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Distinct function of 2 chromatin remodeling complexes that share a common subunit, Williams syndrome transcription factor (WSTF)

Yoshimura, Kimihiro; Kitagawa, Hirochika; Fujiki, Ryoji; Tanabe, Masahiko; Takezawa, Shinichiro; Takada, Ichiro; Yamaoka, Ikuko; Yonezawa, Masayoshi; Kondo, Takeshi; Furutani, Yoshiyuki; Yagi, Hisato; Yoshinaga, Shin; Masuda, Takeyoshi; Fukuda, Toru; Yama
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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A number of nuclear complexes modify chromatin structure and operate as functional units. However, the in vivo role of each component within the complexes is not known. ATP-dependent chromatin remodeling complexes form several types of protein complexes, which reorganize chromatin structure cooperatively with histone modifiers. Williams syndrome transcription factor (WSTF) was biochemically identified as a major subunit, along with 2 distinct complexes: WINAC, a SWI/SNF-type complex, and WICH, an ISWI-type complex. Here, WSTF−/− mice were generated to investigate its function in chromatin remodeling in vivo. Loss of WSTF expression resulted in neonatal lethality, and all WSTF−/− neonates and ≈10% of WSTF+/− neonates suffered cardiovascular abnormalities resembling those found in autosomal-dominant Williams syndrome patients. Developmental analysis of WSTF−/− embryos revealed that Gja5 gene regulation is aberrant from E9.5, conceivably because of inappropriate chromatin reorganization around the promoter regions where essential cardiac transcription factors are recruited. In vitro analysis in WSTF−/− mouse embryonic fibroblast (MEF) cells also showed impaired transactivation functions of cardiac transcription activators on the Gja5 promoter...

Lack ofl-Iduronic Acid in Heparan Sulfate Affects Interaction with Growth Factors and Cell Signaling*

Jia, Juan; Maccarana, Marco; Zhang, Xiao; Bespalov, Maxim; Lindahl, Ulf; Li, Jin-Ping
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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HSEPI (glucuronyl C5-epimerase) catalyzes the conversion of d-glucuronic acid to l-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mice yielded a lethal phenotype with selective organ defects but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi−/− mouse. The HS produced by these cells is devoid of l-iduronic acid residues but shows up-regulated N- and 6-O-sulfation compared with wild type (WT) MEF HS. In medium fortified with 10% fetal calf serum, the Hsepi−/− MEFs proliferated and migrated similarly to WT cells. Under starvation conditions, both cell types showed attenuated proliferation and migration that could be restored by the addition of FGF2 to WT cells, whereas Hsepi−/− cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi−/− compared with WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed a strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor to Hsepi−/− but not to WT HS. glia-derived neurotropic factor has a key role in kidney development...

Use of aluminum films as substrates for enhanced fluorescence in the ultraviolet-blue spectral region

Chowdhury, Mustafa H.; Ray, Krishanu; Lakowicz, Joseph R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/02/2008 Português
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Thermal evaporation was used to deposit particulate aluminum films of varied thicknesses on quartz substrates. These substrates were characterized by scanning electron microscopy (SEM), which reveal that with an increase in aluminum thickness, the films progress from particulate towards smooth surfaces. Until now, metal-enhanced fluorescence (MEF) has primarily been observed in the visible-NIR wavelength region using silver or gold island films and roughened surfaces. We now report that fluorescence can also be enhanced in the ultraviolet-blue region of the spectrum using nano-structured aluminum films. We used two probes, one in the ultraviolet (a DNA base analogue 2-aminopurine: 2-AP) and another one in blue spectral region (a coumarin derivative: 7-HC) for the present study. We observed increased emission, decrease in fluorescence lifetime and increase in photostability of the dyes in a 10 nm spin-casted polyvinyl alcohol film on the Al nanostructured surfaces. We observe that the fluorescence enhancement factor depends on the thickness of the Al films because the size of the nanostructures formed varies with Al thickness. These studies indicate that Al nano-structured substrates can potentially find widespread use in MEF applications particularly in the UV – blue spectral regime. Finite-Difference Time-Domain (FDTD) calculations were performed that revealed enhanced near-fields induced around aluminum nanoparticles by a radiating fluorophore emitting at the emission wavelength of 2-AP. The effect of such enhanced fields on the fluorescence enhancement observed is also discussed.

