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:3.2.1.23 (beta-galactosidase)
14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The hepatic stellate cell (HSC), following a fibrogenic stimulus, is transformed from a quiescent to an activated cell. Cytokines induce NFkappaB activity in activated but not in quiescent HSCs with subsequent expression of NFkappaB-responsive genes, such as intercellular adhesion molecule (ICAM)-1 and interleukin (IL)-6. We investigated the effect of proteasome inhibitors and an IkappaB super-repressor on the cytokine mediated activation of NFkappaB, ICAM-1, and IL-6 in activated HSCs. Culture-activated HSCs were stimulated with IL-1beta or tumor necrosis factor alpha (TNFalpha) in the presence or absence of proteasome inhibitors, ALLN or MG-132, or after infection with an adenovirus expressing the IkappaB super-repressor (Ad5IkappaB) or beta-galactosidase (Ad5LacZ) as a control. NFkappaB activity was evaluated by immunofluorescence and by electrophoretic mobility shift assay. The steady state level of cytoplasmic IkappaB protein was measured by Western Blot. ICAM-1 and IL-6 expression was measured by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbant assay. Proteasome inhibitors, which block the degradation of IkappaB, and the Ad5IkappaB, which provides an exogenous nondegradable IkappaB, block the stimulation of NFkappaB activity by TNFalpha and IL-1beta in activated HSCs. These reagents block the subsequent nuclear translocation of p65 NFkappaB and induction of ICAM-1 and IL-6 by cytokines. The specificities of the proteasome inhibitors and the IkappaB super-repressor are demonstrated by their failure to block c-Jun N-terminal kinase induction by cytokines. Cytokine-induced stimulation of NFkappaB, ICAM-1, and IL-6 is blocked by proteasome inhibitors and Ad5IkappaB in activated HSCs. Inhibition of IkappaBalpha degradation is a potential target for anti-inflammatory therapy in the liver and might influence the activation process of HSCs following fibrotic stimuli.
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PMID:Inhibition of NFkappaB in activated rat hepatic stellate cells by proteasome inhibitors and an IkappaB super-repressor. 958 6

We demonstrated recently that constitutive expression of proinflammatory cytokines interleukin (IL)-1alpha, IL-6, IL-8, and granulocyte-macrophage colony-stimulating factor in head and neck squamous cell carcinoma is correlated with activation of transcription factor nuclear factor (NF)-kappaB/Rel A (p50/p65), which binds the promoter region within each of the genes encoding this repertoire of cytokines. NF-kappaB can be activated after signal-dependent phosphorylation and degradation of inhibitor-kappaBalpha and has been reported to promote cell survival and growth. In the present study, we expressed a phosphorylation site mutant of inhibitor-kappaBalpha (IkappaBalphaM) in head and neck squamous cell carcinoma lines UM-SCC-9, -11B, and -38 to determine the effect of inhibition of NF-kappaB on cytokine expression, cell survival in vitro, and growth in vivo. After transfection with IKBalphaM, only a few UM-SCC-9 clones were obtained that stably expressed the mutant IkappaB, suggesting that expression of a mutant IkappaBalpha may affect survival of the transfected UM-SCC cell lines. After cotransfection of IkappaBalphaM with a Lac-Z reporter, we found that the number of surviving beta-galactosidase-positive cells in the three cell lines was reduced by 70-90% when compared with controls transfected with vector lacking the insert. In UM-SCC-9 cells that stably expressed IkappaBalphaM, inhibition of constitutive and tumor necrosis factor-a induced NF-kappaB activation, and production of all four cytokines was observed. Although UM-SCC-9 IkappaBalphaM-transfected cells proliferated at the same rate as vector-transfected cells in vitro, a significant reduction in growth of tumor xenografts was observed in SCID mice in vivo. The decreased growth of UM-SCC-9 IkappaBalphaM-transfected tumor cells accompanied decreased immunohistochemical detection of the activated form of NF-kappaB in situ. These results provide evidence that NF-KB and IkappaBalpha play an important role in survival, constitutive and inducible expression of proinflammatory cytokines, and growth of squamous cell carcinoma. NF-kappaB could serve as a potential target for therapeutic intervention against cytokine and other immediate-early gene responses that contribute to the survival, growth, and pathogenesis of these cancers.
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PMID:Expression of a dominant-negative mutant inhibitor-kappaBalpha of nuclear factor-kappaB in human head and neck squamous cell carcinoma inhibits survival, proinflammatory cytokine expression, and tumor growth in vivo. 1041 12

