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At least 811 records · Page 45Linked to original sources
PubMed2003

Overexpression of NP95 mRNA by tumor promoters in the promotion phase of a two-stage BALB/3T3 cell transformation assay.

We studied altered gene expressions in BALB/3T3 cells treated by different tumor promoters in the promotion phase of a transformation assay, an in vitro model of a two-stage carcinogenicity test, using fluorescent mRNA differential display analysis. Expression of the NP95 gene, which was previously found to be the gene of a murine nuclear protein associated with cell proliferation, was increased in the cultures treated by 12-O-tetradecanoylphorbol-13-acetate (TPA), okadaic acid, and orthovanadate. The upregulation of NP95 mRNA was confirmed by reverse transcription-PCR, and Northern blot. TPA, okadaic acid, and orthovanadate enhanced cell proliferation as measured by a 5-bromo-2'-deoxyuridine incorporation assay. The expression level of NP95 mRNA was not affected by the treatment with typical carcinogens benzo[a]pyrene and 3-methylcholanthrene at concentrations at which they act as initiators of cell transformation. These facts may imply that the enhancement of cell transformation by these tumor promoters is due, at least in part, to the acceleration of cell proliferation. NP95 mRNA was also increased in the transformed BALB/3T3 cells. Overexpression of NP95 may also participate in the maintenance of the transformed phenotype.

Animals↗
PubMed2004

How elevated oxalate can promote kidney stone disease: changes at the surface and in the cytosol of renal cells that promote crystal adherence and growth.

The present review assesses the mechanisms by which oxalate-induced alterations in renal cell function may promote stone disease focusing on 1) changes in membrane surface properties that promote the attachment of nascent crystals and 2) changes in the expression/secretion of urinary macromolecules that alter the kinetics of crystal nucleation, agglomeration and growth. The general role of renal cellular injury in promoting these responses and the specific role of urinary oxalate in producing injury is emphasized, and the signaling pathways that lead to the observed changes in cell surface properties and in the viability and growth of renal cells are discussed. Particular attention is paid to evidence linking oxalate-induced activation of cytosolic phospholipase A2 to changes in gene expression and to the activation of a second signaling pathway involving ceramide. The effects of the lipid signals, arachidonic acid, lysophosphatidylcholine and ceramide, on mitochondrial function are considered in some detail since many of the actions of oxalate appear to be secondary to increased production of reactive oxygen molecules within these organelles. Data from these studies and from a variety of other studies in vitro and in vivo were used to construct a model that illustrates possible mechanisms by which an increase in urinary oxalate levels leads to an increase in kidney stone formation. Further studies will be required to assess the validity of various aspects of this proposed model and to determine effective strategies for countering these responses in stone-forming individuals.

Animals↗
PubMedDate not supplied

A novel protein (p10) induced by 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and other hyperplasiogenic tumor-promoting and non-promoting agents in murine epidermis.

Treatment of murine epidermis with the tumor promotor 12-O-tetradecanoylphorbol-13-acetate (TPA) shows characteristic and significant changes in protein expression analyzed by 2D PAGE, compared to that of acetone-treated mouse epidermis. Of the seven de novo expressed proteins in TPA treated murine epidermis, one is a 44 kDa protein (p44) located nearby actin, and six proteins are in the low-molecular range between 10-20 kDa (p10, pY, pCa, p1, p2 and p3). Interestingly, the incomplete promoting and inflammative hyperplasiogen 12-O-retinoylphorbol-13-acetate (RPA) and the non-promoting but inflammative and hyperplasiogenic calcium-ionophore A23187 induced the same pattern of proteins observed in the pidermis of mice treated with TPA, with minor quantitative differences. In all cases, p10 expression was quantitative the most abundant. Partial sequencing of this protein has led to the conclusion that it is a novel protein with no such sequences in the database comparisons using FASTA and TFASTA computer programs of Genetics Computer Group. The data presented here do not strictly support the functional role of de novo induced proteins to tumor promotion, but show a causal relationship to hyperplasiogenic potency of TPA, RPA and A23187.

