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At least 379 records · Page 21Linked to original sources
PubMed2003

Sperm DNA fragmentation decreases the pregnancy rate in an assisted reproductive technique.

BACKGROUND: Standard sperm characteristics are poor predictors of the outcome of IVF treatments. On the contrary, sperm genome quality has been emphasized for several years as playing a major role in early embryogenesis, thus in the success of IVF attempt. METHODS: Sperm DNA fragmentation from a selected group of 104 couples undergoing assisted reproductive techniques (ART) (IVF: n = 50; and ICSI: n = 54) was measured by TUNEL assay and correlated with semen and ART outcomes. RESULTS: A negative correlation was found between sperm characteristics and the proportion of sperm showing DNA fragmentation. For fragmentation >10%, a significant decrease of the fertilization rate was observed. No correlation was found between sperm DNA fragmentation and embryo quality. A high proportion of sperm with fragmented DNA was a pejorative factor to obtain pregnancies when ICSI was performed, but there was no relationship when conventional IVF was performed. CONCLUSIONS: The proportion of sperm with DNA fragmentation appears to be potentially useful as a predictor of ICSI outcome, whereas embryo quality based on morphological criteria, appeared unaffected by DNA fragmentation.

DNA Fragmentation↗
PubMed2001

A simple comet assay for archived sperm correlates DNA fragmentation to reduced hyperactivation and penetration of zona-free hamster oocytes.

OBJECTIVE: To correlate sperm variables with sperm DNA fragmentation, as assessed by using a modified alkaline comet assay for sperm smears. DESIGN: The comet assay was adapted for fixed sperm smears (59 cases), and the level of DNA fragmentation was determined. SETTING: Clinical and academic research environment. PATIENT(S): 59 patients undergoing fertility treatment. INTERVENTION(S): Sperm samples leftover from IVF procedures were fixed and processed for the comet assay. MAIN OUTCOME MEASURE(S): Sperm head DNA density and sperm variables. RESULT(S): A correlation was observed between increased sperm head DNA fragmentation and decreased penetration of zona-free hamster oocytes. Heat-induced hyperactive motility decreased as DNA fragmentation increased. The DNA fragmentation did not correlate with percentages of intact acrosome, normality, maturity, and strict normal morphology. CONCLUSION(S): The advantages of the comet assay for archived cells include simplicity, low intraassay coefficient of variation, and low performance cost; in addition, DNA analysis can be carried out at leisure. Low DNA damage was associated with higher hyperactivation and oocyte penetration, suggesting that failed fertilization was linked to compromised DNA integrity in the sperm. Exploration of compounds to repair damaged DNA is warranted.

Acridine Orange↗
PubMed2006

DNA fragmentation in chronic glomerulonephritis: an immunohistological analysis.

BACKGROUND: Experimental data suggest that apoptosis plays an important pathophysiological role in glomerulonephritis by restoring tissue structure after proliferation of intrinsic renal cells and infiltration of leukocytes. Relatively little is known of apoptosis in human glomerulonephritis, particularly in predicting renal function during follow-up. METHODS: In order to colocalize different markers for cell damage in renal tissue from patients with different forms of glomerulonephritis (GN), a series of semithin sections from 34 kidney biopsies were studied retrospectively. Normal kidney from a nephrectomy specimen with a small renal adenocarcinoma served as a control. DNA fragmentation, expression of tissue transglutaminase II, BAX and BCL-2 were visualized immunohistochemically. In some renal biopsies, immunohistochemical staining for activated caspase 3 was performed. Proinflammatory markers (C-reactive protein, leukocytes), serum creatinine, creatinine clearance, total proteinuria, albuminuria, alpha(1)-microglobulin and IgG excretion were determined at the time of biopsy. Serum creatinine and total proteinuria were assessed 6 and 12 months after renal biopsy. RESULTS: Nuclei with different degrees of DNA fragmentation were mainly found in epithelial cells of tubules, but also in glomerular cells, regardless of the form of GN studied. Transglutaminase II expression was found only in cells with a strong staining for DNA fragmentation. DNA fragmentation localized to glomerular cells was more pronounced in proliferative than in non-proliferative forms of GN, being most abundant in patients with rapid progressive GN. Staining for activated caspase 3 in selected biopsies confirmed the presence of apoptosis. BAX and BCL-2 staining was detected within the same cells, but exhibited a different intracellular distribution. In proliferative GN, the extent of DNA damage in tubular epithelial cells significantly corresponds with the concentration of serum creatinine (p < 0.04) and with urinary excretion of alpha(1)-microglobulin (p < 0.01) at the time of biopsy. A significant correlation (p < 0.01) was seen between glomerular DNA fragmentation and follow-up total proteinuria 12 months after biopsy for proliferative forms of GN. The damaged glomerular area (e.g. mesangial sclerosis) significantly correlated with DNA fragmentation in proliferative, but not in nonproliferative GN at the time of biopsy. Furthermore, glomerular damaged showed a significant correlation with tubular DNA damage in proliferative GN. CONCLUSION: In glomerular cells, apoptosis may be important for the clearance of proliferating cells whereas in tubules, cell damage showed dependence on the degree of tubular injury mediated by inflammation and/or proteinuria. Although the degree of apoptosis in tubular cells correlates with serum creatinine in proliferative GN at the time of biopsy, it is of limited use to predict future renal function.

