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Transcription factor Sp1, also known as specificity protein 1 as a therapeutic target.  


Introduction: Specificity protein (Sp) transcription factors (TFs) are members of the Sp/Kruppel-like factor family, and Sp proteins play an important role in embryonic and early postnatal development. Sp1 has been the most extensively investigated member of this family, and expression of this protein decreases with age, whereas Sp1 and other family members (Sp3 and Sp4) are highly expressed in tumors and cancer cell lines. Area covered: The prognostic significance of Sp1 in cancer patients and the functional pro-oncogenic activities of Sp1, Sp3 and Sp4 in cancer cell lines are summarized. Several different approaches have been used to target downregulation of Sp TFs and Sp-regulated genes, and this includes identification of different structural classes of antineoplastic agents including NSAIDs, natural products and their synthetic analogs and several well-characterized drugs including arsenic trioxide, aspirin and metformin. The multiple pathways involved in drug-induced Sp downregulation are also discussed. Expert opinion: The recognition by the scientific and clinical community that experimental and clinically used antineoplastic agents downregulate Sp1, Sp3 and Sp4, and pro-oncogenic Sp-regulated genes will facilitate future clinical applications for individual drug and drug combination therapies that take advantage of their unusual effects. PMID:24793594

Safe, Stephen; Imanirad, Parisa; Sreevalsan, Sandeep; Nair, Vijayalekshmi; Jutooru, Indira



The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System*  

PubMed Central

P2X7 receptors are involved not only in physiological functions but also in pathological brain processes. Although an increasing number of findings indicate that altered receptor expression has a causative role in neurodegenerative diseases and cancer, little is known about how expression of P2rx7 gene is controlled. Here we reported the first molecular and functional evidence that Specificity protein 1 (Sp1) transcription factor plays a pivotal role in the transcriptional regulation of P2X7 receptor. We delimited a minimal region in the murine P2rx7 promoter containing four SP1 sites, two of them being highly conserved in mammals. The functionality of these SP1 sites was confirmed by site-directed mutagenesis and Sp1 overexpression/down-regulation in neuroblastoma cells. Inhibition of Sp1-mediated transcriptional activation by mithramycin A reduced endogenous P2X7 receptor levels in primary cultures of cortical neurons and astrocytes. Using P2rx7-EGFP transgenic mice that express enhanced green fluorescent protein under the control of P2rx7 promoter, we found a high correlation between reporter expression and Sp1 levels in the brain, demonstrating that Sp1 is a key element in the transcriptional regulation of P2X7 receptor in the nervous system. Finally, we found that Sp1 mediates P2X7 receptor up-regulation in neuroblastoma cells cultured in the absence of serum, a condition that enhances chromatin accessibility and facilitates the exposure of SP1 binding sites.

Garcia-Huerta, Paula; Diaz-Hernandez, Miguel; Delicado, Esmerilda G.; Pimentel-Santillana, Maria; Miras-Portugal, M? Teresa; Gomez-Villafuertes, Rosa



The specificity protein factor Sp1 mediates transcriptional regulation of P2X7 receptors in the nervous system.  


P2X7 receptors are involved not only in physiological functions but also in pathological brain processes. Although an increasing number of findings indicate that altered receptor expression has a causative role in neurodegenerative diseases and cancer, little is known about how expression of P2rx7 gene is controlled. Here we reported the first molecular and functional evidence that Specificity protein 1 (Sp1) transcription factor plays a pivotal role in the transcriptional regulation of P2X7 receptor. We delimited a minimal region in the murine P2rx7 promoter containing four SP1 sites, two of them being highly conserved in mammals. The functionality of these SP1 sites was confirmed by site-directed mutagenesis and Sp1 overexpression/down-regulation in neuroblastoma cells. Inhibition of Sp1-mediated transcriptional activation by mithramycin A reduced endogenous P2X7 receptor levels in primary cultures of cortical neurons and astrocytes. Using P2rx7-EGFP transgenic mice that express enhanced green fluorescent protein under the control of P2rx7 promoter, we found a high correlation between reporter expression and Sp1 levels in the brain, demonstrating that Sp1 is a key element in the transcriptional regulation of P2X7 receptor in the nervous system. Finally, we found that Sp1 mediates P2X7 receptor up-regulation in neuroblastoma cells cultured in the absence of serum, a condition that enhances chromatin accessibility and facilitates the exposure of SP1 binding sites. PMID:23139414

García-Huerta, Paula; Díaz-Hernandez, Miguel; Delicado, Esmerilda G; Pimentel-Santillana, María; Miras-Portugal, M Teresa; Gómez-Villafuertes, Rosa



Activation of human papillomavirus type 18 E6-E7 oncogene expression by transcription factor Sp1.  

PubMed Central

The human papillomavirus 18 (HPV18) E6 and E7 proteins are considered to be primarily responsive for the transforming activity of the virus. In order to analyse the molecular mechanisms resulting in viral oncoprotein expression, it is necessary to identify the factors involved in the transcriptional regulation of the E6/E7 genes. Here we define by gel retardation experiments a sequence aberrant Sp1 binding site present in the promoter proximal part of the viral transcriptional control region (Upstream Regulatory Region, URR). Functional analyses employing transient reporter assays reveal that this Sp1 element is required for an efficient stimulation of the HPV18 E6/E7-promoter. Mutation of the Sp1 element in the natural context of the HPV18 URR leads to a strong decrease in the activity of the E6/E7-promoter in several cell lines. The magnitude of reduction varies between different cell types and is higher in cell lines of epithelial origin when compared with nonepithelial cells. Cotransfection assays using Sp1 expression vector systems further define the promoter proximal HPV18 Sp1 binding motif as a functional Sp1 element in vivo and show that its integrity is essential for the stimulation of the E6/E7-promoter by augmented levels of Sp1. These results indicate, that the cellular transcription factor Sp1 plays an important role for the stimulation of the E6/E7-promoter by the viral URR and represents a major determinant for the expression of HPV18 transforming genes E6 and E7. Images

Hoppe-Seyler, F; Butz, K



The oncoprotein HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote the proliferation of breast cancer cells  

SciTech Connect

Highlights: •HBXIP is able to upregulate the expression of PDGFB in breast cancer cells. •HBXIP serves as a coactivator of activating transcription factor Sp1. •HBXIP stimulates the PDGFB promoter via activating transcription factor Sp1. •HBXIP promotes the proliferation of breast cancer cell via upregulating PDGFB. -- Abstract: We have reported that the oncoprotein hepatitis B virus X-interacting protein (HBXIP) acts as a novel transcriptional coactivator to promote proliferation and migration of breast cancer cells. Previously, we showed that HBXIP was able to activate nuclear factor-?B (NF-?B) in breast cancer cells. As an oncogene, the platelet-derived growth factor beta polypeptide (PDGFB) plays crucial roles in carcinogenesis. In the present study, we found that both HBXIP and PDGFB were highly expressed in breast cancer cell lines. Interestingly, HBXIP was able to increase transcriptional activity of NF-?B through PDGFB, suggesting that HBXIP is associated with PDGFB in the cells. Moreover, HBXIP was able to upregulate PDGFB at the levels of mRNA, protein and promoter in the cells. Then, we identified that HBXIP stimulated the promoter of PDGFB through activating transcription factor Sp1. In function, HBXIP enhanced the proliferation of breast cancer cells through PDGFB in vitro. Thus, we conclude that HBXIP upregulates PDGFB via activating transcription factor Sp1 to promote proliferation of breast cancer cells.

Zhang, Yingyi; Zhao, Yu; Li, Leilei; Shen, Yu; Cai, Xiaoli [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)] [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China); Zhang, Xiaodong, E-mail: [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China)] [Department of Cancer Research, Institute for Molecular Biology, College of Life Sciences, Nankai University, Tianjin 300071 (China); Ye, Lihong, E-mail: [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)] [Department of Biochemistry, College of Life Sciences, Nankai University, Tianjin 300071 (China)



TPA-activated transcription of the human MnSOD gene: role of transcription factors Sp-1 and Egr-1.  


Induction of manganese superoxide dismutase (MnSOD) in response to oxidative stress has been well established in animals, tissues, and cell culture. However, the role of the human MnSOD (hMnSOD) promoter in stimulus-dependent activation of transcription is unknown. The hMnSOD promoter lacks both a TATA and a CAAT box but possesses several GC motifs. In a previous study, we showed that the basal promoter contains multiple Sp1 and AP-2 binding sites and that Sp1 is essential for the constitutive expression of the hMnSOD gene. In this study, we identified an Egr-1 binding site in the basal promoter of hMnSOD. We also found that the basal promoter is responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated hMnSOD transcription in the human hepatocarcinoma cell line HepG2. The contributions of these binding sites and the roles of the transcription factors Egr-1, AP-2, and Sp1 in the activation of hMnSOD transcription by TPA were investigated by site-directed mutation analysis, Western blotting, and overexpression of transcription factors. The results showed that Sp1 plays a positive role for both basal and TPA-activated hMnSOD transcription, whereas overexpression of Egr-1 has a negative role in the basal promoter activity without any effect on TPA-mediated activation of hMnSOD transcription. PMID:11560779

Porntadavity, S; Xu, Y; Kiningham, K; Rangnekar, V M; Prachayasittikul, V; Prachayasitikul, V; St Clair, D K



Cytosine methylation does not affect binding of transcription factor Sp1  

SciTech Connect

DNA methylation may be a component of a multilevel control mechanism that regulates eukaryotic gene expression. The authors used synthetic oligonucleotides to investigate the effect of cytosine methylation on the binding of the transcription factor Sp1 to its target sequence (a G+C-rich sequence known as a GC box). Concatemers of double-stranded 14-mers containing a GC box successfully competed with the human metallothionein IIA promoter for binding to Sp1 in DNase I protection experiments. The presence of 5-methylcytosine in the CpG sequence of the GC box did not influence Sp1 binding. The result was confirmed using double-stranded 20-mers containing 16 base pairs of complementary sequence. Electrophoretic gel retardation analysis of annealed 28-mers containing a GC box incubated with an Sp1-containing HeLa cell nuclear extract demonstrated the formation of DNA-protein complexes; formation of these complexes was not inhibited when an oligomer without a GC box was used as a competitor. Once again, the presence of a 5-methylcytosine residue in the GC box did not influence the binding of the protein to DNA. The results therefore preclude a direct effect of cytosine methylation on Sp1-DNA interactions.

Harrington, M.A.; Jones, P.A. (Univ. of Southern California School of Medicine, Los Angeles (USA)); Imagawa, M.; Karin, M. (Univ. of California, San Diego, La Jolla (USA))



Androgen up-regulates vascular endothelial growth factor expression in prostate cancer cells via an Sp1 binding site  

PubMed Central

Background Vascular Endothelial Growth Factor (VEGF) is regulated by a number of different factors, but the mechanism(s) behind androgen-mediated regulation of VEGF in prostate cancer are poorly understood. Results Three novel androgen receptor (AR) binding sites were discovered in the VEGF promoter and in vivo binding of AR to these sites was demonstrated by chromatin immunoprecipitation. Mutation of these sites attenuated activation of the VEGF promoter by the androgen analog, R1881 in prostate cancer cells. The transcription factors AR and Sp1 were shown to form a nuclear complex and both bound the VEGF core promoter in chromatin of hormone treated CWR22Rv1 prostate cancer cells. The importance of the Sp1 binding site in hormone mediated activation of VEGF expression was demonstrated by site directed mutagenesis. Mutation of a critical Sp1 binding site (Sp1.4) in the VEGF core promoter region prevented activation by androgen. Similarly, suppression of Sp1 binding by Mithramycin A treatment significantly reduced VEGF expression. Conclusions Our mechanistic study of androgen mediated induction of VEGF expression in prostate cancer cells revealed for the first time that this induction is mediated through the core promoter region and is dependent upon a critical Sp1 binding site. The importance of Sp1 binding suggests that therapy targeting the AR-Sp1 complex may dampen VEGF induced angiogenesis and, thereby, block prostate cancer progression, helping to maintain the indolent form of prostate cancer.



A crucial role for the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specification.  


Mammalian development is regulated by the interplay of tissue-specific and ubiquitously expressed transcription factors, such as Sp1. Sp1 knockout mice die in utero with multiple phenotypic aberrations, but the underlying molecular mechanism of this differentiation failure has been elusive. Here, we have used conditional knockout mice as well as the differentiation of mouse ES cells as a model with which to address this issue. To this end, we examined differentiation potential, global gene expression patterns and Sp1 target regions in Sp1 wild-type and Sp1-deficient cells representing different stages of hematopoiesis. Sp1(-/-) cells progress through most embryonic stages of blood cell development but cannot complete terminal differentiation. This failure to fully differentiate is not seen when Sp1 is knocked out at later developmental stages. For most Sp1 target and non-target genes, gene expression is unaffected by Sp1 inactivation. However, Cdx genes and multiple Hox genes are stage-specific targets of Sp1 and are downregulated at an early stage. As a consequence, expression of genes involved in hematopoietic specification is progressively deregulated. Our work demonstrates that the early absence of active Sp1 sets a cascade in motion that culminates in a failure of terminal hematopoietic differentiation and emphasizes the role of ubiquitously expressed transcription factors for tissue-specific gene regulation. In addition, our global side-by-side analysis of the response of the transcriptional network to perturbation sheds a new light on the regulatory hierarchy of hematopoietic specification. PMID:24850855

Gilmour, Jane; Assi, Salam A; Jaegle, Ulrike; Kulu, Divine; van de Werken, Harmen; Clarke, Deborah; Westhead, David R; Philipsen, Sjaak; Bonifer, Constanze



A crucial role for the ubiquitously expressed transcription factor Sp1 at early stages of hematopoietic specification  

PubMed Central

Mammalian development is regulated by the interplay of tissue-specific and ubiquitously expressed transcription factors, such as Sp1. Sp1 knockout mice die in utero with multiple phenotypic aberrations, but the underlying molecular mechanism of this differentiation failure has been elusive. Here, we have used conditional knockout mice as well as the differentiation of mouse ES cells as a model with which to address this issue. To this end, we examined differentiation potential, global gene expression patterns and Sp1 target regions in Sp1 wild-type and Sp1-deficient cells representing different stages of hematopoiesis. Sp1?/? cells progress through most embryonic stages of blood cell development but cannot complete terminal differentiation. This failure to fully differentiate is not seen when Sp1 is knocked out at later developmental stages. For most Sp1 target and non-target genes, gene expression is unaffected by Sp1 inactivation. However, Cdx genes and multiple Hox genes are stage-specific targets of Sp1 and are downregulated at an early stage. As a consequence, expression of genes involved in hematopoietic specification is progressively deregulated. Our work demonstrates that the early absence of active Sp1 sets a cascade in motion that culminates in a failure of terminal hematopoietic differentiation and emphasizes the role of ubiquitously expressed transcription factors for tissue-specific gene regulation. In addition, our global side-by-side analysis of the response of the transcriptional network to perturbation sheds a new light on the regulatory hierarchy of hematopoietic specification.

Gilmour, Jane; Assi, Salam A.; Jaegle, Ulrike; Kulu, Divine; van de Werken, Harmen; Clarke, Deborah; Westhead, David R.; Philipsen, Sjaak; Bonifer, Constanze



Suppression of lung cancer cell invasion by LKB1 is due to the downregulation of tissue factor and vascular endothelial growth factor, partly dependent on SP1.  


LKB1 encodes a serine/threonine kinase generally inactivated in many human cancers, which mediates cancer cell proliferation, migration and differentiation. Recent studies indicated that LKB1 exhibits potent anti-metastatic activity. However, the underlying molecular mechanisms of this activity remain unclear. In this study, we re?introduced LKB1 into A549 lung cancer cells that lack the LKB1 gene to investigate how LKB1 affects tumor invasiveness and metastasis. We demonstrated that overexpression of the LKB1 protein in lung cancer cells resulted in significant inhibition of invasion. Furthermore, transfected lung cancer cells with LKB1 suppressed tissue factor (TF) and vascular endothelial growth factor (VEGF) expression at both the mRNA and protein levels. Here, we provided evidence showing that downregulation of TF and VEGF by LKB1 is correlated well with the inhibition of cell invasion. Overexpression of the LKB1 protein in human lung cancer is significantly associated with a decrease in activity and expression of the transcription factor SP1. Constitutive activation of the transcription factor Sp1 plays a critical role in TF and VEGF overexpression. We conclude that suppression of lung cancer cell invasion by LKB1 through downregulation of TF and VEGF may partly depend on its inhibitory effect on the transcription factor Sp1. Collectively, our data provide a novel molecular mechanism for the antitumor activity of LKB1 and may help further improve its effectiveness in controlling lung cancer growth and invasion. PMID:24647869

Liang, Xuan; Li, Zhao-Lun; Jiang, Li-Li; Guo, Qian-Qian; Liu, Meng-Jie; Nan, Ke-Jun



Molecular Characterisation, Evolution and Expression of Hypoxia-Inducible Factor in Aurelia sp.1  

PubMed Central

The maintenance of physiological oxygen homeostasis is mediated by hypoxia-inducible factor (HIF), a key transcriptional factor of the PHD-HIF system in all metazoans. However, the molecular evolutionary origin of this central physiological regulatory system is not well characterized. As the earliest eumetazoans, Cnidarians can be served as an interesting model for exploring the HIF system from an evolutionary perspective. We identified the complete cDNA sequence of HIF-1? (ASHIF) from the Aurelia sp.1, and the predicted HIF-1? protein (pASHIF) was comprised of 674 amino acids originating from 2,025 bp nucleotides. A Pairwise comparison revealed that pASHIF not only possessed conserved basic helix-loop-helix (bHLH) and Per-Arnt-Sim (PAS) domains but also contained the oxygen dependent degradation (ODD) and the C-terminal transactivation domains (C-TAD), the key domains for hypoxia regulation. As indicated by sequence analysis, the ASHIF gene contains 8 exons interrupted by 7 introns. Western blot analysis indicated that pASHIF that existed in the polyps and medusa of Aurelia. sp.1 was more stable for a hypoxic response than normoxia.

Wang, Guoshan; Yu, Zhigang; Zhen, Yu; Mi, Tiezhu; Shi, Yan; Wang, Jianyan; Wang, Minxiao; Sun, Song



5-Azacytidine treatment of HA-A melanoma cells induces Sp1 activity and concomitant transforming growth factor alpha expression.  

PubMed Central

Evidence indicates DNA methylation as a part of the regulatory machinery controlling mammalian gene expression. The human melanoma cell line HA-A expresses low levels of transforming growth factor alpha (TGF-alpha). TGF-alpha mRNA accumulated, however, in response to DNA demethylation induced by a nucleoside analog, 5-azacytidine (5-azaC). The importance of DNA methylation in the TGF-alpha promoter region was examined by a transient transfection assay with luciferase reporter plasmids containing a portion of the TGF-alpha promoter. 5-azaC treatment of HA-A cells before the transfection caused a significant increase in the luciferase activity. Since input plasmids were confirmed to remain unmethylated, DNA demethylation of the TGF-alpha promoter itself does not account for the observed increase in TGF-alpha mRNA. Using an electrophoretic mobility shift assay, enhanced formation of protein-TGF-alpha promoter complex was detected in response to 5-azaC treatment. This 5-azaC-induced complex was shown to contain the transcription factor Sp1 by the following criteria: the protein-DNA complex formed on the TGF-alpha promoter contained immunoreactive Sp1; the mobility of the complex in an electrophoretic mobility shift assay was similar to that formed by recombinant Sp1; and DNase I footprinting analysis demonstrated that the 5-azaC-induced complex produced a footprint on the TGF-alpha promoter identical to that of authentic Sp1. These observations suggest that 5-azaC induces TGF-alpha expression by augmenting the Sp1 activity. However, neither the Sp1 mRNA nor its protein was induced by 5-azaC. These results suggest that in HA-A cells, TGF-alpha expression is down-modulated by DNA methylation. In addition, this process may involve the specific regulation of Sp1 activity without altering the amount of the transcription factor. Images

Shin, T H; Paterson, A J; Grant, J H; Meluch, A A; Kudlow, J E



Transcription Factor E2F-Associated Phosphoprotein (EAPP), RAM2/CDCA7L/JPO2 (R1), and Simian Virus 40 Promoter Factor 1 (Sp1) Cooperatively Regulate Glucocorticoid Activation of Monoamine Oxidase B  

PubMed Central

Glucocorticoid steroid hormones play important roles in many neurophysiological processes such as responses to stress, behavioral adaption, and mood. One mechanism by which glucocorticoids exert functions in the brain is via the modulation of neurotransmission systems. Glucocorticoids are capable of inducing the activities of monoamine oxidases (MAOs), which degrade monoamine neurotransmitters including serotonin, norepinephrine, phenylethylamine, and dopamine. However, the molecular mechanisms for such induction are not yet fully understood. Here, we report that dexamethasone, a synthetic glucocorticoid hormone, stimulates MAO B (an isoform of MAOs) promoter and catalytic activities via both the fourth glucocorticoid response element (GRE) and simian virus 40 promoter factor 1 (Sp1) binding sites in MAO B promoter. Electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation analysis demonstrated that glucocorticoid receptor binds to the fourth GRE in vitro and in vivo. Using Sp1-binding motifs as bait in a yeast one-hybrid system, we identified two novel transcriptional repressors of MAO B, E2F-associated phosphoprotein (EAPP) and R1 (RAM2/CDCA7L/JPO2), that down-regulate MAO B via MAO B core promoter, which contains Sp1 sites. EMSA suggested that EAPP and R1 competed with Sp1 for binding to the Sp1 site in vitro. Moreover, EAPP and R1 reduced Sp1-activated glucocorticoid activation of MAO B promoter. In response to dexamethasone, lower occupancy by EAPP and R1 and higher occupancy by Sp1 were shown at the natural MAO B core promoter. Together, this study uncovers for the first time the molecular mechanisms for glucocorticoid activation of MAO B gene and provides new insights into the hormonal regulation of MAO.

Chen, Kevin; Ou, Xiao-Ming; Wu, Jason Boyang



miR-29b sensitizes multiple myeloma cells to bortezomib-induced apoptosis through the activation of a feedback loop with the transcription factor Sp1  

PubMed Central

MicroRNAs (miRNAs) with tumor-suppressor potential might have therapeutic applications in multiple myeloma (MM) through the modulation of still undiscovered molecular pathways. Here, we investigated the effects of enforced expression of miR-29b on the apoptotic occurrence in MM and highlighted its role in the context of a new transcriptional loop that is finely tuned by the proteasome inhibitor bortezomib. In details, in vitro growth inhibition and apoptosis of MM cells was induced by either transient expression of synthetic miR-29b or its stable lentivirus-enforced expression. We identified Sp1, a transcription factor endowed with oncogenic activity, as a negative regulator of miR-29b expression in MM cells. Since Sp1 expression and functions are regulated via the 26S proteasome, we investigated the effects of bortezomib on miR-29b-Sp1 loop, showing that miR-29b levels were indeed upregulated by the drug. At the same time, the bortezomib/miR-29b combination produced significant pro-apoptotic effects. We also demonstrated that the PI3K/AKT pathway plays a major role in the regulation of miR-29b-Sp1 loop and induction of apoptosis in MM cells. Finally, MM xenografts constitutively expressing miR-29b showed significant reduction of their tumorigenic potential. Our findings indicate that miR-29b is involved in a regulatory loop amenable of pharmacologic intervention and modulates the anti-MM activity of bortezomib in MM cells.

Amodio, N; Di Martino, M T; Foresta, U; Leone, E; Lionetti, M; Leotta, M; Gulla, A M; Pitari, M R; Conforti, F; Rossi, M; Agosti, V; Fulciniti, M; Misso, G; Morabito, F; Ferrarini, M; Neri, A; Caraglia, M; Munshi, N C; Anderson, K C; Tagliaferri, P; Tassone, P



Sp1\\/Sp3 compound heterozygous mice are not viable: Impaired erythropoiesis and severe placental defects  

Microsoft Academic Search

The ubiquitously expressed zinc finger transcription factors Sp1 and Sp3 play critical roles in embryonic development. Sp1 knockout mice die around embryonic day 10.5. Mice lacking Sp3 are postnatal lethal. Mice heterozygous for either Sp1 or Sp3 are apparently normal, although slightly smaller. Here, we show that compound heterozygosity of Sp1 and Sp3 results in embryonic lethality accompanied by a

Imme Krüger; Marion Vollmer; David G. Simmons; Hans-Peter Elsässer; Sjaak Philipsen; Guntram Suske



Unique aspects of transcriptional regulation in neurons - nuances in NF?B and Sp1-related factors  

PubMed Central

The unique physiology and function of neurons create differences in their cellular physiology, including their regulation of gene expression. We began several years ago exploring the relationships between the NF?B transcription factor, neuronal survival, and glutamate receptor activation in telencephalic neurons. These studies led us to conclude that this population of cells is nearly incapable of activating the NF?B that is nonetheless expressed at reasonable levels. A subset of the ?B cis elements are instead bound by members of the Sp1 family in neurons. Also surprising was our discovery that Sp1 itself, typically described as ubiquitous, is severely restricted in expression within forebrain neurons; Sp4 seems to be substituted during neuronal differentiation. These findings and their implications for neuronal differentiation – as well as potential dedifferentiation during degenerative processes – are discussed here.

Mao, Xianrong R; Moerman-Herzog, Andrea M; Chen, Yuzhi; Barger, Steven W



Transcription factor GATA-3 regulates the transcriptional activity of dopamine ?-hydroxylase by interacting with Sp1 and AP4  

PubMed Central

GATA-3 is a zinc finger transcription factor that is expressed in T cell lineages as well as in the nervous system during development. In this study, we report that forced expression of GATA-3 resulted in an increased number of dopamine ?-hydroxylase (DBH)-expressing neurons in primary neural crest stem cell (NCSC) culture, suggesting that the DBH gene may be a downstream target gene of GATA-3. GATA-3 robustly transactivates the promoter function of the noradrenaline (NA)-synthesizing DBH gene, via two specific upstream promoter domains; one at -62 to -32 bp and the other at -891 to -853 bp. Surprisingly, none of these domains contain GATA-3 binding sites but encompass binding motifs for transcription factors Sp1 and AP4, respectively. Protein-protein interaction analyses both in vitro and in vivo and chromatin immunoprecipitation (ChIP) assays showed that GATA-3 effects its transcriptional regulatory function through physical interactions with these transcription factors.

Hong, Seok Jong; Choi, Hyun Jin; Hong, Sunghoi; Huh, Youngbuhm; Chae, Han; Kim, Kwang-Soo



Nerve Growth Factor Regulation of Cyclin D1 in PC12 Cells through a p21RAS Extracellular Signal-regulated Kinase Pathway Requires Cooperative Interactions between Sp1 and Nuclear Factor-?B  

PubMed Central

The PC12 pheochromocytoma cell line responds to nerve growth factor (NGF) by exiting from the cell cycle and differentiating to induce extending neurites. Cyclin D1 is an important regulator of G1/S phase cell cycle progression, and it is known to play a role in myocyte differentiation in cultured cells. Herein, NGF induced cyclin D1 promoter, mRNA, and protein expression via the p21RAS pathway. Antisense- or small interfering RNA to cyclin D1 abolished NGF-mediated neurite outgrowth, demonstrating the essential role of cyclin D1 in NGF-mediated differentiation. Expression vectors encoding mutants of the Ras/mitogen-activated protein kinase pathway, and chemical inhibitors, demonstrated NGF induction of cyclin D1 involved cooperative interactions of extracellular signal-regulated kinase, p38, and phosphatidylinositol 3-kinase pathways downstream of p21RAS. NGF induced the cyclin D1 promoter via Sp1, nuclear factor-?B, and cAMP-response element/activated transcription factor sites. NGF induction via Sp1 involved the formation of a Sp1/p50/p107 complex. Cyclin D1 induction by NGF governs differentiation and neurite outgrowth in PC12 cells.

Marampon, Francesco; Casimiro, Mathew C.; Fu, Maofu; Powell, Michael J.; Popov, Vladimir M.; Lindsay, Jaime; Zani, Bianca M.; Ciccarelli, Carmela; Watanabe, Genichi; Lee, Richard J.



TGF-?1 induction of the adenine nucleotide translocator 1 in astrocytes occurs through Smads and Sp1 transcription factors  

PubMed Central

Background The adenine nucleotide translocator 1 (Ant1) is an inner mitochondrial membrane protein involved with energy mobilization during oxidative phosphorylation. We recently showed that rodent Ant1 is upregulated by transforming growth factor-beta (TGF-?) in reactive astrocytes following CNS injury. In the present study, we describe the molecular mechanisms by which TGF-?1 regulates Ant1 gene expression in cultured primary rodent astrocytes. Results Transcription reporter analysis verified that TGF-?1 regulates transcription of the mouse Ant1 gene, but not the gene encoding the closely related Ant2 isoform. A 69 basepair TGF-?1 responsive element of the Ant1 promoter was also identified. Electrophoretic mobility shift assays demonstrated that astrocyte nuclear proteins bind to this response element and TGF-?1 treatment recruits additional nuclear protein binding to this element. Antibody supershift and promoter deletion analyses demonstrated that Sp1 consensus binding sites in the RE are important for TGF-?1 regulation of Ant1 in astrocytes. Additionally, we demonstrate that Smad 2, 3 and 4 transcription factors are expressed in injured cerebral cortex and in primary astrocyte cultures. TGF-?1 activated Smad transcription factors also contribute to Ant1 regulation since transcription reporter assays in the presence of dominant negative (DN)-Smads 3 and 4 significantly reduced induction of Ant1 by TGF-?1. Conclusion The specific regulation of Ant1 by TGF-?1 in astrocytes involves a cooperative interaction of both Smad and Sp1 binding elements located immediately upstream of the transcriptional start site. The first report of expression of Smads 2, 3 and 4 in astrocytes provided here is consistent with a regulation of Ant1 gene expression by these transcription factors in reactive astrocytes. Given the similarity in TGF-?1 regulation of Ant1 with other genes that are thought to promote neuronal survival, this interaction may represent a general mechanism that underlies the neuroprotective effects of TGF-?1.

Law, Alick KT; Gupta, Deepak; Levy, Shawn; Wallace, Douglas C; McKeon, Robert J; Buck, Charles R



Involvement of PKC{alpha} in insulin-induced PKC{delta} expression: Importance of SP-1 and NF{kappa}B transcription factors  

SciTech Connect

Protein kinase C delta (PKC{delta}) is a key molecule in insulin signaling essential for insulin-induced glucose transport in skeletal muscle. Recent studies in our laboratory have shown that insulin rapidly stimulates PKC{delta} activity and increases PKC{delta} protein and RNA levels, and that the SP-1 transcription factor is involved in insulin-induced transcription of the PKC{delta} gene. Activation of SP-1 involves serine phosphorylation and translocation to the nucleus. In this study we examined the possibility that PKC{alpha} might be involved in serine phosphorylation and activation of SP-1. We found that insulin rapidly phosphorylates and translocates SP-1. In the cytoplasm, SP-1 was constitutively associated with PKC{alpha}, and insulin stimulation caused these proteins to dissociate. In contrast, in the nucleus insulin induced an increase in association between PKC{alpha} and SP-1. PKC{alpha} inhibition blocked insulin-induced serine phosphorylation of SP-1 and its association with PKC{alpha} in the nucleus. Inhibition of PKC{alpha} also reduced the insulin-induced increase in PKC{delta} RNA and protein in the cytoplasmic and nuclear fractions. We also attempted to determine if another transcription factor might be involved in regulation of PKC{delta} expression. We earlier showed that insulin did not affect nuclear NF{kappa}B levels. Inhibition of NF{kappa}B, however, increased insulin-induced increase in PKC{delta} RNA and protein in the cytoplasmic and nuclear fractions. Surprisingly, this inhibition reduced the insulin-induced increase in cytoplasmic and nuclear PKC{alpha} RNA and protein. Inhibition of PKC{delta} reduced I{kappa}B{alpha} phosphorylation as well as NF{kappa}B activation. Thus, PKC{alpha} regulates insulin-induced PKC{delta} expression levels and this regulation involves activation of SP-1 and NF{kappa}B.

Horovitz-Fried, Miriam [Faculty of Life Sciences, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900 (Israel); Sampson, Sanford R. [Faculty of Life Sciences, Department of Life Sciences, Bar-Ilan University, Ramat-Gan 52900 (Israel)]. E-mail:



A Novel Specificity Protein 1 (SP1)-like Gene Regulating Protein Kinase C-1 (Pkc1)-dependent Cell Wall Integrity and Virulence Factors in Cryptococcus neoformans*  

PubMed Central

Eukaryotic cells utilize complex signaling systems to detect their environments, responding and adapting as new conditions arise during evolution. The basidiomycete fungus Cryptococcus neoformans is a leading cause of AIDS-related death worldwide and utilizes the calcineurin and protein kinase C-1 (Pkc1) signaling pathways for host adaptation and expression of virulence. In the present studies, a C-terminal zinc finger transcription factor, homologous both to the calcineurin-responsive zinc fingers (Crz1) of ascomycetes and to the Pkc1-dependent specificity protein-1 (Sp1) transcription factors of metazoans, was identified and named SP1 because of its greater similarity to the metazoan factors. Structurally, the Cryptococcus neoformans Sp1 (Cn Sp1) protein was found to have acquired an additional zinc finger motif from that of Crz1 and showed Pkc1-dependent phosphorylation, nuclear localization, and whole genome epistatic associations under starvation conditions. Transcriptional targets of Cn Sp1 shared functional similarities with Crz1 factors, such as cell wall synthesis, but gained the regulation of processes involved in carbohydrate metabolism, including trehalose metabolism, and lost others, such as the induction of autophagy. In addition, overexpression of Cn Sp1 in a pkc1? mutant showed restoration of altered phenotypes involved in virulence, including cell wall stability, nitrosative stress, and extracellular capsule production. Cn Sp1 was also found to be important for virulence of the fungus using a mouse model. In summary, these data suggest an evolutionary shift in C-terminal zinc finger proteins during fungal evolution, transforming them from calcineurin-dependent to PKC1-dependent transcription factors, helping to shape the role of fungal pathogenesis of C. neoformans.

Adler, Amos; Park, Yoon-Dong; Larsen, Peter; Nagarajan, Vijayaraj; Wollenberg, Kurt; Qiu, Jin; Myers, Timothy G.; Williamson, Peter R.



Functional interactions between Sp1 or Sp3 and the helicase-like transcription factor mediate basal expression from the human plasminogen activator inhibitor-1 gene.  


Basal expression of the human plasminogen activator inhibitor-1 (PAI-1) is mediated by a promoter element named B box that binds the helicase-like transcription factor (HLTF), homologous to SNF/SWI proteins. Electrophoretic mobility shift assays performed on a set of B box point mutants demonstrated two HLTF sites flanking and partially overlapping with a GT box binding Sp1 and Sp3. Mutations affecting either the Sp1/Sp3 or the two HLTF sites inhibited by 6- and 2.5-fold, respectively, transient expression in HeLa cells of a reporter gene fused to the PAI-1 promoter. In Sp1/Sp3-devoid insect cells, co-expression of PAI-1-lacZ with Sp1 or Sp3 led to a 14-26-fold induction while HLTF had no effect. Simultaneous presence of Sp1 or Sp3 and the short HLTF form (initiating at Met-123) provided an additional 2-3-fold synergistic activation suppressed by mutations that prevented HLTF binding. Moreover, a DNA-independent interaction between HLTFMet123 and Sp1/Sp3 was demonstrated by co-immunoprecipitation from HeLa cell extracts and glutathione S-transferase pull-down experiments. The interaction domains were mapped to the carboxyl-terminal region of each protein; deletion of the last 85 amino acids of HLTFMet123 abolished the synergy with Sp1. This is the first demonstration of a functional interaction between proteins of the Sp1 and SNF/SWI families. PMID:10391891

Ding, H; Benotmane, A M; Suske, G; Collen, D; Belayew, A



Overlapping DNA recognition motifs between Sp1 and a novel trans-acting factor within the wt1 tumour suppressor gene promoter.  

PubMed Central

The Wilms' tumor suppressor gene, wt1 , encodes a zinc finger transcription factor which has been shown to regulate the expression of several genes involved in cellular proliferation and differentiation. Expression of wt1 is developmentally regulated and restricted to a small set of tissues which include the fetal urogenital system, mesothelium and spleen. A highly conserved motif within the wt1 promoter, located between nucleotides -34 and -71 relative to the first transcription start site in the murine promoter, harbors consensus binding sites for Sp1 and members of the paired-box transcription factor family. Pax-2 and Pax-8 are known to enhance expression of wt1 through this conserved regulatory element. In this report, we demonstrate that Sp1 is able to bind to two sites within the 38 bp conserved region (CR). By electrophoretic mobility shift assays (EMSAs), we have identified a novel binding activity, referred to as complex D, which recognizes sequences overlapping one of the Sp1 sites in the CR. EMSA competition experiments indicate that binding of complex D and Sp1 to the CR is mutually exclusive and Sp1 is able to displace complex D binding. In situ UV crosslinking and molecular mass determinations indicate that complex D is a complex of approximately 130 kDa, consisting of at least two proteins of approximately 62 and approximately 70 kDa. Transient transfections suggest that complex D may function as an activator.

Discenza, M T; Dehbi, M; Pelletier, J



p53-mediated Down-regulation of the Human DNA Repair Gene O6- Methylguanine-DNA Methyltransferase (MGMT) via Interaction with Sp1 Transcription Factor  

PubMed Central

O6-Methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, reverses mutagenic and cytotoxic effects of O6-alkylguanine in DNA induced by chemotherapeutic N-alkyl N-nitrosourea and procarbazine type drugs by dealkylating the adduct. MGMT expression is down-regulated by wild-type p53 (WTp53) in human tumor cells. Here we report that p53 sequesters the Sp1 transcription factor to prevent its binding to the cognate cis elements in the MGMT promoter and thus inhibits MGMT expression. Sp1 overexpression abrogated the inhibitory effect of p53 on the MGMT promoter activity in a dose-dependent manner. Stable interaction of Sp1 with WTp53 was observed in HCT116 cells. Moreover, WTp53 overexpression reduced the binding of the nuclear extract to the Sp1 consensus sequence, even though recombinant p53 alone did not bind to the same sequence. Taken together, these results suggest that sequestration of Sp1 could be one of the mechanisms by which p53 negatively regulates MGMT expression, thus enhancing sensitivity of tumor cells to O6-alkylguanine generating drugs.

Bocangel, Dora; Sengupta, Shiladitya; Mitra, Sankar; Bhakat, Kishor K.



Heme Oxygenase-1/Carbon Monoxide Induces Vascular Endothelial Growth Factor Expression via p38 Kinase-dependent Activation of Sp1*  

PubMed Central

Heme oxygenase-1 (HO-1) is a stress-inducible enzyme catalyzing the oxidative degradation of heme to free iron, CO, and biliverdin. Previous studies demonstrated that HO-1 overexpression promoted VEGF expression and angiogenesis in the ischemic heart. However, the underlying mechanism remained elusive. Here we show that adenovirus-mediated HO-1 transduction of rat primary cardiomyocytes and H9C2 myocytes resulted in significant induction of VEGF expression, and a similar effect was seen in cells directly exposed to CO gas or a CO-releasing compound, tricarbonyldichlororuthenium (II) dimer. HO-1/CO-induced VEGF expression was significantly suppressed by pharmacological inhibition of p38 kinase, but not of AKT, activation. VEGF promoter-luciferase reporter assays, electrophoretic mobility shift assays, supershift assay, and chromatin immunoprecipitation showed that CO-induced VEGF promoter activation requires the binding of the Sp1 transcriptional factor to a cis-regulatory sequence located at the VEGF promoter. Western blot analysis and immunostaining experiments demonstrated that HO-1/CO induced p38-dependent phosphorylation of Sp1 at Thr-453 and Thr-739 both in vitro and in vivo. Overexpression of Sp1 protein with an alanine mutation at Thr-453 or Thr-739 suppressed CO-induced Sp1 binding to the VEGF promoter and its transcriptional activation. Collectively, these data suggest that p38-dependent phosphorylation of Sp1 at Thr-453/Thr-739 is crucial for HO-1/CO-induced VEGF expression in myocytes.

Lin, Heng-Huei; Lai, Shao-Chuan; Chau, Lee-Young



Sp1 Transcription Factor Interaction with Accumulated Prelamin A Impairs Adipose Lineage Differentiation in Human Mesenchymal Stem Cells: Essential Role of Sp1 in the Integrity of Lipid Vesicles  

PubMed Central

Lamin A (LMNA)-linked lipodystrophies may be either genetic (associated with LMNA mutations) or acquired (associated with the use of human immunodeficiency virus protease inhibitors [PIs]), and in both cases they share clinical features such as anomalous distribution of body fat or generalized loss of adipose tissue, metabolic alterations, and early cardiovascular complications. Both LMNA-linked lipodystrophies are characterized by the accumulation of the lamin A precursor prelamin A. The pathological mechanism by which prelamin A accumulation induces the lipodystrophy associated phenotypes remains unclear. Since the affected tissues in these disorders are of mesenchymal origin, we have generated an LMNA-linked experimental model using human mesenchymal stem cells treated with a PI, which recapitulates the phenotypes observed in patient biopsies. This model has been demonstrated to be a useful tool to unravel the pathological mechanism of the LMNA-linked lipodystrophies, providing an ideal system to identify potential targets to generate new therapies for drug discovery screening. We report for the first time that impaired adipogenesis is a consequence of the interaction between accumulated prelamin A and Sp1 transcription factor, sequestration of which results in altered extracellular matrix gene expression. In fact, our study shows a novel, essential, and finely tuned role for Sp1 in adipose lineage differentiation in human mesenchymal stem cells. These findings define a new physiological experimental model to elucidate the pathological mechanisms LMNA-linked lipodystrophies, creating new opportunities for research and treatment not only of LMNA-linked lipodystrophies but also of other adipogenesis-associated metabolic diseases.

Ruiz de Eguino, Garbine; Infante, Arantza; Schlangen, Karin; Aransay, Ana M.; Fullaondo, Ane; Soriano, Mario; Garcia-Verdugo, Jose Manuel; Martin, Angel G.



Role of cyclooxygenase-2 induction by transcription factor Sp1 and Sp3 in neuronal oxidative and DNA damage response  

Microsoft Academic Search

Cyclooxygenase-2 (COX-2) has been im- plicated in neuronal survival and death. However, the precise regulatory mechanisms involved in COX-2 func- tion are unclear. In the present study we found that COX-2 is induced in response to glutathione depletion- induced oxidative stress in primary cortical neurons. Two proximal specific Sp1 and Sp3 binding sites are responsi- ble for the COX-2 promoter

Junghee Lee; Bela Kosaras; Hossein Aleyasin; Jeong A. Han; David S. Park; Rajiv R. Ratan; Neil W. Kowall; Robert J. Ferrante; Sam W. Lee; Hoon Ryu



Sp4, a Member of the Sp1Family of Zinc Finger Transcription Factors, Is Required for Normal Murine Growth, Viability, and Male Fertility  

Microsoft Academic Search

We report the cloning, characterization, and targeting of an Sp1-related zinc finger transcription factor gene from the distal arm of mouse chromosome 12. This gene, previously identified in rats and humans and designatedsp4,is homologous to theDrosophila buttonhead(btd) gene, which is expressed in the head region of developing flies. Similarly,in situhybridizations show thatsp4is highly expressed in mouse embryos in the developing

Dorothy M. Supp; David P. Witte; William W. Branford; Eric P. Smith; S. Steven Potter



Leishmania donovani activates uncoupling protein 2 transcription to suppress mitochondrial oxidative burst through differential modulation of SREBP2, Sp1 and USF1 transcription factors.  


In order to reside and multiply successfully within the host macrophages, Leishmania parasites impair the generation of cellular as well as mitochondrial reactive oxygen species (ROS), which is a major host defense mechanism against any invading pathogen. Mitochondrial uncoupling protein 2 (UCP2) is strongly induced in Leishmania infection, both at mRNA and protein levels, to suppress the mitochondrial ROS generation. In the present study we have demonstrated that Leishmania donovani infection is associated with strong up-regulation of UCP2 at mRNA level which is the determining factor for its protein level upregulation. The transcriptional activation of UCP2 was mediated by increased nuclear translocation and DNA binding of sterol regulatory element binding protein 2 (SREBP2) and specificity protein 1 (Sp1) transcription factors with concomitant decrease of both the nuclear content and the promoter occupancy of upstream stimulatory factor 1 (USF1). siRNA-mediated silencing of SREBP2 or Sp1 was associated with decreased UCP2 expression in infected macrophages. In contrast, downregulation of USF1 resulted in activated transcription of UCP2. L. donovani infection resulted in degradation of USF1 thereby facilitating SREBP2 binding which in turn assisted in the association of Sp1 with the promoter ultimately culminating in elevated transcription of UCP2. PMID:24417972

Ball, Writoban Basu; Mukherjee, Madhuchhanda; Srivastav, Supriya; Das, Pijush K



Transcriptional regulation of the human cystathionine beta-synthase -1b basal promoter: synergistic transactivation by transcription factors NF-Y and Sp1/Sp3.  

PubMed Central

Cystathionine beta-synthase (CBS) catalyses the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. Human CBS encodes five distinct 5' non-coding exons, the most frequent termed CBS -1a and CBS -1b, each transcribed from its own unique GC-rich TATA-less promoter. The minimal transcriptional region (-3792 to -3667) of the CBS -1b promoter was defined by 5'- and 3'-deletions, and transient transfections of reporter gene constructs in HepG2 cells, characterized by CBS transcription exclusively from the -1b promoter. Included in this 125 bp region are 3 GC-boxes (termed GC-a, GC-b and GC-c), an inverted CAAT-box and an E-box. By gel-shift and supershift assays, binding of specificity protein (Sp)1 and Sp3 to the GC-box elements, upstream stimulatory factor 1 (USF-1) to the E-box, and both nuclear factor (NF)-Y and an NF-1-like factor to the CAAT box could be demonstrated. By transient trans fections and reporter gene assays in HepG2 and Drosophila SL2 cells, a functional interplay was indicated between NF-Y binding to the CAAT-box, or between USF-1 binding to the E-box, and Sp1/Sp3 binding to the GC-box elements. In SL2 cells, NF-Y and Sp1/Sp3 were synergistic. Furthermore, both Sp1 and the long Sp3 isoform transactivated the CBS -1b minimal promoter; however, the short Sp3 isoforms were potent repressors. These results may explain the cell- or tissue-specific regulation of CBS transcription, and clarify the bases for alterations in CBS gene expression in human disease and Down's syndrome.

Ge, Y; Konrad, M A; Matherly, L H; Taub, J W



Brg-1 mediates the constitutive and fenretinide-induced expression of SPARC in mammary carcinoma cells via its interaction with transcription factor Sp1  

PubMed Central

Background Secreted protein, acidic and rich in cysteine (SPARC) is a matricellular protein that mediates cell-matrix interactions. It has been shown, depending on the type of cancer, to possess either pro- or anti-tumorigenic properties. The transcriptional regulation of the SPARC gene expression has not been fully elucidated and the effects of anti-cancer drugs on this process have not been explored. Results In the present study, we demonstrated that chromatin remodeling factor Brg-1 is recruited to the proximal SPARC promoter region (-130/-56) through an interaction with transcription factor Sp1. We identified Brg-1 as a critical regulator for the constitutive expression levels of SPARC mRNA and protein in mammary carcinoma cell lines and for SPARC secretion into culture media. Furthermore, we found that Brg-1 cooperates with Sp1 to enhance SPARC promoter activity. Interestingly, fenretinide [N-4(hydroxyphenyl) retinamide, 4-HPR], a synthetic retinoid with anti-cancer properties, was found to up-regulate the transcription, expression and secretion of SPARC via induction of the Brg-1 in a dose-dependent manner. Finally, our results demonstrated that fenretinide-induced expression of SPARC contributes significantly to a decreased invasion of mammary carcinoma cells. Conclusions Overall, our results reveal a novel cooperative role of Brg-1 and Sp1 in mediating the constitutive and fenretinide-induced expression of SPARC, and provide new insights for the understanding of the anti-cancer effects of fenretinide.



Sulforaphane induction of p21(Cip1) cyclin-dependent kinase inhibitor expression requires p53 and Sp1 transcription factors and is p53-dependent.  


Sulforaphane (SFN) is an important cancer preventive agent derived from cruciferous vegetables. We show that SFN treatment suppresses normal human keratinocyte proliferation via a mechanism that involves increased expression of p21(Cip1). SFN treatment produces a concentration-dependent increase in p21(Cip1) promoter activity via a mechanism that involves stabilization of the p53 protein leading to increased p53 binding to the p21(Cip1) promoter p53 response elements. The proximal p21(Cip1) promoter GC-rich Sp1 factor binding elements are also required, as the SFN-dependent increase is lost when these sites are mutated. SFN treatment increases Sp1 binding to these elements, and the response is enhanced in the presence of exogenous Sp1 and reduced in the presence of ?N-Sp3. CpG island methylation alters p21(Cip1) promoter activity some systems; however, expression in SFN-treated keratinocytes does not involve changes in proximal promoter methylation. The promoter is minimally methylated, and the methylation level is not altered by SFN treatment. This study indicates that SFN increases p21(Cip1) promoter transcription via a mechanism that involves SFN-dependent stabilization of p53 and increased p53 and Sp1 binding to their respective response elements in the p21(Cip1) promoter. These results are in marked contrast to the mechanisms observed in skin cancer cell lines and suggest that SFN may protect normal keratinocytes from damage while causing cancer cells to undergo apoptosis. PMID:22427654

Chew, Yap Ching; Adhikary, Gautam; Wilson, Gerald M; Xu, Wen; Eckert, Richard L



Sulforaphane Induction of p21Cip1 Cyclin-dependent Kinase Inhibitor Expression Requires p53 and Sp1 Transcription Factors and Is p53-dependent*  

PubMed Central

Sulforaphane (SFN) is an important cancer preventive agent derived from cruciferous vegetables. We show that SFN treatment suppresses normal human keratinocyte proliferation via a mechanism that involves increased expression of p21Cip1. SFN treatment produces a concentration-dependent increase in p21Cip1 promoter activity via a mechanism that involves stabilization of the p53 protein leading to increased p53 binding to the p21Cip1 promoter p53 response elements. The proximal p21Cip1 promoter GC-rich Sp1 factor binding elements are also required, as the SFN-dependent increase is lost when these sites are mutated. SFN treatment increases Sp1 binding to these elements, and the response is enhanced in the presence of exogenous Sp1 and reduced in the presence of ?N-Sp3. CpG island methylation alters p21Cip1 promoter activity some systems; however, expression in SFN-treated keratinocytes does not involve changes in proximal promoter methylation. The promoter is minimally methylated, and the methylation level is not altered by SFN treatment. This study indicates that SFN increases p21Cip1 promoter transcription via a mechanism that involves SFN-dependent stabilization of p53 and increased p53 and Sp1 binding to their respective response elements in the p21Cip1 promoter. These results are in marked contrast to the mechanisms observed in skin cancer cell lines and suggest that SFN may protect normal keratinocytes from damage while causing cancer cells to undergo apoptosis.

Chew, Yap Ching; Adhikary, Gautam; Wilson, Gerald M.; Xu, Wen; Eckert, Richard L.



FoxO1 Inhibits Sterol Regulatory Element-binding Protein-1c (SREBP-1c) Gene Expression via Transcription Factors Sp1 and SREBP-1c*  

PubMed Central

Induction of lipogenesis in response to insulin is critically dependent on the transcription factor, sterol regulatory element-binding protein-1c (SREBP-1c). FoxO1, a forkhead box class-O transcription factor, is an important mediator of insulin action, but its role in the regulation of lipid metabolism has not been clearly defined. We examined the effects of FoxO1 on srebp1 gene expression in vivo and in vitro. In vivo studies showed that constitutively active (CA) FoxO1 (CA-FoxO1) reduced basal expression of SREBP-1c mRNA in liver by ?60% and blunted induction of SREBP-1c in response to feeding. In liver-specific FoxO knock-out mice, SREBP-1c expression was increased ?2-fold. Similarly, in primary hepatocytes, CA-FoxO1 suppressed SREBP1-c expression and inhibited basal and insulin-induced SREBP-1c promoter activity. SREBP-1c gene expression is induced by the liver X receptor (LXR), but CA-FoxO1 did not block the activation of SREBP-1c by the LXR agonist TO9. Insulin stimulates SREBP-1c transcription through Sp1 and via “feed forward” regulation by newly synthesized SREBP-1c. CA-FoxO1 inhibited SREBP-1c by reducing the transactivational capacity of both Sp1 and SREBP-1c. In addition, chromatin immunoprecipitation assays indicate that FoxO1 can associate with the proximal promoter region of the srebp1 gene and disrupt the assembly of key components of the transcriptional complex of the SREBP-1c promoter. We conclude that FoxO1 inhibits SREBP-1c transcription via combined actions on multiple transcription factors and that this effect is exerted at least in part through reduced transcriptional activity of Sp1 and SREBP-1c and disrupted assembly of the transcriptional initiation complex on the SREBP-1c promoter.

Deng, Xiong; Zhang, Wenwei; O-Sullivan, InSug; Williams, J. Bradley; Dong, Qingming; Park, Edwards A.; Raghow, Rajendra; Unterman, Terry G.; Elam, Marshall B.



Regulation of the Cyclin-dependent Kinase Inhibitor 1A Gene (CDKN1A) by the Repressor BOZF1 through Inhibition of p53 Acetylation and Transcription Factor Sp1 Binding*  

PubMed Central

The human POZ domain and Krüppel-like zinc finger (POK) family proteins play important roles in the regulation of apoptosis, cell proliferation, differentiation, development, oncogenesis, and tumor suppression. A novel POK family transcription factor, BTB/POZ and zinc finger domains factor on chromosome 1 (BOZF-1; also called ZBTB8A), contains a POZ domain and two C2H2-type Krüppel-like zinc fingers and is localized at nuclear speckles. Compared with paired normal tissues, BOZF1 expression is increased in cancer tissues of the prostate, breast, and cervix. BOZF1 repressed the transcription of p21WAF/CDKN1A by acting on the proximal promoter concentrated with Sp1-binding GC boxes. BOZF1 competed with Sp1 in binding to GC boxes 1–5/6 of the CDKN1A proximal promoter. In addition, BOZF1 interacted with p53 and decreased the acetylation of p53 by p300, which reduced the DNA binding activity of p53 at the far distal p53-binding element. BOZF1 blocked the two major molecular events that are important in both constitutive and inducible transcription activation of CDKN1A. BOZF1 is unique in that it bound to all the proximal GC boxes to repress transcription, and it inhibited p53 acetylation without affecting p53 stability. BOZF1 might be a novel proto-oncoprotein that stimulates cell proliferation.

