Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.30.2 (endonuclease)
18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Mitochondrial DNA (mtDNA) was isolated from liver mitochondria of rats between 2 and 24 months of age. The mtDNA was purified by cesium chloride--ethidium bromide isopycnic density gradient centrifugation. In the gradients, in addition to the two expected bands of ethidium--DNA complex, there was observed a third, more dense band (d = 1.69 g/cm3). This novel band, rarely observed in preparations from younger animals, was present in most preparations from older animals. The latter was characterized using the diphenylamine assay(s) and ascertained to contain DNA and carbohydrate components. Agarose gel electrophoresis revealed the DNA of the novel band to have a migration identical to form I mtDNA. Digestion of the novel band with the restriction endonuclease Bam HI yielded products identical to those obtained upon treatment of form I mtDNA with Bam HI. The observation of mtDNA at a density of 1.69 g/cm3 indicates the presence, predominantly in older animals, of a subclass of mtDNA molecules with altered ethidium binding properties. The significance of this mtDNA and its position in the gradient is unclear at this time.
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PMID:Changes in mitochondrial DNA during aging. 716 20

Apoptosis is a distinct form of cell death characterized by internucleosomal cleavage of DNA, cell membrane blebbing, condensation of nuclear chromatin in the nuclear periphery, and the formation of apoptotic, condensed nuclear bodies. The finding of internucleosomal cleavage of chromatin, perhaps caused by endonuclease activation, has become accepted as a hallmark of this form of cell death. We describe the incidental and artifactual finding of internucleosomal cleavage of chromatin from kidney tissue from normal animals. Nephrectomy was performed in living animals, and renal tissue was digested with proteinase K in 10 mmol/L Tris, 1 mmol/L ethylenediaminetetraacetic acid (EDTA), 10 mmol/L NaCl, and 0.5% sodium dodecyl sulfate. Agarose gel electrophoresis of extracted DNA showed internucleosomal cleavage. Internucleosomal cleavage of DNA was not tissue specific but was evident also in liver DNA from a number of animals. Histologic examination of kidney tissue where DNA exhibited internucleosomal cleavage showed normal morphology, with no evidence of either apoptotic or necrotic cell death. Cleavage was not completely prevented by immediate freezing of kidney tissue in liquid nitrogen before DNA extraction, nor was it prevented by the addition of spermidine, of ethyleneglycol-bis-(beta-aminoethylether)-N,N,N',N'-tetraace tic acid, of phenylmethylsulfonylfluride, or by an increased concentration of NaCl to 100 mmol/L in the digestion buffer. Internucleosomal cleavage of DNA was mostly, although not invariably, inhibited by the use of a digestion buffer containing 10 mmol/L Tris, 25 mmol/L EDTA, and 100 mmol/L NaCl. "Apoptotic" chromatin changes (internucleosomal fragmentation) are not always associated with histologic evidence of apoptosis and may occur artifactually.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Internucleosomal cleavage of DNA as the sole criterion for apoptosis may be artifactual. 803 5

Wistar rats, eight days old, were subjected to permanent bilateral forebrain ischemia, followed by hypoxia for 15 minutes. A cerebral infarct, mainly involving the cerebral neocortex, hippocampus, amygdala, striatum and subcortical white matter was produced. Neurons and glia showing punctate chromatin condensation and karyorrhectic cells were observed 12 hours after hypoxia-ischemia. Their number increased during the first two days and recruitment of cells with degenerating nuclei occurred until day five. In situ labeling of nuclear DNA fragmentation stained many normal-appearing nuclei, as well as punctate chromatin condensations and nuclear fragments in karyorrhectic cells. Delayed neuronal death in the CA1 area of the hippocampus was observed after 20 minutes of transient forebrain ischemia in the adult gerbil. In situ labeling of nuclear DNA fragmentation demonstrated stained punctate chromatin condensation in a few degenerating cells at 48 hours post-ischemia. Substantial labeling of CA1 neurons occurred in the fourth day. Agarose gel electrophoresis of extracted brain DNA from ischemic infant rats and adult gerbils showed a ladder-type pattern which is typical of nuclear DNA fragmentation into oligonucleosomal fragments (internucleosomal cleavage). These findings suggest that endonuclease(s) activation may play a role in cell death induced by different forms of hypoxia-ischemia.
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PMID:Evidence of nuclear DNA fragmentation following hypoxia-ischemia in the infant rat brain, and transient forebrain ischemia in the adult gerbil. 806 57