Adult↗
PubMed2000

Analysis of the chicken DNA fragments that contain structural sites of attachment to the nuclear matrix: DNA-matrix interactions and replication.

Ten short DNA fragments have been selected from a library of the nuclear matrix-attached DNA (nmDNA) from chicken erythrocytes by their ability to hybridize with the fraction of chicken replication origins isolated by nascent DNA strand extrusion. The primary structure of these fragments has been determined. Five of the sequences contained a topoisomerase II recognition site. Most of the studied DNA fragments also have a common eight-nucleotide motif, GCAGACCG/A. A sequence-specific DNA-binding protein with a MW of 55 kDa that interacted with this motif has been identified. Some of the cloned DNA fragments promoted an increased level of transient plasmid replication in transfected chicken cells. The ability of plasmid bearing nmDNA fragments to replicate correlated directly with their ability to target plasmids to the nuclear matrix compartment.

Animals↗
PubMed1999

Inhibition of hepatic cell nuclear DNA fragmentation by zinc in carbon tetrachloride-treated rats.

BACKGROUND/AIMS: The aims of this study were to ascertain: 1) whether hepatic cell DNA fragmentation is increased in rats with early stages of liver disease induced by carbon tetrachloride; 2) whether the inhibition of DNA cleavage is involved in the hepatoprotective effects of zinc; and 3) if relationships exist between DNA fragmentation and the onset of fibrosis in this experimental model. METHODS: Twenty-one treated rats and 23 controls were divided into two groups to receive either a standard diet or one supplemented with zinc. All the animals were sacrificed 1 week later for histological and biochemical assessments, which included a DNA fragmentation index, hepatic zinc and metallothionein concentrations, fibrosis measured by hepatic hydroxyproline concentration and plasma alanine aminotransferase activity. RESULTS: Hepatic cell DNA fragmentation was increased in rats with early hepatic fibrosis and the increase was independent of hepatocytolysis, as measured by alanine aminotransferase activity. Oral zinc administration inhibited hepatic cell DNA fragmentation in the treated rats and was proportional to the hepatic concentration of the metal. The mechanism of the zinc-mediated decrease in DNA cleavage was related to an increase in the hepatic metallothionein concentration. Hepatic cell DNA fragmentation was related to hydroxyproline concentration. CONCLUSIONS: Our results suggest that apoptosis may be involved in the early transformations occurring in the liver and which can lead to the initiation of cirrhosis. As such, the potential therapeutic use of zinc supplementation would warrant further investigation.

Animals↗
PubMed1983

Operational variables in high-performance gel filtration of DNA fragments and RNAs.

