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:2.4.2.30 (PARP)
13,611 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A series of Trypanosoma brucei transfection vectors was constructed in which transcription of the luciferase gene was driven by the procyclic acidic repetitive protein (procyclin) promoter. The untranslated regions surrounding the luciferase gene were derived from the actin, fructose bisphosphate aldolase, or PARP loci. Trans-splicing of the resulting transcripts occurred as expected, but the site of 3' polyadenylation was upstream of the position anticipated. The nature of the 3'-untranslated region was crucial to the level of expression in bloodstream forms.
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PMID:A possible role for the 3'-untranslated region in developmental regulation in Trypanosoma brucei. 825 36

Exoenzyme S of Pseudomonas aeruginosa is an ADP-ribosyltransferase, which is secreted via a type III-dependent secretion mechanism and has been demonstrated to exert cytotoxic effects on eukaryotic cells. Alignment studies predict that the amino-terminus of exoenzyme S has limited primary amino acid homology with the YopE cytotoxin of Yersinia, while biochemical studies have localized the FAS-dependent ADP-ribosyltransferase activity to the carboxyl-terminus. Thus, exoenzyme S could interfere with host cell physiology via several independent mechanisms. The goal of this study was to define the role of the ADP-ribosyltransferase domain in the modulation of eukaryotic cell physiology. The carboxyl-terminal 222 amino acids of exoenzyme S, which represent the FAS-dependent ADP-ribosyltransferase domain (termed deltaN222), and a point mutant, deltaN222-E381A, which possesses a 2000-fold reduction in the capacity to ADP-ribosylate, were transiently expressed in eukaryotic cells under the control of the immediate early CMV promoter. Lysates from cells transfected with deltaN222 expressed ADP-ribosyltransferase activity. Co-transfection of deltaN222, but not deltaN222-E381A, resulted in a decrease in the steady-state levels of two reporter proteins, green fluorescent protein and luciferase, in both CHO and Vero cells. In addition, transfection with deltaN222 resulted in a greater percentage of cells staining with trypan blue than when cells were transfected with either deltaN222-E381A or control plasmid. Together, these data indicate that expression of the ADP-ribosyltransferase domain of exoenzyme S is cytotoxic to eukaryotic cells.
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PMID:Intracellular expression of the ADP-ribosyltransferase domain of Pseudomonas exoenzyme S is cytotoxic to eukaryotic cells. 1009 23

First-generation inducible expression vectors for Trypanosoma brucei utilized a single tetracycline-responsive promoter to drive expression of an experimental gene, in tandem with a drug-resistance marker gene to select for integration (Wirtz E, Clayton CE. Science 1995; 268:1179-1183). Because drug resistance and experimental gene expression both depended upon the activity of the regulated promoter, this approach could not be used for inducible expression of toxic products. We have now developed a dual-promoter approach, for expressing highly toxic products and generating conditional gene knock-outs, using back-to-back constitutive T7 and tetracycline-responsive PARP promoters to drive expression of the selectable marker and test gene, respectively. Transformants are readily obtained with these vectors in the absence of tetracycline, in bloodstream or procyclic T. brucei cell lines co-expressing T7 RNA polymerase and Tet repressor, and consistently show tetracycline-responsive expression through a 10(3)-10(4)-fold range. Uninduced background expression of a luciferase reporter averages no more than one molecule per cell, enabling dominant-negative approaches relying upon inducible expression of toxic products. This tight regulation also permits the production of functional gene knock-outs through regulated expression of an experimental gene in a null-mutant background.
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PMID:A tightly regulated inducible expression system for conditional gene knock-outs and dominant-negative genetics in Trypanosoma brucei. 1021 27

