Apoptosis has been recognized as a type of programmed cell death connected with characteristic morphological and biochemical changes in cells. This programmed cell death plays an important role in the genesis of a number of physiological and pathological processes. Thus, it can be very important to detect the signs of apoptosis in a study of cellular metabolism. The present paper provides an overview of methods often being used for detecting DNA fragmentation as one of the most specific findings in apoptosis. To date, three routine assays have been developed for detecting DNA fragmentation: DNA ladder assay, TUNEL assay, and comet assay. All these methods differ in their principles for detecting DNA fragmentation. DNA ladder assay detects the characteristic "DNA ladder" pattern formed during internucleosomal cleavage of DNA. Terminal deoxynUcleotidyl transferase Nick-End Labeling (TUNEL) assay detects DNA strand breaks using terminal deoxynucleotidyl transferase catalyzing attachment of modified deoxynucleotides on the DNA strand breaks. Comet assay can be used for detecting nucleus breakdown producing single/double-strand DNA breaks. The aim of this review is to describe the present knowledge on these three methods, including optimized approaches, techniques, and limitations.

• Klíčová slova
• Apoptosis, Apoptosis assays, Comet assay, DNA fragmentation, DNA ladder, TUNEL assay,
• MeSH
• apoptóza genetika   fyziologie   MeSH
• biotest metody   MeSH
• DNA analýza   genetika   metabolismus   MeSH
• fragmentace DNA * MeSH
• kometový test metody   MeSH
• koncové značení zlomů DNA in situ metody   MeSH
• lidé MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• přehledy MeSH
• Názvy látek
• DNA MeSH

Role of male factor in recurrent abortion and in vitro fertilization failure has not been fully defined yet and there is much controversy about evaluating male patients with normal semen analysis. One of the factors that might help establish the male role is DNA fragmentation index. However, strong correlation between this factor and quality of semen, has caused many clinicians to believe that it does not help in abortion and implantation failure. We aim to assess this factor in our patients. In a prospective observational study, we assessed age, duration of infertility, undesired fertility related events (assisted reproductive techniques attempts and abortions), semen parameters and DNA fragmentation index in patients with multiple abortions or in vitro fertilization failures and analysed the results by statistical software SPSS version 24. DNA fragmentation index was remarkably correlated with age, duration of infertility and semen parameters. Among all groups in our study, patients with abnormal semen analysis had statistically significant higher level of DNA fragmentation. Ten percent of patients with normal or slightly abnormal semen analysis had abnormally high SDFI (sperm DNA fragmentation index). Checking DNA fragmentation index is recommended in all couples with fertilization problems even in the presence of normal semen analysis. It might be more reasonable to assess it in aged men, long duration of infertility or candidates with remarkable semen abnormality.

• Klíčová slova
• Abortion, DNA fragmentation index, In vitro fertilization, Intracytoplasmic sperm injection, Male, Sperm,
• MeSH
• analýza spermatu MeSH
• fertilizace in vitro metody   MeSH
• fragmentace DNA MeSH
• habituální potrat * MeSH
• lidé MeSH
• mužská infertilita * diagnóza   genetika   MeSH
• senioři MeSH
• sperma MeSH
• spermie MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• senioři MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• pozorovací studie MeSH

A compromised detection of radiation-induced plasmid DNA fragments results in underestimation of calculated damage yields. Electrophoretic methods are easy and cheap, but they can only detect a part of the fragments, neglecting the shortest ones. These can be detected with atomic force microscopy, but at the expense of time and price. Both methods were used to investigate their capabilities to detect the DNA fragments induced by high-energetic heavy ions. The results were taken into account in calculations of radiation-induced yields of single and double strand breaks. It was estimated that the double strand break yield is twice as high when the fragments are at least partially detected with the agarose electrophoresis, compared to when they were completely omitted. Further increase by 13% was observed when the measured fragments were corrected for the fraction of the shortest fragments up to 300 base pairs, as detected with the atomic force microscopy. The effect of fragment detection on the single strand break yield was diminished.

