Home About us Contact
|
Home About us Contact | ||
|
|
||
Transcriptional Activity (transcriptional + activity)
Kinds of Transcriptional Activity Selected AbstractsInduction of Transcriptional Activity of the Cyclic Adenosine Monophosphate Response Element Binding Protein by Parathyroid Hormone and Epidermal Growth Factor in Osteoblastic Cells,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2002John T. Swarthout Abstract Previously, we have shown that parathyroid hormone (PTH) transactivation of cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) requires both serine 129 (S129) and serine 133 (S133) in rat osteosarcoma cells UMR 106-01 (UMR) cells. Furthermore, although protein kinase A (PKA) is responsible for phosphorylation at S133, glycogen synthase kinase 3, (GSK-3,) activity is required and may be responsible for phosphorylation of CREB at S129. Here, we show, using the GAL4-CREB reporter system, that epidermal growth factor (EGF) can transactivate CREB in UMR cells in addition to PTH. Additionally, treatment of UMR cells with both PTH and EGF results in greater than additive transactivation of CREB. Furthermore, using mutational analysis we show that S129 and S133 are required for EGF-induced transcriptional activity. EGF activates members of the MAPK family including p38 and extracellular signal,activated kinases (ERKs), and treatment of UMR cells with either the p38 inhibitor (SB203580) or the MEK inhibitor (PD98059) prevents phosphorylation of CREB at S133 by EGF but not by PTH. Treatment of cells with either SB203580 or PD98059 alone or together significantly inhibits transactivation of CREB by EGF but not by PTH, indicating that EGF regulates CREB phosphorylation and transactivation through p38 and ERKs and PTH does not. Finally, the greater than additive transactivation of CREB by PTH and EGF is significantly inhibited by the PKA inhibitor H-89 or by cotreatment with SB203580 and PD98059. Thus, several different signaling pathways in osteoblastic cells can converge on and regulate CREB activity. This suggests, in vivo, that circulating agents such as PTH and EGF are acting in concert to exert their effects. [source] Transcriptional activities of mutant p53: When mutations are more than a lossJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2004Ella Kim Abstract The dominant oncogenic properties of mutant p53 have been recognized as a phenomenon associated with tumor progression a long time ago, even before it was realized that the major function of wild type p53 is that of a tumor suppressor. Recent advances in this fascinating area in tumor cell biology reveal that the community of mutant p53 proteins is comprised of proteins that are extremely diverse both structurally and functionally, and elicit a multitude of cellular responses that not only are entirely distinct from those mediated by wild type p53, but also vary among different mutant p53 proteins. Aberrant regulation of transcription is one of the mechanisms underlying the ability of some mutant p53 proteins to act as oncogenic factors. Systematic analyses of the transcriptional activities of mutant p53 suggest that not the loss of transcriptional activity as such, but alterations of target DNA selectivity may be the driving force of mutant p53 specific transcription underlying the growth-promoting effects of mutant p53. This article focuses on mechanistic aspects of mutp53 "gain-of-function" with the emphasis on possible mechanisms underlying transcriptional activation by mutp53. © 2004 Wiley-Liss, Inc. [source] Alloplasmic effects on mitochondrial transcriptional activity and RNA turnover result in accumulated transcripts of Arabidopsis orfs in cytoplasmic male-sterile Brassica napusTHE PLANT JOURNAL, Issue 4 2005Matti Leino Summary Mitochondrial transcription was investigated in a cytoplasmic male-sterile (CMS) Brassica napus line with rearranged mitochondrial (mt) DNA mostly inherited from Arabidopsis thaliana. The transcript patterns were compared with the corresponding male-fertile progenitors, B. napus and A. thaliana, and a fertility-restored line. Transcriptional activities, gene stoichiometry and transcript steady-state levels were analysed for all protein and rRNA coding genes and for several orfs present in the A. thaliana mitochondrial genome. The transcriptional activities were highly variable when comparing the parental species, while the CMS and restored lines displayed similar activities. For several ribosomal protein genes transcriptional activity was reduced while it was increased for orf139 in comparison with the parental species. The differences in transcriptional activity observed could be related to differences in relative promoter strength, as gene stoichiometry between lines was very limited. Transcript steady-state levels were more homogenous than the transcriptional activities demonstrating RNA turnover as a compensating mechanism. In the CMS line higher transcript abundance and novel transcript patterns in comparison with the parental lines were found for several genes. Of those, the transcripts for orf139, orf240a and orf294 were less abundant in the fertility-restored line. These putative CMS-associated transcripts were mapped by cRT-PCR. In conclusion we show that (mt) DNA from A. thaliana was non-correctly transcribed and processed/degraded in the B. napus nuclear background. Furthermore, the introgressed nuclear A. thaliana DNA in the fertility-restored line contributes to a more rapid degradation of transcripts accumulated from A. thaliana derived orfs in the CMS line. [source] Transcriptional activity of ecdysone receptor isoforms is regulated by modulation of receptor stability and interaction with Ab- and C-domains of the heterodimerization partner ultraspiracleARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Heike Ruff Abstract The stability of ecdysone receptor (EcR) expressed in a heterologous system is regulated in an isoform-specific manner and modified by ligand and heterodimerization partner. Transcriptional activities of various receptor complexes with Usp and ligand as determined by reporter assays are the result of two effects: change in receptor concentration and altered transcriptional capability. Transcriptional activity of EcR-A is low when compared to EcR-B1 independent of the absence or presence of Ultraspiracle (Usp). Ligand increased the concentration of EcR-A, but had no effect on the transcriptional capability, in contrast to EcR-B1, which is not stabilized by hormone or Usp, but the