Kim, Min-Kyeong; Jeon, Bu-Nam; Koh, Dong-In; Kim, Kyung-Sup; Park, So-Yoon; Yun, Chae-Ok; Hur, Man-Wook



Indole-3-carbinol downregulation of telomerase gene expression requires the inhibition of estrogen receptor-alpha and Sp1 transcription factor interactions within the hTERT promoter and mediates the G1 cell cycle arrest of human breast cancer cells  

PubMed Central

Indole-3-carbinol (I3C), a naturally occurring hydrolysis product of glucobrassicin from cruciferous vegetables such as broccoli, cabbage and Brussels sprouts, is an anticancer phytochemical that triggers complementary sets of antiproliferative pathways to induce a cell cycle arrest of estrogen-responsive MCF7 breast cancer cells. I3C strongly downregulated transcript expression of the catalytic subunit of the human telomerase (hTERT) gene, which correlated with the dose-dependent indole-mediated G1 cell cycle arrest without altering the transcript levels of the RNA template (hTR) for telomerase elongation. Exogenous expression of hTERT driven by a constitutive promoter prevented the I3C-induced cell cycle arrest and rescued the I3C inhibition of telomerase enzymatic activity and activation of cellular senescence. Time course studies showed that I3C downregulated expression of estrogen receptor-alpha (ER?) and cyclin-dependent kinase-6 transcripts levels (which is regulated through the Sp1 transcription factor) prior to the downregulation of hTERT suggesting a mechanistic link. Chromatin immunoprecipitation assays demonstrated that I3C disrupted endogenous interactions of both ER? and Sp1 with an estrogen response element–Sp1 composite element within the hTERT promoter. I3C inhibited 17?-estradiol stimulated hTERT expression and stimulated the production of threonine-phosphorylated Sp1, which inhibits Sp1–DNA interactions. Exogenous expression of both ER? and Sp1, but not either alone, in MCF7 cells blocked the I3C-mediated downregulation of hTERT expression. These results demonstrate that I3C disrupts the combined ER?- and Sp1-driven transcription of hTERT gene expression, which plays a significant role in the I3C-induced cell cycle arrest of human breast cancer cells.

Marconett, Crystal N.; Sundar, Shyam N.; Tseng, Min; Tin, Antony S.; Tran, Kalvin Q.; Mahuron, Kelly M.; Bjeldanes, Leonard F.; Firestone, Gary L.



CRABP2 Promotes Myoblast Differentiation and Is Modulated by the Transcription Factors MyoD and Sp1 in C2C12 Cells  

PubMed Central

Cellular retinoic acid binding protein 2 (CRABP2), a member of a family of specific carrier proteins for Vitamin A, belongs to a family of small cytosolic lipid binding proteins. Our previous study suggested that CRABP2 was involved in skeletal muscle development; however, the molecular function and regulatory mechanism of CRABP2 in myogenesis remained unclear. In this study, we found that the expression of the CRABP2 gene was upregulated during C2C12 differentiation. An over-expression assay revealed that CRABP2 promotes myogenic transformation by regulating the cell cycle during C2C12 differentiation. The region from ?459 to ?4 bp was identified as the core promoter and contains a TATA box, a GC box and binding sites for the transcription factors MyoD and Sp1. Over-expression, site-directed mutagenesis and EMSA assays indicated that the transcription factors MyoD and Sp1 regulate CRABP2 expression and promote myoblast differentiation in C2C12 cells.

Yuan, Jing; Tang, Zhonglin; Yang, Shulin; Li, Kui



Fibroblast growth factor-2 up-regulates the expression of nestin through the Ras–Raf–ERK–Sp1 signaling axis in C6 glioma cells  

SciTech Connect

Highlights: •Nestin expression in C6 glioma cells is induced by FGF-2. •Nestin expression is induced by FGF-2 via de novo RNA and protein synthesis. •The FGFR inhibitor SU5402 blocks the FGF-2-induced nestin expression. •The mRNA of FGFR1 and 3 are detected in C6 glioma cells. •Ras–Raf–ERK–Sp1 signaling pathway is responsibe for FGF-2-induced nestin expression. -- Abstract: Nestin is a 240-kDa intermediate filament protein expressed mainly in neural and myogenic stem cells. Although a substantial number of studies have focused on the expression of nestin during development of the central nervous system, little is known about the factors that induce and regulate its expression. Fibroblast growth factor-2 (FGF-2) is an effective mitogen and stimulates the proliferation and differentiation of a subset of nestin-expressing cells, including neural progenitor cells, glial precursor cells, and smooth muscle cells. To assess whether FGF-2 is a potent factor that induces the expression of nestin, C6 glioma cells were used. The results showed that nestin expression was up-regulated by FGF-2 via de novo RNA and protein synthesis. Our RT-PCR results showed that C6 glioma cells express FGFR1/3, and FGFRs is required for FGF-2-induced nestin expression. Further signaling analysis also revealed that FGF-2-induced nestin expression is mediated through FGFR–MAPK–ERK signaling axis and the transcriptional factor Sp1. These findings provide new insight into the regulation of nestin in glial system and enable the further studies on the function of nestin in glial cells.

Chang, Kai-Wei [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China)] [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China); Huang, Yuan-Li [Department of Biotechnology, College of Health Science, Asia University, Taichung 413, Taiwan (China)] [Department of Biotechnology, College of Health Science, Asia University, Taichung 413, Taiwan (China); Wong, Zong-Ruei; Su, Peng-Han [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China)] [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China); Huang, Bu-Miin [Department of Cell Biology and Anatomy, National Cheng-Kung University, Tainan 701, Taiwan (China)] [Department of Cell Biology and Anatomy, National Cheng-Kung University, Tainan 701, Taiwan (China); Ju, Tsai-Kai [Instrumentation Center, National Taiwan University, Taipei 106, Taiwan (China) [Instrumentation Center, National Taiwan University, Taipei 106, Taiwan (China); Technology Commons, College of Life Science, National Taiwan University, Taipei 106, Taiwan (China); Yang, Hsi-Yuan, E-mail: [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China)] [Institute of Molecular and Cellular Biology, National Taiwan University, Taipei 106, Taiwan (China)



Regulation of Sp1 by cell cycle related proteins  

PubMed Central

Sp1 transcription factor regulates the expression of multiple genes, including the Sp1 gene itself. We analyzed the ability of different cell cycle regulatory proteins to interact with Sp1 and to affect Sp1 promoter activity. Using an antibody array, we observed that CDK4, SKP2, Rad51, BRCA2 and p21 could interact with Sp1 and we confirmed these interactions by co-immunoprecipitation. CDK4, SKP2, Rad51, BRCA2 and p21 also activated the Sp1 promoter. Among the known Sp1-interacting proteins, E2F-DP1, Cyclin D1, Stat3 and Rb activated the Sp1 promoter, whereas p53 and NF?B inhibited it. The proteins that regulated Sp1 gene expression were shown by positive chromatin immunoprecipitation to be bound to the Sp1 promoter. Moreover, SKP2, BRCA2, p21, E2F-DP1, Stat3, Rb, p53 and NF?B had similar effects on an artificial promoter containing only Sp1 binding sites. Transient transfections of CDK4, Rad51, E2F-DP1, p21 and Stat3 increased mRNA expression from the endogenous Sp1 gene in HeLa cells whereas overexpression of NF?B, and p53 decreased Sp1 mRNA levels. p21 expression from a stably integrated inducible promoter in HT1080 cells activated Sp1 expression at the promoter and mRNA levels, but at the same time it decreased Sp1 protein levels due to the activation of Sp1 degradation. The observed multiple effects of cell cycle regulators on Sp1 suggest that Sp1 may be a key mediator of cell cycle associated changes in gene expression.

Tapias, Alicia; Ciudad, Carlos J.; Roninson, Igor B.; Noe, Veronique



Playing styles and possible causative factors in dogs' behaviour when playing with humans  

Microsoft Academic Search

Individual differences and causative factors could modify the behaviour of dogs in object related games played with a human partner. In a two-by-two within-subject design we observed 68 family dogs' behaviour when playing two different types of games (ball game and tugging) with two different play partners (owner or unfamiliar experimenter) in order to categorize each dog's playing style. In

Lilla Toth; M arta Gacsi; Adam Miklosi


Sox9/Sox6 and Sp1 are involved in the insulin-like growth factor-I-mediated upregulation of human type II collagen gene expression in articular chondrocytes.  


Type II collagen is a marker of articular cartilage encoded by the COL2A1 gene. The nature of the trans factors involved in the upregulation of this gene by insulin-like growth factor-I (IGF-I) remains unclear. We found that IGF-I increased type II collagen synthesis by a transcriptional control mechanism involving a 715-bp region within the COL2A1 first-intron specific enhancer. The overproduction of L-Sox5/Sox6/Sox9 and Sp1 and decoy experiments targeting these factors demonstrated their action in concert in IGF-I trans-activation. These results were supported by the data obtained in knockdown experiments in which siRNA against Sox9/Sox6 and Sp1 prevented the IGF-I-induced increase in collagen II production. Indeed, each of these trans-activators increased the expression of others. IGF-I increased the binding of Sox9 and Sp1/Sp3 to their cis elements in the enhancer, and we provide the first evidence of Sox9 interaction with the promoter by chromatin immunoprecipitation. Interactions with COL2A1 were also observed for Sp1, p300/CBP, and Tip60. Finally, a physical interaction between Sox9, p300, Sp3, and Sp1 was detected. These data demonstrate the role of Sox9, Sp1/Sp3, and euchromatin-associated factors (p300, Tip60) in the IGF-I-induced upregulation of COL2A1, indicating possible use of this growth factor in articular cartilage engineering applications to promote repair in patients with degenerative diseases, such as osteoarthritis. PMID:22215151

Renard, Emmanuelle; Porée, Benoît; Chadjichristos, Christos; Kypriotou, Magdalini; Maneix, Laure; Bigot, Nicolas; Legendre, Florence; Ollitrault, David; De Crombrugghe, Benoît; Malléin-Gérin, Frédéric; Moslemi, Safa; Demoor, Magali; Boumediene, Karim; Galéra, Philippe



Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.  


We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells. PMID:12133007

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi



Co-operation of the transcription factor hepatocyte nuclear factor-4 with Sp1 or Sp3 leads to transcriptional activation of the human haem oxygenase-1 gene promoter in a hepatoma cell line.  

PubMed Central

We reported previously that the 5'-flanking region (nucleotides -1976 to -1655) of the human haem oxygenase-1 ( hHO-1 ) gene enhances hHO-1 promoter activity in human hepatoma HepG2 cells, but not in HeLa cells [Takahashi, Takahashi, Ito, Nagano, Shibahara and Miura (1999) Biochim. Biophys. Acta 1447, 231-235]. To define more precisely the regulatory elements involved, in the present study we have functionally dissected this region and localized the enhancer to a 50 bp fragment (-1793 to -1744). Site-direct mutagenesis analysis revealed that two regions were responsible for this enhancer activity, i.e. a hepatocyte nuclear factor-4 (HNF-4) homologous region and a GC box motif homologous region. Mutation in either region alone moderately decreased enhancer activity. However, mutations in both regions reduced promoter activity to the basal level. Electrophoretic mobility-shift assays demonstrated that the P5-2 fragment (-1793 to -1744) interacted with at least two nuclear factors, i.e. HNF-4 and Sp1/Sp3. Co-transfection experiments using Drosophila SL2 cells revealed that HNF-4 and Sp1/Sp3 synergistically stimulated the enhancer activity of the P5-2 fragment. These results indicate that co-operation of HNF-4 with Sp1 or Sp3 leads to the activation of hHO-1 gene expression in hepatoma cells.

Takahashi, Shigeru; Matsuura, Naomi; Kurokawa, Takako; Takahashi, Yuji; Miura, Takashi



Zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1.  


The transcription factor Sp1 is a regulator of TATA-less genes. It belongs to the Cys?-His? zinc finger domain-containing family. A zebrafish cDNA encoding a peptide homologous to mammalian Sp1 was cloned and inserted into a pET43.1a vector and expressed in Escherichia coli Rosetta (DE3) cells as a Nus-His-tag fusion protein. After induction with isopropyl thiogalactoside, the protein was purified with a Ni-Sepharose column, and approximately 5-8 mg of pure protein was obtained per liter of culture. The primary sequence and the predicted partial tertiary structure of the potential recombinant zebrafish Sp1 protein are similar to those of human Sp1. The DNA affinity precipitation assay and dual-luciferase promoter activity assay further confirm the nature of the recombinant zebrafish Sp1 protein as a transcription factor. Our results show that zebrafish Sp1-like protein is structurally and functionally comparable to human Sp1. PMID:21040790

Lin, Cha-Jang; Hsiao, Tsun-Hsien; Chung, Yi-Shao; Chang, Wen-Ni; Yeh, Trai-Ming; Chen, Bing-Hung; Fu, Tzu-Fun



Demographic factors and playing variables in online computer gaming.  


Despite the growing popularity of online game playing, there has been no primary survey of its players. Therefore, an online questionnaire survey was used to examine basic demographic factors of online computer game players who played the popular online game Everquest (i.e., gender, age, marital status, nationality, education level, occupation). The survey also examined playing frequency (i.e., amount of time spent playing the game a week), playing history (i.e., how long they had been playing the game, who they played the game with, whether they had ever gender swapped their game character), the favorite and least favorite aspects of playing the game, and what they sacrifice (if anything) to play the game. Results showed that 81% of online game players were male, and that the mean age of players was 27.9 years of age. For many players, the social aspects of the game were the most important factor in playing. A small minority of players appear to play excessively (over 80 h a week), and results suggest that a small minority sacrifice important activities in order to play (e.g., sleep, time with family and/or partner, work, or schooling). PMID:15331036

Griffiths, Mark D; Davies, Mark N O; Chappell, Darren



Involvement of specific proteins (Sp1/Sp3) and nuclear factor Y in basal transcription of the distal promoter of the rat pyruvate carboxylase gene in {beta}-cells  

SciTech Connect

Pyruvate carboxylase plays diverse roles in different biosynthetic pathways, including glucose-induced insulin secretion in pancreatic {beta}-cells. We have localized the control region of the P2 promoter by generating a series of 5'-nested deletion constructs, and both 25- and 9-bp internal deletion constructs, as well as by performing site-directed mutagenesis. Transient transfections of these constructs into INS-1 cells identified a CCAAT box and a GC box that are located at -65/-61 and -48/-41, respectively, as the important determinants. Disruption of the GC box resulted in a 4-fold reduction of the reporter activity, while disruption of the proximal CCAAT box (-65/-61) but not the distal CCAAT box (-95/-91) increased the reporter activity by 3-fold. Simultaneous disruptions of both the GC box and the CCAAT box reduced the reporter activity to a level that was close to that of the single GC box mutation. Electrophoretic mobility shift assays (EMSAs) and supershift EMSAs using nuclear extract from INS-1 cells demonstrated that Sp1 and Sp3 bind a GC box while the nuclear factor Y was shown to bind the proximal but not the distal CCAAT box.

Sunyakumthorn, Piyanate [Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand); Boonsaen, Thirajit [Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand); Boonsaeng, Vichai [Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand); Wallace, John C. [School of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA 5005 (Australia); Jitrapakdee, Sarawut [Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400 (Thailand)]. E-mail:



Helicobacter pylori stimulates host vascular endothelial growth factor-A (vegf-A) gene expression via MEK\\/ERK-dependent activation of Sp1 and Sp3  

Microsoft Academic Search

VEGF-A is a key regulator of inflammatory and tumor-associated angiogenesis. H. pylori plays a critical role in the pathogenesis of benign and malignant gastric diseases. It has been suggested that H. pylori infection is associated with activation of host angiogenesis, however, underlying mechanisms as well as angiogenic growth factors activated by the bacterium have not yet been identified. Therefore, we

Mathias Z. Strowski; Thorsten Cramer; Georgia Schäfer; Stefan Jüttner; Anna Walduck; Ernestina Schipani; Wolfgang Kemmner; Silja Wessler; Christian Wunder; Matthias Weber; Thomas F. Meyer; Bertram Wiedenmann; Thomas Jöns; Michael Naumann; Michael Höcker



Interleukin-6 induces transcriptional activation of vascular endothelial growth factor (VEGF) in astrocytes in vivo and regulates VEGF promoter activity in glioblastoma cells via direct interaction between STAT3 and Sp1.  


Interleukin-6 (IL-6) expression is strongly correlated with the degree of human glioma malignancy and necessary for tumor formation in a mouse model of spontaneous astrocytomas. Yet, exactly how IL-6 contributes to malignant progression of these brain tumors is still unclear. We have scrutinized the mechanism of transcriptional activation of vascular endothelial growth factor (VEGF) expression by IL-6 in the mouse brain and in glioblastoma cells. We demonstrate here that IL-6 drives transcriptional upregulation of VEGF in astrocytes in vivo using glial fibrillary acidic protein (GFAP)-IL-6/VEGF-green fluorescent protein (GFP) double transgenic mice. We further show that IL-6-induced VEGF transcription and VEGF secretion by human glioblastoma cells is dependent on signal transducer and activator of transcription 3 (STAT3). By progressive 5'-deletion analysis we defined the minimal VEGF promoter region for IL-6-responsiveness to nucleotides -88/-50. Surprisingly, this promoter region is rich in GC-boxes and does not contain STAT3 binding elements. Electrophoretic mobility shift and supershift assays revealed binding of Sp1 and Sp3 to the -88/-50 element upon IL-6 stimulation. Interestingly, preincubation with STAT3 antibody prevented the binding of Sp1 and Sp3 to the -88/-50 element, indicating that STAT3 is involved in IL-6-driven Sp1/Sp3 protein-DNA complex formation. Physical interaction of STAT3 and Sp1 was demonstrated by coimmunoprecipitation. The functional relevance of the STAT3/Sp1 association was corroborated by transient transfection experiments, which showed that overexpression of constitutively active STAT3 increased the minimal VEGF promoter activity. Taken together, our study suggests that IL-6 promotes tumor angiogenesis in gliomas and describes a novel transcriptional activation mechanism for STAT3 in the context of a STAT3 binding element (SBE)-free promoter. PMID:15688401

Loeffler, Sébastien; Fayard, Bérengère; Weis, Joachim; Weissenberger, Jakob




NASA Astrophysics Data System (ADS)

Designing a game with a serious purpose involves considering the worlds of Reality and Meaning yet it is undeniably impossible to create a game without a third world, one that is specifically concerned with what makes a game a game: the play elements. This third world, the world of people like designers and artists, and disciplines as computer science and game design, I call the world of Play and this level is devoted to it. The level starts off with some of the misperceptions people have of play. Unlike some may think, we play all the time, even when we grow old—this was also very noticeable in designing the game Levee Patroller as the team exhibited very playful behavior at many occasions. From there, I go into the aspects that characterize this world. The first concerns the goal of the game. This relates to the objectives people have to achieve within the game. This is constituted by the second aspect: the gameplay. Taking actions and facing challenges is subsequently constituted by a gameworld, which concerns the third aspect. And all of it is not possible without the fourth and final aspect, the type of technology that creates and facilitates the game. The four aspects together make up a “game concept” and from this world such a concept can be judged on the basis of three closely interrelated criteria: engagement, immersion, and fun.

Harteveld, Casper


Nuclear factor ?B2 p52 protein has a role in antiviral immunity through I?B kinase epsilon-dependent induction of Sp1 protein and interleukin 15.  


In this study we describe a previously unreported function for NF?B2, an NF?B family transcription factor, in antiviral immunity. NF?B2 is induced in response to poly(I:C), a mimic of viral dsRNA. Poly(I:C), acting via TLR3, induces p52-dependent transactivation of a reporter gene in a manner that requires the kinase activity of I?B kinase ? (IKK?) and the transactivating potential of RelA/p65. We identify a novel NF?B2 binding site in the promoter of the transcription factor Sp1 that is required for Sp1 gene transcription activated by poly(I:C). We show that Sp1 is required for IL-15 induction by both poly(I:C) and respiratory syncytial virus, a response that also requires NF?B2 and IKK?. Our study identifies NF?B2 as a target for IKK? in antiviral immunity and describes, for the first time, a role for NF?B2 in the regulation of gene expression in response to viral infection. PMID:23873932

Doyle, Sarah L; Shirey, Kari Ann; McGettrick, Anne F; Kenny, Elaine F; Carpenter, Susan; Caffrey, Brian E; Gargan, Siobhan; Quinn, Susan R; Caamaño, Jorge H; Moynagh, Paul; Vogel, Stefanie N; O'Neill, Luke A



Sp1 trans-activates the murine H+-K+-ATPase ?2-subunit gene  

PubMed Central

The H+-K+-ATPase ?2 (HK?2) gene of the renal collecting duct and distal colon plays a central role in potassium and acid-base homeostasis, yet its transcriptional control remains poorly characterized. We previously demonstrated that the proximal 177 bp of its 5?-flanking region confers basal transcriptional activity in murine inner medullary collecting duct (mIMCD3) cells and that NF-?B and CREB-1 bind this region to alter transcription. In the present study, we sought to determine whether the ?144/?135 Sp element influences basal HK?2 gene transcription in these cells. Electrophoretic mobility shift and supershift assays using probes for ?154/?127 revealed Sp1-containing DNA-protein complexes in nuclear extracts of mIMCD3 cells. Chromatin immunoprecipitation (ChIP) assays demonstrated that Sp1, but not Sp3, binds to this promoter region of the HK?2 gene in mIMCD3 cells in vivo. HK?2 minimal promoter-luciferase constructs with point mutations in the ?144/?135 Sp element exhibited much lower activity than the wild-type promoter in transient transfection assays. Overexpression of Sp1, but not Sp3, trans-activated an HK?2 proximal promoter-luciferase construct in mIMCD3 cells as well as in SL2 insect cells, which lack Sp factors. Conversely, small interfering RNA knockdown of Sp1 inhibited endogenous HK?2 mRNA expression, and binding of Sp1 to chromatin associated with the proximal HK?2 promoter without altering the binding or regulatory influence of NF-?B p65 or CREB-1 on the proximal HK?2 promoter. We conclude that Sp1 plays an important and positive role in controlling basal HK?2 gene expression in mIMCD3 cells in vivo and in vitro.

Yu, Zhiyuan; Li, Mei; Zhang, Dongyu; Xu, William; Kone, Bruce C.



Expression of the human endogenous retrovirus (HERV) group HML-2/HERV-K does not depend on canonical promoter elements but is regulated by transcription factors Sp1 and Sp3.  


After fixation in the human genome, human endogenous retroviruses (HERVs) are bona fide cellular genes despite their exogenous origin. To be able to spread within the germ line and the early embryo, the ancient retroviral promoters must have adapted to the requirements for expression in these cell types. We describe that in contrast to the case for current exogenous retroviruses, which replicate in specific somatic cells, the long terminal repeat (LTR) of the human endogenous retrovirus HERV-K acts as a TATA- and initiator element-independent promoter with a variable transcription start site. We present evidence that the HERV-K LTR is regulated by the transcription factors Sp1 and Sp3. Mutating specific GC boxes, which are binding sites for Sp proteins, and knocking down Sp1 and Sp3 by use of small interfering RNA (siRNA) significantly reduced the promoter activity. Binding of Sp1 and Sp3 to the promoter region was confirmed using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP). Our data explain why certain HERV-K proviruses have lost promoter competence. Since vertebrate promoters lacking canonical core promoter elements are common but poorly studied, understanding the HERV-K promoter not only will provide insight into the regulation of endogenous retroviruses but also can serve as a paradigm for understanding the regulation of this class of cellular genes. PMID:21248046

Fuchs, Nina V; Kraft, Martin; Tondera, Christiane; Hanschmann, Kay-Martin; Löwer, Johannes; Löwer, Roswitha



Low SP1 Expression Differentially Affects Intestinal-Type Compared with Diffuse-Type Gastric Adenocarcinoma  

PubMed Central

Specificity protein 1 (SP1) is an essential transcription factor that regulates multiple cancer-related genes. Because aberrant expression of SP1 is related to cancer development and progression, we focused on SP1 expression in gastric carcinoma and its correlation with disease outcomes. Although patient survival decreased as SP1 expression increased (P<0.05) in diffuse-type gastric cancer, the lack of SP1 expression in intestinal-type gastric cancer was significantly correlated with poor survival (P<0.05). The knockdown of SP1 in a high SP1-expressing intestinal-type gastric cell line, MKN28, increased migration and invasion but decreased proliferation. Microarray data in SP1 siRNA-transfected MKN28 revealed that the genes inhibiting migration were downregulated, whereas the genes negatively facilitating proliferation were increased. However, both migration and invasion were decreased by forced SP1 expression in a low SP1-expressing intestinal-type gastric cell line, AGS. Unlike the intestinal-type, in a high SP1-expressing diffuse-type gastric cell line, SNU484, migration and invasion were decreased by SP1 siRNA. In contrast to previous studies that did not identify differences between the 2 histological types, our results reveal that low expression of SP1 is involved in cancer progression and metastasis and differentially affects intestinal-type compared with diffuse-type gastric adenocarcinoma.

Oh, Ensel; Erkin, Ozgur Cem; Jung, Hun Soon; Cho, Mi-Hyun; Kwon, Mi Jeong; Chae, Seoung Wan; Kim, Seok-Hyung; Wang, Li-Hui; Park, Min-Jeong; Lee, Su-Yeon; Yang, Ho Bin; Jia, Lina; Choi, Yoon-La; Shin, Young Kee



Transcription of the catalytic 180-kDa subunit gene of mouse DNA polymerase alpha is controlled by E2F, an Ets-related transcription factor, and Sp1.  


We have isolated a genomic DNA fragment spanning the 5'-end of the gene encoding the catalytic subunit of mouse DNA polymerase alpha. The nucleotide sequence of the upstream region was G/C-rich and lacked a TATA box. Transient expression assays in cycling NIH 3T3 cells demonstrated that the GC box of 20 bp (at nucleotides -112/-93 with respect to the transcription initiation site) and the palindromic sequence of 14 bp (at nucleotides -71/-58) were essential for basal promoter activity. Electrophoretic mobility shift assays showed that Sp1 binds to the GC box. We also purified a protein capable of binding to the palindrome and identified it as GA-binding protein (GABP), an Ets- and Notch-related transcription factor. Transient expression assays in synchronized NIH 3T3 cells revealed that three variant E2F sites near the transcription initiation site (at nucleotides -23/-16, -1/+7 and +17/+29) had no basal promoter activity by themselves, but were essential for growth-dependent stimulation of the gene expression. These data indicate that E2F, GABP and Sp1 regulate the gene expression of this principal replication enzyme. PMID:11004506

Izumi, M; Yokoi, M; Nishikawa, N S; Miyazawa, H; Sugino, A; Yamagishi, M; Yamaguchi, M; Matsukage, A; Yatagai, F; Hanaoka, F



The Play Factor: Effect of Social Skills Group Play Therapy on Adolescent African-American Males  

ERIC Educational Resources Information Center

The purpose of this study was to examine the effectiveness of Social Skills Group Play Therapy on remedying the social skills deficits of adolescent African-American males. Additionally, the study investigated whether age and grade level impacted the outcome of the intervention. The participants were adolescent African-American males ages 10 to…

Earls, Melissa K.



Transcriptional regulation by post-transcriptional modification--role of phosphorylation in Sp1 transcriptional activity.  


Sp1 is a ubiquitously expressed transcription factor involved in the regulation of a large number of genes including housekeeping genes as well as actively regulated genes. Although Sp1 was discovered nearly three decades ago, its functional diversity is still not completely understood. One of the ways that make Sp1 versatile in transcriptional regulation is its post-transcriptional modification, which alters Sp1 structure in different cells and at different times. Compared to other types of modifications of the Sp1 protein, phosphorylation has been studied far more extensively. This review focuses on the inducers, pathways, enzymes, and biological effects of Sp1 phosphorylation. Recent data are beginning to reveal the biological significance and universal presence of Sp1 phosphorylation-related cell/molecular responses. Studies in this field provide a quick glance at how a simple chemical modification of a transcription factor could produce significant functional diversity of the protein. PMID:22835698

Chu, Shijian



Hydrogen Peroxide Decreases Endothelial Nitric Oxide Synthase Promoter Activity through the Inhibition of Sp1 Activity  

PubMed Central

We have previously shown that endothelial nitric oxide synthase (eNOS) promoter activity is decreased in endothelial cells in response to the addition of hydrogen peroxide (H2O2), and this involves, at least in part, the inhibition of AP-1 activity. Thus, the objective of this study was to determine if other cis-element(s) and transcription factor(s) are involved in the oxidant-mediated downregulation of eNOS. Our initial experiments indicated that although H2O2 treatment increased eNOS mRNA levels in ovine pulmonary arterial endothelial cells (OPAECs), there was a significant decrease in the promoter activity of an eNOS promoter construct containing 840?bp of upstream sequence. However, a truncated promoter construct that lacked the AP-1 element (650?bp) was also inhibited by H2O2. A similar effect was observed when the 650?bp human eNOS promoter construct was transfected into human PAECs. We also found that although exposure of the cells to PEG-catalase prevented the inhibitory effect on eNOS promoter activity, the hydroxyl radical scavenger, deferoxamine myslate, did not. Nor could we identify an increase in hydroxyl radical levels in cells exposed to H2O2. Exposure of PAECs caused a significant increase in labile zinc levels in response to H2O2. As the eNOS promoter has a cis-element for Sp1 binding, we evaluated the role of Sp1 in response to H2O2. As previously reported, mutation of the Sp1 consensus lead to the complete loss of eNOS promoter activity, confirming the key role of Sp1 in regulating basal eNOS promoter activity. In addition, we found, using electrophoretic mobility and supershift assays, that H2O2 decreased Sp1 binding. Finally, using chromatin immunoprecipitation analysis, we found a significant decrease in Sp1 binding to the eNOS promoter in vivo in response to treatment with H2O2. Together, these data suggest that the inhibition of Sp1 activity, possibly through loss of zinc in the protein, plays a role in the H2O2-induced inhibition of eNOS promoter activity.

Kumar, Sanjiv; Sun, Xutong; Wiseman, Dean A.; Tian, Jing; Umapathy, Nagavedi S.; Verin, Alexander D.



Video Game Playing and Gambling in Adolescents: Common Risk Factors  

Microsoft Academic Search

Video games and gambling often contain very similar elements with both providing intermittent rewards and elements of randomness. Furthermore, at a psychological and behavioral level, slot machine gambling, video lottery terminal (VLT) gambling and video game playing share many of the same features. Despite the similarities between video game playing and gambling there have been very few studies that have

Richard T. A. Wood; Rina Gupta; Jeffrey L. Derevensky; Mark Griffiths



Lipopolysaccharide induces hypoxia-inducible factor-1 alpha mRNA expression and activation via NADPH oxidase and Sp1-dependent pathway in BV2 murine microglial cells  

Microsoft Academic Search

Hypoxia-inducible factor-1 (HIF-1), the key transcription factor of hypoxia-inducible genes, is known to be involved in inflammation and immune response, but little is known about the regulation of HIF-1 during microglial activation. Thus, we examined effect of lipopolysaccharide (LPS) on HIF-1 activation and its signaling mechanism in BV2 microglial cells. LPS induced HIF-1? mRNA and protein expression as well as

Young Taek Oh; Jung Yeon Lee; Hana Yoon; Eunjoo H. Lee; Hyung Hwan Baik; Sung Soo Kim; Joohun Ha; Kyung-Sik Yoon; Wonchae Choe; Insug Kang



Functional role of post-translational modifications of Sp1 in tumorigenesis  

PubMed Central

Specific protein 1 (Sp1), the first transcription factor to be isolated, regulates the expression of numerous genes involved in cell proliferation, apoptosis, and differentiation. Recent studies found that an increase in Sp1 transcriptional activity is associated with the tumorigenesis. Moreover, post-translational modifications of Sp1, including glycosylation, phosphorylation, acetylation, sumoylation, ubiquitination, and methylation, regulate Sp1 transcriptional activity and modulate target gene expression by affecting its DNA binding activity, transactivation activity, or protein level. In addition, recent studies have investigated several compounds with anti-cancer activity that could inhibit Sp1 transcriptional activity. In this review, we describe the effect of various post-translational modifications on Sp1 transcriptional activity and discuss compounds that inhibit the activity of Sp1.



Regulation of fibrillin-1 gene expression by Sp1.  


Mutations in the fibrillin-1 gene (FBN1) cause Marfan Syndrome (MFS), a hereditary disorder of connective tissue. The transcription of FBN1 has been reported to be driven by a short ultraconserved region (SUPR) in the 5' untranslated exon A of FBN1, but the nature of other factors involved in FBN1 gene regulation has not been clarified. In this study, we characterized the transcription factors involved in FBN1 gene regulation. The results show that Sp1 protein binds to two putative binding sites in the promoter of FBN1. Overexpression of Sp1 resulted in a significant increase in both promoter activity and FBN1 mRNA level in HEK 293 cells, whereas inhibition or knockdown of Sp1 decreased FBN1 gene expression. In addition, we found that Poly [ADP-ribose] polymerase 1 (PARP1) binds to the palindromic sequence TCTCGCGAGA in the ultraconserved region of the FBN1 promoter and that the regulation of FBN1 expression by PARP1 is dependent on Sp1. These results indicate that both Sp1 and PARP1 contribute to FBN1 gene expression. These observations add to our understanding of the transcriptional regulation of FBN1 gene expression. PMID:23860323

Guo, Gao; Rödelsperger, Christian; Digweed, Martin; Robinson, Peter N



Video Game Playing and Gambling in Adolescents: Common Risk Factors  

ERIC Educational Resources Information Center

Video games and gambling often contain very similar elements with both providing intermittent rewards and elements of randomness. Furthermore, at a psychological and behavioral level, slot machine gambling, video lottery terminal (VLT) gambling and video game playing share many of the same features. Despite the similarities between video game…

Wood, Richard T. A.; Gupta, Rina; Griffiths, Mark



Perceived migraine triggers: do dietary factors play a role?  


The present cross-sectional study was designed to assess the frequency of 36 possible triggering factors precipitating a migraine crisis (hormonal, environmental, and dietary) in adult outpatients suffering from migraine attacks. A group of 123 migraine sufferers, aged 43.2 ± 13.9 (mean ± SD) years, including 114 (92.7%) women, 68.3% having migraine without aura, 68.3% reporting pain severe enough to require drug prophylaxis, and 29.3% presenting with hypertension, were evaluated. The most common triggers were stress and fasting, and environmental and hormonal factors were frequently found to precipitate a crisis. More than 90% of the patients reported susceptibility to 5 or more factors, and only 2.4% did not complain about any dietary factor. The large number of triggers detected in the present study emphasises the importance of awareness and avoidance of these factors in the management of patients with migraine. PMID:22732972

Camboim Rockett, F; Castro, K; Rossoni de Oliveira, V; da Silveira Perla, A; Fagundes Chaves, M L; Schweigert Perry, I D



Sp1 modifies leg-to-wing transdetermination in Drosophila  

PubMed Central

During Drosophila development, the transcription factor Sp1 is necessary for proper leg growth and also to repress wing development. Here we test the role of Sp1 during imaginal disc regeneration. Ubiquitous expression of wg induces a regeneration blastema in the dorsal aspect of the leg disc. Within this outgrowth, the wing selector gene vg is activated in some cells, changing their fate to wing identity in a process known as transdetermination. In this report we demonstrate that reducing the gene copy number of Sp1 significantly increases both the frequency and the area of transdetermination in regenerating leg discs. By examining the expression of known Sp1 target genes, we also show that the proximo-distal patterning gene dachshund is downregulated dorsally, leading to a break in its normal ring-shaped expression pattern. We further report that transdetermination, as evidenced by Vg expression, is only observed when there is a broken ring of Dachshund expression. Combined, these studies establish a role for Sp1 in leg-to-wing transdetermination.

Ing, Thomas; Tseng, Alexander; Sustar, Anne; Schubiger, Gerold



Transcriptional regulation of the human manganese superoxide dismutase gene: the role of specificity protein 1 (Sp1) and activating protein-2 (AP-2).  

PubMed Central

Manganese superoxide dismutase (MnSOD) plays an important role in regulating cellular redox conditions. Expression of MnSOD has been shown to protect against damage by oxidative stress and to suppress the malignant phenotype of human cancer cells. We have previously cloned the human MnSOD (SOD2) gene and analysed its 5' proximal promoter, which has been characterized by a lack of a TATA or CAAT box and the presence of multiple GC boxes. To define further the molecular mechanisms for the regulation of MnSOD expression, multiple transcription factor-binding motifs containing overlapping specificity protein 1 (Sp1)- and activator protein (AP)-2-binding sites were identified by DNase I footprinting analysis. Functional studies in three cell lines with different levels of Sp1 and AP-2 proteins suggested that the cellular levels of these proteins may differentially regulate transcription via GC-binding motifs in the human SOD2 promoter. Co-transfection of an Sp1 expression vector resulted in an increase in the transcription of the promoter-driven reporter gene. In contrast, co-transfection of the AP-2 expression vector caused a decrease in transcription. Direct mutagenesis analysis of Sp1- and AP-2-binding sites showed that Sp1 is essential for transcription of the human SOD2 gene, whereas AP-2 plays a negative role in the transcription. Immunoprecipitation of Sp1 and AP-2 proteins demonstrated that Sp1 interacts with AP-2 in vivo. Two-hybrid analysis revealed that interaction between Sp1 and AP-2 plays both a positive and negative role in the transcription of the reporter gene in vivo. Taken together, our data indicate that AP-2 down-regulates transcription of the human SOD2 gene via its interaction with Sp1 within the promoter region. These findings, coupled with our previous observation that several cancer cell lines have mutations in the promoter region of the human MnSOD gene, which lead to an increase in an AP-2-binding site and a decrease in the promoter activity, signal the importance of understanding the promoter structure and the regulation of the human SOD2 gene by Sp1 and AP-2.

Xu, Yong; Porntadavity, Sureerut; St Clair, Daret K



Characterization of the human topoisomerase IIbeta (TOP2B) promoter activity: essential roles of the nuclear factor-Y (NF-Y)- and specificity protein-1 (Sp1)-binding sites.  

PubMed Central

Eukaryotic topoisomerase II (topo II) catalyses topological genomic changes essential for chromosome segregation, chromatin reorganization, DNA replication and transcription. Mammalian topo II exists as two isoforms, designated alpha and beta. Human topo IIalpha is an important cancer drug target, and an established determinant of drug sensitivity and resistance. Human topo IIbeta is also the target of anticancer drugs but its role in drug resistance is less clear. The two human topo II proteins are encoded by the TOP2A and TOP2B genes, respectively, which despite their highly conserved structural organization, are subject to distinctly different modes of regulation. In the present study, we have cloned and characterized the human TOP2B promoter containing a 1.3 kb fragment of the 5'-flanking and untranslated region (-1067 to +193). We found that the promoter activity of this TOP2B fragment was constant throughout the cell cycle, in contrast to the activity of the proximal promoter of TOP2A which was low in resting cells and enhanced during proliferation. Analyses of 5'-serially and internally deleted luciferase reporter constructs revealed that 80% of the TOP2B promoter activity could be attributed to the region between -533 and -481. Mutational analyses of putative regulatory elements indicated that two inverted CCAAT boxes (ICBs) within this region were essential for TOP2B promoter activity and gel mobility-shift assays indicated these sites bound the transcription factor nuclear factor-Y (NF-Y). Co-transfection experiments using a dominant-negative form of subunit A of NF-Y suggested that TOP2B promoter activity required direct interaction of NF-Y with the ICBs. In addition, a specificity protein-1 (Sp1)-binding GC box located just upstream of the ICBs was shown to contribute to TOP2B promoter activity in a synergistic manner with the ICBs. Our results suggest that the binding sites for NF-Y and Sp1 are critical for TOP2B transcription.

Lok, Chun-Nam; Lang, Alexander J; Mirski, Shelagh E L; Cole, Susan P C



Sumoylation differentially regulates Sp1 to control cell differentiation.  


The mammalian small ubiquitin-like modifiers (SUMOs) are actively involved in regulating differentiation of different cell types. However, the functional differences between SUMO isoforms and their mechanisms of action remain largely unknown. Using the ocular lens as a model system, we demonstrate that different SUMOs display distinct functions in regulating differentiation of epithelial cells into fiber cells. During lens differentiation, SUMO1 and SUMO2/3 displayed different expression, localization, and targets, suggesting differential functions. Indeed, overexpression of SUMO2/3, but not SUMO1, inhibited basic (b) FGF-induced cell differentiation. In contrast, knockdown of SUMO1, but not SUMO2/3, also inhibited bFGF action. Mechanistically, specificity protein 1 (Sp1), a major transcription factor that controls expression of lens-specific genes such as ?-crystallins, was positively regulated by SUMO1 but negatively regulated by SUMO2. SUMO2 was found to inhibit Sp1 functions through several mechanisms: sumoylating it at K683 to attenuate DNA binding, and at K16 to increase its turnover. SUMO2 also interfered with the interaction between Sp1 and the coactivator, p300, and recruited a repressor, Sp3 to ?-crystallin gene promoters, to negatively regulate their expression. Thus, stable SUMO1, but diminishing SUMO2/3, during lens development is necessary for normal lens differentiation. In support of this conclusion, SUMO1 and Sp1 formed complexes during early and later stages of lens development. In contrast, an interaction between SUMO2/3 and Sp1 was detected only during the initial lens vesicle stage. Together, our results establish distinct roles of different SUMO isoforms and demonstrate for the first time, to our knowledge, that Sp1 acts as a major transcription factor target for SUMO control of cell differentiation. PMID:24706897

Gong, Lili; Ji, Wei-Ke; Hu, Xiao-Hui; Hu, Wen-Feng; Tang, Xiang-Cheng; Huang, Zhao-Xia; Li, Ling; Liu, Mugen; Xiang, Shi-Hua; Wu, Erxi; Woodward, Zachary; Liu, Yi-Zhi; Nguyen, Quan Dong; Li, David Wan-Cheng



Histone Deacetylase 1 Can Repress Transcription by Binding to Sp1  

Microsoft Academic Search

The members of the Sp1 transcription factor family can act as both negative and positive regulators of gene expression. Here we show that Sp1 can be a target for histone deacetylase 1 (HDAC1)-mediated transcriptional repression. The histone deacetylase inhibitor trichostatin A activates the chromosomally integrated murine thymidine kinase promoter in an Sp1-dependent manner. Coimmunoprecipitation experiments with Swiss 3T3 fibroblasts and




Curcumin Suppresses Metastasis via Sp-1, FAK Inhibition, and E-Cadherin Upregulation in Colorectal Cancer  

PubMed Central

Colorectal cancer (CRC) is a serious public health problem that results due to changes of diet and various environmental stress factors in the world. Curcumin is a traditional medicine used for treatment of a wide variety of tumors. However, antimetastasis mechanism of curcumin on CRC has not yet been completely investigated. Here, we explored the underlying molecular mechanisms of curcumin on metastasis of CRC cells in vitro and in vivo. Curcumin significantly inhibits cell migration, invasion, and colony formation in vitro and reduces tumor growth and liver metastasis in vivo. We found that curcumin suppresses Sp-1 transcriptional activity and Sp-1 regulated genes including ADEM10, calmodulin, EPHB2, HDAC4, and SEPP1 in CRC cells. Curcumin inhibits focal adhesion kinase (FAK) phosphorylation and enhances the expressions of several extracellular matrix components which play a critical role in invasion and metastasis. Curcumin reduces CD24 expression in a dose-dependent manner in CRC cells. Moreover, E-cadherin expression is upregulated by curcumin and serves as an inhibitor of EMT. These results suggest that curcumin executes its antimetastasis function through downregulation of Sp-1, FAK, and CD24 and by promoting E-cadherin expression in CRC cells.

Chen, Chun-Chieh; Sureshbabul, Munisamy; Chen, Huei-Wen; Lin, Yu-Shuang; Lee, Jen-Yi; Hong, Qi-Sheng; Yang, Ya-Chien



Inhibition of Sp1 Functions by Its Sequestration into PML Nuclear Bodies  

PubMed Central

Promyelocytic leukemia nuclear bodies (PML NBs) are comprised of PML and a striking variety of its associated proteins. Various cellular functions have been attributed to PML NBs, including the regulation of gene expression. We report here that induced expression of PML recruits Sp1 into PML NBs, leading to the reduction of Sp1 transactivation function. Specifically, Chromatin immunoprecipitation (ChIP) assay demonstrated that induced expression of PML significantly diminishes the amount of Sp1 binding to its target gene promoter, immunofluorescence staining showed dramatic increase in the co-localization between PML and Sp1 upon induction of PML expression, moreover, PML and Sp1 co-fractionated in the core nuclear matrix. Our study further showed that PML promotes SUMOylation of Sp1 in a RING-motif-dependent manner, SUMOylation of Sp1 facilitates physical interaction between Sp1 and PML and recruitment of Sp1 into the PML NBs, the SUMO binding motif of PML was also important for its interaction with Sp1. The results of this study demonstrate a novel mechanism by which PML regulates gene expression through sequestration of the transcription factor into PML NBs.

Li, June; Zou, Wen-Xin; Chang, Kun-Sang



Early experiences with the IBM SP-1  

SciTech Connect

The IBM SP-1 is IBM`s newest parallel distributed-memory computer. As part of a joint project with IBM, Argonne took delivery of an early system in order to evaluate the software environment and to begin porting programming packages and applications to this machine. This report discusses the results of those early efforts. Despite the newness of the machine and the lack of a fast interprocessor switch (part of the SP-1 but not yet available for the machine), every code that they attempted to port ran on the SP-1 with little or no modification. The report concludes with a discussion of expectations for the fast interconnect.

Gropp, W. [ed.



Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells  

PubMed Central

Retinoic acid (RA)-producing dendritic cells (DCs) play critical roles in gut immunity. Retinal dehydrogenase 2 (RALDH2) encoded by Aldh1a2 is a key enzyme for generating RA in DCs. Granulocyte–macrophage colony-stimulating factor (GM-CSF) potently induces RALDH2 expression in DCs in an RA-dependent manner, and RA alone weakly induces the expression. However, how GM-CSF and RA induce RALDH2 expression remains unclear. Here, we show that GM-CSF-induced activation of the transcription factor Sp1 and RA-dependent signaling via the RA receptor (RAR)/retinoid X receptor (RXR) complex contribute to Aldh1a2 expression. The RAR antagonist LE540 and the Sp1 inhibitor mithramycin A inhibited GM-CSF-induced Aldh1a2 expression in fms-related tyrosine kinase 3 ligand-generated bone marrow-derived DCs (BM-DCs). ERK and p38 MAPK inhibitors suppressed GM-CSF-induced nuclear translocation of Sp1 and Aldh1a2 expression. Sp1 and the RAR?/RXR? complex bound to GC-rich Sp1-binding sites and an RA response element (RARE) half-site, respectively, near the TATA box in the mouse Aldh1a2 promoter. The DNA sequences around these sites were highly conserved among different species. In the presence of RA, ectopic expression of RAR?/RXR? and Sp1 synergistically enhanced Aldh1a2 promoter-reporter activity. GM-CSF did not significantly induce Aldh1a2 expression in plasmacytoid DCs, peritoneal macrophages, or T cells, and the Aldh1a2 promoter in these cells was mostly unmethylated. These results suggest that GM-CSF/RA-induced RALDH2 expression in DCs requires cooperative binding of Sp1 and the RAR/RXR complex to the Aldh1a2 promoter, and can be regulated by a DNA methylation-independent mechanism.

Ohoka, Yoshiharu; Yokota-Nakatsuma, Aya; Maeda, Naoko; Takeuchi, Hajime; Iwata, Makoto



ZEB2-Sp1 cooperation induces invasion by upregulating cadherin-11 and integrin ?5 expression.  


Epithelial-mesenchymal transition (EMT) is a process implicated in invasion and metastasis. EMT is characterized by repression of epithelial markers and induction of mesenchymal markers. ZEB2 is a transcriptional repressor of E-cadherin, leading to EMT. Previously, we have shown that ZEB2 directly upregulates integrin ?5 transcription by cooperating with the transcription factor Sp1. In this study, we investigated the precise mechanism by which ZEB2 modulates invasion and EMT events and the role of Sp1 in ZEB2-induced invasion. We found that ZEB2 directly induced cadherin-11 transcription in an Sp1-dependent, but Smad- and E-box-independent, manner and repressed E-cadherin expression in an Sp1- and Smad-independent manner, leading to cadherin switch. Furthermore, ZEB2 upregulated Sp1 by enhancing Sp1 protein stability, and Sp1 was found to be critical for ZEB2-induced cancer cell invasion, mainly through induction of cadherin-11 and integrin ?5. Expression levels of cadherin-11 and integrin ?5 were interdependent and both modulated c-Jun N-terminal kinase-signaling activity and invasion. Immunofluorescence analysis showed that nuclear expression of ZEB2 was positively correlated with Sp1 expression in human colorectal cancers. Together, these findings demonstrate a previously unrecognized interplay between ZEB2, Sp1, cadherin-11 and integrin ?5 that is, probably, significant in tumor progression and metastasis. PMID:24130169

Nam, Eun-Hee; Lee, Yunhee; Zhao, Xue-Feng; Park, Young-Kyu; Lee, Jung Weon; Kim, Semi



Sp1 and Sp3 control constitutive expression of the human NHE2 promoter by interactions with the proximal promoter and the transcription initiation site  

PubMed Central

We have previously cloned the human Na+/H+ exchanger NHE2 gene and its promoter region. In the present study, the regulatory elements responsible for the constitutive expression of NHE2 were studied. Transient transfection assays revealed that the ?40/+150 promoter region contains the core promoter responsible for the optimal promoter activity. A smaller fragment, ?10/+40, containing the TIS (transcription initiation site) showed minimal activity. We identified a palindrome that overlaps the TIS and binds to the transcription factors Sp1 and Sp3. Mutations in the 5? flank of the palindrome abolished the Sp1/Sp3 interaction and reduced promoter activity by approx. 45%. In addition, a conserved GC-box centered at ?25 was found to play a critical role in basal promoter activity and also interacted with Sp1 and Sp3. An internal deletion in the GC-box severely reduced the promoter activity. Sp1/Sp3 binding to these elements was established using gel-mobility shift assays, confirmed by chromatin immunoprecipitation and co-transfections in Drosophila SL2 cells. Furthermore, we identified two positive regulatory elements in the DNA region corresponding to the 5?-UTR (5?-untranslated region). The results in the present study indicate that Sp1 and Sp3 are required for constitutive NHE2 expression and that the positive regulatory elements of the 5?-UTR may co-operate with the 5?-flanking region to achieve the optimal promoter activity.

Pearse, Ian; Zhu, Ying X.; Murray, Eleanor J.; Dudeja, Pradeep K.; Ramaswamy, Krishnamurthy; Malakooti, Jaleh



Sp1 facilitates DNA double-strand break repair through a nontranscriptional mechanism.  


Sp1 is a ubiquitously expressed transcription factor that is phosphorylated by ataxia telangiectasia mutated kinase (ATM) in response to ionizing radiation and H(2)O(2). Here, we show by indirect immunofluorescence that Sp1 phosphorylated on serine 101 (pSp1) localizes to ionizing radiation-induced foci with phosphorylated histone variant ?H2Ax and members of the MRN (Mre11, Rad50, and Nbs1) complex. More precise analysis of occupancy of DNA double-strand breaks (DSBs) by chromatin immunoprecipitation (ChIP) shows that Sp1, like Nbs1, resides within 200 bp of DSBs. Using laser microirradiation of cells, we demonstrate that pSp1 is present at DNA DSBs by 7.5 min after induction of damage and remains at the break site for at least 8 h. Depletion of Sp1 inhibits repair of site-specific DNA breaks, and the N-terminal 182-amino-acid peptide, which contains targets of ATM kinase but lacks the zinc finger DNA binding domain, is phosphorylated, localizes to DSBs, and rescues the repair defect resulting from Sp1 depletion. Together, these data demonstrate that Sp1 is rapidly recruited to the region immediately adjacent to sites of DNA DSBs and is required for DSB repair, through a mechanism independent of its sequence-directed transcriptional effects. PMID:22826432

Beishline, Kate; Kelly, Crystal M; Olofsson, Beatrix A; Koduri, Sravanthi; Emrich, Jacqueline; Greenberg, Roger A; Azizkhan-Clifford, Jane



Comparative integromics on FZD7 orthologs: conserved binding sites for PU.1, SP1, CCAAT-box and TCF/LEF/SOX transcription factors within 5'-promoter region of mammalian FZD7 orthologs.  


Canonical WNT signals are transduced through Frizzled (FZD) family receptor and LRP5/LRP6 co-receptor to upregulate MYC, CCND1, FGF20, JAG1, WISP1 and DKK1 genes, while non-canonical WNT signals are transduced through FZD family receptor and PTK7/ROR2/RYK co-receptor to activate RHOA/RHOU/RAC/CDC42, JNK, PKC, NFAT and NLK signaling cascades. FZD7, expressed in the normal gastrointestinal tract, is upregulated in esophageal cancer, gastric cancer, colorectal cancer, and hepatocellular carcinoma. Here, chimpanzee FZD7 and cow Fzd7 genes were identified and characterized by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee FZD7 and cow Fzd7 genes were identified within NW_001232110.1 and AC173037.2 genome sequences, respectively. Chimpanzee FZD7 and cow Fzd7 showed 100% and 97.2% total-amino-acid identity with human FZD7. All of the nine amino-acid residues substituted between human FZD7 and human FzE3 were identical to those of human FZD7 in chimpanzee, cow, mouse and rat FZD7 orthologs. Functional analyses using FzE3 with multiple cloning artifacts and/or sequencing errors are invalid. FZD7 orthologs were seven-transmembrane proteins with extracellular Frizzled domain, leucine zipper motif around the 5th transmembrane domain, and cytoplasmic DVL- and PDZ-binding motifs. Ser550 and Ser556 of FZD7 orthologs were putative aPKC phosphorylation sites. Dimerization and Ser550/556 phosphorylation were predicted as regulatory mechanisms for the signaling through FZD7. Transcriptional start site of human FZD7 gene was 735-bp upstream of NM_003507.1 RefSeq 5'-end. In addition to gastrointestinal cancer, hepatocellular cancer and pancreatic cancer, human FZD7 mRNAs were expressed in blastocysts, undifferentiated embryonic stem (ES) cells, ES-derived endodermal progenitors, ES-derived neural progenitors, fetal cochlea, retinal pigment epithelium, olfactory epithelium, regenerating liver, and multiple sclerosis. Comparative genomics analyses revealed that the binding sites for PU.1, SP1/Krüppel-like, CCAAT-box, and TCF/LEF/SOX transcription factors were conserved among 5'-promoter regions of mammalian FZD7 orthologs. PMID:17273804

Katoh, Masuko; Katoh, Masaru



Is video-game playing a risk factor for pathological gambling in Australian adolescents?  


Very little research has been conducted to examine the relationship between video-game playing and gambling in adolescence. In this study, 2,669 adolescents aged 13-17 years were surveyed to obtained details of their involvement in gambling and video-game playing as well as a measure of pathological gambling (the DSM-IV-J). The results showed that, the frequency of video game playing was significantly related to pathological gambling, but that the effect size was very small and largely accounted for by the greater popularity of both activities amongst boys. There was some evidence for stronger associations between technologically similar activities, namely arcade video games and an interest in gaming machines, but other factors discussed in the paper may also account for this association. In summary, the findings suggested that playing video-games is unlikely to be a significant risk factor for pathological gambling during adolescence. PMID:19578983

Delfabbro, Paul; King, Daniel; Lambos, Chrisi; Puglies, Stan



Functional Analyses of Natural Variation in Sp1 Binding Sites of a TATA-Less Promoter  

Microsoft Academic Search

.   Within the lactate dehydrogenase-B (LdhB) proximal promoter is a region with multiple in vivo footprinted sites that resembles the binding site for the transcription\\u000a factor SP1. Like many sequences that regulate transcription rate, these Sp1 binding sites are well conserved among species\\u000a of the teleost fish Fundulus. The only exception is in the northern population of F. heteroclitus, where

Jeff A. Segal; J. Lynn Barnett; Douglas L. Crawford



MYD88-independent growth and survival effects of Sp1 transactivation in Waldenstrom macroglobulinemia.  


