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 measured the binding of highly radioactive (2'-5')pppA4-5[32P]cytidine 3',5'-diphosphate to human, mouse, and rabbit cell and tissue extracts. A binding activity for this oligonucleotide is present in all extracts examined and high levels of this activity are found in some cells of lymphoid origin. In particular, the mouse thymoma cell line W7 has about 10 times higher binding activity than other cell lines. The oligonucleotide is bound with a single high affinity constant, as shown by Scatchard plot analyses. Cell extracts fractionated by centrifugation on glycerol gradients show a single peak of binding activity, which co-sediments with (2'-5')oligo(A)-dependent endoribonuclease (RNase L). This enzyme apparently binds the oligonucleotide, as shown by experiments with an analog of (2'-5')oligo(A) which competes in the binding and inhibits the RNase L. This endonuclease cannot be directly assayed in unfractionated cell or tissue extracts with high levels of other nuclease activities. The binding assay for the oligonucleotide may provide an estimate of RNase L levels in such cell and tissue extracts.
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PMID:2',5'-Oligo(A)-activated endoribonuclease. Tissue distribution and characterization with a binding assay. 728 31

The present studies were undertaken to characterize further the potential role of protein kinase C (PKC) in the regulation of apoptosis in HL-60 promyelocytic leukemia cells. The capacity of acute exposure to specific and nonspecific pharmacological inhibitors of PKC to promote apoptotic DNA fragmentation was examined both quantitatively and qualitatively and correlated with effects on cellular differentiation and proliferation. Incubation of HL-60 cells for 6 h with chelerythrine and calphostin C (highly specific inhibitors that act at the regulatory domain) or H7 and gossypol (nonspecific inhibitors that act at the PKC catalytic domain) produced concentration-dependent increases in DNA fragmentation. Induction of DNA fragmentation by chelerythrine, calphostin C, and gossypol was biphasic, resulting in a sharp decline in effect at concentrations above 5 microM, 0.1 microM, and 100 microM, respectively, whereas maximal and more stable effects were observed in response to H7 (100 microM). A 6-h exposure to staurosporine, a nonspecific but potent PKC inhibitor, failed to induce DNA fragmentation at concentrations generally used to achieve maximal inhibition of enzyme activity (e.g., 50 nM) but promoted fragmentation at considerably higher concentrations (e.g., > or = 200 nM). In contrast, 6-h exposures to the nonspecific protein kinase inhibitor hypericin (0.1 to 100 microM) or to the nonspecific inhibitor of protein kinase A, HA1004 (50 microM), were without effect on DNA fragmentation. DNA obtained from cells exposed to chelerythrine (5 microM), calphostin C (100 nM), H7 (50 microM), gossypol (50 microM), and staurosporine (200 nM)--but not hypericin (25 microM)--exhibited clear evidence of internucleosomal DNA cleavage on agarose gel electrophoresis; moreover, these cells exhibited the classical morphological features of apoptosis (cell shrinkage, nuclear condensation, and the formation of apoptotic bodies). All of the PKC inhibitors that induced apoptosis, and one of the inhibitors that did not (hypericin), substantially inhibited HL-60 cell clonogenicity at the concentrations evaluated. None of the agents tested induced cellular maturation as assessed by nonspecific esterase and nitro-blue tetrazolium positivity. DNA fragments obtained from cells exposed to specific and nonspecific PKC inhibitors possessed predominantly 5'-phosphate termini, consistent with the action of a Ca(2+)-/Mg(2+)-dependent endonuclease. Finally, Northern blot analysis revealed that exposure to calphostin C at a concentration that induced apoptosis (100 nM) failed to alter expression of bcl-2, an oncogene known to block apoptosis in both lymphoid and myeloid leukemia cells.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. 751 Oct 48