Germline-Competent Mouse-Induced Pluripotent Stem Cell Lines Generated on Human Fibroblasts without Exogenous Leukemia Inhibitory Factor

Li, Chunliang; Yu, Hongyao; Ma, Yu; Shi, Guilai; Jiang, Jing; Gu, Junjie; Yang, Ying; Jin, Shibo; Wei, Zhe; Jiang, Hua; Li, Jinsong; Jin, Ying
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 21/08/2009 Português
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Induced pluripotent stem (iPS) cells have attracted enormous attention due to their vast potential in regenerative medicine, pharmaceutical screening and basic research. Most prior established iPS cell lines were derived and maintained on mouse embryonic fibroblast (MEF) cells supplemented with exogenous leukemia inhibitory factor (LIF). Drawbacks of MEF cells impede optimization as well as dissection of reprogramming events and limit the usage of iPS cell derivatives in therapeutic applications. In this study, we develop a reproducible protocol for efficient reprogramming mouse neural progenitor cells (NPCs) on human foreskin fibroblast (HFF) cells via retroviral transfer of human transcriptional factors OCT4/SOX2/KLF4/C-MYC. Two independent iPS cell lines are derived without exogenous LIF. They display typical undifferentiated morphology and express pluripotency markers Oct4 and Sox2. Transgenes are inactivated and the endogenous Oct4 promoter is completely demethylated in the established iPS cell lines, indicating a fully reprogrammed state. Moreover, the iPS cells can spontaneously differentiate or be induced into various cell types of three embryonic germ layers in vitro and in vivo when they are injected into immunodeficient mice for teratoma formation. Importantly...

Aluminum Nanoparticles as Substrates for Metal-Enhanced Fluorescence in the Ultraviolet for the Label-Free Detection of Biomolecules

Chowdhury, Mustafa H.; Ray, Krishanu; Gray, Stephen K.; Pond, James; Lakowicz, Joseph R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/02/2009 Português
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We use finite-difference time-domain calculations to show that aluminum nanoparticles are efficient substrates for metal-enhanced fluorescence (MEF) in the ultraviolet (UV) for the label-free detection of biomolecules. The radiated power enhancement of the fluorophores in proximity to aluminum nanoparticles is strongly dependent on the nanoparticle size, fluorophore-nanoparticle spacing, and fluorophore orientation. Additionally, the enhancement is dramatically increased when the fluorophore is between two aluminum nanoparticles of a dimer. Finally, we present experimental evidence that functionalized forms of amino acids tryptophan and tyrosine exhibit MEF when spin-coated onto aluminum nanostructures.

A Nonhomologous End-joining Pathway Is Required for Protein Phosphatase 2A Promotion of DNA Double-Strand Break Repair12

Wang, Qinhong; Gao, Fengqin; Wang, Ton; Flagg, Tammy; Deng, Xingming
Fonte: Neoplasia Press Inc. Publicador: Neoplasia Press Inc.
Tipo: Artigo de Revista Científica
Publicado em /10/2009 Português
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Protein phosphatase 2A (PP2A) functions as a potent tumor suppressor, but its mechanism(s) remains enigmatic. Specific disruption of PP2A by either expression of SV40 small tumor antigen or depletion of endogenous PP2A/C by RNA interference inhibits Ku DNA binding and DNA-PK activities, which results in suppression of DNA double-strand break (DSB) repair and DNA end-joining in association with increased genetic instability (i.e., chromosomal and chromatid breaks). Overexpression of the PP2A catalytic subunit (PP2A/C) enhances Ku and DNA-PK activities with accelerated DSB repair. Camptothecin-induced DSBs promote PP2A to associate with Ku 70 and Ku 86. PP2A directly dephosphorylates Ku as well as the DNA-PK catalytic subunit (DNA-PKcs) in vitro and in vivo, which enhances the formation of a functional Ku/DNA-PKcs complex. Intriguingly, PP2A promotes DSB repair in wild type mouse embryonic fibroblast (MEF) cells but has no such effect in Ku-deficient MEF cells, suggesting that the Ku 70/86 heterodimer is required for PP2A promotion of DSB repair. Thus, PP2A promotion of DSB repair may occur in a novel mechanism by activating the nonhomologous end-joining pathway through direct dephosphorylation of Ku and DNA-PKcs, which may contribute to maintenance of genetic stability.