Current paradigms in cancer therapy suggest that activation of nuclear factor-kappaB (NF-kappaB) by a variety of stimuli, including some cytoreductive agents, may inhibit apoptosis. Thus, inhibiting NF-kappaB activation may sensitize cells to anticancer therapy, thereby providing a more effective treatment for certain cancers. E-1-deleted adenoviral (Ad) vectors encoding a "superrepressor" form of the NF-kappaB inhibitor IkappaBalpha (AdIkappaBalphaSR) or beta-galactosidase (AdLacZ) were tested alone and in combination with tumor necrosis factor-alpha (TNF-alpha) in lung cancer cells for sensitization of the cells to death. Following transduction with AdIkappaBalphaSR, lung cancer cells expressed IkappaBalphaSR in a dose-dependent manner. Probing nuclear extracts of lung cancer cells with NF-kappaB-sequence-specific oligonucleotides indicated that there was a minimal amount of NF-kappaB in the nucleus at baseline and an expected and dramatic increase in nuclear NF-kappaB following exposure of cells to TNF-alpha. Control E-1-deleted AdLacZ did not promote NF-kappaB activation. Importantly, AdIkappaBalphaSR-mediated gene transfer resulted in the complete block of nuclear translocation of NF-kappaB by specific binding of its p65/relA component with transgenic IkappaBalphaSR. At the cellular level, transduction with AdIkappaBalphaSR resulted in increased cytotoxicity in lung cancer cells as opposed to transduction with equivalent doses of AdLacZ. In addition, whereas the parental cells were resistant to TNF-alpha-mediated cytotoxicity, IkappaBalphaSR-transduced cells could be sensitized to TNF-alpha. Consequently, AdIkappaBalphaSR transduction followed by exposure to TNF-alpha uniformly resulted in the death of non-small-cell lung cancer cells. These data suggest that novel approaches incorporating IkappaBalpha gene therapy may have a role in the treatment of lung cancer.
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PMID:IkappaBalpha gene transfer is cytotoxic to squamous-cell lung cancer cells and sensitizes them to tumor necrosis factor-alpha-mediated cell death. 1042 7

Three major inhibitors of the NF-kappaB/Rel family of transcription factors, IkappaBalpha, IkappaBbeta, and IkappaBepsilon, have been described. To examine the in vivo role of the most recently discovered member of the IkappaB family, IkappaBepsilon, we generated a null allele of the murine IkappaBepsilon gene by replacement of all coding sequences with nlslacZ. Unlike IkappaBalpha nullizygous mice, mice lacking IkappaBepsilon are viable, fertile, and indistinguishable from wild-type animals in appearance and histology. Analysis of beta-galactosidase expression pattern revealed that IkappaBepsilon is mainly expressed in T cells in the thymus, spleen, and lymph nodes. Flow cytometric analysis of immune cell populations from the bone marrow, thymus, spleen, and lymph nodes did not show any specific differences between the wild-type and the mutant mice, with the exception of a reproducible 50% reduction of the CD44-CD25+ T cell subspecies. The IkappaBepsilon-null mice present constitutive up-regulation of IgM and IgG1 Ig isotypes together with a further increased synthesis of these two isotypes after immunization against T cell-dependent or independent Ags. The failure of observable augmentation of constitutive nuclear NF-kappaB/Rel-binding activity is probably due to compensatory mechanisms involving IkappaBalpha and IkappaBbeta, which are up-regulated in several organs. RNase-mapping analysis indicated that IL-1alpha, IL-1beta, IL-1Ra, and IL-6 mRNA levels are constitutively elevated in thioglycolate-elicited IkappaBepsilon-null macrophages in contrast to GM-CSF, G-CSF, and IFN-gamma, which remain undetectable.
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PMID:IkappaBepsilon-deficient mice: reduction of one T cell precursor subspecies and enhanced Ig isotype switching and cytokine synthesis. 1057 Feb 87

