked DNA fragmentation, but not cell death induced by anti-APO-1. These data suggest that DNA degradation and nuclear signaling are not required for induction of apoptotic cell death.

Animals↗
PubMed1982

A Dictyostelium discoideum DNA fragment complements a Saccharomyces cerevisiae ura3 mutant.

Dictyostelium discoideum DNA fragments have been inserted into the chimeric bacterium-yeast plasmid YEp13. Recombinant plasmids were used to transform yeast using a strain of Saccharomyces cerevisiae deficient in OMP decarboxylase activity. Several clones were selected for growth in uracil-free medium. One clone was further analysed and contains a plasmid with a segment of D. discoideum DNA which complements a yeast ura3 mutation.

DNA, Fungal↗
PubMed1997

Frequency and distribution of DNA fragmentation as a marker of cell death in chronic liver diseases.

To study the early stages of cell death in various types of chronic liver injury, liver biopsies from a total of 26 patients, including 7 with chronic hepatitis C(CHC), 4 with chronic hepatitis B(CHB), 7 with alcoholic liver disease (ALD), 4 with autoimmune or drug hepatitis (AI/DH), and 4 with primary biliary cirrhosis(PBC), were examined by an in situ nucleotidyl transferase assay (ISNTA), which detects DNA fragmentation. Positive nuclei in hepatocytes and sinusoidal lining cells were counted in all parenchymal areas, excluding triads and areas of fibrosis, using a computer with Sigmascan software. The number of positive hepatocytes/mm2 was similar in the biopsies of patients with CHC, CHB, ALD and AI/DH, but significantly lower in PBC. The number of positive sinusoidal lining cells/mm2 was significantly greater in biopsies with CHC compared to CHB, ALD, AI/DH and PBC. Double staining revealed that the ISNTA-positive sinusoidal lining cells were also CD68 positive, indicating that they were Kupffer cells. The frequency of ISNTA positivity did not correlate with serum AST or ALT levels, steatosis, cell swelling or cirrhosis. ISNTA-positive hepatocytes were more frequent than acidophilic bodies in every disease category. We conclude that apoptosis may be a common pathway of cell death in different liver diseases, that the high frequency of DNA fragmentation in Kupffer cells in CHC suggests that during chronic hepatitis C infection activated Kupffer cells may be subject to regulatory control by apoptosis and that ISNTA is more sensitive than acidophilic bodies in assessing the degree of cell injury in the liver.

Adult↗
PubMed2001

Damaged myocytes as detected by the colocalization of DNA fragmentation and tissue transglutaminase and their prognostic significance in enterovirus-associated dilated cardiomyopathy.

BACKGROUND: Apoptotic cardiac myocytes have been described in chronic heart failure, but no data exist on the relationship between these 'damaged' myocytes and myocardial detection of enterovirus RNA often associated with dilated cardiomyopathy (DCM). DESIGN: In patients with idiopathic DCM, endomyocardial biopsy samples were studied for enteroviral RNA by one step reverse transcription-polymerase chain reaction (PCR) and a subsequent hybridization of the PCR product using a Southern blot technique. The endomyocardial biopsies were further investigated for markers of cell damage and apoptosis: DNA fragmentation and expression of tissue-transglutaminase (TTG) in the myocytes using the in-situ endlabelling method or an anti-TTG-staining, respectively. To assess the prognostic significance of these two markers the correlation between the percentage of myocytes positive both for DNA fragmentation and TTG (the index of damaged myocytes) and the hemodynamic course of the patients during a mean follow-up period of 15.9 +/- 6.2 months was investigated prospectively by echocardiography. RESULTS: In 14 (45%) of the 31 patients with idiopathic DCM, enteroviral RNA was found in the endomyocardial biopsy samples, while 17 patients (55%) were enterovirus-negative. In enterovirus-positive patients, the index of 'damaged' myocytes was significantly lower (10.7 +/- 4.9% vs. 19.2 +/- 8.8%, P = 0.002) and the left ventricular ejection fraction (LVEF) improved significantly (P = 0.00017 vs. P = 0.13) during long-term follow-up. In addition, a weak negative correlation was seen between the index of damaged myocytes and the changes in LVEF in all patients during long-term follow-up (r = - 0.48, P = 0.004). CONCLUSION: Our results favour the view that enterovirus-positive patients with DCM have less damaged myocytes and a better haemodynamic course than enterovirus-negative patients.

Adult↗
PubMed1983

Transfer of small plasmid DNA fragments from polyacrylamide gels onto nitrocellulose paper.

The incorporation into a 7% polyacrylamide gel of a nucleic acid-specific photochemical reagent 4,5',8-trimethylpsoralen at a concentration of 1 mg/dl of acrylamide solution improves both the sensitivity and the efficiency of the transfer of plasmid DNA fragments from the gel onto nitrocellulose filters. This improvement allows detection by blot hybridization of DNA fragments as small as 57 bp long.

Collodion↗
PubMed1990

Synthesis of well-defined phosphate-methylated DNA fragments: the application of potassium carbonate in methanol as deprotecting reagent.

A new deprotection procedure in the synthesis of (partially) phosphate-methylated oligodeoxynucleotides has been developed, involving treatment of fully protected DNA fragments with methanolic potassium carbonate. It is shown that base deprotection can be accomplished in potassium carbonate/methanol without affecting the methyl phosphotriesters. This methodology enables us to synthesize, both in solution and on a solid support, DNA fragments which are phosphate-methylated at defined positions. The solid phase synthesis, however, turns out to be accompanied by considerable demethylation of the phosphotriesters. It is demonstrated that this demethylation does not occur during the deprotection or work-up procedure. Furthermore, it was found that the latter side-reaction is suppressed when the standard capping procedure with acetic anhydride is included.

Carbonates↗
PubMed1995

Two-dimensional DNA typing: A cost-effective way of analyzing complex mixtures of DNA fragments for sequence variations.

By two-dimensional (2D) DNA typing, multiple DNA fragments can be analyzed in parallel for all possible sequence variations. The method involves electrophoretic separation in polyacrylamide gels, first by size and subsequently on the basis of basepair sequence in a denaturing gradient. The system has been automated partly and a dedicated image analysis system for rapid interpretation of the often complex spot patterns was developed. In this review, the basic principles of 2D DNA typing, its methodology, and some major applications in genetic studies of animals, plants, and microorganisms will be discussed, with the emphasis on human genetics.

Cost-Benefit Analysis↗