Reduction of Lysyl Hydroxylase 3 Causes Deleterious Changes in the Deposition and Organization of Extracellular Matrix*

Risteli, Maija; Ruotsalainen, Heli; Salo, Antti M.; Sormunen, Raija; Sipilä, Laura; Baker, Naomi L.; Lamandé, Shireen R.; Vimpari-Kauppinen, Leena; Myllylä, Raili
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Lysyl hydroxylase 3 (LH3) is a multifunctional enzyme possessing lysyl hydroxylase, collagen galactosyltransferase, and glucosyltransferase (GGT) activities. We report here an important role for LH3 in the organization of the extracellular matrix (ECM) and cytoskeleton. Deposition of ECM was affected in heterozygous LH3 knock-out mouse embryonic fibroblasts (MEF+/−) and in skin fibroblasts collected from a member of a Finnish epidermolysis bullosa simplex (EBS) family known to be deficient in GGT activity. We show the GGT deficiency to be due to a transcriptional defect in one LH3 allele. The ECM abnormalities also lead to defects in the arrangement of the cytoskeleton in both cell lines. Ultrastructural abnormalities were observed in the skin of heterozygous LH3 knock-out mice indicating that even a moderate decrease in LH3 has deleterious consequences in vivo. The LH3 null allele in the EBS family member and the resulting abnormalities in the organization of the extracellular matrix, similar to those found in MEF+/−, may explain the correlation between the severity of the phenotype and the decrease in GGT activity reported in this family.

U19/Eaf2 Binds to and Stabilizes von Hippel-Lindau Protein

Xiao, Wuhan; Ai, Junkui; Habermacher, Geoffrey; Volpert, Olga; Yang, Ximing; Zhang, Ai-yuan; Hahn, Junghyun; Cai, Xiaoyan; Wang, Zhou
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Studies have firmly established a key regulatory role for the tumor suppressor pVHL in the regulation of the vascular system and normal spermatogenesis. Here, we report that knockout of the newly identified tumor suppressor U19/Eaf2 also caused vascular system abnormalities and aspermatogenesis, suggesting a potential link between U19/Eaf2 and pVHL. Coimmunoprecipitation and in vitro binding assays showed an association between U19/Eaf2 and pVHL, whereas deletion mutagenesis revealed the requirement of the NH2 terminus of U19/Eaf2 and both the α and β domains of pVHL for this binding. U19/Eaf2 stabilizes pVHL, as shown by protein stability and pulse-chase studies. Testes and mouse embryonic fibroblasts (MEF) derived from U19/Eaf2 knockout mice expressed reduced levels of pVHL, indicating that full in vivo expression of pVHL indeed requires U19/Eaf2. As expected, U19/Eaf2 knockout MEF cells exhibited an increased level and activity of hypoxia-inducible factor 1α (HIF1α), a protein typically regulated via a pVHL-mediated degradation pathway. Furthermore, angiogenesis in a Matrigel plug assay was significantly increased in U19/Eaf2 knockout mice. The above observations argue that U19/Eaf2 can modulate HIF1α and angiogenesis, possibly via direct binding and stabilization of pVHL.