Sp1 transcription factor controls a pleiotropic group of genes and its aberrant activation has been reported in a number of malignancies, including multiple myeloma. In this study, we investigate and report its aberrant activation in Waldenström macroglobulinemia (WM). Both loss of and gain of Sp1 function studies have highlighted a potential oncogenic role of Sp1 in WM. We have further investigated the effect of a small molecule inhibitor, terameprocol (TMP), targeting Sp1 activity in WM. Treatment with TMP inhibited the growth and survival and impaired nuclear factor-?B and signal transducer and activator of transcription activity in WM cells. We next investigated and observed that TMP treatment induced further inhibition of WM cells in MYD88 knockdown WM cells. Moreover, we observed that Bruton's tyrosine kinase, a downstream target of MYD88 signaling pathway, is transcriptionally regulated by Sp1 in WM cells. The combined use of TMP with Bruton's tyrosine kinase or interleukin-1 receptor-associated kinase 1 and 4 inhibitors resulted in a significant and synergistic dose-dependent antiproliferative effect in MYD88-L265P-expressing WM cells. In summary, these results demonstrate Sp1 as an important transcription factor that regulates proliferation and survival of WM cells independent of MYD88 pathway activation, and provide preclinical rationale for clinical development of TMP in WM alone or in combination with inhibitors of MYD88 pathway. PMID:24622324

Fulciniti, Mariateresa; Amodio, Nicola; Bandi, Rajya Lakshmi; Munshi, Mansa; Yang, Guang; Xu, Lian; Hunter, Zachary; Tassone, Pierfrancesco; Anderson, Kenneth C; Treon, Steven P; Munshi, Nikhil C



Regulated expression of the alpha isoform of the human thromboxane A2 receptor during megakaryocyte differentiation: a coordinated role for WT1, Egr1, and Sp1.  


Thromboxane plays an essential role in hemostasis, regulating platelet aggregation and vessel tone. In humans, it signals through the TPalpha and TPbeta isoforms that are transcriptionally regulated by distinct promoters Prm1 and Prm3, respectively. Herein, the consequence of megakaryocytic differentiation on Prm1-directed TPalpha expression was investigated. Phorbol 12-myristate 13-acetate (PMA) treatment substantially increased TPalpha mRNA and Prm1-directed gene expression in human erythroleukemia and K562 cells. Deletional analyses localized the major responsive element(s) to the upstream -8500 to -7504 region while mutation of four WT1/Egr1/Sp1 cis elements therein established that each contributes to the induction. Moreover, PMA increased Egr1, but not WT1 or Sp1, expression while the NGFI-A-binding protein 1 co-repressor impaired PMA induction of Egr1- and Prm1-directed gene expression. Chromatin immunoprecipitations established that WT1 is predominantly bound in vivo to the 5' Prm1 region in non-differentiated human erythroleukemia cells. In response to PMA, there was initial induction in Egr1 and associated reduction in WT1 binding to Prm1 in vivo, which was displaced by Sp1 following sustained treatment. Collectively, data establish that regulated WT1 followed by sequential Egr1 and Sp1 binding to elements within Prm1 mediate repression and subsequent induction of TPalpha during differentiation into the megakaryocytic phenotype, shedding significant insights into factors regulating TPalpha expression therein. PMID:19747485

Gannon, AnneMarie M; Turner, Elizebeth C; Reid, Helen M; Kinsella, B Therese



Auto-Regulation of the Sohlh1 Gene by the SOHLH2/SOHLH1/SP1 Complex: Implications for Early Spermatogenesis and Oogenesis  

PubMed Central

Tissue-specific basic helix-loop-helix (bHLH) transcription factor proteins often play essential roles in cellular differentiation. The bHLH proteins SOHLH2 and SOHLH1 are expressed specifically in spermatogonia and oocytes and are required for early spermatogonial and oocyte differentiation. We previously reported that knocking out Sohlh2 causes defects in spermatogenesis and oogenesis similar to those in Sohlh1-null mice, and that Sohlh1 is downregulated in the gonads of Sohlh2-null mice. We also demonstrated that SOHLH2 and SOHLH1 can form a heterodimer. These observations led us to hypothesize that the SOHLH2/SOHLH1 heterodimer regulates the Sohlh1 promoter. Here, we show that SOHLH2 and SOHLH1 synergistically upregulate the Sohlh1 gene through E-boxes upstream of the Sohlh1 promoter. Interestingly, we identified an SP1-binding sequence, called a GC-box, adjacent to these E-boxes, and found that SOHLH1 could bind to SP1. Furthermore, chromatin-immunoprecipitation analysis using testes from mice on postnatal day 8 showed that SOHLH1 and SP1 bind to the Sohlh1 promoter region in vivo. Our findings suggest that an SOHLH2/SOHLH1/SP1 ternary complex autonomously and cooperatively regulates Sohlh1 gene transcription through juxtaposed E- and GC-boxes during early spermatogenesis and oogenesis.

Mizuta, Junya; Miyazaki, Jun-ichi



Assessment of pretend play in preschool-aged children: validation and factor analysis of the affect in play scale-preschool version.  


The Affect in Play Scale-Preschool (APS-P) and Affect in Play Scale-Preschool-Brief Rating (APS-P-BR) versions assess cognitive and affective play processes during a 5-min standardized play task. In this study, construct validity, external validity, and factor analyses for each scale were examined in 107 preschoolers. Reliability and validity were supported. Unlike results found with school-aged samples, positive affect loaded with the cognitive variables on factor analyses of the APS-P and APS-P-BR, suggesting that negative and undefined affect might represent a separate factor in preschool-aged children. Developmental significance and implications for use of the 2 scoring versions are discussed. PMID:24090344

Fehr, Karla K; Russ, Sandra W



Crosstalk of Sp1 and Stat3 signaling in pancreatic cancer pathogenesis  

PubMed Central

Pancreatic cancer progression is attributed to genetic and epigenetic alterations and a chaotic tumor microenvironment. Those diverse “upstream signal” factors appear to converge on specific sets of central nuclear regulators, namely, transcription factors. Specificity Protein 1 (Sp1) and signal transducer and activator of transcription 3 (Stat3) are central transcription factors that regulate a number of pathways important to tumorigenesis, including tumor cell-cycle progression, apoptosis, angiogenesis, metastasis, and evasion of the immune system. Recently, researchers demonstrated many types of crosstalk of Sp1 and Stat3 in tumor signal transduction and that these factors function cooperatively to activate targeted genes and promote tumorigenesis in pancreatic cancer. Therefore, targeting both Sp1 and Stat3 is a potential preventive and therapeutic strategy for pancreatic cancer.

Huang, Chen; Xie, Keping



Zac1, an Sp1-like protein, regulates human p21{sup WAF1/Cip1} gene expression in HeLa cells  

SciTech Connect

Zac1 functions as both a transcription factor and a transcriptional cofactor for p53, nuclear receptors (NRs) and NR coactivators. Zac1 might also act as a transcriptional repressor via the recruitment of histone deacetylase 1 (HDAC1). The ability of Zac1 to interact directly with GC-specific elements indicates that Zac1 possibly binds to Sp1-responsive elements. In the present study, our data show that Zac1 is able to interact directly with the Sp1-responsive element in the p21{sup WAF1/Cip1} gene promoter and enhance the transactivation activity of Sp1 through direct physical interaction. Our data further demonstrate that Zac1 might enhance Sp1-specific promoter activity by interacting with the Sp1-responsive element, affecting the transactivation activity of Sp1 via a protein-protein interaction, or competing the HDAC1 protein away from the pre-existing Sp1/HDAC1 complex. Finally, the synergistic regulation of p21{sup WAF1/Cip1} gene expression by Zac1 and Sp1 is mediated by endogenous p53 protein and p53-responsive elements in HeLa cells. Our work suggests that Zac1 might serve as an Sp1-like protein that directly interacts with the Sp1-responsive element to oligomerize with and/or to coactivate Sp1.

Liu, Pei-Yao [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC (China)] [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC (China); Hsieh, Tsai-Yuan [Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan, ROC (China)] [Division of Gastroenterology, Department of Internal Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan, ROC (China); Liu, Shu-Ting; Chang, Yung-Lung [Department of Biochemistry, National Defense Medical Center, Taipei 114, Taiwan, ROC (China)] [Department of Biochemistry, National Defense Medical Center, Taipei 114, Taiwan, ROC (China); Lin, Wei-Shiang [Division of Cardiology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan, ROC (China)] [Division of Cardiology, Department of Medicine, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan, ROC (China); Wang, Wei-Ming, E-mail: [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC (China) [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC (China); Department of Dermatology, Tri-Service General Hospital, National Defense Medical Center, Taipei 114, Taiwan, ROC (China); Huang, Shih-Ming, E-mail: [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC (China) [Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan, ROC (China); Department of Biochemistry, National Defense Medical Center, Taipei 114, Taiwan, ROC (China)



Hepatitis B virus X protein upregulates Lin28A/Lin28B through Sp-1/c-Myc to enhance the proliferation of hepatoma cells.  


Hepatitis B virus X protein (HBx) plays critical roles in the pathogenesis of hepatocellular carcinoma (HCC). Here, we were interested in knowing whether the oncogene Lin28A and its homolog Lin28B are involved in the hepatocarcinogenesis mediated by HBx. We showed that the expression levels of Lin28A and Lin28B were increased in clinical HCC tissues, HepG2.2.15 cell line and liver tissues of p21-HBx transgenic mice. Interestingly, the expression levels of HBx were positively associated with those of Lin28A/Lin28B in clinical HCC tissues. Moreover, the overexpression of HBx resulted in the upregulation of Lin28A/Lin28B in hepatoma HepG2/H7402 cell lines by transient transfection, suggesting that HBx was able to upregulate Lin28A and Lin28B. Then, we examined the mechanism by which HBx upregulated Lin28A and Lin28B. We identified that the promoter region of Lin28A regulated by HBx was located at nt -235/-66 that contained Sp-1 binding element. Co-immunoprecipitation showed that HBx was able to interact with Sp-1 in HepG2-X cells. Moreover, chromatin immunoprecipitation (ChIP) demonstrated that HBx could bind to the promoter of Lin28A, which failed to work when Sp-1 was silenced. Electrophoretic mobility shift assay (EMSA) further identified that HBx was able to interact with Sp-1 element in Lin28A promoter via transcription factor Sp-1. In addition, we found that c-Myc was involved in the activation of Lin28B mediated by HBx. In function, Lin28A/Lin28B played important roles in HBx-enhanced proliferation of hepatoma cells in vitro and in vivo. In conclusion, HBx activates Lin28A/Lin28B through Sp-1/c-Myc in hepatoma cells. Lin28A/Lin28B serves as key driver genes in HBx-induced hepatocarcinogenesis. PMID:23318446

You, X; Liu, F; Zhang, T; Lv, N; Liu, Q; Shan, C; Du, Y; Kong, G; Wang, T; Ye, L; Zhang, X



Control of intestinal promoter activity of the cellular migratory regulator gene ELMO3 by CDX2 and SP1.  


An important aspect of the cellular differentiation in the intestine is the migration of epithelial cells from the crypt to the villus tip. As homeodomaine transcription factor CDX2 has been suggested to influence cell migration, we performed a genome-wide promoter analysis for CDX2 binding in the differentiated human intestinal cancer cell line Caco-2 in order to identify CDX2-regulated genes involved in cellular migration. The engulfment and cell motility 3 (ELMO3) gene was identified as a potential CDX2 target gene. ELMO3 is an essential upstream regulator of the GTP-binding protein RAC during cell migration. However, no information is available about the transcriptional regulation of the ELMO3 gene. The aim of this study was to investigate the potential role of CDX2 in the regulation of the ELMO3 promoter activity. Electrophoretic mobility shift assays showed that CDX2 bound to conserved CDX2 sequences and mutations of the CDX2-binding sites, significantly reduced the promoter activity. Reporter gene assays demonstrated that the region mediating ELMO3 basal transcriptional activity to be located between -270 and -31 bp. Sequence analysis revealed no typical TATA-box, but four GC-rich sequences. In vitro analyses (electrophoretic mobility shift assays and promoter analyses) demonstrate that the SP1-binding sites are likely to play an important role in regulating the ELMO3 promoter activity. Furthermore, we showed here that CDX2 and SP1 can activate the ELMO3 promoter. Taken together, the present study reports the first characterization of the ELMO3 promoter and suggests a significant role of CDX2 in the basal transcriptional regulation of the intestine-specific expression of ELMO3, possibly through interaction with SP1. PMID:20127720

Coskun, Mehmet; Boyd, Mette; Olsen, Jørgen; Troelsen, Jesper T



Variation in Sp1 binding sites correlates with expression of survivin in breast cancer.  


Survivin is the smallest member of the inhibitor of apoptosis (IAP) family and is deregulated in breast cancer, where it is associated with a poor overall prognosis. It is well established that survivin overexpression predominately occurs at the transcriptional level. Numerous transcription factors bind to specific sequences in the promoter regions of genes and are involved in transcriptional regulation. Specificity protein (Sp1 binding sites have been found in the promoter region of the survivin gene. The present study aimed to investigate whether variations in Sp1 binding sites affect survivin expression. Nested polymerase chain reaction followed by DNA sequencing were performed to analyze the survivin gene promoter region in 42 breast cancer tissue samples. Furthermore, survivin expression was assessed using immunohistochemistry. High survivin protein expression was found in 66.7% (28/42) of breast cancer tissue samples. In addition, 15 variations in seven Sp1 binding sites were detected in 12 samples and Sp1 binding site variation was found to be associated with low survivin expression in the 42 samples. These findings suggested that variations in Sp1 binding sites may be associated with survivin expression. PMID:25018047

Xu, Qing; Liu, Mei; Xu, Ningzhi; Zhu, Hongxia



Combined treatment of pancreatic cancer with mithramycin A and tolfenamic acid promotes Sp1 degradation and synergistic antitumor activity.  


Mithramycin (MIT) and tolfenamic acid (TA) inhibit the activity of the transcription factor Sp1. In the present study, we investigated whether pancreatic cancer treatment with a combination of these compounds has a synergistic effect on Sp1 activity, tumor growth, and their underlying response mechanisms. Treatment of pancreatic tumor xenografts with MIT and TA produced dose-dependent antitumor activity, and significant antitumor activity of either compound alone was directly associated with systemic side effects. Combination treatment with nontoxic doses of both compounds produced synergistic antitumor activity, whereas treatment with a nontoxic dose of either compound alone lacked a discernible antitumor effect. Synergistic therapeutic effects correlated directly with synergistic antiproliferation and antiangiogenesis in vitro. Moreover, combination treatment resulted in Sp1 protein degradation, drastically downregulating expression of Sp1 and vascular endothelial growth factor. Our findings established that Sp1 is a critical target of TA and MIT in human pancreatic cancer therapy, rationalizing clinical studies to determine the effect of existing pancreatic cancer therapy regimens on Sp1 signaling in tumors and normal pancreatic tissue, and the ability of Sp1-targeting strategies to modify cancer responses. PMID:20086170

Jia, Zhiliang; Gao, Yong; Wang, Liwei; Li, Qiang; Zhang, Jun; Le, Xiangdong; Wei, Daoyan; Yao, James C; Chang, David Z; Huang, Suyun; Xie, Keping



Krüppel-like factor KLF8 plays a critical role in adipocyte differentiation.  


KLF8 (Krüppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer-binding protein ? (C/EBP?). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPAR?2 and C/EBP? promoter activity, suggesting that KLF8 is an upstream regulator of PPAR? and C/EBP?. The KLF8 binding sites were localized by site mutation analysis to -191 region in C/EBP? promoter and -303 region in PPAR? promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis. PMID:23285057

Lee, Haemi; Kim, Hyo Jung; Lee, Yoo Jeong; Lee, Min-Young; Choi, Hyeonjin; Lee, Hyemin; Kim, Jae-woo



Kr?ppel-Like Factor KLF8 Plays a Critical Role in Adipocyte Differentiation  

PubMed Central

KLF8 (Krüppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor ? (PPAR?) and CCAAT/enhancer-binding protein ? (C/EBP?). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPAR?2 and C/EBP? promoter activity, suggesting that KLF8 is an upstream regulator of PPAR? and C/EBP?. The KLF8 binding sites were localized by site mutation analysis to ?191 region in C/EBP? promoter and ?303 region in PPAR? promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis.

Lee, Yoo Jeong; Lee, Min-Young; Choi, Hyeonjin; Lee, Hyemin; Kim, Jae-woo



Bortezomib induces DNA hypomethylation and silenced gene transcription by interfering with Sp1/NF-?B-dependent DNA methyltransferase activity in acute myeloid leukemia  

PubMed Central

Bortezomib reversibly inhibits 26S proteasomal degradation, interferes with NF-?B, and exhibits antitumor activity in human malignancies. Zinc finger protein Sp1 transactivates DNMT1 gene in mice and is functionally regulated through protein abundance, posttranslational modifications (ie, ubiquitination), or interaction with other transcription factors (ie, NF-?B). We hypothesize that inhibition of proteasomal degradation and Sp1/NF-?B–mediated transactivation may impair aberrant DNA methyltransferase activity. We show here that, in addition to inducing accumulation of polyubiquitinated proteins and abolishment of NF-?B activities, bortezomib decreases Sp1 protein levels, disrupts the physical interaction of Sp1/NF-?B, and prevents binding of the Sp1/NF-?B complex to the DNMT1 gene promoter. Abrogation of Sp1/NF-?B complex by bortezomib causes transcriptional repression of DNMT1 gene and down-regulation of DNMT1 protein, which in turn induces global DNA hypomethylation in vitro and in vivo and re-expression of epigenetically silenced genes in human cancer cells. The involvement of Sp1/NF-?B in DNMT1 regulation is further demonstrated by the observation that Sp1 knockdown using mithramycin A or shRNA decreases DNMT1 protein levels, which instead are increased by Sp1 or NF-?B overexpression. Our results unveil the Sp1/NF-?B pathway as a modulator of DNA methyltransferase activity in human cancer and identify bortezomib as a novel epigenetic-targeting drug.

Liu, Shujun; Liu, Zhongfa; Xie, Zhiliang; Pang, Jiuxia; Yu, Jianhua; Lehmann, Esther; Huynh, Lenguyen; Vukosavljevic, Tamara; Takeki, Mitsui; Klisovic, Rebecca B.; Baiocchi, Robert A.; Blum, William; Porcu, Pierluigi; Garzon, Ramiro; Byrd, John C.; Perrotti, Danilo; Caligiuri, Michael A.; Chan, Kenneth K.; Wu, Lai-Chu



Differential Regulation of Bvg-Activated Virulence Factors Plays a Role in Bordetella pertussis Pathogenicity  

PubMed Central

Bordetella pertussis, the causative agent of whooping cough, regulates expression of many virulence factors via a two-component signal transduction system encoded by the bvgAS regulatory locus. It has been shown by transcription activation kinetics that several of the virulence factors are differentially regulated. fha is transcribed within 10 min following a bvgAS-inducing signal, while prn is transcribed after 1 h and ptx is not transcribed until 2 to 4 h after induction. These genes therefore represent early, intermediate, and late classes of bvg-activated promoters, respectively. Although there have been many insightful studies into the mechanisms of BvgAS-mediated regulation, the role that differential regulation of virulence genes plays in B. pertussis pathogenicity has not been characterized. We provide evidence that alterations to the promoter regions of bvg-activated genes can alter the kinetic pattern of expression of these genes without changing steady-state transcription levels. In addition, B. pertussis strains containing these promoter alterations that express either ptx at an early time or fha at a late time demonstrate a significant reduction in their ability to colonize respiratory tracts in an intranasal mouse model of infection. These data suggest a role for differential regulation of bvg-activated genes, and therefore for the BvgAS regulatory system, in the pathogenicity of B. pertussis.

Kinnear, Susan M.; Marques, Ryan R.; Carbonetti, Nicholas H.



Functional and physical interactions between the estrogen receptor Sp1 and nuclear aryl hydrocarbon receptor complexes.  

PubMed Central

17beta-Estradiol (E2) induces cathepsin D gene expression in MCF-7 human breast cancer cells and previous analyses of the proximal promoter region of this gene identified two functional enhancer sequences; namely an Sp1(N)23estrogen-responsive element (ERE) half-site (-199 to -165) and an imperfect palindromic ERE (-119 to -107). A third region of the cathepsin D gene promoter (CD/L, -145 to -119) was also E2 responsive in transient transfection assays. A GC-rich sequence which contains two overlapping Sp1 binding sites (-145 to -135) was responsible for ER-mediated transactivation and required formation of an ER/Sp1 complex in which only the Sp1 protein bound DNA. E2 responsiveness of the CD/L sequence was also dependent on an adjacent overlapping GCGTG motif corresponding to the dioxin-responsive element (DRE) core binding sequence, which is the cognate response element for the heterodimeric aryl hydrocarbon receptor (AhR)/AhR nuclear translocator (ARNT) transcription factor complex. The results show that ER-mediated transactivation of CD/L was associated with the Sp1(N)2-4DRE (core) motif and involved formation of a multiprotein ER/Sp1-AhR/ARNT complex. These results illustrate a unique example of an endogenous role for AhR/ARNT in the absence of added AhR agonist and indicate that the cathepsin D gene proximal promoter region contains at least three different functional motifs associated with ER-mediated transactivation.

Wang, F; Hoivik, D; Pollenz, R; Safe, S



Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury  

PubMed Central

Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1?-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1? deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1? mRNA levels. Furthermore, partial HIF-1? deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1?+/? mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1? protein levels nor caused significant gut and lung injury in wild-type or HIF-1?+/? mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role.

Kannan, Kolenkode B.; Colorado, Iriana; Reino, Diego; Palange, David; Lu, Qi; Qin, Xiaofa; Abungu, Billy; Watkins, Anthony; Caputo, Francis J.; Xu, Da-Zhong; Semenza, Gregg L.; Deitch, Edwin A.



Hypoxia-inducible factor plays a gut-injurious role in intestinal ischemia reperfusion injury.  


Gut injury and loss of normal intestinal barrier function are key elements in the paradigm of gut-origin systemic inflammatory response syndrome, acute lung injury, and multiple organ dysfunction syndrome (MODS). As hypoxia-inducible factor (HIF-1) is a critical determinant of the physiological and pathophysiological response to hypoxia and ischemia, we asked whether HIF-1 plays a proximal role in the induction of gut injury and subsequent lung injury. Using partially HIF-1?-deficient mice in an isolated superior mesenteric artery occlusion (SMAO) intestinal ischemia reperfusion (I/R) injury model (45 min SMAO followed by 3 h of reperfusion), we showed a direct relationship between HIF-1 activation and intestinal I/R injury. Specifically, partial HIF-1? deficiency attenuated SMAO-induced increases in intestinal permeability, lipid peroxidation, mucosal caspase-3 activity, and IL-1? mRNA levels. Furthermore, partial HIF-1? deficiency prevented the induction of ileal mucosal inducible nitric oxide synthase (iNOS) protein levels after SMAO and iNOS deficiency ameliorated SMAO-induced villus injury. Resistance to SMAO-induced gut injury was also associated with resistance to lung injury, as reflected by decreased levels of myeloperoxidase, IL-6 and IL-10 in the lungs of HIF-1?(+/-) mice. In contrast, a short duration of SMAO (15 min) followed by 3 h of reperfusion neither induced mucosal HIF-1? protein levels nor caused significant gut and lung injury in wild-type or HIF-1?(+/-) mice. This study indicates that intestinal HIF-1 activation is a proximal regulator of I/R-induced gut mucosal injury and gut-induced lung injury. However, the duration and severity of the gut I/R insult dictate whether HIF-1 plays a gut-protective or deleterious role. PMID:21183660

Kannan, Kolenkode B; Colorado, Iriana; Reino, Diego; Palange, David; Lu, Qi; Qin, Xiaofa; Abungu, Billy; Watkins, Anthony; Caputo, Francis J; Xu, Da-Zhong; Semenza, Gregg L; Deitch, Edwin A; Feinman, Rena



Activation of Sp1-mediated transcription by Rta of Epstein-Barr virus via an interaction with MCAF1  

PubMed Central

Rta is a transcription factor encoded by BRLF1 of the Epstein–Barr virus (EBV). This factor is expressed during the immediate-early stage of the lytic cycle to activate the genes required for EBV lytic development. Although transcription activation by Rta is frequently associated with the binding of Rta to the Rta-response element (RRE) in promoters, Rta sometimes activates promoters without an RRE. Here we show that Rta interacts with an Sp1-interacting protein, MBD1-containing chromatin-associated factor 1 (MCAF1). This interaction is critical to the formation of an Sp1–MCAF1–Rta complex at Sp1 sites. Therefore, following lytic induction and the expression of Rta, Rta increases Sp1-mediated transcription. The genes that are thus activated include p16, p21, SNRPN and BRLF1. However, the binding of Rta to RRE prevents the interaction between Rta and MCAF1; therefore, transcription activation by RRE depends only on Rta, and not on MCAF1 or Sp1. Furthermore, this study finds that MCAF1 promotes the expression of Rta and Zta from EBV, indicating that MCAF1 participates EBV lytic activation. Our study documents the critical role of Rta in regulating the transcription of the genes that are mediated by Sp1.

Chang, Li-Kwan; Chung, Jian-Ying; Hong, Yi-Ren; Ichimura, Takaya; Nakao, Mitsuyoshi; Liu, Shih-Tung



Users guide for the ANL IBM SP1  

SciTech Connect

This guide presents the features of the IBM SP1 installed in the Mathematics and Computer Science Division at Argonne National Laboratory. The guide describes the available hardware and software, access policies, and hints for using the system productively.

Gropp, W.; Lusk, E.; Pieper, S.C.



The Factors of Design on Playing Equipment in Young Children Schools by Viewpoint of Young Children Behavioral Development  

ERIC Educational Resources Information Center

The main purpose of this study was to explore the care-givers of preschool education institutions whose cognition on playing equipment functions, conditions of both setting and using, and the main factors which should beware of design. Besides, not only constructed the factors of design, but also provided suggestions about setting and designing of…

Kuo, Chuen-tzay



On play and playing.  


The paper offers a review of the development of the concept of play and playing. The true beginnings of the development of the theories of play are set as late as in the 19th century. It is difficult to define play as such; it may much more easily be defined through its antipode--work. In the beginning, play used to be connected with education; it was not before Freud's theory of psychoanalysis and Piaget's developmental psychology that the importance of play in a child's development began to be explained in more detail. The paper further tackles the role of play in the adult age. Detailed attention is paid to psychodynamic and psychoanalytic authors, in particular D. W. Winnicott and his understanding of playing in the intermediary (transitional) empirical or experiential space. In other words, playing occupies a space and time of its own. The neuroscientific concept of playing is also tackled, in the connection with development as well. PMID:24611363

Rudan, Dusko



Butyrate suppresses expression of neuropilin I in colorectal cell lines through inhibition of Sp1 transactivation  

PubMed Central

Background Neuropilin is a transmembrane receptor for vascular endothelial growth factor (VEGF) and is expressed in normal endothelial cells and upregulated in cancer cells. Neuropilin-1 (NRP-1) has been shown to promote tumour cell migration and survival in colon cancer in response to VEGF binding. The expression profiles of neuropilins, associated co-receptors and known ligands have been mapped in three colorectal cell lines: Caco-2, HCT116 & HT29. We have previously shown that butyrate, a naturally occurring histone deacetylase inhibitor (HDACi) produced by fermentation of fibre in the colon, causes apoptosis of colon cancer cell lines. Results Here we demonstrate that butyrate down-regulates NRP-1 and VEGF at the mRNA and protein level in colorectal cancer cell lines. NRP-1 is a known transcriptional target of Sp1, whose activity is regulated by acetylation. NRP-1 down-regulation by butyrate was associated with decreased binding affinity of Sp1 for canonical Sp-binding sites in the NRP-1 promoter. siRNA-mediated knock-down of Sp1 implied that Sp1 may have strong DNA binding activity but weak transactivation potential. Conclusion The downregulation of the key apoptotic and angiogenesis regulator NRP-1 by butyrate suggests a novel contributory mechanism to the chemopreventive effect of dietary fibre.



Platelet-derived growth factor plays a critical role to convert bone marrow cells into glomerular mesangial-like cells  

Microsoft Academic Search

Platelet-derived growth factor plays a critical role to convert bone marrow cells into glomerular mesangial-like cells.BackgroundDespite increasing interest in bone marrow–derived stem cells, little is known about critical factors that determine their fates both in vitro and in vivo. Recently, we have reported that bone marrow is a reservoir for glomerular mesangial cells in rats. To find a key factor




Yes and Lyn play a role in nuclear translocation of the Epidermal Growth Factor Receptor  

PubMed Central

The epidermal growth factor receptor (EGFR) is a central regulator of tumor progression in human cancers. Cetuximab is an anti-EGFR antibody that has been approved for use in oncology. Previously we investigated mechanisms of resistance to cetuximab using a model derived from the non-small cell lung cancer line NCI-H226. We demonstrated that cetuximab-resistant clones (CtxR) had increased nuclear localization of the EGFR. This process was mediated by Src family kinases (SFK), and nuclear EGFR played a role in resistance to cetuximab. To better understand SFK mediated nuclear translocation of EGFR, we investigated which SFK member(s) controlled this process as well as the EGFR tyrosine residues that are involved. Analyses of mRNA and protein expression indicated up-regulation of the SFK members Yes and Lyn in all CtxR clones. Further, immunoprecipitation analysis revealed that EGFR interacts with Yes and Lyn in CtxR clones, but not in cetuximab-sensitive (CtxS) parental cells. Using RNAi interference, we found that knockdown of either Yes or Lyn led to loss of EGFR translocation to the nucleus. Conversely, overexpression of Yes or Lyn in low nuclear EGFR expressing CtxS parental cells led to increased nuclear EGFR. Chromatin immunoprecipitation (ChIP) assays confirmed nuclear EGFR complexes associated with the promoter of the known EGFR target genes B-Myb and iNOS. Further, all CtxR clones exhibited up-regulation of B-Myb and iNOS at the mRNA and protein levels. siRNAs directed at Yes or Lyn led to decreased binding of EGFR complexes to the B-Myb and iNOS promoters based on ChIP analyses. SFKs have been shown to phosphorylate EGFR on tyrosines 845 and 1101 (Y845 and Y1101) and mutation of Y1101, but not Y845, impaired nuclear entry of the EGFR. Taken together, our findings demonstrate that Yes and Lyn phosphorylate EGFR at Y1101 which influences EGFR nuclear translocation in this model of cetuximab resistance.

Iida, Mari; Brand, Toni M.; Campbell, David A; Li, Chunrong; Wheeler, Deric L.



Growth factor delivery methods in the management of sports injuries: the state of play  

Microsoft Academic Search

In recent years there have been rapid developments in the use of growth factors for accelerated healing of injury. Growth factors have been used in maxillo-facial and plastic surgery with success and the technology is now being developed for orthopaedics and sports medicine applications. Growth factors mediate the biological processes necessary for repair of soft tissues such as muscle, tendon

L Creaney



SP1 protein-based nanostructures and arrays.  


Controlled formation of complex nanostructures is one of the main goals of nanoscience and nanotechnology. Stable Protein 1 (SP1) is a boiling-stable ring protein complex, 11 nm in diameter, which self-assembles from 12 identical monomers. SP1 can be utilized to form large ordered arrays; it can be easily modified by genetic engineering to produce various mutants; it is also capable of binding gold nanoparticles (GNPs) and thus forming protein-GNP chains made of alternating SP1s and GNPs. We report the formation and the protocols leading to the formation of those nanostructures and their characterization by transmission electron microscopy, atomic force microscopy, and electrostatic force microscopy. Further control over the GNP interdistances within the protein-GNP chains may lead to the formation of nanowires and structures that may be useful for nanoelectronics. PMID:18193911

Medalsy, Izhar; Dgany, Or; Sowwan, Mukhles; Cohen, Hezy; Yukashevska, Alevtyna; Wolf, Sharon G; Wolf, Amnon; Koster, Abraham; Almog, Orna; Marton, Ira; Pouny, Yehonathan; Altman, Arie; Shoseyov, Oded; Porath, Danny



An Axis Involving SNAI1, microRNA-128 and SP1 Modulates Glioma Progression  

PubMed Central

Background Glioblastoma is an extraordinarily aggressive disease that requires more effective therapeutic options. Snail family zinc finger 1, dysregulated in many neoplasms, has been reported to be involved in gliomas. However, the biological mechanisms underlying SNAI1 function in gliomas need further investigation. Methods Quantitative real-time PCR was used to measure microRNA-128 (miR-128) expression level and western blot was performed to detect protein expression in U87 and U251 cells and human brain tissues. Cell cycle, CCK-8, transwell and wound-healing assays were performed. Dual-luciferase reporter assay was used for identifying the mechanism of SNAI1 and miR-128b regulation. The mechanism of miR-128 targeting SP1 was also tested by luciferase reporter assay. Immunohistochemistry and in situ hybridisation staining were used for quantifying SNAI1, SP1 and miR-128 expression levels in human glioma samples. Results The Chinese Glioma Genome Atlas (CGGA) data revealed that SNAI1 was up-regulated in glioma and we confirmed the findings in normal and glioma tissues. SNAI1 depletion by shRNA retarded the cell cycle and suppressed proliferation and invasion in glioma cell lines. The CGGA data showed that the Pearson correlation index between SNAI1 and miR-128 was negatively correlated. SNAI1 suppressed miR-128b expression by binding to the miR-128b specific promoter motif, and miR-128 targeted SP1 via binding to the 3?-untranslated region of SP1. Moreover, introduction of miR-128 anti-sense oligonucleotide alleviated the cell cycle retardation, proliferation and invasion inhibition induced by SNAI1 shRNA. Immunohistochemistry and in situ hybridisation analysis of SNAI1, SP1 and miR-128 unraveled their expression levels and correlations in glioma samples. Conclusions We propose that the SNAI1/miR-128/SP1 axis, which plays a vital role in glioma progression, may come to be a clinically relevant therapeutic target.

Zhang, Rui; Yan, Wei; Li, Rui; Zhang, Junxia; Luo, Hui; Shi, Yan; Luan, Wenkang; Zhang, Yaxuan; You, Yongping; Wang, Yingyi; Liu, Ning



Factors Related to Play Therapists' Social Justice Advocacy Attitudes  

ERIC Educational Resources Information Center

The authors used a correlational research design to examine how belief in a just world, political ideology, socioeconomic status of family of origin, and percentage of racial minority clients were related to social justice advocacy attitudes among play therapists. A multiple regression was used to analyze the data. Results indicated that belief in…

Parikh, Sejal B.; Ceballos, Peggy; Post, Phyllis



Specificity Protein 1 (Sp1) Oscillation Is Involved in Copper Homeostasis Maintenance by Regulating Human High-Affinity Copper Transporter 1 Expression  

PubMed Central

Copper is an essential micronutrient for cell growth but is toxic in excess. Copper transporter (Ctr1) plays an important role in regulating adequate copper levels in mammalian cells. We have shown previously that expression of the human high-affinity copper transporter (hCtr1) was transcriptionally up-regulated under copper-depleted conditions and down-regulated under replete conditions; moreover, elevated hCtr1 levels suppress hCtr1 expression. Specificity protein 1 (Sp1) regulates expression of hCtr1 under copper-stressed conditions. In this study, we made the following important observations: 1) Sp1 expression is down-regulated under copper-replete conditions but up-regulated under copper-depleted conditions. These up- and down-regulations of Sp1 in turn regulate hCtr1 expression to control copper homeostasis. 2) Copper-regulated Sp1 expression involved Sp1 binding to its own promoter as demonstrated by the chromatin immunoprecipitation assay; therefore, Sp1 is also transcriptionally self-regulated via hCtr1/copper intermediation. 3) Both zinc finger and glutamine-rich transactivation domains of Sp1 are involved in the Sp1-mediated hCtr1 and Sp1 regulation by copper stresses. 4) Although Sp3 expression is also regulated by copper availability, Sp3 does not regulate hCtr1 homeostasis. Collectively, our results demonstrated that mammalian cells use Sp1 oscillation in response to copper availability to regulate copper homeostasis through hCtr1 expression in a tripartite inter-regulatory relationship. These findings have important implications in mammalian copper physiology regulation.

Liang, Zheng D.; Tsai, Wen-Bin; Lee, Mei-Yi; Savaraj, Niramol



Suppression of survivin promoter activity by YM155 involves disruption of Sp1-DNA interaction in the survivin core promoter  

PubMed Central

YM155, a novel survivin suppressant, shows potent antitumor activity against various human cancers and is currently in phase II clinical trials. In this study, we investigated whether YM155 selectively inhibits survivin transcription. We hypothesize that inhibition of survivin transcription plays a role in YM155-mediated survivin inhibition. We found that YM155 inhibited survivin promoter activity, while it showed minimal inhibitory effect on four control gene promoters in transfection and luciferase activity assay experiments, indicating its selectivity. Transfection of various survivin promoter-luciferase constructs followed by luciferase assays revealed that the survivin core promoter (269 bp) plays a major role in YM155-mediated inhibitory effects. However, flow cytometry analysis indicated that inhibition of survivin promoter activity by YM155 is cell cycle-independent without G1 cell arrests. Electrophoretic mobility shift assays (EMSA) identified that YM155 abrogates nuclear proteins binding to the region of -149 to -71, in which Sp1 is a major candidate, and that YM155 treatment induces Sp1 re-subcellular localization without inhibiting its expression. Forced expression of Sp1 neutralized YM155-mediated downregulation of survivin promoter activity. Consistently, mutation of the identified Sp1 sites in the oligonucleotide probe diminished DNA-protein interactions in EMSA experiments, and mutation of the Sp1 sites in the survivin promoter-luciferase construct diminished survivin promoter activity. These findings indicate that YM155 inhibition of survivin expression is at least in part through its inhibition of survivin transcription by disruption of Sp1 interaction with the region of -149 to -71 in the survivin core promoter.

Cheng, Qiuying; Ling, Xiang; Haller, Andrew; Nakahara, Takahito; Yamanaka, Kentaro; Kita, Aya; Koutoku, Hiroshi; Takeuchi, Masahiro; Brattain, Michael G; Li, Fengzhi



SP1 mediates the link between methylation of the tumour suppressor miR-149 and outcome in colorectal cancer.  


Although recent studies indicate that DNA methylation contributes to the down-regulation of microRNAs (miRNAs) in colorectal cancer (CRC), this field remains largely unexplored. To identify methylation-silenced miRNAs and clarify their role in CRC, we performed a microarray analysis and screened for miRNAs that were induced in CRC cells by 5-aza-2'-deoxycytidine treatment or by the knockdown of DNA methyltransferases. The DNA methylation status of the candidate miRNA was analysed by bisulphite sequencing PCR and methylation-specific PCR. We found that miRNA-149 (miR-149) was epigenetically silenced in CRC and down-regulation of miR-149 was associated with hypermethylation of the neighbouring CpG island (CGI). Quantitative RT-PCR analysis demonstrated that the miR-149 level was markedly reduced in 51.6% of the CRC tissues compared with matched non-cancerous tissues. In addition, low expression of miR-149 was associated with a greater depth of invasion (p = 0.012), lower 5-year survival rate (p = 0.025), and was found to be an independent prognostic factor for overall survival (p = 0.016) in a multivariate analysis. Moreover, transfection of miR-149 inhibited cell growth and invasion of CRC cells in vitro. We also identified mRNA for Specificity Protein 1 (SP1, Sp1), a potential oncogenic protein, as a target of miR-149. Our data suggest that, as a methylation-sensitive miRNA, miR-149 may play an important role as a tumour suppressor in CRC, which has prognostic and therapeutic implications. PMID:22821729

Wang, Feng; Ma, Yan-Lei; Zhang, Peng; Shen, Tong-Yi; Shi, Chen-Zhang; Yang, Yong-Zhi; Moyer, Mary-Pat; Zhang, Hui-Zhen; Chen, Hong-Qi; Liang, Yong; Qin, Huan-Long



The role played by key transcription factors in activated mast cells.  


The network of transcription factors in mast cells has not been investigated as widely as it has been in other differentiated hematopoietic cells. There are still many mechanisms of transcriptional regulation that need to be fully elucidated to understand how mast cell external stimuli lead to the appropriate physiological responses. Such information could be used to determine potential therapeutic targets for the control of mast cell activation in inflammatory diseases, allergy, and asthma. The aim of this article is to review hallmark studies in the field of transcription factor regulation in mast cells. We elaborate especially on several transcription factors studied in our laboratory in the past decade, including activator protein-1, microphthalmia-associated transcription factor, upstream stimulating factor-2, and signal transducer and activator of transcription 3. PMID:17498066

Carmi, Irit; Razin, Ehud



Improved Adhesives Containing CNT\\/SP1 Nano Fillers  

Microsoft Academic Search

Carbon nanotubes (CNT) have stimulated research due to their wide range of applications. However, their existence as aggregates and the difficulty in debundling and dispersion limits the improvement of properties when used as fillers. Many techniques have been employed to obtain such dispersions including mechanical, ultrasonic, and solution mixing, resulting in limited effect. Attaching a protein moiety such as SP1

A. Wolf; A. Buchman; A. Eitan; T. Fine; Y. Nevo; A. Heyman; O. Shoseyov



Functional role for Sp1 in the transcriptional amplification of a cell cycle regulated histone H4 gene.  


The promoter of the cell cycle regulated histone FO108 H4 gene is mediated by two in vivo protein/DNA interaction domains, sites I and II. We have shown previously that site II mediates the cell cycle controlled enhancement of H4 gene transcription at the G1/S phase boundary. Here we show that site I, an element containing both G-rich and ATF-like consensus sequences, confers maximal levels of transcription in proliferating cells. By the combined application of gel shift assays with site-directed mutagenesis, DNase I footprinting, oligonucleotide competition, in vitro expression of recombinant proteins, and specific antibody supershift studies, we demonstrate that the proximal G-rich sequence within site I interacts with the transcription factor Sp1, while the distal portion of site I interacts with members of the ATF family of proteins, including ATF-1. In vitro transcription studies as well as expression assays of transiently and stably transfected genes in HeLa cells reveal that the deletion of site I causes a dramatic decrease in expression. Mutation of the Sp1 element, which abolishes Sp1 binding, results in a 6-10-fold reduction in reporter activity. In addition, overexpression of Sp1 in Sp1-deficient cells results in the dramatic activation of the histone promoter. In contrast, mutation of the asymmetric ATF binding site, located distally within site I, has a more limited effect upon expression. Interestingly, the contribution of the Sp1 site to maximal transcription was cell type dependent. Thus, we demonstrate that the Sp1 binding site of the site I histone H4 promoter in particular is critical for maximal expression in living cells and postulate that this site may act to amplify the cell cycle response. PMID:7779811

Birnbaum, M J; Wright, K L; van Wijnen, A J; Ramsey-Ewing, A L; Bourke, M T; Last, T J; Aziz, F; Frenkel, B; Rao, B R; Aronin, N



A review of platelet derived growth factor playing pivotal role in bone regeneration.  


This article is focused on the literature review and study of recent advances in the field of bone grafting, which involves platelet-derived growth factor (PDGF) as one of the facilitating factors in bone regeneration. This article includes a description of the mechanism of PDGF for use in surgeries where bone grafting is required, which promotes future application of PDGF for faster bone regeneration or inhibition of bone growth if required as in osteosarcoma. The important specific activities of PDGF include mitogenesis (increase in the cell populations of healing cells), angiogenesis (endothelial mitoses into functioning capillaries), and macrophage activation (debridement of the wound site and a second phase source of growth factors for continued repair and bone regeneration). Thus PDGF can be utilized in wound with bone defect to conceal the wound with repair of bony defect. PMID:24914921

Shah, Prasun; Keppler, Louise; Rutkowski, James



Interplay between proximal and distal promoter elements is required for squamous differentiation marker induction in the bronchial epithelium: role for ESE-1, Sp1, and AP-1 proteins.  


Overexpression of SPRR1B in bronchial epithelial cells is a marker for early metaplastic changes induced by various toxicants/carcinogens. Previously, we have shown that the transcriptional stimulation of SPRR1B expression by phorbol 12-myristate 13-acetate (PMA) is mainly mediated by a -150/-94 bp enhancer harboring two critical 12-O-tetradecanoylphorbol-13-acetate-responsive elements (TREs) and by Jun.Fra-1 dimers. Here, we show that a region between -54 and -39 bp containing an ETS-binding site (EBS) and a GC box is essential for both basal and PMA-inducible SPRR1B transcription. In vivo footprinting demonstrated binding of transcription factors to these elements. However, unlike enhancer TREs, exposure of cells to PMA did not significantly alter the footprinting pattern at these elements. Mutations that crippled both the EBS and GC box suppressed both basal and PMA-inducible SPRR1B transcription. Consistent with this, overexpression of EBS-binding proteins ESE-1 and ESE-3 significantly stimulated SPRR1B promoter activity. Furthermore, preceding SPRR1B transcription, PMA up-regulated mRNA expression of ETS family members such as ESE-1 and ESE-3. Although ESE-1 synergistically activated c-Jun- and PMA-enhanced SPRR1B transcription, coexpression of Sp1 and ESE-1 showed no synergistic or additive effect on promoter activity, indicating an obligatory role for AP-1 proteins in such regulation. In support of this notion, deletion or mutation of two functional TREs inhibited ESE-1- and Sp1-enhanced promoter activation. Thus, the interaction between ESE-1 and Sp1, and AP-1 proteins that bind to the proximal and distal promoter regions, respectively, play a critical role in the induction of squamous differentiation marker expression in bronchial epithelial cells. PMID:12682075

Reddy, Sekhar P M; Vuong, Hue; Adiseshaiah, Pavan



Inherited Factors Play an Important Role in Breast Cancer Progression According to New Study in Mice

New research in mice and five independent collections of human breast tumors has enabled NCI scientists to confirm that genes for factors contributing to susceptibility for breast cancer metastasis can be inherited. The new findings support earlier results from the same laboratory.


Sp1 acetylation is associated with loss of DNA binding at promoters associated with cell cycle arrest and cell death in a colon cell line  

PubMed Central

Butyrate, a known histone deacetylase inhibitor (HDACi) and product of fibre fermentation, is postulated to mediate the protective effect of dietary fibre against colon cancer. The transcription factor Sp1 is a target of acetylation and is known to be associated with class I HDACs, including HDAC1. Sp1 is a ubiquitous transcription factor and Sp1-regulated genes include those involved in cell cycle regulation, apoptosis and lipogenesis: all major pathways in cancer development. The only known acetylated residue of Sp1 is lysine703 which resides in the DNA binding domain. Here we show that acetylated Sp1 loses p21- and bak-promoter -binding function in vitro. Furthermore treatment with a panel of HDAC inhibitors showed clustering of activities for a subset of inhibitors, causing G2 cell cycle arrest, Sp1 acetylation, p21 and Bak over-expression, all with very similar EC50 concentrations. These HDACi activities were not distributed according to the molecular class of compound. In order to mimic loss of binding, an siRNA strategy was used to reduce Sp1 expression. This resulted in altered expression of multiple elements of the p53/p21 pathway. Taken together our data suggest a mechanistic model for the chemopreventive actions of butyrate in colon epithelial cells, and provide new insight into the differential activities some classes of HDAC inhibitors.



Nuclear factor of activated T-cells (NFAT) plays a role in SV40 infection  

SciTech Connect

Recent evidence highlighted a role for the transcription factor, nuclear factor of activated T-cells (NFAT), in the transcription of the human polyomavirus JCV. Here we show that NFAT is also important in the transcriptional control of the related polyomavirus, Simian Virus 40 (SV40). Inhibition of NFAT activity reduced SV40 infection of Vero, 293A, and HeLa cells, and this block occurred at the stage of viral transcription. Both NFAT3 and NFAT4 bound to the SV40 promoter through {kappa}B sites located within the 72 bp repeated enhancer region. In Vero cells, NFAT was involved in late transcription, but in HeLa and 293A cells both early and late viral transcription required NFAT activity. SV40 large T-Ag was found to increase NFAT activity and provided a positive feedback loop to transactivate the SV40 promoter.

Manley, Kate [Graduate Program in Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912 (United States); O'Hara, Bethany A. [Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912 (United States); Atwood, Walter J. [Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI 02912 (United States)], E-mail:



The Cytotoxic Necrotizing Factor 1 from E. Coli: A Janus Toxin Playing with Cancer Regulators  

PubMed Central

Certain strains of Escherichia coli have been indicated as a risk factor for colon cancer. E. coli is a normal inhabitant of the human intestine that becomes pathogenic, especially in extraintestinal sites, following the acquisition of virulence factors, including the protein toxin CNF1. This Rho GTPases-activating toxin induces dysfunctions in transformed epithelial cells, such as apoptosis counteraction, pro-inflammatory cytokines’ release, COX2 expression, NF-kB activation and boosted cellular motility. As cancer may arise when the same regulatory pathways are affected, it is conceivable to hypothesize that CNF1-producing E. coli infections can contribute to cancer development. This review focuses on those aspects of CNF1 related to transformation, with the aim of contributing to the identification of a new possible carcinogenic agent from the microbial world.

Fabbri, Alessia; Travaglione, Sara; Ballan, Giulia; Loizzo, Stefano; Fiorentini, Carla



Stress incontinence in females: which factors play a role in operative results?  

Microsoft Academic Search

During the years 1981–1985, 251 female patients with stress incontinence consulted the department. The average age was 47.3 years. Surgical treatment consisting of cystourethropexy ad modem Marshall-Marchetti-Krantz, Stamey-Pereyra, or by fascial sling and\\/or periurethral injection with Teflon was performed in 185 patients, with a total of 215 procedures. In a retrospective study, factors which could possibly predict the results of

M. A. Affandi; A. E. J. L. Kramer; U. Jonas



Splase: a new class IIS zinc-finger restriction endonuclease with specificity for Sp1 binding sites.  


A new restriction endonuclease, named Splase, was constructed by genetically fusing the DNA-cleavage domain of the restriction endonuclease Fok1 with the zinc-finger DNA-binding domain of the transcription factor Sp1. The resulting protein was expressed in Escherichia coli., partially purified, and shown to selectively digest plasmid DNA harboring consensus Sp1 sites. Splase was also shown to selectively digest the long terminal repeat of the HIV-1 DNA at Sp1 sites. Splase recognizes a 10-bp DNA sequence and hydrolyzes phosphodiester bonds upstream of the binding sequence. The binding specificity of Splase makes this a "rare cutter" restriction enzyme which could be valuable in creating large DNA fragments for genome sequencing projects. The result also presents the opportunity to create other restriction enzymes by altering the binding specificity of the zinc-finger recognition helix. PMID:8895094

Huang, B; Schaeffer, C J; Li, Q; Tsai, M D



Basal Transcription Factor 3 Plays an Important Role in Seed Germination and Seedling Growth of Rice  

PubMed Central

BTF3 has been recognized to be involved in plant growth and development. But its function remains mostly unknown during seed germination and seedling stage. Here, we have analyzed OsBTF3-related sequences in Oryza sativa L. subspecies, japonica, which resembles with the conserved domain of a nascent polypeptide associated complex (NAC) with different homologs of OsBTF3 and human BTF3. Inhibition of Osj10gBTF3 has led to considerable morphological changes during seed germination and seedling growth. Germination percentage was not influenced by the application of GA3, ABA, and NaCl but all concentrations caused wild-type (WT) seeds to germinate more rapidly than the RNAi (Osj10gBTF3Ri) transgenic lines. Seedling inhibition was more severe in the Osj10gBTF3Ri seedlings compared with their WT especially when treated with 100 or 200??M GA3; 50% reduction in shoots was observed in Osj10gBTF3Ri seedlings. The expression of Osj3g1BTF3, Osj3g2BTF3 and Osj10gBTF3 was primarily constitutive and generally modulated by NaCl, ABA, and GA3 stresses in both Osj10gBTF3Ri lines and WT at the early seedling stage, suggesting that Osj3g1BTF3 and Osj10gBTF3 are much similar but different from Osj3g2BTF3 in biological function. These results show that OsBTF3 plays an important role in seed germination and seedling growth gives a new perception demonstrating that more multifaceted regulatory functions are linked with BTF3 in plants.

Wang, Wenyi; Xu, Mengyun; Wang, Ya



The 'Perfect Storm' and Acute Coronary Syndrome Onset: Do Psychosocial Factors Play a Role?  

PubMed Central

The revolution in cardiac care over the past two decades, characterized by emergent revascularization, drug eluting stents, anti-platelet medications, and advanced imaging has had little impact on overall ACS recurrence, or ACS prevention. The “Perfect Storm” refers to a confluence of events and processes, including atherosclerotic plaque, coronary flow dynamics, hemostatic and fibrinolytic function, metabolic and inflammatory conditions, neurohormonal dysregulation, and environmental events that give rise to, and result in an ACS event. In this article we illustrate the limits of the traditional main effect research model, giving a brief description of the current state of knowledge regarding the development of atherosclerotic plaque and the rupturing of these plaques that defines an ACS event. We then apply the Perfect Storm conceptualization to describe a program of research concerning a psychosocial vulnerability factor that contributes to increased risk of recurrent ACS and early mortality, and that has defied our efforts to identify underlying pathophysiology and successfully mount efforts to fully mitigate this risk.

Burg, Matthew M.; Edmondson, Donald; Shimbo, Daichi; Shaffer, Jonathan; Kronish, Ian M.; Whang, William; Alcantara, Carmela; Schwartz, Joseph E.; Muntner, Paul; Davidson, Karina W.



ADP Ribosylation Factor 1 Plays an Essential Role in the Replication of a Plant RNA Virus  

PubMed Central

Eukaryotic positive-strand RNA viruses replicate using the membrane-bound replicase complexes, which contain multiple viral and host components. Virus infection induces the remodeling of intracellular membranes. Virus-induced membrane structures are thought to increase the local concentration of the components that are required for replication and provide a scaffold for tethering the replicase complexes. However, the mechanisms underlying virus-induced membrane remodeling are poorly understood. RNA replication of red clover necrotic mosaic virus (RCNMV), a positive-strand RNA plant virus, is associated with the endoplasmic reticulum (ER) membranes, and ER morphology is perturbed in RCNMV-infected cells. Here, we identified ADP ribosylation factor 1 (Arf1) in the affinity-purified RCNMV RNA-dependent RNA polymerase fraction. Arf1 is a highly conserved, ubiquitous, small GTPase that is implicated in the formation of the coat protein complex I (COPI) vesicles on Golgi membranes. Using in vitro pulldown and bimolecular fluorescence complementation analyses, we showed that Arf1 interacted with the viral p27 replication protein within the virus-induced large punctate structures of the ER membrane. We found that inhibition of the nucleotide exchange activity of Arf1 using the inhibitor brefeldin A (BFA) disrupted the assembly of the viral replicase complex and p27-mediated ER remodeling. We also showed that BFA treatment and the expression of dominant negative Arf1 mutants compromised RCNMV RNA replication in protoplasts. Interestingly, the expression of a dominant negative mutant of Sar1, a key regulator of the biogenesis of COPII vesicles at ER exit sites, also compromised RCNMV RNA replication. These results suggest that the replication of RCNMV depends on the host membrane traffic machinery.

Hyodo, Kiwamu; Mine, Akira; Taniguchi, Takako; Kaido, Masanori; Mise, Kazuyuki; Taniguchi, Hisaaki



Survivin plays as a resistant factor against tamoxifen-induced apoptosis in human breast cancer cells.  


