A classic feature of apoptotic cell death is chromatin condensation and fragmentation, as DNA is broken up into smaller fragments by enzymes that are activated as part of the apoptotic program. This DNA fragmentation produces a characteristic pattern that can be visualized by gel electrophoresis, referred to as DNA laddering The apoptotic DNA fragmentation is being used as a marker of apoptosis and for identification of apoptotic cells either via the DNA laddering assay, the TUNEL assay, or the by detection of cells with fractional DNA content (sub G 1 cells) on DNA content frequency histograms e.g. as in the Nicoletti assay DNA fragmentation There are certain characteristics of apoptotic cells that can be identified and used to detect apoptotic cells in an otherwise healthy population of cells. Technically the easiest characteristic to detect is loss of DNA from permeabilised cells due to DNA fragmentation A cellular response that is characteristic of apoptosis is fragmentation of the nuclear genome to create a nucleosomal ladder. This activity is conducted by multiple nucleases activated by apoptotic signaling pathways. One nuclease involved in apoptosis is DNA fragmentation factor (DFF), a caspase-activated DNAse (CAD) . Recent observations also suggest large DNA fragments and even single-strand cleavage events occur during cell death..
DNA fragmentation during apoptosis induced by ROS or other triggers. Large DNA and internucleosomal DNA fragment cleavage Wyllie  has popularized the notion that DNA fragmentation is a component of apoptosis. Studies using glucocorticoids and rat thymocytes as a model system showed that DNA was fragmented into 180-200 bp prior to cell death. . Elegant biochemical work identified the DNA fragmentation factor (DFF) as a major apoptotic endonuclease for DNA fragmentation in vitro. Genetic studies in mice support the importance of DFF in DNA fragmentation and possibly in apoptosis in vivo This videos reviews various methods to detect apoptosis in cells, including DNA fragmentation assay, TUNEL assay, and DAPI staining in microscopy
The complexity of DNA degradation during apoptosis is poorly understood. So far, we have focused our discussion on the role of CAD in DNA fragmentation . CAD belongs to the cell-autonomous nucleases, which cleave the DNA in a cell during apoptosis (Figure 3A ). While CAD is the main cell-autonomous nuclease in apoptotic cell Apoptosis- normal and pathologic. DNA or proteins are damaged beyond repair, the cell kills itself characterized by nuclear dissolution, fragmentation of the cell without complete loss of membrane integrity. Autophagy- normal and pathologic. Oxygen Deprivation
In addition to morphological changes, the late phase of apoptosis can also be characterized by DNA fragmentation. DNA fragmentation can be visualized by flow cytometry using DNA binding dyes such as PI, 7-AAD, DAPI and Hoechst 33342 (Table 7). Cells in the late phase of apoptosis can be fixed and permeabilized using 70% ethanol The enzyme which fragments DNA during apoptosis is the caspase activated DNAases also known as DNA fragmentation factor subunit beta (DFFB/DFF40). DFF is a heterodimeric protein of 40 kDa (DFFB) and 45-kD subunits. DFFA is the substrate for caspase-3 and triggers DNA fragmentation during apoptosis Recently, examination of apoptosis (cell death) in frozen-thawed bovine oocytes and embryos using a Fluorescein-FragEL DNA fragmentation detection kit and a caspase-3 detection kit was reported (Hongshen et al., Reference Hongsheng, Monson, Parrish and Rutledge 2003). However, there has been no information elucidating apoptosis-related gene. - DNA fragmentation in apoptotic cells facilitated by a caspase activated DNase. - This enzyme cleaves DNA at internucleosomal linker sites. - This is the DNA that is found between nucleosomes within chromatin. - This cleavage typically occurs at ~180 bp intervals, resulting in a ladder effect when apoptotic DNA is visualized by gel.
