scholarly journals Differences in DNA Fragmentation following Transient Cerebral or Decapitation Ischemia in Rats

1995 ◽  
Vol 15 (5) ◽  
pp. 728-737 ◽  
Author(s):  
J. P. MacManus ◽  
I. E. Hill ◽  
E. Preston ◽  
I. Rasquinha ◽  
T. Walker ◽  
...  
Keyword(s):  
Dna Damage ◽  
Gel Electrophoresis ◽  
Time Course ◽  
Apoptotic Cell ◽  
Forebrain Ischemia ◽  
Dna Breaks ◽  
Double Labeling ◽  
Ca1 Region ◽  
Cell Death Pathway

The time course of appearance of cells with DNA damage was studied in rats following transient severe forebrain ischemia. This DNA damage could be detected by in situ end-labeling on brain sections. The breaks in DNA appeared selectively by day 1 in the striatum and later in the CA1 region of the hippocampus. It was possible by double labeling to show that there was no DNA damage in astrocytes. The DNA breaks consisted of laddered DNA fragments indicative of an ordered apoptotic type of internucleosomal cleavage, which persisted without smearing for up to 7 days of reperfusion. In contrast, the DNA breaks following ischemia induced by decapitation were random and, after gel electrophoresis, consisted of smeared fragments of multiple sizes. There was some early regional cellular death, restricted to the dentate of the hippocampus, prior to the pannecrotic degeneration. It is concluded that transient forebrain ischemia leads to a type of neuronal destruction that is not random necrosis but that shares some component of the apoptotic cell death pathway.

scholarly journals Apoptosis and Necrosis after Reversible Focal Ischemia: An in Situ DNA Fragmentation Analysis

1996 ◽  
Vol 16 (2) ◽  
pp. 186-194 ◽  
Author(s):  
C. Charriaut-Marlangue ◽  
I. Margaill ◽  
A. Represa ◽  
T. Popovici ◽  
M. Plotkine ◽  
...  
Keyword(s):  
Cell Death ◽  
Dna Fragmentation ◽  
Cortical Neurons ◽  
Apoptotic Cell ◽  
Chromatin Condensation ◽  
Focal Ischemia ◽  
Dna Breaks ◽  
Ischemic Lesion ◽  
In Cells

Apoptosis is one of the two forms 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. Early activation of an endonuclease has been previously demonstrated after a transient focal ischemia in the rat brain ( Charriaut-Marlangue C, Margaill I, Plotkine M, Ben-Ari Y (1995) Early endonuclease activation following reversible focal ischemia. J Cereb Blood Flow Metab 15:385–388). We now show that a significant number of striatal and cortical neurons exhibited chromatin condensation, nucleus segmentation, and apoptotic bodies increasing with recirculation time, as demonstrated by in situ labeling of DNA breaks in cryostat sections. Apoptotic nuclei were also detected in the horizontal limb diagonal band, accumbens nucleus and islands of Calleja. Several necrotic neurons, in which random DNA fragmentation occurs, were also shown at 6 h recirculation, in the ischemic core. Further investigation with hematoxylin/eosin staining revealed that apoptotic nuclei were present in cells with a large and swelled cytoplasm and in cells with an apparently well-preserved cytoplasm. These two types of cell death were reminiscent of those described in developmental cell death. Our data suggested that apoptosis may contribute to the expansion of the ischemic lesion.

scholarly journals STRIDE—a fluorescence method for direct, specific in situ detection of individual single- or double-strand DNA breaks in fixed cells

2019 ◽  
Vol 48 (3) ◽  
pp. e14-e14 ◽  
Author(s):  
Magdalena M Kordon ◽  
Mirosław Zarębski ◽  
Kamil Solarczyk ◽  
Hanhui Ma ◽  
Thoru Pederson ◽  
...  
Keyword(s):  
Dna Damage ◽  
Single Cells ◽  
Dna Lesions ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Dna Breaks ◽  
Damage Site ◽  
Double Strand Dna Breaks ◽  
In Situ Detection ◽  
Double Strand Dna

Abstract We here describe a technique termed STRIDE (SensiTive Recognition of Individual DNA Ends), which enables highly sensitive, specific, direct in situ detection of single- or double-strand DNA breaks (sSTRIDE or dSTRIDE), in nuclei of single cells, using fluorescence microscopy. The sensitivity of STRIDE was tested using a specially developed CRISPR/Cas9 DNA damage induction system, capable of inducing small clusters or individual single- or double-strand breaks. STRIDE exhibits significantly higher sensitivity and specificity of detection of DNA breaks than the commonly used terminal deoxynucleotidyl transferase dUTP nick-end labeling assay or methods based on monitoring of recruitment of repair proteins or histone modifications at the damage site (e.g. γH2AX). Even individual genome site-specific DNA double-strand cuts induced by CRISPR/Cas9, as well as individual single-strand DNA scissions induced by the nickase version of Cas9, can be detected by STRIDE and precisely localized within the cell nucleus. We further show that STRIDE can detect low-level spontaneous DNA damage, including age-related DNA lesions, DNA breaks induced by several agents (bleomycin, doxorubicin, topotecan, hydrogen peroxide, UV, photosensitized reactions) and fragmentation of DNA in human spermatozoa. The STRIDE methods are potentially useful in studies of mechanisms of DNA damage induction and repair in cell lines and primary cultures, including cells with impaired repair mechanisms.

scholarly journals In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.

