Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the development of a PCR-based assay for the detection of microsporidia in clinical specimens. A single primer pair complementary to conserved sequences of the small-subunit rRNA enabled amplification of DNA from the four major microsporidian pathogens of humans: Encephalitozoon cuniculi, Encephalitozoon hellem, Enterocytozoon bieneusi, and Septata intestinalis. The extraction method allowed PCR amplification of E. bieneusi and S. intestinalis DNA from sodium hypochlorite-treated stool specimens. Differentiation of the microsporidian gastrointestinal pathogens E. bieneusi and S. intestinalis could be accomplished by restriction endonuclease digestion of PCR products using PstI and HaeIII.
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PMID:Identification of microsporidia in stool specimens by using PCR and restriction endonucleases. 766 39

Apurinic endonuclease (APE; also known as Ref-1 protein) is a key enzyme in base excision repair, cleaving apurinic sites that arise spontaneously because of the activity of DNA glycosylases. To address the question of whether APE can be modulated by genotoxic stress affecting cellular protection, we analyzed the expression of APE in Chinese hamster ovary (CHO) cells after treatment with various genotoxic agents. We show that treatment of CHO cells with hydrogen peroxide (H2O2) or sodium hypochlorite (NaOCl) increases the levels of APE mRNA and protein. APE induction was observed 3-9 h after treatment and was accompanied by an increase in APE activity. We also show that the cloned human APE promoter transfected into CHO cells is stimulated by the oxidants, indicating transcriptional activation of the APE gene. When cells were pretreated with NaOCl, inducing APE, and then challenged with H2O2, the clastogenic effect of the challenge dose was significantly reduced, suggesting clastogenic adaptation due to APE induction. To further prove the involvement of APE in adaptation against induced chromosomal breakage, we transfected human APE cDNA driven by an inducible promoter into CHO cells and observed that transient induction of APE reduced the clastogenic effect of H2O2. Overall, the data demonstrate that the APE gene can be activated by oxidative agents, resulting in a transient increase in APE repair activity, which reduces the clastogenic response of cells to an oxidative agent. The protection of cells from chromosomal aberrations seen after prior exposure to oxidants is attributed to an adaptive response to oxidative stress.
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PMID:Apurinic endonuclease (Ref-1) is induced in mammalian cells by oxidative stress and involved in clastogenic adaptation. 976 71

Reactive oxygen species (ROS) arise through normal cellular aerobic respiration, and, in combination with external sources such as ionizing radiation, cigarette tar and smoke, and particulate matter generated by combustion, can have a profound negative effect on cellular macromolecules such as DNA that may lead to a number of human pathological disorders including accelerated aging and cancer. A major end product of ROS damage to DNA is the formation of apurinic/apyrimidinic (AP) sites, which without removal are known to halt mRNA and DNA synthesis, or act as non-coding lesions resulting in the increased generation of DNA mutations. In human cells, the major enzyme in correcting the deleterious effects of AP sites in DNA is through the participation of AP endonuclease (APE), which initiates the removal of baseless sites in DNA through the catalytic scission of the phosphodiester bond 5' and adjacent to an AP site. Interestingly, APE also possesses an activity (Ref-1) that controls the redox status of a number of transcription factors including Fos and Jun. The means by which APE/Ref-1 is directed to carry out such disparate roles are unknown. The presence of a number of phosphorylation sites scattered throughout both functional domains of APE/Ref-1 however offered one possible mechanism that we reasoned could play a role in dictating how this protein responds to different stimuli. Here we show that the in vitro redox activity of APE/Ref-1 is stimulated by PKC phosphorylation. Furthermore, when human cells were exposed to the PKC activator phorbol 12-myristate 13-acetate, an increase in redox activity was observed that corresponded to an increase in the phosphorylation status of APE/Ref-1. Importantly, human cells exposed to the oxidizing agent hypochlorite, followed by methyl methanesulfanate, responded with an increase in redox activity by APE/Ref-1 that also involved an increase in PKC activity and a corresponding increase in the phosphorylation of APE/Ref-1. These results suggest that the ability of APE/Ref-1 to perform its in vivo redox function is correlated to its susceptibility to PKC phosphorylation that notably occurs in response to DNA damaging agents.
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PMID:Activation of APE/Ref-1 redox activity is mediated by reactive oxygen species and PKC phosphorylation. 1145 37

Although a large body of evidence supports a causative link between oxidative stress and neurodegeneration, the mechanisms are still elusive. We have recently demonstrated that hydrogen peroxide (H(2)O(2)), the major mediator of oxidative stress triggers higher order chromatin degradation (HOCD), i.e. excision of chromatin loops at the matrix attachment regions (MARs). The present study was designed to determine the specificity of H(2)O(2) in respect to HOCD induction. Rat glioma C6 cells were exposed to H(2)O(2) and other oxidants, and the fragmentation of genomic DNA was assessed by field inversion gel electrophoresis (FIGE). S1 digestion before FIGE was used to detect single strand fragmentation. The exposure of C6 cells to H(2)O(2) induced a rapid and extensive HOCD. Thus, within 30 min, total chromatin was single strandedly digested into 50 kb fragments. Evident HOCD was elicited by H(2)O(2) at concentrations as low as 5 micro M. HOCD was mostly reversible during 4-8h following the removal of H(2)O(2) from the medium indicating an efficient relegation of the chromatin fragments. No HOCD was induced by H(2)O(2) in isolated nuclei indicating that HOCD-endonuclease is activated indirectly by cytoplasmic signal pathways triggered by H(2)O(2). The exposure of cells to a synthetic peroxide, i.e. tert-butyrylhydroperoxide (tBH) also induced HOCD, but to a lesser extent than H(2)O(2). Contrary to the peroxides, the exposure of cells to equitoxic concentration of hypochlorite and spermine NONOate, a nitric oxide generator, failed to induce rapid HOCD. These results indicate that rapid HOCD is not a result of oxidative stress per se, but is rather triggered by signaling cascades initiated specifically by H(2)O(2). Furthermore, the rapid and extensive HOCD was observed in several rat and human cell lines challenged with H(2)O(2), indicating that the process is not restricted to glial cells, but rather represents a general response of cells to H(2)O(2).
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PMID:Hydrogen peroxide mediates higher order chromatin degradation. 1242 92