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)

We have isolated a rabbit neuronal nitric oxide synthase (nNOS) cDNA encoding a protein of 1,435 amino acids. Using the cDNA clones as probes, the 5'-flanking region of the nNOS gene was isolated from a rabbit genomic DNA library. 5'RACE and primer extension analysis of rabbit brain total RNA mapped multiple transcription initiation sites localized 474-487 bp upstream from the translation start codon. Analysis of 5,197 bp of the 5'-flanking sequence revealed that the rabbit nNOS gene promoter lacks canonical TATA or CCAAT boxes and, instead, contains a GC-rich region and multiple Sp1 sites. Farther from the +1start, various putative cis-elements including AP-1, AP-4, NF-kappaB, STAT, CREB, C/EBP and c-Myc were observed. The functional promoter activity of the 5'-flanking region was demonstrated by its ability to drive the expression of a beta-galactosidase reporter gene in several cell types. Serial deletion analysis of the promoter region revealed that the -291 to -172 region, which contains two Sp1 sites, is essential for basal transcriptional activity. These results suggest that the rabbit nNOS promoter contains characteristics of inducible genes.
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PMID:5'-Flanking sequence and promoter activity of the rabbit neuronal nitric oxide synthase (nNOS) gene. 1110 Nov 49

Interleukin 1alpha (IL-1alpha) is an important regulatory cytokine, the release of which after an injury can induce activation of transcription factors nuclear factor (NF)kappaB and activator protein (AP-1), which promote expression of genes involved in cell survival, proliferation, and angiogenesis. IL-1alpha is expressed autonomously by head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers, raising the possibility that IL-1alpha may serve as an autocrine factor that stimulates the activation of prosurvival transcription factors and target genes in cancer. In this study, we examined the role of IL-1alpha in the activation of NFkappaB and AP-1, the expression of proangiogenic cytokine IL-8, and in the survival and proliferation of HNSCC cell lines. HNSCCs were found to secrete and respond to functional IL-1alpha, in that culture supernatant from a high IL-1alpha-secreting line, UM-SCC-11B, could induce secretion of cytokine IL-8 by a low IL-1alpha-secreting line, UM-SCC-9; and the induction of IL-8 secretion could be blocked by the anti-IL-1alpha-neutralizing antibody or the IL-1 receptor antagonist (IL-1RA). Furthermore, IL-1alpha could induce the expression of IL-8 through an autocrine mechanism, in that transfection of UM-SCC-9 cells with a plasmid encoding IL-1alpha resulted in the increased coexpression of IL-1alpha and IL-8; whereas transfection with a plasmid encoding IL-1RA lacking the secretory leader sequence led to the decreased coexpression of IL-1alpha and IL-8. IL-1alpha was found to induce coexpression of IL-8 through the activation of NFkappaB and AP-1, in that mutation of the NFkappaB site within the IL-8 promoter abolished autocrine- and recombinant IL-1alpha-induced IL-8 reporter gene activity, whereas mutation in AP-1 partially decreased IL-8 reporter gene activity in UM-SCC-9 cells. Intracellular expression of IL-1RA decreased NFkappaB reporter gene activity, indicating that endogenously expressed IL-1alpha contributes to constitutive NFkappaB activation in this HNSCC line. Expression of IL-1alpha affected survival of UM-SCC-9, inasmuch as transfection of cells with plasmid encoding IL-1alpha or IL-1RA led to the increased or decreased survival of cells cotransfected with a beta-galactosidase reporter gene, respectively. IL-1alpha was also found to promote the increased growth of UM-SCC-9 cells in vitro. We demonstrate that exogenous and endogenous IL-1alpha contributes to the transcriptional activation of NFkappaB and AP-1, to the expression of IL-8, and to cell survival and the growth of HNSCC in vitro.
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PMID:IL (interleukin)-1alpha promotes nuclear factor-kappaB and AP-1-induced IL-8 expression, cell survival, and proliferation in head and neck squamous cell carcinomas. 1141 May 24

