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)

The ability of glucocorticoids to induce apoptosis or programmed cell death in mouse thymocytes is well-established. Measurement of apoptosis-associated internucleosomal DNA fragmentation through determination of the percentage of fragmented DNA by electrophoresis or centrifugation of whole cell lysates is by far the most common means of quantifying apoptosis. Since these methods measure DNA fragmentation in whole cell lysates rather than intact cells, they have severe limitations, particularly with heterogeneous cell populations. When mouse thymocytes were incubated with glucocorticoids, fixed, stained with propidium iodide and analysed flow cytometrically for cell cycle distribution, a distinct subpopulation of cells was observed to form below the G0/G1 region, denoted as the A0 region. The presence of cells in this region was consistent with the presence of internucleosomal DNA fragments as determined by gel electrophoresis. Inhibitors of transcription, translation and endonuclease activity, and a glucocorticoid receptor antagonist prevented accumulation of cells in this region. Irradiation of mouse thymocytes also produced a population in the A0 region. Cells in this region are believed to have undergone glucocorticoid-induced DNA fragmentation. This method represents a useful alternative to whole cell lysate assays, since apoptosis can be evaluated on an individual cell basis.
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PMID:Evaluation of glucocorticoid-induced DNA fragmentation in mouse thymocytes by flow cytometry. 165 18

Restriction endonuclease fragment length variations (RFLV) were detected in mice with DNA probes for myelin basic protein (Mbp), glucocorticoid receptor-1 (Grl-1), and Friend MuLV integration site-2 (Fim-2). RFLV of the Mbp gene were found in SacI restriction patterns, RFLV of the Grl-1 gene were found in EcoRV patterns, and RFLV of the Fim-2 were found in BglII patterns. A three-point backcross was carried out by the backcross mating (C57BL/KsJ-spm/spm x MOL-MIT)F1 males x C57BL/KsJ-spm/spm; spm is an autosomal recessive gene causing sphingomyelinosis. From the results, spm, Grl-1, Fim-2, and Mbp loci were mapped on chromosome 18, and the following order of genes is proposed, with distances between genes in parentheses: centromere--spm--(7.8 cM)--Grl-1--(7.8 cM)--Fim-2--(39.1 cM)--Mbp--telomere. All laboratory strains and two European subspecies (Mus mus domesticus and M. m. brevirostris) carry the Grl-1a, Fim-2a, and Mbpa alleles. In contrast, another wild subspecies from Europe (M. m. musculus) and some Asian subspecies (M. m. molossinus, Chinese mice of wild origin, and M. m. yamashinai) carry the Grl-1b, Fim-2b, and Mbpb alleles. Only castaneus strains carry the intermediate combination of the Grl-1b, Fim-2a, and Mbpb alleles.
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PMID:A molecular genetic linkage map of mouse chromosome 18, including spm, Grl-1, Fim-2/c-fms, and Mbp. 167 25

Apoptosis is a programmed form of cell death that occurs under numerous physiological conditions, including endocrine regulation of specific cell populations. We have investigated the biochemical mechanisms involved in glucocorticoid-induced apoptosis in rat thymocytes. Internucleosomal cleavage of chromatin into oligonucleosomal fragments is common to all forms of apoptosis and precedes the onset of cell death. To identify the endonuclease that is responsible for the specific pattern of DNA degradation in glucocorticoid-induced apoptosis, we have developed an assay to measure internucleosomal cleavage activity in thymocyte nuclear extracts. This assay uses nuclei from cells resistant to hormone-induced DNA fragmentation (HeLa cells) as a substrate for nuclear extracts prepared from thymocytes of adrenalectomized rats treated with either dexamethasone (dex) or vehicle (control). After incubation at room temperature for 90 min, the HeLa DNA is purified, and its integrity is analyzed by agarose gel electrophoresis. The appearance of internucleosomal fragments of HeLa DNA is indicative of nuclease activity in the thymocyte nuclear extract. Nuclear extracts prepared from thymocytes of rats treated with dex for 5 h caused internucleosomal cleavage of HeLa DNA, whereas extracts from control rats did not result in any DNA fragmentation. Regulation of nuclease activity by dex was time dependent. Internucleosomal cleavage activity in thymocyte extract from dex-treated animals was detected as early as 2 h after hormone treatment and occurred before any detectable change in cell viability. Maximal extractable nuclease activity was coincident with decreased thymocyte viability and thymic involution. In contrast, extracts from medullary thymocytes, which are the only thymocytes that survive 72 h of glucocorticoid treatment, did not contain nuclease activity by this assay. Regulation of internucleosomal cleavage activity by dex was dose dependent and was specific for the glucocorticoid class of steroid hormones. Furthermore, the dex-induced response was inhibited by pretreating rats with the glucocorticoid receptor antagonist RU486, indicating that receptor-mediated processes are involved in the regulation of nuclease activity. The similarities between the regulation of internucleosomal cleavage activity reported here and the previously described degradation of thymocyte DNA in vivo makes this nuclease a likely constituent of the apoptotic process.
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PMID:Internucleosomal deoxyribonucleic acid cleavage activity in apoptotic thymocytes: detection and endocrine regulation. 198 52