• MeSH
• elektroforéza metody   MeSH
• fragmentace DNA účinky záření   MeSH
• lineární přenos energie MeSH
• mikroskopie atomárních sil metody   MeSH
• plazmidy MeSH
• těžké ionty MeSH
• zlomy DNA účinky záření   MeSH
• Publikační typ
• časopisecké články MeSH

Specific DNA fragmentation into oligonucleosomal units occurs during programmed cell death (PCD) in both animal and plant cells, usually being regarded as an indicator of its apoptotic character. This internucleosomal DNA fragmentation is demonstrated in tobacco suspension and leaf cells, which were killed immediately by freezing in liquid nitrogen, and homogenization or treatment with Triton X-100. Although these cells could not activate and realize the respective enzymatic processes in a programmed manner, the character of DNA fragmentation was similar to that in the cells undergoing typical gradual PCD induced by 50 microM CdSO4. This internucleosomal DNA fragmentation was connected with the action of cysteine proteases and the loss of membrane, in particular tonoplast, integrity. The mechanisms of DNase activation in the rapidly killed cells, hypothetical biological relevance, and implications for the classification of cell death are discussed.

• MeSH
• apoptóza * účinky léků   MeSH
• buněčné jádro genetika   MeSH
• fragmentace DNA * účinky léků   MeSH
• inhibitory proteas farmakologie   MeSH
• kultivované buňky MeSH
• tabák cytologie   účinky léků   genetika   fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• inhibitory proteas MeSH

Using cryopreservation techniques can increase the effectiveness of reproducing cultured fish species by ensuring a dependable supply of sperm, although the quality of the sperm could be impacted by the procedures involved. The goal of this study was to investigate the effect of purified seminal plasma transferrin (Tf), bovine serum albumin (BSA), and antifreeze protein (AFP) types I and III at 1 µg mL-1 on relevant characteristics of cryopreserved sperm from common carp Cyprinus carpio. We compared oxidative stress indices, antioxidant activity, and DNA fragmentation of fresh sperm to that frozen with extender only or with Tf, BSA, or AFP types I and III. Fresh sperm had significantly lower levels of thiobarbituric acid reactive substances (TBARS) compared to samples that underwent cryopreservation without protein treatment, which resulted in 0.54 ± 0.06 nmol/108 cells of TBARS. Carbonyl derivatives of proteins (CP) decreased significantly (ANOVA; P > 0.05) in carp sperm with addition of Tf, AFPI, and AFPIII. Significant differences in superoxide dismutase (SOD), glutathione reductase (GR), and glutathione peroxidase (GPx) activity were seen in sperm supplemented with Tf, BSA, AFPI, and AFPIII from those without. Significantly less DNA damage, expressed as percent tail DNA (11.56 ± 1.34) and olive tail moment (0.59 ± 0.13), was recorded in samples cryopreserved with Tf. The findings indicated that addition of Tf, BSA, AFPI, or AFPIII to cryopreservation medium is beneficial to sperm preservation. The mechanisms through which these proteins act positively on sperm need to be further investigated.

• Klíčová slova
• Cyprinidae, Freeze/thaw, Proteins, Reactive oxygen species, Sperm DNA fragmentation,
• MeSH
• alfa-fetoproteiny MeSH
• antioxidancia MeSH
• fragmentace DNA MeSH
• kapři * MeSH
• kryoprezervace veterinární   metody   MeSH
• kryoprotektivní látky MeSH
• kryoprotektivní proteiny MeSH
• látky reagující s kyselinou thiobarbiturovou MeSH
• motilita spermií MeSH
• oxidační stres MeSH
• sperma MeSH
• spermie MeSH
• uchování spermatu * veterinární   metody   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• alfa-fetoproteiny MeSH
• antioxidancia MeSH
• kryoprotektivní látky MeSH
• kryoprotektivní proteiny MeSH
• látky reagující s kyselinou thiobarbiturovou MeSH