Intracellular signaling by mitogen-activated protein (MAP) kinase cascades plays an essential role in the cellular response to environmental stress. In the yeast Saccharomyces cerevisiae, the PKC1-regulated, stress-activated MAP kinase pathway, the MPK1 cascade, is activated by heat and by a decrease in osmolarity. The genes WSC1, WSC2 and WSC3 encode putative receptors that maintain cell wall integrity under conditions of heat stress. Genetic studies place the function of the WSC genes upstream of the MPK1 kinase cascade. To further define the role of the WSC family in the stress response we determined whether: (1) the wscdelta mutants are sensitive to other environmental stress conditions, in addition to heat shock; (2) expression from four transcriptional control elements, known to be activated by stress, is impaired in wscdelta mutants; and (3) Wsc4, a Wsc homolog, has functions that overlap with those of the other Wsc family members. We report here that deletion of WSC and PKC1 causes hypersensitivity to ethanol, hydrogen peroxide and DNA-damaging drugs. In wscdelta mutants expression of beta-galactosidase from the AP-1 response element (ARE), the heat shock response element (HSE) or the stress response element (STRE) is not reduced. In contrast, expression of a reporter gene placed under the control of the Rlm1 (transcription factor)-dependent response element is significantly reduced in wscdelta mutants. This suggests that the lysis defect of wscdelta mutants is at least in part caused by a defect in transcriptional regulation by Rlm1. Phenotypic analysis of the effect of deleting WSC4 in a wsc1delta mutant show that, unlike WSC2 or WSC3, deletion of WSC4 does not exacerbate the lysis defect of a wsc1delta strain. In contrast, deletion of WSC4 enhances the sensitivity of the wsc1delta mutant to heat shock, ethanol, and a DNA-damaging drug, suggesting that WSC4 plays a role in the response to environmental stress but that its function may differ from those of the other WSC family members.
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PMID:Mutations in WSC genes for putative stress receptors result in sensitivity to multiple stress conditions and impairment of Rlm1-dependent gene expression in Saccharomyces cerevisiae. 1158 72

Recent studies have shown that the presence of tumor suppressors such as p53 or p16 account for the lack of transformation in primary cells. To investigate a potential role of active Ras in atherosclerosis, we infected bovine aortic endothelial cells with a replication-deficient, recombinant adenovirus containing the activated H-Ras61L gene. Ras overexpression led after 72 hours to G1- and G2/M-cell cycle arrest due to induction of p21(Cip1/Waf1). Treatment of Ras-infected endothelial cells with 40 ng/ml TNF-alpha for 20 hours augmented apoptosis 8-fold in comparison to Ad-Con (control virus with empty expression cassette) infected cells (36.2% vs. 4.3%, p < 0.001), while Ras itself did not cause any cell death. Furthermore, more than 58% of Ras-infected cells stained positive for senescence-associated beta-galactosidase activity as opposed to 2% in control vector-infected cells (p < 0.001), strongly suggesting a senescent phenotype in the Ras-infected population. We found further features of senescence in Ras-transduced endothelial cells, such as growth arrest and the lack of AP-1 serum inducibility. Finally, we evaluated the role of p21(Cip1/Waf1) in this process of senescence. Adenoviral overexpression of p21 led to growth arrest by induction of G1- and G2/M-cell cycle arrest. In addition, p21-overexpressing endothelial cells were highly sensitive for TNF-alpha induced-apoptosis. Surprisingly, senescence-associated beta-galactosidase activity was not apparant in p21-infected endothelial cells, suggesting further signaling events necessary for the senescent morphology of endothelial cells. Our results demonstrate a novel way to render primary endothelial cells senescent by overexpressing oncogenic Ras. Increased sensitivity of senescent endothelial cells for cytotoxic stimuli seemed to be due to Ras-induced upregulation of p21(Cip1/Waf1). Future studies have to investigate a potential role of Ras in human vascular biology.
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PMID:Oncogenic ras induces premature senescence in endothelial cells: role of p21(Cip1/Waf1). 1200 58