Amino Acid Sequence↗
PubMed2000

Nitric oxide synthase is induced in tumor promoter-sensitive, but not tumor promoter-resistant, JB6 mouse epidermal cells cocultured with interferon-gamma-stimulated RAW 264.7 cells: the role of tumor necrosis factor-alpha.

Expression of inducible nitric oxide synthase (iNOS) has been reported to be involved in certain organs of potential tumorigenesis, including the stomach and colon. The mechanisms for iNOS expression in epithelial cells, however, are not fully understood. In the present study, we investigated the role of macrophages in epithelial iNOS expression by coculturing a stimulated murine macrophage-like cell line, RAW 264.7, with either tumor promoter-sensitive (P+) or promoter-resistant (P-) JB6 murine epidermal cells. After monoculture, treatment of RAW 264.7 cells with IFN-gamma for 24 h generated a large amount of nitrite (NO2-), as reported previously, whereas no increase in NO2- concentration was observed in the IFN-gamma-treated P+ or P-subclones. Interestingly, when IFN-gamma-treated RAW 264.7 cells were cocultured with P+ but not P- cells, we observed a marked increase in NO2- concentration (30.8+/-3.6 microM), which significantly exceeded (P < 0.01) the sum of the concentrations (20.0+/-2.3 microM) added from each cell line monoculture. Western blotting analysis revealed that, after coculture, iNOS protein was up-regulated 55-fold more than the control in JB6 P+ but not in P- cells. IFN-gamma-treated RAW 264.7 cells secreted proinflammatory cytokines, including tumor necrosis factor (TNF)-alpha and interleukin (IL)-1beta. The addition of IFN-gamma-treated RAW 264.7 cell-conditioned media to P+ subclones led to a significant enhancement of NO2- formation that was diminished by the TNF-alpha-specific but not IL-1beta-specific antibody. When combined with IFN-gamma, the recombinant TNF-alpha (1-100 ng/ml) enhanced NO2- formation in JB6 P+ cells, whereas IL-1beta (1-100 ng/ml) did not. These results led us to conclude that IFN-gamma-treated RAW 264.7 cells release TNF-alpha to induce iNOS expression in promoter-sensitive JB6 cells. Thus, we propose the hypothesis that macrophages stimulate neoplastic cells with TNF-alpha via a paracrine loop to induce epithelial iNOS protein expression.

Animals↗
PubMed1989

Analysis of the B-cell growth-promoting activity of human IL-4, the co-stimulatory assay with anti-immunoglobulin antibodies. Comparison with the B-cell growth-promoting activity of other lymphokines.

Human recombinant interleukin-4 (rIL-4) was assessed for its ability to promote the proliferative response of purified human B cells co-stimulated with submitogenic concentrations of soluble F(ab')2 fragments of anti-immunoglobulin (Ig) antibodies. The growth-promoting activity of rIL-4 was usually as potent as, or even more potent than, that of recombinant interleukin-2 (rIL-2), and more potent than that of recombinant interferon-gamma (rIFN-gamma). Preincubation with rIL-4 did not cause enhancement of the proliferative response of B cells to the subsequent addition of rIL-4 and anti-IgM antibody. In contrast, the proliferative response of B cells preincubated with anti-IgM antibody and rIL-4 was potentiated by the subsequent addition of rIL-4. The simultaneous addition of rIFN-gamma and rIL-2 or rIFN-gamma and rIL-4 had an additive effect in comparison with the response induced by rIL-2 or rIL-4 alone, respectively, whereas simultaneous addition of rIL-2 and rIL-4 induced a response equal or lower than that stimulated by rIL-2 or rIL-4 alone. The addition of rIFN-gamma at the beginning of culture or preincubation of B cells with rIFN-gamma and anti-IgM antibody potentiated the proliferative response of B cells to the subsequent addition of either rIL-2 or rIL-4. Taken together, these data suggest that rIL-4 acts as a growth factor for activated human B cells and displays on such cells a growth-promoting activity similar to that of rIL-2.

Adjuvants, Immunologic↗
PubMed1988

P-450 enzyme induction by 5-ethyl-5-phenylhydantoin and 5,5-diethylhydantoin, analogues of barbiturate tumor promoters phenobarbital and barbital, and promotion of liver and thyroid carcinogenesis initiated by N-nitrosodiethylamine in rats.