The endoplasmic reticular Ca(2+)-ATPase inhibitor, thapsigargin, was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition, confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus, we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis.
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PMID:Intracellular Ca2+ signals activate apoptosis in thymocytes: studies using the Ca(2+)-ATPase inhibitor thapsigargin. 817 45

The effects of selenite on DNA integrity, cell viability, and long-term proliferative potential of mouse leukemic L1210 cells were examined in this study. Selenite treatment resulted in concentration-dependent increases in DNA single-strand breaks and double-strand breaks, as detected by a modified filter elution assay. A time-course experiment showed that DNA single-strand breaks preceded DNA double-strand breaks. Agarose gel electrophoresis of DNA extracted from selenite-treated cells displayed a nucleosomal fragmentation pattern that is characteristic of apoptotic cell death. The involvement of a Ca2+,Mg(2+)-dependent endonuclease responsible for DNA double-strand fragmentation was implied by the observation that two inhibitors of endonuclease activity, i.e. aurintricarboxylic acid and zinc, blocked selenite-induced DNA double-strand breaks. These inhibitors also prevented selenite-induced cell death as defined by loss of ability to exclude trypan blue dye. Selenite treatment severely impaired the colony-forming ability of cells capable of trypan blue exclusion. The induction of DNA strand breaks and commitment to apoptosis may explain the selenite-mediated growth inhibition and loss of long-term proliferative potential.
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PMID:Selenite induction of DNA strand breaks and apoptosis in mouse leukemic L1210 cells. 818 64

Camptothecin (CPT) has been recognized as a topoisomerase I (Topo I) inhibitor. However, the mechanism of cytotoxicity of this agent remains unknown. In the present study, we analyzed the kinetics of Topo I-mediated DNA single-strand breaks and internucleosomal DNA cleavage produced by CPT and its derivative, 7-ethyl-10-hydroxycamptothecin (SN-38), in HL-60 cells. DNA single-strand breaks were detected using alkaline sucrose gradient centrifugation when HL-60 cells were incubated with 10 microM CPT or 10 microM SN-38 for 30 min. These DNA single-strand breaks were rapidly repaired after drug removal, while the cytotoxic action of these drugs was sustained. Treatment of HL-60 cells with CPT or SN-38 for 3 h produced extensive degradation of DNA. Agarose gel electrophoresis showed a ladder of DNA fragments consisted of multimers of approximately 200 base pairs, characteristic of apoptosis. Interestingly, this type of DNA fragmentation was also induced within 4 h after repair of DNA single-strand breaks, and subsequently loss of cell viability was observed. When zinc ion, a potent inhibitor of endonuclease, was added to drug-free medium after treatment with CPT or SN-38, internucleosomal DNA cleavage was abolished. Furthermore, addition of zinc ion reduced the loss of cell viability. These data suggest that Topo I-mediated DNA single-strand breaks may be necessary but are not sufficient for cell death, and the endonuclease involved in induction of internucleosomal DNA cleavage may play an important role in HL-60 cell death induced by Topo I inhibitor.
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PMID:DNA damage and cell killing by camptothecin and its derivative in human leukemia HL-60 cells. 839 26

The appearance of G- and C-banding patterns on metaphase chromosomes from the Don cell line (Cricetulus griseus) was accompanied by extraction of chromosomal DNA. A microdensitometric technique was employed to measure the loss of DNA produced by Hin dIII, Hae III and Eco RI endonuclease digestion on fixed chromosomes, from 0.5 to 24 h of treatment. Agarose gel electrophoresis was carried out to estimate the size of the DNA fragments extracted after digestion by Alu I and the restriction endonucleases already mentioned. Results obtained show that Alu I and Hae III, which possess 4 base pair recognition sequences, caused higher DNA extraction than enzymes with recognition targets of 6 base pairs (Hin dIII and Eco RI). Incubation buffers induced G-banding patterns which were accompanied by DNA extraction, although this loss was lower than that produced by endonuclease treatments.
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PMID:Chromosomal DNA extraction after restriction endonuclease treatments: a study by microdensitometry and electrophoresis. 840 28