Recombinant adeno-associated virus (rAAV) transduction is limited in vivo, yet can be enhanced by hydroxyurea, ultraviolet-irradiation, or adenovirus coinfection, possibly via mechanisms involving stress in the host cell. Because chronic ethanol induces oxidative stress, it was hypothesized that chronic ethanol would increase rAAV transduction in vivo. To test this hypothesis, rAAV encoding beta-galactosidase was given to Wistar rats that later received either ethanol diet or high-fat control diet via an enteral-feeding protocol for 3 weeks. Expression and activity of beta-galactosidase in the liver were increased nearly 5-fold by ethanol. The increase in transgene expression was inhibited by antioxidant diphenylene iodonium (DPI), which is consistent with the hypothesis that ethanol causes an increase in rAAV transduction via oxidative stress. Ethanol increased DNA synthesis only slightly; however, it increased the nuclear transcription factor kappaB (NFkappaB) 4-fold, a phenomenon also sensitive to DPI. Moreover, a 6-fold increase in rAAV transgene expression was observed in an acute ischemia-reperfusion model of oxidative stress. Transgene expression was transiently increased 24 hours after ischemia-reperfusion 3 days and 3 weeks after rAAV infection. Further, adenoviral expression of superoxide dismutase or IkappaBalpha superrepressor inhibited rAAV transgene expression caused by ischemia-reperfusion. Therefore, it is concluded that ethanol increases rAAV transgene expression via mechanisms dependent on oxidative stress, and NFkappaB likely through enhancement of cytomegaloviral (CMV) promoter elements. Alcoholic liver disease is an attractive target for gene therapy because consumption of ethanol could theoretically increase expression of therapeutic genes (e.g., superoxide dismutase). Moreover, this study has important implications for rAAV gene therapy and potential enhancement and regulation of transgene expression in liver.
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PMID:Chronic ethanol increases adeno-associated viral transgene expression in rat liver via oxidant and NFkappaB-dependent mechanisms. 1105 56

Dorsal-ventral polarity within the Drosophila syncytial blastoderm embryo is determined by the maternally encoded dorsal group signal transduction pathway that regulates nuclear localization of the transcription factor Dorsal. Nuclear uptake of Dorsal, a Rel/NFkappaB homolog, is controlled by the interaction with its cognate IkappaB inhibitor protein Cactus, which is degraded on the ventral side of the embryo in response to dorsal group signaling. Previous studies have suggested that an N-terminally located kinase target motif similar to that found in IkappaB proteins is involved in the spatially controlled degradation of Cactus. We report studies of the in vivo function and distribution of fusion proteins comprising segments of Cactus attached to Escherichia coli beta-galactosidase (lacZ). Full-length Cactus-lacZ expressed in vivo normalizes the ventralized phenotype of embryos that lack Cactus and faithfully reconstitutes dorsal group-regulated degradation, while fusion protein constructs that lack the first 125 amino acids of Cactus escape dorsal group-dependent degradation. Furthermore, Cactus-lacZ constructs that lack only the putative IkappaB-dependent kinase target-like motif can nevertheless undergo spatially regulated dorsal group-dependent degradation and we have identified the regulatory determinant responsible for dorsal group-dependent degradation of Cactus in the absence of this motif. Taken together, our studies indicate the presence of two distinct redundantly acting determinants in the N terminus of Cactus that direct dorsal group-dependent degradation. Strikingly, the regulatory domain of human IkappaBalpha can also direct polarized degradation of Cactus-lacZ fusion protein.
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PMID:Separable and redundant regulatory determinants in Cactus mediate its dorsal group dependent degradation. 1153 19