Male F344/NCr rats, 6 wk old, were fed 500 ppm of phenobarbital (PB) or equimolar doses of either 5-ethyl-5-phenylhydantoin (EPH) or 5,5-diethylhydantoin (EEH) in diet for 2 wk and hepatic cytochrome P-450-mediated alkoxyresorufin O-dealkylase and aminopyrine N-demethylase activities were determined. Both PB and EPH greatly increased P-450-mediated enzyme activities in rat liver while EEH was ineffective. To evaluate the hydantoins as tumor promoters, 5-wk-old male F344 rats were given a single i.p. injection of 75 mg N-nitrosodiethylamine/kg body weight. Beginning 2 wk later, they were placed either on normal diet or diet containing 500 ppm of PB or equimolar doses of EPH or EEH for the remaining experimental period. Control groups received an i.p. injection of saline followed by each of the test diets. Animals were sacrificed at either 52 or 78 wk. PB and EPH significantly enhanced the development of hepatocellular foci and hepatocellular adenomas at 52 wk and hepatocellular carcinomas at 78 wk in N-nitrosodiethylamine-initiated rats. Neither the incidence of hepatocellular neoplasms nor the number and size of hepatocellular foci was significantly increased by EEH. At 78 wk, both PB and EPH enhanced the development of thyroid follicular cell neoplasms in N-nitrosodiethylamine-initiated rats while no such enhancement was observed with EEH. Thus, EPH, a long-acting sedative/anticonvulsant, like the structurally similar PB, promoted hepatocellular and thyroid follicular cell carcinogenesis and induced the PB-inducible form(s) of cytochrome P-450 (P-450b) in rats. In contrast, EEH unlike barbital failed to promote hepatocellular and thyroid follicular cell carcinogenesis and also failed to induce PB-inducible form(s) of cytochrome P-450 in rats.

Animals↗
PubMed1985

UV cross-linking of the Bacillus subtilis RNA polymerase to DNA in promoter and non-promoter complexes.

Complexes between Bacillus subtilus RNA polymerase and 32P-labeled DNA were irradiated with UV light and digested with nuclease; electrophoresis and autoradiography were used to identify the polymerase subunits cross-linked to DNA. These experiments showed: 1) that cross-linkage of promoter complexes yielded predominantly the beta and sigma subunits; 2) that beta, beta', and sigma were detected in non-promoter complexes; 3) that addition of the delta subunit or high concentrations of NaCl decreased cross-linkage of all subunits, especially the cross-linkage of the sigma subunit in non-promoter complexes and the binding of polymerase at DNA ends; 4) that different patterns of cross-linkage were obtained at 0 degrees C (conditions favoring the formation of closed complexes) and 37 degrees C (conditions favoring the formation of open complexes); and 5) predominantly beta and possibly alpha were cross-linked by irradiation of core-DNA complexes whereas similar experiments with core-delta complexed to DNA showed the efficient cross-linkage of beta' and beta.

Bacillus subtilis↗
PubMed1998

Altered expression of epidermal growth factor receptor ligands in tumor promoter-treated mouse epidermis and in primary mouse skin tumors induced by an initiation-promotion protocol.

Multiple epidermal growth factor receptor (EGFr) ligands have been identified, including transforming growth factor alpha (TGFalpha), heparin-binding epidermal growth factor (HB-EGF), amphiregulin (AR), and betacellulin (BTC). Previous work from our laboratory demonstrated that TGFalpha mRNA and protein are upregulated in epidermis during tumor-promoter treatment of mouse skin and in skin tumors produced by initiation-promotion regimens. The purpose of the study described here was to explore the role of other EGFr ligands in multistage skin carcinogenesis. A single topical treatment of either 12-O-tetradecanoylphorbol-13-acetate (TPA) or chrysarobin or a single full-thickness wound induced the expression of HB-EGF and AR in mRNA samples isolated from whole mouse skin. However, only full-thickness wounding significantly elevated expression of the BTC transcript. The levels of HB-EGF and AR transcripts were significantly elevated in skin tumors (both papillomas and squamous cell carcinomas) induced by initiation-promotion protocols. BTC transcript levels were low and barely detectable in all skin tumors examined. The level of keratinocyte growth factor (KGF) mRNA was also examined as a possible mechanism for upregulation of EGFr ligands. Only full-thickness wounding significantly elevated KGF transcript levels in whole-skin RNA samples. Furthermore, no evidence for upregulation of KGF mRNA in skin tumors was obtained. The results are discussed in terms of the role of EGFr activation in skin carcinogenesis and the mechanisms for altered regulation of EGFr ligands.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2002