The lectin concanavalin-A signals MT1-MMP catalytic independent induction of COX-2 through an IKKγ/NF-κB-dependent pathway

Sina, Asmaa; Proulx-Bonneau, Sébastien; Roy, Alain; Poliquin, Laurent; Cao, Jian; Annabi, Borhane
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
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The lectin from Canavalia ensiformis (Concanavalin-A, ConA), one of the most abundant lectins known, enables one to mimic biological lectin/carbohydrate interactions that regulate extracellular matrix protein recognition. As such, ConA is known to induce membrane type-1 matrix metalloproteinase (MT1-MMP) which expression is increased in brain cancer. Given that MT1-MMP correlated to high expression of cyclooxygenase (COX)-2 in gliomas with increasing histological grade, we specifically assessed the early proinflammatory cellular signaling processes triggered by ConA in the regulation of COX-2. We found that treatment with ConA or direct overexpression of a recombinant MT1-MMP resulted in the induction of COX-2 expression. This increase in COX-2 was correlated with a concomitant decrease in phosphorylated AKT suggestive of cell death induction, and was independent of MT1-MMP’s catalytic function. ConA- and MT1-MMP-mediated intracellular signaling of COX-2 was also confirmed in wild-type and in Nuclear Factor-kappaB (NF-κB) p65−/− mutant mouse embryonic fibroblasts (MEF), but was abrogated in NF-κB1 (p50)−/− and in I kappaB kinase (IKK) γ−/− mutant MEF cells. Collectively, our results highlight an IKK/NF-κB-dependent pathway linking MT1-MMP-mediated intracellular signaling to the induction of COX-2. That signaling pathway could account for the inflammatory balance responsible for the therapy resistance phenotype of glioblastoma cells...

Wild-type and Hupki (Human p53 Knock-in) Murine Embryonic Fibroblasts: p53/ARF PATHWAY DISRUPTION IN SPONTANEOUS ESCAPE FROM SENESCENCE*

Whibley, Catherine; Odell, Adam F.; Nedelko, Tatiana; Balaburski, Gregor; Murphy, Maureen; Liu, Zhipei; Stevens, Louisa; Walker, John H.; Routledge, Michael; Hollstein, Monica
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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Research on cell senescence and immortalization of murine embryonic fibroblasts (MEFs) has revealed important clues about genetic control of senescence in humans. To investigate senescence and genetic alterations in the p53 pathway that lead to senescence bypass in culture, we compared the behavior of MEFs from wild-type mice with MEFs from Hupki mice, which harbor a humanized p53 gene. We found that humanizing the p53 gene in mice preserved major features of the MEF senescence/immortalization process. In both genotypes, a significant proportion of spontaneously arising cell lines had sustained either a p53 point mutation or p19/ARF biallelic deletion. The p53 mutations selected for during Hupki MEF immortalization have been found in human tumors and are classified in the yeast transactivation assay as transcriptionally defunct, suggesting that disabling this component of p53 activity is crucial in senescence bypass. Surprisingly, in spontaneously immortalized cell lines from both wild-type and Hupki MEFs, the predominant type of p53 mutation was a G to C transversion, rather than the G to T substitutions expected from the raised oxygen levels characteristic of standard culture conditions. Over half of the cell lines did not reveal evidence of p53 mutation or loss of p19/ARF and retained a robust wild-type p53 response to DNA damage...

Antimicrobial susceptibility and serotype distribution of Streptococcus pneumoniae isolated from patients with community-acquired pneumonia and molecular analysis of multidrug-resistant serotype 19F and 23F strains in Japan