Pseudomonas aeruginosa delivers exoenzyme S (ExoS) into the intracellular compartment of eukaryotic cells via a type III secretion pathway. Intracellular delivery of ExoS is cytotoxic for eukaryotic cells and has been shown to ADP-ribosylate Ras in vivo and uncouple a Ras-mediated signal transduction pathway. Functional mapping has localized the FAS-dependent ADP-ribosyltransferase domain to the carboxyl-terminus of ExoS. A transient transfection system was used to examine cellular responses to the amino-terminal 234 amino acids of ExoS (DeltaC234). Intracellular expression of DeltaC234 elicited the rounding of Chinese hamster ovary (CHO) cells and the disruption of actin filaments in a dose-dependent manner. Expression of DeltaC234 did not inhibit the expression of two independent reporter proteins, GFP and luciferase, or induce trypan blue uptake, which indicated that expression of DeltaC234 was not cytotoxic to CHO cells. Carboxyl-terminal deletion proteins of DeltaC234 were less efficient in the elicitation of CHO cell rounding than DeltaC234. Cytoskeleton rearrangement elicited by DeltaC234 was blocked and reversed by the addition of cytotoxic necrotizing factor 1 (CNF-1). CNF-1 catalyses the deamidation of Gln-63 of members of the Rho subfamily of small-molecular-weight GTP-binding proteins, resulting in protein activation. This implies a role for small-molecular-weight GTP-binding proteins in the disruption of actin by DeltaC234. Together, these data identify ExoS as a cytotoxin that possesses two functional domains. Intracellular expression of the amino-terminal domain of ExoS elicits the disruption of actin, while expression of the carboxyl-terminal domain of ExoS possesses FAS-dependent ADP-ribosyltransferase activity and is cytotoxic to eukaryotic cells.
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PMID:The amino-terminal domain of Pseudomonas aeruginosa ExoS disrupts actin filaments via small-molecular-weight GTP-binding proteins. 1023 94

Phorbol 12-myristate 13-acetate (PMA)-induced HIV-1 production in U1 cells was markedly suppressed by inhibitors of poly (ADP-ribose) polymerase (PARP). Northern blot analysis revealed that the PARP-inhibitors suppressed the virus production at a level of transcription. In order to examine the effect of PARP on transcriptional regulation of HIV-1 genes, we transfected a reporter plasmid containing HIV-1-LTR-promoted luciferase gene to L-1210 cell clones, which expressed varying decreased level of PARP. In wild type L-1210 cells, the expression of LTR-promoted luciferase gene was stimulated approximately 4-fold in response to PMA, whereas the PMA-dependent response was almost abolished in mutant cells, which expressed only 8% of PARP of the wild type cells. The effect of decrease in PARP content on the function of HIV-1-LTR was confirmed also in human wild type cells, Jurkat and J111, which were co-transfected with the reporter plasmid and a plasmid expressing a PARP-antisense RNA: Down-regulation of PARP in the cells by the expression of the antisense RNA significantly suppressed the PMA-dependent, LTR-function of the reporter plasmid in both Jurkat and J111 cells. NF-kappaB, which is known to mediate the PMA-induced activation of HIV-1 in U1 cells, was found to be activated approximately 5-fold in PMA-treated U1 cells. PARP-inhibitor, unexpectedly, did not suppress but rather stimulated (approximately 2-fold) the NF-kappaB activation. Combining the results with the finding that the LTR-function was minimum in a PARP-defective mutant cells in spite of a very high level of the activated NF-kappaB in the cells, we suggest that PARP, in addition to activated NF-kappaB, is essential for the function of HIV-1 LTR.
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PMID:Poly (ADP-ribose) polymerase is involved in PMA-induced activation of HIV-1 in U1 cells by modulating the LTR function. 1044 6