Sixty Serratia marcescens isolates were obtained from patient specimens from three different hospitals in the city of Riyadh. These were tested for their antibiotic resistance factors using eleven different antibiotics. Their ability to transfer antibiotic resistance plasmids to a sensitive E. coli strain (RR1) was tested by transformation and conjugation experiments. Agarose gel electrophoresis was used to determine the size and number of the R-plasmids. Southern blotting was used to assess homologies between antibiotic resistance plasmids from different isolates. Among the isolates tested, 36.7% contained plasmids, and all these were from strains isolated from two hospitals. No R-plasmids could be detected among the multiple resistant strains isolated from the third hospital. Among the strains that contained plasmids, approximately 63.6% transferred multiple antibiotic resistance to E. coli and the rest transferred only one antibiotic resistance marker. The majority of strains carrying out the plasmids showed similarities in band number and size. In view of the similarities this group was denoted the predominant group, and selected for further molecular investigations. Restriction endonuclease digests of plasmids from this group gave the same restriction pattern which confirmed that they were closely related. Hybridization experiments using these plasmids and nick-translated 32p-labelled pBR-322 DNA probe, showed that all the large bands (36 kb) are related and exhibit homology with pBR-322.
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PMID:Non-transmissible and self-transmissible plasmids conferring drug resistance in clinical isolates of Serratia marcescens from hospitals in Riyadh, Saudi Arabia. 846 72

Previous approaches to mutation detection in mRNA from the neurofibromatosis 1 (NF1) locus have required the PCR amplification of five or more overlapping cDNA segments to screen the entire 8.5-kb open reading frame (ORF). Systematically, these assays do not detect deletions that span the region of overlap (usually 1-3 exons) of any two consecutive target segments. In such cases, amplification from the mutant region of the disease-causing allele fails because binding sites for the PCR primers are missing, but amplification from the normal allele proceeds, yielding only the normal product. To alleviate this problem, we have developed a protocol to reverse transcribe and amplify the entire protein-coding sequence of NF1 as a single PCR product, starting with total RNA from lymphoblast cell lines or from whole blood. The 8.7-kb RT-PCR product was prepared from nine NF1 patients with known deletions or insertions, ranging in size from a 30-bp deletion within 1 exon to a 2.4-kb deletion that removes 12 exons. Agarose gel analysis of the initial products detected deletions as small as 341 bp. Restriction endonuclease digestion with Asel and Fspl, followed by agarose gel electrophoresis, revealed the predicted abnormal bands in all nine patients. All mutant bands were identified readily by observers with no knowledge of the patients' mutations. This simple assay should detect a great variety of insertion/deletion mutations in the NF1mRNA internal to the primer binding sites, including all possible single and multiple exon dropouts and approximately 30% of all previously reported NF1 mutations.
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PMID:Long RT-PCR of the entire 8.5-kb NF1 open reading frame and mutation detection on agarose gels. 868 Nov 40

The apparent biological significance of DNA double-strand breaks (DSBs) has stimulated considerable effort toward quantification of this lesion. The neutral (or nondenaturing) filter elution assay at pH 7.2 or 9.6 has long been a standard method for the measurement of double-strand breakage and rejoining in eukaryotic cells, with a threshold dose for detection of DSBs of 5-10 Gy. Agarose gel electrophoresis, either pulsed- or constant-field, can detect DSBs induced by as little as 1 Gy of ionizing radiation, but electrophoresis assays may have inherent problems in measurement of break rejoining, and may be more susceptible than elution to factors other than break frequency, such as cell cycle stage or bromodeoxyuridine substitution. We report here that filter elution performed at pH 11.1 can detect DSBs produced by only 1 Gy of ionizing radiation, but is insensitive to the single-strand breaks that are formed when cells are exposed to hydrogen peroxide. Double-strand breaks produced in permeabilized cells by the restriction endonuclease HaeIII were used to demonstrate that the increase in the pH of the eluting solution from 9.6 to 11.1, although increasing assay sensitivity by a factor of five, converts few additional alkali-labile sites to DSBs. Thus validated, the pH 11.1 filter elution assay was applied to a low-dose measurement of induction and rejoining of DSBs in 9L cells.
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PMID:Subdenaturing (pH 11.1) filter elution: more sensitive quantification of DNA double-strand breaks. 914 2


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