The cytosolic untransformed molybdate-stabilized glucocorticoid-receptor complex from rat liver was eluted as a heterogenous peak containing two components with Stokes radii (Rs) of 8.3 nm and 7.1 nm when analyzed by size-exclusion HPLC even in the absence of molybdate. In contrast, the highly purified glucocorticoid receptor yielded a sharp symmetrical peak of Rs = 7.1 nm. We demonstrate that the 7.1-nm component could not result from a proteolytic degradation of the 8.3-nm receptor form. The same receptor heterogeneity was observed in thymus cytosol which contains less proteases than liver. After labeling with [3H]dexamethasone 21-mesylate and SDS/PAGE the same 94-kDa receptor band was revealed in both the 8.3-nm and 7.1-nm forms. Immunoblotting experiments showed that both the 94-kDa hormone-binding subunit and the 90-kDa heat-shock protein were present in the two different receptor forms. The 8.3-nm receptor form was converted to the 7.1-nm receptor form after treatment by ribonuclease A in the presence of molybdate and this effect was dose-dependent, being completely prevented by placental ribonuclease inhibitor (RNasin). In contrast, in the presence of molybdate, the 7.1-nm receptor form was ribonuclease-insensitive. Treatment of cytosol with RNase A in the absence of molybdate, partially shifted the untransformed receptor towards the 5.2-nm transformed receptor form. This effect was abolished by placental ribonuclease inhibitor. RNase S protein, an enzymatically inactive proteolytic fragment of RNase A, or S1 nuclease, which is specific for single-stranded nucleic acids, were ineffective when used instead of RNase A. In contrast, cobra venom endonuclease, which preferentially attacks double-stranded regions of small RNAs, caused a complete conversion of the 7-8-nm untransformed receptor to the 5.2-nm transformed receptor form. These results were not observed in the presence of molybdate. Addition of RNasin prior to heating cytosol in the absence of molybdate did not prevent the receptor from dissociating to the 5.2-nm form, suggesting that an endogenous RNase is not involved in the transformation process. The 7.1-nm receptor form was shifted to a 9.2-nm complex when incubated with an excess of GR 49 antireceptor antibody, whereas the 8.3-nm receptor form did not bind to the antibody.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:RNA binding to the untransformed glucocorticoid receptor. Sensitivity to substrate-specific ribonucleases and characterization of a ribonucleic acid associated with the purified receptor. 246 3

In the IL-2-dependent T cell clone CTLL-2, dexamethasone, a synthetic glucocorticoid, induces a suicide program characterized by the early degradation of chromatin in oligonucleosome-length fragments which precedes the loss of cell viability by 2 to 4 h. These effects are most likely mediated through the interaction with a specific glucocorticoid receptor as suggested by the structure-activity relationship of the various steroids tested. Incubation of nuclei of glucocorticoid-untreated cells in the presence of calcium and magnesium ions induces the cleavage of DNA in the linker region between nucleosomes, suggesting that fragmentation of chromatin in intact cells by glucocorticoids may involve the activation of a preexisting endonuclease. Interestingly, the presence of a saturating dose of IL-2 during the treatment of CTLL-2 cells with glucocorticoids completely blocks the cell death program.
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PMID:IL-2 protects T lymphocytes from glucocorticoid-induced DNA fragmentation and cell death. 255 79

Glucocorticoid-induced apoptosis in the murine interleukin-2-dependent T-cell line CTLL-2 and in freshly isolated thymocytes was studied. It was demonstrated here that in CTLL-2 cells, dexamethasone (methyl in position 16 alpha) was more efficient in inducing apoptosis than betamethasone (methyl in position 16 beta) or triamcinolone (hydroxyl in position 16). In contrast, no such difference between these three molecules was found in murine thymocytes. In addition, we showed that glucocorticoid-induced apoptosis on the two models was mediated through interaction with the glucocorticoid receptor and did not occur in the presence of inhibitors of transcription, translation or an endonuclease-inhibitor. Furthermore, in CTLL-2 cells, apoptosis took place in the presence of EGTA whereas it was prevented in murine thymocytes, thus indicating that calcium plays a different role in these two models. Finally, higher concentrations of interleukin-2 were needed to protect CTLL-2 cells against dexamethasone-induced apoptosis than that induced by betamethasone or triamcinolone. Thus, structural differences at position 16 of the steroid nucleus correlate with a different apoptosis-inducing activity by glucocorticoids which, however, is only evidenced in the calcium-independent CTLL-2 model.
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PMID:Structure-activity relationships in glucocorticoid-induced apoptosis in T lymphocytes. 760 35