E2F-1, a transcription factor implicated in the activation of genes required for S phase such as DNA pol alpha, is regulated by interactions with Rb and by cell-cycle dependent alterations in E2F-1 abundance. We have shown that depletion of poly(ADP-ribose) polymerase (PARP) by antisense RNA expression downregulates pol alpha and E2F-1 expression during early S phase. To examine the role of PARP in the regulation of pol alpha and E2F-1 gene expression, we utilized immortalized mouse fibroblasts derived from wild-type and PARP knockout (PARP-/-) mice as well as PARP-/- cells stably transfected with PARP cDNA [PARP-/-(+PARP)]. After release from serum deprivation, wild-type and PARP-/-(+PARP) cells, but not PARP-/- cells, exhibited a peak of cells in S phase by 16 h and had progressed through the cell cycle by 22 h. Whereas [3H]thymidine incorporation remained negligible in PARP-/- cells, in vivo DNA replication maximized after 18 h in wild-type and PARP-/-(+PARP) cells. To investigate the effect of PARP on E2F-1 promoter activity, a construct containing the E2F-1 gene promoter fused to a luciferase reporter gene was transiently transfected into these cells. E2F-1 promoter activity in control and PARP-/-(+PARP) cells increased eightfold after 9 h, but not in PARP-/- cells. PARP-/- cells did not show the marked induction of E2F-1 expression during early S phase apparent in control and PARP-/-(+PARP) cells. RT - PCR analysis and pol alpha activity assays revealed the presence of pol alpha transcripts and a sixfold increase in activity in both wild-type and PARP-/-(+PARP) cells after 20 h, but not in PARP-/- cells. These results suggest that PARP plays a role in the induction of E2F-1 promoter activity, which then positively regulates both E2F-1 and pol alpha expression, when quiescent cells reenter the cell cycle upon recovery from aphidicolin exposure or removal of serum.
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PMID:Poly(ADP-ribose) polymerase upregulates E2F-1 promoter activity and DNA pol alpha expression during early S phase. 1049 Aug 38

An expression vector was constructed to express foreign genes in Trypanosoma congolense. The foreign gene and a neomycin phosphotransferase (NPT) gene are flanked by glutamate and alanine rich protein (GARP) gene processing signals and their expression is driven by a ribosomal RNA gene promoter. The plasmid is not maintained as an episome in T. congolense, but the NPT gene permits selection of cells in which the plasmid has integrated into the genome. We used this plasmid to express luciferase, green fluorescent protein and a surface protein of Trypanosoma brucei, glycine-proline-glutamate glutamate threonine procyclic acidic repetitive protein (GPEET PARP). The plasmid-derived GPEET PARP is expressed on the surface of procyclic T. congolense and comigrates on a polyacrylamide gel with native GPEET PARP from T. brucei procyclic cells. We also attempted to use the plasmid to overexpress a previously identified T. congolense cysteine protease. The plasmid-derived cysteine protease mRNA species occurs in the transfected cells, but we were unable to detect increased levels of protein or protease activity.
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PMID:Expression of foreign proteins in Trypanosoma congolense. 1058 80

We have investigated the effects of hydrogen peroxide (H(2)O(2)), a potent naturally occurring oxidant on cell signaling and viability in the pluripotent HT29 intestinal cell line. There was a dose-dependent reduction in cell viability upon exposure to H(2)O(2) as measured by the XTT assay. Features of apoptosis were indicated by the findings of PARP and caspase 3 cleavage, as well as changes in cell morphology using phase contrast and nuclear fragmentation using fluorescence microscopy. There was a dose-dependent increase in the activation of p45-JNK, p42/p44-ERK, and p38-HOG. Surprisingly, oxidant-induced cell injury could be attenuated by preincubation with PD98059 to 50% of untreated control cells (P = 0.002). This and UO126, another MEK inhibitor were ably to reproducibly inhibit p45-JNK activation induced by hydrogen peroxide. Transfection with kinase-inactive constructs of JNK and ERK revealed that the improvement in cell viability was due to inhibition of JNK and not ERK. Transient transfections with AP-1 and NF-kappaB luciferase reporter constructs did not reveal any transcriptional activation due to hydrogen peroxide exposure however, in both cases the basal levels of transcriptional activity were suppressed in the presence of PD98059. It is concluded that JNK mediates H(2)O(2)-induced cellular injury in the HT29 cell line, and additionally, we report for the first time that JNK activation can be inhibited by both PD98059 and UO126 at conventional doses used to inhibit MEK.
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PMID:PD98059 attenuates hydrogen peroxide-induced cell death through inhibition of Jun N-Terminal Kinase in HT29 cells. 1128 30