Ethanol is known to cause both tolerance and sensitization to endotoxin (lipopolysaccharide). It is also known that ethanol modulates the expression and activity of several intracellular signaling molecules and transcription factors in monocytes and Kupffer cells, the resident hepatic macrophages. Expression of CD14, the endotoxin receptor, is up-regulated following chronic exposure to endotoxin and ethanol. Ethanol-induced oxidative stress is important in the regulation of transcription factor activation and cytokine production by Kupffer cells. Thus, it was hypothesized that acute ethanol increases CD14 expression through a mechanism dependent upon oxidant production. This hypothesis was tested by overexpression of superoxide dismutase via recombinant adenovirus. Mice were infected with adenovirus (3 x 10(9) plaque-forming units, intravenously) containing either Cu,Zn superoxide dismutase (Ad.SOD1) or beta-galactosidase (Ad.lacZ), which caused significant expression of Cu,Zn-SOD in hepatocytes and Kupffer cells. Three days post-infection, mice were given saline or ethanol (5 g/kg, intragastrically). A significant increase in CD14 mRNA was observed 3 h after ethanol, and this increase was almost completely blocked in mice overexpressing Cu,Zn-SOD. Additionally, overexpression of SOD also blunted ethanol-induced activation of redox-sensitive transcription factors NFkappaB and AP-1 and production of cytokines. However, only inhibition of AP-1 with dominant-negative TAK1 but not NFkappaB by dominant-negative IkappaBalpha significantly blunted ethanol-induced increases in CD14, suggesting that AP-1 is important for CD14 transcriptional regulation. It is also shown here that NADPH oxidase is important in the increase in CD14 due to ethanol. Moreover, these data suggest that acute ethanol causes sensitization to endotoxin through mechanisms dependent upon oxidative stress.
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PMID:Up-regulation of CD14 in liver caused by acute ethanol involves oxidant-dependent AP-1 pathway. 1248 56

We used a molecular genetics approach to investigate the role of nuclear factor-kappaB (NF-kappaB) in neointimal hyperplasia induced by flow interruption of carotid artery in mice. Wild type mice (WT mice) and mice rendered deficient in p105, the precursor of p50, one of the components of the multimeric transcription factor NF-kappaB (NF-kappaB knockout mice; KO mice), were subjected to a complete ligation of the left common carotid artery. Morphometric analysis of the structural alteration caused by the disruption of the arterial blood flow was performed 14 days after surgery. Furthermore the expression of intercellular adhesion molecule-1 (ICAM-1) in injured arteries was evaluated 4 days after artery ligation by the means of reverse transcriptase polymerase chain reaction (RT-PCR) and quantification of the ICAM-1 protein levels. In a separate experiment normal mice were randomly assigned to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-kappaB inhibitory protein IkappaBalpha (rAAV-IkappaBalpha), or the beta-galactosidase gene (rAAV-LacZ), both at a dose of 10(11) copies and 2 weeks later were subjected to the complete ligation of the left carotid artery. NF-kappaB activity (studied by means of electrophoretic mobility shift assay-EMSA), IkappaBalpha expression (evaluated by Western blot analysis) ICAM-1 evaluation (RT-PCR and quantification of the protein levels) and a morphometric analysis were evaluated in the injured arteries. Disruption of the arterial blood flow caused a marked neointimal hyperplasia. The mean intimal area was 0.023+/-0.002 mm(2) in wild type mice compared with 0.002+/-0.001 mm(2) in NF-kappaB knockout mice. ICAM-1 expression was 1.7+/-0.8 relative amount of ICAM-1 mRNA in wild type mice compared with 0.4+/-0.06 relative amount of ICAM-1 mRNA in NF-kappaB knockout mice. ICAM-1 protein levels were also significantly reduced in NF-kappaB knockout mice. Injured arteries treated with rAAV-IkappaBalpha had a greater expression of IkappaBalpha and lower NF-kappaB activity, when compared with vessels treated with rAAV-LacZ. Furthermore, ICAM-1 expression was markedly attenuated by the treatment with rAAV-IkappaBalpha (rAAV-LacZ=1.6+/-0.8 relative amount of ICAM-1 mRNA; rAAV-IkappaBalpha=0.55+/-0.04 relative amount of ICAM-1 mRNA). ICAM-1 protein levels were also significantly decreased in rAAV-IkappaBalpha treated mice. Finally the mean intimal area was 0.028+/-0.003 mm(2) in left carotid arteries treated with rAAV-LacZ whereas it was 0.003+/-0.004 mm(2) in vessels treated with rAAV-IkappaBalpha. Our data indicate that NF-kappaB plays a crucial role in neointimal hyperplasia induced by flow cessation in the mouse carotid artery, and in addition suggest that rAAV-mediated gene transfer of IkappaBalpha might represent a novel therapeutic approach to the treatment of restenosis.
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PMID:Crucial role of nuclear factor-kappaB in neointimal hyperplasia of the mouse carotid artery after interruption of blood flow. 1253 35