The present study was undertaken to determine whether specific interactions between cAMP and glucocorticoids regulate apoptosis in thymocytes. Incubation of murine thymocytes with agents that elevate the cAMP level resulted in enhancement of glucocorticoid-induced Ca2+ increases, DNA fragmentation, and cell death compared to levels observed in thymocytes treated with steroid alone. cAMP did not affect DNA fragmentation in thymocytes treated with Ca2+ ionophore, a compound that induces endonuclease activation via an independent mechanism. Treatment with cAMP also increased glucocorticoid potency by lowering the concentration of steroid required for induction of apoptosis. The mechanism of cAMP action appeared to involve the glucocorticoid receptor, since the glucocorticoid antagonist RU-486 abrogated the cAMP response in animals treated with the adenosine analog NECA in vivo. Analysis of cellular glucocorticoid binding and receptor protein levels revealed modest cAMP-stimulated increases that appeared insufficient to account for the effects of cAMP on endogenous endonuclease activation, suggesting the possible involvement of a posttranslational mechanism in the response. These results demonstrate that cAMP and glucocorticoids synergize to promote apoptosis in thymocytes via a mechanism that appears to involve modification of glucocorticoid receptor activity.
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PMID:Cyclic AMP potentiates glucocorticoid-induced endogenous endonuclease activation in thymocytes. 838 20

We tested the hypothesis that the previously observed loss of thymic lymphocytes in mice after treatment with time-release morphine pellets was occurring through the process of apoptosis. Apoptosis is a form of cell death, distinct from necrosis, which involves a specific endonuclease that fragments the cell's own DNA. Forty-eight hours after implantation of a time-release morphine pellet in B6C3F1 mice, thymus weight and cellularity was reduced to 30% of that observed in placebo-treated mice. Thymocytes from morphine pellet-treated mice were found to have a significantly greater percentage of their DNA fragmented than did thymocytes from either placebo pellet-implanted or naive control mice. The peak level of DNA fragmentation was found to occur approximately 12 hr postpellet implant. When separated on agarose gels, the sizes of the DNA fragments observed corresponded to the multiples of 180 base pairs which are characteristic of apoptosis. In vivo, the use of either the opiate receptor antagonist naloxone, or the glucocorticoid receptor antagonist RU-38486, was able to block completely the morphine mediated increase in thymocyte apoptosis. In vitro experiments in which thymocytes were cultured with morphine concentrations as high as 10(-4) M showed no evidence of an increased rate of DNA fragmentation. These data indicate that both opiate and glucocorticoid receptors are involved in morphine-induced apoptosis and that the opiate receptor responsible is not located on the thymic lymphocytes.
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PMID:Morphine induces apoptosis in murine thymocytes in vivo but not in vitro: involvement of both opiate and glucocorticoid receptors. 839 61

In recent years much has been learned about the cellular and molecular events underlying cerebral hypoxia-ischemia (HI). We review, from a molecular standpoint, the main pathogenetic theories in hypoxic-ischemic cerebral injury, including excitotoxicity, free radical damage, and the role of growth factors, proto-oncogenes and heat shock proteins. The various forms of cell death in the developing and adult brain (necrosis, apoptosis and delayed neuronal death) are reviewed, with an emphasis on gene regulation of naturally-occurring and HI-associated cell death. We report the expression of the immediate early gene c-fos and c-jun mRNAs and of HSP72 mRNA and protein in several models of cerebral HI. Gel agarose electrophoresis of extracted DNA and in situ end-labeling of fragmented DNA revealed that cell death in these models was associated with endonuclease(s) activation. We also pre-treated some animals with dexamethasone, a neuroprotective drug in a model of perinatal HI. High-dose dexamethasone prevented c-fos induction in cerebral regions sensitive to HI. This effect may be due to a functional antagonism, at the transcriptional level, between Fos and the glucocorticoid receptor.
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PMID:[Molecular factors of cerebral hypoxia-ischemia]. 868 Dec 2

Apoptosis is accompanied by major changes in ion compartmentalization and transmembrane potentials. Thymocyte apoptosis is characterized by an early dissipation of the mitochondrial transmembrane potential, with transient mitochondrial swelling and a subsequent loss of plasma membrane potential (DeltaP sip) related to the loss of cytosolic K+, cellular shrinkage, and DNA fragmentation. Thus, a gross perturbation of DeltaPsip occurs at the postmitochondrial stage of apoptosis. Unexpectedly, we found that blockade of plasma membrane K+ channels by tetrapentylammonium (TPA), which leads to a DeltaP sip collapse, can prevent the thymocyte apoptosis induced by exposure to the glucocorticoid receptor agonist dexamethasone, the topoisomerase inhibitor etoposide, gamma-irradiation, or ceramide. The TPA-mediated protective effect extends to all features of apoptosis, including dissipation of the mitochondrial transmembrane potential, loss of cytosolic K+, phosphatidylserine exposure on the cell surface, chromatin condensation, as well as caspase and endonuclease activation. In strict contrast, TPA is an ineffective inhibitor when cell death is induced by the potassium ionophore valinomycin, the specific mitochondrial benzodiazepine ligand PK11195, or by primary caspase activation by Fas/CD95 cross-linking. These results underline the importance of K+ channels for the regulation of some but not all pathways leading to thymocyte apoptosis.
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PMID:Plasma membrane potential in thymocyte apoptosis. 1035 69


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