transcriptional capability is enhanced by heterodimerization and ligand. Exchange of the AB-domain of Usp by the activation domain (AD) of Vp16 revealed that the N-terminus of Usp inhibited transcriptional activity only with EcR-B isoforms, whereas the hexapeptide in the AB-domain of wild type Usp adjacent to the C-domain of Usp harbours an activating function. Deletion of the C-domain of Usp did not affect the stability of the receptor complex, but reduced the transcriptional capability of heterodimers with all EcR-isoforms, indicating that the stability of the receptor, which is important for termination of the hormone signal transduction, is regulated in a cooperative manner by the AB-domains of EcR and Usp, and ligand. We show the active role of Usp in modulation of the transcriptional activity of the heterodimer in an isoform-specific manner by the inhibitory N-terminus, the activating hexapeptide in the AB-domain, and the C-domain of Usp. © 2009 Wiley Periodicals, Inc. [source] Transcriptional activity of paddy soil bacterial communitiesENVIRONMENTAL MICROBIOLOGY, Issue 4 2009Pravin Malla Shrestha Summary Bulk mRNA was used to explore the transcriptional activity of bacterial communities in oxic versus anoxic paddy soil. Two microbial cDNA libraries were constructed from composite samples using semi-randomly primed RT-PCR. cDNAs averaged 500,600 bp in length and were treated as expressed sequence tags (ESTs). Clustering analysis of 805 random cDNAs resulted in 179 and 155 different ESTs for the oxic and anoxic zones respectively. Using an E -value threshold of e,10, a total of 218 different ESTs could be assigned by blastx, while 116 ESTs were predicted novel. Both the proportion and significance of the EST assignments increased with cDNA length. Taxonomic assignment was more powerful in discriminating between the aerobic and anaerobic bacterial communities than functional inference, as most ESTs in both oxygen zones were putative indicators of similar housekeeping functions, in particular ABC-type transporters. A few ESTs were putative indicators for community function in a biogeochemical context, such as ,-oxidation of long-chain fatty acids specifically in the oxic zone. Expressed sequence tags assigned to Alpha- and Betaproteobacteria were predominantly found in the oxic zone, while those affiliated with Deltaproteobacteria were more frequently detected in the anoxic zone. At the genus level, multiple assignments to Bradyrhizobium and Geobacter were unique to the oxic and anoxic zones respectively. The phylum-level affiliations of 93 16S rRNA sequences corresponded well with two taxonomically distinct EST patterns. Expressed sequence tags affiliated with Acidobacteria and Chloroflexi were frequently detected in both oxygen zones. In summary, the soil metatranscriptome is accessible for global analysis and such studies have great potential in elucidating the taxonomic and functional status of soil bacterial communities, but study significance depends on the number and length of cDNAs being randomly analysed. [source] Transcriptional activity of interferon regulatory factor (IRF)-3 depends on multiple protein,protein interactionsFEBS JOURNAL, Issue 24 2002Hongmei Yang Virus infection results in the activation of a set of cellular genes involved in host antiviral defense. IRF-3 has been identified as a critical transcription factor in this process. The activation mechanism of IRF-3 is not fully elucidated, yet it involves a conformational change triggered by the virus-dependent phosphorylation of its C-terminus. This conformational change leads to nuclear accumulation, DNA binding and transcriptional transactivation. Here we show that two distinct sets of Ser/Thr residues of IRF-3, on phosphorylation, synergize functionally to achieve maximal activation. Remarkably, we find that activated IRF-3 lacks transcriptional activity, but activates transcription entirely through the recruitment of the p300/CBP coactivators. Moreover, we show that two separate domains of IRF-3 interact with several distinct regions of p300/CBP. Interference with any of these interactions leads to a complete loss of transcriptional activity, suggesting that a bivalent interaction is essential for coactivator recruitment by IRF-3. [source] Differential response to phytoestrogens in endocrine sensitive and resistant breast cancer cells in vitroINTERNATIONAL JOURNAL OF CANCER, Issue 3 2006Jane L. Limer Abstract Women approaching menopause increasingly investigate alternatives to hormone replacement therapy. Plant phytoestrogens are being promoted as "natural" alternatives but there is a lack of substantive data to advocate their safe use in breast cancer patients receiving tamoxifen (TAM), or in those who have relapsed. The aim of our study was to investigate the proliferative effects and mode of action of the phytoestrogens genistein, daidzein and coumestrol on TAM-sensitive (-s) and resistant (-r) breast cancer cells under in vitro conditions designed to mimic the hormonal environment of the pre- and post-menopausal breast. At physiological concentrations (<10 ,M) and under reduced estrogen (E2) conditions, genistein was mitogenic to TAM-s cells with TAM-r cells generally refractory. Daidzein and coumestrol were growth stimulatory irrespective of TAM sensitivity. Transcriptional activity was ERE-mediated. Combining phytoestrogens with E2 (simulating the pre-menopausal breast environment) had no effect on growth of TAM-s or TAM-r cells. Addition of 4-HT mimicked the hormonal environment in post-menopausal breast cancer patients receiving TAM. The growth inhibitory effects of 4-HT were abrogated in TAM-s cells when combined with genistein and coumestrol, and to a lesser extent, daidzein, where significant growth stimulatory effects were observed. In TAM-r cells, proliferation did not exceed control values. At phytoestrogen concentrations above 10 ,M, growth inhibitory effects were seen, irrespective of estrogenic environment or cell sensitivity to TAM. Our in vitro data suggests that phytoestrogens could have potentially adverse mitogenic effects on tumour cells and should probably be avoided by patients who remain sensitive to TAM or in those with pre-existing and possibly undiagnosed breast tumours. © 2006 Wiley-Liss, Inc. [source] Single nucleotide polymorphisms of 17,-hydroxysteroid dehydrogenase type 7 gene: Mechanism of estramustine-related adverse reactions?INTERNATIONAL JOURNAL OF UROLOGY, Issue 10 2009Takeshi Ozeki Objectives: To investigate