QIN, L.; WATANABE, H.; YOSHIMINE, H.; GUIO, H.; WATANABE, K.; KAWAKAMI, K.; IWAGAKI, A.; NAGAI, H.; GOTO, H.; KURIYAMA, T.; FUKUCHI, Y.; MATSUSHIMA, T.; KUDOH, S.; SHIMADA, K.; MATSUMOTO, K.; NAGATAKE, T.; MIZOTA, T.; OISHI, K.
Fonte: Cambridge University Press Publicador: Cambridge University Press
Tipo: Artigo de Revista Científica
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A nationwide study was undertaken to determine the susceptibility to penicillin and serotypes of Streptococcus pneumoniae in Japan. S. pneumoniae was isolated from 114 adult patients with community-acquired pneumonia over 22 months at 20 hospitals and medical centres in different regions in Japan. All but five isolates were from sputum. Forty-eight isolates (42·1%) were susceptible, 40 (35·1%) showed intermediate resistance (MIC, 0·12–1·0 μg/ml) and 26 (22·8%) were resistant (MIC, ⩾2·0 μg/ml) to penicillin G. All isolates were susceptible to ceftriaxone (breakpoint 1 μg/ml), imipenem (4 μg/ml) and vancomycin (4 μg/ml). Most were resistant to erythromycin, clarithromycin and azithromycin; only two were resistant to levofloxacin. Differences were found in the distribution of serotypes among isolates showing susceptibility to penicillin (predominant types 3, 6B, and 19F), intermediate resistance (6B, 14, 19F, and 23F) and full resistance (19F and 23F). PFGE typing showed that 14 of the 25 strains of serotype 19F had a single DNA profile, pattern A, a pattern closely similar to that of the Taiwan multidrug-resistant 19F clone. Twelve pattern A strains were not susceptible to penicillin but carried the macrolide resistance gene mef(A). The DNA profiles of the 15 strains of 23F were also heterogeneous but six were highly similar (pattern b) yet distinct from the Spanish multidrug-resistant 23F clone although possibly related to the Taiwan multidrug-resistant 23F clone. The pattern b strains were not susceptible to penicillin and also harboured either mef(A) or erm(B). Our results indicate that multidrug-resistant pneumococci are spreading rapidly in Japan. Efforts to prevent the spread of the pandemic multidrug-resistant serotypes should be intensified.

Human haem oxygenase-1 induction by nitro-linoleic acid is mediated by cAMP, AP-1 and E-box response element interactions

Wright, Marcienne M.; Kim, Junghyun; Hock, Thomas D.; Leitinger, Norbert; Freeman, Bruce A.; Agarwal, Anupam
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 13/08/2009 Português
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Nitro-fatty acid products of oxidative inflammatory reactions mediate anti-inflammatory cell signalling responses. LNO2 (nitro-linoleic acid) induces expression of HO-1 (haem oxygenase-1), an enzyme that catabolizes haem into products exhibiting potent anti-inflammatory properties. In the present manuscript, the molecular mechanisms underlying HO-1 induction by LNO2 were examined in HAEC (human aortic endothelial cells), HEK-293 (human embryonic kidney 293) cells, and in transcription factor-deficient MEF (mouse embryonic fibroblasts). LNO2 induced HO-1 expression in Nrf2 [NF-E2 (nuclear factor-erythroid 2)-related factor 2]-deficient MEF and in HEK-293 cells transfected with Nrf2-specific shRNA (small-hairpin RNA), supporting the fact that LNO2-mediated HO-1 induction can be regulated by Nrf2-independent mechanisms. LNO2 activated expression of a − 4.5 kb human HO-1 promoter construct, whereas a − 4.0 kb construct with deletion of 500 bp from the 5′ region was unresponsive. Site-directed mutagenesis of a CRE (cAMP-response element) or of a downstream NF-E2/AP-1 (activating protein-1) element, individually, within this 500 bp region modestly reduced activation of the HO-1 promoter by LNO2. Mutations of both the CRE and the NF-E2/AP-1 site also attenuated LNO2-mediated HO-1 promoter expression...