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

In these experiments precision-cut tissue slices from two existing transgenic mouse strains, with transgenes that couple promoting or binding elements to a reporter protein, were used for determination of reporter induction. This approach combines the power of transgenic animals with the practicality of in vitro systems to investigate the biological impact of xenobiotics. Additionally, the normal cellular architecture and heterogeneity is retained in precision-cut tissue slices. Two transgenic mouse strains, one of which couples the promoting region of CYP 1A1 to beta-galactosidase, and another which couples two forward and two backward 12-O-tetradecanoyl phorbol-13-acetate (TPA) repeat elements (TRE) to luciferase (termed AP-1/luciferase), were used to determine the feasibility of this approach. Precision-cut kidney and liver slices from both transgenic strains remain viable as determined by slice K(+) ion content and LDH enzyme release. Liver slices harvested from the CYP 1A1/beta-galactosidase transgenic mice exhibit a 14-fold increase in beta-galactosidase activity when incubated with beta-napthoflavone for 24 h. Kidney and liver slices obtained from the AP-1/luciferase transgenic mice demonstrate induction of luciferase (up to 2.5-fold) when incubated with phorbol myristate acetate (PMA or TPA) up to 4 h. These data indicate that precision-cut tissue slices from transgenic mice offer a novel in vitro method for toxicity evaluation while maintaining normal cell heterogeneity.
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PMID:Precision-cut tissue slices from transgenic mice as an in vitro toxicology system. 1265 Jun 74

FRNK, the autonomously expressed carboxyl-terminal region of focal adhesion kinase (FAK), is expressed in tissues that are rich in vascular smooth muscle cells (VSMCs). Here we report the generation of transgenic mice harboring the putative FRNK promoter fused to LacZ and examine the promoter activity in situ via expression of beta-galactosidase. The transgenic mice exhibited expression of beta-galactosidase predominantly in arterial VSMCs in large and small blood vessels of major organs. Upregulation of beta-galactosidase activity was observed in tunica media following carotid injury, indicating that the FRNK promoter is activated in VSMCs in response to injury. Robust expression of beta-galactosidase in blood vessels was also detected in the developing embryo. However, expression was also observed in the midline, the nose and skin epidermis, indicating distinct transcriptional regulation of the FRNK promoter in embryogenesis. To analyze FRNK expression in vitro, we identified a 116 bp sequence in the FRNK promoter that was sufficient to function as an enhancer when fused to the minimal actin promoter and expressed in cultured smooth muscle cells. Mutation of AP-1 and NF-E2 binding consensus sequences within this element abrogated enhancer activity, supporting the involvement of this promoter element in VSMC expression of FRNK.
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PMID:FRNK, the autonomously expressed C-terminal region of focal adhesion kinase, is uniquely regulated in vascular smooth muscle: analysis of expression in transgenic mice. 1596 14

Islet neogenesis associated protein (INGAP) promotes the generation of new islet mass in adult animal models. It is not understood what factors control the expression of INGAP. In this study, factors that regulate the expression of INGAP promoter activity are reported. To determine factors that regulate INGAP expression, we previously cloned the promoter region for INGAP. Analysis of the INGAP promoter suggested that candidate regulators of INGAP expression include the transcription factors PDX-1, NeuroD, PAN-1, STAT and AP-1. Using gene addition experiments in the 293 cell line the activity of these transcription factors on an INGAP-promoter construct linked to the beta-galactosidase reporter has been determined. Induction of AP-1 activity or STAT activity using PMA or LIF stimulation respectively, or direct expression of PAN-1 specifically up-regulates INGAP promoter activity. In contrast, co-expression of PDX-1 but not NeuroD inhibits activation of the INGAP-promoter driven by PAN-1, PMA or LIF stimulation. PDX-1 binds directly to the INGAP promoter as determined in electromobility shift and antibody supershift assays. Expression of the INGAP-promoter-reporter construct in the HIT-T15 beta-cell line, a cell line that expresses endogenous PDX-1, did not reveal PMA-mediated stimulation of INGAP promoter activity. HIT-T15 cells however did efficiently transfect (> 68%) and respond (2-fold) to PMA-induced signal transduction to a transfected AP-1-CAT reporter. Partial reduction of PDX-1 expression in HIT-T15 cells was associated with recovery of PMA induced INGAP promoter activity. These data suggest that expression of PDX-1 is associated with a repression of stimulus-induced INGAP promoter activity that appears to be mediated by a direct DNA interaction. These findings implicate PDX-1 in a possible feedback loop to block unbridled islet expansion.
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PMID:PDX-1 can repress stimulus-induced activation of the INGAP promoter. 1652 40