Double-stranded DNA fragments and ribosomal and transfer RNAs were measured by high-performance gel filtration on TSK-GEL G2000SW, G3000SW, G4000SW and G5000PW columns to investigate the separation range and resolution of these columns and the effects of eluent ionic strength and flow-rate on retention and resolution. These columns could separate double-stranded DNA fragments up to ca 1 X 10(6) and rRNAs up to ca 5 X 10(6) daltons in molecular weight. However, it was found that the selection of the column is very important to achieve optimum separation, depending on the molecular weight of the sample. Elution is delayed as the eluent ionic strength is increased. An eluent ionic strength of 0.3-0.5 seemed appropriate in most cases. Resolution is greatly increased as the flow-rate is decreased.

Chromatography, Gel↗
PubMed2004

Increased DNA fragmentation and ultrastructural changes in fibromyalgic muscle fibres.

OBJECTIVE: To determine whether there is evidence of increased DNA fragmentation and ultrastructural changes in muscle tissue of patients with fibromyalgia (FM) compared with healthy controls. METHODS: Muscle tissues from 10 community residents with FM and 10 age and sex matched healthy controls were examined "blindly" for the presence of DNA fragmentation by two different methods: terminal deoxynucleotidyl transferase (TdT) staining (TUNEL) and the FragEL-Klenow DNA fragmentation detection kit. Ultrastructural analysis of tissue was performed by electron microscopy. RESULTS: DNA fragmentation was detected by both methods in 55.4 (SEM 2.5)% of the nuclei in muscle tissue of patients with FM compared with 16.1 (4.1)% (p<0.001) of the nuclei in healthy controls. Contrary to expectation, no typical features of apoptosis could be detected by electron microscopy. The myofibres and actin filaments were disorganised and lipofuscin bodies were seen; glycogen and lipid accumulation were also found. The number of mitochondria was significantly lower in patients with FM than in controls and seemed to be morphologically altered. CONCLUSION: The ultrastructural changes described suggest that patients with FM are characterised by abnormalities in muscle tissue that include increased DNA fragmentation and changes in the number and size of mitochondria. These cellular changes are not signs of apoptosis. Persistent focal contractions in muscle may contribute to ultrastructural tissue abnormalities as well as to the induction and/or chronicity of nociceptive transmission from muscle to the central nervous system.

Adult↗
PubMed2005

Correction of frameshift mutations with single-stranded and double-stranded DNA fragments prepared from phagemid/plasmid DNAs.

We recently found that a heat-denatured, double-stranded DNA fragment, prepared from plasmid DNA (dsHES), and a sense single-stranded DNA fragment, prepared from single-stranded phagemid DNA (fSense), corrected an inactivated hygromycin-resistance and enhanced green fluorescence protein fusion (Hyg-EGFP) gene containing a base substitution (G:C to C:G) mutation 2-fold and more than 10-fold, respectively, more efficiently than the conventional PCR fragment (pcrHES), in the small fragment homologous replacement method. In this study, we tested the abilities of these new DNA fragments to correct Hyg-EGFP genes inactivated by one base insertion (+G) and deletion (-C) mutations. In contrast to its activity with the substitution mutation, the fSense fragment showed similar efficiencies to those of the dsHES fragment in the correction of frameshift mutations. For the correction of the insertion mutation, the efficiencies were in the order of dsHES (0.21%)>or=fSense (0.18%)>pcrHES (0.08%). In the case of the correction of the deletion mutation, the efficiencies were in the order of fSense (0.27%)>or=dsHES (0.19%)>pcrHES (0.12%). These results suggest that sense single- and double-stranded DNA fragments prepared from phagemid and plasmid DNAs, respectively, have the potential to correct frameshift mutations.

Animals↗
PubMedDate not supplied

1-beta-D-arabinosylcytosine and 5-azacytidine induce internucleosomal DNA fragmentation and cell death in thymocytes.