the influence of single nucleotide polymorphisms (SNP) on transcription of the 17,-hydroxysteroid dehydrogenase (HSD17B7) gene. Methods: Luciferase reporter genes containing a 5,-flanking of the HSD17B7 gene, as well as the sequence around the SNP, were transfected into LNCaP and DU145 cells. Then, luciferase assays were carried out. Results: The presence of the G allele resulted in an increase of transcriptional activity derived from the 5,-flanking region of the HSD17B7 gene by 270% and 370% in LNCaP and DU145 cells, respectively. Transcriptional activity of the HSD17B7 gene containing the G allele was higher than that of the C allele. Conclusions: The transcriptional activity of the HSD17B7 gene containing the G allele is higher than that of the C allele. This difference in HSD17B7 expression may regulate the risk of peripheral edema as an adverse reaction induced by estramustine phosphate sodium. [source] Decreased Triadin and Increased Calstabin2 Expression in Great Danes with Dilated CardiomyopathyJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 5 2009M.A. Oyama Background: Dilated cardiomyopathy (DCM) is a common cardiac disease of Great Dane dogs, yet very little is known about the underlying molecular abnormalities that contribute to disease. Objective: Discover a set of genes that are differentially expressed in Great Dane dogs with DCM as a way to identify candidate genes for further study as well as to better understand the molecular abnormalities that underlie the disease. Animals: Three Great Dane dogs with end-stage DCM and 3 large breed control dogs. Methods: Prospective study. Transcriptional activity of 42,869 canine DNA sequences was determined with a canine-specific oligonucleotide microarray. Genome expression patterns of left ventricular tissue samples from affected Great Dane dogs were evaluated by measuring the relative amount of complementary RNA hybridization to the microarray probes and comparing it with expression from large breed dogs with noncardiac disease. Results: Three hundred and twenty-three transcripts were differentially expressed (,2-fold change). The transcript with the greatest degree of upregulation (+61.3-fold) was calstabin2 (FKBP12.6), whereas the transcript with the greatest degree of downregulation (,9.07-fold) was triadin. Calstabin2 and triadin are both regulatory components of the cardiac ryanodine receptor (RyR2) and are critical to normal intracellular Ca2+ release and excitation-contraction coupling. Conclusion and clinical importance: Great Dane dogs with DCM demonstrate abnormal calstabin2 and triadin expression. These changes likely affect Ca2+ flux within cardiac cells and may contribute to the pathophysiology of disease. Microarray-based analysis identifies calstabin2, triadin, and RyR2 function as targets of future study. [source] Transcriptional activity of ecdysone receptor isoforms is regulated by modulation of receptor stability and interaction with Ab- and C-domains of the heterodimerization partner ultraspiracleARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Heike Ruff Abstract The stability of ecdysone receptor (EcR) expressed in a heterologous system is regulated in an isoform-specific manner and modified by ligand and heterodimerization partner. Transcriptional activities of various receptor complexes with Usp and ligand as determined by reporter assays are the result of two effects: change in receptor concentration and altered transcriptional capability. Transcriptional activity of EcR-A is low when compared to EcR-B1 independent of the absence or presence of Ultraspiracle (Usp). Ligand increased the concentration of EcR-A, but had no effect on the transcriptional capability, in contrast to EcR-B1, which is not stabilized by hormone or Usp, but the transcriptional capability is enhanced by heterodimerization and ligand. Exchange of the AB-domain of Usp by the activation domain (AD) of Vp16 revealed that the N-terminus of Usp inhibited transcriptional activity only with EcR-B isoforms, whereas the hexapeptide in the AB-domain of wild type Usp adjacent to the C-domain of Usp harbours an activating function. Deletion of the C-domain of Usp did not affect the stability of the receptor complex, but reduced the transcriptional capability of heterodimers with all EcR-isoforms, indicating that the stability of the receptor, which is important for termination of the hormone signal transduction, is regulated in a cooperative manner by the AB-domains of EcR and Usp, and ligand. We show the active role of Usp in modulation of the transcriptional activity of the heterodimer in an isoform-specific manner by the inhibitory N-terminus, the activating hexapeptide in the AB-domain, and the C-domain of Usp. © 2009 Wiley Periodicals, Inc. [source] DNA-binding properties of Drosophila ecdysone receptor isoforms and their modification by the heterodimerization partner ultraspiracleARCHIVES OF INSECT BIOCHEMISTRY AND PHYSIOLOGY (ELECTRONIC), Issue 3 2009Simone Braun Abstract Transcriptional activity of ecdysone receptor (EcR) isoforms varies considerably and is modified further by the heterodimerization partner and hormone treatment. To investigate whether differences in DNA binding of receptor complexes are responsible for these variations in transcriptional activity, interaction of Drosophila EcR isoforms, and variants of Ultraspiracle (Usp), the orthologue of RXR, with the ecdysone response elements (EcRE) hsp 27, PAL-1, and DR-1, were determined by electrophoretic mobility shift assays. Receptor proteins were expressed in vertebrate cells (CHO-K1) in order to rule out an influence of endogenous receptor proteins. In the absence of a heterodimerization partner, weak DNA binding of EcR was detected even without hormone with EcR-A and -B1, but not EcR-B2. In the presence of hormone, all three isoforms show increased binding to the hsp 27 EcRE. The heterodimerization partner Usp increased DNA binding considerably. The hormone effect of heterodimers is more pronounced with both EcR-B isoforms compared to EcR-A. Two specific bands were obtained for EcR-A and B1 but only one band is visible with EcR-B2. Deletion of the C-domain of Usp still allows basal DNA binding of the heterodimer, but in contrast to full-length Usp, addition of hormone decreases the intensity of the retarded receptor band of all EcR isoforms and the EcREs hsp27 and DR-1 considerably, whereas interaction with the EcRE PAL-1 is only slightly affected. Synergistic effects on transcriptional activity are