Efficiency of Iε promoter-directed switch recombination in GFP expression-based switch constructs works synergistically with other promoter and/or enhancer elements but is not tightly linked to the strength of transcription.

One key unresolved issue in immunoglobulin class switch recombination (CSR) is how the accessibility of the switch region for CSR is controlled. To better understand the nature of accessibility control for human Ig CSR, we developed a novel inducible switch recombination assay based on expression of green fluorescence protein (GFP) from switch constructs undergoing substrate switch recombination (SSR). Efficient SSR depends on the cytokine-inducible Iepsilon promoter and co-stimulation with IL-4+anti-CD40. Characterization of SSR reveals that both S-S deletional recombination and S-S inversion occur. We show that the IL-4-inducible Iepsilon promoter (pIepsilon) selectively determines the efficiency of the accessibility for SSR. However, the pIepsilon-induced transcription, by itself,is not sufficient to direct efficient SSR. For efficient SSR, both pIepsilon-driven transcriptional activity and an additional promoter/enhancer-derived activity are required. The efficiency of SSR is not tightly correlated with the strength of the combined transcriptional activity. Our results suggest that the mechanism(s) underlying the transcriptional activity, e.g. DNA modification is important for controlling the accessibility for efficient switch recombination.

Animals↗
PubMed1986

Structure of RNA polymerase II promoters. Conformational alterations and template properties of circularized Saccharomyces cerevisiae GAL1-GAL10 divergent promoters.

A DNA fragment encompassing the Saccharomyces cerevisiae GAL1--GAL10 divergent promoters (914 bp) has been circularized in vitro with T4 DNA ligase. We have defined a set of conditions that allows the production of a series of nine topoisomers covering a range from relaxed to highly negatively supercoiled DNA. Topoisomers were recovered in pure form from agarose gels and were analysed singly for the presence of sites sensitive to the single strand-specific endonuclease Pl. In this way, the occurrence of conformational alterations as a function of the linking deficiency of the closed DNA domain has been determined. Interestingly, sites of Pl hypersensitivity localize on the three sequences identified as relevant for the in vitro transcription of the GAL1 moiety of the divergent promoter: the upstream activator sequence (UAS), the TATA sequence, and the RNA initiation site (RIS). In vitro transcription with purified S. cerevisiae RNA polymerase II shows that activation of transcription parallels the appearance of conformational alterations on the UAS, the TATA and the RIS sequences.

Base Composition↗
PubMed2004

Expression of the bovine aromatase cytochrome P450 gene (Cyp19) is primarily regulated by promoter 2 in bovine follicles and by promoter 1.1 in corpora lutea.

Follicular development and differentiation are closely associated with increasing steroidogenesis. During the present study transcript concentration of Cyp19, Cyp11A1, and 3beta-hydroxysteroid dehydrogenase delta (3beta-HSD) encoding the steroidogenic enzymes P450(arom), P450(SCC), and 3beta-HSD were determined by real-time PCR in bovine granulosa cells (GC) as potential markers for follicular differentiation. Ovaries were collected from a local abattoir (experiment 1) and from synchronized animals at day 4 of estrus cycle (experiment 2). To study effects of luteinization, steroidogenic transcripts were also quantified in corpora lutea (CL) 4 and 20 days after fertilization. In most follicles, all three steroidogenic transcripts were detected, however, at very different concentration. Expression of 3beta-HSD and Cyp11A1 was highly significantly co-regulated and showed a significant correlation with follicular size. Contrary, Cyp19 expression was extremely variable even in follicles of similar size. Cyp19 transcripts were derived predominantly from promoter P2 and less abundant from promoters P1.1 and P1.5. After luteinization, the concentration of 3beta-HSD and Cyp11A1 transcripts increased (75-fold and fivefold, respectively) whereas the Cyp19 transcript level dropped (160-fold). Residual Cyp19 transcripts in CL were almost exclusively derived from P1.1. The data indicate that Cyp19 expression in GC is predominantly regulated by P2 and to a minor extend by P1.1, whereas P1.1 is almost exclusively responsible for residual Cyp19 expression in CL. Correlation analyses suggest that the expression of 3beta-HSD and Cyp11A1 primarily depend on the size of follicles whereas the concentration of P2 derived Cyp19 transcripts in GC is a marker for follicular differentiation towards selection and dominance.