Incubation of mouse thymocytes with arabinosylcytosine or 5-azacytidine induced dose-dependent internucleosomal DNA cleavage followed by cell death. This process was RNA- and protein synthesis-dependent, since DNA fragmentation and cell death was inhibited by actinomycin D and cycloheximide. The results suggest that the cytidine analogs induce apoptosis, a programmed cell death, in thymocytes. The DNA cleavage induced by arabinosylcytosine and 5-azacytidine was inhibited by deoxycytidine and cytidine, respectively, suggesting that phosphorylation of these antimetabolites is required to induce DNA cleavage. DNA fragmentation was unaffected by the addition of aphidicolin or 3-aminobenzamide, indicating that DNA cleavage is not due to the inhibition of DNA synthesis or repair. Other antimetabolites, including methotrexate, fluoropyrimidines and thiopurines, failed to induce DNA fragmentation. Arabinosylguanine induced DNA fragmentation similar to that produced by the cytidine analogs, suggesting similarity to the selective sensitivity of T lymphocytes to deoxyguanosine toxicity. The precise mechanism by which DNA cleavage is induced remains unclear, but the present study shows that certain antimetabolites act on cells not only by inhibiting proliferation, but by inducing apoptosis with internucleosomal DNA fragmentation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMedDate not supplied

1-beta-D-arabinosylcytosine and 5-azacytidine induce internucleosomal DNA fragmentation and cell death in thymocytes.

Incubation of mouse thymocytes with arabinosylcytosine or 5-azacytidine induced dose-dependent internucleosomal DNA cleavage followed by cell death. This process was RNA and protein synthesis-dependent, since DNA fragmentation and cell death was inhibited by actinomycin D and cycloheximide. The results suggest that the cytidine analogs induce apoptosis, a programmed cell death, in thymocytes. The DNA cleavage induced by arabinosylcytosine and 5-azacytidine was inhibited by deoxycytidine and cytidine, respectively, suggesting that phosphorylation of these antimetabolites is required to induce DNA cleavage. DNA fragmentation was unaffected by the addition of aphidicolin or 3-aminobenzamide, indicating that DNA cleavage is not due to the inhibition of DNA synthesis or repair. Other antimetabolites including methotrexate, fluoropyrimidines and thiopurines failed to induce DNA fragmentation. Arabinosylguanine induced DNA fragmentation similar to that produced by the cytidine analogs, suggesting similarity to the selective sensitivity of T lymphocytes to deoxyguanosine toxicity. The precise mechanism by which DNA cleavage is induced remains unclear, but the present study shows that certain antimetabolites act on cells not only by inhibiting proliferation, but by inducing apoptosis with internucleosomal DNA fragmentation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed2005

Longitudinal study of sperm DNA fragmentation as measured by terminal uridine nick end-labelling assay.

BACKGROUND: One major limitation in the use of sperm DNA fragmentation as measured by the TdT (terminal deoxynucleotidyl transferase)-mediated dUTP nick-end labelling (TUNEL) assay is the paucity of solid data on the stability of this parameter. METHODS: The objective of our study was to evaluate variations in the degree of sperm DNA fragmentation, as measured by the TUNEL assay, over a 6 month period. Five donors provided semen samples (total 107) on the average three times per month, and 10 infertility patients provided semen samples every 4 weeks (total 58). RESULTS: The mean percentage of sperm DNA fragmentation for donors was 13.18%, the within-donor standard deviation (SD(W) = 3.79%) was small compared to between-donor (SD(B) = 17.56%). For the group of patients, the mean percentage of sperm DNA fragmentation was 22.44%, with SD(W) of 4.43% within patients and SD(B) of 29.48% between patients. No seasonal rhythm was observed during the study. The intra-class correlation coefficient for all subjects combined was 0.83. Compared to sperm concentration, individual coefficients of variation for sperm DNA fragmentation indicated less variability in four subjects, but were similar in the others. CONCLUSION: This longitudinal study shows that sperm DNA fragmentation is a parameter with good stability (repeatability) over time; it can be taken as a baseline both in healthy fertile men and in patients from infertility couples.

Adult↗
PubMed1998

A study of DNA fragmentation patterns in cells irradiated with charged particles: evidence for non-random distributions.