associated with the formation of different receptor DNA-complexes observed with 1×hsp27 and 3×hsp27. Comparison of DNA-binding properties of EcR isoforms and EcR/Usp heterodimers revealed that binding of receptor complexes to hsp 27 EcRE is dependent on the AB domain of EcR and the AB-, C-, and D-domains of the heterodimerization partner. Interaction with the hsp 27 EcRE correlates neither with ligand binding nor with transcriptional activity of the various receptor complexes. We, therefore, conclude that the different receptor functions are regulated separately, for example, by interaction with co-modulators or post-transcriptional modifications. © 2009 Wiley Periodicals, Inc. [source] Analysis of genetic control elements in eukaryotes: Transcriptional activity or nuclear hitchhiking?BIOESSAYS, Issue 12 2001Muriel Zohar A common way to analyse basal and stimulated activity of eukaryotic genetic control elements, such as promoters and enhancers, is to introduce them into cells via DNA vectors containing an easily assayable reporter gene. Activity is then studied by measurement of transiently produced mRNA or reporter protein. In such assays, it is assumed that the variable measured is proportional to the transcriptional activity of the control element under investigation. Here we question the validity of this generally accepted assumption. Specifically, recent observations indicate that control elements, in addition to modulating transgene transcription, can facilitate the nuclear uptake of their carrier plasmids. This process is mediated by transcription factors or other nuclear proteins harbouring nuclear localisation signals, which bind to the control elements in the cytoplasm and transport the DNA into the nucleus through the protein nuclear import machinery. As the number of mRNA transcripts produced for an epi-chromosomally expressed transgene is directly related to its copy number inside the nucleus, such transport activity may lead to substantial overestimation of the transcriptional potency of the control element(s) studied. BioEssays 23:1176,1179, 2001. © 2001 John Wiley & Sons, Inc. [source] Links between methane flux and transcriptional activities of methanogens and methane oxidizers in a blanket peat bogFEMS MICROBIOLOGY ECOLOGY, Issue 1 2010Thomas E. Freitag Abstract The relationship between biogeochemical process rates and microbial functional activity was investigated by analysis of the transcriptional dynamics of the key functional genes for methanogenesis (methyl coenzyme M reductase; mcrA) and methane oxidation (particulate methane monooxygenase; pmoA) and in situ methane flux at two peat soil field sites with contrasting net methane-emitting and -oxidizing characteristics. qPCR was used to quantify the abundances of mcrA and pmoA genes and transcripts at two soil depths. Total methanogen and methanotroph transcriptional dynamics, calculated from mcrA and pmoA gene : transcript abundance ratios, were similar at both sites and depths. However, a linear relationship was demonstrated between surface mcrA and pmoA transcript dynamics and surface flux rates at the methane-emitting and methane-oxidizing sites, respectively. Results indicate that methanotroph activity was at least partially substrate-limited at the methane-emitting site and by other factors at the methane-oxidizing site. Soil depth also contributed to the control of surface methane fluxes, but to a lesser extent. Small differences in the soil water content may have contributed to differences in methanogen and methanotroph activities. This study therefore provides a first insight into the regulation of in situ, field-level surface CH4 flux at the molecular level by an accurate reflection of gene : transcript abundance ratios for the key genes in methane generation and consumption. [source] The interaction of KCTD1 with transcription factor AP-2, inhibits its transactivation,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Xiaofeng Ding Abstract AP-2 is a transcription factor implicated in mammalian development, cell proliferation, apoptosis, and carcinogenesis. To identify potential AP-2,-interacting partners, a yeast two-hybrid screen was performed in human brain cDNA library. One of the identified clones encodes potassium channel tetramerization domain-containing 1 (KCTD1). We demonstrated the novel KCTD1,AP-2, interaction in vitro by GST pull-down assays and in vivo by co-immunoprecipitation assays and mapped the interaction domains to the N-termini of both proteins. In addition, we observed that the two proteins were completely co-localized in the nuclei of mammalian cells. Transient transfection assays using four promoters containing AP-2-binding sites confirmed that KCTD1 significantly repressed AP-2,-mediated transactivation through the BTB domain, whereas KCTD1 siRNA strongly relieved KCTD1-mediated repression of AP-2, transcriptional activity, and other BTB domain proteins such as PDIP1, KCTD10, and TNFAIP1 did not markedly inhibit the transcriptional activity of AP-2,, suggesting that KCTD1 specifically acts as a negative regulator of AP-2,. Finally, we found that KCTD1 interacted with three major members of the AP-2 family and inhibited their transcriptional activities. Taken together, our results indicate the novel function of KCTD1 as the transcriptional repressor for AP-2 family, especially for AP-2,. J. Cell. Biochem. 106: 285,295, 2009. © 2008 Wiley-Liss, Inc. [source] Transcriptional activities of mutant p53: When mutations are more than a lossJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 5 2004Ella Kim Abstract The dominant oncogenic properties of mutant p53 have been recognized as a phenomenon associated with tumor progression a long time ago, even before it was realized that the major function of wild type p53 is that of a tumor suppressor. Recent advances in this fascinating area in tumor cell biology reveal that the community of mutant p53 proteins is comprised of proteins that are extremely diverse both structurally and functionally, and elicit a multitude of cellular responses that not only are entirely distinct from those mediated by wild type p53, but also vary among different mutant p53 proteins. Aberrant regulation of transcription is one of the mechanisms underlying the ability of some mutant p53 proteins to act as oncogenic factors. Systematic analyses of the transcriptional activities of mutant p53 suggest that not the loss of transcriptional activity as such, but alterations of target DNA selectivity may be the