Tamoxifen has been the mainstay of endocrine therapy for estrogen receptor-positive breast cancer. However, approximately 40% of breast cancer patients do not respond to tamoxifen treatment. Further, most tumors eventually acquire tamoxifen resistance. Therefore, it is necessary to develop effective modalities to enhance the efficacy of tamoxifen in breast cancer treatment. In this study, we investigated the mechanism by which breast cancer cells develop resistance against tamoxifen from the viewpoint of tamoxifen-induced apoptosis. Overexpression of the anti-apoptotic molecule survivin rendered the human breast cancer cells MCF-7 resistant to tamoxifen-induced apoptosis. To examine whether the down-regulation of survivin can enhance tamoxifen-induced apoptosis, we introduced siRNA targeting the survivin gene (survivin-siRNA) into MCF-7 cells. Survivin-siRNA transfection not only induced apoptosis without tamoxifen treatment but also augmented the tamoxifen-induced apoptosis. We have previously demonstrated that 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (HRIs), which are widely used to reduce the serum cholesterol levels in hypercholesterolemia patients, decreases survivin expression in colon cancer cells. To develop a pharmacological approach for improving the efficacy of tamoxifen treatment, we determined whether HRIs can enhance tamoxifen-induced apoptosis. Lovastatin, an HRI, down-regulated the expression of survivin protein in MCF-7 cells in a dose-dependent manner. In addition, the proportion of apoptotic cells induced by the tamoxifen and lovastatin combination was greater than the theoretical additive effect. These results suggest that survivin may function as a factor inducing resistance against tamoxifen-induced apoptosis, and the combined use of tamoxifen and HRI may be a novel approach to overcome tamoxifen resistance in breast cancer. PMID:18815881

Moriai, Ryosuke; Tsuji, Naoki; Moriai, Mikako; Kobayashi, Daisuke; Watanabe, Naoki



Tumor necrosis factor plays an essential role in determining hypersensitivity pneumonitis in a mouse model.  


We examined the importance of the cytokine tumor necrosis factor-alpha (TNF-alpha) in a mouse model of hypersensitivity pneumonitis (HP). Mice of the C57BL/6 strain were instilled intranasally 3 days/wk for 3 wk with 150 micrograms of the actinomycete Faenia rectivirgula (Micropolyspora faeni) to induce HP as a model of farmer's lung. This experimental model was associated with a progressive inflammation in the lungs of challenged mice, seen histologically as cellular infiltrates of large quantities of macrophages and lymphocytes and some neutrophils. The disease in challenged mice treated with a control rabbit serum was also associated with a substantial release of tumor TNF-alpha (up to 80 U/ml of TNF-alpha in the bronchoalveolar lavage [BAL] at 3 wk after beginning of treatment) and interleukin-1, which peaked at 1 wk (approximately 300 U/ml) and diminished thereafter. A very large increase in BAL cell number (11-fold increase versus saline controls) and an enhanced release potential for TNF-alpha by alveolar macrophages was also seen. Lung fibrosis was also evident in challenged animals, as demonstrated by a 2-fold increase in hydroxyproline levels. Infusion of challenged mice with a rabbit polyclonal antibody against TNF-alpha (2 mg/wk) completely abrogated the disease, as mice so treated had normal lung histology. Anti-TNF-alpha blocked cellular recruitment in the lungs (only a 2-fold increase at week 3); it also completely abolished TNF-alpha secretion in the BAL and drastically reduced interleukin-1 levels in this fluid. Anti-TNF-alpha also abolished lung index increases and lung fibrosis, with both parameters similar to that of saline-instilled mice.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1931076

Denis, M; Cormier, Y; Fournier, M; Tardif, J; Laviolette, M



Important roles of multiple Sp1 binding sites and epigenetic modifications in the regulation of the methionine sulfoxide reductase B1 (MsrB1) promoter  

PubMed Central

Background Methionine sulfoxide reductases (Msrs) are enzymes that catalyze the reduction of oxidized methionine residues. Most organisms that were genetically modified to lack the MsrA gene have shown shortening of their life span. Methionine sulfoxide reductases B (MsrB) proteins codified by three separate genes, named MsrB1, MsrB2, and MsrB3, are included in the Msrs system. To date, the mechanisms responsible for the transcriptional regulation of MsrB genes have not been reported. The aim of this study was to investigate the regulation of MsrB1 selenoprotein levels through transcriptional regulation of the MsrB1 gene in MDA-MB231 and MCF-7 breast carcinoma cell lines. Results A MsrB1 gene promoter is located 169 base pairs upstream from the transcription start site. It contains three Sp1 binding sites which are sufficient for maximal promoter activity in transient transfection experiments. High levels of MsrB1 transcript, protein and promoter activity were detected in low metastatic MCF7 human breast cancer cells. On the contrary, very low levels of both MsrB1 transcript and promoter activity were detected in the highly metastatic counterpart MDA-MB231 cells. A pivotal role for Sp1 in the constitutive expression of the MsrB1 gene was demonstrated through transient expression of mutant MsrB1 promoter-reporter gene constructs and chromatin immunoprecipitation experiments. Since Sp1 is ubiquitously expressed, these sites, while necessary, are not sufficient to explain the patterns of gene expression of MsrB1 in various human breast cancer cells. MDA-MB231 cells can be induced to express MsrB1 by treatment with 5-Aza-2'-deoxycytidine, a demethylating agent. Therefore, the MsrB1 promoter is controlled by epigenetic modifications. Conclusion The results of this study provide the first insights into the transcriptional regulation of the human MsrB1 gene, including the discovery that the Sp1 transcription factor may play a central role in its expression. We also demonstrated that the MsrB1 promoter activity appears to be controlled by epigenetic modifications such as methylation.

De Luca, Antonella; Sacchetta, Paolo; Nieddu, Marzia; Di Ilio, Carmine; Favaloro, Bartolo



Functional polymorphisms in carboxylesterase1A2 (CES1A2) gene involves specific protein 1 (Sp1) binding sites.  


Carboxylesterase 1 (CES1) is involved in metabolic activation of a variety of prodrugs into active derivatives and plays an important role in pharmacokinetics. We previously reported that a single nucleotide polymorphism (SNP), -816A/C of the CES1A2 gene associates with the responsiveness to an angiotensin-converting enzyme (ACE) inhibitor, imidapril, whose activity is achieved by CES1. To identify relevant functional polymorphisms, we re-sequenced the CES1A2 promoter region ( approximately 1kb) in 100 Japanese hypertensive patients. Altogether 10 SNPs and one insertion/deletion (I/D) were identified, among which seven SNPs and one I/D residing between -62 and -32 were in almost complete linkage disequilibrium (D'=1.00, r2=0.97). They consisted a minor and a major haplotype, the allele frequencies of which were 22% and 74%, respectively. The minor haplotype possessed two putative Sp1 binding sites while the major haplotype did not have any Sp1 binding site. The minor haplotype had a higher transcription and Sp1 binding activities than the major haplotype, invitro. The original -816A/C was in high linkage disequilibrium with these haplotypes (D'=0.92, r2=0.85), and well agreed with the efficacy of imidapril medication. These results suggest that the Sp1 binding site variation in the CES1A2 promoter is functional, and are good candidates for the pharmacogenetic studies of CES1-activated drugs. PMID:18328811

Yoshimura, Mika; Kimura, Tomomi; Ishii, Miho; Ishii, Keisuke; Matsuura, Tadashi; Geshi, Eiichi; Hosokawa, Masakiyo; Muramatsu, Masaaki



The p65 Subunit of NF-?B Inhibits COL1A1 Gene Transcription in Human Dermal and Scleroderma Fibroblasts through Its Recruitment on Promoter by Protein Interaction with Transcriptional Activators (c-Krox, Sp1, and Sp3)*  

PubMed Central

Transcriptional mechanisms regulating type I collagen genes expression in physiopathological situations are not completely known. In this study, we have investigated the role of nuclear factor-?B (NF-?B) transcription factor on type I collagen expression in adult normal human (ANF) and scleroderma (SF) fibroblasts. We demonstrated that NF-?B, a master transcription factor playing a major role in immune response/apoptosis, down-regulates COL1A1 expression by a transcriptional control involving the ?112/?61 bp sequence. This 51-bp region mediates the action of two zinc fingers, Sp1 (specific protein-1) and Sp3, acting as trans-activators of type I collagen expression in ANF and SF. Knockdown of each one of these trans factors by siRNA confirmed the trans-activating effect of Sp1/Sp3 and the p65 subunit of NF-?B trans-inhibiting effect on COL1A1 expression. Despite no existing ?B consensus sequence in the COL1A1 promoter, we found that Sp1/Sp3/c-Krox and NF-?B bind and/or are recruited on the proximal promoter in chromatin immunoprecipitation (ChIP) assays. Attempts to elucidate whether interactions between Sp1/Sp3/c-Krox and p65 are necessary to mediate the NF-?B inhibitory effect on COL1A1 in ANF and SF were carried out; in this regard, immunoprecipitation assays revealed that they interact, and this was validated by re-ChIP. Finally, the knockdown of Sp1/Sp3/c-Krox prevents the p65 inhibitory effect on COL1A1 transcription in ANF, whereas only the siRNAs targeting Sp3 and c-Krox provoked the same effect in SF, suggesting that particular interactions are characteristic of the scleroderma phenotype. In conclusion, our findings highlight a new mechanism for COL1A1 transcriptional regulation by NF-?B, and these data could allow the development of new antifibrotic strategies.

Beauchef, Gallic; Bigot, Nicolas; Kypriotou, Magdalini; Renard, Emmanuelle; Poree, Benoit; Widom, Russell; Dompmartin-Blanchere, Anne; Oddos, Thierry; Maquart, Francois-Xavier; Demoor, Magali; Boumediene, Karim; Galera, Philippe



SP-1 regulation of MMP-9 expression requires serine-586 in the PEST domain  

PubMed Central

SYNOPSIS Rac1, a small GTPase, regulates macrophage matrix metalloproteinase-9 (MMP-9) in an ERK- and SP-1-dependent manner. SP-1 contains a PEST domain that may modulate protein stability. We hypothesize that T578, S586, and/or S587 in the PEST domain are required for SP-1 stability and MMP-9 expression secondary to activation of ERK, a serine/threonine kinase. We determined the effects of Rac1 and ERK on MMP-9 expression driven by SP-1WT and SP-1 mutants, T578A, S586A and S587A. Expression of WT and mutant SP-1 increased MMP-9 promoter activity in alveolar macrophages. However, constitutively active Rac1 suppressed MMP-9 promoter activity in cells expressing SP-1WT, SP-1T578A, and SP-1S587A, but not SP-1S586A. Furthermore, constitutive ERK activation, which was inhibited by Rac1, significantly increased MMP-9 transcription in cells expressing SP-1WT but not SP-1S586A. Because Rac1 activation and ERK inactivation increased degradation of SP-1WT and not SP-1S586A, our results suggest that SP-1 stability mediated at S586 regulates MMP-9 transcription.. In vivo, alveolar macrophages obtained from asbestosis patients had less MMP-9 that was associated with decreased SP-1 expression and ERK activation. These observations demonstrate that S586 in the PEST domain of SP-1 is important for MMP-9 gene expression in alveolar macrophages and highlight the importance of these proteins in pulmonary fibrosis.

Murthy, Shubha; Ryan, Alan J.; Carter, A. Brent



Transcriptional Protein Sp1 Regulates LEDGF Transcription by Directly Interacting with Its Cis-Elements in GC-Rich Region of TATA-Less Gene Promoter  

PubMed Central

LEDGF/p75 interacts with DNA/protein to regulate gene expression and function. Despite the recognized diversity of function of LEDGF/p75, knowledge of its transregulation is in its infancy. Here we report that LEDGF/p75 gene is TATA-less, contains GC-rich cis elements and is transcriptionally regulated by Sp1 involving small ubiquitin-like modifier (Sumo1). Using different cell lines, we showed that Sp1 overexpression increased the level of LEDGF/p75 protein and mRNA expression in a concentration-dependent fashion. In contrast, RNA interference depletion of intrinsic Sp1 or treatment with artemisinin, a Sp1 inhibitor, reduced expression of LEDGF/p75, suggesting Sp1-mediated regulation of LEDGF/p75. In silico analysis disclosed three evolutionarily conserved, putative Sp1 sites within LEDGF/p75 proximal promoter (?170/+1 nt). DNA-binding and transactivation assays using deletion and point mutation constructs of LEDGF/p75 promoter-CAT revealed that all Sp1 sites (?50/?43, ?109/?102 and ?146/?139) differentially regulate LEDGF/p75. Cotransfection studies with Sp1 in Drosophila cells that were Sp1-deficient, showed increased LEDGF/p75 transcription, while in lens epithelial cells (LECs) promoter activity was inhibited by artemisinin. These events were correlated with levels of endogenous Sp1-dependent LEDGF/p75 expression, and higher resistance to UVB-induced cell death. ChIP and transactivation assays showed that Sumoylation of Sp1 repressed its transcriptional activity as evidenced through its reduced binding to GC-box and reduced ability to activate LEDGF/p75 transcription. As whole, results revealed the importance of Sp1 in regulating expression of LEDGF/p75 gene and add to our knowledge of the factors that control LEDGF/p75 within cellular microenvironments, potentially providing a foundation for LEDGF/p75 expression-based transcription therapy.

Singh, Dhirendra P.; Bhargavan, Biju; Chhunchha, Bhavana; Kubo, Eri; Kumar, Anil; Fatma, Nigar



Scaffolding, Analysis and Materials: Contributing Factors in an Unexpected Finding of Advanced Infant/Toddler Pretend Play?  

ERIC Educational Resources Information Center

As part of a longitudinal study, infant/toddler pretend play development and maternal play modelling were investigated in dyadic context. A total of 21 children were videotaped in monthly play sessions with their mothers, from age 8 to 17 months. Child and mother pretend play frequencies and levels were measured using Brown's Pretend Play

Morrissey, Anne-Marie



Phosphorylated Sp1 is the regulator of DNA-PKcs and DNA ligase IV transcription of daunorubicin-resistant leukemia cell lines.  


Multidrug resistance (MDR) is a serious problem faced in the treatment of malignant tumors. In this study, we characterized the expression of non-homologous DNA end joining (NHEJ) components, a major DNA double strand break (DSB) repair mechanism in mammals, in K562 cell and its daunorubicin (DNR)-resistant subclone (K562/DNR). K562/DNR overexpressed major enzymes of NHEJ, DNA-PKcs and DNA ligase IV, and K562/DNR repaired DSB more rapidly than K562 after DNA damage by neocarzinostatin (MDR1-independent radiation-mimetic). Overexpressed DNA-PKcs and DNA ligase IV were also observed in DNR-resistant HL60 (HL60/DNR) cells as compared with parental HL60 cells. Expression level of DNA-PKcs mRNA paralleled its protein level, and the promoter activity of DNA-PKcs of K562/DNR was higher than that of K562, and the 5'-region between -49bp and the first exon was important for its activity. Because this region is GC-rich, we tried to suppress Sp1 family transcription factor using mithramycin A (MMA), a specific Sp1 family inhibitor, and siRNAs for Sp1 and Sp3. Both MMA and siRNAs suppressed DNA-PKcs expression. Higher serine-phosphorylated Sp1 but not total Sp1 of both K562/DNR and HL60/DNR was observed compared with their parental K562 and HL60 cells. DNA ligase IV expression of K562/DNR was also suppressed significantly with Sp1 family protein inhibition. EMSA and ChIP assay confirmed higher binding of Sp1 and Sp3 with DNA-PKcs 5'-promoter region of DNA-PKcs of K562/DNR than that of K562. Thus, the Sp1 family transcription factor affects important NHEJ component expressions in anti-cancer drug-resistant malignant cells, leading to the more aggressive MDR phenotype. PMID:24530422

Nishida, Yayoi; Mizutani, Naoki; Inoue, Minami; Omori, Yukari; Tamiya-Koizumi, Keiko; Takagi, Akira; Kojima, Tetsuhito; Suzuki, Motoshi; Nozawa, Yoshinori; Minami, Yosuke; Ohnishi, Kazunori; Naoe, Tomoki; Murate, Takashi



Sp1 Expression Is Disrupted in Schizophrenia; A Possible Mechanism for the Abnormal Expression of Mitochondrial Complex I Genes, NDUFV1 and NDUFV2  

PubMed Central

Background The prevailing hypothesis regards schizophrenia as a polygenic disease, in which multiple genes combine with each other and with environmental stimuli to produce the variance of its clinical symptoms. We investigated whether the ubiquitous transcription factor Sp1 is abnormally expressed in schizophrenia, and consequently can affect the expression of genes implicated in this disorder. Methodology/Principal Findings mRNA of Sp1 and of mitochondrial complex I subunits (NDUFV1, NDUFV2) was analyzed in three postmortem brain regions obtained from the Stanley Foundation Brain Collection, and in lymphocytes of schizophrenic patients and controls. Sp1 role in the transcription of these genes was studied as well. Sp1 was abnormally expressed in schizophrenia in both brain and periphery. Its mRNA alteration pattern paralleled that of NDUFV1 and NDUFV2, decreasing in the prefrontal cortex and the striatum, while increasing in the parieto-occipital cortex and in lymphocytes of schizophrenic patients as compared with controls. Moreover, a high and significant correlation between these genes existed in normal subjects, but was distorted in patients. Sp1 role in the regulation of complex I subunits, was demonstrated by the ability of the Sp1/DNA binding inhibitor, mithramycin, to inhibit the transcription of NDUFV1 and NDUFV2, in neuroblastoma cells. In addition, Sp1 activated NDUFV2 promoter by binding to its three GC-boxes. Both activation and binding were inhibited by mithramycin. Conclusions/Significance These findings suggest that abnormality in Sp1, which can be the main activator/repressor or act in combination with additional transcription factors and is subjected to environmental stimuli, can contribute to the polygenic and clinically heterogeneous nature of schizophrenia.

Ben-Shachar, Dorit; Karry, Rachel



v-Jun downregulates the alpha 2 (I) collagen target gene indirectly through Sp1/3.  


Transformation of chick embryo fibroblasts (CEFs) by the v-Jun oncoprotein correlates with a downregulation of the alpha 2 (I) collagen gene. To investigate whether this gene constitutes a direct target of v-Jun, an analysis of a large proximal fragment of the promoter, extending from position -1080 to +109, was performed. Transient transfections with -1080/+109 and deleted derivatives revealed that a short proximal fragment, -433/+11, is the target for repression by v-Jun. Extensive analysis, conducted in CEFs and in Sp1/3-deficient Drosophila SL2 cells, further showed that (i) high constitutive activity of -433/+11 requires a direct binding of the ubiquitous Sp1 and/or Sp3 transcription factors acting on two distinct motifs, that is, a proximal TCC-rich region and an upstream GC box, and that (ii) repression by v-Jun does not require any direct binding of the oncoprotein to the DNA, but an indirect binding within a v-Jun-Sp1/3-DNA chromatin-associated complex. This situation is reminiscent of a situation previously reported with the tata-less, SPARC (secreted protein, acidic, and rich in cysteine) target promoter that regulates the expression of another extracellular matrix component in the same model of cell transformation. Taken together, these data reinforce the view that, at least in CEFs, v-Jun downregulates a family of direct target genes by binding to the DNA indirectly through Sp1/3. PMID:15735704

Chamboredon, Sandrine; Castellazzi, Marc



A formal anthropological view of motivation models of problematic MMO play: achievement, social, and immersion factors in the context of culture.  


Yee (2006) found three motivational factors-achievement, social, and immersion-underlying play in massively multiplayer online role-playing games ("MMORPGs" or "MMOs" for short). Subsequent work has suggested that these factors foster problematic or addictive forms of play in online worlds. In the current study, we used an online survey of respondents (N?=?252), constructed and also interpreted in reference to ethnography and interviews, to examine problematic play in the World of Warcraft (WoW; Blizzard Entertainment, 2004-2013). We relied on tools from psychological anthropology to reconceptualize each of Yee's three motivational factors in order to test for the possible role of culture in problematic MMO play: (a) For achievement, we examined how "cultural consonance" with normative understandings of success might structure problematic forms of play; (b) for social, we analyzed the possibility that developing overvalued virtual relationships that are cutoff from offline social interactions might further exacerbate problematic play; and (c) in relation to immersion, we examined how "dissociative" blurring of actual- and virtual-world identities and experiences might contribute to problematic patterns. Our results confirmed that compared to Yee's original motivational factors, these culturally sensitive measures better predict problematic forms of play, pointing to the important role of sociocultural factors in structuring online play. PMID:23690445

Snodgrass, Jeffrey G; Dengah, H J Francois; Lacy, Michael G; Fagan, Jesse



An Active Factor from Tomato Root Exudates Plays an Important Role in Efficient Establishment of Mycorrhizal Symbiosis  

PubMed Central

Root exudates play an important role in the early signal exchange between host plants and arbuscular mycorrhizal fungi. M161, a pre-mycorrhizal infection (pmi) mutant of the tomoto (Solanum lycopersicum) cultivar Micro-Tom, fails to establish normal arbuscular mycorrhizal symbioses, and produces exudates that are unable to stimulate hyphal growth and branching of Glomus intraradices. Here, we report the identification of a purified active factor (AF) that is present in the root exudates of wild-type tomato, but absent in those of M161. A complementation assay using the dual root organ culture system showed that the AF could induce fungal growth and branching at the pre-infection stage and, subsequently, the formation of viable new spores in the M161 background. Since the AF-mediated stimulation of hyphal growth and branching requires the presence of the M161 root, our data suggest that the AF is essential but not sufficient for hyphal growth and branching. We propose that the AF, which remains to be chemically determined, represents a plant signal molecule that plays an important role in the efficient establishment of mycorrhizal symbioses.

Sun, Shubin; Wang, Jingjing; Zhu, Lingling; Liao, Dehua; Gu, Mian; Ren, Lixuan; Kapulnik, Yoram; Xu, Guohua



Differential regulation of MMPs by E2F1, Sp1 and NF-kappa B controls the small cell lung cancer invasive phenotype  

PubMed Central

Background E2F1 transcription factor plays a vital role in the regulation of diverse cellular processes including cell proliferation, apoptosis, invasion and metastasis. E2F1 overexpression has been demonstrated in small cell lung cancer (SCLC), and extensive metastasis in early phase is the most important feature of SCLC. In this study, we investigated the involvement of E2F1 in the process of invasion and metastasis in SCLC by regulating the expression of matrix metalloproteinases (MMPs). Methods Immunohistochemistry was performed to evaluate the expression of E2F1 and MMPs in SCLC samples in a Chinese Han population. The impact of E2F1 on invasion and metastasis was observed by transwell and wound healing experiments with depletion of E2F1 by specific siRNA. The target genes regulated by E2F1 were identified by chromatin immunoprecipitation (ChIP)-to-sequence, and the expressions of target genes were detected by real time PCR and western blotting. The dual luciferase reporter system was performed to analyze the regulatory relationship between E2F1 and MMPs. Results E2F1 is an independent and adverse prognosis factor that is highly expressed in SCLC in a Chinese Han population. Knockdown of E2F1 by specific siRNA resulted in the downregulation of migration and invasion in SCLC. The expressions of MMP-9 and ?16 in SCLC were higher than other MMPs, and their expressions were most significantly reduced after silencing E2F1. ChIP-to-sequence and promoter-based luciferase analysis demonstrated that E2F1 directly controlled MMP-16 expression via an E2F1 binding motif in the promoter. Although one E2F1 binding site was predicted in the MMP-9 promoter, luciferase analysis indicated that this binding site was not functionally required. Further study demonstrated that E2F1 transcriptionally controlled the expression of Sp1 and p65, which in turn enhanced the MMP-9 promoter activity in SCLC cells. The associations between E2F1, Sp1, p65, and MMP-9 were validated by immunohistochemistry staining in SCLC tumors. Conclusions E2F1 acts as a transcriptional activator for MMPs and directly enhances MMP transcription by binding to E2F1 binding sequences in the promoter, or indirectly activates MMPs through enhanced Sp1 and NF-kappa B as a consequence of E2F1 activation in SCLC.



Cadmium down-regulates human OGG1 through suppression of Sp1 activity.  


Cadmium is a well known human and animal carcinogen and is a ubiquitous contaminant in the environment. Although the carcinogenic mechanism of cadmium is a multifactorial process, oxidative DNA damage is believed to be of prime importance. In particular, cadmium suppresses the capacity of cells to repair oxidative DNA damage. In this study, cadmium treatment led to a significant increase in gamma-ray-induced 8-oxoguanine (8-oxoG) formation. Western blotting and semiquantitative reverse transcription-PCR revealed that cadmium treatment caused a decrease in the expression level of human OGG1 (8-oxoguanine-DNA glycosylase-1; hOGG1) in human fibroblast GM00637 and HeLa S3 cells. In addition, the cadmium-mediated decrease in hOGG1 transcription was the result of decreased binding of the transcription factor Sp1 to the hOGG1 promoter. Finally, we show that an increase in the functional hOGG1 expression level could inhibit the cadmium-mediated increase in gamma-ray-induced 8-oxoG accumulation as well as in gamma-radiation-induced mutation frequency at the HPRT (hypoxanthine-guanine phosphoribosyltransferase) gene locus. These results suggest that cadmium attenuates removal of gamma-ray-induced 8-oxoG adducts, which in turn increases the mutation frequency, and that this effect might, at least in part, result from suppression of hOGG1 transcription via inactivation of Sp1 as a result of cadmium treatment. PMID:15760895

Youn, Cha-Kyung; Kim, Soo-Hyun; Lee, Do Young; Song, Seung Hee; Chang, In-Youb; Hyun, Jin-Won; Chung, Myung-Hee; You, Ho Jin



ERK3 Promotes Endothelial Cell Functions by Upregulating SRC-3/SP1-Mediated VEGFR2 Expression.  


Despite a regain of interest recently in ERK3 kinase signaling, the molecular regulations of both ERK3 gene expression and protein kinase activity are still largely unknown. While it is shown that disruption of ERK3 gene causes neonatal lethality, cell type-specific functions of ERK3 signaling remain to be explored. In this study, we report that ERK3 gene expression is upregulated by cytokines through c-Jun in endothelial cells; c-Jun binds to the ERK3 gene and regulates its transcription. We further reveal a new role for ERK3 in regulating endothelial cell migration, proliferation and tube formation by upregulating SRC-3/SP-1-mediated VEGFR2 expression. The underlying molecular mechanism involves ERK3-stimulated formation of a transcriptional complex involving coactivator SRC-3, transcription factor SP-1 and the secondary coactivator CBP. Taken together, our study identified a molecular regulatory mechanism of ERK3 gene expression and revealed a previously unknown role of ERK3 in regulating endothelial cell functions. J. Cell. Physiol. 229: 1529-1537, 2014. © 2014 Wiley Periodicals, Inc. PMID:24585635

Wang, Wei; Bian, Ka; Vallabhaneni, Sreeram; Zhang, Bin; Wu, Ray-Chang; O'Malley, Bert W; Long, Weiwen



Occupancy and synergistic activation of the FMR1 promoter by Nrf-1 and Sp1 in vivo  

Microsoft Academic Search

Fragile X syndrome is due to mutation of the FMR1 gene. The most common mutation is an expansion of a CGG repeat in the 50 UTR that triggers dense DNA methylation and formation of a heterochromatin-like structure which lead to transcriptional silencing. In vitro experiments have identified several transcription factors, including Sp1, Nrf-1 and USF1\\/2, as potential regulators of normal

Karen T. Smith; Daniel Reines



Myogenic effect of SP-1f and SP-1h two novel ?3-adrenoceptor (?3-AR) agonists in human colonic circular smooth muscle.  


The effect of two novel ?3-adrenoceptor (?3-AR) agonists SP-1f and SP-1h on human colon circular smooth muscle contractility and ?3-AR mRNA expression have been determined. ?3-AR is ascertained co-participates to the control of the gut motility. Isometric tension on human colon muscle strips was measured in response to increasing concentrations of SP-1f, SP-1h and (-)-isoprenaline, alone and in the presence of Betaxolol, ICI 11,855 and SR 59230A (?1-, ?2- and ?3-AR antagonists, respectively). (-)-Isoprenaline concentration-dependently relaxed circular muscle strips with an EC50=0.32±0.06?M. Such an effect was antagonized either by the contemporaneously presence of Betaxolol and ICI 11,855 [(-)-isoprenaline EC50=1.75±0.35?M, pKB=7.88±0.10] or by Betaxolol, ICI 11,855 and SR 59230A [(-)-isoprenaline EC50=3.49±0.38?M, pKB=8.51±0.14]. Besides, SP-1f and SP-1h concentration-dependently relaxed circular muscle strips with an EC50=0.35±0.07?M and 0.45±0.12?M, respectively. These values remained unchanged by blocking the ?1- and ?2-AR. The presence of SR 59230A antagonized the relaxing effect of SP-1f (EC50=3.51±0.94?M, pKB=8.93±0.16) and did not modify the SP-1h relaxing potency. In colon circular smooth muscle and in mucosa, ?3-AR mRNA expression levels were found to be 0.39±0.70 and 0.26±0.12 (P<0.05), respectively. Such results provide further evidence of the ?3-adrenoceptor functional role in the human colon and the crucial contribution of SP-1f to the control of the gut dysmotility. PMID:24275352

Maselli, Maria Antonietta; Trisolini, Piero; Ignazzi, Antonia; Notarnicola, Maria; Lorusso, Dionigi; Pezzolla, Francesco; Iacobazzi, Dominga; Scilimati, Antonio; Perrone, Maria Grazia



Genetic Variant of C-5312T Can Change Binding Pattern of Sp1 to Renin Enhancer that are Very Likely to Affect Renin Gene Expression  

PubMed Central

Renin distal enhancer plays a pivotal role in renin gene expression, and the genetic variants C-5312T of renin enhancer can affect renin gene transcription level. However, the mechanism associated with the transcription level changes remains unknown. Therefore, it is of interest to investigate the possible role of distal enhancer in regulating the expression of renin gene. Single nucleotide polymorphism in renin distal enhancer was identified in 34 hypertensive patients by automatic sequencing. The data showed that the renin enhancer from the patients have genetic variants C-5312T or C-5312T SNP. Hence, the functionality of the renin enhancer and influence of the genetic variants C-5312T on binding to Sp1 is studied. These results from the binding study suggested that Sp1 binds to the DNA in GC rich region. Thus, the genetic variant C-5312T has changed the binding pattern of Sp1 to renin enhancer. This is likely to influence Sp1 activity to stimulate the expression of renin gene. The binding of Sp1 to the cis-element will enhance transcription of renin gene. Thus, polymorphism within C-5312T might contribute to the reduction of renin transcription.

Lukitasari, Mifetika; Putri, Jayarani F; Choiriyah, Muladefi; Rohman, Mohammad Saifur; Widodo, Nashi



Transcription Factor ?B Plays an Important Role in the Production of Extracellular Membrane-Derived Vesicles in Listeria monocytogenes  

PubMed Central

Gram-negative bacteria produce extracellular outer membrane vesicles (OMVs) that interact with host cells. Unlike Gram-negative bacteria, less is known about the production and role of extracellular membrane vesicles (MVs) in Gram-positive bacteria. The food-borne pathogen Listeria monocytogenes can survive under extreme environmental and energy stress conditions and the transcription factor ?B is involved in this survival ability. Here, we first determined the production of MVs from L. monocytogenes and evaluated whether general stress transcription factor ?B affected production of MVs in L. monocytogenes. L. monocytogenes secreted MVs during in vitro broth culture. The wild-type strain actively produced MVs approximately nine times more and also produced more intact shapes of MVs than those of the isogenic ?sigB mutant. A proteomic analysis showed that 130 and 89 MV proteins were identified in the wild-type and ?sigB mutant strains, respectively. Wild-type strain-derived MVs contained proteins regulated by ?B such as transporters (OpuCA and OpuCC), stress response (Kat), metabolism (LacD), translation (InfC), and cell division protein (FtsZ). Gene Ontology (GO) enrichment analysis showed that wild-type-derived MV proteins corresponded to several GO terms, including response to stress (heat, acid, and bile resistance) and extracellular polysaccharide biosynthetic process, but not the ?sigB mutant. Internalin B (InlB) was almost three times more contained in MVs derived from the wild-type strain than in MVs derived from the ?sigB mutant. Taken together, these results suggest that ?B plays a pivotal role in the production of MVs and protein profiles contained in MVs. L. monocytogenes MVs may contribute to host infection and survival ability under various stressful conditions.

Lee, Jung Hwa; Choi, Chi-Won; Lee, Taewon; Kim, Seung Il; Lee, Je-Chul; Shin, Ji-Hyun



Mitochondria-Localized Glutamic Acid-Rich Protein (MGARP) Gene Transcription Is Regulated by Sp1  

PubMed Central

Background Mitochondria-localized glutamic acid-rich protein (MGARP) is a novel mitochondrial transmembrane protein expressed mainly in steroidogenic tissues and in the visual system. Previous studies showed that MGARP functions in hormone biosynthesis and its expression is modulated by the HPG axis. Methodology/Principal Findings By bioinformatics, we identified two characteristic GC-rich motifs that are located proximal to the transcription start site (TSS) of MGARP, and each contains two Specificity protein 1 (Sp1) binding elements. We then determined that the ?3 kb proximal MGARP promoter is activated in a Sp1-dependent manner using reporter assays and knockdown of Sp1 led to decreased expression of endogenous MGARP messages. We also demonstrated that one of the two GC-rich motifs, GC-Box1, harbors prominent promoter activity mediated by Sp1, and that it requires both GC boxes for full transcriptional activation. These findings suggest a dominant role for these GC boxes and Sp1 in activating the MGARP promoter through a synergistic mechanism. Consistently, the results of an Electrophoretic Mobility Gel Shift Assay (EMSA) and Chromatin Immunoprecipitation (ChIP) confirmed that Sp1 specifically interacts with the GC-rich region. We further found that estrogen receptor ? (ER?), a known Sp1 co-activator, could potentiate GC-boxes containing MGARP promoter activity and this effect is mediated by Sp1. Knockdown of Sp1 significantly diminished the MGARP promoter transactivation and the expression of endogenous MGARP mediated by both Sp1 and ER?. Conclusions/Significance The present study identified a proximal core sequence in the MGARP promoter that is composed of two enriched Sp1 binding motifs and established Sp1 as one major MGARP transactivator whose functions are synergistic with ER?, providing a novel understanding of the mechanisms of MGARP gene transcriptional regulation.

Jin, Da; Li, Rui; Mao, Dongxue; Luo, Nan; Wang, Yifeng; Chen, Shaoyong; Zhang, Shuping



First functional polymorphism in CFTR promoter that results in decreased transcriptional activity and Sp1/USF binding  

SciTech Connect

Growing evidences show that functionally relevant polymorphisms in various promoters alter both transcriptional activity and affinities of existing protein-DNA interactions, and thus influence disease progression in humans. We previously reported the -94G>T CFTR promoter variant in a female CF patient in whom any known disease-causing mutation has been detected. To investigate whether the -94G>T could be a regulatory variant, we have proceeded to in silico analyses and functional studies including EMSA and reporter gene assays. Our data indicate that the promoter variant decreases basal CFTR transcriptional activity in different epithelial cells and alters binding affinities of both Sp1 and USF nuclear proteins to the CFTR promoter. The present report provides evidence for the first functional polymorphism that negatively affects the CFTR transcriptional activity and demonstrates a cooperative role of Sp1 and USF transcription factors in transactivation of the CFTR gene promoter.

Taulan, M. [INSERM U827, Laboratoire de Genetique de Maladies Rares, Montpellier (France); Universite Montpellier1, UFR de Medecine, Montpellier (France); CHU Montpellier, Hopital Arnaud de Villeneuve, Laboratoire de Genetique Moleculaire, Montpellier F-34000 (France)], E-mail:; Lopez, E. [INSERM U827, Laboratoire de Genetique de Maladies Rares, Montpellier (France); Universite Montpellier1, UFR de Medecine, Montpellier (France); Guittard, C. [INSERM U827, Laboratoire de Genetique de Maladies Rares, Montpellier (France); CHU Montpellier, Hopital Arnaud de Villeneuve, Laboratoire de Genetique Moleculaire, Montpellier F-34000 (France); Rene, C. [INSERM U827, Laboratoire de Genetique de Maladies Rares, Montpellier (France); Universite Montpellier1, UFR de Medecine, Montpellier (France); CHU Montpellier, Hopital Arnaud de Villeneuve, Laboratoire de Genetique Moleculaire, Montpellier F-34000 (France); Baux, D.; Altieri, J.P.; DesGeorges, M. [INSERM U827, Laboratoire de Genetique de Maladies Rares, Montpellier (France); CHU Montpellier, Hopital Arnaud de Villeneuve, Laboratoire de Genetique Moleculaire, Montpellier F-34000 (France); Claustres, M. [INSERM U827, Laboratoire de Genetique de Maladies Rares, Montpellier (France); Universite Montpellier1, UFR de Medecine, Montpellier (France); CHU Montpellier, Hopital Arnaud de Villeneuve, Laboratoire de Genetique Moleculaire, Montpellier F-34000 (France); Romey, M.C. [INSERM U827, Laboratoire de Genetique de Maladies Rares, Montpellier (France); Universite Montpellier1, UFR de Medecine, Montpellier (France)



City Play.  

ERIC Educational Resources Information Center

Today, fewer city blocks preserve the confidence of lifestyle and urban geography that sustain traditional games and outdoor play. Large groups of children choosing sides and organizing Red Rover games are no longer commonplace. Teachers must encourage free play; urban planners must build cities that are safe play havens. (MLH)

Dargan, Amanda; Zeitlin, Steve



A Novel SP1/SP3 Dependent Intronic Enhancer Governing Transcription of the UCP3 Gene in Brown Adipocytes  

PubMed Central

Uncoupling protein (UCP) 3 is a mitochondrial inner membrane protein implicated in lipid handling and metabolism of reactive oxygen species. Its transcription is mainly regulated by peroxisome proliferator-activated receptors (PPAR), a family of nuclear hormone receptors. Employing bandshift assays, RNA interference and reporter gene assays we examine an intronic region in the UCP3 gene harboring a cis-element essential for expression in brown adipocytes. We demonstrate binding of SP1 and SP3 to this element which is adjacent to a direct repeat 1 element mediating activation of UCP3 expression by PPAR? agonists. Transactivation mediated by these elements is interdependent and indispensable for UCP3 expression. Systematic deletion uncovered a third binding element, a putative NF1 site, in close proximity to the SP1/3 and PPAR? binding elements. Data mining demonstrated binding of MyoD and Myogenin to this third element in C2C12 cells, and, furthermore, revealed recruitment of p300. Taken together, this intronic region is the main enhancer driving UCP3 expression with SP1/3 and PPAR? as the core factors required for expression.

Hoffmann, Christoph; Zimmermann, Anika; Hinney, Anke; Volckmar, Anna-Lena; Jarrett, Harry W.; Fromme, Tobias; Klingenspor, Martin



Association of Sp1 tandem repeat polymorphism of ALOX5 with coronary artery disease in Indian subjects.  


Lipoxygenases have been implicated in the pathogenesis of coronary artery disease (CAD) for its potent proinflammatory role. The Sp1 addition/deletion polymorphism in promoter region of the 5-lipoxygenase gene (ALOX5) has been associated with increased risk of carotid atherosclerosis and myocardial infarction. To determine the role of this polymorphism in our population we performed a case-control-genetic association study on 117 healthy controls and 119 angiographically verified CAD patients. Biochemical analysis was performed using standard automated assays. High-density lipoprotein cholesterol (HDL-C) and LDL-C subfraction levels were estimated using precipitation methods. Genotyping of polymorphism in the ALOX5 (Sp1 variants) was done using PCR-based heteroduplex analysis and automated sequencing. The Sp1 promoter repeat variants were found to be associated with CAD (p < 0.0001, OR = 4.47, 95% confidence interval = 2.58-7.74). Furthermore, the 5/5 genotype of the ALOX5 polymorphism in the healthy subjects was found to be associated with elevated HDL-C (p= 0.004), HDL(3) -C (p= 0.04), apo A1 (p= 0.011) and sdLDL (p= 0.001). We conclude that this polymorphism influences LDL and HDL subfraction levels and is a risk factor for CAD in our population. Clin Trans Sci 2012; Volume 5: 408-411. PMID:23067353

Todur, Seema P; Ashavaid, Tester F



Mutant MT-SP1 proteases with altered substrate specificity or activity  

US Patent & Trademark Office Database

MT-SP1 mutein proteases with altered specificity for the target molecules they cleave can be used to treat human diseases, such as cancer. Cleaving VEGF or VEGFR at certain substrate sequences with wild-type and mutein MT-SP1 proteases can be used to treat pathologies associated with angiogenesis.



Serum concentration of pregnancy-specific ? -1-glycoprotein (SP1) in normal and pathologic pregnancies  

Microsoft Academic Search

In 50 females with normal pregnancies serum concentrations of the pregnancy-specific protein SP-1 were assayed by single radial immunodiffusion (Mancini) from the 20th week of gestation onwards. The total of 283 single tests revealed that serum SP-1 concentrations steadily increased after the 20th week of gestation to reach a plateau in the 37th week.

G. Tatra; G. Breitenecker; W. Gruber



Cyclin A regulates a cell-cycle-dependent expression of CKAP2 through phosphorylation of Sp1  

SciTech Connect

Highlights: Black-Right-Pointing-Pointer We identified a GC box and a CHR element in human CKAP2 minimal promoter. Black-Right-Pointing-Pointer The CHR element repressed the CKAP2 minimal promoter activity at the G1/S phase. Black-Right-Pointing-Pointer The GC box was essential for the basic promoter activity of human CKAP2. Black-Right-Pointing-Pointer The GC box was also essential for the cyclic expression of human CKAP2. Black-Right-Pointing-Pointer The phosphorylation of Sp1, mediated by Cyclin A, underlies the cyclic expression. -- Abstract: CKAP2 plays crucial roles in proper chromosome segregation and maintaining genomic stability. CKAP2 protein showed cell-cycle-dependent expression, which reached a maximum level at the G2/M phase and disappeared at the onset of G1 phase. To elucidate the mechanisms underlying cell cycle-dependent expression of CKAP2, we cloned and analyzed the human CKAP2 promoter. The upstream 115-bp region from the transcription start site was sufficient for minimal CKAP2 promoter activity. We identified 2 regulatory sequences; a CHR (-110 to -104 bp) and a GC box (-41 to -32 bp). We confirmed Sp1 bound to the GC box using a supershift assay and a ChIP assay. Mutation in the GC box resulted in a near complete loss of CKAP2 promoter activity while mutation in the CHR decreased the promoter activity by 50%. The CHR mutation showed enhanced activity at the G1/S phase, but still retained cyclic activity. The Chromatin IP revealed that the amount of Sp1 bound to the GC box gradually increased and reached a maximum level at the G2/M phase. The amount of Sp1 bound to the GC box was greatly reduced when Cyclin A was depleted, which was restored by adding Cyclin A/Cdk2 complex back into the nuclear extracts. Together, we concluded that the GC box was responsible for the cyclic activity of human CKAP2 promoter through the phosphorylation of Sp1, possibly by Cyclin A/Cdk complex.

Kang, Du-Seock [Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440-746 (Korea, Republic of)] [Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440-746 (Korea, Republic of); Hong, Kyeong-Man [Research Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang 410-769 (Korea, Republic of)] [Research Institute, National Cancer Center, 323 Ilsan-ro, Ilsandong-gu, Goyang 410-769 (Korea, Republic of); Park, Joobae [Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440-746 (Korea, Republic of)] [Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440-746 (Korea, Republic of); Bae, Chang-Dae, E-mail: [Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440-746 (Korea, Republic of)] [Department of Molecular Cell Biology and Samsung Biomedical Research Institute, Sungkyunkwan University School of Medicine, 300 Chunchundong, Jangangu, Suwon 440-746 (Korea, Republic of)



Alu elements contain many binding sites for transcription factors and may play a role in regulation of developmental processes  

PubMed Central

Background The human genome contains over one million Alu repeat elements whose distribution is not uniform. While metabolism-related genes were shown to be enriched with Alu, in structural genes Alu elements are under-represented. Such observations led researchers to suggest that Alu elements were involved in gene regulation and were selected to be present in some genes and absent from others. This hypothesis is gaining strength due to findings that indicate involvement of Alu elements in a variety of functions; for example, Alu sequences were found to contain several functional transcription factor (TF) binding sites (BSs). We performed a search for new putative BSs on Alu elements, using a database of Position Specific Score Matrices (PSSMs). We searched consensus Alu sequences as well as specific Alu elements that appear on the 5 Kbp regions upstream to the transcription start site (TSS) of about 14000 genes. Results We found that the upstream regions of the TSS are enriched with Alu elements, and the Alu consensus sequences contain dozens of putative BSs for TFs. Hence several TFs have Alu-associated BSs upstream of the TSS of many genes. For several TFs most of the putative BSs reside on Alu; a few of these were previously found and their association with Alu was also reported. In four cases the fact that the identified BSs resided on Alu went unnoticed, and we report this association for the first time. We found dozens of new putative BSs. Interestingly, many of the corresponding TFs are associated with early markers of development, even though the upstream regions of development-related genes are Alu-poor, compared with translational and protein biosynthesis related genes, which are Alu-rich. Finally, we found a correlation between the mouse B1 and human Alu densities within the corresponding upstream regions of orthologous genes. Conclusion We propose that evolution used transposable elements to insert TF binding motifs into promoter regions. We observed enrichment of biosynthesis genes with Alu-associated BSs of developmental TFs. Since development and cell proliferation (of which biosynthesis is an essential component) were proposed to be opposing processes, these TFs possibly play inhibitory roles, suppressing proliferation during differentiation.

Polak, Paz; Domany, Eytan



Activation of nuclear factor erythroid 2-related factor 2 and PPAR? plays a role in the genistein-mediated attenuation of oxidative stress-induced endothelial cell injury.  


We investigate the cytoprotective effects and the molecular mechanism of genistein in oxidative stress-induced injury using an endothelial cell line (EA.hy926). An oxidative stress model was established by incubating endothelial cells with H?O?. According to the present results, genistein pretreatment protected endothelial cells against H?O?-induced decreases in cell viability and increases in apoptosis. Genistein also prevented the inhibition of B-cell lymphoma 2 and the activation of caspase-3 induced by H?O?. Genistein increased superoxide dismutase (SOD), catalase (CAT) and glutathione (GSH) levels and attenuated the decrease in these antioxidants during oxidative stress. We also found that genistein induced the promoter activity of both nuclear factor erythroid 2-related factor 2 (Nrf2) and PPAR?. Additionally, genistein induced the nuclear translocation of Nrf2 and PPAR?. While genistein caused the up-regulation of both Nrf2 and PPAR?, it also activated and up-regulated the protein expression and transcription of a downstream protein, haem oxygenase-1 (HO-1). Moreover, the use of Nrf2 small interfering RNA transfection and HO-1- or PPAR?-specific antagonists (Znpp and GW9662, respectively) blocked the protective effects of genistein on endothelial cell viability during oxidative stress. Therefore, we conclude that oxidative stress-induced endothelial cell injury can be attenuated by treatment with genistein, which functions via the regulation of the Nrf2 and PPAR? signalling pathway. Additionally, the endogenous antioxidants SOD, CAT and GSH appear to play a role in the antioxidant activity of genistein. The present findings suggest that the beneficial effects of genistein involving the activation of cytoprotective antioxidant genes may represent a novel strategy in the prevention and treatment of cardiovascular endothelial damage. PMID:22716961

Zhang, Ting; Wang, Fan; Xu, Hong-Xia; Yi, Long; Qin, Yu; Chang, Hui; Mi, Man-Tian; Zhang, Qian-Yong



Playing Shakespeare.  

ERIC Educational Resources Information Center

Describes a yearlong project at 12 Catholic middle schools in the Diocese of Arlington, Virginia, to incorporate the plays of William Shakespeare into the curriculum. Teachers attended university lectures and directed students in performances of the plays. Concludes that Shakespeare can be understood and enjoyed by middle school students. (BCY)

Bashian, Kathleen Ryniker



Shadow Play  

ERIC Educational Resources Information Center

A bunny rabbit playfully hops across the wall. Then hands realign and fingers shift to make a hawk soar toward the ceiling. Most children have enjoyed the delightful experience of playing with shadow puppets. The authors build on this natural curiosity to help students link shadows to complex astronomical concepts such as seasons. The…

Trundle, Kathy Cabe; Hilson, Margilee P.



Playing war  

Microsoft Academic Search

This paper argues that war video games are transitional spaces that connect players to the ‘war on terror’. It explores the pervasive influence of militarism in video games and how the US Army is enlisting play as an active force in blurring the distinctions between civilian and soldier. The paper begins by theorizing what exactly it means to ‘play’, and

Ian Graham Ronald Shaw



Why people continue to play online games: in search of critical design factors to increase customer loyalty to online contents.  


As people increasingly play online games, numerous new features have been proposed to increase players' log-on time at online gaming sites. However, few studies have investigated why people continue to play certain online games or which design features are most closely related to the amount of time spent by players at particular online gaming sites. This study proposes a theoretical model using the concepts of customer loyalty, flow, personal interaction, and social interaction to explain why people continue to play online network games. The study then conducts a large-scale survey to validate the model. Finally, it analyzes current online games to identify design features that are closely related to the theoretical concepts. The results indicate that people continue to play online games if they have optimal experiences while playing the games. This optimal experience can be attained if the player has effective personal interaction with the system or pleasant social interactions with other people connected to the Internet. Personal interaction can be facilitated by providing appropriate goals, operators and feedback; social interaction can be facilitated through appropriate communication places and tools. This paper ends with the implications of applying the study results to other domains such as e-commerce and cyber communities. PMID:15006164

Choi, Dongseong; Kim, Jinwoo



Triplex formation prevents Sp1 binding to the dihydrofolate reductase promoter.  


The human dihydrofolate reductase (DHFR) promoter sequence contains two consensus binding sites for the Sp1 regulatory protein. We have determined the effect of intermolecular triplex DNA formation on Sp1 binding to the DHFR promoter. The DHFR Sp1 binding site I (-39 to -48 relative to the DHFR transcription start site) demonstrates concentration-dependent triplex formation with a 19-base pair G-rich oligonucleotide (GR19) which is complementary to the polypyrimidine strand. DNase I footprint analysis demonstrates that GR19 forms a DNA triplex structure with the DHFR promoter fragment in a sequence-specific manner. DNase I footprinting analysis also indicates that the orientation of binding of these G-rich oligonucleotides is antiparallel. CR19, a C-rich complementary oligonucleotide, on the other hand, does not form triplex. The DNase I protection pattern of DHFR promoter fragment incubated with both recombinant Sp1 and triplex-forming oligonucleotide suggests that triplex formation prevents Sp1 binding. This is confirmed by gel shift analysis which demonstrates that triplex formation by the Sp1 binding sequences of the DHFR promoter prevents recombinant Sp1 binding in a concentration-dependent manner. These results demonstrate that intermolecular triplex formation prevents regulatory protein binding in a sequence-specific manner. PMID:1597451

Gee, J E; Blume, S; Snyder, R C; Ray, R; Miller, D M



Nuclear Factor-kB Plays an Essential Role in the Late Phase of Ischemic Preconditioning in Conscious Rabbits  

Microsoft Academic Search

Although it is recognized that late preconditioning (PC) results from upregulation of cardioprotective genes, the specific transcription factor(s) that govern this genetic adaptation remains unknown. The aim of this study was to test the hypothesis that the development of late PC is mediated by nuclear factor-kB (NF-kB) and to elucidate the mechanisms that control the activation of NF-kB after an

Yu-Ting Xuan; Xian-Liang Tang; Supratim Banerjee; Hitoshi Takano; Richard C. X. Li; Hui Han; Yumin Qiu; Jian-Jun Li; Roberto Bolli



Interplay Between Egr-1 and Sp1 Critical for 13-cis Retinoic Acid Mediated Transcriptional Repression of Angiotensin Type 1A Receptor  

PubMed Central

Recently, we have demonstrated that 13-cis retinoic acid (13cRA) down-regulates rat angiotensin type 1A receptor (AT1AR) gene transcription through a MAP Kinase (ERK1/2) dependent mechanism in rat liver epithelial and aortic smooth muscle cells. However, the exact mechanism remained unknown. In this study, we determined the signaling intermediates activated by ERK1/2 involved in 13cRA mediated AT1AR down-regulation. Serially deleted rat AT1AR promoter CAT constructs indicate fragments containing a region ?2541 and ?1836 bp upstream of the 5 prime possess an Sp1 consensus sequence (5?-TGGGGCGGGGCGGGG-3?) have reduced CAT activity. Mobility shift analysis using untreated nuclear extracts in the presence of mithramycin A suggest the trans-acting factor binding to this cis-acting element is Sp1. 13cRA significantly reduced specific binding without any change in Sp1 protein expression. Studies showed 13cRA maximally phosphorylates and tranlocates to the nucleus ERK1/2 within 5–10 minutes, activating Egr-1 mRNA expression at 20 minutes followed by de novo protein synthesis, leading to an Egr-1/Sp1 interaction. siRNA silencing of Egr-1 restored AT1AR mRNA and protein expression in 13cRA treated cells, and Sp1 silencing results in complete loss of AT1AR expression. Our study suggests that 13cRA mediated activation of ERK1/2, through Egr-1, is capable of disrupting Sp1, the requisite transactivator for AT1AR expression, providing a novel paradigm in AT1AR gene transcription.

Snyder, Russell; Thekkumkara, Thomas



Adiponectin upregulates prolyl-4-hydroxylase ?1 expression in interleukin 6-stimulated human aortic smooth muscle cells by regulating ERK 1/2 and Sp1.  


Adiponectin is an anti-atherogenic adipokine that inhibits the development of plaque by mechanisms that are not completely understood. Extracellular matrix (ECM) may have a role in the pathogenesis of atherosclerosis. We explored the effect and mechanisms of adiponectin on the synthesis of prolyl-4-hydroxylase (P4H) in interleukin 6 (IL-6)-stimulated human aortic smooth muscle cells (HASMCs). P4H?1 mRNA level was quantified by RT-PCR, the protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and P4H?1 were quantified by western blot analysis, and activation of specific protein 1 (Sp1) was determined by electrophoretic mobility shift assay and subcellular localization of Sp1 by immunofluorescence analysis. Adiponectin significantly increased P4H?1 mRNA and protein levels in IL-6-stimulated HASMCs in a dose- and time-dependent manner. As well, ERK1/2 and Sp1 played a crucial role in the effect of adiponectin upregulating P4H?1 expression in IL-6-stimulated HASMCs. Adiponectin abrogated the effects of IL-6 on collagen III level, which may indicate that P4H?1 is essential for folding the procollagen polypeptide chains into stabilized collagen. Adiponectin attenuates IL-6-inhibited P4H?1 synthesis and stabilizes collagen formation in HASMCs through a Sp1-ERK1/2-P4H?1-dependent pathway. PMID:21829524

Li, Li; Zhang, Ke; Cai, Xiao-Jun; Feng, Min; Zhang, Yun; Zhang, Mei



Adiponectin Upregulates Prolyl-4-Hydroxylase ?1 Expression in Interleukin 6-Stimulated Human Aortic Smooth Muscle Cells by Regulating ERK 1/2 and Sp1  

PubMed Central

Adiponectin is an anti-atherogenic adipokine that inhibits the development of plaque by mechanisms that are not completely understood. Extracellular matrix (ECM) may have a role in the pathogenesis of atherosclerosis. We explored the effect and mechanisms of adiponectin on the synthesis of prolyl-4-hydroxylase (P4H) in interleukin 6 (IL-6)-stimulated human aortic smooth muscle cells (HASMCs). P4H?1 mRNA level was quantified by RT-PCR, the protein levels of phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and P4H?1 were quantified by western blot analysis, and activation of specific protein 1 (Sp1) was determined by electrophoretic mobility shift assay and subcellular localization of Sp1 by immunofluorescence analysis. Adiponectin significantly increased P4H?1 mRNA and protein levels in IL-6-stimulated HASMCs in a dose- and time-dependent manner. As well, ERK1/2 and Sp1 played a crucial role in the effect of adiponectin upregulating P4H?1 expression in IL-6-stimulated HASMCs. Adiponectin abrogated the effects of IL-6 on collagen III level, which may indicate that P4H?1 is essential for folding the procollagen polypeptide chains into stabilized collagen. Adiponectin attenuates IL-6–inhibited P4H?1 synthesis and stabilizes collagen formation in HASMCs through a Sp1-ERK1/2-P4H?1-dependent pathway.