1996 ◽  
Vol 44 (9) ◽  
pp. 959-968 ◽  
Author(s):  
A Negoescu ◽  
P Lorimier ◽  
F Labat-Moleur ◽  
C Drouet ◽  
C Robert ◽  
...  
Keyword(s):  
Apoptotic Cell ◽  
Rapid Identification ◽  
Cell Labeling ◽  
Cell Fraction ◽  
Terminal Deoxynucleotidyl Transferase ◽  
Current Debate ◽  
Double Labeling ◽  
Apoptotic Morphology ◽  
Induced Apoptosis

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

scholarly journals Expression of the HPV18/E6 oncoprotein induces DNA damage

2017 ◽  
Author(s):  
Elva I. Cortés Gutiérrez ◽  
Catalina Garcí­a-Vielma ◽  
Adriana Aguilar-Lemarroy ◽  
Veronica Vallejo-Ruí­z ◽  
Patricia Piña-Sánchez ◽  
...  
Keyword(s):  
Dna Damage ◽  
Hela Cells ◽  
Dna Hybridization ◽  
Dna Breaks ◽  
Dna Breakage ◽  
Hpv E6 ◽  
E6 Oncoprotein ◽  
Normal Expression ◽  
Hpv18 E6

This study investigated possible variations in DNA damage in HeLa cells with silenced expression of the HPV/E6 oncogene compared with HeLa cells with normal expression of the E6 oncogene using the DNA breakage detection-fluorescence in situ hybridization (DBD-FISH) technique and a whole human genome DNA probe. The variable levels of DNA breaks present were measured quantitatively using image analysis after whole-genome DNA hybridization. HeLa cells with silenced expression of the HPV18/E6 oncogene showed a significant decrease in DNA damage compared with parental cells with normal expression of the E6 oncogene. These results were confirmed by alkaline comet assay. In conclusion, we demonstrated a decrease in DNA damage in HeLa clones associated with low expression of the HPV/E6 oncogene. The significance of this decrease regarding the HPV life cycle and carcinogenesis requires further exploration.

scholarly journals Characterization of DNA Damage in Yeast Apoptosis Induced by Hydrogen Peroxide, Acetic Acid, and Hyperosmotic Shock

2006 ◽  
Vol 17 (10) ◽  
pp. 4584-4591 ◽  
Author(s):  
Gabriela F. Ribeiro ◽  
Manuela Côrte-Real ◽  
Björn Johansson
Keyword(s):  
Hydrogen Peroxide ◽  
Dna Damage ◽  
Acetic Acid ◽  
Dna Fragmentation ◽  
Tunel Staining ◽  
Chromosomal Dna ◽  
Dna Breaks ◽  
Hyperosmotic Shock ◽  
Double Strand Dna Breaks

Saccharomyces cerevisiae has been reported to die, under certain conditions, from programmed cell death with apoptotic markers. One of the most important markers is chromosomal DNA fragmentation as indicated by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. We found TUNEL staining in S. cerevisiae to be a consequence of both single- and double-strand DNA breaks, whereas in situ ligation specifically stained double-strand DNA breaks. Cells treated with hydrogen peroxide or acetic acid staining positively for TUNEL assay stained negatively for in situ ligation, indicating that DNA damage in both cases mainly consists of single-strand DNA breaks. Pulsed field gel electrophoresis of chromosomal DNA from cells dying from hydrogen peroxide, acetic acid, or hyperosmotic shock revealed DNA breakdown into fragments of several hundred kilobases, consistent with the higher order chromatin degradation preceding DNA laddering in apoptotic mammalian cells. DNA fragmentation was associated with death by treatment with 10 mM hydrogen peroxide but not 150 mM and was absent if cells were fixed with formaldehyde to eliminate enzyme activity before hydrogen peroxide treatment. These observations are consistent with a process that, like mammalian apoptosis, is enzyme dependent, degrades chromosomal DNA, and is activated only at low intensity of death stimuli.