Many studies have shown that the effectiveness of radiations of varying LET is similar when yields of dsb have been measured, despite large differences in biological response. Recent evidence has suggested however, that current techniques underestimate the yields of dsb. By monitoring the fragmentation of DNA over a wide range of fragment sizes (< 10 kbp > 6 Mbp) by pulsed field electrophoresis, RBE values greater than 1.0 for radiations of around 100 keV/mm have been determined. The data provide evidence for the production of correlated breaks produced within cells as particle tracks traverse the nucleus. The highly ordered structure of DNA within mammalian cells may lead to clustering of breaks over distances related to the repeating unit structures of the chromatin. As well as these regionally damaged sites, a major contributor to radiation effectiveness will be the localised clustering of damage in the 1-20 bp region. A major effort is required to elucidate the relative importance of these levels of clustering and their importance in biological response.

Alpha Particles↗
PubMed1995

Cadmium-induced DNA fragmentation is inhibitable by zinc in porcine kidney LLC-PK1 cells.

DNA fragmentation was induced by the addition of cadmium (10 microM) to cultured LLC-PK1 cells, resulting in cell death. The cells were able to survive exposures of 10 microM cadmium without change in morphology, but most had rounded by 40 microM. Other metals tested such as Cu2+, Co2+, Ni2+, and Pb2+ had much lower ability to induce DNA fragmentation in LLC-PK1 cells under the same conditions as used for cadmium, indicating that the fragmentation seen in kidney cells is cadmium-specific. DNA fragmentation induced by 10 microM cadmium was completely inhibited using an equimolar concentration of a cell-permeant chelator specific for heavy metals, N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine. Furthermore, the apoptotic mechanism evoked by 10 microM cadmium was blocked by 50 microM zinc which when used alone has no capacity for DNA fragmentation.

Animals↗
PubMed1994

T-cassette ligation: a method for direct sequencing and cloning of PCR-amplified DNA fragments.

We describe a method to ligate a PCR-amplified DNA fragment with T-protruding cassettes, which have multiple sites for endonuclease, promoter sequences of T3 and T7, and annealing sites for the universal M13 forward and reverse primers. This method, which we named T-cassette ligation, substantially facilitated direct sequencing and subcloning of PCR products. Two T-cassettes with a protruding T at their 3' ends were obtained by annealing adaptor oligomers to XbaI- or XhoI-digested pBluescript. A DNA fragment amplified by the first PCR was ligated separately to one of the two T-cassettes. The second PCR was performed using a primer complementary to one of the two T-cassettes and one of the two primers used for the first PCR to amplify the ligation product in low abundance. The resultant two DNA fragments had the T3 and T7 promoter, and an annealing site for the universal M13 forward and reverse primer, respectively. These DNA fragments were applicable to direct sequencing. For the purpose of subcloning, the third PCR was carried out with two primers each complementary to each of two T-cassettes and two PCR-amplified DNAs by the second PCR, as templates. The PCR product of the third PCR had multiple sites of the pBluescript polycloning site at both ends, facilitating its subcloning into pBluescript.

Base Sequence↗
PubMed1991

Characterization of DNA fragment from Chlamydia psittaci avian strain which shows high homology with hypB gene of Chlamydia.

A study was performed to characterize DNA fragment No. 17 of C. psittaci strain P-1041 which encoded 42 KD beta-galactosidase fusion protein with type-specific antigenicity. Sequence determination identified a partial open reading frame that spanned about 1,200b. p. nucleotides. Screening the literatures for the nucleotide and deduced amino acid sequences revealed extensive similarity between the DNA fragment of P-1041 and two chlamydial hypB genes. This DNA showed 91.5% homology with C. psittaci GPIC hypB gene in nucleotide sequence and 96.4% homology in deduced amino acid sequence. The hypB gene of C. trachomatis serovar A and the P-1041 DNA fragment showed 81.2% and 91.3% homology in nucleotide and amino acid sequences, respectively. Dot enzyme-linked immunosorbent assay, for the products of deleted DNA fragments defined the coding region for type-specific antigenic polypeptide. In addition, the P-1041 DNA fragment carried a sequence highly homologous (greater than 49%) with other bacterial and plant genes called chaperonin which responds to various stress in cells. From these results, the P-1041 DNA fragment was found to be a part of hypB gene and to encode the region critical for type-specific antigenicity.