Feng, Min; Zhang, Yun; Zhang, Mei



DNA dynamics play a role as a basal transcription factor in the positioning and regulation of gene transcription initiation.  


We assess the role of DNA breathing dynamics as a determinant of promoter strength and transcription start site (TSS) location. We compare DNA Langevin dynamic profiles of representative gene promoters, calculated with the extended non-linear PBD model of DNA with experimental data on transcription factor binding and transcriptional activity. Our results demonstrate that DNA dynamic activity at the TSS can be suppressed by mutations that do not affect basal transcription factor binding-DNA contacts. We use this effect to establish the separate contributions of transcription factor binding and DNA dynamics to transcriptional activity. Our results argue against a purely 'transcription factor-centric' view of transcription initiation, suggesting that both DNA dynamics and transcription factor binding are necessary conditions for transcription initiation. PMID:20019064

Alexandrov, Boian S; Gelev, Vladimir; Yoo, Sang Wook; Alexandrov, Ludmil B; Fukuyo, Yayoi; Bishop, Alan R; Rasmussen, Kim Ø; Usheva, Anny



ER?-dependent effects on uterine endothelial cells are cell specific and mediated via Sp1  

PubMed Central

STUDY QUESTION What are the in vitro effects of estrogen receptor ? (ER?) activation on the function of endothelial cells (ECs) from different vascular beds: human endometrial ECs (HEECs; endometrium), uterine myometrial microvascular ECs (UtMVECs; myometrium) and human umbilical vein ECs (HUVECs)? SUMMARY ANSWER Studies conducted in vitro demonstrate that the ER? agonist 2,3-bis(4-hydroxy-phenyl)-propionitrile (DPN) has EC type-specific effects on patterns of gene expression and network formation. Identification of a key role for the transcription factor Sp1 in ER?-dependent signaling in uterine ECs offers new insights into cell-specific molecular mechanisms of estrogen action in the human uterus. WHAT IS KNOWN ALREADY Estrogens, acting via ERs (ER? and ER?), have important, body-wide impacts on the vasculature. The human uterus is an estrogen target organ, the endometrial lining of which exhibits physiological, cyclical angiogenesis. In fixed tissue sections, human endometrial ECs are immunopositive for ER?. STUDY DESIGN, SIZE, DURATION Cells were treated with a vehicle control or the ER? agonist, DPN, for 2 h or 24 h (n = 5) followed by gene expression analysis. Functional assays were analyzed after a 16 h incubation with ligand (n = 5). PARTICIPANT/MATERIALS, SETTING, METHODS Analysis of DPN-treated ECs using Taqman gene array cards focused on genes involved in angiogenesis and inflammation identified cell type-specific ER?-dependent changes in gene expression, with validation using qPCR and immunohistochemistry. Molecular mechanisms involved in ER? signaling were investigated using bioinformatics, reporter assays, immunoprecipitation, siRNA and a specific inhibitor blocking Sp1-binding sites. The endometrium and myometrium from women with regular menses were used to validate the protein expression of candidate genes. MAIN RESULTS AND THE ROLE OF CHANCE HEECs and UtMVECs were ER?+/ER??. Treatment of ECs with DPN had opposite effects on network formation: a decrease in network formation in HEECs (P ? 0.001) but an increase in UtMVECs (P ? 0.05). Genomic analysis identified opposite changes in ER? target gene expression with only three common transcripts (HEY1, ICAM1, CASP1) in all three ECs; a unique profile was observed for each. An important role for Sp1 was identified, consistent with the regulation of ER? target genes via association with the transcription factor (‘tethered’ mechanism). LIMITATIONS, REASONS FOR CAUTION The study was mainly carried out in vitro using ECs of which one type was immortalized. Although the analysis of the protein expression of candidate genes was carried out using intact tissue samples from patients, investigations into in vivo angiogenesis were not carried out. WIDER IMPLICATIONS OF THE FINDINGS These results have implications for our understanding of the mechanisms responsible for ER?-dependent changes in EC gene expression in hormone-dependent disorders. STUDY FUNDING/COMPETEING INTEREST(S) The study was funded by a Medical Research Council Programme Grant. E.G. is the recipient of an MRC Career Development Fellowship. The authors have nothing to disclose.

Greaves, Erin; Collins, Frances; Critchley, Hilary O.D.; Saunders, Philippa T.K.



Sweet Play  

ERIC Educational Resources Information Center

This article features Sweet play math, a "math by the month" activity that involves decorating and making sugar cubes. Teachers may want to substitute straws, paper squares, alphabet blocks, or such commercially made manipulatives as Unifix[R] cubes for the real sweets. Given no allergy concerns, teachers and students alike would enjoy some sweet…

Leung, Shuk-kwan S.; Lo, Jane-Jane



CRiSP1 Passage: Columbia River Salmon Passage Model and Data  

NSDL National Science Digital Library

The CRiSP1 Passage Columbia River Salmon Passage Model, developed by researchers at the University of Washington, "predicts downstream migration and survival of individual stocks of wild and hatchery spawned juvenile fish from the tributaries and dams of the Columbia and Snake rivers to the estuary." At the site, viewers may download the CRiSP1 Passage model (v1.6.0) in addition to PATH Spring and Fall Chinook Data Files (self-extracting files).


Sp1 transcriptionally regulates BRK1 expression in non-small cell lung cancer cells.  


Following a previous study reporting that BRK1 is upregulated in non-small cell lung cancer (NSCLC), the present study sought to clarify the role of specificity protein 1 (Sp1) in the transcriptional regulation of the BRK1 gene. Therefore, a construct, named F8, consisting of the -1341 to -1nt sequence upstream of the start codon of the BRK1 gene inserted into pGL4.26 was made. A series of truncated fragments was then constructed based on F8. Segment S831, which contained the -84 to -1nt region, displayed the highest transcriptional activity in the A549, H1299 and H520 NSCLC cell lines. Bioinformatic analysis showed a potential Sp1-binding element at -73 to -64nt, and a mutation in this region suppressed the transcriptional activity of S831. Then the RNAi assays of Sp1 and its coworkers Sp3 and Sp4 were performed, and suppression of Sp1 by siRNA inhibited the mRNA expression of BRK1. Both an electrophoretic mobility shift assay (EMSA) and a chromatin immunoprecipitation (ChIP) assay demonstrated that Sp1 bound to the promoter area of the BRK1 gene. Our data identified a functional and positive Sp1 regulatory element from -73 to -64nt in the BRK1 promoter, which may likely explain the overexpression of BRK1 in NSCLC. PMID:24680773

Li, Meng; Ling, Bing; Xiao, Ting; Tan, Jinjing; An, Ning; Han, Naijun; Guo, Suping; Cheng, Shujun; Zhang, Kaitai



The Role Played by the Interaction between Genetic Factors and Attachment in the Stress Response in Infancy  

ERIC Educational Resources Information Center

Background: The importance of understanding which environmental and biological factors are involved in determining individual differences in physiological response to stress is widely recognized, given the impact that stress has on physical and mental health. Methods: The child-mother attachment relationship and some genetic polymorphisms…

Frigerio, Alessandra; Ceppi, Elisa; Rusconi, Marianna; Giorda, Roberto; Raggi, Maria Elisabetta; Fearon, Pasco



Systematic RNAi studies on the role of Sp/KLF factors in globin gene expression and erythroid differentiation.  


Sp/KLF family of factors regulates gene expression by binding to the CACCC/GC/GT boxes in the DNA through their highly conserved three zinc finger domains. To investigate the role of this family of factors in erythroid differentiation and globin gene expression, we first measured the expression levels of selected Sp/KLF factors in primary cells of fetal and adult stages of erythroid development. This quantitative analysis revealed that their expression levels vary significantly in cells of either stages of the erythroid development. Significant difference in their expression levels was observed between fetal and adult erythroid cells for some Sp/KLF factors. Functional studies using RNA interference revealed that the silencing of Sp1 and KLF8 resulted in elevated level of gamma globin expression in K562 cells. In addition, K562 cells become visibly red after Sp1 knockdown. Benzidine staining revealed significant hemoglobinization of these cells, indicating erythroid differentiation. Moreover, the expression of PU.1, ETS1 and Notch1 is significantly down-regulated in the cells that underwent erythroid differentiation following Sp1 knockdown. Overexpression of PU.1 or ETS1 efficiently blocked the erythroid differentiation caused by Sp1 knockdown in K562 cells. The expression of c-Kit, however, was significantly up-regulated. These data indicate that Sp1 may play an important role in erythroid differentiation. PMID:17224162

Hu, Jie Hong; Navas, Patrick; Cao, Hua; Stamatoyannopoulos, George; Song, Chao-Zhong



NF-Y and Sp1/Sp3 are involved in the transcriptional regulation of the peptidylarginine deiminase type III gene (PADI3) in human keratinocytes  

PubMed Central

Human peptidylarginine deiminase type III gene (PADI3) encodes a crucial post-translational modification enzyme that converts protein-bound arginine residues into citrulline residues. Its expression is restricted to a few cell types, including keratinocytes in the granular layer of the epidermis and in the inner root sheath of hair follicles. In these cells, the enzyme is involved in terminal processing of intermediate filament-binding proteins such as filaggrin and trichohyalin. To study the molecular mechanisms that control the expression of PADI3 in human keratinocytes at the transcriptional level, we characterized its promoter region using human keratinocytes transfected with variously deleted fragments of the 5?-upstream region of PADI3 coupled to the luciferase gene. We found that as few as 129 bp upstream from the transcription initiation site were sufficient to direct transcription of the reporter gene. Electrophoretic mobility-shift and chromatin immunoprecipitation assays revealed that NF-Y (nuclear factor Y) and Sp1/Sp3 (specificity protein 1/3) bind to this region in vitro and in vivo. Moreover, mutation of the Sp1- or NF-Y-binding motif markedly reduced PADI3 promoter activity. Furthermore, Sp1 or NF-YA (NF-Y subunit) small interfering RNAs effectively diminished PADI3 expression in keratinocytes cultured in both low- and high-calcium medium. These data indicate that PADI3 expression is driven by Sp1/Sp3 and NF-Y binding to the promoter region.

Dong, Sijun; Kanno, Takuya; Yamaki, Ayako; Kojima, Toshio; Shiraiwa, Masakazu; Kawada, Akira; Mechin, Marie-Claire; Chavanas, Stephane; Serre, Guy; Simon, Michel; Takahara, Hidenari



Msn2- and Msn4Like Transcription Factors Play No Obvious Roles in the Stress Responses of the Fungal Pathogen Candida albicans  

Microsoft Academic Search

In Saccharomyces cerevisiae, the (C2H2)2 zinc finger transcription factors Msn2 and Msn4 play central roles in responses to a range of stresses by activating gene transcription via the stress response element (STRE; CCCCT). The pathogen Candida albicans displays stress responses that are thought to help it survive adverse environmental conditions encountered within its human host. However, these responses differ from

Susan Nicholls; Melissa Straffon; Brice Enjalbert; A. Nantel; S. Macaskill; M. Whiteway; A. J. P. Brown



Characterization of an Essential Orc2p-Associated Factor That Plays a Role in DNA Replication  

Microsoft Academic Search

TheSaccharomyces cerevisiaeOrc2 protein is a subunit of the origin recognition complex, ORC, which binds in a sequence-specific manner to yeast origins of DNA replication. With screens for orc2-1 synthetic lethal mutations and Orc2p two-hybrid interactors, a novel Orc2p-associated factor (Oaf1p) was identified.OAF1is essential,itsgeneproductislocalizedtothenucleus,andanoaf1temperature-sensitivemutantarrestsaslarge budded cells with a single nucleus. The mutantoaf1-2, isolated in the synthetic lethal screen, loses plasmids containing




Spatial factors play a major role as determinants of endemic ground beetle beta diversity of Madeira Island Laurisilva.  


The development in recent years of new beta diversity analytical approaches highlighted valuable information on the different processes structuring ecological communities. A crucial development for the understanding of beta diversity patterns was also its differentiation in two components: species turnover and richness differences. In this study, we evaluate beta diversity patterns of ground beetles from 26 sites in Madeira Island distributed throughout Laurisilva--a relict forest restricted to the Macaronesian archipelagos. We assess how the two components of ground beetle beta diversity (?(repl)--species turnover and ?(rich)--species richness differences) relate with differences in climate, geography, landscape composition matrix, woody plant species richness and soil characteristics and the relative importance of the effects of these variables at different spatial scales. We sampled 1025 specimens from 31 species, most of which are endemic to Madeira Island. A spatially explicit analysis was used to evaluate the contribution of pure environmental, pure spatial and environmental spatially structured effects on variation in ground beetle species richness and composition. Variation partitioning showed that 31.9% of species turnover (?(repl)) and 40.7% of species richness variation (?(rich)) could be explained by the environmental and spatial variables. However, different environmental variables controlled the two types of beta diversity: ?(repl) was influenced by climate, disturbance and soil organic matter content whilst ?(rich) was controlled by altitude and slope. Furthermore, spatial variables, represented through Moran's eigenvector maps, played a significant role in explaining both ?(repl) and ?(rich), suggesting that both dispersal ability and Madeira Island complex orography are crucial for the understanding of beta diversity patterns in this group of beetles. PMID:23724065

Boieiro, Mário; Carvalho, José C; Cardoso, Pedro; Aguiar, Carlos A S; Rego, Carla; de Faria e Silva, Israel; Amorim, Isabel R; Pereira, Fernando; Azevedo, Eduardo B; Borges, Paulo A V; Serrano, Artur R M



Physician perspectives and compliance with patient advance directives: the role external factors play on physician decision making  

PubMed Central

Background Following passage of the Patient Self Determination Act in 1990, health care institutions that receive Medicare and Medicaid funding are required to inform patients of their right to make their health care preferences known through execution of a living will and/or to appoint a surrogate-decision maker. We evaluated the impact of external factors and perceived patient preferences on physicians’ decisions to honor or forgo previously established advance directives (ADs). In addition, physician views regarding legal risk, patients’ ability to comprehend complexities involved with their care, and impact of medical costs related to end-of-life care decisions were explored. Methods Attendees of two Mayo Clinic continuing medical education courses were surveyed. Three scenarios based in part on previously court-litigated matters assessed impact of external factors and perceived patient preferences on physician compliance with patient-articulated wishes regarding resuscitation. General questions measured respondents’ perception of legal risk, concerns over patient knowledge of idiosyncrasies involved with their care, and impact medical costs may have on compliance with patient preferences. Responses indicating strength of agreement or disagreement with statements were treated as ordinal data and analyzed using the Cochran Armitage trend test. Results Three hundred eighty-eight of 951 surveys were completed (41% response rate). Eighty percent reported they were likely to honor a patient’s AD despite its 5 year age. Fewer than half (41%) would honor the AD of a patient in ventricular fibrillation who had expressed a desire to “pass away in peace.” Few (17%) would forgo an AD following a family’s request for continued resuscitative treatment. A majority (52%) considered risk of liability to be lower when maintaining someone alive against their wishes than mistakenly failing to provide resuscitative efforts. A large percentage (74%) disagreed that patients could not appreciate complexities surrounding their care while 69% agreed that costs should never impact a physician’s decision as to whether to comply with a patient’s AD. Conclusions Our findings highlight the impact, albeit small, external factors have on physician AD compliance. Most respondents based their decision on the clinical situation at hand and interpretation of the patient’s initial wishes and preferences expressed by the AD.



Negative guidance factor-induced macropinocytosis in the growth cone plays a critical role in repulsive axon turning  

PubMed Central

Macropinocytosis is a type of poorly characterized fluid-phase endocytosis which results in formation of relatively large vesicles. We report that Sonic hedgehog (Shh) protein induces macropinocytosis in the axons, through activation of a noncanonical signaling pathway including Rho GTPase and nonmuscle myosin II. Macropinocytosis induced by Shh is independent of clathrin-mediated endocytosis, but dependent on dynamin, myosin II and Rho GTPase activities. Inhibitors of macropinocytosis also abolished the negative effects of Shh on axonal growth including growth cone collapse and chemorepulsive axon turning, but not turning per se. On the other hand, activation of myosin II or treatment of phorbol ester induces macropinocytosis in the axons, elicits growth cone collapse and repulsive axon turning. Furthermore, macropinocytosis is also induced by ephrin-A2 and inhibition of dynamin abolished repulsive axon turning induced by ephrin-A2. Macropinocytosis can be induced ex vivo by high Shh, correlating with axon retraction. These results demonstrate that macropinocytosis-mediated membrane trafficking is an important cellular mechanism involved in axon chemorepulsion induced by negative guidance factors.

Kolpak, Adrianne L.; Jiang, Jun; Guo, Daorong; Standley, Clive; Bellve, Karl; Fogarty, Kevin; Bao, Zheng-Zheng



Activation of PPAR{gamma} negatively regulates O-GlcNAcylation of Sp1  

SciTech Connect

O-GlcNAcylation is a kind of post-translational modification and many nuclear and cytoplasmic proteins are O-GlcNAcylated. In this study, we demonstrated that thiazolidinediones (TZDs), which are used as insulin sensitizer, specifically inhibited the O-GlcNAcylation of Sp1 but did not affect the O-GlcNAcylation of the total proteins in cell culture systems and mouse models. This effect was mediated by peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) activation and probably by synthesis of a specific protein induced by PPAR{gamma} activation. In addition, we demonstrated that the O-GlcNAcylation sites in the zinc-finger domain were involved in the transcriptional activation of Sp1 and that rosiglitazone, a member of TZDs, affected Sp1 transcriptional activity partially by regulating the O-GlcNAcylation level of these sites. Considering the role of hexosamine biosynthesis pathway in hyperglycemia-induced insulin resistance and Sp1 in the hyperglycemia-induced gene expression, the regulation of Sp1 O-GlcNAcylation by TZDs may help to explain the function of TZDs as a treatment for insulin resistance and diabetes.

Chung, Sung Soo; Kim, Ji Hyun; Park, Ho Seon; Choi, Hye Hun; Lee, Kyeong Won; Cho, Young Min; Lee, Hong Kyu [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 (Korea, Republic of); Park, Kyong Soo [Department of Internal Medicine, Seoul National University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-744 (Korea, Republic of)], E-mail:



Sp1 Regulates Osteopontin Expression in SW480 Human Colon Adenocarcinoma Cells  

PubMed Central

Introduction Osteopontin (OPN) mediates cancer metastatis. Mechanisms regulating OPN expression in human colorectal cancer are unknown. Using SW480 colon adenocarcinoma cells, we hypothesized that transcription determines OPN expression. Methods SW480 constitutively express OPN. Transient transfection and deletion analysis of human OPN promoter (full-length 2.1 kb)-luciferase constructs identified cis-regulatory regions. Gelshift and chromatin immunoprecipitation (ChIP) assays identified the trans-regulatory nuclear protein. Using in vitro adhesion, migration and invasion studies, siRNA was utilized to determine the functional effect of decreased nuclear protein expression. Results A cis-regulatory promoter region, nt-80 to nt-108, upregulated OPN transcription. Gelshift assays demonstrated specific binding of nuclear proteins. Competition with unlabelled mutant oligonucleotides indicated that the region, nt-94 to nt-104 (TGGGCTGGGC), was essential for protein binding in gelshift assays. Confirmatory ChIP assays showed the corresponding nuclear protein to be Sp1. Sp1 expression was ablated with siRNA (si-Sp1) resulting in decreased OPN dependent adhesion, migration and invasion by 50%, 70% and 65%, respectively. Exogenous addition of OPN to si-Sp1 cells restored adhesion, migration and invasion indices. Conclusions In SW480 human colon cancer cells, we conclude that Sp1 mediated expression of the tumor metastasis protein, OPN, regulates in vitro functional correlates of tumor metastasis.

Takami, Yoji; Russell, Michael B.; Gao, Chengjiang; Mi, Zhiyong; Guo, Hongtao; Mantyh, Christopher R.; Kuo, Paul C.



Sp1-mediated microRNA-182 expression regulates lung cancer progression  

PubMed Central

Our recent study indicated that overexpression of Sp1 enhances the proliferation of lung cancer cells, while represses metastasis. In this study, we found that the transcriptional activity of FOXO3 was increased, but its protein levels decreased following Sp1 expression. Sp1 increased expression of miR-182, which was then recruited to the 3'-untranslated region of FOXO3 mRNA to silence its translational activity. Knockdown of miR-182 inhibited lung cancer cells growth, but enhanced the invasive and migratory abilities of these cells through increased N-cadherin expression. Repression of FOXO3 expression in the miR-182 knockdown cells partially reversed this effect, suggesting that miR-182 promotes cancer cell growth and inhibits cancer metastatic activity by regulating the expression of FOXO3. The expression of several cancer metastasis-related genes such as ADAM9, CDH9 and CD44 was increased following miR-182 knockdown. In conclusion, in the early stages of lung cancer progression, Sp1 stimulates miR-182 expression, which in turn decreases FOXO3 expression. This stimulates proliferation and tumor growth. In the late stages, Sp1 and miR-182 decline, thus increasing FOXO3 expression, which leads to lung metastasis.

Yang, Wen-Bin; Chen, Ping-Hsin; Hsu, Tsung-I; Fu, Tzu-Fun; Su, Wu-Chou; Liaw, Hungjiun; Chang, Wen-Chang; Hung, Jan-Jong



Sp1-mediated microRNA-182 expression regulates lung cancer progression.  


Our recent study indicated that overexpression of Sp1 enhances the proliferation of lung cancer cells, while represses metastasis. In this study, we found that the transcriptional activity of FOXO3 was increased, but its protein levels decreased following Sp1 expression. Sp1 increased expression of miR-182, which was then recruited to the 3'-untranslated region of FOXO3 mRNA to silence its translational activity. Knockdown of miR-182 inhibited lung cancer cells growth, but enhanced the invasive and migratory abilities of these cells through increased N-cadherin expression. Repression of FOXO3 expression in the miR-182 knockdown cells partially reversed this effect, suggesting that miR-182 promotes cancer cell growth and inhibits cancer metastatic activity by regulating the expression of FOXO3. The expression of several cancer metastasis-related genes such as ADAM9, CDH9 and CD44 was increased following miR-182 knockdown. In conclusion, in the early stages of lung cancer progression, Sp1 stimulates miR-182 expression, which in turn decreases FOXO3 expression. This stimulates proliferation and tumor growth. In the late stages, Sp1 and miR-182 decline, thus increasing FOXO3 expression, which leads to lung metastasis. PMID:24519909

Yang, Wen-Bin; Chen, Ping-Hsin; Hsu, Tsung; Fu, Tzu-Fun; Su, Wu-Chou; Liaw, Hungjiun; Chang, Wen-Chang; Hung, Jan-Jong



Transforming growth factor beta (TGF?) plays a crucial role in prolonging allograft survival in an allodepletion ("pruning") skin transplant model.  


Adoptive cell therapies involving cell manipulation to achieve tolerance are increasingly being studied in animal models and in human trials. We have demonstrated that the specific removal of allo-stimulated dividing cells (or "pruning") promotes long-term allograft survival across a major MHC mismatch in transplant models including skin, heart and islet transplants. In this study, we examine the role of transforming growth factor beta (TGF?), an important regulatory cytokine, on allograft survival in our allodepletion or "pruning" skin transplant model. Increased proliferation of CD4(+) T cells was observed following allo-stimulation of BALB/c spleen cells (labeled with CFSE) in the presence of the regulatory cytokines TGF? and (interleukin-2) IL-2 in a mixed lymphocyte culture (MLC). Expression of the regulatory gene forkhead box-3 (FoxP3) was increased in both the allo-stimulated non-dividing (ND) (CFSE(high)) and dividing (D) (CFSE(low)) CD4(+) T cell populations, with the highest expression found in the D CD4(+) T cell population. Mice reconstituted with allo-stimulated ND CD4(+) T cells following TGF?/IL-2 stimulation showed prolonged allograft survival, similar to previous data. Significantly, TGF?/IL-2 stimulation prevented acute rejection of allografts across a major MHC mismatch in the presence of highly activated allo-stimulated D CD4(+) T cells. Blockade of TGF? promoted rejection of allografts even following depletion of allo-stimulated D CD4(+) T cells. These studies support a crucial role for TGF? in the survival of allografts and shows that regulatory cytokines TGF?/IL2 can delay the rejection of allografts, even in the presence of highly activated alloreactive T cells. PMID:24746800

Watson, Debbie; Zhang, Geoff Yu; Hu, Min; Wang, Yuan-Min; Fletcher, Jeffery; Sartor, Mary; Alexander, Stephen I



Licochalcone A, a natural chalconoid isolated from Glycyrrhiza inflata root, induces apoptosis via Sp1 and Sp1 regulatory proteins in oral squamous cell carcinoma.  


Licochalcone A (LCA), a chalconoid derived from root of Glycyrrhiza inflata, has been known to possess a wide range of biological functions such as antitumor, anti-angiogenesis, antiparasitic, anti-oxidant, antibacterial and anti-inflammatory effects. However, the anticancer effects of LCA on oral squamous cell carcinoma (OSCC) have not been reported. Our data showed that LCA inhibited OSCC cell (HN22 and HSC4) growth in a concentration- and time-dependent manner. Mechanistically, it was mediated via downregulation of specificity protein 1 (Sp1) expression and subsequent regulation of Sp1 downstream proteins such as p27, p21, cyclin D1, Mcl-1 and survivin. Here, we found that LCA caused apoptotic cell death in HSC4 and HN22 cells, as characterized by sub-G1 population, nuclear condensation, Annexin V staining, and multi-caspase activity and apoptotic regulatory proteins such as Bax, Bid, Bcl?xl, caspase-3 and PARP. Consequently, this study strongly suggests that LCA induces apoptotic cell death of OSCC cells via downregulation of Sp1 expression, prompting its potential use for the treatment of human OSCC. PMID:24858379

Cho, Jung Jae; Chae, Jung-Il; Yoon, Goo; Kim, Ka Hwi; Cho, Jin Hyoung; Cho, Seung-Sik; Cho, Young Sik; Shim, Jung-Hyun



Histone deacetylase 3 represses p15{sup INK4b} and p21{sup WAF1/cip1} transcription by interacting with Sp1  

SciTech Connect

Histone deacetylase 3 (HDAC3) has been implicated to play roles in governing cell proliferation. Here we demonstrated that the overexpression of HDAC3 repressed transcription of p15{sup INK4b} and p21{sup WAF1/cip1} genes in 293T cells, and that the recruitment of HDAC3 to the promoter regions of these genes was critical to this repression. We also showed that HDAC3 repressed GAL4-Sp1 transcriptional activity, and that Sp1 was co-immunoprecipitated with FLAG-tagged HDAC3. We conclude that HDAC3 can repress p15{sup INK4b} and p21{sup WAF1/cip1} transcription by interacting with Sp1. Furthermore, knockdown of HDAC3 by RNAi up-regulated the transcriptional expression of p15{sup INK4b}, but not that of p21{sup WAF1/cip1}, implicating the different roles of HDAC3 in repression of p15{sup INK4b} and p21{sup WAF1/cip1} transcription. Data from this study indicate that the inhibition of p15{sup INK4b} and p21{sup WAF1/cip1} may be one of the mechanisms by which HDAC3 participates in cell cycle regulation and oncogenesis.

Huang Weifeng [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China); Tan Dapeng [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China); Wang Xiuli [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China); Han Songyan [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China); Tan Jiang [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China); Zhao Yanmei [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China); Lu Jun [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China)]. E-mail:; Huang Baiqu [Institute of Genetics and Cytology, Northeast Normal University, Changchun 130024 (China)



Factor XII (Hageman factor) is a missing link between stress and hypercoagulability and plays an important role in the pathophysiology of ischemic stroke.  


A new hypothesis is presented on the function of factor XII, which is postulated to be a "missing link" between acute stress and transient hypercoagulability. The implications of this idea are developed to show how chronic stress, which involves activation of hypertension and migraine as well as hypercoagulability, can cause of cerebrovascular disease. "Acute stress" is defined as "the normal short-term physiological response to the perception of major threats or demands". "Chronic stress" is "the abnormal ongoing physiological response to the continuing perception of unresolvable major threats or demands". The factor XII hypothesis is as follows: Acute stress includes release of epinephrine by the adrenal medulla. Epinephrine activates platelets by binding to alpha-2A adrenergic receptors. Activated platelets convert pre-bound factor XII to its active form, which then initiates the intrinsic coagulation cascade. This can be called the "activated platelet initiation pathway" for coagulation. Neither tissue factor nor pre-formed thrombin is required. Thrombosis proceeds to completion, but only a minute amount of thrombin is formed, and the process normally stops at this point. In people who lapse into a state of chronic stress, essential hypertension, which is also a manifestation of stress, synergizes with hypercoagulability: there is both a baseline rise in blood pressure and systemic platelet activation as well as superimposed labile rises of both. Upregulation of these two stress parameters is atherogenic: epinephrine-activated platelets stimulating thrombin formation interact with endothelial cells activated by angiotensin II to cause, first, smooth muscle cell proliferation, which is a histological hallmark of atherosclerosis, and, lastly, a symptomatic thrombotic occlusion-the stroke. The migraine symptoms which often accompany this process are a marker of chronic stress and ongoing pathophysiologic damage. Therapeutic predictions are made regarding novel ways of blocking stress-induced hypercoagulability and hypertension. Hypercoagulability could be targeted by monoclonal antibodies directed against the platelet-specific alpha-2 adrenergic receptor or the (putative) platelet receptor for Factor XII; hypertension could be treated with monoclonal antibodies directed against the beta-adrenergic receptor in the juxtaglomerular apparatus or by surgical denervation of the kidneys, either of which would decrease the renin release which helps drive the hypertension. PMID:16757126

Eggers, Arnold E



A critical Sp1 element in the rhesus rhadinovirus (RRV) Rta promoter confers high-level activity that correlates with cellular permissivity for viral replication.  


KSHV establishes characteristic latent infections in vitro, while RRV, a related macaque rhadinovirus, establishes characteristic permissive infections with virus replication. We identified cells that are not permissive for RRV replication and recapitulate the latent KSHV infection and reactivation processes. The RRV replication and transactivator (Rta) promoter was characterized in permissive and non-permissive cells and compared to the KSHV Rta promoter. Both promoters contained a critical Sp1 element, had equivalent activities in different cell types, and were inhibited by LANA. RRV and KSHV infections were non-permissive in cells with low Rta promoter activity. While RRV infections were permissive in cells with high basal promoter activity, KSHV infections remained non-permissive. Our studies suggest that RRV lacks the Rta-inducible LANA promoter that is responsible for LANA inhibition of the KSHV Rta promoter and induction of latency during KSHV infection. Instead, the outcome of RRV infection is determined by host factors, such as Sp1. PMID:24314650

DeMaster, Laura K; Rose, Timothy M




PubMed Central

Small cholangiocytes proliferate via activation of Ca2+-dependent signaling in response to pathological conditions that trigger the damage of large cAMP-dependent cholangiocytes. Although our previous studies suggest that small cholangiocyte proliferation is regulated by the activation of Ca2+-dependent signaling, the intracellular mechanisms regulating small cholangiocyte proliferation are undefined. Therefore, we sought to address the role and mechanisms of action by which phenylephrine, an ?1-adrenergic agonist stimulating intracellular IP3/Ca2+ levels, regulates small cholangiocyte proliferation. Small and large bile ducts and cholangiocytes expressed all AR receptor subtypes. Small (but not large) cholangiocytes respond to phenylephrine with increased proliferation via the activation of IP3/Ca2+-dependent signaling. Phenylephrine stimulated the production of intracellular IP3. The Ca2+-dependent transcription factors, NFAT2 and NFAT4, were predominantly expressed by small bile ducts and small cholangiocytes. Phenylephrine stimulated the Ca2+-dependent DNA-binding activities of NFAT2, NFAT4, and Sp1 (but not Sp3) and the nuclear translocation of NFAT2 and NFAT4 in small cholangiocytes. To determine the relative roles of NFAT2, NFAT4, or Sp1, we knocked down the expression of these transcription factors with shRNA. We observed an inhibition of phenylephrine-induced proliferation in small cholangiocytes lacking the expression of NFAT2 or Sp1. Phenylephrine stimulated small cholangiocyte proliferation is regulated by Ca2+-dependent activation of NFAT2 and Sp1. Selective stimulation of Ca2+-dependent small cholangiocyte proliferation may be key to promote the repopulation of the biliary epithelium when large bile ducts are damaged during cholestasis or by toxins.

Alpini, Gianfranco; Franchitto, Antonio; DeMorrow, Sharon; Onori, Paolo; Gaudio, Eugenio; Wise, Candace; Francis, Heather; Venter, Julie; Kopriva, Shelley; Mancinelli, Romina; Carpino, Guido; Stagnitti, Franco; Ueno, Yoshiyuki; Han, Yuyan; Meng, Fanyin; Glaser, Shannon



NtbZIP60, an endoplasmic reticulum-localized transcription factor, plays a role in the defense response against bacterial pathogens in Nicotiana tabacum.  


A spermine-based signal transduction pathway plays a defensive role against incompatible pathogens. We identified a novel spermine-responsive cDNA from Nicotiana tabacum that encodes a basic region/leucine zipper protein with a putative transmembrane domain. Identity to Arabidopsis thaliana AtbZIP60 was sufficiently high to name the novel cDNA NtbZIP60. Expression analysis revealed that NtbZIP60 is a component of the spermine-signal pathway, and is also involved in the unfolded protein response (UPR), as demonstrated for AtbZIP60. The gene product, NtbZIP60, localizes to the endoplasmic reticulum (ER) in plant cells; once the putative transmembrane domain is eliminated from the intact protein, it targets the nucleus. The putative processed form of NtbZIP60 transactivates target genes through binding to plant-specific UPR cis-elements. Expression of NbbZIP60, an NtbZIP60 ortholog in Nicotiana benthamiana, was significantly up-regulated at 6 h and later time points upon infection with the non-host pathogen Pseudomonas cichorii, while it was unaffected by infection with the compatible pathogen Pseudomonas syringae pv. tabaci. Furthermore, NbbZIP60-silenced N. benthamiana plants allowed higher multiplication of P. cichorii compared to the control plants. Taken together, the results suggest that this ER-localized transcription factor is involved in the spermine-signal transduction pathway and plays an important role in plant innate immunity. PMID:18758894

Tateda, Chika; Ozaki, Rei; Onodera, Yu; Takahashi, Yoshihiro; Yamaguchi, Koji; Berberich, Thomas; Koizumi, Nozomu; Kusano, Tomonobu



Orai1-mediated calcium entry plays a critical role in osteoclast differentiation and function by regulating activation of the transcription factor NFATc1  

PubMed Central

Bone diseases such as postmenopausal osteoporosis are primarily caused by excessive formation and activity of osteoclasts (OCLs). Receptor activator of nuclear factor-?B ligand (RANKL) is a key initiating cytokine for OCL differentiation and function. RANKL induces calcium (Ca2+) oscillations, resulting in selective and robust induction of nuclear factor of activated T cells c1 (NFATc1), a Ca2+-responsive transcription factor that drives osteoclastogenesis. Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in most nonexcitable cell types and is activated by any stimulus that depletes Ca2+ stores in the endoplasmic reticulum. Although the role of Orai1, a SOCE channel in the plasma membrane, in maintaining Ca2+ oscillations and transactivation of NFAT in other cell types is well known, its contribution to osteoclastogenesis remains unclear. We show here that silencing of the Orai1 gene with viral delivery of shRNA reduces SOCE and inhibits RANKL-induced osteoclastogenesis of RAW264.7 cells, a murine monocyte/macrophage cell line, by suppressing the induction of NFATc1. This was accompanied by defective induction of OCL-specific genes, such as tartrate-resistant acid phosphatase and immunoreceptor OCL-associated receptor, which are known to be direct transcriptional targets of NFATc1 during osteoclastogenesis. In addition, maturation of OCLs was abrogated by defective cell fusion of pre-OCLs depleted of Orai1, consistent with defective RANKL-mediated induction of d2 isoform of vacuolar ATPase Vo domain that is involved in cell fusion of pre-OCLs. We found that the functional bone resorbing capacity was severely impaired in OCLs depleted of Orai1, potentially related to the observed decrease in the induction of cathepsin K, a major bone matrix degrading protease. Our results indicate that Orai1 plays a critical role in the differentiation and function of OCLs, suggesting that Orai1 might be a potential therapeutic target for the treatment or prevention of bone loss caused by OCLs.—Hwang, S.-Y., Putney, J. W. Orai1-mediated calcium entry plays a critical role in osteoclast differentiation and function by regulating activation of the transcription factor NFATc1.

Hwang, Sung-Yong; Putney, James W.



Involvement of the GC-rich sequence and specific proteins (Sp1/Sp3) in the basal transcription activity of neurogranin gene  

SciTech Connect

Neurogranin (Ng), a neuronal protein implicated in learning and memory, contains a TATA-less promoter. Analysis of 5'-deletion mutations and site-directed mutations of the mouse Ng promoter revealed that a 258 bp 5'-flanking sequence (+3 to +260) conferred the basal transcription activity, and that the GC-rich sequence (+22 to +33) served as an important determinant of the promoter activity. Transient transfection of the Sp1 expression plasmid transactivated the reporter activity in neuroblastoma N2A cells while knocking down of endogenous Sp1 expression resulted in a 2.5-fold reduction of the reporter activity in HEK 293 cells. Exogenous expression of Sp3 in HEK 293 cells, however, repressed the reporter activity by 50%. Nevertheless, by gel shift assays, Sp1 and Sp3 were not found to be responsible for the protein-DNA complexes formed by the GC-rich sequence. Moreover, a nuclear factor from the mouse brain tissues was discovered to bind to multiple AT-rich regions in Ng promoter.

Gui Jingang [Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 (Singapore); Song Yan [Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 (Singapore); Han, N.-L.R. [Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 (Singapore); Zhou Shufeng [Department of Pharmacy, National University of Singapore, 18 Science Drive 4, Singapore 117543 (Singapore); Sheu, F.-S. [Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543 (Singapore) and University Scholars Program, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260 (Singapore)]. E-mail:



HMGI(Y) and Sp1 in addition to NF-kappa B regulate transcription of the MGSA/GRO alpha gene.  


Expression of the chemokine MGSA/GRO is upregulated as melanocytes progress to melanoma cells. We demonstrate that constitutive and cytokine induced MGSA/GRO alpha expression requires multiple DNA regulatory regions between positions -143 to -62. We have previously shown that the NF-kappa B element at -83 to -65 is essential for basal and cytokine induced MGSA/GRO alpha promoter activity in the Hs294T melanoma and normal retinal pigment epithelial (RPE) cells, respectively. Here, we have determined that the Sp1 binding element located approximately 42 base pairs upstream from the NF-kappa B element binds Sp1 and Sp3 constitutively and this element is necessary for basal MGSA/GRO alpha promoter activity. We demonstrate that the high mobility group proteins HMGI(Y) recognize the AT-rich motif nested within the NF-kappa B element in the MGSA/GRO alpha promoter. Loss of either NF-kappa B or HMGI(Y) complex binding by selected point mutations in the NF-kappa B element results in decreased basal and cytokine induced MGSA/GRO alpha promoter activity. Thus, these results indicate that transcriptional regulation of the chemokine MGSA/GRO alpha requires at least three transcription factors: Sp1, NF-kappa B and HMGI(Y). PMID:7479086

Wood, L D; Farmer, A A; Richmond, A



Capsaicin sensitizes TRAIL-induced apoptosis through Sp1-mediated DR5 up-regulation: Involvement of Ca{sup 2+} influx  

SciTech Connect

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various malignant cells, several cancers including human hepatocellular carcinoma (HCC) exhibit potent resistance to TRAIL-induced cell death. The aim of this study is to evaluate the anti-cancer potential of capsaicin in TRAIL-induced cancer cell death. As indicated by assays that measure phosphatidylserine exposure, mitochondrial activity and activation of caspases, capsaicin potentiated TRAIL-resistant cells to lead to cell death. In addition, we found that capsaicin induces the cell surface expression of TRAIL receptor DR5, but not DR4 through the activation Sp1 on its promoter region. Furthermore, we investigated that capsaicin-induced DR5 expression and apoptosis are inhibited by calcium chelator or inhibitors for calmodulin-dependent protein kinase. Taken together, our data suggest that capsaicin sensitizes TRAIL-mediated HCC cell apoptosis by DR5 up-regulation via calcium influx-dependent Sp1 activation. Highlights: ? Capsaicin sensitizes TRAIL-induced apoptosis through activation of caspases. ? Capsaicin induces expression of DR5 through Sp1 activation. ? Capsaicin activates calcium signaling pathway.

Moon, Dong-Oh [Department of Biology Education, Daegu University, Gyungsan, Gyeongbuk 712–714 (Korea, Republic of)] [Department of Biology Education, Daegu University, Gyungsan, Gyeongbuk 712–714 (Korea, Republic of); Kang, Chang-Hee; Kang, Sang-Hyuck [Department of Marine Life Sciences, Jeju National University, Jeju 690–756 (Korea, Republic of)] [Department of Marine Life Sciences, Jeju National University, Jeju 690–756 (Korea, Republic of); Choi, Yung-Hyun [Department of Biochemistry, College of Oriental Medicine, Dongeui University, Busan 614–054 (Korea, Republic of)] [Department of Biochemistry, College of Oriental Medicine, Dongeui University, Busan 614–054 (Korea, Republic of); Hyun, Jin-Won; Chang, Weon-Young; Kang, Hee-Kyoung; Koh, Young-Sang; Maeng, Young-Hee; Kim, Young-Ree [School of Medicine, Jeju National University, Jeju-si 690–756 (Korea, Republic of)] [School of Medicine, Jeju National University, Jeju-si 690–756 (Korea, Republic of); Kim, Gi-Young, E-mail: [Department of Marine Life Sciences, Jeju National University, Jeju 690–756 (Korea, Republic of)] [Department of Marine Life Sciences, Jeju National University, Jeju 690–756 (Korea, Republic of)



CpG methylation regulates allelic expression of GDF5 by modulating binding of SP1 and SP3 repressor proteins to the osteoarthritis susceptibility SNP rs143383.  


GDF5 encodes an extracellular signalling molecule that is essential for normal skeletal development. The rs144383 C to T SNP located in the 5'UTR of this gene is functional and has a pleiotropic effect on the musculoskeletal system, being a risk factor for knee-osteoarthritis (OA), congenital hip dysplasia, lumbar disc degeneration and Achilles tendon pathology. rs143383 exerts a joint-wide effect on GDF5 expression, with expression of the OA-associated T allele being significantly reduced relative to the C allele, termed allelic expression imbalance. We have previously reported that the GDF5 locus is subject to DNA methylation and that allelic imbalance of rs143383 is mediated by SP1, SP3 and DEAF1 transcriptional repressors. In this study, we have assayed GDF5 methylation in normal and osteoarthritic cartilage, and investigated the effect of methylation on the allelic imbalance of rs143383. We observed demethylation of the GDF5 5'UTR in OA knee cartilage relative to both OA (p = 0.009) and non-OA (p = 0.001) hip cartilage, with the most significant demethylation observed at the highly conserved +37 CpG site located 4 bp upstream of rs143383. Methylation modulates the level and direction of allelic imbalance of rs143383, with methylation of the +37 CpG dinucleotide within the SP1/SP3 binding site having an allele-specific effect on SP1 and SP3 binding. Furthermore, methylation attenuated the repressive effects of SP1, SP3 and DEAF1 on GDF5 promoter activity. This data suggest that the differential methylation of the +37 CpG site between osteoarthritic hip and knee cartilage may be responsible for the knee-specific effect of rs143383 on OA susceptibility. PMID:24861163

Reynard, Louise N; Bui, Catherine; Syddall, Catherine M; Loughlin, John



Aspen SP1, an exceptional thermal, protease and detergent-resistant self-assembled nano-particle.  


Stable protein 1 (SP1) is a homo-oligomeric protein isolated from aspen (Populus tremula aspen) plants which forms a ring-shape dodecameric particle with a central cavity. The oligomeric form of SP1 is an exceptionally stable structure that is resistant to proteases (e.g., trypsin, V8, and proteinase K), high temperatures, organic solvents, and high levels of ionic detergent. Analytical ultra-centrifugation, chemical cross-linking, matrix-assisted laser-desorption time-of-flight mass spectrometry (MALDI-TOF-MS), and transmission electron microscopy were used to further characterize the SP1 dodecamer. Introduction of a single cysteine at the N-terminus of SP1 enabled the formation of disulfide bridges within the SP1 dodecamer, concurrent with increased melting point. A six-histidine tag was introduced at the N-terminus of SP1 to generate 6HSP1, and the DeltaNSP1 mutant was generated by a deletion of amino acids 2-6 at the N-terminus. Both 6HSP1 and DeltaNSP1 maintained their ability to assemble a stable dodecamer. Remarkably, these SP1 homo-dodecamers were able to re-assemble into stable hetero-dodecamers following co-electro-elution from SDS-PAGE. The exceptional stability of the SP1-nano ring and its ability to self-assemble hetero-complexes paves the way to further research in utilizing this unique protein in nano-biotechnology. PMID:16732592

Wang, Wang-Xia; Dgany, Or; Wolf, Sharon Grayer; Levy, Ilan; Algom, Rachel; Pouny, Yehonathan; Wolf, Amnon; Marton, Ira; Altman, Arie; Shoseyov, Oded



Gender-based blood transcriptomes and interactomes in multiple sclerosis: involvement of SP1 dependent gene transcription.  


In this study we investigated the contribution of gender to global gene expression in peripheral blood mononuclear cells from multiple sclerosis (MS) patients and healthy controls. We observed that, in contrast to the conventional approach, gender-based case-control comparisons resulted in genelists with significantly reduced heterogeneity in human populations. In addition, MS was characterized by significant changes both in the quantity and in the quality of the sex-specific genes. Application of stringent statistics defined gender-based signatures which classified a second independent MS population with high precision. The global unsupervised cluster analyses for 60 subjects showed that 29/31 female and 27/29 male samples were properly identified. Notably, MS was associated in women and in men with distinct gene signatures which however shared several molecular functions, biological processes and interactors. Issues regarding epigenetic control of gene expression appeared as the main common theme for disease, with a central role for the functional modules related to histone deacetylase, NF-kappaB and androgen receptor signaling. Moreover, in silico analyses predicted that the differential expression in MS women and men were depending on the transcription factor SP1. Specific targeting of this pathway by the bis-anthracycline WP631 impaired T cell responses in vitro and in vivo, and reduced the incidence and the severity of experimental autoimmune encephalomyelitis, indicating that SP1 dependent gene transcription sustains neuroinflammation. Thus, the gender-based approach with its reduced heterogeneity and the systems biology tools with the identification of the molecular and functional networks successfully uncovered the differences but also the commonalities associated to multiple sclerosis in women and men. In conclusion, we propose gender-based systems biology as a novel tool to gain fundamental information on disease-associated functional processes. PMID:22119415

Menon, Ramesh; Di Dario, Marco; Cordiglieri, Chiara; Musio, Silvia; La Mantia, Loredana; Milanese, Clara; Di Stefano, Anna Luisa; Crabbio, Massimo; Franciotta, Diego; Bergamaschi, Roberto; Pedotti, Rosetta; Medico, Enzo; Farina, Cinthia



Filamin B Plays a Key Role in Vascular Endothelial Growth Factor-induced Endothelial Cell Motility through Its Interaction with Rac-1 and Vav-2*  

PubMed Central

Actin-binding proteins filamin A (FLNA) and B (FLNB) are expressed in endothelial cells and play an essential role during vascular development. In order to investigate their role in adult endothelial cell function, we initially confirmed their expression pattern in different adult mouse tissues and cultured cell lines and found that FLNB expression is concentrated mainly in endothelial cells, whereas FLNA is more ubiquitously expressed. Functionally, small interfering RNA knockdown of endogenous FLNB in human umbilical vein endothelial cells inhibited vascular endothelial growth factor (VEGF)-induced in vitro angiogenesis by decreasing endothelial cell migration capacity, whereas FLNA ablation did not alter these parameters. Moreover, FLNB-depleted cells increased their substrate adhesion with more focal adhesions. The molecular mechanism underlying this effect implicates modulation of small GTP-binding protein Rac-1 localization and activity, with altered activation of its downstream effectors p21 protein Cdc42/Rac-activated kinase (PAK)-4/5/6 and its activating guanine nucleotide exchange factor Vav-2. Moreover, our results suggest the existence of a signaling complex, including FLNB, Rac-1, and Vav-2, under basal conditions that would further interact with VEGFR2 and integrin ?v?5 after VEGF stimulation. In conclusion, our results reveal a crucial role for FLNB in endothelial cell migration and in the angiogenic process in adult endothelial cells.

del Valle-Perez, Beatriz; Martinez, Vanesa Gabriela; Lacasa-Salavert, Cristina; Figueras, Agnes; Shapiro, Sandor S.; Takafuta, Toshiro; Casanovas, Oriol; Capella, Gabriel; Ventura, Francesc; Vinals, Francesc



The importance of play and playfulness  

Microsoft Academic Search

This paper focuses on the roots of play and playfulness in children's early experience of relationships. The paper seeks to argue that the current debate about the loss of safe outdoor play spaces and the dangers of over-reliance on digital entertainments sometimes neglects the fact that play and playfulness are developmental achievements and that there are huge variations in children's

Biddy Youell




PubMed Central

The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA·protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn2+ and readily competes with zinc-finger peptides for Zn2+ resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc-finger protein·DNA adducts. This paper examines the chemistry that permits the detection of zinc-finger·DNA complexes in the presence of EDTA, using Zn3-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1·GC1 was shown to depend on three properties: the inertness of Zn-Sp1·GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn3-Sp1 under the conditions of the assay that permit some Zn3-Sp1·GC1 to form. Inquiring about the mechanism of stabilization of Zn3-Sp1 by GC1, EDTA readily reacted with Zn3-Sp1 bound to a non-specific DNA, poly(dI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn3-Sp1 failed to compete for zinc bound in the Zn3-Sp1·GC-1 adduct. On the basis of these and other results indicated that the stability of Zn3-Sp1·GC-1 has a thermodynamic not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved.

Kothinti, Rajendra; Tabatabai, Niloofar M.; Petering, David H.



Electrophoretic mobility shift assay of zinc finger proteins: competition for Zn(2+) bound to Sp1 in protocols including EDTA.  


The electrophoretic mobility shift assay (EMSA) offers a principal method to detect specific DNA-protein interactions. As commonly conducted, the reaction and electrophoresis running buffers contain large concentrations of EDTA. EDTA has large affinity for Zn(2+) and readily competes with zinc finger peptides for Zn(2+) resulting in protein unfolding. Nevertheless, EMSA is routinely used to detect zinc finger protein-DNA adducts. This paper examines the chemistry that permits the detection of zinc finger-DNA complexes in the presence of EDTA, using Zn(3)-Sp1 and a cognate DNA binding site, GC1. Twice as much adduct was detected when the reaction was conducted in the absence than in the presence of EDTA. The observation of Zn-Sp1-GC1 was shown to depend on three properties: the inertness of Zn-Sp1-GC1 to reaction with EDTA and the comparatively similar rates of reaction of EDTA and GC1 with Zn(3)-Sp1 under the conditions of the assay that permit some Zn(3)-Sp1-GC1 to form. Inquiring about the mechanism of stabilization of Zn(3)-Sp1 by GC1, EDTA readily reacted with Zn(3)-Sp1 bound to a non-specific DNA, (polydI-dC). Two structurally similar but oppositely charged chelators, nitrilotriacetate (NTA) and tris-(2-ethylaminoethyl) amine (TREN), that react with free Zn(3)-Sp1 failed to compete for zinc bound in the Zn(3)-Sp1-GC-1 adduct. On the basis of these, other results indicated that the stability of Zn(3)-Sp1-GC-1 has a thermodynamic, not a kinetic origin. It is concluded that the observation of zinc finger proteins in the EMSA rests on a fortuitous set of chemical properties that may vary depending on the structures involved. PMID:21396899

Kothinti, Rajendra; Tabatabai, Niloofar M; Petering, David H



Localization of q-form fields on AdSp+1 branes  

NASA Astrophysics Data System (ADS)

In this paper, we investigate localization of a free massless q-form bulk field on thin and thick AdSp+1 branes with codimension one. It is found that the zero mode of the q-form field with q>(p+2)/2 can be localized on the thin negative tension brane, which is different from the flat brane case given in Fu et al. (2012) [57]. For the thick AdSp+1 branes, the q-form field with q>(p+2)/2 also has a localized zero mode under some conditions. Furthermore, we find that there are massive bound KK modes of the q-form field, which are localized on this type p-branes.

Fu, Chun-E.; Liu, Yu-Xiao; Guo, Heng; Chen, Feng-Wei; Zhang, Sheng-Li



The CACC box upstream of human embryonic epsilon globin gene binds Sp1 and is a functional promoter element in vitro and in vivo.  


DNA sequences 179 base pairs upstream and 23 base pairs downstream of the cap site of human embryonic epsilon globin gene exhibit promoter activity in transfected cell cultures. The nuclear factor binding in vitro of this epsilon promoter region was studied by DNase I foot-printing, methylation interference, and gel mobility shift assay. Four major nuclear factor-binding sites are detected in complexes formed with unfractionated nuclear extracts: NF-E1 at -163, epsilon F1 at -143, CACC box at -111, and CBF at -81, respectively. Of these, NF-E1 is an erythroid-specific factor. epsilon F1 is probably a ubiquitous factor because it is present in both erythroid K562 cells and nonerythroid HeLa cells. The epsilon F1-binding site exhibits sequence similarity to that of the cAMP response element-binding protein family of transcription factors. Finally, the CCAAT box-binding protein (CBF)-binding site centers around the CCAAT promoter box. Comparative binding studies with unfractionated nuclear extracts and affinity purified HeLa Sp1 demonstrated that the epsilon-globin CACC box at -111 is a binding site of Sp1. The spatial arrangements of the NF-E1-binding site, the CACC box, and CCAAT box, with respect to their mutual separations by approximately integral numbers of helical turns, is well conserved in all mammalian embryonic epsilon globin promoters. Transient expression assay, using human growth hormone gene as the receptor, demonstrated that similar to the other two human beta-like globin genes (beta and gamma), the CACC box of epsilon globin gene is an essential promoter element for epsilon globin gene expression in vivo in K562 cells. This CACC promoter box also functions in vitro in nuclear extracts prepared from K562 cells. These data, together with Sp1-binding studies of the human beta and gamma globin CACC boxes, suggest that the general transcription factor Sp1, through its differential interactions with different forms of CACC promoter boxes, is an essential component of the machinery that controls the developmental program of mammalian globin gene regulation. PMID:2026603

Yu, C Y; Motamed, K; Chen, J; Bailey, A D; Shen, C K



Involvement of Sp1 and Sp3 in differential regulation of human NHE3 promoter activity by sodium butyrate and IFN-gamma/TNF-alpha.  