Apoptosis is an active process that requires energy (ATP). Necrosis is a passive process that doesn't need energy. The DNA fragmentation during apoptosis occurs in a non-random way, forming mon- or oligonucleotide lengths. The degradation of DNA during necrosis is random. A series of reactions of activation (cascade) of proteins takes place DNA fragmentation is one of the last phases in apoptosis, resulting from the activation of endonucleases during the apoptotic process. There are several established methods for the study of DNA fragmentation including isolation and separation of DNA fragments by agarose gel electrophoresis and end labeling Other causes of sperm DNA fragmentation can be defective apoptosis, excessive reactive oxygen species (ROS) production and decreased seminal antioxidants [13-23]. Also toxic effects of drugs, cigarette smoking, pollution, and factors as xenobiotics, high testicular temperature (fever, varicocele) and advanced age have been associated with. Evaluating sperm DNA fragmentation, apoptosis course and OS biomarkers on the same semen sample gives an overview of the impact of varicocele on sperm quality that allows to go further than investigating only standard semen parameters. However, the present study has some limitations mainly linked to the sample size in each group In one study of 360 couples undergoing IVF, higher sperm DNA fragmentation levels were associated with lower fertilization rates, embryo quality, and pregnancy rates. The couples who were not able to achieve pregnancy had an average of 51.7% sperm with DNA fragmentation, as opposed to 39.5% in the pregnant couples
DNA fragmentation is associated with apoptosis and can be identified with the use of a molecular biological immunohistochemical method. 21 Since extensive DNA fragmentation is an important characteristic of apoptosis, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells The execution phase of apoptosis occurs through the activation and function of caspases which cleave key substrates that orchestrate the death process. DNA fragmentation and cell death in T. Apoptosis Protocols. Explore our collection of apoptosis assay flow cytometry protocols and protocols for imaging, high-content analysis, and microplate readers. Apoptosis protocols are available for apoptosis induction, caspase activity, mitochondrial membrane potential and transition pore opening, annexin V staining, TUNEL assays, chromatin.
The assay of DNA agarose gel electrophoresis provides good results for the definition of cell apoptosis. A typical ladder pattern of DNA fragmentation should be observed in most apoptotic cells. 3.5. Time consideration Apoptotic DNA fragmentation, visualised by the DNA laddering assay (left). A 1 kb marker (middle) and control DNA (right) are included for comparison.. Apoptotic DNA fragmentation is a key feature of apoptosis, a type of programmed cell death.Apoptosis is characterized by the activation of endogenous endonucleases, particularly the caspase-3 activated DNase (CAD),  with subsequent cleavage.
The Calbiochem ® TdT-FragEL™ DNA Fragmentation Detection Kit is a non-isotopic system for the labeling of DNA breaks in apoptotic nuclei in paraffin-embedded tissue sections, tissue cryosections, and in cell preparations fixed on slides. Cell death has been shown to occur by two major mechanisms, necrosis, and apoptosis no clear evidence has been presented so far that DNA degra- dation plays a primary and causative role in apoptotic cell death. In contrast, recent reports have described that cell death with key morphological features of apoptosis can be induced in the absence of detectable DNA fragmentation (4, 9, 20, 30) Sperm DNA fragmentation is referred to as one of the main causes of male infertility. Failures in the protamination process, apoptosis and action of reactive oxygen species (ROS) are considered the most important causes of DNA fragmentation. Action of ROS or changes in sperm protamination would increase the susceptibility of sperm DNA to. Apoptosis is characterised by a series of typical morphological events, such as shrinkage of the cell, fragmentation into membrane-bound apoptotic bodies and rapid phagocytosis by neighbouring cells . Internucleosomal fragmentation of genomic DNA has been the biochemical hallmark of apoptosis for many years  DNA Fragmentation Subject Areas on Research.