Discordant morphology between lymph node or extra-nodal site and bone marrow (BM) involvement by non-Hodgkin's malignant lymphoma (NHL) is a common occurrence, causing diagnostic difficulties. Additional diagnostic problems are posed by lymphoid aggregates commonly found in the BM of elderly patients, the age group with the highest incidence of lymphoma. Morphologic features are used to distinguish between benign and malignant lesions but no feature is diagnostic and exceptions are numerous. Immunophenotyping is helpful for detecting B cell monoclonality, but it cannot detect T cell monoclonality. Unique B and T cell gene rearrangement patterns, the molecular "signature" of the lymphoma, can be used to detect monoclonal lymphoid populations. Finding the same rearrangement pattern in the BM as in the primary mass is proof of BM involvement by the same clone of malignant cells. We used B/T and Bcl-2 gene rearrangements to help diagnose cases with discordant morphology between primary site and BM. One hundred and seventy-five specimens, obtained from patients undergoing staging or restaging for NHL, were analyzed for B/T cell and Bcl-2 gene rearrangements by multiple restriction endonuclease digestion and Southern hybridization with 32P labeled JH, JK, CT beta, and Bcl-2 probes. Forty-two specimens (24%) from 24 patients showed discordant morphology: of 13 specimens with atypical lymphoid aggregates, only one had B cell gene rearrangement; of 15 specimens with morphologically benign lymphoid aggregates, one demonstrated B cell gene rearrangement; and of 14 specimens positive for NHL with different morphology than the lymph node, 13 were positive for B cell gene rearrangements. Molecular analysis can aid in the diagnosis of NHL, can establish a "baseline" for detection of recurrence, and is useful in monitoring therapy. These data suggest that it is also a tool for the pathologist in cases of discordant morphology between the primary tumor and BM, and should be strongly considered for each site.
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PMID:Discordant morphologic features in bone marrow involvement by malignant lymphomas: use of gene rearrangement patterns for diagnosis. 763 75

The previously established fact of low activity of Ca, Mg-dependent endonucleolysis of cell nucleus DNA in lymphoproliferative diseases (CME-activity) brought the authors to study intranuclear characteristics of lymphoid cells in childhood acute lymphoblastic leukemia (ALL). The intensity of DNA-endonucleolysis was measured in 0.7% agarose gel using electrophoresis. Peripheral blood and bone marrow samples from 13 untreated ALL patients and 23 children in remission were examined. The age of the patients ranged from 4 to 14 years. CME-activity before treatment appeared to be 2-10 times less than normal in 8 out of 13 patients. In bone marrow cell nuclei CME-activity was universally reduced 3-20-fold. In ALL remission endonuclease activity in blood and bone marrow cells returned to normal.
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PMID:[Changes in the activity of endonucleolysis of nuclear DNA in children with acute leukemia]. 764 86

In a number of experimental systems in which lymphocyte depletion was induced by apoptosis-inducing manipulations, no apoptotic morphology and ladder-type DNA fragmentation were detected among freshly isolated peripheral lymphocytes ex vivo. Here we report that one alteration that can be detected among splenocytes stimulated with lymphocyte-depleting doses of dexamethasone (DEX) in vivo is a reduced uptake of 3,3'dihexyloxacarbocyanine iodide (DiOC6[3]), a fluorochrome which incorporates into cells dependent upon their mitochondrial transmembrane potential (delta psi m). In contrast, ex vivo isolated splenocytes still lacked established signs of programmed cell death (PCD):DNA degradation into high or low molecular weight fragments, ultrastructural changes of chromatin arrangement and endoplasmatic reticulum, loss in viability, or accumulation of intracellular peroxides. Moreover, no changes in cell membrane potential could be detected. A reduced delta psi m has been observed in response to different agents inducing lymphoid cell depletion in vivo (superantigen and glucocorticoids [GC]), in mature T and B lymphocytes, as well as their precursors. DEX treatment in vivo, followed by cytofluorometric purification of viable delta psi mlow splenic T cells ex vivo, revealed that this fraction of cells is irreversibly committed to undergoing DNA fragmentation. Immediately after purification neither delta psi mlow, nor delta psi mhigh cells, exhibit detectable DNA fragmentation. However, after short-term culture (37 degrees C, 1 h) delta psi mlow cells show endonucleolysis, followed by cytolysis several hours later. Incubation of delta psi mlow cells in the presence of excess amount of the GC receptor antagonist RU38486 (which displaces DEX from the GC receptor), cytokines that inhibit DEX-induced cell death, or cycloheximide fails to prevent cytolysis. The antioxidant, N-acetylcysteine, as well as linomide, an agent that effectively inhibits DEX or superantigen-induced lymphocyte depletion in vivo, also stabilize the DiOC6(3) uptake. In contrast, the endonuclease inhibitor, aurintricarboxylic acid acts at later stages of apoptosis and only retards the transition from the viable delta psi mlow to the nonviable fraction. Altogether, these data suggest a sequence of PCD-associated events in which a reduction in delta psi m constitutes an obligate irreversible step of ongoing lymphocyte death, preceding other alterations of cellular physiology, and thus allowing for the ex vivo assessment of PCD.
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PMID:Reduction in mitochondrial potential constitutes an early irreversible step of programmed lymphocyte death in vivo. 772 46