Photodynamic therapy (PDT) is an alternative method of tumour treatment. It is based on a photochemical reaction of a photosensitizer, irradiation, and O(2) which converts to cytotoxic (1)O(2) and other forms of reactive oxygen species (ROS). The comet assay (also called single-cell gel electrophoresis, SCGE) is a sensitive, simple and quantitative technique for detection of DNA damage. In our study we investigated the phototoxicity of the two porphyrin photosensitizers, TPPS4 and MgTPPS4, on HeLa cells. Three different radiation doses and six different concentrations of the photosensitizers were used. Our results show that the DNA of the cells treated with the TPPS(4) and MgTPPS(4) at the concentrations higher than 5 μM was highly fragmented indicating a strong phototoxic effect resulting in a cell apoptosis. On the base of our results we can hypothesize that even the irradiation dose of 1 J cm(-2) is sufficient enough to provoke the DNA fragmentation.

• MeSH
• apoptóza účinky léků   MeSH
• dávka záření MeSH
• fotochemoterapie metody   MeSH
• fotosenzibilizující látky aplikace a dávkování   farmakologie   MeSH
• fragmentace DNA účinky léků   MeSH
• HeLa buňky MeSH
• hořčík chemie   MeSH
• kometový test MeSH
• lidé MeSH
• metaloporfyriny aplikace a dávkování   chemie   farmakologie   MeSH
• porfyriny aplikace a dávkování   chemie   farmakologie   MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• fotosenzibilizující látky MeSH
• hořčík MeSH
• magnesium 5,10,15,20-tetrakis(4-sulphophenyl)-porphine dodecahydrate MeSH Prohlížeč
• metaloporfyriny MeSH
• porfyriny MeSH
• tetraphenylporphine sulfonate MeSH Prohlížeč

AIM OF STUDY: To assess cell free fetal DNA (cffDNA) fragmentation rate in pregnant women during the course of gravidity. STUDY DESIGN: QF PCR efficiency in cffDNA and quantitative analyses in particular cffDNA molecular size fractions. SETTING: The study was performed at Department of Medical Genetics and Fetal Medicine, University Hospital Olomouc. METHOD: 1. 363 plasma DNA samples from women in different week of pregnancy (from 4th w.g. to 37th w.g.) were tested for QF PCR efficiency in particular STRs and AMELX/Y. 2. Size fractionated cff DNA (150-300 bp, 300-500 bp, 500-760 bp) was quantified by QF PCR in 91 pregnant women (from 9th w.g. to 40th w.g.). 3. Size fractionated cff DNA from male fetuses was quantified by real time PCR (SRY/internal control) in 22 pregnant women (from 9th w.g. to 36th w.g.). RESULTS: 1. QF PCR efficiency decreased from longer to shorter molecules. 2. The only 500 -760 bp fraction showed cffDNA increase in relation to week of gravidity. 3. Indirect relation between amount of cffDNA and week of gravidity was found in 150-300 bp fraction by Real-time PCR. CONCLUSION: Assembling of all 3 approaches indicates increase of longer cffDNA molecules during the gravidity while level of the short cffDNA molecule fragments probably remains from the approximately 9th w.g. the same.

• MeSH
• DNA krev   MeSH
• elektroforéza kapilární MeSH
• fragmentace DNA * MeSH
• lidé MeSH
• lidské chromozomy X genetika   MeSH
• lidský chromozom Y genetika   MeSH
• mikrosatelitní repetice genetika   MeSH
• plod * MeSH
• polymerázová řetězová reakce MeSH
• těhotenství MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• těhotenství MeSH
• ženské pohlaví MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH
• Názvy látek
• DNA MeSH