When DNA is cleaved, which is usually a late event, fragments are random in size (smear pattern in gels) APOPTOSIS Nuclear chromatin marginates and chromatin condenses, becoming very compact Chromatin condensation and DNA fragmentation occur together; DNA cleaved into multiples of 200 base pair unit To determine if the DNA pieces resulted from the DNA fragmentation that occurs during apoptosis, we analyzed mutants known to affect apoptosis in C. elegans (reviewed in , ). Genes essential for the killing phase of apoptosis, whose mutation completely eliminates apoptosis, were devoid of the small DNA pieces ( Figure 2A ) ApoBrdU-IHC DNA Fragmentation Assay Kit. Select up to 5 products from above to compare or request more information. This kit was used to detect and quantify the apoptotic cells in a cell population treated with anticancer compounds compared to control cells. The product is simple to use and has high throughput DNA Fragmentation (10) Apoptotic DNA Ladder Assays (3) Assay Kits (3) TUNEL Assays (4) Mitochondrial Assays (21) Necroptosis Inhibitors (6) Non-Caspase Proteases (118) Calpain (7) Calpain Antibodies & Peptides (3) Calpain Assay Kits (2) Calpain Enzymes (Active) (1) Calpain Inhibitors (1) Cathepsin (79) Cathepsin Antibodies & Peptides (25.
Annexin V-FITC staining of apoptosis in Jurkat T cells. Annexin V-FITC staining of apoptosis in Jurkat T cells. Apoptosis was induced with 200 ng/ml anti-Fas (lgM,CH11). At the indicated times, cells were incubated for 5 minutes with 1.0 μg/ml Annexin V-FITC in binding buffer, and 5,000 cells were examined by BD FACS analysis DNA fragmentation is the separation of DNA strands by pieces. It occurs naturally by the cells, with the activation of executioner caspase, which leads to the activation of endonucleases. 2. Based on your understanding of Bcl-2 in apoptosis and DNA fragmentation, which cell type does 'A' and 'B' on the gel above correspond to? Justify. 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. KEY WORDS: Apoptosis · White spot syndrome virus · WSSV · Penaeus monodon Resale or republication not permitte DNA FRAGMENTATION IN APOPTOSIS. Sei-ichi TANUMA 1) 2) 1) Department of Biochemistry, Faculty of Pharmaceutical Science, Science University of Tokyo 2) Research Institute for Bioscience, Science University of Tokyo Released 20121113 Received 19950526 Accepted 19950605.
The Quick Apoptotic DNA Ladder Detection Kit offers a fast and sensitive detection of DNA fragmentation in apoptotic cells. Unlike other similar kits in the market which require 1-2 days to prepare nucleic acids, the new procedure requires less than 90 minutes to prepare chromosomal DNA in a single tube Introduction. Apoptosis is a crucial cellular mechanism that is involved in inflammation, cell differentiation and cell proliferation. As a form of cell death, it is characterized by distinctive morphological and biochemical changes, including plasma membrane blebbing, phosphatidylserine exposure, nuclear condensation and DNA fragmentation [].These cellular changes are largely mediated by. Detection of DNA Fragmentation in Apoptotic Cells by TUNEL. Lisa C. Crowley 1, Degradation of DNA into oligonucleosomal-sized fragments is a unique event in apoptosis that is orchestrated by caspase-activated DNase. Traditionally, this event is observed by resolving cellular DNA by gel electrophoresis, which results in a characteristic. the small DNA pieces are products of the DNA fragmentation that occurs during apoptosis, and correspondingly, are absent in mutant strains incapable of apoptosis. In contrast, the small DNA pieces are present in strains defective for the engulfment process of apoptosis, suggesting they are produced in the dying cell prior to engulfment DNA fragmentation assay Apoptosis can be visualized as a ladder pattern of 180-200 bp due to DNA cleavage by the activation of a nuclear endonuclease by standard agarose gel electrophoresis.10, 11 Thus, we showed the formation of the DNA ladder in gel electrophoresis by induction of apoptosis in NIH-3T3 cell line (Fig. 1). Flow cytometry assa