driving force of mutant p53 specific transcription underlying the growth-promoting effects of mutant p53. This article focuses on mechanistic aspects of mutp53 "gain-of-function" with the emphasis on possible mechanisms underlying transcriptional activation by mutp53. © 2004 Wiley-Liss, Inc. [source] WDR26: A novel G,-like protein, suppresses MAPK signaling pathway,JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2004Ying Zhu Abstract WD40 repeat proteins play important roles in a variety of cellular functions, including cell growth, proliferation, apoptosis, and intracellular signal transduction. Mitogen-activated protein kinases (MAPKs) are evolutionary conserved enzymes in cell signal transduction connecting cell-surface receptors to critical regulatory targets within cells and control cell survival, adaptation, and proliferation. Previous studies revealed that G-protein coupled receptors (GPCRs) play important roles in the signal transduction from extracellular stimuli to MAPKs and the WD40-containing G, proteins as well as G,-like proteins are involved in the stimulation and regulation of the MAPK signaling pathways. Here we report the identification and characterization of a novel human WD40 repeat protein, WD40 repeat protein 26 (WDR26). The cDNA of WDR26 is 3,729 bp, encoding a G,-like protein of 514 amino acids in the cytoplasm. The protein is highly conserved in evolution across different species from yeast, Drosophila, mouse, to human. Northern blot analysis indicates that WDR26 is expressed in most of the examined human tissues, especially at a high level in skeletal muscle. Overexpression of WDR26 in the cell inhibits the transcriptional activities of ETS proteins, ELK-1 and c-fos serum response element (SRE), mediated by MEKK1. These results suggest that WDR26 may act as a negative regulator in MAPK signaling pathway and play an important role in cell signal transduction. © 2004 Wiley-Liss, Inc. [source] Ets protein Elf-1 bidirectionally suppresses transcriptional activities of the tumor suppressor Tsc2 gene and the repair-related Nth1 geneMOLECULAR CARCINOGENESIS, Issue 3 2003Satoshi Honda Abstract Alterations in the rat tuberous sclerosis gene (Tsc2) cause renal cell carcinomas (RCCs) with complete penetrance. In this study, it was shown that the minimal core promoters of the rat Tsc2 and endonuclease III 1 (Nth1) genes, lying in a 5,-to-5, arrangement, were localized in a 0.11-kb region containing two Ets binding sites (EBSs). This region worked as a bidirectional promoter in a single reporter plasmid. Mutational inactivation of each of the two EBSs significantly reduced promoter activity. Moreover, gel shift assays revealed the presence of specific EBSs-protein complexes. These results demonstrate that some members of the Ets family positively regulate the promoter activities of the Tsc2/Nth1 genes by binding to the EBSs. We identified Elf-1 as a binding factor for EBSs through super-shift assays, and detected ,35 kDa bands with an EBSs-containing DNA probe by Southwestern blot analysis. Forced expression of Elf-1 in cells, however, bidirectionally suppressed the activities of the Tsc2/Nth1 promoters. Elf-1 may be a negative regulator of Tsc2/Nth1 gene expression and may compete against positive regulators for binding to the EBSs. Our observations suggest that mechanisms that inactivate Tsc2 gene expression, such as promoter suppression, may exist. © 2003 Wiley-Liss, Inc. [source] Inositol hexaphosphate inhibits ultraviolet B,induced signal transductionMOLECULAR CARCINOGENESIS, Issue 3 2001Nanyue Chen Abstract Inositol hexaphosphate (InsP6) has an effective anticancer action in many experimental models in vivo and in vitro. Ultraviolet B (UVB) radiation is believed to be responsible for many of the carcinogenic effects related to sun exposure, and alteration in UVB-induced signal transduction is associated with UVB-induced carcinogenesis. Here we report the effects of InsP6 on UVB-induced signal transduction. InsP6 strongly blocked UVB-induced activator protein-1 (AP-1) and NF-,B transcriptional activities in a dose-dependent manner. InsP6 also suppressed UVB-induced AP-1 and nuclear factor ,B (NF-,B) DNA binding activities and inhibited UVB-induced phosphorylation of extracellular signal-regulated protein kinases (Erks) and c-Jun NH2-terminal kinases (JNKs). Phosphorylation of p38 kinases was not affected. InsP6 also blocked UVB-induced phosphorylation of I,B-,, which is known to result in the inhibition of NF-,B transcriptional activity. InsP6 does not block UVB-induced phosphotidylinositol-3, (PI-3) kinase activity, suggesting that the inhibition of UVB-induced AP-1 and NF-,B activities by InsP6 is not mediated through PI-3 kinase. Because AP-1 and NF-,B are important nuclear transcription factors that are related to tumor promotion, our work suggests that InsP6 prevents UVB-induced carcinogenesis by inhibiting AP-1 and NF-,B transcription activities. © 2001 Wiley-Liss, Inc. [source] Stable transgene expression in human embryonic stem cells after simple chemical transfectionMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 6 2009Jun Liu In this study we used plasmid-based vectors to investigate the transcriptional activities of three commonly used promoters in transient and stable transfection of MEL-1, a human embryonic stem (ES) cell line, using ExGen500, Fugene HD, and Lipofectamine. We demonstrated that cytomegalovirus (CMV), phosphoglycerate kinase (PGK) and human elongation factor-1, (EF1,) promoters all resulted in robust activity of a reporter gene in MEL-1 ES cell transient transfections regardless of the transfection reagent. Stable transfection outcomes varied, depending on the promoter and the transfection reagent used in the study. The phenomenon of transgene silencing was observed, most notably with the CMV vector, with which no positive stably transfected clones were obtained. Of the methods used in the study, Fugene HD resulted in the highest stable transfection rate, estimated by antibiotic selection, with plasmids containing genes under the control of the EF1, or PGK promoters. Stably transfected cells maintained typical hES cell morphology, with immunostaining exhibiting expression of the hES cell markers: Oct4, SSEA4, Tra-1-60, and Tra-1-81. Further, embryoid bodies formed by suspension culture retained reporter gene expression. Following injection into immunodeficient mice, the transfected cell lines showed robust formation of teratomas with cell types representative of the