The detection of doxorubicin-induced apoptosis in individual cardiomyocytes was performed on a microfluidic device. Microstructures integrated on a CD-like plastic disk were adapted for the selection of individual cells their lysis in an alkaline environment and the separation of released apoptotic DNA fragments. The fragments with typical 180 base pairs ladder pattern were electrophoretically resolved in a 2% solution of linear polyacrylamide with 0.1 M NaOH on a migration distance of 6 mm. The laser-induced fluorescence of fragments labeled by ethidium bromide was monitored by a photomultiplier tube mounted on a confocal microscope. The causal relation between the enhanced doxorubicin concentration and the extent of DNA fragmentation in a single cell was confirmed. The results show that the extent of DNA fragmentation is proportional to the time of a cell treatment. Onset of necrosis was evident in cardiomyocytes treated by doxorubicin for more than 24 h. The adverse effect of doxorubicin, an important cytostatics used for the treatment of many solid tumors, leads to the destruction of cardiomyocytes and, consequently, may result in the heart failure of treated individuals. Therefore, the monitoring of the extent of apoptotic DNA damage of cardiac myocytes represents critical step toward understanding of the development of chronic doxorubicin-induced cardiomyopathy.

• MeSH
• apoptóza MeSH
• DNA chemie   genetika   izolace a purifikace   MeSH
• elektroforéza přístrojové vybavení   metody   MeSH
• krysa rodu Rattus MeSH
• mikrofluidika metody   MeSH
• myokard cytologie   MeSH
• separace buněk přístrojové vybavení   metody   MeSH
• svalové buňky cytologie   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA MeSH

The possibility of the study of a quantitative correlation between short-term tests and carcinogenicity, instead of a qualitative one, is discussed. Four tests related to the target organ, rat liver, were considered: alkaline DNA fragmentation, DNA repair, DNA adducts and the formation of preneoplastic nodules. All the four tests showed a similar level of correlation with carcinogenic potency (r approximately equal to 0.4). With this level of correlation, the dispersion of the data appeared too large to offer a meaningful degree of quantitative predictivity of carcinogenicity, in reference to a single test. It appeared however, that the use of a battery of two or three independent short-term tests, with the above level of simple correlation, could generate a multiple correlation high enough to be potentially useful for some degree of quantitative predictivity of carcinogenic potency.

• MeSH
• aminy toxicita   MeSH
• azosloučeniny toxicita   MeSH
• DNA metabolismus   MeSH
• halogenované uhlovodíky toxicita   MeSH
• hydraziny toxicita   MeSH
• játra účinky léků   metabolismus   MeSH
• karcinogeny metabolismus   toxicita   MeSH
• krysa rodu Rattus MeSH
• mykotoxiny toxicita   MeSH
• nádory jater patologie   MeSH
• nitrosaminy toxicita   MeSH
• oprava DNA * MeSH
• polycyklické sloučeniny toxicita   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aminy MeSH
• azosloučeniny MeSH
• DNA MeSH
• halogenované uhlovodíky MeSH
• hydraziny MeSH
• karcinogeny MeSH
• mykotoxiny MeSH
• nitrosaminy MeSH
• polycyklické sloučeniny MeSH

The great complexity of extracellular freezing stress, involving mechanical, osmotic, dehydration and chemical perturbations of the cellular milieu, hampers progress in understanding the nature of freezing injury and the mechanisms to cope with it in naturally freeze-tolerant insects. Here, we show that nuclear DNA fragmentation begins to occur in larval haemocytes of two fly species, Chymomyza costata and Drosophila melanogaster, before or at the same time as the sub-zero temperature is reached that causes irreparable freezing injury and mortality in freeze-sensitive larval phenotypes. However, when larvae of the freeze-tolerant phenotype (diapausing-cold acclimated-hyperprolinemic) of C. costata were subjected to severe freezing stress in liquid nitrogen, no DNA damage was observed. Artificially increasing the proline concentration in freeze-sensitive larvae of both species by feeding them a proline-enriched diet resulted in a decrease in the proportion of nuclei with fragmented DNA during freezing stress. Our results suggest that proline accumulated in diapausing C. costata larvae during cold acclimation may contribute to the protection of nuclear DNA against fragmentation associated with freezing stress.

• Klíčová slova
• Comet assay, DNA fragmentation, Freeze tolerance, Insects, Proline,
• MeSH
• aklimatizace MeSH
• Drosophila melanogaster * MeSH
• hmyz * MeSH
• larva MeSH
• nízká teplota MeSH
• prolin MeSH
• zmrazování MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• prolin MeSH