This review summarizes the evidence that apoptosis is modulated by intracellular excess or deficiency of Zn2+, considers mechanisms whereby Zn2+ may influence apoptosis, and delineates gaps in current knowledge and opportunities for research. The experimental evidence supports four major conclusions: [1] Zinc deficiency, resulting from dietary deprivation of mice, or exposure of cultured cells to membrane-permeable Zn(2+)-chelators, can induce apoptosis; [2] Zinc supplementation, either by pretreating mice with ZnSO4, or adding Zn2+ to the media of cell cultures, can prevent apoptotic death. Zn2+ protects against the apoptosis induced by diverse physical, chemical, or immunologic stimuli in cultured cells of lymphoid, hepatic, or neoplastic origin; [3] Zn2+ does not affect the triggering events or earliest signs of apoptosis, but acts later in the apoptotic pathway, preventing endonucleosomal fragmentation and subsequent cytolysis; and [4] An intracellular pool of chelatable Zn2+ plays a critical role in apoptosis, possibly by modulating the activation or activity of endonuclease(s). These conclusions should alert pharmacologists and physicians to the potential therapeutic applications of zinc compounds and zinc chelators in clinical disorders and diseases that involve apoptosis, and to the relevance of zinc nutrition in such conditions.
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PMID:The influence of zinc on apoptosis. 778 63

The development of a reliable polymerase chain reaction (PCR) technique for the routine detection of clonal immunoglobulin heavy chain (IgH) gene rearrangements would represent an attractive alternative to Southern hybridization analysis because of the relative simplicity of PCR protocols, and because the requirements for both quality and quantity of DNA would be considerably less stringent. To assess the utility of PCR for the routine detection of clonal IgH gene rearrangements, samples from 123 adult patients were evaluated and analysis by PCR amplification using IgH Framework 1 or Framework 3 variable region consensus primers was compared with analysis by restriction endonuclease digestion and Southern hybridization with genomic, IgH probes. The authors found that 90% of IgH genes found to be rearranged by Southern hybridization are detected by the PCR technique. An additional 9 patient samples had clonal IgH gene rearrangements that were detectable by PCR alone. Eight of these nine patients had a history of a clonal hematopoietic process at either the morphologic or molecular level, and six had a history of a B-cell malignancy. It is likely that these specimens contained clonal lymphoid populations undetected by the Southern hybridization technique. Thus, the diagnostic sensitivity and specificity of the PCR method for the detection of B-cell tumors were 91% and 95%, respectively. The combination of improved analytical sensitivity and specimen flexibility of the IgH PCR assay could make it the method of choice for the routine detection of clonal IgH gene rearrangements, if minor improvements in the diagnostic sensitivity of the assay can be achieved.
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PMID:Comparison of PCR with southern hybridization for the routine detection of immunoglobulin heavy chain gene rearrangements. 785 59

Treatment of experimental animals with T-2 toxin has been found to markedly decrease thymic cellularity and to suppress cell-mediated immune function. Although T-2 toxin readily crosses the placenta, little is known about its effect on development of immunity following gestational exposure. In the present report, prenatal T-2 toxin resulted in significant fetal thymic atrophy in mice. In vitro exposure to T-2 toxin resulted in decreased thymocyte proliferation, as well as significant but transient increases in thymocyte viability. Cycloheximide increased thymocyte viability parallel to that seen after T-2 toxin, indicating that enhanced viability after T-2 toxin may be the result of inhibited endonuclease synthesis. These findings suggest that direct cytotoxic effects of T-2 toxin make limited contribution to thymic atrophy production. In support of this conclusion, in vivo T-2 toxin exposure resulted in only limited alteration of thymocyte development, as evidenced by expression of CD4, CD8, and alpha beta TCR cell-surface antigens. These data further indicate that antiproliferative effects of T-2 toxin on thymocytes may contribute limitedly to thymic atrophy observed in vivo. In vivo T-2 toxin treatment did not affect total numbers of CD44+, CD45+, or Mac-1+ fetal liver cells. However, such exposure resulted in significant decreases in CD44lo and CD45lo fetal liver prolymphoid cell subpopulations. Subsequent in vitro T-2 toxin exposure of fetal liver cells enriched for lymphoid precursors resulted in both decreased cell viability and highly significant decreased proliferation. Taken together, these data suggest that lymphocyte progenitors, in contrast to thymocytes, represent highly sensitive targets of T-2 toxin exposure, responsible for thymic atrophy.
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PMID:Fetal thymic atrophy after exposure to T-2 toxin: selectivity for lymphoid progenitor cells. 833 3