three germ layers. Mol. Reprod. Dev. 76: 580,586, 2009. © 2008 Wiley-Liss, Inc. [source] Study of PfMyb1 Transcription Factor Regulation Network during Plasmodium falciparum Erythrocytic CycleTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 2 2005M. GISSOT During the complex life cycle of Plasmodium falciparum, the regulation of events that occur during the erythrocytic cycle, such as proliferation and differentiation, implies a fine control of transcriptional activities governing the expression profiles of each gene. However, transcriptional regulation and notably its actors, transcription factors and regulation motifs, are poorly described in Plasmodium. In order to decipher the mechanisms implicated in transcriptional regulation, we studied a transcription factor belonging to the trytophan family and showed that the PfMyb1 protein contained in nuclear extracts has a specific DNA binding activity. We took advantage of long pfmyb1 double stranded RNA (dsRNA) to inactivate the cognate messenger and understand the role of PfMyb1 during the erythrocytic cycle. Culture treated with pfmyb1 dsRNA exhibited a 40% growth inhibition and mortality during trophozoite to schizont transition when compared to either untreated control or culture treated with unrelated long dsRNA. We have further demonstrated that pfmyb1 transcript and protein decreased up to 80% in treated trophozoite culture at the time of pfmyb1 expression peak. Thus, we investigated the effect of this partial loss of transcript and protein using a thematic DNA microarray containing PCR products, representative of P. falciparum genes involved in cell cycle and transcriptional regulation. SAM software enabled us to identify several genes over and under-expressed, potentially directly or indirectly regulated by PfMyb1. These alterations of expression were verified by qPCR and Western blotting. We are currently working on the promoters of those genes to decode determinants of gene regulation by Pfmyb1. [source] Immune-compromised state in the rat pancreas after chronic alcohol exposure: the role of peroxisome proliferator-activated receptor ,,THE JOURNAL OF PATHOLOGY, Issue 4 2007F Fortunato Abstract Alcohol exposure is known to sensitize acinar cells to various insults but the pathophysiological mechanisms of alcoholic pancreatitis remain unknown. Alcohol abuse has been shown to mediate an anti-inflammatory response and periods of immune suppression seem to be associated with organ injury and mortality. The purpose of this study was to determine the mechanisms by which alcohol exerts transcriptional activities in the rat pancreas and how alcohol alters the inflammatory response. Using the Lieber,DeCarli alcohol/control diet, rats that were fed with alcohol over 14 weeks demonstrated a decrease of inflammatory cells in pancreatic tissue compared to controls. The anti-inflammatory effects of alcohol were confirmed by decreased expression of pro-inflammatory cytokines including TNF,, IL-1,, IL-18, TGF,, and MCP-1. In addition, alcohol significantly increased the activity of PPAR,, which is a known anti-inflammatory transcription factor, while pro-inflammatory factors including AP-2 and EGR-1 were significantly suppressed. NF,B binding showed a tendency towards a reduction. Electron microscopy studies revealed enlarged and injured mitochondria and lysosomes, accompanied by peri-cellular fibrosis. Furthermore, alcohol exposure increased the activities of trypsin and cathepsin B, both known to be critical in initiating acinar cell injury and pancreatitis. Despite the known alcohol-mediated acinar cell and mitochondrial injury, the mitochondrial-mediated apoptotic pathway was attenuated. These data demonstrate that the pancreas exposed to alcohol maintains an anti-inflammatory state by activating PPAR,. Intracellular mitochondrial and lysosomal damage after chronic alcohol exposure induces premature activation of digestive enzymes and establishment of peri-cellular fibrosis in the absence of inflammation. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source] Tobacco Mg protoporphyrin IX methyltransferase is involved in inverse activation of Mg porphyrin and protoheme synthesisTHE PLANT JOURNAL, Issue 2 2005Ali E. Alawady Summary Protoporphyrin, a metabolic intermediate of tetrapyrrole biosynthesis, is metabolized by Mg chelatase and ferrochelatase and is directed into the Mg-branch for chlorophyll synthesis and in the Fe-branch for protoheme synthesis respectively. Regulation of the enzyme activities at the beginning of this branchpoint ensures accurate partition of protoporphyrin, but is still not entirely understood. Transgenic tobacco plants were generated that express antisense or sense RNA for inhibited and excessive expression of Mg protoporphyrin methyltransferase (MgPMT) respectively. This enzyme accepts Mg protoporphyrin from Mg chelatase and catalyses the transfer of a methyl group to the carboxyl group of the C13-propionate side chain. Low MgPMT activity is correlated with reduced Mg chelatase activity and a low synthesis rate of 5-aminolevulinate, but with enhanced ferrochelatase activity. In contrast, high MgPMT activity leads to inverse activity profiles: high activities of Mg chelatase and for 5-aminolevulinate synthesis, but reduced activity of ferrochelatase, indicating a direct influence of MgPMT in combination with Mg chelatase on the metabolic flux of ALA and the distribution of protoporphyrin into the branched pathway. The modified enzyme activities in tetrapyrrole biosynthesis in the transgenic plants can be explained with changes of certain corresponding mRNA contents: increased 5-aminolevulinate synthesis and Mg chelatase activity correlate with enhanced transcript levels of the HemA, Gsa, and CHLH gene encoding glutamyl-tRNA reductase, glutamate-1-semialdehyde aminotransferase and a Mg chelatase subunit respectively. It is proposed that reduced and increased MgPMT activity in chloroplasts is communicated to the cytoplasm for modulating transcriptional activities of regulatory enzymes of the pathway. [source] Apoptosis signal-regulating kinase 1-mediated sustained p38 mitogen-activated protein kinase activation regulates mycoplasmal lipoprotein- and staphylococcal peptidoglycan-triggered Toll-like receptor 2 signalling pathwaysCELLULAR MICROBIOLOGY, Issue 9 2005Takeshi Into Summary Toll-like receptor (TLR) 2 functions as a sensor for detecting various microbial components conserved in bacteria or fungi in innate immunity. TLR2 induces several signalling