Animals↗
PubMed2000

Promoting health or promoting pleasure? A contingency approach to the effect of informational and emotional appeals on food liking and consumption.

Here, we suggest that the relative effectiveness of informational and emotional appeals in persuasive communications may depend on the dominant basis of attitude towards the focal item (affective or cognitive), and on the precise response being targeted (i.e. influencing affect-based food liking or cognitive-based consumption). Ninety-five participants (milk drinking adults) participated in an experiment in which they were presented with a persuasive communication promoting milk consumption. A mixed design combined as between-participants factors attitude bases (two: affective/cognitive) and persuasive appeals (two: informational/emotional) with, as a within-participants measure, types of effect (two: liking and consumption change intent). We also measured immediate feelings and thoughts responses to the communication. As expected, cognition-based attitudes were not sensitive to a match between persuasive appeals and types of effect. For affect-based attitudes, results confirmed the predicted superiority of a match between the affective and cognitive bases of the targeted responses and that of the persuasive appeal. Food liking was particularly sensitive to an emotional appeal while an informational appeal tended to be more influential on food consumption. The underlying mechanism of these effects and their implication for the practice of health promotion and food marketing are discussed.

Adult↗
PubMed1995

Isolation of the human interleukin 10 promoter. Characterization of the promoter activity in Burkitt's lymphoma cell lines.

Interleukin 10 (IL-10) is a pleiotropic growth and differentiation factor with potent suppressor functions on macrophages, T cells and NK cells and contributes to the regulation of proliferation and differentiation of B cells. The expression of IL-10 appears to be tightly regulated, as the levels of constitutive expression in normal cells is extremely low. In contrast to normal haematopoetic cells, Epstein-Barr virus (EBV)-immortalized B cells and EBV-positive Burkitt's lymphoma cells express high levels of IL-10 constitutively. In this report we have cloned and sequenced IL-10 promoter fragments and analysed their activity in EBV-positive Burkitt's lymphoma cells. A nested set of DNA fragments from the IL-10 gene 5'-flanking region was placed upstream of the luciferase gene and assayed for their ability to direct luciferase expression in Burkitt's lymphoma cells. We have identified elements within the 5'-flanking region of the human IL-10 gene which can activate or suppress the constitutive expression of IL-10. The essential promoter of the IL-10 gene, which induces low levels of luciferase expression, was found to require the major start site of transcription (+1), a TATA-box (-77) and up to 150 additional 5' nucleotides. Positive regulatory sequences are located between -1100/-900. Negative regulatory elements which abolish luciferase activity were identified between -800/-300.

Base Sequence↗
PubMed1995

Characterization of the "promoter region" of the enolase-encoding gene enol from the anaerobic fungus Neocallimastix frontalis: sequence and promoter analysis.

The sequence of the Neocallimastix frontalis enolase gene promoter was determined up to 1800 nucleotides 5' to the major transcriptional start point. The base composition of the enolase upstream sequence revealed a very A + T-rich profile (13.5% G + C) leading to many putative hairpin structures. The functional organization of the N. frontalis enolase promoter was investigated by heterologous transient-expression assays. DNA fragments obtained by the sequential removal of sequences upstream of the translation start codon were fused to the Escherichia coli lacZ gene and the resulting plasmids were used to transform the ascomycetes Aspergillus nidulans and Penicillium roqueforti and the oomycete Saprolegnia monoica. Transient expression of the lacZ reporter gene was observed in regenerating proteoplasts of S. monoica when using the 0.3 kb or 1 kb upstream of the enolase coding region. In contrast no beta-galactosidase activity was detected in ascomycete protoplasts. DNA hybridization analysis revealed the integration of vector DNA in the genomic DNA of S. monoica and the presence of free copies of the transformation plasmid which could be rescued in E. coli. Our results indicate that the transcriptional machinery of the anaerobic chytrid N. frontalis may differ significantly from that of ascomycetes but that enough conservation exists within the lower fungi to allow a transient-driven expression of a reporter gene in an oomycete fungus.