Previously, we reported that IFN-gamma and TNF-alpha downregulate the expression of the human Na(+)/H(+) exchanger (NHE)3 gene by modulating Sp1/Sp3 transcription factors in C2BBe1 cells. It is reported that butyrate inhibits IFN-gamma and TNF-alpha signaling pathways. In this study, we have investigated the effect of sodium butyrate (NaB) and IFN-gamma/TNF-alpha on human NHE3 promoter activity. In transient transfection studies, NaB (5 mM) led to 10-fold stimulation of NHE3 promoter activity after incubation for 24 h. With 5'-deletion analysis, the NaB-responsive region was mapped to the NHE3 core promoter, bp -95 to + 5, which we had shown previously to confer responsiveness to IFN-gamma/TNF-alpha. The stimulatory effect of NaB on the NHE3 promoter was reduced by 60% in the presence of IFN-gamma/TNF-alpha. Mutually, the repressive effect of these cytokines was attenuated by NaB. Knockdown of Sp1 and Sp3 expression with small interfering RNA (siRNA) resulted in a significant resistance to NaB effects. NaB treatment showed no effect on Sp1 and Sp3 protein expression as assessed by Western blot analyses. Gel mobility shift assays with nuclear proteins from NaB-treated cells showed enhanced binding of Sp1 and Sp3 to the NHE3 promoter. The phosphatase inhibitor okadaic acid (200 nM) blocked the stimulatory effect of NaB on the NHE3 promoter. NaB effects on the NHE3 promoter were significantly attenuated by protein phosphatase (PP)1alpha- and PP2Aalpha-specific siRNA transfection. Our data suggest that the differential regulation of NHE3 gene expression by NaB and IFN-gamma/TNF-alpha is mediated through alternative pathways that converge on Sp1/Sp3. PMID:17540780

Amin, Md Ruhul; Dudeja, Pradeep K; Ramaswamy, Krishnamurthy; Malakooti, Jaleh



Characterization of a gene (sp1) encoding a secreted protein from Leptosphaeria maculans, the blackleg pathogen of Brassica napus.  


SUMMARY A gene (sp1) encoding a 12.3 kDa protein with a predicted secretion signal has been characterized from Leptosphaeria maculans, the dothideomycete that causes blackleg disease of canola (Brassica napus). This protein (SP1) contains four cysteine residues and shows a high sequence similarity to proteins from other ascomycetes. L. maculans sp1 has been placed on genetic and physical maps. This gene is expressed during the infection of B. napus cotyledons 10 days post-inoculation, coinciding with detection of the constitutively expressed fungal gene, beta-tubulin. L. maculans sp1, along with opsin and glyceraldehyde phosphate dehydrogenase, is light regulated. A recombinant SP1 protein expressed in Escherichia coli and a crude protein fraction secreted by L. maculans induced an autofluorescence response on B. napus leaves. The sp1 gene was mutated by targeted gene disruption whereby a hygromycin resistance gene was inserted. Such mutants caused similar-sized lesions on B. napus cotyledons as those caused by the wild-type isolate, indicating that sp1 is not crucial for pathogenicity of L. maculans on B. napus. This is the first report of disruption of this gene in any fungus. PMID:20569355

Wilson, Leanne M; Idnurm, Alexander; Howlett, Barbara J



Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway  

SciTech Connect

Highlights: {yields} Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. {yields} Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. {yields} Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

Chuang, Jian-Ying [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China)] [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China); Hung, Jan-Jong, E-mail: [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China) [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China); Institute of Bioinformatics and Biosignal Transduction, National Cheng-Kung University, Tainan 701, Taiwan (China)



The Theobroma cacao B3 domain transcription factor TcLEC2 plays a duel role in control of embryo development and maturation  

PubMed Central

Background The Arabidopsis thaliana LEC2 gene encodes a B3 domain transcription factor, which plays critical roles during both zygotic and somatic embryogenesis. LEC2 exerts significant impacts on determining embryogenic potential and various metabolic processes through a complicated genetic regulatory network. Results An ortholog of the Arabidopsis Leafy Cotyledon 2 gene (AtLEC2) was characterized in Theobroma cacao (TcLEC2). TcLEC2 encodes a B3 domain transcription factor preferentially expressed during early and late zygotic embryo development. The expression of TcLEC2 was higher in dedifferentiated cells competent for somatic embryogenesis (embryogenic calli), compared to non-embryogenic calli. Transient overexpression of TcLEC2 in immature zygotic embryos resulted in changes in gene expression profiles and fatty acid composition. Ectopic expression of TcLEC2 in cacao leaves changed the expression levels of several seed related genes. The overexpression of TcLEC2 in cacao explants greatly increased the frequency of regeneration of stably transformed somatic embryos. TcLEC2 overexpressing cotyledon explants exhibited a very high level of embryogenic competency and when cultured on hormone free medium, exhibited an iterative embryogenic chain-reaction. Conclusions Our study revealed essential roles of TcLEC2 during both zygotic and somatic embryo development. Collectively, our evidence supports the conclusion that TcLEC2 is a functional ortholog of AtLEC2 and that it is involved in similar genetic regulatory networks during cacao somatic embryogenesis. To our knowledge, this is the first detailed report of the functional analysis of a LEC2 ortholog in a species other then Arabidopsis. TcLEC2 could potentially be used as a biomarker for the improvement of the SE process and screen for elite varieties in cacao germplasm.



The N-terminal 20-Amino Acid Region of Guanine Nucleotide Exchange Factor Vav1 Plays a Distinguished Role in T Cell Receptor-mediated Calcium Signaling*  

PubMed Central

Vav1 is a guanine nucleotide exchange factor (GEF) specifically expressed in hematopoietic cells. It consists of multiple structural domains and plays important roles in T cell activation. The other highly conserved isoforms of Vav family, Vav2 and Vav3, are ubiquitously expressed in human tissues including lymphocytes. All three Vav proteins activate Rho family small GTPases, which are involved in a variety of biological processes during T cell activation. Intensive studies have demonstrated that Vav1 is indispensable for T cell receptor (TCR)-mediated signal transduction, whereas Vav2 and Vav3 function as GEFs that overlap with Vav1 on TCR-induced cytoskeleton reorganization. T cells lacking Vav1 exhibited severe defect in TCR-mediated calcium elevation, indicating that the co-existing Vav2 and Vav3 did not compensate Vav1 in calcium signaling. What is the functional particularity of Vav1 in lymphocytes? In this study, we identified the N-terminal 20 amino acids of Vav1 in the calponin homology (CH) domain to be essential for its interaction with calmodulin (CaM) that leads to TCR-induced calcium mobilization. Substitution of the 1–20 amino acids of Vav1 with those of Vav2 or Vav3 abolished the association with CaM, and the N-terminal mutations of Vav1 failed to potentiate normal TCR-induced calcium mobilization, that in turn, suspended nuclear factor of activated T cells (NFAT) activation and IL-2 production. This study highlights the importance of the N-terminal 20 aa of Vav1 for CaM binding, and provides new insights into the distinguished and irreplaceable role of Vav1 in T cell activation and signal transduction.

Li, Shi-Yang; Du, Ming-Juan; Wan, Ya-Juan; Lan, Bei; Liu, Yao-Hui; Yang, Yin; Zhang, Cui-Zhu; Cao, YouJia



Dephosphorylation of Sp1 at Ser-59 by protein phosphatase 2A (PP2A) is required for induction of CYP1A1 transcription after treatment with 2,3,7,8-tetrachlorodibenzo-p-dioxin or omeprazole.  


The aryl hydrocarbon receptor (AhR) is a transcription factor that is activated by either 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or omeprazole (OP). Activated AhR can induce CYP1A1 transcription by binding to the xenobiotic responsive element (XRE). However, the mechanism of activation of the CYP1A1 promoter region is poorly understood. Previous reports showed that Sp1 could bind to a GC-rich region near the CYP1A1 promoter. This study sought to clarify the function of Sp1 in CYP1A1 transcription. Phosphorylation of Sp1 at Ser-59 (pSer-59) was previously reported to be closely related to transcriptional regulation. We used a site-specific phospho-antibody to show that treatment with TCDD or OP drastically reduced the level of pSer-59 in Sp1 from HepG2 cells. This reduction was too much, we hypothesized that the reduced phosphorylation level resulted from activation of phosphatase activity. Given that pSer-59 is dephosphorylated by PP2A, we examined the effect of a PP2A inhibitor, okadaic acid (OA), on pSer-59 and transcription of CYP1A1. The results showed that OA blocked dephosphorylation of Ser-59 and drastically inhibited transcription of CYP1A1. Similar results were obtained after knockdown of PP2A. Treatment with OA had no effect on the expression of AhR, its nuclear translocation, or its ability to bind to the XRE. Furthermore, dephosphorylation of Sp1 at Ser-59 was not affected by knockdown of AhR. These results indicate that the signals from TCDD or OP caused PP2A-mediated dephosphorylation of Sp1 at Ser-59 and induced CYP1A1 transcription. This signaling pathway was independent of the AhR-mediated pathway. PMID:24382322

Shimoyama, Shuji; Kasai, Shuya; Kahn-Perlès, Brigitte; Kikuchi, Hideaki



Actin-depolymerizing factor and cofilin-1 play overlapping roles in promoting rapid F-actin depolymerization in mammalian nonmuscle cells.  


Actin-depolymerizing factor (ADF)/cofilins are small actin-binding proteins found in all eukaryotes. In vitro, ADF/cofilins promote actin dynamics by depolymerizing and severing actin filaments. However, whether ADF/cofilins contribute to actin dynamics in cells by disassembling "old" actin filaments or by promoting actin filament assembly through their severing activity is a matter of controversy. Analysis of mammalian ADF/cofilins is further complicated by the presence of multiple isoforms, which may contribute to actin dynamics by different mechanisms. We show that two isoforms, ADF and cofilin-1, are expressed in mouse NIH 3T3, B16F1, and Neuro 2A cells. Depleting cofilin-1 and/or ADF by siRNA leads to an accumulation of F-actin and to an increase in cell size. Cofilin-1 and ADF seem to play overlapping roles in cells, because the knockdown phenotype of either protein could be rescued by overexpression of the other one. Cofilin-1 and ADF knockdown cells also had defects in cell motility and cytokinesis, and these defects were most pronounced when both ADF and cofilin-1 were depleted. Fluorescence recovery after photobleaching analysis and studies with an actin monomer-sequestering drug, latrunculin-A, demonstrated that these phenotypes arose from diminished actin filament depolymerization rates. These data suggest that mammalian ADF and cofilin-1 promote cytoskeletal dynamics by depolymerizing actin filaments and that this activity is critical for several processes such as cytokinesis and cell motility. PMID:15548599

Hotulainen, Pirta; Paunola, Eija; Vartiainen, Maria K; Lappalainen, Pekka



Poly(ADP-ribose) Polymerase 1 Interacts with Nuclear Respiratory Factor 1 (NRF-1) and Plays a Role in NRF-1 Transcriptional Regulation.  


Nuclear respiratory factor 1 (NRF-1) is one of the key transcriptional activators for nuclear-coded genes involved in mitochondrial biogenesis and function as well as for many housekeeping genes. A transcriptional co-activator PGC-1 and its related family member PRC have previously been shown to interact with NRF-1 and co-activate NRF-1. We show here that NRF-1 can also directly interact with poly(ADP-ribose) polymerase 1 (PARP-1) and co-purify the PARP-1.DNA-PK.Ku80.Ku70.topoisomerase IIbeta-containing protein complex. Our in vitro binding experiments show that DNA-binding/dimerization domain of NRF-1 and the N-terminal half of PARP-1, which contains two Zinc fingers and the auto-modification domain, are responsible for the interaction, and that this interaction occurs with or without PARP-1 poly(ADP-ribosyl)ation (PARylation). DNA-bound NRF-1 can form a complex with PARP-1, suggesting that NRF-1 can recruit the PARP-1.DNA-PK.Ku80.Ku70.topoisomerase IIbeta-containing protein complex to the promoter. PARP-1 can also PARylate the DNA-binding domain of NRF-1 and negatively regulate NRF-1.PARP-1 interaction. Transient transfection and chromatin immunoprecipitation experiments suggest that PARP-1 plays a role during transcriptional activation by NRF-1. Our finding identifies a new aspect of transcriptional regulation used by NRF-1. PMID:19181665

Hossain, Mohammad B; Ji, Ping; Anish, Ramakrishnan; Jacobson, Raymond H; Takada, Shinako



Transcriptional activation of the murine Muc5ac mucin gene in epithelial cancer cells by TGF-beta/Smad4 signalling pathway is potentiated by Sp1.  

PubMed Central

The nucleotide sequence of the pMS1 clone was submitted to the GenBank Nucleotide Sequence Database under accession number AF288076. Changes in the expression of mucin genes in gastrointestinal cancers is thought to contribute to the development of the disease. In our laboratory we have shown previously that MUC5AC is aberrantly expressed in rectosigmoid villous adenomas. However, the regulatory mechanisms underlying that altered profile of expression is unknown. In order to study its regulation at the transcriptional level, we have isolated and characterized 5.5 kb of the 5'-flanking region of the mouse Muc5ac mucin gene. The promoter is flanked by a TATA box and a transcriptional start site is located 22 bp downstream of the TATA box. Analysis of the sequence showed a high density of binding sites for Smad4, an essential factor in the signalling cascade activated by TGF-beta (transforming growth factor-beta), and Sp1, an important factor in the regulation of MUC5AC. This led us to study Muc5ac regulation by TGF-beta. We show that exogenous addition of TGF-beta to the cells induces Muc5ac endogenous expression, promoter activity and Smad4 binding to the promoter. By co-transfection studies we show that Smad4 is essential for Muc5ac promoter activation and that it does not synergize with Smad2 or Smad3. By gel-retardation and co-transfection assays, we identified Sp1 and Sp3 as important regulators of Muc5ac expression and showed that Smad4 and Sp1 act in a co-operative manner to transactivate Muc5ac promoter activity. Altogether these results bring new insights into the molecular mechanisms of TGF-beta-mediated up-regulation of Muc5ac and enhance our understanding as to how Muc5ac is regulated in certain pathologies of the gastrointestinal tract.

Jonckheere, Nicolas; Van Der Sluis, Maria; Velghe, Amelie; Buisine, Marie-Pierre; Sutmuller, Marjolein; Ducourouble, Marie-Paule; Pigny, Pascal; Buller, Hans A; Aubert, Jean-Pierre; Einerhand, Alexandra W C; Van Seuningen, Isabelle



Interferon regulatory factor 3 plays an anti-inflammatory role in microglia by activating the PI3K/Akt pathway  

PubMed Central

Background Microglia are the principal cells involved in the innate immune response in the CNS. Activated microglia produce a number of proinflammatory cytokines implicated in neurotoxicity but they also are a major source of anti-inflammatory cytokines, antiviral proteins and growth factors. Therefore, an immune therapy aiming at suppressing the proinflammatory phenotype while enhancing the anti-inflammatory, growth promoting phenotype would be of great benefit. In the current study, we tested the hypothesis that interferon regulatory factor 3 (IRF3), a transcription factor required for the induction of IFN? following TLR3 or TLR4 activation, is critical to the microglial phenotype change from proinflammatory to anti-inflammatory, and that this phenotype change can be greatly facilitated by IRF3 gene transfer. Methods Cultures of primary human fetal microglia were transduced with IRF3 using recombinant adenovirus (Ad-IRF3) and subjected to microarray analysis, real-time PCR, immunoblotting and ELISA to determine inflammatory gene expression. Two different types of immune stimuli were tested, the TLR ligands, poly IC (PIC) and LPS, and the proinflammatory cytokines, IL-1/IFN?. In addition, the role of the PI3K/Akt pathway was examined by use of a pharmacological inhibitor, LY294002. Results Our results show that Ad-IRF3 suppressed proinflammatory genes (IL-1?, IL-1?, TNF?, IL-6, IL-8 and CXCL1) and enhanced anti-inflammatory genes (IL-1 receptor antagonist, IL-10 and IFN?) in microglia, regardless of the cell stimuli applied. Furthermore, Ad-IRF3 activated Akt, and LY294002 reversed the effects of Ad-IRF3 on microglial inflammatory gene expression. pAkt was critical in LPS- or PIC-induced production of IL-10 and IL-1ra. Significantly, microglial IFN? protein production was also dependent on pAkt and required both Ad-IRF3 and immunological stimuli (PIC > IL-1/IFN?). pAkt played much less prominent and variable roles in microglial proinflammatory gene expression. This anti-inflammatory promoting role of PI3K/Akt appeared to be specific to microglia, since astrocyte proinflammatory gene expression (as well as IFN? expression) required PI3K/Akt. Conclusions Our results show a novel anti-inflammatory role for the PI3K/Akt signaling pathway in microglia. They further suggest that IRF3 gene therapy could facilitate the microglial phenotype switch from proinflammatory ("M1-like") to anti-inflammatory and immunomodulatory ("M2-like"), in part, by augmenting the level of pAkt.



Propensity for HBZ-SP1 isoform of HTLV-I to inhibit c-Jun activity correlates with sequestration of c-Jun into nuclear bodies rather than inhibition of its DNA-binding activity  

SciTech Connect

HTLV-I bZIP factor (HBZ) contains a C-terminal zipper domain involved in its interaction with c-Jun. This interaction leads to a reduction of c-Jun DNA-binding activity and prevents the protein from activating transcription of AP-1-dependent promoters. However, it remained unclear whether the negative effect of HBZ-SP1 was due to its weak DNA-binding activity or to its capacity to target cellular factors to transcriptionally-inactive nuclear bodies. To answer this question, we produced a mutant in which specific residues present in the modulatory and DNA-binding domain of HBZ-SP1 were substituted for the corresponding c-Fos amino acids to improve the DNA-binding activity of the c-Jun/HBZ-SP1 heterodimer. The stability of the mutant, its interaction with c-Jun, DNA-binding activity of the resulting heterodimer, and its effect on the c-Jun activity were tested. In conclusion, we demonstrate that the repression of c-Jun activity in vivo is mainly due to the HBZ-SP1-mediated sequestration of c-Jun to the HBZ-NBs.

Clerc, Isabelle, E-mail: isabelle.clerc@univ-montp1.f [Universite Montpellier 1, Centre d'etudes d'agents Pathogenes et Biotechnologies pour la Sante (CPBS) (France); CNRS, UM5236, CPBS, F-34965 Montpellier (France); Universite Montpellier 2, CPBS, F-34095 Montpellier (France); Hivin, Patrick, E-mail: patrick.hivin@cea.f [Universite Montpellier 1, Centre d'etudes d'agents Pathogenes et Biotechnologies pour la Sante (CPBS) (France); CNRS, UM5236, CPBS, F-34965 Montpellier (France); Universite Montpellier 2, CPBS, F-34095 Montpellier (France); Rubbo, Pierre-Alain, E-mail: [Universite Montpellier 1, Centre d'etudes d'agents Pathogenes et Biotechnologies pour la Sante (CPBS) (France); CNRS, UM5236, CPBS, F-34965 Montpellier (France); Universite Montpellier 2, CPBS, F-34095 Montpellier (France); Lemasson, Isabelle, E-mail: LEMASSONI@ecu.ed [East Carolina University, Department of Microbiology and Immunology, North Carolina (United States); Barbeau, Benoit, E-mail: barbeau.benoit@uqam.c [Universite du Quebec a Montreal, Departement des sciences biologiques, Montreal (Canada); Mesnard, Jean-Michel, E-mail: jean-michel.mesnard@univ-montp1.f [Universite Montpellier 1, Centre d'etudes d'agents Pathogenes et Biotechnologies pour la Sante (CPBS) (France); CNRS, UM5236, CPBS, F-34965 Montpellier (France); Universite Montpellier 2, CPBS, F-34095 Montpellier (France)



Playing It Safe: Risk Management for Games Play.  

ERIC Educational Resources Information Center

Considering the players, the game, and the environment in which games are played can make games play at camp safer, more successful, and more enjoyable. Presents factors concerning the players, the game, roughhousing, excessive competitiveness, the environment, space, boundaries, weather, time, and overall game objectives that need to be…

Halliday, Nancy



Cloning and characterization of the human trefoil factor 3 gene promoter.  


Human trefoil factor 3 (hTFF3) is a small-molecule peptide with potential medicinal value. Its main pharmacological function is to alleviate gastrointestinal mucosal injuries caused by various factors and promote the repair of damaged mucosa. However, how its transcription is regulated is not yet known. The aim of this study was to clone the hTFF3 gene promoter region, identify the core promoter and any transcription factors that bind to the promoter, and begin to clarify the regulation of its expression. The 5' flanking sequence of the hTFF3 gene was cloned from human whole blood genomic DNA by PCR. Truncated promoter fragments with different were cloned and inserted into the pGL3-Basic vector to determine the position of the core hTFF3 promoter. Transcription element maintaining basic transcriptional activity was assessed by mutation techniques. Protein-DNA interactions were analyzed by chromatin immunoprecipitation (ChIP). RNA interference and gene over-expression were performed to assay the effect of transcription factor on the hTFF3 expression. The results showed that approximately 1,826 bp of the fragment upstream of hTFF3 was successfully amplified, and its core promoter region was determined to be from -300 bp to -280 bp through analysis of truncated mutants. Mutation analysis confirmed that the sequence required to maintain basic transcriptional activity was accurately positioned from -300 bp to -296 bp. Bioinformatic analysis indicated that this area contained a Sp1 binding site. Sp1 binding to the hTFF3 promoter was confirmed by ChIP experiments. Sp1 over-expression and interference experiments showed that Sp1 enhanced the transcriptional activity of the hTFF3 promoter and increased hTFF3 expression. This study demonstrated that Sp1 plays an important role in maintaining the transcription of hTFF3. PMID:24743382

Sun, Yong; Wang, Liangxi; Zhou, Yifang; Mao, Xuefei; Deng, Xiangdong



An Sp1-NF-Y/progesterone receptor DNA binding-dependent mechanism regulates progesterone-induced transcriptional activation of the rabbit RUSH/SMARCA3 gene.  


Steroids regulate alternative splicing of rabbit RUSH/SMARCA3, an SWI/SNF-related transcription factor. Transactivation was evaluated in 2057 bp of genomic sequence. Truncation analysis identified a minimal 252-bp region with strong basal promoter activity in transient transfection assays. The size of the 5'-untranslated region (233 bp) and the transcription start site were determined by primer extension analysis. The transcription start site mapped to a consensus initiator (Inr) element in a TATA-less region with a downstream promoter element (+29). These elements were authenticated by mutation/deletion analysis. The Inr/downstream promoter element combination is conserved in the putative core promoter (-35/+35) of the human ortholog, suggesting that transcription initiation is similarly conserved. Two Sp1 sites that are also conserved in the putative promoter of human SMARCA3 and a RUSH binding site (-616/-611) that is unique to the rabbit promoter repress basal transcription. These sites were variously authenticated by gel shift and chromatin immunoprecipitation assays. Analysis of the proximal promoter showed the -162/+90 region was required for progesterone responsiveness in transient transfection assays. Subsequent mutation/deletion analysis revealed a progesterone receptor half-site mediated induction by progesterone. An overlapping Y-box (in the reverse ATTGG orientation) repressed basal transcription and progesterone-induced transcriptional activation in the presence of the Sp1 sites. The specificity of progesterone receptor and transcription factor NF-Y binding were authenticated by gel shift assays. Chromatin immunoprecipitation assays confirmed the Y-box effects were mediated in a DNA binding-dependent fashion. This represents a unique regulatory scenario in which ligand-dependent transactivation by the progesterone receptor is subject to Sp1/NF-Y repression. PMID:12890680

Hewetson, Aveline; Chilton, Beverly S



Proto-oncogene FBI-1 Represses Transcription of p21CIP1 by Inhibition of Transcription Activation by p53 and Sp1*S?  

PubMed Central

Aberrant transcriptional repression through chromatin remodeling and histone deacetylation has been postulated as the driving force for tumorigenesis. FBI-1 (formerly called Pokemon) is a member of the POK family of transcriptional repressors. Recently, FBI-1 was characterized as a critical oncogenic factor that specifically represses transcription of the tumor suppressor gene ARF, potentially leading indirectly to p53 inactivation. Our investigations on transcriptional repression of the p53 pathway revealed that FBI-1 represses transcription of ARF, Hdm2 (human analogue of mouse double minute oncogene), and p21CIP1 (hereafter indicated as p21) but not of p53. FBI-1 showed a more potent repressive effect on p21 than on p53. Our data suggested that FBI-1 is a master controller of the ARF-Hdm2-p53-p21 pathway, ultimately impinging on cell cycle arrest factor p21, by inhibiting upstream regulators at the transcriptional and protein levels. FBI-1 acted as a competitive transcriptional repressor of p53 and Sp1 and was shown to bind the proximal Sp1–3 GC-box and the distal p53-responsive elements of p21. Repression involved direct binding competition of FBI-1 with Sp1 and p53. FBI-1 also interacted with corepressors, such as mSin3A, NCoR, and SMRT, thereby deacetylating Ac-H3 and Ac-H4 histones at the promoter. FBI-1 caused cellular transformation, promoted cell cycle proliferation, and significantly increased the number of cells in S phase. FBI-1 is aberrantly overexpressed in many human solid tumors, particularly in adenocarcinomas and squamous carcinomas. The role of FBI-1 as a master controller of the p53 pathway therefore makes it an attractive therapeutic target.

Choi, Won-Il; Jeon, Bu-Nam; Yun, Chae-Ok; Kim, Pyung-Hwan; Kim, Sung-Eun; Choi, Kang-Yell; Kim, Se Hoon; Hur, Man-Wook



Fatty Acid Desaturation and Elongation Reactions of Trichoderma sp. 1OH2-3  

Microsoft Academic Search

The fatty acid desaturation and elongation reactions catalyzed by Trichoderma sp. 1-OH-2-3 were investigated. This strain converted palmitic acid (16:0) mainly to stearic acid (18:0), and further to\\u000a oleic acid (c9-18:1), linoleic acid (c9,c12-18:2), and ?-linolenic acid (c9,c12,c15-18:3) through elongation, and ?9, ?12, and ?15 desaturation reactions, respectively. Palmitoleic acid (c9-16:1) and cis-9,cis-12-hexadecadienoic acid were also produced from 16:0 by

Akinori Ando; Jun Ogawa; Shigenobu Kishino; Taiyo Ito; Norifumi Shirasaka; Eiji Sakuradani; Kenzo Yokozeki; Sakayu Shimizu



Play Therapy: A Review  

ERIC Educational Resources Information Center

This article discusses the current issues in play therapy and its implications for play therapists. A brief history of play therapy is provided along with the current play therapy approaches and techniques. This article also touches on current issues or problems that play therapists may face, such as interpreting children's play, implementing…

Porter, Maggie L.; Hernandez-Reif, Maria; Jessee, Peggy



The Antimetastatic Effects of Resveratrol on Hepatocellular Carcinoma through the Downregulation of a Metastasis-Associated Protease by SP-1 Modulation  

PubMed Central

Background The mortality and morbidity rates from cancer metastasis have not declined in Taiwan, especially because of hepatocellular carcinoma (HCC). Resveratrol has been shown to have benefits such as cardioprotection, providing antioxidative, anti-inflammatory, anti-cancer properties in previous studies. Therefore, HCC cells were subjected to treatment with resveratrol and then analyzed to determine the effects of resveratrol on the migration and invasion. Methodology and Principal Findings Modified Boyden chamber assays revealed that resveratrol treatment significantly inhibited cell migration and invasion capacities of Huh7 cell lines that have low cytotoxicity in vitro, even at a high concentration of 100 µM. The results of casein zymography and western blotting revealed that the activities and protein levels of the urokinase-type plasminogen activator (u-PA) were inhibited by resveratrol. Western blot analysis also showed that resveratrol inhibits phosphorylation of JNK1/2. Tests of the mRNA level, real-time PCR, and promoter assays evaluated the inhibitory effects of resveratrol on u-PA expression in HCC cells. The chromatin immunoprecipitation (ChIP) assay showed that reactive in transcription protein of nuclear factor SP-1 was inhibited by resveratrol. Conclusions Resveratrol inhibits u-PA expression and the metastasis of HCC cells and is a powerful chemopreventive agent. The inhibitory effects were associated with the downregulation of the transcription factors of SP-1 signaling pathways.

Yeh, Chao-Bin; Hsieh, Ming-Ju; Lin, Chiao-Wen; Chiou, Hui-Ling; Lin, Pen-Yuan; Chen, Tzy-Yen; Yang, Shun-Fa



A Feedback Loop Consisting of MicroRNA 23a/27a and the ?-Like Globin Suppressors KLF3 and SP1 Regulates Globin Gene Expression  

PubMed Central

The developmental stage-specific expression of the human ?-like globin genes has been studied for decades, and many transcriptional factors as well as other important cis elements have been identified. However, little is known about the microRNAs that potentially regulate ?-like globin gene expression directly or indirectly during erythropoiesis. In this study, we show that microRNA 23a (miR-23a) and miR-27a promote ?-like globin gene expression in K562 cells and primary erythroid cells through targeting of the transcription factors KLF3 and SP1. Intriguingly, miR-23a and miR-27a further enhance the transcription of ?-like globin genes through repression of KLF3 and SP1 binding to the ?-like globin gene locus during erythroid differentiation. Moreover, KLF3 can bind to the promoter of the miR-23a?27a?24-2 cluster and suppress this microRNA cluster expression. Hence, a positive feedback loop comprised of KLF3 and miR-23a promotes the expression of ?-like globin genes and the miR-23a?27a?24-2 cluster during erythropoiesis.

Ma, Yanni; Wang, Bin; Jiang, Fengbing; Wang, Dongsheng; Liu, Huiwen; Yan, Yunmeng; Dong, He; Wang, Fang; Gong, Bei; Zhu, Yong; Dong, Lei; Yin, Haixin; Zhang, Zhongzu; Zhao, Hualu; Wu, Zhikui; Zhang, Junwu



Genetic Selection for Context-Dependent Stochastic Phenotypes: Sp1 and TATA Mutations Increase Phenotypic Noise in HIV-1 Gene Expression  

PubMed Central

The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly ‘switch’ between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify ‘Switching’ phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a methodology to identify and characterize promoter elements that affect the distribution of stochastic phenotypes over genomic contexts, and advances our understanding of how promoter mutations may control the frequency of latent HIV infection.

Shah, Priya S.; Arkin, Adam P.; Schaffer, David V.



Evaluating Playfulness: Construct Validity of the Children's Playfulness Scale.  

ERIC Educational Resources Information Center

Examined the underlying structure and factorial validity of the Children's Playfulness Scale in evaluating preschool children's behavior. Found that factor loadings, factor variances/covariances, and error variances/covariances are invariant across calibration and validation groups, indicating the good cross-generalizability of the scale. (JPB)

Trevlas, Efthimios; Grammatikopoulous, Vasilios; Tsigilis, Nikolaos; Zachopoulou, Evridiki



Scalability of Parallel Spatial Direct Numerical Simulations on Intel Hypercube and IBM SP1 and SP2  

NASA Technical Reports Server (NTRS)

The implementation and performance of a parallel spatial direct numerical simulation (PSDNS) approach on the Intel iPSC/860 hypercube and IBM SP1 and SP2 parallel computers is documented. Spatially evolving disturbances associated with the laminar-to-turbulent transition in boundary-layer flows are computed with the PSDNS code. The feasibility of using the PSDNS to perform transition studies on these computers is examined. The results indicate that PSDNS approach can effectively be parallelized on a distributed-memory parallel machine by remapping the distributed data structure during the course of the calculation. Scalability information is provided to estimate computational costs to match the actual costs relative to changes in the number of grid points. By increasing the number of processors, slower than linear speedups are achieved with optimized (machine-dependent library) routines. This slower than linear speedup results because the computational cost is dominated by FFT routine, which yields less than ideal speedups. By using appropriate compile options and optimized library routines on the SP1, the serial code achieves 52-56 M ops on a single node of the SP1 (45 percent of theoretical peak performance). The actual performance of the PSDNS code on the SP1 is evaluated with a "real world" simulation that consists of 1.7 million grid points. One time step of this simulation is calculated on eight nodes of the SP1 in the same time as required by a Cray Y/MP supercomputer. For the same simulation, 32-nodes of the SP1 and SP2 are required to reach the performance of a Cray C-90. A 32 node SP1 (SP2) configuration is 2.9 (4.6) times faster than a Cray Y/MP for this simulation, while the hypercube is roughly 2 times slower than the Y/MP for this application. KEY WORDS: Spatial direct numerical simulations; incompressible viscous flows; spectral methods; finite differences; parallel computing.

Joslin, Ronald D.; Hanebutte, Ulf R.; Zubair, Mohammad



Children's Play and Television.  

ERIC Educational Resources Information Center

Discusses adverse effects of FCC deregulation of children's television programming on children's play behavior. Discusses the difference between play and imitation, the role of high quality dramatic play in healthy child development, the popularity of war play, and use of toys to increase dramatic play. Considers ways to help children gain control…

Powell, Mark



All the World's a Stage? Consequences of a Role-Playing Pedagogy on Psychological Factors and Writing and Rhetorical Skill in College Undergraduates  

ERIC Educational Resources Information Center

Reacting to the Past is a pedagogy involving collaborative role playing in history-based games over a semester. This article presents results from a systematic assessment of this novel pedagogy conducted in 3 phases following student focus group interviews. Interviews indicated that the method was generally popular compared with traditional…

Stroessner, Steven J.; Beckerman, Laurie Susser; Whittaker, Alexis



Work in progress — Using interactivity video games factors to define Role Playing Games as a supporting tool for learning by doing  

Microsoft Academic Search

Edutainment has emerged as a computational area that combines entertainment and education in order to engage students to learn in creative ways. Role Playing Games (RPG) are a type of games where players take the role of a character within a story and make decisions to advance and reach a given goal. RPG's are highly interactive entertainment tools that propose

Gilberto Huesca; Julieta Noguez; Luis Neri; Víctor Robledo-Rella



The Nuclear Factor Y subunits NF-YB2 and NF-YB3 play additive roles in the promotion of flowering by inductive long-day photoperiods in Arabidopsis  

Microsoft Academic Search

Accumulating evidence supports a role for members of the plant Nuclear Factor Y (NF-Y) family of CCAAT-box binding transcription\\u000a factors in the regulation of flowering time. In this study we have used a genetic approach to show that the homologous proteins\\u000a NF-YB3 and NF-YB2 have comparable activities and play additive roles in the promotion of flowering, specifically under inductive\\u000a photoperiodic

Roderick W. Kumimoto; Luc Adam; Graham J. Hymus; Peter P. Repetti; T. Lynne Reuber; Colleen M. Marion; Frederick D. Hempel; Oliver J. Ratcliffe



Isolation and partial characterization of ?SP-1, a Salmonella specific lytic phage from intestinal content of broiler chicken.  


Salmonella enterica subsp. enterica serovar Enteritidis is a major causative agent of gastroenteritis with contaminated eggs and chicken meat being the major source of infection. Phages are seriously being considered as a safe and cheaper alternative to antibiotics. The intestinal content of chicken was used as source for isolating phages. Phage designated as ?SP-1 was selected for the study. Transmission electron microscopy (TEM) of phage ?SP-1 revealed that it belonged to family Podoviridae. The optimal multiplicity of infection (MOI) was 5 phages/cell. Latent and rise period were calculated to be 30 and 55 minutes respectively, while burst size was 44 phages/bacterial cell. The genome size of ?SP-1 was estimated to be 86?kb from pulsed-field gel electrophoresis analysis (PFGE). The effect of different physical and chemical parameters like temperature, pH, salinity and CaCl? were analyzed to optimize the conditions for large scale production of phages and to check the viability of ?SP-1 under different physiochemical conditions. A temperature of 40?°C, pH 8 and 0.25?M NaCl were found to be optimum for phage adsorption and it was able to survive up to a temperature of 50?°C for 3?min. Capability to survive under hostile environmental conditions, absence of virulence genes in genome and genus specificity suggest suitability of ?SP-1 to be used as a biocontrol agent. PMID:22733367

Augustine, Jeena; Louis, Linda; Varghese, Siju M; Bhat, Sarita G; Kishore, Archana



miR-145 sensitizes ovarian cancer cells to paclitaxel by targeting Sp1 and Cdk6.  


Multidrug resistance (MDR) remains a major obstacle to effective chemotherapy treatment in ovarian cancer. In our study, paclitaxel-resistant ovarian cancer patients and cell lines had decreased miR-145 levels and expressed high levels of Sp1 and Cdk6. Introducing miR-145 into SKOV3/PTX and A2780/PTX cells led to a reduction in Cdk6 and Sp1 along with downregulation of P-gp and pRb. These changes resulted in increased accumulation of antineoplastic drugs and G1 cell cycle arrest, which rendered the cells more sensitive to paclitaxel in vitro and in vivo. These effects could be reversed by reintroducing Sp1 or Cdk6 into cells expressing high levels of miR-145, resulting in restoration of P-gp and pRb levels. Furthermore, we confirmed that both Cdk6 and Sp1 are targets of miR-145. Intriguingly, demethylation with 5-aza-dC led to reactivation of miR-145 expression in drug-resistant ovarian cancer cell lines, which also resulted in increased sensitivity to paclitaxel. Collectively, these findings begin to elucidate the role of miR-145 as an important regulator of chemoresistance in ovarian cancer by controlling both Cdk6 and Sp1. PMID:24510775

Zhu, Xiaolan; Li, Yuefeng; Xie, Chanjuan; Yin, Xinming; Liu, Yueqin; Cao, Yuan; Fang, Yue; Lin, Xin; Xu, Yao; Xu, Wenlin; Shen, Huiling; Wen, Jian



Electron cryotomography of immature HIV-1 virions reveals the structure of the CA and SP1 Gag shells  

PubMed Central

The major structural elements of retroviruses are contained in a single polyprotein, Gag, which in human immunodeficiency virus type 1 (HIV-1) comprises the MA, CA, spacer peptide 1 (SP1), NC, SP2, and p6 polypeptides. In the immature HIV-1 virion, the domains of Gag are arranged radially with the N-terminal MA domain at the membrane and C-terminal NC-SP2-p6 region nearest to the center. Here, we report the three-dimensional structures of individual immature HIV-1 virions, as obtained by electron cryotomography. The concentric shells of the Gag polyprotein are clearly visible, and radial projections of the different Gag layers reveal patches of hexagonal order within the CA and SP1 shells. Averaging well-ordered unit cells leads to a model in which each CA hexamer is stabilized by a bundle of six SP1 helices. This model suggests why the SP1 spacer is essential for assembly of the Gag lattice and how cleavage between SP1 and CA acts as a structural switch controlling maturation.

Wright, Elizabeth R; Schooler, Jordan B; Ding, H Jane; Kieffer, Collin; Fillmore, Christopher; Sundquist, Wesley I; Jensen, Grant J



Sp1 mediates cell proliferation-dependent regulation of rat DNA topoisomerase IIalpha gene promoter.  

PubMed Central

DNA topoisomerase IIalpha (topo IIalpha) is an essential nuclear enzyme required for chromosome segregation during mitosis. Consistent with its critical role in cell division is the fact that the expression of the gene for topo IIalpha is strongly regulated by the proliferation state of cells. Using a transient expression system, we determined the contribution of putative cis-acting elements in its promoter region to its basal level and cell proliferation-dependent transcription. Experiments with 5' and/or 3' serial deletion and site-directed mutation revealed that (1) maximal promoter activity resides in the fragment extending to position -663 bp from the ATG initiation codon, (2) minimal promoter activity is harboured at -195 bp, (3) the defined minimal promoter contains only two putative elements, inverted CCAAT box 4 (ICB4) (-166 to -162 bp) and the most proximal GC-rich box in the promoter (GC2) (-149 to -143 bp), and (4) ICB4 is most important in the basal-level transcription of the gene for rat topo IIalpha. The luciferase activities of the mutated reporter plasmids in G(0)-arrested and exponentially growing cells showed that proliferation-specific regulation is controlled mainly by GC2. Electrophoretic mobility-shift assays indicated that Sp1 binds specifically to the GC2 site. The extent of DNA-protein complex formation increases after the stimulation of cells to proliferate. These results indicate that the increased binding activity of Sp1 to GC2 is important in the up-regulation of the gene for topo IIalpha in growing cells.

Yoon, J H; Kim, J K; Rha, G B; Oh, M; Park, S H; Seong, R H; Hong, S H; Park, S D



Basal expression of the human MAPEG members microsomal glutathione transferase 1 and prostaglandin E synthase genes is mediated by Sp1 and Sp3.  


Microsomal glutathione transferase (MGST1) and prostaglandin E synthase (PGES) are both members of the MAPEG (Membrane Associated Proteins involved in Eicosanoid and Glutathione metabolism) superfamily. In humans, their organ distribution is quite distinct with the former being widely and constitutively expressed whereas PGES is largely inducible. In order to study the basal expression of these genes, we characterized the promoter regions and identified the elements and the transcription factors required using in vitro assays, including reporter analysis of deletion and mutant clones and EMSA. The results indicate that Sp1 is the protein mediating the basal transcription of MGST1. It appears that both the Sp1 and Sp3 proteins are important for the basal expression of PGES. In addition, mutational analysis of two Barbie-box elements in the PGES promoter showed that these were not involved in the down-regulation of PGES by phenobarbital (PB). These results provide the first description of the basal regulation of these genes in humans. PMID:12818425

Ekström, Lena; Lyrenäs, Louise; Jakobsson, Per-Johan; Morgenstern, Ralf; Kelner, Michael J



Cytosine methylation of an Sp1 site contributes to organ-specific and cell-specific regulation of expression of the lung epithelial gene t1alpha.  

PubMed Central

Several recent observations have suggested that cytosine methylation has a role in the in vivo transcriptional regulation of cell-specific genes in normal cells. We hypothesized that methylation regulates T1alpha, a gene expressed primarily in lung in adult rodents. In fetuses T1alpha is expressed in several organs, including the entire nervous system, but during development its expression is progressively restricted to lung alveolar type I epithelial cells, some osteoblasts and choroid plexus. Here we report that T1alpha is methylated at a key Sp1 site in the proximal promoter in cells and organs, including brain, where no gene expression is detectable. Conversely, in T1alpha-expressing cells, these sites are not methylated. In embryonic brain T1alpha is unmethylated and expressed; in adult brain the gene is methylated and not expressed. In lung epithelial cell lines, methylation of the T1alpha promoter in vitro decreases expression by approx. 50% (the maximum suppression being 100%). Analysis of mutated promoter constructs indicates that a single Sp1 site in the proximal promoter provides all or most of the methylation-sensitive gene silencing. We conclude that, in addition to regulation by transcription factors, cytosine methylation has a role in the complex expression patterns of this gene in intact animals and primary cells.

Cao, Y X; Jean, J C; Williams, M C



Des Regles et du Jeu. Complementarite des facteurs genetiques et epigenetiques dans le developpement cerebral (Of Rules and of Play. The Complementary Nature of Genetic and Epigenetic Factors in Brain Development).  

ERIC Educational Resources Information Center

Discusses the importance of genetic and epigenetic factors in the development of the nervous system and the performances it conditions. From the perspective of rules, play, and relaxation of rules, learning and education are not considered as a kind of conditioning but as providing a content in which the cumulative expression of potential can take…

Lambert, Jean-Francois



Understanding Playful Pedagogies, Play Narratives and Play Spaces  

ERIC Educational Resources Information Center

This paper is a tentative attempt to unwrap and understand one aspect of playful practice and the influences which determine its existence in early years settings. "Storying" events, those occasions when teachers and children together "make up" stories or parts of stories, develop roles or co-construct fantasies, occur moment by moment in some…

Goouch, Kathy



Musical Instrument Choice and Playing History in Post-Secondary Level Music Students: Some Descriptive Data, Some Causes and Some Background Factors  

ERIC Educational Resources Information Center

Why do musicians specialize in the specific instruments that they do? Research has shown effects of such factors as the perceived masculinity/femininity of instruments and musician's personality but there are little background data on other factors. The present study had two major aims. The first aim was to gather some useful background data on…

Chen, Simy Meng-Yu; Howard, Robert W.



Children's empowerment in play  

Microsoft Academic Search

This article examines the level of empowerment and autonomy children can create in their play experiences. It examines the play discourses that children build and maintain and considers the importance of play contexts in supporting children's emotional and social development. These aspects of play are often unseen or misunderstood by the adult observer. The article emphasises the importance of adult?free

Natalie Canning




PubMed Central

We previously reported up-regulation of aryl hydrocarbon receptor (AhR) expression as a mechanism by which overexpression of Cu/Zn-superoxide dismutase (SOD) and/or catalase accelerates benzo(a)pyrene (BaP) detoxification in mouse aorta endothelial cells (MAECs). The objective of this study was to investigate the regulatory role of specificity protein-1 (Sp1) in AhR expression in MAECs that overexpress Cu/Zn-SOD and/or catalase. Our data demonstrated comparable levels of nuclear Sp1 protein in the transgenic and wild-type MAECs; however, binding of Sp1 protein to the AhR promoter region was more than 2-fold higher in MAECs overexpressing Cu/Zn-SOD and/or catalase than in wild-type cells. Inhibition of Sp1 binding to the AhR promoter by mithramycin A reduced AhR expression and eliminated the differences between wild-type MAECs, and three lines of transgenic cells. Functional promoter analysis indicated that AhR promoter activity was significantly higher in MAECs overexpressing catalase than in wild-type cells. Mutation of an AhR promoter Sp1-binding site or addition of hydrogen peroxide to the culture medium reduced AhR promoter activity, and decreased the differences between wild-type MAECs and transgenic cells overexpressing catalase. These results suggest that increased Sp1 binding to the AhR promoter region is an underlying mechanism for up-regulation of AhR expression in MAECs that overexpress Cu/Zn-SOD and/or catalase.

Tang, Tian; Lin, Xinghua; Yang, Hong; Zhou, LiChun; Wang, Zefen; Shan, Guang; Guo, ZhongMao



Child's Play: Therapist's Narrative  

PubMed Central

Play has been recognized as an essential component to children's healthy development. Schools of play therapy differ philosophically and technically, but they all embrace the therapeutic and developmental properties of play. This case report is an illustration of how a 6-year-old child with emotional disorder was facilitated to express concerns in child-centered play therapy. The paper discusses the therapist's narration of the child's play.

Reddy, Rajakumari P.; Hirisave, Uma



The interactomes of influenza virus NS1 and NS2 proteins identify new host factors and provide insights for ADAR1 playing a supportive role in virus replication.  


Influenza A NS1 and NS2 proteins are encoded by the RNA segment 8 of the viral genome. NS1 is a multifunctional protein and a virulence factor while NS2 is involved in nuclear export of viral ribonucleoprotein complexes. A yeast two-hybrid screening strategy was used to identify host factors supporting NS1 and NS2 functions. More than 560 interactions between 79 cellular proteins and NS1 and NS2 proteins from 9 different influenza virus strains have been identified. These interacting proteins are potentially involved in each step of the infectious process and their contribution to viral replication was tested by RNA interference. Validation of the relevance of these host cell proteins for the viral replication cycle revealed that 7 of the 79 NS1 and/or NS2-interacting proteins positively or negatively controlled virus replication. One of the main factors targeted by NS1 of all virus strains was double-stranded RNA binding domain protein family. In particular, adenosine deaminase acting on RNA 1 (ADAR1) appeared as a pro-viral host factor whose expression is necessary for optimal viral protein synthesis and replication. Surprisingly, ADAR1 also appeared as a pro-viral host factor for dengue virus replication and directly interacted with the viral NS3 protein. ADAR1 editing activity was enhanced by both viruses through dengue virus NS3 and influenza virus NS1 proteins, suggesting a similar virus-host co-evolution. PMID:23853584

de Chassey, Benoît; Aublin-Gex, Anne; Ruggieri, Alessia; Meyniel-Schicklin, Laurène; Pradezynski, Fabrine; Davoust, Nathalie; Chantier, Thibault; Tafforeau, Lionel; Mangeot, Philippe-Emmanuel; Ciancia, Claire; Perrin-Cocon, Laure; Bartenschlager, Ralf; André, Patrice; Lotteau, Vincent



Stromal factor plays an essential role in protein integration into thylakoids that cannot be replaced by unfolding or by heat shock protein Hsp70.  

PubMed Central

The light-harvesting chlorophyll a/b protein (LHCP) is an integral thylakoid membrane protein. It is made in the cytosol as a precursor (pLHCP), imported into chloroplasts, and subsequently integrated into thylakoids. Integration of pLHCP into thylakoids requires a stromal protein factor that functions in part to maintain the solubility and integration competence of pLHCP. Recently, it was reported that unfolded pLHCP was sufficient for integration and that the stromal factor, identified as the plastid Hsp70, was required only to prevent pLHCP refolding [Yalovsky, S., Paulsen, H., Michaeli, D., Chitnis, P. R. & Nechushtai, R. (1992) Proc. Natl. Acad. Sci. USA 89, 5616-5619]. Our studies, using more rigorous criteria for integration, show that unfolded pLHCP is not sufficient; stromal factor is an absolute requirement for integration. Furthermore, experiments with purified Hsp70 as well as Hsp70-depleted stromal extract demonstrate that Hsp70 is not the stromal factor. These results plus the finding that pLHCP diluted out of urea is relatively stable as a substrate for integration point to an additional role for the stromal factor in targeting and/or membrane translocation. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 6

Yuan, J; Henry, R; Cline, K



The concept of play  

Microsoft Academic Search

Recent criticisms of the concept of play do not go far enough. After noting the possible roles of generalization, thresholds and learning in play activities, it is suggested that the facts subsummed under the term \\

Harold Schlosberg



Adlerian Play Therapy.  

ERIC Educational Resources Information Center

Describes Adlerian method of play therapy. Claims Adlerian therapy represents an integration of the concepts and techniques of individual psychology into a method of using play to help troubled children. (Author/ABL)

Kottman, Terry; Warlick, Jayne



Play the Electrocardiogram Game  


... and Work Teachers' Questionnaire Electrocardiogram Play the ECG Game About the game ECG is used for diagnosing heart conditions by ... last will in Paris. Play the Blood Typing Game Try to save some patients and learn about ...


Play the MRI Game  


... Teachers' Questionnaire MRI Play MRI the Magnetic Miracle Game About the game In the MRI imaging technique, strong magnets and ... last will in Paris. Play the Blood Typing Game Try to save some patients and learn about ...


Role-Playing Mitosis.  

ERIC Educational Resources Information Center

Introduces a role playing activity that actively engages students in the learning process of mitosis. Students play either chromosomes carrying information, or cells in the cell membrane. (Contains 11 references.) (Author/YDS)

Wyn, Mark A.; Stegink, Steven J.



Playing With Fire?  


... Injuries Jellyfish The Pink Locker Society Playing With Fire? KidsHealth > Kids > Staying Safe > Playing It Safe Outdoors ... night, or a professional fireworks display. Continue Why Fire Fascinates Fire is a tool and a fascinating ...


Synthesis and biological evaluation of a new derivative of bevirimat that targets the Gag CA-SP1 cleavage site.  


Bevirimat (2), the first-in-class HIV-1 maturation inhibitor, shows a low efficacy due essentially to the natural polymorphism of its target, the CA-SP1 junction. Moreover, its low hydrosolubility makes it difficult to study its interaction with the CA-SP1 junction. We have synthesized new derivatives of bevirimat by adding different hydrophilic substituents at the C-28 position to improve their hydrosolubility and perform the structural study of a complex by NMR. Synthesis of the new derivatives, the effect of substituents at the C-28 position and their hydrosolubility are discussed. The ability of these molecules to inhibit viral infection and their cytotoxicity is assessed. Compared to the well-known bevirimat (2), one of our compounds (16) shows a higher hydrosolubility associated with a 2.5 fold increase in activity, a higher selectivity index and a better antiviral profile. Moreover, for the first time a direct interaction between a derivative of bevirimat (16) and the domain CA-SP1-NC is shown by NMR. Information from this study should allow us to decipher the mechanism by which bevirimat inhibits HIV-1 maturation and how the natural polymorphism of the spacer peptide SP1 triggers resistance to inhibitors. PMID:23399723

Coric, Pascale; Turcaud, Serge; Souquet, Florence; Briant, Laurence; Gay, Bernard; Royer, Jacques; Chazal, Nathalie; Bouaziz, Serge



Quantitative Analysis of Estrogen Receptor Expression Shows SP1 antibody is more sensitive than 1D5  

PubMed Central

Studies comparing rabbit monoclonal SP1 antibody to 1D5 for ER immunohistochemical (IHC) testing show conflicting results. Here we use a standardized quantitative immunofluorescent (QIF) ER assay to determine the level and significance of discordance between antibodies. Both antibodies are assessed by QIF on our Index TMA of cell lines and case controls, followed by QIF and IHC on two retrospective cohorts from Yale. On the Index TMA, SP1 displayed stronger signal-to-noise than 1D5. On the patient cohorts, the range of discrepancy between the two antibodies is 8% to 16.9%, with the majority of discrepant cases being SP1-positive/1D5-negative. Kaplan Meier analysis of the discrepant cases shows outcome comparable to double positive cases, suggesting that SP1 is more sensitive than 1D5. A series of cases with high levels of ER-beta shows that neither antibody cross-reacts, suggesting equivalent specificity. Future efforts are needed to determine if response to endocrine therapies show superiority of either antibody as a companion diagnostic test.

Welsh, Allison W.; Harigopal, Malini; Wimberly, Hallie; Prasad, Manju; Rimm, David L.



Quantitative analysis of estrogen receptor expression shows SP1 antibody is more sensitive than 1D5.  


Studies comparing rabbit monoclonal SP1 antibody with 1D5 for estrogen receptor (ER) immunohistochemical testing show conflicting results. Here we use a standardized quantitative immunofluorescent (QIF) ER assay to determine the level and significance of discordance between the antibodies. Both antibodies were assessed by QIF on our Index TMA of cell lines and case controls, followed by QIF and immunohistochemical analysis on 2 retrospective cohorts from Yale. On the Index TMA, SP1 displayed stronger signal-to-noise ratio compared with 1D5. On the patient cohorts, the range of discrepancy between the 2 antibodies was 8% to 16.9%, with the majority of discrepant cases being SP1 positive/1D5 negative. Kaplan-Meier analysis of the discrepant cases showed outcomes comparable to those of double-positive cases, suggesting that SP1 is more sensitive than 1D5. A series of cases with high levels of ER-? shows that neither antibody cross-reacts, suggesting equivalent specificity. Future efforts are needed to determine whether response to endocrine therapies show superiority of either antibody as a companion diagnostic test. PMID:22820659

Welsh, Allison W; Harigopal, Malini; Wimberly, Hallie; Prasad, Manju; Rimm, David L



Economic-Statistical Design of Effective Variance Control Charts, /Sp/1/p  

NASA Astrophysics Data System (ADS)

Multivariate Statistical Process Control (MSPC) is to control several quality characteristics simultaneously during a production process. When a process is statistically under control, only common causes of variability affect the process. The MSPC techniques help to detect the special causes of variability on the process. In the last few years multivariate quality control has been thoroughly studied and there are different but those charts don't let control de multivariate dispersion on process. Recently the generalized variance control chart /S/, has been develop to control multivariate dispersion. Nevertheless, this chart is unhelpful to work with incomplete databases or with missing data, that are commonly found in a great deal of processes due to the failure of certain sensors. In this sense, Garcia-Diaz developed the effective variance control chart, /Sp/1/p, to control multivariate dispersion with missing data. To design the economic-statistical of the Variance Effective control chart, attempting to obtain the values of the parameters in the sampling plan that minimize the expected operation costs by time unit, according to the restrictions of a minimum ARL value under control, and a maximum ARL value when the process is out of control.

García-Díaz, J. Carlos; Martínez-Gómez, M.



The Pedagogy of Play  

ERIC Educational Resources Information Center

Play is important. Environmental educators Sobel and Louv write about the relationship between children and outside play and suggest that early transcendental experiences within nature allow children to develop empathetic orientations towards the natural world. Children who play out-of-doors develop an appreciation for the environment and…

Giesbrecht, Sheila



Playful Teaching Practices  

ERIC Educational Resources Information Center

In physical education, playful teaching practices are essential to relationship building and creating "connections" for successful group dynamics. Perhaps most importantly, playful teachers develop positive attitudes in their students and help students understand that learning can be fun and joyful. Playful teaching practices also greatly enhance…

Michaelis, Bill



Dramatic Play and Print.  