Amino Acid Sequence↗
PubMed1996

Nuclear DNA fragmentation during postnatal tooth development of mouse and hamster and during dentin repair in the rat.

The TUNEL (transferase-mediated, dUTP-biotin nick end labeling) method for in situ labeling of DNA strands was utilized to localize DNA fragmentation in cells involved in tooth formation in the neonatal mouse and hamster. Positive reactions for the presence of DNA fragments were obtained in some epithelial cells of the cervical loop region of incisors, late secretory, transitional and early maturation stage ameloblasts, stratum intermedium cells and in shortened ameloblasts just before eruption. Also, cells of the periodontal ligament of the continuously erupting incisors stained positive shortly before eruption. Odontoblasts were negative but became strongly positive during the formation of physiological osteodentin at the tip of developing incisors. Osteodentin matrix and the surfaces of unerupted enamel and cementum just prior to eruption stained for DNA fragments as well. DNA fragmentation could be elicited in odontoblasts and underlying pulpal tissues of mature erupted molars after mechanical injury to the odontoblast processes during cavity preparation. We conclude that, in rodents, DNA fragmentation and cell death are biological processes which take place in a variety of cells involved in formation of teeth. The TUNEL staining technique is a simple but powerful tool to examine the fate of cells and tissues undergoing either programmed cell death (apoptosis) or fragmentation of nuclear DNA induced by external factors leading to pathological changes.

Amelogenesis↗
PubMed1985

Structural studies of DNA fragments: the G.T wobble base pair in A, B and Z DNA; the G.A base pair in B-DNA.

The crystal structures of five double helical DNA fragments containing non-Watson-Crick complementary base pairs are reviewed. They comprise four fragments containing G.T base pairs: two deoxyoctamers d(GGGGCTCC) and d(GGGGTCCC) which crystallise as A type helices; a deoxydodecamer d(CGCGAATTTGCG) which crystallises in the B-DNA conformation; and the deoxyhexamer d(TGCGCG), which crystallises as a Z-DNA helix. In all four duplexes the G and T bases form wobble base pairs, with bases in the major tautomer forms and hydrogen bonds linking N1 of G with O2 of T and O6 of G with N3 of T. The X-ray analyses establish that the G.T wobble base pair can be accommodated in the A, B or Z double helix with minimal distortion of the global conformation. There are, however, changes in base stacking in the neighbourhood of the mismatched bases. The fifth structure, d(CGCGAATTAGCG), contains the purine purine mismatch G.A where G is in the anti and A in the syn conformation. The results represent the first direct structure determinations of base pair mismatches in DNA fragments and are discussed in relation to the fidelity of replication and mismatch recognition.

Base Composition↗
PubMed2002

A theoretical study of an empirical function for the mobility of DNA fragments in sieving matrices.

The separation of DNA fragments by gel electrophoresis has been studied extensively over the last two decades. More recently, similar studies have been carried out to characterize the separation achieved by the current capillary array electrophoresis systems and their sieving polymer solutions. In all cases, at least three different mobility regimes have been shown to exist: the Ogston regime when the radius of gyration of the DNA fragment is smaller than the pore size, the reptation regime when the DNA is larger than the pore size but remains in a random coil conformation, and finally the reptation-with-orientation regime where the DNA orients in the field direction and essentially all resolution is lost. Unfortunately, although theory helps us understand the different regimes and how to properly exploit them, we still have no theory-based general equations that would apply to all regimes. Such equations would be especially useful to analyze data, optimize separation systems and interpolate mobilities to estimate unknown molecular sizes. Recently, van Winkle, Beheshti and Rill (Electrophoresis 2002, 23, 15-19) proposed an intriguing empirical formula that seems to adequately fit the mobility of dsDNA fragments across all three regimes. In this paper, I investigate the relation between this empirical formula and the known theories of gel electrophoresis, and I study the dependence of its fitting parameters upon the experimental conditions. Finally, I examine how this equation may need to be modified to capture the more subtle details predicted by fundamental theories of DNA gel electrophoresis.

DNA↗