Subtle Neuronal Death in Striatum After Short Forebrain Ischemia in Rats Detected by In Situ End-Labeling for DNA Damage

Stroke ◽  
1997 ◽  
Vol 28 (1) ◽  
pp. 163-170 ◽  
Author(s):  
Rainald Schmidt-Kastner ◽  
Henry Fliss ◽  
Antoine M. Hakim
Keyword(s):  
Dna Damage ◽  
Neuronal Death ◽  
Forebrain Ischemia

scholarly journals HPF1 remodels the active site of PARP1 to enable the serine ADP-ribosylation of histones

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Fa-Hui Sun ◽  
Peng Zhao ◽  
Nan Zhang ◽  
Lu-Lu Kong ◽  
Catherine C. L. Wong ◽  
...  
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Active Site ◽  
Negative Charge ◽  
Structural Data ◽  
Dna Breaks ◽  
Adp Ribosylation ◽  
Local Conformation ◽  
Key Residues ◽  
Structural Insights

AbstractUpon binding to DNA breaks, poly(ADP-ribose) polymerase 1 (PARP1) ADP-ribosylates itself and other factors to initiate DNA repair. Serine is the major residue for ADP-ribosylation upon DNA damage, which strictly depends on HPF1. Here, we report the crystal structures of human HPF1/PARP1-CAT ΔHD complex at 1.98 Å resolution, and mouse and human HPF1 at 1.71 Å and 1.57 Å resolution, respectively. Our structures and mutagenesis data confirm that the structural insights obtained in a recent HPF1/PARP2 study by Suskiewicz et al. apply to PARP1. Moreover, we quantitatively characterize the key residues necessary for HPF1/PARP1 binding. Our data show that through salt-bridging to Glu284/Asp286, Arg239 positions Glu284 to catalyze serine ADP-ribosylation, maintains the local conformation of HPF1 to limit PARP1 automodification, and facilitates HPF1/PARP1 binding by neutralizing the negative charge of Glu284. These findings, along with the high-resolution structural data, may facilitate drug discovery targeting PARP1.

scholarly journals DHX9-dependent recruitment of BRCA1 to RNA promotes DNA end resection in homologous recombination

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Prasun Chakraborty ◽  
Kevin Hiom
Keyword(s):  
Dna Damage ◽  
Homologous Recombination ◽  
Rna Polymerase Ii ◽  
Critical Role ◽  
Dna Breaks ◽  
Double Stranded Dna ◽  
Recombination Repair ◽  
Dna End Resection ◽  
End Resection ◽  
Rna Polymerase Ii Transcription

AbstractDouble stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.

scholarly journals Increased Calbindin D28k Expression via Long-Term Alternate-Day Fasting Does Not Protect against Ischemia-Reperfusion Injury: A Focus on Delayed Neuronal Death, Gliosis and Immunoglobulin G Leakage

2021 ◽  
Vol 22 (2) ◽  
pp. 644
Author(s):  
Hyejin Sim ◽  
Tae-Kyeong Lee ◽  
Yeon Ho Yoo ◽  
Ji Hyeon Ahn ◽  
Dae Won Kim ◽  
...  
Keyword(s):  
Immunoglobulin G ◽  
Pyramidal Neurons ◽  
Short Term Memory ◽  
Forebrain Ischemia ◽  
Short Term ◽  
Term Memory ◽  
Transient Forebrain Ischemia ◽  
Ca1 Pyramidal Neurons ◽  
Ca1 Region ◽  
Calbindin D28k

Calbindin-D28k (CB), a calcium-binding protein, mediates diverse neuronal functions. In this study, adult gerbils were fed a normal diet (ND) or exposed to intermittent fasting (IF) for three months, and were randomly assigned to sham or ischemia operated groups. Ischemic injury was induced by transient forebrain ischemia for 5 min. Short-term memory was examined via passive avoidance test. CB expression was investigated in the Cornu Ammonis 1 (CA1) region of the hippocampus via western blot analysis and immunohistochemistry. Finally, histological analysis was used to assess neuroprotection and gliosis (microgliosis and astrogliosis) in the CA1 region. Short-term memory did not vary significantly between ischemic gerbils with IF and those exposed to ND. CB expression was increased significantly in the CA1 pyramidal neurons of ischemic gerbils with IF compared with that of gerbils fed ND. However, the CB expression was significantly decreased in ischemic gerbils with IF, similarly to that of ischemic gerbils exposed to ND. The CA1 pyramidal neurons were not protected from ischemic injury in both groups, and gliosis (astrogliosis and microgliosis) was gradually increased with time after ischemia. In addition, immunoglobulin G was leaked into the CA1 parenchyma from blood vessels and gradually increased with time after ischemic insult in both groups. Taken together, our study suggests that IF for three months increases CB expression in hippocampal CA1 pyramidal neurons; however, the CA1 pyramidal neurons are not protected from transient forebrain ischemia. This failure in neuroprotection may be attributed to disruption of the blood–brain barrier, which triggers gliosis after ischemic insults.

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