Cell death occurring by apoptosis has become widely recognized as an integral component of the life cycle of many cell types. Apoptosis can be induced in many tissues by a wide variety of endogenous signals, including glucocorticoids. However, even though there are glucocorticoid receptors present in almost all cells, only certain lymphoid cells are susceptible to glucocorticoid-induced apoptosis. The basis for this selective regulation of programmed cell death is unknown. Internucleosomal chromatin degradation is an integral characteristic of apoptosis, common to all cells undergoing this form of programmed cell death. Thus, we have developed an in vitro assay that recapitulates apoptotic DNA degradation in isolated nuclei and have identified a nuclease activity with internucleosomal specificity that is present in nuclear extracts of thymocytes undergoing glucocorticoid-induced apoptosis. We have now extended these studies to analyze the molecular properties of the crude enzyme and to further elucidate the mechanism of its regulation during the tissue-specific induction of apoptosis. In vitro, optimal internucleosomal cleavage activity was detected in the presence of millimolar concentrations of calcium and magnesium or manganese. Nuclease activity was inhibited by three agents previously shown to block apoptosis (zinc, aurintricarboxylic acid, and sodium), suggesting that the nuclease we detected in apoptotic thymocytes is responsible for the DNA degradation associated with apoptosis. Size-fractionation analysis of thymocyte nuclear extract under native conditions revealed a protein with an apparent molecular mass of 22.7 kilodaltons and a sedimentation coefficient of 3.5S. Interestingly, when extracts from control thymocytes, inactive in internucleosomal cleavage activity, were subjected to gel filtration or sucrose density gradient fractionation, internucleosomal cleavage activity was detected. The physical characteristics of this endonuclease activity were identical to those found in thymocyte extract from glucocorticoid-treated rats. Repressed or latent internucleosomal cleavage activity was also detected in hepatocyte nuclear extracts, but this activity was not activated by glucocorticoid treatment. These data suggest that glucocorticoid-induced apoptosis involves cell-specific activation of a latent endonuclease, rather than nuclease induction.
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PMID:Mechanism of tissue-specific induction of internucleosomal deoxyribonucleic acid cleavage activity and apoptosis by glucocorticoids. 839 69

Hemopoietic cells have been reported to synthesize insulin-like growth factor-I (IGF-I) messenger RNA (mRNA), but the relative contribution of specific cell lineages that express these transcripts remains unknown. Reverse transcription and amplification of complementary DNA (cDNA) by the polymerase chain reaction were used to characterize full-length IGF-I mRNA transcripts in murine hemopoietic cells. The identity of transcripts encoding the entire prepropeptide was confirmed by restriction endonuclease digestion, Southern blotting, cloning, and Sanger sequencing. Abundance of IGF-I mRNA transcripts was assessed both by Northern blotting and sensitive ribonuclease protection assays followed by quantification with Phosphor-Imager analysis. Whereas IGF-I cDNA transcripts could be detected in a variety of leukocytes after polymerase chain reaction amplification, IGF-I mRNA was negligible or nondetectable in T and B cell lines and in those tissues containing a predominance of these cell types (e.g. spleen and thymus) by Northern blotting and ribonuclease protection assays. In contrast, elicited peritoneal macrophages, a macrophage cell line, microglia, and bone marrow macrophages differentiated in vitro expressed abundant IGF-I mRNA transcripts, whereas neither a premyeloid cell line nor freshly isolated bone marrow cells expressed significant transcripts. The 5'-identity of macrophage IGF-I transcripts was established using an exon 2-derived IGF-I cDNA probe. All protected transcripts were foreshortened, indicating transcript initiation exclusively within exon 1, characteristic of extra-hepatic IGF-I mRNA. However, at the 3'-end, both IGF-I Ea (lacking exon 5) and IGF-I Eb (containing exon 5) mRNA transcripts were evident, with the Eb product being detected at levels similar to those present in hepatic cellular RNA. A large molecular size (26 kilodaltons) prepro-IGF-I peptide was also detected in macrophage cell lysates by Western blotting. Collectively, our observations show that: 1) among hemopoietic cells, myeloid rather than lymphoid cells are the major source of IGF-I; 2) macrophage IGF-I mRNA consists of class I Ea and Eb transcripts; 3) these transcripts are translated into protein; and 4) expression of IGF-I is directly associated with differentiation of bone marrow macrophages.
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PMID:Murine macrophages express abundant insulin-like growth factor-I class I Ea and Eb transcripts. 840 86


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