Besides the release of cytochrome c from the intramembrane space, the intramembrane content released also contains apoptosis inducing factor (AIF) to facilitate DNA fragmentation, and Smac/Diablo proteins to inhibit the inhibitor of apoptosis (IAP) . Figure 2. The intrinsic and extrinsic pathways leading to apoptosis. Cance Results Curcumin Induces Cell Death but Protects Human T Cells against Apoptotic DNA Fragmentation. Curcumin has been shown to induce apoptosis in many cancer cells (reviewed in ref. 6).However, in our hands, 50 μmol/L curcumin induced death in Jurkat cells without the oligonucleosomal DNA fragmentation and morphology typical for apoptotic cells (13, 18)
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 Sperm DNA fragmentation (sDF) represents a threat to male fertility, human reproduction and the health of the offspring. The causes of sDF are still unclear, even if apoptosis, oxidative assault and defects in chromatin maturation are hypothesized. Using multicolor flow cytometry and sperm sorting, we challenged the three hypothesized mechanisms by simultaneously evaluating sDF and signs of. Fragmentation of this length might be indicative of apoptotic DNA fragmentation, suggesting that apoptosis may be the primary method of ctDNA release. Circulating tumor DNA - Wikipedia These changes include blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, chromosomal DNA fragmentation, and global mRNA decay
As the elevations in Ca +2 Mg +2 ‐dependent endonuclease activity and DNA fragmentation are initiated at a time well before the cell is morphologically dead, as defined by apoptosis, these changes in DNA metabolism must not be the consequences of cell death but instead are early causal events in an active process of programmed cell death Apoptosis assays, based on methodology, can be classified into six major groups: 1. Cytomorphological alterations 2. DNA fragmentation 3. Detection of caspases, cleaved substrates, regulators and inhibitors 4. Membrane alterations 5. Detection of apoptosis in whole mounts 6. Mitochondrial assays 37 38
Quantitation of DNA Fragmentation in Apoptosis. Overview of attention for article published in Nucleic Acids Research, January 1996. Altmetric Badge. About this Attention Score Good Attention Score compared to outputs of the same age (65th percentile) Average Attention Score compared to outputs of the same age and source. Mentioned b DNA‐fragmentation and expression of apoptosis‐related proteins in muscular dystrophies DNA‐fragmentation and expression of apoptosis‐related proteins in muscular dystrophies Tews, D. S.; Goebel, H. H. 1997-08-01 00:00:00 Footnotes Correspondence: Dr Dominique S. Tews, Division of Neuropathology, Mainz University Medical Centre, Langenbeckstrasse 1, D‐55131 Mainz, Germany gated DNA damage induced by benzene metabohites l,4-BQ and l,4-HQ in relation to apoptosis and carcinogenesis. DNA strand breaks in human cultured cells were investigated with pulsed-field gel electrophoresis. Internucleosomal DNA fragmentation and morpho logical changes of apoptotic cells were examined. The cells treate
On the contrary, DNA fragmentation in S. cerevisiae undergoing apoptosis-like PCD has so far only been evident by TUNEL-positive phenotype. We detected large DNA fragments (several hundred kilobases) in cells dying from treatment with acetic acid, hydrogen peroxide, or from hyperosmotic shock apoptosis  and defective maturation  correlate with the role of intrinsic fac-tors in testicular SDF. Moreover, evidence show more DNA fragmentation in epi - didymal and ejaculated sperm than testicular sperm, signifying the impact of extrinsic factors . Presence of large amount of polyunsaturated fatty acids (PUFA) in th DNA fragmentation is usually associated with ultrastructural changes in cellular morphology in apoptosis (26, 38). In a number of well-researched model systems, large fragments of 300 kb and 50 kb are first produced by endonucleolytic degradation of higher-order chromatin structural organization / Two in situ labeling techniques reveal different patterns of DNA fragmentation during spontaneous apoptosis in vivo and induced apoptosis in vitro. In: Anticancer research. 1995 ; Vol. 15, No. 5 B. pp. 1895-1904 Given that, excessive damage in sperm DNA may result in early reproductive failures and during the 4 to 8 cell stage, when the paternal genome is switched on, the development of the embryo will be affected by sperm DNA integrity causing apoptosis, fragmentation, and difficulty to reach the blastocyst stage [19, 76]