Metal Enhanced Intrinsic Fluorescence of Proteins and Label-Free Bioassays

Ray, Krishanu; Szmacinski, Henryk; Chowdhury, Mustafa H.; Lakowicz, Joseph R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/2010 Português
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Most of the applications of fluorescence require the use of labeled drugs and labeled biomolecules. Due to the need of labeling biomolecules with extrinsic fluorophores, there is a rapidly growing interest in methods which provide label-free detection (LFD). Proteins are highly fluorescent, which is due primarily to tryptophan residues. However, since most proteins contain tryptophan, this emission is not specific for proteins of interest in a biological sample. This is one of the reasons of not utilizing intrinsic tryptophan emission from proteins to detect specific proteins. Here, we present the intrinsic fluorescence for several proteins bound to the silver or aluminum metal nanostructured surfaces. We demonstrate the metal enhanced fluorescence (MEF) of proteins with different numbers of tryptophan residues. Large increases in fluorescence intensity and decreases in lifetime provide the means of direct detection of bound protein without separation from the unbound. We present specific detection of individual types of proteins and measure the binding kinetics of proteins such as IgG and streptavidin. Additionally, specific detection of IgG and streptavidin has been accomplished in the presence of large concentrations of other proteins in sample solutions. These results will allow design of surface-based assays with biorecognitive layer that specifically bind the protein of interest and thus enhance its intrinsic fluorescence. The present study demonstrates the occurrence of MEF in the UV region and thus opens new possibilities to study tryptophan-containing proteins without labeling with longer wavelength fluorophores and provides an approach to label-free detection of biomolecules.

Enhanced Nrf2-Dependent Induction of Glutathione in Mouse Embryonic Fibroblasts by Isoselenocyanate Analog of Sulforaphane

Emmert, Sans W.; Desai, Dhimant; Amin, Shantu; Richie, John P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Epidemiological and laboratory studies have highlighted the potent chemopreventive effectiveness of both dietary selenium and cruciferous vegetables, particularly broccoli. Sulforaphane (SFN), an isothiocyanate, was identified as the major metabolite of broccoli responsible for its anti-cancer properties. An important mechanism for SFN chemoprevention is through the enhancement of glutathione (GSH), the most abundant antioxidant in animals and an important target in chemoprevention. Enhancement of GSH biosynthetic enzymes including the rate-limiting glutamate cysteine ligase (GCL), as well as other Phase II detoxification enzymes results from SFN-mediated induction of the nuclear factor-erythroid 2-related factor 2 (Nrf2)/antioxidant response elements (ARE) signaling pathway. While isothiocyanate compounds such as SFN are among the most potent Nrf2 inducers known, we hypothesized that substitution of sulfur with selenium in the isothiocyanate functional group of SFN would result in an isoselenocyanate compound (SFN-isoSe) with enhanced Nrf2 induction capability. Here we report that SFN-isoSe activated an ARE-luciferase reporter in HepG2 cells more potently than SFN. It was also found that SFN-isoSe induced GCL and GSH in MEF cells in an Nrf2-dependent manner. Finally...

HIF-1α Promotes A Hypoxia-Independent Cell Migration

Li, Liyuan; Madu, Chikezie O.; Lu, Andrew; Lu, Yi
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/01/2010 Português
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Hypoxia-inducible factor-1α (HIF-1α) is known as a transactivator for VEGF gene promoter. It can be induced by hypoxia. However, no study has been done so far to dissect HIF-1α-mediated effects from hypoxia or VEGF-mediated effects. By using a HIF-1α knockout (HIF-1α KO) cell system in mouse embryonic fibroblast (MEF) cells, this study analyzes cell migration and HIF-1α, hypoxia and VEGF activation. A hypoxia-mediated HIF-1α induction and VEGF transactivation were observed: both HIF-1α WT lines had significantly increased VEGF transactivation, as an indicator for HIF-1α induction, in hypoxia compared to normoxia; in contrast, HIF-1α KO line had no increased VEGF transactivation under hypoxia. HIF-1α promotes cell migration: HIF-1α-KO cells had a significantly reduced migration compared to that of the HIF-1α WT cells under both normoxia and hypoxia. The significantly reduced cell migration in HIF-1α KO cells can be partially rescued by the restoration of WT HIF-1α expression mediated by adenoviral-mediated gene transfer. Interestingly, hypoxia has no effect on cell migration: the cells had a similar cell migration rate under hypoxic and normoxic conditions for both HIF-1α WT and HIF-1α KO lines, respectively. Collectively...