pathways linking to activation of the transcriptional factors NF-,B and AP-1 as well as induction of cell death. In human embryonic kidney 293 cells expressed human TLR2, mycoplasmal lipoproteins (MLP) or staphylococcal peptidoglycans (PGN) induced sustained phosphorylation of p38 mitogen-activated protein kinase (MAPK), accompanied by generation of reactive oxygen species. This observation encouraged us to examine roles of apoptosis signal-regulating kinase 1 (ASK1) in TLR2 signalling, because ASK1 is an upstream activator of p38 MAPK during exposure to oxidative stress and other stressful stimuli. A kinase-inactive mutant of ASK1 greatly impaired the sustained phosphorylation of p38 MAPK induced by MLP or PGN. This mutant also attenuated MLP- or PGN-induced transcriptional activities of NF-,B and AP-1 via inhibition of p38 MAPK activation. MLP- or PGN-induced cell death reactions, including DNA fragmentation and caspase-3/7 activation, were also downregulated by the ASK1 mutant via p38 MAPK inhibition. Furthermore, TLR2 signalling had a potential to phosphorylate and dephosphorylate ASK1 at Ser83 residue. Thus, MLP and PGN have capabilities to induce ASK1-dependent signalling pathways which regulate p38 MAPK activation through TLR2, leading to activation of NF-,B and AP-1 as well as induction of cell death. [source] Mitogen-activated protein kinase signal transduction in skeletal muscle: effects of exercise and muscle contractionACTA PHYSIOLOGICA, Issue 3 2001U. Widegren Exercise has numerous growth and metabolic effects in skeletal muscle, including changes in glycogen metabolism, glucose and amino acid uptake, protein synthesis and gene transcription. However, the mechanism(s) by which exercise regulates intracellular signal transduction to the transcriptional machinery in the nucleus, thus modulating gene expression, is largely unknown. This review will provide insight on potential intracellular signalling mechanisms by which muscle contraction/exercise leads to changes in gene expression. Mitogen-activated protein kinase (MAPK) cascades are associated with increased transcriptional activity. The MAPK family members can be separated into distinct parallel pathways including the extracellular signal-regulated kinase (ERK) 1/2, the stress-activated protein kinase cascades (SAPK1/JNK and SAPK2/p38) and the extracellular signal-regulated kinase 5 (ERK5). Acute exercise elicits signal transduction via MAPK cascades in direct response to muscle contraction. Thus, MAPK pathways appear to be potential physiological mechanisms involved in the exercise-induced regulation of gene expression in skeletal muscle. [source] Transgenic analysis of the medaka mesp-b enhancer in somitogenesisDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 3 2006Harumi Terasaki Somitogenesis is a critical step during the formation of metameric structures in vertebrates. Recent studies in mouse, chick, zebrafish and Xenopus have revealed that several factors, such as T-box genes, Notch/Delta, Wnt, retinoic acid and FGF signaling, are involved in the specification of nascent somites. By interacting with these pathways, the Mesp2-like bHLH transcription factors are transiently expressed in the anterior presomitic mesoderm and play a crucial role in somite formation. The regulatory mechanisms of Mesp2 and its related genes during somitogenesis have been studied in mouse and Xenopus. However, the precise mechanism that regulates the transcriptional activity of Mesp2 has yet to be determined. In our current report, we identify the essential enhancer element of medaka mesp-b, an orthologue of mouse Mesp2, using transgenic techniques and embryo manipulation. Our results demonstrate that a region of approximately 2.8 kb, upstream of the mesp-b gene, is responsible for both the initiation and anterior localization of mesp-b transcription within a somite primordium. Furthermore, putative motifs for both T-box transcription factors and Notch/Delta signaling are present in this enhancer region and are essential for activity. [source] Global analysis of gene expression in Xenopus hindlimbs during stage-dependent complete and incomplete regenerationDEVELOPMENTAL DYNAMICS, Issue 10 2006Matthew Grow Abstract Xenopus laevis tadpoles are capable of limb regeneration after amputation, in a process that initially involves the formation of a blastema. However, Xenopus has full regenerative capacity only through premetamorphic stages. We have used the Affymetrix Xenopus laevis Genome Genechip microarray to perform a large-scale screen of gene expression in the regeneration-complete, stage 53 (st53), and regeneration-incomplete, stage 57 (st57), hindlimbs at 1 and 5 days postamputation. Through an exhaustive reannotation of the Genechip and a variety of comparative bioinformatic analyses, we have identified genes that are differentially expressed between the regeneration-complete and -incomplete stages, detected the transcriptional changes associated with the regenerating blastema, and compared these results with those of other regeneration researchers. We focus particular attention on striking transcriptional activity observed in genes associated with patterning, stress response, and inflammation. Overall, this work provides the most comprehensive views yet of a regenerating limb and different transcriptional compositions of regeneration-competent and deficient tissues. Developmental Dynamics 235:2667,2685, 2006. © 2006 Wiley-Liss, Inc. [source] Analysis of regulatory elements of E-cadherin with reporter gene constructs in transgenic mouse embryosDEVELOPMENTAL DYNAMICS, Issue 2 2003Marc P. Stemmler Abstract Proper regulation of E-cadherin,mediated cell adhesion is important during early embryonic development and in organogenesis. In mice, E-cadherin is expressed from the fertilized egg onward and becomes down-regulated during gastrulation in mesoderm and its derivatives, but its expression is maintained in all epithelia. E-cadherin promoter analyses led to the identification of binding sites for two transcriptional repressors, Snail and SIP1, which are able to mediate down-regulation in vitro, but little is known about the regulatory elements that govern E-cadherin transcriptional activity in vivo. Here, we compared the developmentally regulated expression of a series of lacZ -reporter transgenes fused to different sequences of the murine E-cadherin gene between ,6 kb, including the promoter, and +16 kb, covering one third of intron 2. Four different segments with distinct