We found that Drosophila inhibitor of apoptosis dBruce is degraded by autophagy and this degradation promotes DNA fragmentation and subsequent nurse cell death. These studies demonstrate that autophagic degradation of an inhibitor of apoptosis is a novel mechanism of triggering cell death While studying small noncoding RNA in C. elegans, we discovered that protocols used for isolation of RNA are contaminated with small DNA pieces. After electrophoresis on a denaturing gel, the DNA fragments appear as a ladder of bands, 10 nucleotides apart, mimicking the pattern of nuclease digestion of DNA wrapped around a nucleosome. Here we show that the small DNA pieces are products of the. The signaling pathway for DNA damaging drug-triggered apoptosis was examined in a chemosensitive human neuroblastoma cell line, SH-SY5Y. Doxorubicin and etoposide induce rapid and extensive apoptosis in SH-SY5Y cells. After the drug treatment, p53 protein levels increase in the nucleus, leading to the induction of its transcription targets p21 Waf1/Cip1 and MDM2
T1 - Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at serine 139. AU - Rogakou, Emmy P. AU - Nieves-Neira, Wilberto. AU - Boon, Chye. AU - Pommier, Yves. AU - Bonner, William M. PY - 2000/3/31. Y1 - 2000/3/3 Furthermore, the increased cell apoptosis rate and DNA fragmentation were closely related to the increased dose and exposure duration of GNPs in SH-SY5Y cells. The reduction in MMP with a simultaneous increase in the expression of bax/bcl2 gene ratio indicated that mitochondria-mediated pathway involved in GNPs induced apoptosis strated that HMW DNA fragmentation was a key committed step of apoptosis (26), which was inhibited by in vivo administration of cycloheximide (28). In earlier studies, we demonstrated that, in con trast to the Ca2@-dependent nucleosomal DNA fragmentation, the cleavage of DNA into 11MW fragments was a Ca2tindependen Liu et al. (1997) determined that caspase-3 cleaves the 45-kD subunit at 2 sites to generate an active factor that produces DNA fragmentation without further requirement of caspase-3 or other cytosolic proteins. In cells undergoing apoptosis, the 45-kD subunit was cleaved in the same pattern as it is cleaved by caspase-3 in vitro. These results delineated a direct signal transduction pathway.
We report the purification from a rat natural killer (RNK) large granular lymphocyte leukemia of a 32-kD granule protein that induces rapid DNA fragmentation and apoptosis. The protein, which we have called fragmentin, was capable of causing DNA from intact YAC-1 cells to be cleaved into oligonucleosomal-sized fragments and producing severe. DNA fragmentation could result in initiation of apoptosis (natural cellular death) and mutations resulting in blastocyst arrest, miscarriage and abnormalities in the offspring Sperms with high DNA fragmentation fertilising younger oocytes than older oocytes carry a better prognosis of successful pregnancy, as they are much more efficient at DNA. Our aim was to develop a quantitative assay for DNA fragmentation in apoptosis by enzymatically labeling DNA with a fluorescent dideoxynucleotide. Terminal deoxynucleotidyl transferase was used to enzymatically label 3′-OH DNA ends with fluorescein-12-dideoxyuridine triphosphate in an assay referred to as fluorophore end-labeling 188.8.131.52.1 Ability to Detect Nuclear Changes to Drive the Demand for DNA Fragmentation Assay Kits 184.108.40.206 Mitochondrial Assays 220.127.116.11.1 Merck, Thermo Fisher , Bio-Rad, and BD are Among the Key. We report here the reconstitution of a pathway that leads to the apoptotic changes in nuclei by using recombinant DNA fragmentation factor (DFF), a heterodimeric protein of 40 and 45 kDa. Coexpression of DFF40 and DFF45 is required to generate recombinant DFF, which becomes activated when DFF45 is cleaved by caspase-3
DNA fragmentation factor (DFF) is one of the major endonucleases responsible for internucleosomal DNA cleavage during apoptosis. Understanding the regulatory checkpoints involved in safeguarding non-apoptotic cells against accidental activation of this nuclease is as important as elucidating its activation mechanisms during apoptosis