Transcriptional regulation of ferritin and antioxidant genes by HIPK2 under genotoxic stress

Hailemariam, Kiros; Iwasaki, Kenta; Huang, Bo-Wen; Sakamoto, Kensuke; Tsuji, Yoshiaki
Fonte: Company of Biologists Publicador: Company of Biologists
Tipo: Artigo de Revista Científica
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ATF1 (activating transcription factor 1), a stimulus-induced CREB family transcription factor, plays important roles in cell survival and proliferation. Phosphorylation of ATF1 at Ser63 by PKA (cAMP-dependent protein kinase) and related kinases was the only known post-translational regulatory mechanism of ATF1. Here, we found that HIPK2 (homeodomain-interacting protein kinase 2), a DNA-damage-responsive nuclear kinase, is a new ATF1 kinase that phosphorylates Ser198 but not Ser63. ATF1 phosphorylation by HIPK2 activated ATF1 transcription function in the GAL4-reporter system. ATF1 is a transcriptional repressor of ferritin H, the major intracellular iron storage gene, through an ARE (antioxidant-responsive element). HIPK2 overrode the ATF1-mediated ARE repression in a kinase-activity-dependent manner in HepG2 cells. Furthermore, DNA-damage-inducing agents doxorubicin, etoposide and sodium arsenite induced ferritin H mRNA expression in HIPK2+/+ MEF cells, whereas it was significantly impaired in HIPK2−/− MEF cells. Induction of other ARE-regulated detoxification genes such as NQO1 (NADPH quinone oxidoreductase 1), GST (glutathione S-transferase) and HO1 (heme oxygenase 1) by genotoxic stress was also decreased in HIPK2-deficient cells. Taken together...

RNA editing enzyme adenosine deaminase is a restriction factor for controlling measles virus replication that also is required for embryogenesis

Ward, Simone V.; George, Cyril X.; Welch, Megan J.; Liou, Li-Ying; Hahm, Bumsuk; Lewicki, Hanna; de la Torre, Juan C.; Samuel, Charles E.; Oldstone, Michael B.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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Measles virus (MV), a member of the family Paramyxoviridae and an exclusively human pathogen, is among the most infectious viruses. A progressive fatal neurodegenerative complication, subacute sclerosing panencephalitis (SSPE), occurs during persistent MV infection of the CNS and is associated with biased hypermutations of the viral genome. The observed hypermutations of A-to-G are consistent with conversions catalyzed by the adenosine deaminase acting on RNA (ADAR1). To evaluate the role of ADAR1 in MV infection, we selectively disrupted expression of the IFN-inducible p150 ADAR1 isoform and found it caused embryonic lethality at embryo day (E) 11–E12. We therefore generated p150-deficient and WT mouse embryo fibroblast (MEF) cells stably expressing the MV receptor signaling lymphocyte activation molecule (SLAM or CD150). The p150−/− but not WT MEF cells displayed extensive syncytium formation and cytopathic effect (CPE) following infection with MV, consistent with an anti-MV role of the p150 isoform of ADAR1. MV titers were 3 to 4 log higher in p150−/− cells compared with WT cells at 21 h postinfection, and restoration of ADAR1 in p150−/− cells prevented MV cytopathology. In contrast to infection with MV, p150 disruption had no effect on vesicular stomatitis virus...