Nuclear factor-kappaB (NF-kappaB) plays a central role in myocardial ischemia-reperfusion (MI/R) injury. The inhibitory protein IkappaBalpha prevents its activation. We investigated the effects of adeno-associated viral vector-mediated IkappaBalpha gene transfer in MI/R injury. Male C57BL/6 mice were randomized to receive a recombinant adeno-associated virus (rAAV) encoding the gene for the NF-kappaB inhibitory protein IkappaBalpha (rAAV- IkappaBalpha) or the beta-galactosidase gene (a control and inert gene; rAAV-LacZ), both at a dose of 10(11) copies. Four weeks later anesthetized animals were subjected to total occlusion (45 minutes) of the left main coronary artery followed by 5 hours of reperfusion. MI/R produced a wide infarct size (IF/area-at-risk = 56 +/- 8%; IF/left ventricle = 44 +/- 5%) and tissue neutrophil infiltration, studied by means of elastase activity (area-at-risk = 2.5 +/- 0.4 micro g/gm tissue; infarct area = 2.9 +/- 0.6 micro g/gm tissue). Furthermore MI/R caused peak message for intercellular adhesion molecule-1 (ICAM-1) in the area-at-risk at 3 hours of reperfusion (1.2 +/- 0.4 relative amount of cardiac ICAM-1 mRNA). NF-kappaB activation was evident at 0.5 hours of reperfusion and reached its maximum increase at 2 hours of reperfusion. rAAV-IkappaBalpha injection reduced infarct size (IF/area-at-risk = 19 +/- 3%; IF/left ventricle = 10 +/- 2%; p < 0.001), blocked NF-kappaB activation, diminished cardiac ICAM-1 expression (0.4 +/- 0.02 relative amount of cardiac ICAM-1 mRNA; p < 0.001), and blunted leukocyte accumulation (area-at-risk = 0.6 +/- 0.05 micro g/gm tissue; infarct area = 0.4 +/- 0.02 micro g/gm tissue; p < 0.001). Our data indicate that rAAV-IkappaBalpha may be useful for MI/R gene therapy.
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PMID:Gene transfer of IkappaBalpha limits infarct size in a mouse model of myocardial ischemia-reperfusion injury. 1292 Feb 39

Around 200-600 million Asians chew areca (also called betel), which contains a mixture of areca nut and other ingredients. Epidemiological evidences indicated that areca use is tightly linked to oral carcinogenesis. This study investigated the effects of ripe areca nut extract (ANE) on cultured normal human oral keratinocyte (NHOK). Acute subtoxic ANE treatment inhibited DNA synthesis and induced cell cycle arrest at G1 phase in early passage (< 4th passage) cells. This was accompanied by a slight increase in the sub-G1 cellular fraction. O6-Methylguanine-DNA methyltransferase (MGMT), Hsp27 and p38MAPK was upregulated. p16 and p21 were remarkably upregulated early and declined afterwards. In contrast, the increase of dephosphorylated Rb seemed to be secondary to the episodes of p16 and p21 upregulation. To simulate the chronic areca exposure in vivo, constant ANE treatment in serial NHOK culture was performed. It resulted in a significant decrease in the population doubling, increase in senescence-associated beta-galactosidase (SA-beta-Gal) and decrease in cell proliferation in NHOK of late passages (> or = 4th passage). Induction of senescence-associated phenotypes, G2/M accumulation and genomic instability following long-term ANE treatment were also observed in a low-grade oral carcinoma cell. ANE-treated NHOK also had a higher nuclear factor-kappaB (NF-kappaB) fraction and a lower cytosolic IkappaBalpha level relative to the control in late passages. Moreover, electrophoretic mobility shift assay (EMSA) indicated that ANE treatment shifted the NF-kappaB complex from high mobility position to lower mobility position in late-passaged NHOK. ANE treatment also upregulated IL-6 and cyclooxygenase-2 (COX-2) mRNA expressions in late-passaged NHOK. In summary, our findings suggest that ANE induces the cell cycle arrest at G1/S phase and the occurrence of senescence-associated phenotypes of NHOK. The upregulation of p38MAPK, p16, p21, NF-kappaB, IL-6 and COX-2 are likely to participate in the control of these impacts.
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PMID:Ripe areca nut extract induces G1 phase arrests and senescence-associated phenotypes in normal human oral keratinocyte. 1647 77


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