ERIC Educational Resources Information Center

Discusses a project that examined literacy development within the context of play in two Reykjavik preschool classrooms. Suggests that preschool teachers should enrich dramatic play with literacy props and print materials. Indicates that incorporating print materials in dramatic play provides a natural extension of children's preschool work and…

Einarsdottir, Johanna



Understanding through Play.  

ERIC Educational Resources Information Center

Examines practice play, symbolic play, games with rules, and constructions and their relation to Piaget's active education, the intentional social process of constructing understanding involving interest, experimentation, and cooperation within the play context. Recommendations for identifying the type of knowledge being constructed (physical,…

Chaill, Christine; Silvern, Steven B.



Play, Policy & Practice.  

ERIC Educational Resources Information Center

In 1992, the U.S.-Israel Binational Science Foundation (BSF), in conjunction with Wheelock College (Boston), sponsored its second workshop on children's play, entitled "Play and Cognitive Ability: The Cultural Context." This volume reflects the presentations and discussions held at the workshop, offering perspectives on children's play that, taken…

Klugman, Edgar, Ed.


Rsk2 Knockdown in PC12 Cells Results in Sp1 Dependent Increased Expression of the Gria2 Gene, Encoding the AMPA Receptor Subunit GluR2  

PubMed Central

The RSK2 protein is a member of the RSK serine-threonine protein kinase family and is encoded by the X-linked rps6ka3 gene in human. Highly heterogeneous loss-of-function mutations affecting this gene are responsible for a severe syndromic form of cognitive impairment, Coffin-Lowry syndrome. RSK2, which is highly conserved in mammals, acts at the distal end of the Ras-ERK signaling pathway and is activated in response to growth factors and neurotransmitters. RSK2 is highly expressed in the hippocampus, and Rsk2-KO mice display spatial learning and memory impairment. We recently showed that ERK1/2 activity is abnormally increased in the hippocampus of Rsk2-KO mice as well as the expression of the AMPA receptor subunit GluR2. The mechanism via which RSK2 deficiency affects the expression of GluR2 in neural cells was unknown. To address this issue we constitutively suppressed the expression of RSK2 in PC12 cells via vector-based shRNA in the present study. We show that Rsk2 silencing leads also to an elevation of ERK1/2 phosphorylation as well as of GluR2 expression and that the increased level of GluR2 expression results from the increased ERK1/2 activity on the transcription factor Sp1. Our results provide evidence that RSK2 modulates ERK1/2 activity on Sp1, which regulates GluR2 expression through transcriptional activation.

Mehmood, Tahir; Schneider, Anne; Pannetier, Solange; Hanauer, Andre



Activation of TGF-?1 Promoter by Hepatitis C Virus-Induced AP-1 and Sp1: Role of TGF-?1 in Hepatic Stellate Cell Activation and Invasion  

PubMed Central

Our previous studies have shown the induction and maturation of transforming growth factor-beta 1 (TGF-?1) in HCV-infected human hepatoma cells. In this study, we have investigated the molecular mechanism of TGF-?1 gene expression in response to HCV infection. We demonstrate that HCV-induced transcription factors AP-1, Sp1, NF-?B and STAT-3 are involved in TGF-?1 gene expression. Using chromatin immunoprecipitation (ChIP) assay, we further show that AP-1 and Sp1 interact with TGF-b1 promoter in vivo in HCV-infected cells. In addition, we demonstrate that HCV-induced TGF-?1 gene expression is mediated by the activation of cellular kinases such as p38 MAPK, Src, JNK, and MEK1/2. Next, we determined the role of secreted bioactive TGF-?1 in human hepatic stellate cells (HSCs) activation and invasion. Using siRNA approach, we show that HCV-induced bioactive TGF-?1 is critical for the induction of alpha smooth muscle actin (?-SMA) and type 1 collagen, the markers of HSCs activation and proliferation. We further demonstrate the potential role of HCV-induced bioactive TGF-?1 in HSCs invasion/cell migration using a transwell Boyden chamber. Our results also suggest the role of HCV-induced TGF-?1 in HCV replication and release. Collectively, these observations provide insight into the mechanism of TGF-?1 promoter activation, as well as HSCs activation and invasion, which likely manifests in liver fibrosis associated with HCV infection.

Presser, Lance D.; McRae, Steven; Waris, Gulam



Intrauterine Growth Retardation and Consequences for Endocrine and Cardiovascular Diseases in Adult Life: Does Insulin-Like Growth Factor-I Play a Role?  

Microsoft Academic Search

Low birth weight has been associated with an increased incidence of ischaemic heart disease (IHD) and type 2 diabetes. Endocrine regulation of fetal growth by growth hormone (GH) and insulin-like growth factor (IGF)-I is complex. Placental GH is detectable in maternal serum from the 8th to the 12th gestational week, and rises gradually during pregnancy where it replaces pituitary GH

Rikke Bodin Beck Jensen; Marla Chellakooty; Signe Vielwerth; Allan Vaag; Torben Larsen; Gorm Greisen; Niels E. Skakkebæk; Thomas Scheike; Anders Juul




NSDL National Science Digital Library

Inter-Play is a Web-based, multilingual index to plays published as part of collections, anthologies, or periodicals, and compiles many citations to plays not indexed by standard print indexes. Inter-Play contains about 16,130 bibliographic citations and spans in date from the late nineteenth century to the present. Although the index is multilingual, the search interface is in English. Users may search the index by author or title only. Inter-Play is updated frequently by its co-editors: Robert Westover and Janet Wright, humanities librarians at Portland State University.


African oil plays  

SciTech Connect

The vast continent of Africa hosts over eight sedimentary basins, covering approximately half its total area. Of these basins, only 82% have entered a mature exploration phase, 9% have had little or no exploration at all. Since oil was first discovered in Africa during the mid-1950s, old play concepts continue to bear fruit, for example in Egypt and Nigeria, while new play concepts promise to become more important, such as in Algeria, Angola, Chad, Egypt, Gabon, and Sudan. The most exciting developments of recent years in African oil exploration are: (1) the Gamba/Dentale play, onshore Gabon; (2) the Pinda play, offshore Angola; (3) the Lucula/Toca play, offshore Cabinda; (4) the Metlaoui play, offshore Libya/Tunisia; (5) the mid-Cretaceous sand play, Chad/Sudan; and (6) the TAG-I/F6 play, onshore Algeria. Examples of these plays are illustrated along with some of the more traditional oil plays. Where are the future oil plays likely to develop No doubt, the Saharan basins of Algeria and Libya will feature strongly, also the presalt of Equatorial West Africa, the Central African Rift System and, more speculatively, offshore Ethiopia and Namibia, and onshore Madagascar, Mozambique, and Tanzania.

Clifford, A.J. (BHP Petroleum, Melbourne, Victoria (Australia))



Activation of heat shock factor 1 plays a role in pyrrolidine dithiocarbamate-mediated expression of the co-chaperone BAG3  

PubMed Central

Adaptive responses to physical and inflammatory stressors are mediated by transcription factors and molecular chaperones. The transcription factor heat shock factor 1 (HSF1) has been implicated in extending lifespan in part by increasing expression of heat shock response genes. Pyrrolidine dithiocarbamate (PDTC) is a small thiol compound that exerts in vivo and in vitro anti-inflammatory properties through mechanisms that remain unclear. Here we report that PDTC induced the release of monomeric HSF1 from the molecular chaperone heat shock protein 90 (Hsp90), with concomitant increase in HSF1 trimer formation, translocation to the nucleus, and binding to promoter of target genes in human HepG2 cells. siRNA-mediated silencing of HSF1 blocked BAG3 gene expression by PDTC. The protein levels of the co-chaperone BAG3 and its interaction partner Hsp72 were stimulated by PDTC in a dose-dependent fashion, peaking at 6 hours. Inhibition of Hsp90 function by geldanamycin derivatives and novobiocin elicited a pattern of HSF1 activation and BAG3 expression that was similar to PDTC. Chromatin immunoprecipitation studies showed that PDTC and the inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin enhanced the binding of HSF1 to the promoter of several target genes, including BAG3, HSPA1A, HSPA1B, FKBP4, STIP1 and UBB. Cell treatment with PDTC increased significantly the level of Hsp90? thiol oxidation, a posttranslational modification known to inhibit its chaperone function. These results unravel a previously unrecognized mechanism by which PDTC and related compounds could confer cellular protection against inflammation through HSF1-induced expression of heat shock response genes.

Song, Shaoming; Kole, Sutapa; Precht, Patricia; Pazin, Michael J.; Bernier, Michel



Control of Stochastic Gene Expression by Host Factors at the HIV Promoter  

PubMed Central

The HIV promoter within the viral long terminal repeat (LTR) orchestrates many aspects of the viral life cycle, from the dynamics of viral gene expression and replication to the establishment of a latent state. In particular, after viral integration into the host genome, stochastic fluctuations in viral gene expression amplified by the Tat positive feedback loop can contribute to the formation of either a productive, transactivated state or an inactive state. In a significant fraction of cells harboring an integrated copy of the HIV-1 model provirus (LTR-GFP-IRES-Tat), this bimodal gene expression profile is dynamic, as cells spontaneously and continuously flip between active (Bright) and inactive (Off) expression modes. Furthermore, these switching dynamics may contribute to the establishment and maintenance of proviral latency, because after viral integration long delays in gene expression can occur before viral transactivation. The HIV-1 promoter contains cis-acting Sp1 and NF-?B elements that regulate gene expression via the recruitment of both activating and repressing complexes. We hypothesized that interplay in the recruitment of such positive and negative factors could modulate the stability of the Bright and Off modes and thereby alter the sensitivity of viral gene expression to stochastic fluctuations in the Tat feedback loop. Using model lentivirus variants with mutations introduced in the Sp1 and NF-?B elements, we employed flow cytometry, mRNA quantification, pharmacological perturbations, and chromatin immunoprecipitation to reveal significant functional differences in contributions of each site to viral gene regulation. Specifically, the Sp1 sites apparently stabilize both the Bright and the Off states, such that their mutation promotes noisy gene expression and reduction in the regulation of histone acetylation and deacetylation. Furthermore, the NF-?B sites exhibit distinct properties, with ?B site I serving a stronger activating role than ?B site II. Moreover, Sp1 site III plays a particularly important role in the recruitment of both p300 and RelA to the promoter. Finally, analysis of 362 clonal cell populations infected with the viral variants revealed that mutations in any of the Sp1 sites yield a 6-fold higher frequency of clonal bifurcation compared to that of the wild-type promoter. Thus, each Sp1 and NF-?B site differentially contributes to the regulation of viral gene expression, and Sp1 sites functionally “dampen” transcriptional noise and thereby modulate the frequency and maintenance of this model of viral latency. These results may have biomedical implications for the treatment of HIV latency.

Burnett, John C.; Miller-Jensen, Kathryn; Shah, Priya S.; Arkin, Adam P.; Schaffer, David V.



Proto-oncogene FBI-1 (Pokemon/ZBTB7A) Represses Transcription of the Tumor Suppressor Rb Gene via Binding Competition with Sp1 and Recruitment of Co-repressors*S?  

PubMed Central

FBI-1 (also called Pokemon/ZBTB7A) is a BTB/POZ-domain Krüppel-like zinc-finger transcription factor. Recently, FBI-1 was characterized as a proto-oncogenic protein, which represses tumor suppressor ARF gene transcription. The expression of FBI-1 is increased in many cancer tissues. We found that FBI-1 potently represses transcription of the Rb gene, a tumor suppressor gene important in cell cycle arrest. FBI-1 binds to four GC-rich promoter elements (FREs) located at bp –308 to –188 of the Rb promoter region. The Rb promoter also contains two Sp1 binding sites: GC-box 1 (bp –65 to –56) and GC-box 2 (bp –18 to –9), the latter of which is also bound by FBI-1. We found that FRE3 (bp –244 to –236) is also a Sp1 binding element. FBI-1 represses transcription of the Rb gene not only by binding to the FREs, but also by competing with Sp1 at the GC-box 2 and the FRE3. By binding to the FREs and/or the GC-box, FBI-1 represses transcription of the Rb gene through its POZ-domain, which recruits a co-repressor-histone deacetylase complex and deacetylates histones H3 and H4 at the Rb gene promoter. FBI-1 inhibits C2C12 myoblast cell differentiation by repressing Rb gene expression.

Jeon, Bu-Nam; Yoo, Jung-Yoon; Choi, Won-Il; Lee, Choong-Eun; Yoon, Ho-Geun; Hur, Man-Wook



Evidence that tumor necrosis factor plays a pathogenetic role in the paraneoplastic syndromes of cachexia, hypercalcemia, and leukocytosis in a human tumor in nude mice.  

PubMed Central

Recently, we have established a human squamous cell carcinoma of the maxilla (called MH-85) associated with hypercalcemia, leukocytosis, and cachexia in culture. MH-85 tumor cells caused the same paraneoplastic syndromes in tumor-bearing nude mice. We found that there was a sixfold increase in splenic size in MH-85 tumor-bearing mice. This increase paralleled tumor growth and was reversed by surgical removal of the tumor. Splenectomy in nude mice 1 wk before or 6 wk after tumor inoculation resulted in a decrease in tumor growth, and impairment of hypercalcemia, leukocytosis, and cachexia. In MH-85 tumor-bearing animals that had been pretreated by splenectomy, intravenous injection of fresh normal spleen cells caused an immediate reversal of leukocytosis, hypercalcemia, and cachexia. Since the presence of cachexia in both the patient and the mice carrying the tumor suggested tumor necrosis factor (TNF) may be overproduced, we injected polyclonal neutralizing antibodies raised against murine TNF into tumor-bearing mice. There was a rapid and reproducible decrease in blood ionized calcium, accompanied by suppression of osteoclast activity. No changes in blood ionized calcium were seen in mice injected with normal immune sera. In addition, there was an increase in body weight and decrease in white cell count. Plasma immunoreactive TNF was increased almost fourfold in tumor-bearing nude mice compared with control nude mice. Although TNF activity was undetectable in MH-85 culture supernatants, cells of the macrophage lineage, including spleen cells, released increased amounts of TNF when cultured with MH-85 tumor-conditioned media. These results suggest that splenic cytokines such as TNF may influence the development of the paraneoplastic syndromes of hypercalcemia, leukocytosis, and cachexia in these animals, as well as tumor growth. They also show that paraneoplastic syndromes may be due to factors produced by normal host cells stimulated by the presence of the tumor. Images

Yoneda, T; Alsina, M A; Chavez, J B; Bonewald, L; Nishimura, R; Mundy, G R



A Single-Base Substitution in the Proximal Sp1 Site of the Human Low Density Lipoprotein Receptor Promoter as a Cause of Heterozygous Familial Hypercholesterolemia  

Microsoft Academic Search

We have identified a Finnish family with a typical phenotype of heterozygous familial hypercholesterolemia (FH) due to a single-base substitution in the proximal Sp1 binding site of the low density lipoprotein (LDL) receptor gene promoter. The mutation, a C --> T substitution at nucleotide -43, cosegregated with the FH phenotype in six available family members and abolished binding of Sp1

Ulla-Maija Koivisto; Jorma J. Palvimo; Olli A. Janne; Kimmo Kontula



CD1d induction in solid tumor cells by histone deacetylase inhibitors through inhibition of HDAC1/2 and activation of Sp1  

PubMed Central

CD1d is a MHC class-like molecule that presents glycolipids to natural killer T (NKT) cells, then regulates innate and adaptive immunity. The regulation of CD1d gene expression in solid tumors is still largely unknown. Gene expression can be epigenetically regulated by DNA methylation and histone acetylation. We found that histone deacetylase inhibitors, trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA), induced CD1d gene expression in human (A549 and NCI-H292) and mouse (TC-1 and B16/F0) cancer cells. Simultaneous knockdown of HDAC1 and 2 induced CD1d gene expression. Sp1 inhibitor mitramycin A (MTM) blocked TSA- and SAHA-induced CD1d mRNA expression and Sp1 luciferase activity. Co-transfection of GAL4-Sp1 and Fc-luciferase reporters demonstrated that TSA and SAHA induced Sp1 luciferase reporter activity by enhancing Sp1 transactivation activity. The binding of Sp1 to CD1d promoter and histone H3 acetylation on Sp1 sites were increased by TSA and SAHA. These results indicate that TSA and SAHA could up-regulate CD1d expression in tumor cells through inhibition of HDAC1/2 and activation of Sp1.

Yang, Pei-Ming; Lin, Pei-Jie; Chen, Ching-Chow



Enhanced NF-?B Activity Impairs Vascular Function Through PARP-1-, SP-1-, and COX-2-Dependent Mechanisms in Type 2 Diabetes  

PubMed Central

Type 2 diabetes (T2D) is associated with vascular dysfunction. We hypothesized that increased nuclear factor-?B (NF-?B) signaling contributes to vascular dysfunction in T2D. We treated type 2 diabetic (db?/db?) and control (db?/db+) mice with two NF-?B inhibitors (6 mg/kg dehydroxymethylepoxyquinomicin twice a week and 500 ?g/kg/day IKK-NBD peptide) for 4 weeks. Pressure-induced myogenic tone was significantly potentiated, while endothelium-dependent relaxation (EDR) was impaired in small coronary arterioles and mesenteric resistance artery from diabetic mice compared with controls. Interestingly, diabetic mice treated with NF-?B inhibitors had significantly reduced myogenic tone potentiation and improved EDR. Importantly, vascular function was also rescued in db?/db?p50NF-?B?/? and db?/db?PARP-1?/? double knockout mice compared with db?/db? mice. Additionally, the acute in vitro downregulation of NF-?B–p65 using p65NF-?B short hairpin RNA lentivirus in arteries from db?/db? mice also improved vascular function. The NF-?B inhibition did not affect blood glucose level or body weight. The RNA levels for Sp-1 and eNOS phosphorylation were decreased, while p65NF-?B phosphorylation, cleaved poly(ADP-ribose) polymerase (PARP)-1, and cyclooxygenase (COX)-2 expression were increased in arteries from diabetic mice, which were restored after NF-?B inhibition and in db?/db?p50NF-?B?/? and db?/db?PARP-1?/? mice. In the current study, we provided evidence that enhanced NF-?B activity impairs vascular function by PARP-1–, Sp-1–, and COX-2–dependent mechanisms in male type 2 diabetic mice. Therefore, NF-?B could be a potential target to overcome diabetes-induced vascular dysfunction.

Kassan, Modar; Choi, Soo-Kyoung; Galan, Maria; Bishop, Alexander; Umezawa, Kazuo; Trebak, Mohamed; Belmadani, Souad; Matrougui, Khalid



Nuclear factor-?B plays a critical role in both intrinsic and acquired resistance against endocrine therapy in human breast cancer cells.  


Since more than 75% of breast cancers overexpress estrogen receptors (ER), endocrine therapy targeting ER has significantly improved the survival rate. Nonetheless, breast cancer still afflicts women worldwide and the major problem behind it is resistance to endocrine therapy. We have previously shown the involvement of nuclear factor-?B (NF-?B) in neoplastic proliferation of human breast cancer cells; however, the association with the transformation of ER-positive cells remains unclear. In the current study, we focused on roles of NF-?B in the hormone dependency of breast cancers by means of ER-positive MCF-7 cells. Blocking of NF-?B signals in ER-negative cells stopped proliferation by downregulation of D-type cyclins. In contrast, the MCF-7 cells were resistant to NF-?B inhibition. Under estrogen-free conditions, the ER levels were reduced when compared with the original MCF-7 cells and the established cell subline exhibited tamoxifen resistance. Additionally, NF-?B participated in cell growth instead of the estrogen-ER axis in the subline and consequently, interfering with the NF-?B signals induced additive anticancer effects with tamoxifen. MMP-9 production responsible for cell migration, as well as the cell expansion in vivo, were suppressed by NF-?B inhibition. Therefore, we suggest that NF-?B is a master switch in both ER-positive and ER-negative breast cancers. PMID:24531845

Oida, Kumiko; Matsuda, Akira; Jung, Kyungsook; Xia, Yan; Jang, Hyosun; Amagai, Yosuke; Ahn, Ginnae; Nishikawa, Sho; Ishizaka, Saori; Jensen-Jarolim, Erika; Matsuda, Hiroshi; Tanaka, Akane



Evidence that E-box promoter elements and MyoD transcription factors play a role in the induction of cathepsin B gene expression during human myoblast differentiation.  


HB13 human myoblasts express physiological and biochemical markers associated with myoblast differentiation in non-human cell culture model systems. During differentiation, HB13 myoblasts also demonstrate fusion-related increases in cathepsin B activity and protein levels. These increases are associated with an increase in levels of cathepsin B mRNA suggesting the involvement of transcriptional regulatory mechanisms. To examine these mechanisms human myoblasts were transfected with cathepsin B nested deletion promoter constructs within the 1.8 kb 5' promoter 1 region of the human catB gene. Transfected myoblasts that were maintained under differentiating conditions demonstrated higher promoter activity than those maintained in proliferating conditions. The highest activity was obtained with pSCB2-3 (-1279/+56 bp), a construct containing two putative upstream E-box elements. Co-transfection experiments demonstrated that MyoD and myogenin transactivate cathepsin B promoter activity. Electrophoretic mobility shift assays of nuclear extracts incubated with an oligonucleotide containing two upstream E-box elements found within the cathepsin B promoter demonstrated two band shifts. The band shifts were abolished using an oligonucleotide with mutations in both E-box elements. Moreover, the shifted bands were super-shifted and abolished when incubated with anti-myogenin and anti-MyoD, respectively. Collectively, these data support myogenic transcription factor-mediated activation of cathepsin B expression during myogenesis. PMID:12553720

Jane, Derek T; Morvay, Leslie C; Koblinski, Jennifer; Yan, Shiqing; Saad, Fawzy A; Sloane, Bonnie F; Dufresne, Michael J



Translation factor LepA contributes to tellurite resistance in Escherichia coli but plays no apparent role in the fidelity of protein synthesis  

PubMed Central

LepA is a translational GTPase highly conserved in bacterial lineages. While it has been shown that LepA can catalyze reverse ribosomal translocation in vitro, the role of LepA in the cell remains unclear. Here, we show that deletion of the lepA gene (?lepA) in E. coli causes hypersensitivity to potassium tellurite and penicillin G, but has no appreciable effect on growth under many other conditions. ?lepA does not increase miscoding or frameshifting errors under normal or stress conditions, indicating that LepA does not contribute to the fidelity of translation. Overexpression of LepA interferes with tmRNA-mediated peptide tagging and A-site mRNA cleavage, suggesting that LepA is a bona fide translation factor that can act on stalled ribosomes with a vacant A site in vivo. Together these results lead us to hypothesize that LepA is involved in co-translational folding of proteins that are otherwise vulnerable to tellurite oxidation.

Shoji, Shinichiro; Janssen, Brian D.; Hayes, Christopher S.; Fredrick, Kurt



Clinical value of pregnancy-specific beta 1-glycoprotein (SP1) and beta-hCG determination in serum in suspected ectopic pregnancy.  


We compared the clinical value of pregnancy-specific beta 1-glycoprotein (SP1) determination in serum by means of a highly sensitive enzyme-linked immunosorbent assay (ELISA) versus that of beta human chorionic gonadotropin (beta-hCG) determination in suspected ectopic pregnancy. The study comprised 58 women admitted consecutively with suspected ectopic pregnancy but without signs warranting immediate surgical intervention. Both SP1 and beta-hCG were found in 11 patients with ectopic pregnancy and in 8 patients with early intra-uterine pregnancy, whereas beta-hCG was detected in 4 and SP1 in 7 of 8 women with a recent abortion. Of 31 women presenting a non-pregnant condition, 2 were positive for both SP1 and beta-hCG. The measurement of SP1 in serum thus appears to be an alternative to beta-hCG measurement when ectopic pregnancy is suspected. PMID:2433888

Sørensen, S; Blaabjerg, J



Fibroblast Growth Factor Receptors-1 and -3 Play Distinct Roles in the Regulation of Bladder Cancer Growth and Metastasis: Implications for Therapeutic Targeting  

PubMed Central

Fibroblast growth factor receptors (FGFRs) are activated by mutation and overexpressed in bladder cancers (BCs), and FGFR inhibitors are currently being evaluated in clinical trials in BC patients. However, BC cells display marked heterogeneity in their responses to FGFR inhibitors, and the biological mechanisms underlying this heterogeneity are not well defined. Here we used a novel inhibitor of FGFRs 1–3 and RNAi to determine the effects of inhibiting FGFR1 or FGFR3 in a panel of human BC cell lines. We observed that FGFR1 was expressed in BC cells that also expressed the “mesenchymal” markers ZEB1 and vimentin, whereas FGFR3 expression was restricted to the E-cadherin- and p63-positive “epithelial” subset. Sensitivity to the growth-inhibitory effects of BGJ-398 was also restricted to the “epithelial” BC cells and it correlated directly with FGFR3 mRNA levels but not with the presence of activating FGFR3 mutations. In contrast, BGJ-398 did not strongly inhibit proliferation but did block invasion in the “mesenchymal” BC cells in vitro. Similarly, BGJ-398 did not inhibit primary tumor growth but blocked the production of circulating tumor cells (CTCs) and the formation of lymph node and distant metastases in mice bearing orthotopically implanted “mesenchymal” UM-UC3 cells. Together, our data demonstrate that FGFR1 and FGFR3 have largely non-overlapping roles in regulating invasion/metastasis and proliferation in distinct “mesenchymal” and “epithelial” subsets of human BC cells. The results suggest that the tumor EMT phenotype will be an important determinant of the biological effects of FGFR inhibitors in patients.

Cheng, Tiewei; Roth, Beat; Choi, Woonyoung; Black, Peter C.; Dinney, Colin; McConkey, David J.



Fibroblast growth factor receptor 3 activation plays a causative role in urothelial cancer pathogenesis in cooperation with Pten loss in mice.  


Although somatic mutations and overexpression of the tyrosine kinase fibroblast growth factor receptor 3 (FGFR3) are strongly associated with bladder cancer, evidence for their functional involvement in the pathogenesis remains elusive. Previously we showed that activation of Fgfr3 alone is not sufficient to initiate urothelial tumourigenesis in mice. Here we hypothesize that cooperating mutations are required for Fgfr3-dependent tumourigenesis in the urothelium and analyse a mouse model in which an inhibitor of Pi3k-Akt signalling, Pten, is deleted in concert with Fgfr3 activation (UroIICreFgfr3(+/) (K644E) Pten(flox) (/flox) ). Two main phenotypical characteristics were observed in the urothelium: increased urothelial thickness and abnormal cellular histopathology, including vacuolization, condensed cellular appearance, enlargement of cells and nuclei, and loss of polarity. These changes were not observed when either mutation was present individually. Expression patterns of known urothelial proteins indicated the abnormal cellular differentiation. Furthermore, quantitative analysis showed that Fgfr3 and Pten mutations cooperatively caused cellular enlargement, while Pten contributed to increased cell proliferation. Finally, FGFR3 overexpression was analysed along the level of phosphorylated mTOR in 66 T1 urothelial tumours in tissue microarray, which supported the occurrence of functional association of these two signalling pathways in urothelial pathogenesis. Taken together, this study provides evidence supporting a functional role of FGFR3 in the process of pathogenesis in urothelial neoplasms. Given the wide availability of inhibitors specific to FGF signalling pathways, our model may open the avenue for FGFR3-targeted translation in urothelial disease. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. PMID:24519156

Foth, Mona; Ahmad, Imran; van Rhijn, Bas Wg; van der Kwast, Theodorus; Bergman, Andre M; King, Louise; Ridgway, Rachel; Leung, Hing Y; Fraser, Sioban; Sansom, Owen J; Iwata, Tomoko



Promoting Early Play.  

ERIC Educational Resources Information Center

A toy-play intervention program is described that was initially used in a research setting with 24 young Dutch children, using outcome measures and a 1-year follow-up assessment. The original intervention was successful. However, improvements were not maintained unless children continued to receive play support. (Contains references.) (Author/CR)

Van Berckelaer-Onnes, I. A.



Children Playing with Fire  

MedlinePLUS Videos and Cool Tools

... associated civilian fire deaths. Video: Children playing with fire cause hundreds of deaths and injuries each year. NFPA's Lisa Braxton talks about how clear rules and consequences must be established for children about fire misuse. Source: NFPA's " Children Playing with Fire " report ...


Playful Communication in Mentoring  

ERIC Educational Resources Information Center

Freshmen in an orientation course at a state university answered questionnaires about their peer mentors' playful communication and about how well their peer mentors help them ease tensions of socialization. Results showed that a mentor's perceived playful communication helped the protege ease tensions of socialization. Proteges liked mentors who…

Young, Raymond W.; Cates, Carl M.



Growing Up with Play  

ERIC Educational Resources Information Center

Many adults are afraid of boys' play today, believing that the aggression that is so common in boys' fantasies is dangerous and might make them become violent men. This personal reflection describes the importance of multiage play in showing little boys how to become big boys while encouraging empathy and emotional growth in older boys. The author…

Katch, Jane



The Fear of Play  

ERIC Educational Resources Information Center

Real play--play that is initiated and directed by children and that bubbles up from within the child rather than being imposed by adults--has largely disappeared from the landscape of childhood in the United States. There are many reasons for this, such as the long hours spent in front of screens each day or in activities organized by adults. In…

Almon, Joan



Play and Digital Media  

ERIC Educational Resources Information Center

This article examines how play is affected by computers and digital toys. Research indicates that when computer software targeted at children is problem-solving oriented and open-ended, children tend to engage in creative play and interact with peers in a positive manner. On the other hand, drill-and-practice programs can be quite boring and limit…

Johnson, James E.; Christie, James F.



Return to Play  

ERIC Educational Resources Information Center

Call it physical activity, call it games, or call it play. Whatever its name, it's a place we all need to return to. In the physical education, recreation, and dance professions, we need to redesign programs to address the need for and want of play that is inherent in all of us.

Mangan, Marianne



Specificity protein, Sp1-mediated increased expression of Prdx6 as a curcumin-induced antioxidant defense in lens epithelial cells against oxidative stress  

PubMed Central

Peroxiredoxin 6 (Prdx6) is a pleiotropic oxidative stress-response protein that defends cells against reactive oxygen species (ROS)-induced damage. Curcumin, a naturally occurring agent, has diversified beneficial roles including cytoprotection. Using human lens epithelial cells (hLECs) and Prdx6-deficient cells, we show the evidence that curcumin protects cells by upregulating Prdx6 transcription via invoking specificity protein 1 (Sp1) activity against proapoptotic stimuli. Curcumin enhanced Sp1 and Prdx6 mRNA and protein expression in a concentration-dependent manner, as evidenced by western and real-time PCR analyses, and thereby negatively regulated ROS-mediated apoptosis by blunting ROS expression and lipid peroxidation. Bioinformatic analysis and DNA–protein binding assays disclosed three active Sp1 sites (?19/27, ?61/69 and ?82/89) in Prdx6 promoter. Co-transfection experiments with Sp1 and Prdx6 promoter–chloramphenicol acetyltransferase (CAT) constructs showed that CAT activity was dramatically increased in LECs or Sp1-deficient cells (SL2). Curcumin treatment of LECs enhanced Sp1 binding to its sites, consistent with curcumin-dependent stimulation of Prdx6 promoter with Sp1 sites and cytoprotection. Notably, disruption of Sp1 sites by point mutagenesis abolished curcumin transactivation of Prdx6. Also, curcumin failed to activate Prdx6 expression in the presence of Sp1 inhibitors, demonstrating that curcumin-mediated increased expression of Prdx6 was dependent on Sp1 activity. Collectively, the study may provide a foundation for developing transcription-based inductive therapy to reinforce endogenous antioxidant defense by using dietary supplements.

Chhunchha, B; Fatma, N; Bhargavan, B; Kubo, E; Kumar, A; Singh, D P



Negative regulation of the oncogenic transcription factor FoxM1 by thiazolidinediones and mithramycin.  


The Forkhead Box transcription factor FoxM1 regulates expression of genes that promote cell cycle progression, and it plays essential roles in the development of liver, lung, prostate and colorectal tumors. Thiazolidinediones (TZDs) activate the peroxisome proliferator-activated receptor gamma (PPAR?), a ligand-activated nuclear receptor transcription factor. We found that treatment of the human hepatoma cell lines HepG2 and PLC/PRF/5 cells with TZDs leads to inhibition of FoxM1 gene expression. No PPAR?/retinoid X receptor (RXR) consensus DNA binding sites were detected in the FoxM1 promoter extending to -10 kb upstream, and knockdown of PPAR? had no impact on TZD mediated downregulation of FoxM1 expression. Previously, others showed that PPAR? agonists inhibit the expression and DNA-binding activity of the Sp1 transcription factor. Here we show that Sp1 binds to the FoxM1 promoter region and positively regulates FoxM1 transcription, while mithramycin, a chemotherapy drug that specifically binds GC rich sequences in the DNA and inhibits activities of Sp1, inhibits expression of FoxM1. Our data suggest that TZD mediated suppression of Sp1 is responsible for downregulation of FoxM1 gene expression. Inhibition of FoxM1 expression by TZDs provides a new mechanism for TZD mediated negative regulation of cancer cell growth. FoxM1 expression and activity in cancer cells can be targeted using PPAR? agonists or the anti-neoplastic antibiotic mithramycin. PMID:20372080

Petrovic, Vladimir; Costa, Robert H; Lau, Lester F; Raychaudhuri, Pradip; Tyner, Angela L



Transcriptional Repression of Telomerase RNA Gene Expression by c-Jun-NH2Kinase and Sp1\\/Sp3  

Microsoft Academic Search

Telomerase is essential for immortalization of most human cancer cells. Expression of the core telomerase RNA (hTR) and reverse transcriptase (hTERT) subunits is mainly regulated by transcription. However, hTR transcriptional regulation remains poorly understood. We previously showed that the core hTR promoter is activated by Sp1 and is repressed by Sp3. Here, we show that the mitogen-activated protein kinase kinase

Alan E. Bilsland; Katrina Stevenson; Stuart Atkinson; Walter Kolch; W. Nicol Keith



The prototype HIV-1 maturation inhibitor, bevirimat, binds to the CA-SP1 cleavage site in immature Gag particles  

PubMed Central

Background Bevirimat, the prototype Human Immunodeficiency Virus type 1 (HIV-1) maturation inhibitor, is highly potent in cell culture and efficacious in HIV-1 infected patients. In contrast to inhibitors that target the active site of the viral protease, bevirimat specifically inhibits a single cleavage event, the final processing step for the Gag precursor where p25 (CA-SP1) is cleaved to p24 (CA) and SP1. Results In this study, photoaffinity analogs of bevirimat and mass spectrometry were employed to map the binding site of bevirimat to Gag within immature virus-like particles. Bevirimat analogs were found to crosslink to sequences overlapping, or proximal to, the CA-SP1 cleavage site, consistent with previous biochemical data on the effect of bevirimat on Gag processing and with genetic data from resistance mutations, in a region predicted by NMR and mutational studies to have ?-helical character. Unexpectedly, a second region of interaction was found within the Major Homology Region (MHR). Extensive prior genetic evidence suggests that the MHR is critical for virus assembly. Conclusions This is the first demonstration of a direct interaction between the maturation inhibitor, bevirimat, and its target, Gag. Information gained from this study sheds light on the mechanisms by which the virus develops resistance to this class of drug and may aid in the design of next-generation maturation inhibitors.



Play the Tuberculosis Game  


... Malaria MRI Nerve Signaling Pavlov's Dog Split Brain Experiments The Cell and its Organelles The Genetic Code ... Life and Work Teachers' Questionnaire Tuberculosis Play Tuberculosis Experiments & Discoveries About the game Discover and experience some ...


The Play Activity Questionnaire: A parent report measure of children's play preferences  

Microsoft Academic Search

Three studies describe the development and validation of the Play Activity Questionnaire (PQ), a parent report measure of children's play preferences. In Study 1, the 15-item PQ was completed by parents of 239 6- to 8-year-old children, and exploratory factor analysis revealed four play factors: Active and Adventurous, Athletic, Rough-and-Tumble, and Quiet. In Study 2, the factor structure was replicated

Jo-Anne K. Finegan; G. Alison Niccols; James E. Zacher; Jane E. Hood



4-hexylresorcinol stimulates the differentiation of SCC-9 cells through the suppression of E2F2, E2F3 and Sp3 expression and the promotion of Sp1 expression.  


The dormancy-inducing factors of bacteria inhibit tumor cell growth. In the present study, we evaluated the antitumor effects of the dormancy-inducing factor 4-hexylresorcinol (4-HR) using real-time cell electronic sensing (RT-CES) in SCC-9 cells (tongue squamous cell carcinoma cells). Treatment with 4-HR suppressed the growth of SCC-9 cells in a dose-dependent manner. We used a DNA microarray to identify genes that showed a significant change in expression upon 4-HR administration in SCC-9 cells. Among the differentially expressed genes, the protein expression of several cell proliferation related factors, including E2F1, E2F2, E2F3, E2F4, E2F5, E2F6, Sp1 and Sp3, were determined by western blot analyses. Treatment with 4-HR strongly suppressed E2F2 and slightly suppressed E2F3 but did not change the expression of E2F1, E2F4, E2F5 and E2F6 relative to no treatment. Furthermore, 4-HR increased Sp1 expression in a dose-dependent manner and decreased Sp3 expression. Therefore, the ratio of Sp1 to Sp3, an important driving force of epithelial cell differentiation, was drastically increased. Consistent with this observation, 4-HR increased the expression of the epithelial cell differentiation markers involucrin and keratin 10. Together, our results indicate that 4-HR induces the differentiation of SCC-9 via the modulation of the E2F-mediated signaling pathway. PMID:22664654

Kim, Seong-Gon; Kim, An-Sook; Jeong, Jae-Hwan; Choi, Je-Yong; Kweon, Haeyong



Expression of the hypoxia marker carbonic anhydrase IX is critically dependent on SP1 activity. Identification of a novel type of hypoxia-responsive enhancer.  


In the present study, we further studied mechanisms of transcriptional regulation of the tumor-associated carbonic anhydrase IX (CAIX). We identified PR5 in the CA9 promoter as another SP1/SP3-binding site. As shown by electromobility shift assays and block-replacement mutagenesis, PR5 is functionally equivalent to the SP1/SP3-binding PR1 identified previously. However, there is a strong requirement for SP1/SP3 activity in the PR1 position, and SP1/SP3 activity from the PR5 position cannot compensate for this. In various cell lines, the expression of endogenous CAIX and activity of CA9 promoter constructs depend on SP1/SP3 activity as demonstrated by the dose-dependent inhibitory effect of the SP1 inhibitor mithramycin A. The two conditions of the induction of CAIX expression described previously differ in their sensitivity to mithramycin A inhibition; the hypoxia-mimic-induced expression is less sensitive than the cell density (mild hypoxia)-induced expression. Our present study highlights the importance of SP1/SP3 activity for CAIX expression and provides additional evidence for distinct mechanisms responsible for true and mild hypoxia-induced CAIX expression. The presence of a SP1/SP3-binding element in the PR1 position is absolutely required for mild hypoxia-induced activity, and it significantly up-regulates the true hypoxic induction. The SP1/SP3 and hypoxia-response element in the CA9 promoter thus may represent a novel type of enhancer capable of mounting responses to a wider range of hypoxic conditions. PMID:12615703

Kaluz, Stefan; Kaluzová, Milota; Stanbridge, Eric J



Regulation of Neph3 gene in podocytes – key roles of transcription factors NF-?B and Sp1  

Microsoft Academic Search

BACKGROUND: Neph3 (filtrin) is expressed in the glomerular podocytes where it localizes at the specialized cell adhesion structures of the foot processes called slit diaphragms which form the outermost layer of the glomerular filtration barrier. Neph3 protein shows homology and structural similarity to Neph1, Neph2 and nephrin, which all are crucial for maintaining the normal glomerular ultrafiltration function. The exact

Mervi Ristola; Satu Arpiainen; Moin A Saleem; Peter W Mathieson; Gavin I Welsh; Sanna Lehtonen; Harry Holthöfer



Play Dough Planets  

NSDL National Science Digital Library

This is a lesson about planet sizes. Learners will demonstrate the size (volume) differences between Earth, Earthâs Moon, and Mars. An extension to estimate the distance between the Earth and the Moon, and the Earth and Mars, using the scale of the play dough planets' sizes is provided. Advance preparation of the play dough (recipie provided) is required. This is lesson 3 of 16 in the MarsBots learning module. It was adapted from 3-D Model of the Earth and Moon, an activity in The Universe at Your Fingertips.


TGF? Signaling Regulates the Timing of CNS Myelination by Modulating Oligodendrocyte Progenitor Cell Cycle Exit through SMAD3/4/FoxO1/Sp1.  


Research on myelination has focused on identifying molecules capable of inducing oligodendrocyte (OL) differentiation in an effort to develop strategies that promote functional myelin regeneration in demyelinating disorders. Here, we show that transforming growth factor ? (TGF?) signaling is crucial for allowing oligodendrocyte progenitor (OP) cell cycle withdrawal, and therefore, for oligodendrogenesis and postnatal CNS myelination. Enhanced oligodendrogenesis and subcortical white matter (SCWM) myelination was detected after TGF? gain of function, while TGF? receptor II (TGF?-RII) deletion in OPs prevents their development into mature myelinating OLs, leading to SCWM hypomyelination in mice. TGF? signaling modulates OP cell cycle withdrawal and differentiation through the transcriptional modulation of c-myc and p21 gene expression, mediated by the interaction of SMAD3/4 with Sp1 and FoxO1 transcription factors. Our study is the first to demonstrate an autonomous and crucial role of TGF? signaling in OL development and CNS myelination, and may provide new avenues in the treatment of demyelinating diseases. PMID:24899714

Palazuelos, Javier; Klingener, Michael; Aguirre, Adan



Predicting elections: child's play!  


In two experiments, children and adults rated pairs of faces from election races. Naïve adults judged a pair on competence; after playing a game, children chose who they would prefer to be captain of their boat. Children's (as well as adults') preferences accurately predicted actual election outcomes. PMID:19251621

Antonakis, John; Dalgas, Olaf



Playing It Safe.  

ERIC Educational Resources Information Center

Offers tips for avoiding sports-related injuries: (1) expect more of coaches; (2) develop an athletic-safety plan; (3) consider hiring an athletic trainer; (4) check facilities and equipment regularly; (5) recognize athletes' limitations; (6) take precautions beyond the playing field; and (7) check liability coverage and obtain informed consent.…

Jones, Rebecca



"Playing" with Science  

ERIC Educational Resources Information Center

When faced with a multitude of tasks, any opportunity to "kill two birds with one stone" is welcome. Drama has always excited the author: as a child performing in plays, later as a student and now as a teacher directing performances and improvising within lessons. The author was lucky enough to have inspirational teachers during his primary and…

Allen, Dave



Games Professors Play  

ERIC Educational Resources Information Center

The games are Build a Reputation (REP), Confuse the Student (CON), Blame the Opposition (BOP), and Pass the Buck (BUCK). Professors play these games because they "want to show off on occasion, . . . want to get off the hook and avoid responsibility, . . . are prone to blame others, or simply because they are lazy. (WM)

Kenny, James A.; Herzing, Thomas W.



Want to Play Geometry?  

ERIC Educational Resources Information Center

Students enter the geometry classroom with a strong concept of fairness and a sense of what it means to "play by the rules," yet many students have difficulty understanding the postulates, or rules, of geometry and their implications. Although they may never have articulated the properties of an axiomatic system, they have gained a practical…

Kaufmann, Matthew L.; Bomer, Megan A.; Powell, Nancy Norem



Electrostatic charges instigate 'concertina-like' mechanisms of molecular toughening in MaSp1 (spider silk) proteins.  


According to a recent article authored by Ortega-Jimenez and Dudley [1], the capture success of spiders is in part due to electrostatic charges on the surfaces of insects that macroscopically deform the spider web and increase the chances of insect-web contact. In this brief communication, we further show that electrostatic charges instigate a molecular 'concertina-like' mechanism of deformation in MaSp1 protein, which effectively begins the toughening-up of dragline silk threads prior to insect-web contact. PMID:24907767

Pahlevan, Mahdi; Toivakka, Martti; Alam, Parvez



The Collagen Ia1 SP1 Polymorphism is Associated With Differences in Ultrasound Transmission Velocity in the Calcaneus in Postmenopausal Women  

Microsoft Academic Search

Bone mineral density (BMD) and fracture risk are under genetic control. An association of a G to T polymorphism in the Sp1 binding site of the collagen Ia1 (COLIa1) gene with the risk for fractures has been previously reported. This association is only partly explained by differences in BMD. Thus, we analyzed the relationship between the COLIa1 Sp1 polymorphism and

P. Kann; A. P. Bergink; Y. Fang; P. L. A. van Daele; A. Hofman; J. Beyer; A. G. Uitterlinden; H. A. P. Pols



The Autophagy-related Gene 14 (Atg14) Is Regulated by Forkhead Box O Transcription Factors and Circadian Rhythms and Plays a Critical Role in Hepatic Autophagy and Lipid Metabolism*  

PubMed Central

Autophagy plays a critical role in cell survival from prolonged starvation and recycling of aggregated proteins and damaged organelles. One of the essential genes involved in the autophagic initiation is autophagy-related 14 (Atg14), also called Barkor for Beclin 1-associated autophagy-related key regulator. Although its crucial role in the autophagic process has been reported, the gene regulation of Atg14 and its metabolic functions remain unclear. In this work we have identified that the Atg14 gene is regulated by forkhead box O (FoxO) transcription factors and circadian rhythms in the mouse liver. Luciferase reporter analyses and chromatin immunoprecipitation assays have revealed well conserved cis-elements for FoxOs and Clock/Bmal1 in the proximal promoter of the Atg14 gene. To examine the functions of hepatic Atg14, we have performed the gene knockdown and overexpression in the mouse livers. Remarkably, knockdown of Atg14 leads to elevated levels of triglycerides in the liver and serum as well. Conversely, overexpression of Atg14 improves hypertriglyceridemia in both high fat diet-treated wild-type mice and FoxO1/3/4 liver-specific knock-out mice. In summary, our data suggest that Atg14 is a new target gene of FoxOs and the core clock machinery, and this gene plays an important role in hepatic lipid metabolism.

Xiong, Xiwen; Tao, Rongya; DePinho, Ronald A.; Dong, X. Charlie



Development through Work and Play.  

ERIC Educational Resources Information Center

Five proposals are made for incorporating a work-play perspective in career development research: (1) fuse work and play conceptually over the life course; (2) imbue developmental career theory with a work-play fusion; (3) study work and play across the life span; (4) investigate work and play within the life space; and (5) consider a work-play

Hartung, Paul J.



Time perspective as a predictor of massive multiplayer online role-playing game playing.  


This article focuses on the relationship between the time perspective (TP) personality trait and massive multiplayer online role-playing game (MMORPG) playing. We investigate the question of frequency of playing. The TP was measured with Zimbardo's TP Inventory (ZTPI), which includes five factors-past negative, past positive, present hedonistic, present fatalistic, and future. The study used data from 154 MMORPG players. We demonstrated that TP partially explained differences within a group of players with respect to the frequency of playing. Significant positive correlations were found between present factors and the amount of time spent playing MMORPGs, and significant negative correlation was found between the future factor and the time spent playing MMORPGs. Our study also revealed the influence of future-present balance on playing time. Players who scored lower in future-present balance variables (their present score was relatively high compared with their future score) reported higher values in playing time. In contrast to referential studies on TP and drug abuse and gambling, present fatalistic TP was demonstrated to be a stronger predictor of extensive playing than present hedonistic TP, which opened the question of motivation for playing. The advantage of our study compared with other personality-based studies lies in the fact that TP is a stable but malleable personality trait with a direct link to playing behavior. Therefore, TP is a promising conceptual resource for excessive playing therapy. PMID:22032796

Lukavska, Katerina



Complete genome sequence of Terriglobus saanensis type strain SP1PR4T, an Acidobacteria from tundra soil  

PubMed Central

Terriglobus saanensis SP1PR4T is a novel species of the genus Terriglobus. T. saanensis is of ecological interest because it is a representative of the phylum Acidobacteria, which are dominant members of bacterial soil microbiota in Arctic ecosystems. T. saanensis is a cold-adapted acidophile and a versatile heterotroph utilizing a suite of simple sugars and complex polysaccharides. The genome contained an abundance of genes assigned to metabolism and transport of carbohydrates including gene modules encoding for carbohydrate-active enzyme (CAZyme) family involved in breakdown, utilization and biosynthesis of diverse structural and storage polysaccharides. T. saanensis SP1PR4T represents the first member of genus Terriglobus with a completed genome sequence, consisting of a single replicon of 5,095,226 base pairs (bp), 54 RNA genes and 4,279 protein-coding genes. We infer that the physiology and metabolic potential of T. saanensis is adapted to allow for resilience to the nutrient-deficient conditions and fluctuating temperatures of Arctic tundra soils.

Rawat, Suman R.; Mannisto, Minna K.; Starovoytov, Valentin; Goodwin, Lynne; Nolan, Matt; Hauser, Lauren; Land, Miriam; Davenport, Karen Walston; Woyke, Tanja; Haggblom, Max M.



Concentration-dependent effects of endogenous S-nitrosoglutathione on gene regulation by specificity proteins Sp3 and Sp1.  

PubMed Central

The activities of certain nuclear regulatory proteins are modified by high concentrations of S-nitrosothiols associated with nitrosative stress. In the present study, we have studied the effect of physiological (low microM) concentrations of the endogenous S-nitrosothiol, GSNO (S-nitrosoglutathione), on the activities of nuclear regulatory proteins Sp3 and Sp1 (specificity proteins 3 and 1). Low concentrations of GSNO increased Sp3 binding, as well as Sp3-dependent transcription of the cystic fibrosis transmembrane conductance regulatory gene, cftr. However, higher GSNO levels prevented Sp3 binding, augmented Sp1 binding and prevented both cftr transcription and CFTR (cystic fibrosis transmembrane conductance regulator) expression. We conclude that low concentrations of GSNO favour Sp3 binding to 'housekeeping' genes such as cftr, whereas nitrosative stress-associated GSNO concentrations shut off Sp3-dependent transcription, possibly to redirect cellular resources. Since low micromolar concentrations of GSNO also increase the maturation and activity of a clinically common CFTR mutant, whereas higher concentrations have the opposite effect, these observations may have implications for dosing of S-nitrosylating agents used in cystic fibrosis clinical trials.

Zaman, Khalequz; Palmer, Lisa A; Doctor, Allan; Hunt, John F; Gaston, Benjamin



Spps, a Drosophila Sp1/KLF family member, binds to PREs and is required for PRE activity late in development  

PubMed Central

The Polycomb group of proteins (PcG) is important for transcriptional repression and silencing in all higher eukaryotes. In Drosophila, PcG proteins are recruited to the DNA by Polycomb-group response elements (PREs), regulatory sequences whose activity depends on the binding of many different sequence-specific DNA-binding proteins. We previously showed that a binding site for the Sp1/KLF family of zinc-finger proteins is required for PRE activity. Here, we report that the Sp1/KLF family member Spps binds specifically to Ubx and engrailed PREs, and that Spps binds to polytene chromosomes in a pattern virtually identical to that of the PcG protein, Psc. A deletion of the Spps gene causes lethality late in development and a loss in pairing-sensitive silencing, an activity associated with PREs. Finally, the Spps mutation enhances the phenotype of pho mutants. We suggest that Spps may work with, or in parallel to, Pho to recruit PcG protein complexes to PREs.

Brown, J. Lesley; Kassis, Judith A.



Serum Starvation-Induced Voltage-Gated Potassium Channel Kv7.5 Expression and Its Regulation by Sp1 in Canine Osteosarcoma Cells  

PubMed Central

The KCNQ gene family, whose members encode Kv7 channels, belongs to the voltage-gated potassium (Kv) channel group. The roles of this gene family have been widely investigated in nerve and muscle cells. In the present study, we investigated several characteristics of Kv7.5, which is strongly expressed in the canine osteosarcoma cell line, CCL-183. Serum starvation upregulated Kv7.5 expression, and the Kv7 channel opener, flupirtine, attenuated cell proliferation by arresting cells in the G0/G1 phase. We also showed that Kv7.5 knockdown helps CCL-183 cells to proliferate. In an effort to find an endogenous regulator of Kv7.5, we used mithramycin A to reduce the level of the transcription factor Sp1, and it strongly inhibited the induction of Kv7.5 in CCL-183 cells. These results suggest that the activation of Kv7.5 by flupirtine may exert an anti-proliferative effect in canine osteosarcoma. Therefore, Kv7.5 is a possible molecular target for canine osteosarcoma therapy.

Lee, Bo Hyung; Ryu, Pan Dong; Lee, So Yeong



BMP-2 and TGF-? Stimulate Expression of ?1,3-Glucuronosyl Transferase 1 (GlcAT-1) in Nucleus Pulposus Cells Through AP1, TonEBP, and Sp1: Role of MAPKs  

PubMed Central

The goal of the study was to investigate bone morphogenetic protein 2 (BMP-2) and transforming growth factor ? (TGF-?) control of the expression of ?1,3-glucuronosyl transferase 1 (GlcAT-1), an important regulator of chondroitin sulfate synthesis in cells of the nucleus pulposus. Treatment with both growth factors resulted in induction of GlcAT-1 expression and promoter activity. Deletion analysis indicated that promoter constructs lacking AP1 and TonE sites were unresponsive to growth factor treatment. Experiments using dominant-negative proteins showed that these transcription factors along with Sp1 were required for induction of GlcAT-1 promoter activity. Moreover, when either AP1 or TonE binding sites were mutated, induction was suppressed. Both BMP-2 and TGF-? increased c-Jun and TonEBP expression and phosphorylation of transactivation domains. We investigated the role of the mitogen-activated protein kinase (MAPK) signaling pathway following growth factor treatment; a robust and transient activation of ERK1/2, p38, and JNK was noted. Treatment with MAPK inhibitors blocked BMP-2- and TGF-?-induced AP1 reporter function, GlcAT-1 expression, and GAG accumulation. We found that DN-ERK1 but not DN-ERK2 resulted in suppression of growth factor–mediated induction of GlcAT-1 promoter activity; we also showed that p38? was important in GlcAT-1 activation. Results of these studies demonstrate that BMP-2 and TGF-? regulate GlcAT-1 expression in nucleus pulposus cells through a signaling network comprising MAPK, AP1, Sp1, and TonEBP. It is concluded that by controlling both GAG and aggrecan synthesis, these growth factors positively influence disk cell function. © 2010 American Society for Bone and Mineral Research.

Hiyama, Akihiko; Gogate, Shilpa S; Gajghate, Sachin; Mochida, Joji; Shapiro, Irving M; Risbud, Makarand V



Curcumin Down-Regulates DNA Methyltransferase 1 and Plays an Anti-Leukemic Role in Acute Myeloid Leukemia  

PubMed Central

Bioactive components from dietary supplements such as curcumin may represent attractive agents for cancer prevention or treatment. DNA methylation plays a critical role in acute myeloid leukemia (AML) development, and presents an excellent target for treatment of this disease. However, it remains largely unknown how curcumin, a component of the popular Indian spice turmeric, plays a role in DNA hypomethylation to reactivate silenced tumor suppressor genes and to present a potential treatment option for AML. Here we show that curcumin down-regulates DNMT1 expression in AML cell lines, both in vitro and in vivo, and in primary AML cells ex vivo. Mechanistically, curcumin reduced the expression of positive regulators of DNMT1, p65 and Sp1, which correlated with a reduction in binding of these transcription factors to the DNMT1 promoter in AML cell lines. This curcumin-mediated down-regulation of DNMT1 expression was concomitant with p15INK4B tumor suppressor gene reactivation, hypomethylation of the p15INK4B promoter, G1 cell cycle arrest, and induction of tumor cell apoptosis in vitro. In mice implanted with the human AML MV4–11 cell line, administration of curcumin resulted in remarkable suppression of AML tumor growth. Collectively, our data indicate that curcumin shows promise as a potential treatment for AML, and our findings provide a basis for future studies to test the clinical efficacy of curcumin – whether used as a single agent or as an adjuvant – for AML treatment.