Transient expression of the tumor suppressor gene p53 via adenoviral-mediated gene transfer induces apoptosis in glioma cells expressing mutant p53, while causing cell cycle arrest in cells with wild-type p53. To determine whether a change in p53 status of a wild-type p53-expressing cell line such as U-87 MG would alter its apoptotic resistant phenotype in response to Ad-p53 infection, we generated cell lines U-87-175.4 and U-87-175.13 via retroviral-mediated gene transfer of the p53 (175H) mutant into the U-87 MG parental line. Control cell lines U-87-Lux.6 and U-87-Lux.8 were also generated and express the reporter gene luciferase. Both U-87-175.4 and U-87-175.13, but not control cell lines, exhibited morphology characteristic of apoptosis after Ad-p53 infection. Furthermore, expression of other p53 mutants (248W, 273H) in U-87 MG also sensitized cells to Ad-p53-induced apoptosis. Apoptosis was confirmed by TUNEL and cell cycle analysis. Several p53 response genes were examined in cells infected with Ad-p53, and among these, BCL2, p21WAF1/CIP1, CPP32/caspase 3, and PARP showed differences in expression between U87-175 and U87-Lux cell lines. Taken together, our data demonstrate that the introduction of p53 mutants in U-87 MG promotes an apoptotic response in association with adenoviral-mediated wild-type p53 gene transfer. These results underscore the importance of glioma p53 genotype for predicting tumor response to p53-based gene therapy.
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PMID:Introduction of mutant p53 into a wild-type p53-expressing glioma cell line confers sensitivity to Ad-p53-induced apoptosis. 1129 82

Because tumor necrosis factor-alpha (TNF-alpha) and some chemotherapeutic agents activate both apoptosis and NF-kappaB-dependent antiapoptotic genes, they may neutralize their own antitumor effects. The cell-signaling mechanisms for such chemoresistance are not clear but may involve phosphotidylinositol-3' kinase (PI3K). To clarify this we examined whether cross-signaling between PI3K and NF-kappaB enhances the antitumor effect of TNF-alpha in human pancreatic cancer cells. Quiescent pancreatic cancer cells (Panc-1, MiaPaCa-2) with TNF-alpha, Ly294002 (PI3K inhibitor), alone or combined, were restimulated with mitogen (10% fetal calf serum [FCS] to induce cell cycle entry). Proliferation (monotetrazolium), cell cycle progression (ApoBrDU and fluorescence-activated cell sorter analysis), and apoptosis (PARP cleavage; caspase-3 activation) were measured. Akt activation (Akt kinase assay) and IkappaBalpha degradation were determined by Western blot analysis. Translocation of NF-kappaB into the nucleus was examined by EMSA, whereas an NF-kappaB/luciferase reporter gene was used to quantify NF-kappaB-dependent gene expression. Statistical analysis was carried out by means of two-tailed t test (P <0.05). PI3K inhibition significantly enhanced the antiproliferative and proapoptotic effects of TNF-alpha in both cell lines, Ly294002 also blocked TNF-alpha-induced Akt activation but failed to alter cytoplasmic IkappaBalpha degradation or subsequent NF-kappaB nuclear translocation. NF-kappaB-dependent gene expression, however, was ultimately suppressed by Ly294002, suggesting that PI3k-dependent activation of NF-kappaB is IkappaBalpha independent. PI3K inhibition can block NF-kappaB-dependent gene expression regardless of cytoplasmic IkappaBalpha/NF-kappaB activation. Because it also regulates the antitumor effects of TNF-alpha, PI3K may in part determine NF-kappaB-induced chemoresistance in human pancreatic cancer.
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PMID:PI-3' kinase and NF-kappaB cross-signaling in human pancreatic cancer cells. 1208 98


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