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)

We have isolated and characterized several overlapping clones from two human genomic libraries constructed in cosmid and bacteriophage vectors. They span about 80 kbp and include the entire human cytosolic aldehyde dehydrogenase (ALDH1) gene. Restriction endonuclease mapping, Southern blotting with cDNA and specific oligonucleotide probes, and DNA sequencing were performed to analyze the cloned genomic DNA. The ALDH1 gene is about 53 kbp long and is divided into 13 exons which encode 501 amino acid residues. Primer extension results defined the transcription initiation site to 53 bp upstream from the A of the initiation codon ATG. The promoter region of the gene contains an ATA box and a CCAAT box, which are located 32 and 74 bp upstream, respectively, from the transcription initiation site. The possible functional domains of the protein encoded by exons are discussed. A similar intron-exon organization between the genes of cytosolic ALDH1 and its mitochondrial ALDH2 isozyme in which both enzymes are encoded by 13 exons and 9 of the 12 introns interrupt the coding sequence at homologous positions was observed. This is consistent with the model that the two isozyme genes evolved after the duplication of a common ancestor gene.
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PMID:Genomic structure of the human cytosolic aldehyde dehydrogenase gene. 259 67

The PvuII restriction-modification system has been found to contain three genes which code for a DNA methyltransferase (MTase), a restriction endonuclease (ENase) and a small protein required for expression of the ENase-encoding gene. In addition, there is a small open reading frame (ORF) within and opposite to the MTase-encoding gene. The region containing this ORF is transcribed, and the ORF has an excellent Shine-Dalgarno sequence with an ATA start codon. A closely related ORF is present in the SmaI system. The 28-amino-acid (aa) predicted peptide from the PvuII ORF resembles a region of the PvuII ENase at the dimer interface. We have cloned this ORF, giving it an ATG start codon and putting it under the control of an inducible promoter: induction leads to a slight but significant decrease in restriction of bacteriophage lambda. We also have obtained the 28-aa synthetic peptide, and are exploring the possibility that it modulates ENase subunit association. While this peptide has no detectable effect on dimeric PvuII ENase, it inhibits renaturation of urea-denatured ENase in a concentration-dependent manner. The ORF may represent an additional safeguard during establishment of the PvuII restriction-modification system in a new host cell, helping to delay the appearance of active ENase dimers, while the MTase accumulates and protects the host chromosome.
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PMID:Gene pvuIIW: a possible modulator of PvuII endonuclease subunit association. 760 91

A murine fibrosarcoma clone, Gc-4 SD, grows depending on fetal calf serum. In MTT assay, protein-free cultivation resulted in a reduction of the viable cell number time-dependently. Electron-microscopic and flow-cytometric analyses revealed that the reduction in growth was accompanied by the appearance of apoptotic cells. However, no internucleosomal fragmentation was observed even after SI-nuclease treatment. On the other hand, pulse field gel electrophoresis revealed that cleavage of DNA into high-molecular-weight fragments estimated as 50 to 150 kilobase pairs (kbp), with a peak of 100 kbp, was found in the serum-deprived cells. Large fragments disappeared from the DNA extracts when the smaller cells with high blue fluorescence with Hoechst 33342 were removed by flow cytometry, suggesting direct correlation between the large DNA fragmentation and apoptosis. The addition of aurintricarboxylic acid neither abolished the large DNA fragmentation nor inhibited the reduction in the number of viable cells. Both cycloheximide and actinomycin D enhanced the reduction in the number of viable cells as well as the large DNA fragmentation. These results suggest that apoptosis of a fibrosarcoma induced by protein-free culture involves a specific endogenous endonuclease, which may be distinct from and independent of the ATA-sensitive endonuclease producing internucleosomal DNA fragmentation.
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PMID:Apoptosis of a fibrosarcoma induced by protein-free culture involves DNA cleavage to large fragments but not internucleosomal fragmentation. 762 95

Serum-free PC12 cell cultures have been used to study the mechanisms of neuronal death after neurotrophic factor deprivation. We previously reported that PC12 cells undergo "apoptotic" internucleosomal DNA cleavage after withdrawal of trophic support. Here, we have used a sensitive method to detect PC12 cell DNA fragmentation within three hrs of serum removal and have exploited this assay to examine several aspects regarding the mechanisms of neuronal survival/death. Major advantages of this assay are that it permits acute experiments to be performed well before other manifest signs of cell death and under conditions that cannot be applied chronically. We find that this apopotic DNA fragmentation is distinct from the random DNA degradation that occurs during necrotic death. Major observations include the following: (a) There is a good correlation between the ability of trophic substances to promote PC12 cell survival and to inhibit early DNA fragmentation. (b) Phorbol ester, an activator of PKC, acutely suppresses DNA fragmentation, but does not promote long-term survival or inhibition of endonuclease activity when applied chronically due to its downregulation of PKC. (c) Cells undergoing apoptosis within 3 h of serum withdrawal have a "commitment point" of only 1.0-1.5 h beyond which they can no longer be rescued by NGF. (d) Aurin, a non-carboxylic analog of the endonuclease inhibitor ATA, also inhibits DNA fragmentation and promotes short-term survival of PC12 cells. (e) Macromolecular synthesis is not required for DNA fragmentation or for NGF to prevent this event. (f) Extracellular Ca2+ is not required for internucleosomal DNA cleavage caused by serum withdrawal or for suppression of this by NGF. (g) DNA fragmentation can also be detected in cultures of rat sympathetic neurons as early as 10 h after removal of NGF. As in PC12 cell cultures, this precedes morphological signs of cell death.
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PMID:Internucleosomal DNA cleavage and neuronal cell survival/death. 768 3