Yu, Jianhua; Peng, Yong; Wu, Lai-Chu; Xie, Zhiliang; Deng, Youcai; Hughes, Tiffany; He, Shun; Mo, XiaoKui; Chiu, Ming; Wang, Qi-En; He, Xiaoming; Liu, Shujun; Grever, Michael R.; Chan, Kenneth K.; Liu, Zhongfa



Baroreceptor Reflex Role Play  

NSDL National Science Digital Library

In this activity about the baroreceptor reflex (BR) arc (page 123 of the PDF), learners discover the importance of maintaining adequate arterial blood pressure through a role playing exercise. This activity will model how the brain processes information and sends out signals to the heart and arteries. Learners can also consider how this affects astronauts in the microgravity environment of space. The lesson guide, part of NASA's "The Brain in Space: A Teacher's Guide with Activities for Neuroscience," includes background information and evaluation strategies. Note: this activity requires 9 learners per group.

Macleish, Marlene Y.; Mclean, Bernice R.



Disease Role Play  

NSDL National Science Digital Library

Students in collaborative groups will develop an action plan to address a new disease. This activity provides 3 roles for student participation: scientist, public health official and community leader. Each group member will be required to remain within the parameters described by the scenario during the role play. For example, the scientists will be given a data sheet the they will need to interpret. This group member will be the only one with knowledge of the disease. Only this person will act as a disease expert. Once the groups have an opportunity to read their scenarios and prepare for a committee meeting, they will meet and devise an action plan.

Chris Kuka (Bend Senior High School REV)



PlayStation purpura.  


A 16-year-old boy presented with a number of asymptomatic pigmented macules on the volar aspect of his index fingers. Dermoscopy of each macule revealed a parallel ridge pattern of homogenous reddish-brown pigment. We propose that these lesions were induced by repetitive trauma from a Sony PlayStation 3 (Sony Corporation, Tokyo, Japan) vibration feedback controller. The lesions completely resolved following abstinence from gaming over a number of weeks. Although the parallel ridge pattern is typically the hallmark for early acral lentiginous melanoma, it may be observed in a limited number of benign entities, including subcorneal haematoma. PMID:20695869

Robertson, Susan J; Leonard, Jane; Chamberlain, Alex J



Playing Connect Three  

NSDL National Science Digital Library

This activity promotes students to explore and analyze the number of different ways of achieving each of the specific outcomes when adding and subtracting positive and negative integers while playing the game, "Connect Three." By answering key questions, the players work out a strategy for improving their chances of winning the game. The Teachers' Notes page offers suggestions for implementation, discussion questions, ideas for support, extension and answers to questions are provided. A pdf of the game board and a spreadsheet to simulate tossing the dice are linked.



Farm Hall: The Play  

NASA Astrophysics Data System (ADS)

It's July 1945. Germany is in defeat and the atomic bombs are on their way to Japan. Under the direction of Samuel Goudsmit, the Allies are holding some of the top German nuclear scientists--among them Heisenberg, Hahn, and Gerlach--captive in Farm Hall, an English country manor near Cambridge, England. As secret microphones record their conversations, the scientists are unaware of why they are being held or for how long. Thinking themselves far ahead of the Allies, how will they react to the news of the atomic bombs? How will these famous scientists explain to themselves and to the world their failure to achieve even a chain reaction? How will they come to terms with the horror of the Third Reich, their work for such a regime, and their behavior during that period? This one-act play is based upon the transcripts of their conversations as well as the author's historical work on the subject.[4pt] Discussion of the play with the playwright follows after the staged reading.

Cassidy, David C.



Hyperosmolarity-induced up-regulation of claudin-4 mediated by NADPH oxidase-dependent H2O2 production and Sp1/c-Jun cooperation.  


Claudin-4 is exclusively localized in the tight collecting ducts in the renal tubule. We examined what molecular mechanism is involved in the regulation of claudin-4 expression. In Madin-Darby canine kidney cells, hyperosmolarity increased the expression level of claudin-4 and the production of reactive oxygen species, which were inhibited by diphenyleneiodonium (DPI), an NADPH oxidase inhibitor, and manganese (III) tetrakis (4-benzoic acid)porphyrin (MnTBAP), a scavenger of H2O2. Both hyperosmolarity and H2O2 increased p-ERK1/2 and p-JNK, which were inhibited by U0126, a MEK inhibitor, and SP600125, a JNK inhibitor, respectively. Immunoprecipitation assay showed that hyperosmolarity increased the association of nuclear Sp1 with c-Jun, which was inhibited by U0126 and SP600125. In mouse inner medullary collecting duct cells and rat kidney slices, hyperosmolarity increased the expression level of claudin-4, which was inhibited by DPI, MnTBAP, U0126, and SP600125. Hyperosmolarity increased luciferase reporter activity of claudin-4, which was inhibited by U0126, SP600125, Sp1 siRNA, and c-Jun siRNA. The activity was inhibited by the mutation in the Sp1 binding site. Chromatin immunoprecipitation assay and avidin-biotin conjugated DNA assay showed that Sp1 and c-Jun are associated with the Sp1 binding site. These results suggest that hyperosmolarity increases nuclear Sp1/c-Jun complex and the association of the complex with the Sp1 binding site, resulting in the segment-specific expression of claudin-4 in the kidney. PMID:23816505

Ikari, Akira; Atomi, Kosuke; Yamazaki, Yasuhiro; Sakai, Hideki; Hayashi, Hisayoshi; Yamaguchi, Masahiko; Sugatani, Junko



The promoter activity of long terminal repeats of the HERV-H family of human retrovirus-like elements is critically dependent on Sp1 family proteins interacting with a GC/GT box located immediately 3' to the TATA box.  

PubMed Central

The HERV-H family of endogenous retrovirus-like elements is widely distributed in the human genome, with about 1,000 full-length elements and a similar number of solitary long terminal repeats (LTRs). HERV-H LTRs have been shown to direct the transcription of both HERV-H-encoded and adjacent cellular genes. Transcripts of HERV-H elements are especially abundant in placenta, teratocarcinoma cell lines, and cell lines derived from testicular and lung tumors. Here we report that only a subset of HERV-H LTRs display promoter activity in human cell lines and that these LTRs are characterized by the presence of a GC/GT box immediately downstream of the TATA box. This GC/GT box is required for promoter activity, while, surprisingly, the TATA box is dispensable. The ubiquitously expressed transcription factors Sp1 and Sp3 bound to this GC/GT box and stimulated transcription from the promoter-active LTRs in the teratocarcinoma cell line NTera2-D1. However, in HeLa and Drosophila SL-2 cells, Sp1 acted as a transcriptional activator of the LTRs, while Sp3 acted as a repressor of Sp1-mediated transcriptional activation. Cotransfection studies also revealed that the tissue-specific Sp1-related protein BTEB bound to this GC/GT box and stimulated transcription from the LTR promoters in NTera2-D1 cells. These results show that members of the Sp1 protein family are crucial determinants for transcriptional activation of HERV-H LTR promoters and suggest that these proteins may also be involved in determining the tissue-specific expression pattern of HERV-H elements.

Sj?ttem, E; Anderssen, S; Johansen, T



Inhibitory effect of acetyl-11-keto-beta-boswellic acid on androgen receptor by interference of Sp1 binding activity in prostate cancer cells.  


Androgen receptor (AR)-mediated signaling is crucial for the development and progression of prostate cancer (PCa). Naturally occurring phytochemicals that target the AR signaling offer significant protection against this disease. Acetyl-11-keto-beta-boswellic acid (AKBA), a compound isolated from the gum-resin of Boswellia carterii, caused G1-phase cell cycle arrest with an induction of p21(WAF1/CIP1), and a reduction of cyclin D1 as well in prostate cancer cells. AKBA-mediated cellular proliferation inhibition was associated with a decrease of AR expression at mRNA and protein levels. Furthermore, the functional biomarkers used in evaluation of AR transactivity showed suppressions of prostate-specific antigen promoter-dependent and androgen responsive element-dependent luciferase activities. Additionally, down-regulation of an AR short promoter mainly containing a Sp1 binding site suggested the essential role of Sp1 for the reduction of AR expression in cells exposed to AKBA. Interruption effect of AKBA on Sp1 binding activity but not Sp1 protein levels was further confirmed by EMSA and transient transfection with a luciferase reporter driven by three copies of the Sp1 binding site of the AR promoter. Therefore, anti-AR properties ascribed to AKBA suggested that AKBA-containing drugs could be used for the development of novel therapeutic chemicals. PMID:18430409

Yuan, Hui-Qing; Kong, Feng; Wang, Xiao-Ling; Young, Charles Y F; Hu, Xiao-Yan; Lou, Hong-Xiang



Child's Play: Revisiting Play in Early Childhood Settings.  

ERIC Educational Resources Information Center

Noting that play is an essential aspect of learning for young children, this book presents a collection of articles on children's play in Australia. Part 1, "Play, Development, and Learning," contains the following chapters: (1) "The Role of Play in Development and Learning" (Ann Glover); (2) "Stop, Look, and Listen: Adopting an Investigative…

Dau, Elizabeth, Ed.; Jones, Elizabeth, Ed.


Asbestos in play sand  

SciTech Connect

A letter in the New England Journal of Medicine (Oct. 2 issue) stated that a carbonate sand marketed in New Jersey was contaminated with 2 to 4 percent tremolite asbestos. The authors were called on by one of the federal agencies to repeat the analysis of this sand, specifically for its asbestos content. The sand was pulverized and immersed in oils with known refractive indexes, and the predominant amphibole was characterized by polarized light microscopy. The optical characteristics were noted, and the indexes of refraction were measured and found to be consistent with tremolite. On the basis of optical characterization, the authors concluded that all the tremolite visualized with light microscopy consisted of large, single cleavage fragments and was not asbestiform. They used the technique of x-ray diffraction, as did the author of the original report, which showed the presence of an amphibole mineral (probably tremolite) in the carbonate sand. The technique was not used, and cannot be used, to distinguish between the tremolite habits (asbestiform or nonasbestiform). An acid-insoluble residue, recovered from the carbonate sand, was examined by analytic electron microscopy. The tremolite grains were observed to consist of single untwinned, crystalline fragments. Few defects were noted. Selected area electron diffraction nets were indicative of fragments lying near or at the common amphibole cleavage plane. These characteristics are consistent with cleavage fragments and not asbestos. Aspect ratios reflected short particles (less than 5.1). On the basis of their examination of the carbonate play sand, they conclude that it did not contain tremolite asbestos.

Langer, A.M.; Nolan, R.P.



Play in evolution and development  

Microsoft Academic Search

In this paper we examine the role of play in human ontogeny and phylogeny, following Surplus Resource Theory. We consider how juveniles use play to sample their environment in order to develop adaptive behaviors. We speculate about how innovative behaviors developed in play in response to environmental novelty may influence subsequent evolutionary processes. Play during this period of immaturity is

Anthony D. Pellegrini; Danielle Dupuis; Peter K. Smith



Literacy Learning during Block Play.  

ERIC Educational Resources Information Center

Researchers long have sought a connection between play and literacy behaviors. Most agree that play is a basis for developing literacy skills. This study investigated the use of block play in a first-grade classroom. Results showed that the students increased the number of literacy behaviors after having been exposed to the enriched block-play

Pickett, Linda



Effect of abasic linker substitution on triplex formation, Sp1 binding, and specificity in an oligonucleotide targeted to the human Ha-ras promoter.  

PubMed Central

A region of the human Ha-ras promoter (-8 to -28) which contains two of the three Sp1 binding sites essential for transcriptional activity forms a sequence specific oligonucleotide-directed pur*pur:pyr triple helix. The relative binding of oligonucleotides containing different substitutions, including an abasic propanediol linker, over three potentially destabilizing C:G interruptions in the otherwise poly G:poly C target was examined. DNase I footprint titrations reveal that substitution of the positively charged abasic propanediol linker results in approximately ten fold greater binding than cytosine substitution which in turn provides greater sequence specific binding than substitution of a guanine in the third strand oligonucleotide over the C:G interruptions. Protein binding assays demonstrate that triplex formation by the linker substituted oligomer (HR21Xap) is less effective in inhibiting Sp1 binding than the cytosine substituted oligomer (HR21ap) both to the target sequence as well as an upstream sequence. As an indication of the effect of linker substitution and targeting consensus Sp1 sites on triplex specificity, the relative ability of the Ha-ras promoter targeted oligonucleotides to interact with non-target Sp1 sequences within the Ha-ras promoter as well as in the DHFR promoter and HIV-1 LTR was also investigated. At concentrations which afford complete DNase I protection of the target sequence, HR21ap does not bind to the non-target sequences while HR21Xap interacts weakly only at a distal site in the DHFR promoter. Also, HR21ap as well as HR21Xap are specific in their inhibition of Sp1 binding. These results suggest that the propanediol linker is able to skip over interruptions in a target sequence thereby stabilizing triplex but, slightly compromises sequence specificity and the ability to inhibit Sp1 binding to the Ha-ras promoter. Images

Mayfield, C; Miller, D



Rough and Tumble Play 101  

ERIC Educational Resources Information Center

Many people fear that play-fighting or rough and tumble play is the same as real fighting. There is also a fear that this rough play will become real fighting if allowed to continue. Most of all, parents and teachers fear that during the course of rough and tumble play a child may be hurt. To provide for and allow children to play rough without…

Carlson, Frances



Can I Play?: Classroom-Based Interventions for Teaching Play Skills to Children with Autism.  

ERIC Educational Resources Information Center

This article discusses teaching play skills to children with autism. Factors considered include developmental appropriateness, language development, peer involvement, motivational techniques, and setting and intervention methods. Methods for teaching play skills are then reviewed, including script training, peer trainers, and pivotal response…

Terpstra, Judith E.; Higgins, Kyle; Pierce, Tom



Characterization of a marine-isolated mercury-resistant Pseudomonas putida strain SP1 and its potential application in marine mercury reduction  

Microsoft Academic Search

The Pseudomonas putida strain SP1 was isolated from marine environment and was found to be resistant to 280 ?M HgCl2. SP1 was also highly resistant to other metals, including CdCl2, CoCl2, CrCl3, CuCl2, PbCl2, and ZnSO4, and the antibiotics ampicillin (Ap), kanamycin (Kn), chloramphenicol (Cm), and tetracycline (Tc). mer operon, possessed by most mercury-resistant bacteria, and other diverse types of resistant

Weiwei Zhang; Lingxin Chen; Dongyan Liu


A Re-examination of the Smilansky-Parten Matrix of Play Behavior.  

ERIC Educational Resources Information Center

Describes factor analysis of preschool children's play behavior, as measured by the Smilansky-Parten play matrix. Varimax rotation solution suggests three factors: Dramatic-Constructive Play, Solitary Behavior, and Functional-Constructive Play. Study suggests that these factors represent constructs of preschoolers' play. (Author/RWB)

Pellegrini, A. D.; Perlmutter, Jane C.



Play, Play Therapy, Play Research. Proceedings of the International Symposium (Amsterdam, the Netherlands, September 1985).  

ERIC Educational Resources Information Center

After an introduction which briefly discusses emotional, therapeutic, phenomenological, cognitive, and developmental perspectives on play, this volume presents the complete texts of all the main lectures and a few short papers that were given at the International Symposium on Play, Play Therapy, and Play Research. Papers in part 1 concern certain…

Kooij, Rimmert van der; Hellendoorn, Joop


Solar Power at Play  

NASA Astrophysics Data System (ADS)

For the very first time, astronomers have witnessed the speeding up of an asteroid's rotation, and have shown that it is due to a theoretical effect predicted but never seen before. The international team of scientists used an armada of telescopes to discover that the asteroid's rotation period currently decreases by 1 millisecond every year, as a consequence of the heating of the asteroid's surface by the Sun. Eventually it may spin faster than any known asteroid in the solar system and even break apart. ESO PR Photo 11a/07 ESO PR Photo 11a/07 Asteroid 2000 PH5 "The Yarkovsky-O'Keefe-Radzievskii-Paddack (YORP) effect is believed to alter the way small bodies in the Solar System rotate," said Stephen Lowry (Queens University Belfast, UK), lead-author of one of the two companion papers in which this work is reported [1, 2]. "The warming caused by sunlight hitting the surfaces of asteroids and meteoroids leads to a gentle recoil effect as the heat is released," he added. "By analogy, if one were to shine light on a propeller over a long enough period, it would start spinning." Although this is an almost immeasurably weak force, its effect over millions of years is far from negligible. Astronomers believe the YORP effect may be responsible for spinning some asteroids up so fast that they break apart, perhaps leading to the formation of double asteroids. Others may be slowed down so that they take many days to complete a full turn. The YORP effect also plays an important role in changing the orbits of asteroids between Mars and Jupiter, including their delivery to planet-crossing orbits, such as those of near-Earth asteroids. Despite its importance, the effect has never been seen acting on a solar system body, until now. Using extensive optical and radar imaging from powerful Earth-based observatories, astronomers have directly observed the YORP effect in action on a small near-Earth asteroid, known as (54509) 2000 PH5. Shortly after its discovery in 2000, it was realised that asteroid 2000 PH5 would be the ideal candidate for such a YORP detection. With a diameter of just 114 metres, it is relatively small and so more susceptible to the effect. Also, it rotates very fast, with one 'day' on the asteroid lasting just over 12 Earth minutes, implying that the YORP effect may have been acting on it for some time. With this in mind, the team of astronomers undertook a long term monitoring campaign of the asteroid with the aim of detecting any tiny changes in its rotation speed. Over a 4-year time span, Stephen Lowry, Alan Fitzsimmons and colleagues took images of the asteroid at a range of telescope sites including ESO's 8.2-m Very Large Telescope array and 3.5-m New Technology Telescope in Chile, the 3.5-m telescope at Calar Alto, Spain, along with a suite of other telescopes from the Czech Republic, the Canary Islands, Hawaii, Spain and Chile. With these facilities the astronomers measured the slight brightness variations as the asteroid rotated. ESO PR Photo 11b/07 ESO PR Photo 11b/07 Radar Images of 2000 PH5 Over the same time period, the radar team led by Patrick Taylor and Jean-Luc Margot of Cornell University employed the unique capabilities of the Arecibo Observatory in Puerto Rico and the Goldstone radar facility in California to observe the asteroid by 'bouncing' a radar pulse off the asteroid and analysing its echo. "With this technique we can reconstruct a 3-D model of the asteroid's shape, with the necessary detail to allow a comparison between the observations and theory," said Taylor. After careful analysis of the optical data, the asteroid's spin rate was seen to steadily increase with time, at a rate that can be explained by the YORP theory. Critically, the effect was observed year after year, for more than 4 years. Furthermore, this number was elegantly supported via analysis of the combined radar and optical data, as it was required that the asteroid is increasing its spin rate at exactly this rate in order for a satisfactory 3-D shape model to be determined. ESO PR Video



Valproic acid induces functional heat shock protein 70 via Class I histone deacetylase inhibition in cortical neurons: a potential role of Sp1 acetylation  

PubMed Central

Neuroprotective properties of the mood stabilizer valproic acid (VPA) are implicated in its therapeutic efficacy. Heat shock protein 70 (HSP70) is a molecular chaperone, neuroprotective and anti-inflammatory agent. The present study aimed to investigate underlying mechanisms and functional significance of HSP70 induction by VPA in rat cortical neurons. VPA treatment markedly upregulated HSP70 protein levels, and this was accompanied by increased HSP70 mRNA levels and promoter hyperacetylation and activity. Other HDAC inhibitors - sodium butyrate, trichostatin A and Class I HDAC-specific inhibitors MS-275 and apicidin, - all mimicked the ability of VPA to induce HSP70. Pretreatment with PI3-kinase inhibitors or an Akt inhibitor attenuated HSP70 induction by VPA and other HDAC inhibitors. VPA treatment increased Sp1 acetylation, and a Sp1 inhibitor, mithramycin, abolished the induction of HSP70 by HDAC inhibitors. Moreover, VPA promoted the association of Sp1 with the histone acetyltransferases p300 and recruitment of p300 to the HSP70 promoter. Further, VPA-induced neuroprotection against glutamate excitotoxicity was prevented by blocking HSP70 induction. Taken together, the data suggest that the PI3-kinase/Akt pathway and Sp1 are likely involved in HSP70 induction by HDAC inhibitors, and induction of HSP70 by VPA in cortical neurons may contribute to its neuroprotective and therapeutic effects.

Marinova, Zoya; Ren, Ming; Wendland, Jens R.; Leng, Yan; Liang, Min-Huei; Yasuda, Shigeru; Leeds, Peter; Chuang, De-Maw



Play Memories and Place Identity.  

ERIC Educational Resources Information Center

This retrospective study examined play memories from childhood to adulthood of 478 university students between ages 20 and 62 as exhibited in drawings of play memories and questionnaire responses. The study focused on the role of the physical environment and place identity in play memories and individual identity development. Findings showed that…

Sandberg, Anette



Preschoolers' Thinking during Block Play  

ERIC Educational Resources Information Center

Children build foundations for mathematical thinking in early play and exploration. During the preschool years, children enjoy exploring mathematical concepts--such as patterns, shape, spatial relationships, and measurement--leading them to spontaneously engage in mathematical thinking during play. Block play is one common example that engages…

Piccolo, Diana L.; Test, Joan



Play in Evolution and Development  

ERIC Educational Resources Information Center

In this paper we examine the role of play in human ontogeny and phylogeny, following Surplus Resource Theory. We consider how juveniles use play to sample their environment in order to develop adaptive behaviors. We speculate about how innovative behaviors developed in play in response to environmental novelty may influence subsequent evolutionary…

Pellegrini, Anthony D.; Dupuis, Danielle; Smith, Peter K.



Enhancing Learning through Sociodramatic Play.  

ERIC Educational Resources Information Center

The role of sociodramatic play in children's cognitive, social, and physical development is discussed, drawing on observations of work with parents and their children ages 3-5. The paper focuses on the way the teachers guided and facilitated play taking cues from the children. A training session was provided to the parents on ways to play with…

Rosberg, Merilee


Meanings of Play among Children  

ERIC Educational Resources Information Center

The purpose of this study was to examine meanings of play among children. Thirty-eight students aged 7-9 years from a suburban public school in Western Canada participated in focus groups. Data analysis revealed participants saw almost anything as an opportunity for play and would play almost anywhere with anyone. However, they perceived parents…

Glenn, Nicole M.; Knight, Camilla J.; Holt, Nicholas L.; Spence, John C.



The oncoprotein hepatitis B X-interacting protein promotes the migration of ovarian cancer cells through the upregulation of S-phase kinase-associated protein 2 by Sp1.  


Hepatitis B X-interacting protein (HBXIP) is a novel oncoprotein. We have previously reported that HBXIP promotes the proliferation and migration of breast cancer cells. S-phase kinase-associated protein 2 (Skp2) is another oncoprotein which is important for migration. In this study, we investigated whether Skp2 is involved in the migration enhanced by HBXIP in ovarian cancer. The expression of HBXIP and Skp2 in ovarian cancer tissues was examined by immunohistochemistry using tissue microarrays. The role of HBXIP and Skp2 in the migration of ovarian cancer cells was investigated by wound-healing assay and Transwell migration assay. The effect of HBXIP on Skp2 was assessed by reverse transcription polymerase chain reaction (RT-PCR), western blot analysis, luciferase reporter gene assays and chromatin immunoprecipitation in ovarian cancer cells (SKOV3 and CAOV3). We found that both HBXIP and Skp2 were highly expressed in ovarian cancer tissues. We observed that the overexpression of HBXIP enhanced the migration of ovarian cancer cells, while Skp2 siRNAs decreased the cell migration enhanced by HBXIP. The HBXIP siRNAs inhibited ovarian cancer cell migration and Skp2 rescued the migration inhibition induced by HBXIP siRNA. HBXIP could upregulate Skp2 at the levels of mRNA and protein in ovarian cancer cells. Moreover, HBXIP increased the activity of Skp2 promoter via binding to the transcription factor Sp1. HBXIP is highly expressed in ovarian cancer tissues. HBXIP enhances the migration of ovarian cancer cells. HBXIP was able to stimulate the activity of Skp2 promoter via transcription factor Sp1 thus promoting the migration of ovarian cancer cells. PMID:24788380

Xu, Fuqiang; Zhu, Xiaoming; Han, Tao; You, Xiaona; Liu, Fabao; Ye, Lihong; Zhang, Xiaodong; Wang, Xiaohong; Yao, Yuanqing



Repression of integrin-linked kinase by antidiabetes drugs through cross-talk of PPAR?- and AMPK?-dependent signaling: role of AP-2? and Sp1.  


Nasopharyngeal carcinoma (NPC) is one of the most common cancers of the head and neck, particularly in Southern China and Southeast Asia with high treatment failure due to the development of local recurrence and distant metastasis. The molecular mechanisms related to the progression of NPC have not been fully understood. In this study, we showed that antidiabetes drugs rosiglitazone and metformin inhibit NPC cell growth through reducing the expression of integrin-linked kinase (ILK). Blockade of PPAR? and AMPK? overcame the effects of rosiglitazone and metformin on ILK protein. Importantly, overexpression of ILK abrogated the effect of rosiglitazone and metformin on NPC cell growth. Furthermore, these agents reduced ILK promoter activity, which was not observed in AP-2?, but not Sp1 site mutation in ILK gene promoter. In addition, silencing of AP-2? or overexpression of Sp1 reversed the effect of these agents on ILK protein expression and cell growth. Chromatin immunoprecipitation (ChIP) assay showed that rosiglitazone induced AP-2?, while metformin reduced Sp1 protein binding to the DNA sequences in the ILK gene promoter. Intriguingly, overexpression of Sp1 abolished the effect of rosiglitazone on AP-2? protein expression. Collectively, we show that rosiglitazone and metformin inhibit ILK gene expression through PPAR?- and AMPK?-dependent signaling pathways that are involved in the regulation of AP-2? and Sp1 protein expressions. The effect of combination of rosiglitazone and metformin demonstrates greater extent than single agent alone. The cross-talk of PPAR? and AMPK? signaling enhances the synergistic effects of rosiglitazone and metformin. This study unveils novel mechanisms by which oral antidiabetes drugs inhibit the growth of human NPC cells. PMID:24361375

Hahn, Swei Sunny; Tang, Qing; Zheng, Fang; Zhao, Shunyu; Wu, Jingjing; Chen, Jianping



Playing Safe: Eliminating Hazards on Children's Playgrounds.  

ERIC Educational Resources Information Center

Discusses typical playground hazards, environmental factors affecting safety in play areas, playground safety guidelines and standards established by the Consumer Product Safety Commission and the American Society of Testing Materials, and playground surfacing and equipment. Offers suggestions for reducing the number and severity of playground…

Wallach, Frances



Mammalian Krüppel-like transcription factors: more than just a pretty finger  

Microsoft Academic Search

The transcription factor SP1 contains three Krüppel-like zinc fingers. Recently, several related proteins, including erythroid, lung and gut-enriched Krüppel-like factors, have been identified. Together with SP1, these proteins form a sizeable family of transcription factors that share homology in their zinc-finger domains but differ elsewhere. Analysis of these differences is illuminating specific mechanisms by which transcription is regulated.

Jeremy Turner; Merlin Crossley





... confidence to try new experiences and explore new environments. Occupational therapists have expertise in evaluating children’s neurological, muscular, and emotional development; and determining the effects of infant and childhood ...


Children's active play: self-reported motivators, barriers and facilitators  

PubMed Central

Background Physical activity has important benefits for children's physical health and mental wellbeing, but many children do not meet recommended levels. Research suggests that active play has the potential to make a valuable contribution to children's overall physical activity, whilst providing additional cognitive, social and emotional benefits. However, relatively little is known about the determinants of UK children's active play. Understanding these factors provides the critical first step in developing interventions to increase children's active play, and therefore overall physical activity. Methods Eleven focus groups were conducted with 77, 10-11 year old children from four primary schools in Bristol, UK. Focus groups examined: (i) factors which motivate children to take part in active play; (ii) factors which limit children's active play and (iii) factors which facilitate children's active play. All focus groups were audio-taped and transcribed verbatim. Data were analysed using a thematic approach. Results Children were motivated to engage in active play because they perceived it to be enjoyable, to prevent boredom, to have physical and mental health benefits and to provide freedom from adult control, rules and structure. However, children's active play was constrained by a number of factors, including rainy weather and fear of groups of teenagers in their play spaces. Some features of the physical environment facilitated children's active play, including the presence of green spaces and cul-de-sacs in the neighbourhood. Additionally, children's use of mobile phones when playing away from home was reported to help to alleviate parents' safety fears, and therefore assist children's active play. Conclusions Children express a range of motivational and environmental factors that constrain and facilitate their active play. Consideration of these factors should improve effectiveness of interventions designed to increase active play.



Behavioral approaches to promoting play.  


A variety of techniques grounded in behavioral psychology, and more specifically in applied behavior analysis, have been established to increase and improve play skills in children with autistic spectrum disorders. This article introduces a set of efficacious methods, which range from highly structured techniques to more naturalistic strategies. It focuses on object play as other authors in the issue discuss social play in greater depth. Behavioral techniques that are reviewed include: discrete trial training, use of stereotyped behaviors to increase play skills, pivotal response training, reciprocal imitation training, differential reinforcement of appropriate behavior, in vivo modeling and play scripts, and video modeling. A discussion of expanding behavior techniques to teach more complex play as well as training in varied environments is also presented. References are provided to allow the reader to obtain more in-depth information about each technique. PMID:14678679

Stahmer, Aubyn C; Ingersoll, Brooke; Carter, Cynthia



ErbB2 upregulates the Na+,HCO3(-)-cotransporter NBCn1/SLC4A7 in human breast cancer cells via Akt, ERK, Src, and Kruppel-like factor 4.  


Misregulation of acid-base transport plays central roles in cancer development. We previously demonstrated the strong up-regulation of the Na(+),HCO3(-) cotransporter NBCn1 (SLC4A7) in MCF-7 breast cancer cells by a truncated, constitutively active ErbB2 (HER2) receptor, ?NErbB2, and showed that NBCn1 expression and activity are increased in breast cancer tissue from patients. Here, we present the first in-depth characterization of an SLC4A7 promoter and identify its minimal ?NErbB2-sensitive region. Inhibition or siRNA-mediated knockdown of PI3K, Akt1, ERK1/2, or Src decreased the NBCn1 protein level in ?NErbB2-expressing MCF-7 cells by ~50, 60, 30 and 35%, respectively. Further, knockdown of the transcription factor Krüppel-like factor 4 (KLF4) reduced NBCn1 protein expression by ~40%, and KLF4 overexpression increased NBCn1 expression by 50-80%. In contrast, knockdown of the closely related transcription factor specificity protein 1 (Sp1) or transfection with dominant-negative Sp1 increased NBCn1 expression by ~35 and ~50%, respectively. NBCn1 expression was also increased by stimulation of full-length ErbB1, -2, and -3 receptors in SKBr3 cells (1.5- and 2-fold by NRG1 or EGF, respectively) or after their exogenous expression in MCF-7 cells. Finally, stimulation with NRG1 or EGF more than doubled acid extrusion capacity in SKBr3 cells. In conclusion, NBCn1 is strongly upregulated by ErbB receptor signaling in a manner involving opposite effects of KLF4 and Sp1, transcription factors with central roles in cancer development. ErbB-induced up-regulation of NBCn1-mediated acid extrusion may play important physiological and pathophysiological roles in the breast epithelium and other tissues with high ErbB receptor levels. PMID:24088818

Gorbatenko, Andrej; Olesen, Christina W; Mørup, Nina; Thiel, Gerald; Kallunki, Tuula; Valen, Eivind; Pedersen, Stine F



Understanding Young Children's Learning through Play: Building Playful Pedagogies  

ERIC Educational Resources Information Center

This timely and accessible text introduces, theorises and practically applies two important concepts which now underpin early years practice: those of "playful learning" and "playful pedagogies". Pat Broadhead and Andy Burt draw upon filmed material, conversations with children, reflection, observation, and parental and staff interviews, in their…

Broadhead, Pat; Burt, Andy



Well Played: The Origins and Future of Playfulness  

ERIC Educational Resources Information Center

In this article, the author synthesizes research from several disciplines to shed light on play's central role in healthy development. Gordon builds on research in attachment theory that correlates secure attachment in infancy with adult well-being to demonstrate how playfulness might be a lifelong outcome of secure attachment and a primary…

Gordon, Gwen



The Influence of Play Context and Adult Attitudes on Young Children's Physical Risk-Taking during Outdoor Play  

ERIC Educational Resources Information Center

Many children naturally seek challenging physically active play which may involve injury-risk. Prior studies have attempted to describe the characteristics of risky play but to date none have considered factors that impact on opportunities for risky play or the likely resultant outcomes. Using semi-structured interviews and naturalistic…

Little, Helen; Wyver, Shirley; Gibson, Frances



NRF-1 is the major transcription factor regulating the expression of the human TOMM34 gene.  


The human TOMM34 gene encodes a cytosolic protein with chaperone-like activity that helps import some preproteins to the mitochondria by keeping them in an unfolded, import-compatible state. TOMM34 was found to be upregulated frequently in colorectal tumors, suggesting that it also has a role in the growth of cancer cells. In this context, TOMM34 is a potential target for novel anticancer drugs, and it might also be used in the diagnosis of colorectal cancer. Nuclear respiratory factors (NRFs) play an important role in governing the nuclear-mitochondrial interactions implicated in mitochondrial biogenesis. Our previous studies revealed that NRFs promote the expression of the major members of the mitochondrial transport machinery, TOMM70 and TOMM20. Here we report the existence of binding sites for NRF-1, Sp1, and NRF-2 in the 5' region of the human TOMM34 gene. We determined the effects of mutations at these sites on promoter activity in HeLa S3 and A204 cells, in conjunction with chromatin immunoprecipitation experiments, electrophoretic mobility shift assays, and in vivo methylation analysis of the promoter region. We conclude that NRF-1 is the main transcription factor regulating the expression of TOMM34. Sp1 interacts with NRF-1 to stimulate the promoter's full activity. PMID:18364745

Blesa, José R; Prieto-Ruiz, Jesús A; Abraham, Beth A; Harrison, Bridget L; Hegde, Anita A; Hernández-Yago, José



Playful biometrics: controversial technology through the lens of play.  


This article considers the role of play in the context of technological emergence and expansion, particularly as it relates to recently emerging surveillance technologies. As a case study, I consider the trajectory of automated face recognition—a biometric technology of numerous applications, from its more controversial manifestations under the rubric of national security to a clearly emerging orientation toward play. This shift toward “playful” biometrics—or from a technology traditionally coded as “hard” to one now increasingly coded as “soft”—is critical insofar as it renders problematic the traditional modes of critique that have, up until this point, challenged the expansion of biometric systems into increasingly ubiquitous realms of everyday life. In response to this dynamic, I propose theorizing the expansion of face recognition specifically in relation to “play,” a step that allows us to broaden the critical space around newly emerging playful biometrics, as well as playful surveillance more generally. In addition, play may also have relevance for theorizing other forms of controversial technology, particularly given its potential role in processes of obfuscation, normalization, and marginalization. PMID:22175066

Ellerbrok, Ariane



Learning Academic Skills through Play.  

ERIC Educational Resources Information Center

The purposes of this study are (1) to identify the relationship between play and achievement in preschool and kindergarten students and (2) to compare academic skill mastery levels between two types of school groups: play curriculum and direct teacher instruction. A total of 48 preschool and kindergarten students, half enrolled in a curriculum…

Gallegos, Margaret


Sand and Water Table Play  

ERIC Educational Resources Information Center

The authors observed preschoolers engaged at the sand and water table to determine if math could be found within their play. Wanting to understand how children interact with provided materials and what kinds of math ideas they explore during these interactions, the authors offer practical examples of how such play can promote mathematical…

Wallace, Ann H.; White, Mary J.; Stone, Ryan



Play Therapy with Special Populations.  

ERIC Educational Resources Information Center

This paper notes that therapists often feel unqualified to deal with special populations of children because of a lack of understanding of the universalness of play therapy. Suggestions are offered for beginning play therapists who may work with a number of special populations of children. It is recommended that the social learning approach to…

Carmichael, Karla D.


Playground Play: Educational and Inclusive  

ERIC Educational Resources Information Center

It is easy to understand that fun is one of the key ingredients to any playground activity. But what one may not realize is that play systems--including slides, tunnels, activity panels, and more--encourage a lot more than just fun: there is learning at work in playground play, as well as the opportunity to include children of all abilities in…

Moore, Lisa



Let's Play Three on Three!  

ERIC Educational Resources Information Center

Over the course of nine years as a supervisor of intern teachers, the first author collected observations of game play during lessons taught by intern teachers or their mentors. In general, the observations indicated that the majority of students got limited practice opportunities during game play. A close look at the data revealed an interesting…

Kern, Jack; Calleja, Paul



Niger Delta play types, Nigeria  

SciTech Connect

Exploration databases can be more valuable when sorted by play type. Play specific databases provide a system to organize E & P data used in evaluating the range of values of parameters for reserve estimation and risk assessment. It is important both in focusing the knowledge base and in orienting research effort. A play in this context is any unique combination of trap, reservoir and source properties with the right dynamics of migration and preservation that results in hydrocarbon accumulation. This definitions helps us to discriminate the subtle differences found with these accumulation settings. About 20 play types were identified around the Niger Delta oil province in Nigeria. These are grouped into three parts: (1) The proven plays-constituting the bulk of exploration prospects in Nigeria today. (2) The unproven or semi-proven plays usually with some successes recorded in a few tries but where knowledge is still inadequate. (3) The unproven or analogous play concept. These are untested but geologically sound ideas which may or may not have been tried elsewhere. With classification and sub grouping of these play types into specific databases, intrinsic attributes and uniqueness of each of them with respect to the four major risk elements and the eight parameters for reserve estimation can be better understood.

Akinpelu, A.O. [Chevron Nigeria Limited, Lagos (Nigeria)



Puzzle Play Improves Math Skills  

NSDL National Science Digital Library

This brief press release from the National Science Foundation summarizes the results of a University of Chicago study linking puzzle play with math skills. The study found that puzzle play proved to be a significant predictor of spatial skills. The study also found gender differences in child/parent interactions and in acquired skills.



Playing the Game of School.  

ERIC Educational Resources Information Center

Ways to succeed in school and advice on reading books and textbooks are covered in four essays. The analogy of playing the "game of school" and preparing for and playing a sport is developed by Charles I. Brown in two essays. Study activities that are designed to be done before, during, and after class are suggested to help the student maximize…

Brown, Charles I.; And Others


The Play of Socratic Dialogue  

ERIC Educational Resources Information Center

Proponents of philosophy for children generally see themselves as heirs to the "Socratic" tradition. They often claim too that children's aptitude for play leads them naturally to play with abstract, philosophical ideas. However in Plato's dialogues we find in the mouth of "Socrates" many warnings against philosophising with the young. Those…

Smith, Richard



Empowering Groups that Enable Play  

ERIC Educational Resources Information Center

Creating play environments for children usually requires groups of adults working together. An extensive scientific literature describes how groups function to achieve shared goals in general terms, and groups attempting to empower play may find this literature useful. Design principles for managing natural resources, identified by Elinor Ostrom…

Wilson, David Sloan; Marshall, Danielle; Iserhott, Hindi



Role-Playing and Playing Roles: The Person, Player, and Persona in Fantasy Role-Playing  

Microsoft Academic Search

In fantasy role-playing games, participants collectively create and play fan- tasy personas in an imaginary universe by using a vast system of rules that function as guidelines for make-believe action and interaction. Conse- quently, role-playing games obligate participants to occupy a liminal role located in the boundaries of persona, player, and person. This study, based on approximately ninety hours of

Dennis Waskul; Matt Lust



Regulation of poly(ADP-ribose) polymerase-1 (PARP1) gene expression through the post-translational modification of Sp1: a nuclear target protein of PARP1  

Microsoft Academic Search

BACKGROUND: Poly(ADP-ribose) polymerase-1 (PARP-1) is a nuclear enzyme that plays critical functions in many biological processes, including DNA repair and gene transcription. The main function of PARP-1 is to catalyze the transfer of ADP-ribose units from nicotinamide adenine dinucleotide (NAD+) to a large array of acceptor proteins, which comprises histones, transcription factors, as well as PARP-1 itself. We have previously

Karine Zaniolo; Serge Desnoyers; Steeve Leclerc; Sylvain L Guérin



Possible Synthesis of Pregnancy‘Specific’ Beta1Glycoprotein (SP1) and Placenta‘Specific’ Tissue Proteins (PP10, PP12) by Human and Cynomolgus Monkey Leukocytes  

Microsoft Academic Search

Using an enzyme-bridge immunoperoxidase (PAP) technique, it was revealed that pregnancy- and\\/or placenta-‘specific’ proteins, SP1 PP10 and PP12 were localized in human and cynomolgus monkey (Macaca fascicularis) polymorphonuclear neutrophils, and PP10, in mononuclear phagocytes. There was no difference in staining results, irrespective of whether the specimens were from human or monkey males, or pregnant or nonpregnant females. The results suggest

Noriyuki Inaba; Thomas Renk; Wolfgang Ax; Sabine Schottler; Ernst Weinmann; Hans Bohn



Downregulation of Sp1 is involved in honokiol-induced cell cycle arrest and apoptosis in human malignant pleural mesothelioma cells.  


Malignant pleural mesothelioma (MPM) is an extremely aggressive type of cancer and is associated with a poor patient prognosis due to its rapid progression. Novel therapeutic agents such as honokiol (HNK) improve the clinical outcomes of cancer therapy, yet the mechanisms involved have not been fully elucidated. The present study examined the regulatory effects of HNK on the growth and apoptosis of MSTO-211H mesothelioma cells and investigated its anticancer mechanism. The results revealed that HNK significantly reduced the cell viability and increased the sub-G1 population in MSTO-211H cells and suppressed the expression of the specificity protein 1 protein (Sp1). HNK reduced the transcriptional activity of Sp1 regulatory proteins, including cyclin D1, Mcl-1 and survivin, and, thus, induced apoptosis signaling pathways by increasing Bax, reducing Bid and Bcl-xl and activating caspase-3 and PARP in mesothelioma cells. The results suggest that Sp1, a novel molecular target of HNK, may be related to cell cycle arrest and apoptosis induction through the modulation of signal transduction pathways in MPM. PMID:23525508

Chae, Jung-Il; Jeon, Young-Joo; Shim, Jung-Hyun



The Plays of William Shakespeare  

NSDL National Science Digital Library

This e-text site offers the complete plays of William Shakespeare with introductions, concordances, a character guide, and well-known quotations for each play. The online concordance allows users to instantly search for all occurences of any particular word or phrase in each play -- a useful means of examining motifs and themes. The extensive, searchable glossary is also helpful for looking up unfamiliar Elizabethan vocabulary, and an online forum allows users to share their thoughts on the Bard with other readers. According to the site, an e-text of the Sonnets is forthcoming.


Elementary GLOBE: Earth System Play  

NSDL National Science Digital Library

The class will brainstorm, write, create, and produce a play in which they represent how all the Earth systems are interconnected. This play can be based on the Elementary GLOBE book "All About Earth: Our World on Stage" or on other student-generated topics representing interconnections of the Earth systems. The purpose of the play is to serve as a performance assessment providing students with the opportunity to display what they have learned about the Earth as a system in a creative manner. Through this activity, students will demonstrate their knowledge of how the hydrosphere, atmosphere, geosphere and biosphere interact.



Game Experience May Vary: Understanding Play  

Microsoft Academic Search

Why do we call it game research and not play research? For the last decade of videogame studies, most of the attention has\\u000a been paid to games as formal entities. At first, games seem easier to understand: they generally have clear rules and goals.\\u000a They would be perfect machineries with formal mechanics if it was not for one factor: humans

Gonzalo Frasca



Analytical Chemistry Role Playing Experiments  

NSDL National Science Digital Library

This page features a number of laboratory experiments (available for download in PDF format) which allow students the opportunity to role play in groups to solve problems. Experiments involve titrations, gravimetry, atomic absorption, chromatography.

Walters, John P.



The Play Dough Evaluation Project  

NSDL National Science Digital Library

Fourth-grade students gain a deeper understanding of the meaning of "properties" by evaluating and comparing play doughs. Students also hone their observation and reporting skills and enrich their vocabulary, integrating language arts into this activity.

Heckscher, Mary




NSDL National Science Digital Library

This interactive Java applet helps students explore the relationship between area and multiplication. First, users are asked to input all factor pairs of a given number. Then, selecting each of those factor pairs, the user draws the respective rectangular array by clicking and dragging across a grid. Options include the use of the commutative property (e.g., user must enter both 2x4 and 4x2 for factors of 8 and represent them with different arrays), entering a number of the user's own choice, and an optional scoring feature allowing the user to keep track of the number correct.



The core promoter of human thioredoxin reductase 1: cloning, transcriptional activity, and Oct-1, Sp1, and Sp3 binding reveal a housekeeping-type promoter for the AU-rich element-regulated gene.  


The selenoprotein thioredoxin reductase 1 (TrxR1) carries many vital antioxidant and redox regulatory functions. Its mRNA levels are known to be post-transcriptionally modulated via AUUUA motifs (AU-rich elements (AREs)), but the promoter yet remains unknown. Here we have cloned and determined the sequence of a 0.8-kilobase pair human genomic fragment containing the proximal promoter for TrxR1, which has transcriptional activity in several different cell types. The core promoter (-115 to +167) had an increased GC content and lacked TATA or CCAAT boxes. It contained a POU motif binding the Oct-1 transcription factor and two sites binding Sp1 and Sp3, which were identified with electrophoretic mobility shift assays using crude nuclear extracts of A549 cells. The TrxR1 promoter fulfills the typical criteria of a housekeeping gene. To our knowledge this is the first housekeeping-type promoter characterized for a gene with post-transcriptional regulation via ARE motifs generally possessed by transiently expressed proto-oncogenes, nuclear transcription factors, or cytokines and influencing mRNA stability in response to diverse exogenous factors. Expression of TrxR1 as an ARE-regulated housekeeping gene agrees with a role for the enzyme to maintain a balance between intracellular signaling via reactive oxygen species and protection of cells from excessive oxidative damage. PMID:11375392

Rundlöf, A K; Carlsten, M; Arnér, E S



Transcriptional activation of p21(WAF1/CIP1) is mediated by increased DNA binding activity and increased interaction between p53 and Sp1 via phosphorylation during replicative senescence of human embryonic fibroblasts.  


Although p21(WAF1/CIP1) is known to be elevated during replicative senescence of human embryonic fibroblasts (HEFs), the mechanism for p21 up-regulation has not been elucidated clearly. In order to explore the mechanism, we analyzed expression of p21 mRNA and protein and luciferase activity of full-length p21 promoter. The result demonstrated that p21 up-regulation was accomplished largely at transcription level. The promoter assay using serially-deleted p21 promoter constructs revealed that p53 binding site was the most important site and Sp1 binding sites were necessary but not sufficient for transcriptional activation of p21. In addition, p53 protein was shown to interact with Sp1 protein. The interaction was increased in aged fibroblasts and was regulated by phosphorylation of p53 and Sp1. DNA binding activity of p53 was significantly elevated in aged fibroblasts but that of Sp1 was not. DNA binding activities of p53 and Sp1 were also regulated by phosphorylation. Phosphorylation of p53 at serine-15 and of Sp1 at serines appears to be involved. Taken together, the result demonstrated that p21 transcription during replicative senescence of HEFs is up-regulated by increase in DNA binding activity and interaction between p53 and Sp1 via phosphorylation. PMID:24445528

Kim, Hyun-Seok; Heo, Jee-In; Park, Seong-Hoon; Shin, Jong-Yeon; Kang, Hong-Jun; Kim, Min-Ju; Kim, Sung Chan; Kim, Jaebong; Park, Jae-Bong; Lee, Jae-Yong



Interferon-? Induced microRNA-129-5p Down-Regulates HPV-18 E6 and E7 Viral Gene Expression by Targeting SP1 in Cervical Cancer Cells  

PubMed Central

Infection by human papillomavirus (HPV) can cause cervical intraepithelial neoplasia (CIN) and cancer. Down-regulation of E6 and E7 expression may be responsible for the positive clinical outcomes observed with IFN treatment, but the molecular basis has not been well determined. As miRNAs play an important role in HPV induced cervical carcinogenesis, we hypothesize that IFN-? can regulate the expressions of specific miRNAs in cervical cancer cells, and that these miRNAs can mediate E6 and E7 expression, thus modulate their oncogenic potential. In this study, we found that miR-129-5p to be a candidate IFN-? inducible miRNA. MiR-129-5p levels gradually decrease with the development of cervical intraepithelial lesions. Manipulation of miR-129-5p expression in Hela cells modulates HPV-18 E6 and E7 viral gene expression. Exogenous miR-129-5p inhibits cell proliferation in Hela cells, promotes apoptosis and blocks cell cycle progression in Hela cells. SP1 is a direct target of miR-129-5p in Hela cells. This study is the first report of a cellular miRNA with anti-HPV activity and provides new insights into regulatory mechanisms between the HPV and the IFN system in host cells at the miRNA level.

Zhang, Jiarong; Li, Shuangdi; Yan, Qin; Chen, Xiaoyue; Yang, Yixia; Liu, Xuelian; Wan, Xiaoping



Guided Play: Where Curricular Goals Meet a Playful Pedagogy  

ERIC Educational Resources Information Center

Decades of research demonstrate that a strong curricular approach to preschool education is important for later developmental outcomes. Although these findings have often been used to support the implementation of educational programs based on direct instruction, we argue that "guided play" approaches can be equally effective at delivering content…

Weisberg, Deena Skolnick; Hirsh-Pasek, Kathy; Golinkoff, Roberta Michnick



Conserved POU-binding site linked to SP1-binding site within FZD5 promoter: Transcriptional mechanisms of FZD5 in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer.  


Canonical WNT signals are transduced through Frizzled (FZD) family receptor and LRP5/LRP6 co-receptor to upregulate FGF20, JAG1, DKK1, WISP1, CCND1 and MYC genes for cell-fate determination, while non-canonical WNT signals are transduced through FZD family receptor and ROR2/PTK7/RYK co-receptor to activate RHOA/RHOU/RAC/CDC42, JNK, PKC, NLK and NFAT signaling cascades for the regulation of tissue polarity, cell movement, and adhesion. We previously reported molecular cloning and characterization of human FZD5, which showed six amino-acid substitutions with human Hfz5. FZD5, functioning as WNT5A receptor, is the key molecule in the fields of oncology, regenerative medicine, cardiology, rheumatology, diabetology, and gastroenterology. Here, comparative integromics analyses on FZD5 orthologs were performed by using bioinformatics (Techint) and human intelligence (Humint). Chimpanzee FZD5 and cow Fzd5 genes were identified within NW_104292.1 and AC166656.2 genome sequences, respectively. FZD5 orthologs were seven-transmembrane proteins with extracellular Frizzled domain, leucine zipper motif around the 5th transmembrane domain, and cytoplasmic DVL- and PDZ-binding motifs. Ser523 and Ser529 around the DVL-binding motif of FZD5 orthologs were putative aPKC phosphorylation sites. POU5F1 (OCT4)-binding site linked to SP1-binding site within the 5'-promoter region of human FZD5 gene was evolutionarily conserved among mammalian FZD5 orthologs. POU5F1 was more related to POU2F and POU3F subfamily members. POU5F1 was preferentially expressed in undifferentiated human embryonic stem (ES) cells, pancreatic islet, and diffuse-type gastric cancer. POU2F1 (OCT1) was expressed in ES cells, fetal liver/spleen, adult colon, POU2F2 in ES cells, fetal liver/spleen, and POU2F3 in diffuse-type gastric cancer. Multiple SP1/KLF family members, other than KLF2 or KLF4, were expressed in undifferentiated human ES cells. Together, these facts indicate that POU5F1 and POU2F subfamily members play a pivotal role for the FZD5 expression in undifferentiated human ES cells, fetal liver/spleen, adult colon, pancreatic islet, and diffuse-type gastric cancer. PMID:17273778

Katoh, Yuriko; Katoh, Masaru



"Playing God" and germline intervention.  


The phrase "playing God" so popular with journalists takes on a serious meaning in the debate over germline genetic intervention. While guarding against the dangers of human pride implied in the phrase "playing God," special attention is given here to the Christian concept of the human being as created in the divine image, the imago dei. Human beings are dubbed "created co-creators." In this light ethical arguments proscribing germline intervention are examined and refuted, leaving the door open for creative responsibility on the part of the present generation for our future progeny. PMID:8568437

Peters, T



Teaching Technical Skills through Play.  

ERIC Educational Resources Information Center

The value of light-hearted play in teaching technical recreational sport skills is immense. Children as well as adults can learn more quickly and completely with a games-oriented approach. Often without realizing the hidden goal of excellent skiing or paddling, participants respond to intriguing tasks in a game, immerse themselves in good…

Gullion, Laurie


Playing Videogames: The Electronic Friend.  

ERIC Educational Resources Information Center

Concluded that the children in this study (ages 10-14) played video games in arcades for some of the same reasons they watched television: (1) escape; (2) a sense of personal involvement in the action; and (3) a source of or substitute for companionship. (PD)

Selnow, Gary W.



Strengthening Play through Father Involvement  

ERIC Educational Resources Information Center

In his essay exploring the latest research finding on the importance of men in the lives of young children, the author describes two ongoing empirical studies that are proving particularly instructive in understanding the significance of paternal contributions to improving young child outcomes. Both projects are encouraging direct paternal play

Pruett, Kyle



Teaching Shakespeare Through Play Production.  

ERIC Educational Resources Information Center

A performance-oriented approach to teaching William Shakespeare's literature has been found to be effective and enthusiastically received by college students. Ten years of teaching Shakespeare through full play production has shown that the rewards, eloquently expressed in the testimony of students, more than compensate for extra work required of…

Stodder, Joseph H.



Interpretive Reproduction in Children's Play  

ERIC Educational Resources Information Center

The author looks at children's play from the perspective of interpretive reproduction, emphasizing the way children create their own unique peer cultures, which he defines as a set of routines, artifacts, values, and concerns that children engage in with their playmates. The article focuses on two types of routines in the peer culture of preschool…

Corsaro, William A.



Learning, Play, and Your Newborn  


... newborn to learn and play: Put on soothing music, and hold your baby, gently swaying to the tune. Pick a soothing song or lullaby and softly sing it often to your baby. The familiarity of the sound and words will have a soothing effect, particularly during fussy times. Smile, stick out your ...


Playing Piano across the Curriculum.  

ERIC Educational Resources Information Center

Contends that the amount of piano study required of music education majors to pass the piano proficiency examination is insufficient. States that keyboarding across the curriculum will enable music education majors to become proficient in playing the piano. Offers suggestions for including the keyboard within other courses. (CMK)

Hamel, Barbara L.



Learning to Plan through Play.  

ERIC Educational Resources Information Center

Presents strategies to make planning-through-play activities a fun and integral part of the preschool developmental curriculum. Strategies are influenced by a Piagetian approach and an information processing approach that focuses on development of decision making. Specific suggestions are based on a model preschool program in Brookline,…

Casey, M. Beth; Lippman, Marjory



Fort Play Children Recreate Recess  

ERIC Educational Resources Information Center

Recess beckons well before it actually arrives. Its allure can be heard in children's lunchtime conversations as they discuss imaginary roles, plans, alliances and teams, with an obvious appetite for play and its unbounded possibility. For some children, recess provides the most important reasons to come to school. In team sports, games of chase…

Powell, Mark