Differential utilization of decapping enzymes in mammalian mRNA decay pathways

Li, You; Song, Mangen; Kiledjian, Megerditch
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /03/2011 Português
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mRNA decapping is a crucial step in the regulation of mRNA stability and gene expression. Dcp2 is an mRNA decapping enzyme that has been widely studied. We recently reported the presence of a second mammalian cytoplasmic decapping enzyme, Nudt16. Here we address the differential utilization of the two decapping enzymes in specified mRNA decay processes. Using mouse embryonic fibroblast (MEF) cell lines derived from a hypomorphic knockout of the Dcp2 gene with undetectable levels of Dcp2 or MEF cell lines harboring a Nudt16-directed shRNA to generate reduced levels of Nudt16, we demonstrate the distinct roles for Dcp2 and Nudt16 in nonsense-mediated mRNA decay (NMD), decay of ARE-containing mRNA and miRNA-mediated silencing. Our results indicated that NMD preferentially utilizes Dcp2 rather than Nudt16; Dcp2 and Nudt16 are redundant in miRNA-mediated silencing; and Dcp2 and Nudt16 are differentially utilized for ARE-mRNA decay. These data demonstrate that the two distinct decapping enzymes can uniquely function in specific mRNA decay processes in mammalian cells.

Mouse Embryonic Stem Cells Inhibit Murine Cytomegalovirus Infection through a Multi-Step Process

Kawasaki, Hideya; Kosugi, Isao; Arai, Yoshifumi; Iwashita, Toshihide; Tsutsui, Yoshihiro
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 02/03/2011 Português
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In humans, cytomegalovirus (CMV) is the most significant infectious cause of intrauterine infections that cause congenital anomalies of the central nervous system. Currently, it is not known how this process is affected by the timing of infection and the susceptibility of early-gestational-period cells. Embryonic stem (ES) cells are more resistant to CMV than most other cell types, although the mechanism responsible for this resistance is not well understood. Using a plaque assay and evaluation of immediate-early 1 mRNA and protein expression, we found that mouse ES cells were resistant to murine CMV (MCMV) at the point of transcription. In ES cells infected with MCMV, treatment with forskolin and trichostatin A did not confer full permissiveness to MCMV. In ES cultures infected with elongation factor-1α (EF-1α) promoter-green fluorescent protein (GFP) recombinant MCMV at a multiplicity of infection of 10, less than 5% of cells were GFP-positive, despite the fact that ES cells have relatively high EF-1α promoter activity. Quantitative PCR analysis of the MCMV genome showed that ES cells allow approximately 20-fold less MCMV DNA to enter the nucleus than mouse embryonic fibroblasts (MEFs) do, and that this inhibition occurs in a multi-step manner. In situ hybridization revealed that ES cell nuclei have significantly less MCMV DNA than MEF nuclei. This appears to be facilitated by the fact that ES cells express less heparan sulfate...

Differential requirements for the Ets transcription factor Elf-1 in the development of NKT cells and NK cells

Choi, Hak-Jong; Geng, Yanbiao; Cho, Hoonsik; Li, Sha; Giri, Pramod Kumar; Felio, Kyrie; Wang, Chyung-Ru
Fonte: American Society of Hematology Publicador: American Society of Hematology
Tipo: Artigo de Revista Científica
Publicado em 10/02/2011 Português
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E26 Transformation specific (Ets) family transcription factors control the expression of a large number of genes regulating hematopoietic cell development and function. Two such transcription factors, Ets-1 and myeloid Elf-1–like factor (MEF), have been shown to play critical roles in both natural killer (NK)– and NKT-cell development, but not in the development of conventional T cells. In this study, we address the role of E74-like factor 1 (Elf-1), another Ets family transcription factor that is closely related to MEF but divergent from Ets-1, in NK- and NKT-cell development using Elf-1–deficient (Elf-1−/−) mice. Whereas the proportion of NK cells in Elf-1−/− mice was normal, the proportion of NKT cells was significantly reduced in the thymus and periphery of Elf-1−/− mice compared with wild-type (WT) mice. Although Ets-1–deficient mice lack NKT cells altogether, Elf-1−/− mice exhibited only a partial block in NKT-cell development caused by a cell-intrinsic defect in the selection, survival, and maturation of NKT cells. In addition, residual NKT cells found in Elf-1−/− mice produced less cytokine upon antigen stimulation compared with WT NKT cells. Our data demonstrate that Elf-1 plays an important and nonredundant role in the development and function of NKT cells...