The effect of cisplatin and its non-cross resistant analogue, liposomal cis-bis-neodecanoato-trans-R,R-1,2-diamiconocyclohexaneplatinum (II) (L-NDDP) on inducing internucleosomal DNA fragmentation and cell death was examined in A2780 and A2780/PDD cells. In A2780 cells, both drugs were markedly effective in inducing DNA fragmentation, whereas in A2780/PDD cells, only L-NDDP produced significant DNA fragmentation, in good correlation with the observed cytotoxicity. The endonuclease inhibitor (ATA) prevented the DNA fragmentation caused by high (30-60 microM) or low (3-10 microM) concentrations of each drug in A2780 cells. In contrast, the protein synthesis inhibitor (CHX) displayed only a significant inhibitory effect on the DNA fragmentation caused by low concentrations at 48 h post-treatment. These results indicate that there are at least two different pathways leading to both drugs induced-cell death depending on drug concentrations in this cell line.
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PMID:Cell death and DNA fragmentation induced by liposomal platinum(II) complex, L-NDDP in A2780 and A2780/PDD cells. 801 41

Cyanide inhibits the mitochondrial respiratory chain enzyme cytochrome oxidase causing histotoxic hypoxia. It is primarily considered as a neurotoxin but its other toxic manifestations are also well documented. Cyanide-induced apoptosis in neuronal cells has also been demonstrated recently. At the same time we also reported that potassium cyanide (KCN) produces extensive cytotoxicity and DNA fragmentation in rat thymocytes. The DNA damage was sensitive to elevated levels of extracellular Ca2+ and was attenuated by Zn2+ (modulator of Ca2+ dependent endonuclease), N-acetylcysteine (free radical scavenger) and diltiazem (Ca2+ channel blocker). In a continuation of this work, in the present study we have shown that the cytotoxicity and DNA fragmentation induced by 5 mM KCN was preceded by loss of mitochondrial integrity (MTT assay and rhodamine-123 staining) and nuclear viability (propidium iodide uptake) which were mediated by generation of reactive oxygen species (DCHF-DA staining). The DNA damage was also accompanied by nuclear fragmentation (Hoechst 33342 staining), a phenomenon that characterises the 'apoptotic' type of cell death. The in vitro toxic insult of KCN was challenged by pre-treatment (0.5 h), simultaneous treatment or post-treatment (0.5-3 h) of various pharmacological agents viz., Trolox (antioxidant), EGTA (Ca2+ modulator) and aurintricarboxylic acid (ATA; Ca2+/Mg2+ dependent endonuclease inhibitor). In addition, Quercetin (antioxidant) was tested as simultaneous treatment alone and was found to be ineffective. On the basis of various biochemical indices and DNA fragmentation (quantitative and qualitative), simultaneous treatment of Trolox was found to be the most effective in attenuating cyanide toxicity in vitro. This protection can be attributed to interventions in oxidative stress-mediated cell injury which is an early event preceding DNA damage. Both EGTA and ATA could not prevent this damage. Trolox also increased the LD(50) of KCN in mice 2.5-fold as compared to 1.8- and 1.6-fold for EGTA and ATA, respectively.
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PMID:Pharmacological interventions of cyanide-induced cytotoxicity and DNA damage in isolated rat thymocytes and their protective efficacy in vivo. 1127 22

Diethyldithiocarbamate (DDTC) has been shown to induce cytotoxicity in several different systems. We examined whether the DDTC-induced cytotoxicity was via apoptosis, or in relation to intracellular glutathione (GSH) in various murine and human leukemia cell lines. The cells most sensitive to DDTC-induced cytotoxicity were P388 lymphoid neoplasma cells and NALM-6, a B cell line of acute lymphocytic leukemia (ALL). The next level of susceptible cells included J774.1, having a macrophage function, HL-60 premyelocytic leukemia cells, MOLT-4, an acute lymphoblastic leukemia cell, and Jurkat, a T-cell leukemia. U937 (expressing many monocyte-like characteristics), K562 erythroleukemia and K562/DXR (a multidrug-resistant clone derived from K562) were almost unaffected by DDTC. P388 was also highly susceptible to H(2)O(2), a most useful exogenous reactive oxygen species generator, and was lower in intracellular total GSH content than other leukemia cells. DDTC-induced cytotoxicity was closely related to intracellular GSH, but the level of cellular GSH did not always correlate with H(2)O(2)-induced cytotoxicity in this experiment. K562 had a higher intracellular total GSH content and showed lower susceptibility to DDTC and H(2)O(2), but with the combination of DDTC and DL-buthionine-(S,R)-sulfoximine (BSO), cytotoxicity increased significantly. The ratio of GSH/GSSG in P388 was reduced by DDTC or H(2)O(2). H(2)O(2)-induced cytotoxicity was completely blocked by catalase (CAT), while it was enhanced by superoxide dismutase (SOD). CAT or SOD did not affect DDTC-induced cytotoxicity. N-Acetylcysteine (NAC: 1 mM), a vanguard substance of GSH, and aurintricarboxylic acid (ATA: 100 microM), an endonuclease inhibitor, ameliorated DDTC-induced cytotoxicity and apoptosis. In conclusion, we suggest that DDTC-induced cytotoxicity was via an oxidative shift in the intracellular redox state, and accompanied the activation of endonuclease through apoptosis in leukemia cell lines.
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PMID:Diethyldithiocarbamate-induced cytotoxicity and apoptosis in leukemia cell lines. 1284 19

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