Base Sequence↗
PubMed1995

The Gnb5 gene is a novel beta-transducin homolog transcribed from a divergent promoter located immediately upstream of the Syrian hamster p53 P1 promoter.

Regulatory regions controlling p53 gene transcription in Syrian hamster embryo cells were characterized by use of chloramphenicol acetyl-transferase (CAT) constructs encompassing various subfragments of its 5'-flanking sequences. This analysis identified a 961 bp PstI-SacI (PS) fragment upstream from the p53 P1 promoter, which exhibited promoter activity only in the reverse orientation relative to the p53 gene. Northern hybridizations of mRNA from hamster embryo cells with genomic probes containing the PS fragment detected a 2.1-kb transcript expressed at much lower levels than the p53 mRNA. Steady-state levels of the 2.1-kb mRNA were threefold higher in actively growing cells than in cells from confluent cultures. Library screenings with PS-containing probes resulted in the isolation from exponentially growing cells of a cDNA, the nucleotide sequence of which showed no significant homology to genes previously described. This novel gene, named Gnb5, for guanine nucleotide-binding protein, beta 5, codes for a protein of 538 amino acids with a highly acidic amino terminus containing a proline-rich domain, followed by a neutral domain with five repeat units of the beta-transducin (WD-40) motif. The homology with beta subunits of G proteins and with other WD-40 repeat-containing proteins was restricted to the repeats. The Gnb5 gene is well conserved in rodents and primates, as the hamster Gnb5 cDNA recognized, under high stringency conditions, the human and mouse counterparts in Southern and Northern hybridizations. Expression of Gnb5 in adult tissues was detected preferentially in testes, in both hamsters and humans.

Adult↗
PubMed1987

Replacement of the promoter of the yeast plasma membrane ATPase gene by a galactose-dependent promoter and its physiological consequences.

In order to probe the physiological role of the yeast plasma membrane ATPase we have replaced the constitutive promoter of its gene by a galactose-dependent promoter. The resulting cells stop growing on glucose medium when the preformed ATPase is diluted to 20% of normal. There is a correlation between ATPase activity and both proton efflux from the cells and amino acid transport. A large proportion of growth-arrested cells appear enlarged and with several buds containing nuclei.

Adenosine Triphosphatases↗
PubMed1996

Stimulation of mitotic recombination upon transcription from the yeast GAL1 promoter but not from other RNA polymerase I, II and III promoters.

Homologous recombination in Saccharomyces cerevisiae and other organisms can be stimulated by transcription. Consistent with this, we find that recombination of a chromosomal ade1 allele with a plasmid-borne ADE1 ORF under the control of the GAL1 promoter increased from 6.1x10(-6) to 1.7x10(-4) when transcription of the plasmid locus was induced by growing the cells in the presence of galactose. Recombination could also be stimulated by over-expressing the Gal4 transcription factor in the presence of the GAL1-ADE1 plasmid, while culturing the cells in dextrose medium. However, when transcription of the same ORF was driven from the highly active promoters of the rDNA (RNA polymerase I), and ADH1 (RNA polymerase II) genes, only background levels of recombination (5-10x10(-6)) were observed, irrespective of the carbon source. Recombination was found to involve integration of the whole plasmid and to depend on RAD51, RAD52 and RAD54. The results indicate that increased accessibility of transcriptionally active chromatin is not sufficient to cause increased rates of this kind of reciprocal exchange.

Alleles↗
PubMed2004

FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster.

Foldback ( FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w(67C23), a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w(67C23) locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.

ATP-Binding Cassette Transporters↗