regulatory properties were identified. The promoter fragment from +0.1 to ,1.5 kb remains inactive in most cases but occasionally induces ectopic expression in mesodermal tissues, although it contains binding sites for the repressors Snail and SIP1. This promoter fragment also lacks positive elements needed for the activation of transcription in ectoderm and endoderm. Sequences from ,1.5 to ,6 kb harbor regulatory elements for brain-specific expression and, in addition, insulator or silencer elements, because they are consistently inactive in the mesoderm. Only if sequences from +0.1 to +11 kb are combined with the promoter fragments is E-cadherin,specific transgene expression observed in endoderm and certain epithelia. Sequences between +11 and +16 kb contain cis -active elements that generally enhance transcription. Our analyses show that E-cadherin expression is governed by a complex interplay of multiple regulatory regions dispersed throughout large parts of the locus. Developmental Dynamics 227:238,245, 2003. © 2003 Wiley-Liss, Inc. [source] Activity of nAChRs containing ,9 subunits modulates synapse stabilization via bidirectional signaling programsDEVELOPMENTAL NEUROBIOLOGY, Issue 14 2009Vidya Murthy Abstract Although the synaptogenic program for cholinergic synapses of the neuromuscular junction is well known, little is known of the identity or dynamic expression patterns of proteins involved in non-neuromuscular nicotinic synapse development. We have previously demonstrated abnormal presynaptic terminal morphology following loss of nicotinic acetylcholine receptor (nAChR) ,9 subunit expression in adult cochleae. However, the molecular mechanisms underlying these changes have remained obscure. To better understand synapse formation and the role of cholinergic activity in the synaptogenesis of the inner ear, we exploit the nAChR ,9 subunit null mouse. In this mouse, functional acetylcholine (ACh) neurotransmission to the hair cells is completely silenced. Results demonstrate a premature, effusive innervation to the synaptic pole of the outer hair cells in ,9 null mice coinciding with delayed expression of cell adhesion proteins during the period of effusive contact. Collapse of the ectopic innervation coincides with an age-related hyperexpression pattern in the null mice. In addition, we document changes in expression of presynaptic vesicle recycling/trafficking machinery in the ,9 null mice that suggests a bidirectional information flow between the target of the neural innervation (the hair cells) and the presynaptic terminal that is modified by hair cell nAChR activity. Loss of nAChR activity may alter transcriptional activity, as CREB binding protein expression is decreased coincident with the increased expression of N-Cadherin in the adult ,9 null mice. Finally, by using mice expressing the nondesensitizing ,9 L9,T point mutant nAChR subunit, we show that increased nAChR activity drives synaptic hyperinnervation. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009 [source] The sexually dimorphic expression of L7/SPA, an estrogen receptor coactivator, in zebra finch telencephalonDEVELOPMENTAL NEUROBIOLOGY, Issue 14 2007Kelli A. Duncan Abstract Sex differences in the zebra finch (Taeniopygia guttata) brain are robust and include differences in morphology (song control nuclei in males are significantly larger) and behavior (only males sing courtship songs). In zebra finches, hormonal manipulations during development fail to reverse sex differences in song nuclei size and suggest that the classical model of sexual differentiation is incomplete for birds. Coactivators act to initiate transcriptional activity of steroid receptors, and may help explain why hormonal manipulations alone are not sufficient to demasculinize the male zebra finch brain. The present study investigated the expression and localization of L7/SPA (an estrogen receptor coactivator) mRNA and protein expression across the development of zebra finch song nuclei from males and females collected on P1 (song nuclei not yet formed), P10 (posthatch day 10, song nuclei formed), P30 (30 days posthatch, sexually immature but song nuclei formed and birds learning to sing), and adult birds (older than 65 days and sexually mature). Northern blot analysis showed a significant sex difference in P1 and adult L7/SPA mRNA expression while Western blot analysis also showed enhanced expression in the male brain at all age points. Both in situ hybridization and immunohistochemistry demonstrated that L7/SPA mRNA and protein were located in the song nuclei as well as expressed globally. Elevated coactivator expression may be a possible mechanism controlling the development of male song control nuclei, and coactivators such as L7/SPA may be important regulators of the masculinizing effects of estradiol on brain sexual differentiation. © 2007 Wiley Periodicals, Inc. Develop Neurobiol, 2007. [source] Dendritic growth induced by BMP-7 requires Smad1 and proteasome activityDEVELOPMENTAL NEUROBIOLOGY, Issue 2 2001Xin Guo Abstract Bone morphogenetic proteins (BMPs) induce dendritic growth in cultured sympathetic neurons; however, the signaling pathways that mediate this dendrite-promoting activity have not been previously characterized. Here we report studies of the signaling events that regulate the growth of these afferent processes. We find that Smad1 is expressed in sympathetic neurons and that BMPs rapidly induce its phosphorylation and translocation from the cytoplasm to the nucleus. Furthermore, a dominant negative form of Smad1 inhibits BMP-7-induced dendritic growth, suggesting a requirement for Smad1 activation in this biological activity of BMP-7. A physical interaction between Smad1 and components involved in the proteasome-mediated degradation system was detected with a yeast two-hybrid screen, thereby prompting an examination of the effects of proteasome inhibitors on dendritic growth. Lactacystin and ALLN (N -acetyl-Leu-Leu-norleucinal) selectively blocked BMP-7-induced dendritic growth without adversely affecting either cell viability or axonal growth. Moreover, studies of transfected P19 cells suggest that the proteasome inhibitors directly block the effects of Smad1 on the transcriptional activity of the Tlx-2 promoter. These data indicate that BMP-induced dendritic growth requires Smad1 activation and involves proteasome-mediated degradation events. © 2001 John Wiley & Sons, Inc. J Neurobiol 48: 120,130, 2001 [source] | ||||||||||||||||||||||||||||||||||||||||