Hepatic stellate cells (HSCs) play a pivotal role in hepatic fibrogenesis, and are considered a cellular target for therapeutic intervention. We recently established that a 2.2-kb hGFAP (human glial fibrillary acidic protein) promoter could be used to specifically target cultured HSCs. In the current study, we aimed to investigate whether the same transgene (2.2-kb hGFAP-lacZ) can be used as a biomarker for studying the inhibition of HSC activation. HSC-T6 cells stably transfected with the transgene were treated with two natural anti-fibrotic compounds, epigallocatechin gallate (EGCG) and genistein separately. Results showed that both transgenic beta-galactosidase activity and endogenous GFAP expression (mRNA and protein) were attenuated by EGCG or genistein treatment in a dose- and time-dependent manner. Our data further demonstrated that the suppression of fibrogenic end-points was primarily mediated through the inhibition of AP-1 signaling (and to a lesser degree through the NFkappaB pathway) onto the GFAP promoter. In conclusion, the current findings provide a proof-of-concept for using GFAP for studying HSC activation and inhibition. It could be envisioned that a HSC-based high-throughput system can be constructed using the GFAP promoter in conjunction with a real-time reporter for the screening of anti-fibrotic and anti-inflammatory agents.
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PMID:Epigallocatechin gallate and genistein attenuate glial fibrillary acidic protein elevation induced by fibrogenic cytokines in hepatic stellate cells. 1708 19

Upregulation of the MDR1 (multidrug resistance 1) gene is involved in the development of resistance to antifungal agents in clinical isolates of the pathogen Candida albicans. To better understand the molecular mechanisms underlying the phenomenon, the cis-acting regulatory elements present in the MDR1 promoter were characterized using a beta-galactosidase reporter system. In an azole-susceptible strain, transcription of this reporter is transiently upregulated in response to either benomyl or H(2)O(2), whereas its expression is constitutively high in an azole-resistant strain (FR2). Two cis-acting regulatory elements within the MDR1 promoter were identified that are necessary and sufficient to confer the same transcriptional responses on a heterologous promoter (CDR2). One, a benomyl response element (BRE), is situated at position -296 to -260 with respect to the ATG start codon. It is required for benomyl-dependent MDR1 upregulation and is also necessary for constitutive high expression of MDR1. A second element, termed H(2)O(2) response element (HRE), is situated at position -561 to -520. The HRE is required for H(2)O(2)-dependent MDR1 upregulation, but dispensable for constitutive high expression. Two potential binding sites (TTAG/CTAA) for the bZip transcription factor Cap1p (Candida AP-1 protein) lie within the HRE. Moreover, inactivation of CAP1 abolished the transient response to H(2)O(2). Cap1p, which has been previously implicated in cellular responses to oxidative stress, may thus play a trans-acting and positive regulatory role in the H(2)O(2)-dependent transcription of MDR1. A minimal BRE (-290 to -273) that is sufficient to detect in vitro sequence-specific binding of protein complexes in crude extracts prepared from C. albicans was also defined. Interestingly, the sequence includes a perfect match to the consensus binding sequence of Mcm1p, raising the possibility that MDR1 may be a direct target of this MADS box transcriptional activator. In conclusion, while the identity of the trans-acting factors that bind to the BRE and HRE remains to be confirmed, the tools developed during this characterization of the cis-acting elements of the MDR1 promoter should now serve to elucidate the nature of the components that modulate its activity.
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PMID:Identification of promoter elements responsible for the regulation of MDR1 from Candida albicans, a major facilitator transporter involved in azole resistance. 1715 23


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