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1.
Biochem Biophys Res Commun ; 662: 18-25, 2023 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-37094429

RESUMO

The number of patients with end-stage renal failure is increasing annually worldwide and the problem is compounded by a shortage of renal transplantation donors. In our previous research, we have shown that transplantation of renal progenitor cells into the nephrogenic region of heterologous fetuses can induce the development of nephrons. We have also developed transgenic mice in which specific renal progenitor cells can be removed by drugs. By combining these two technologies, we have succeeded in generating human-mouse chimeric kidneys in fetal mice. We hope to apply these technologies to regenerative medicine. The quality of nephron progenitor cells (NPCs) derived from human pluripotent stem cells is important for the generation of chimeric kidneys, but there is currently no simple evaluation system for the chimerogenic potential of human NPCs. In this study, we focused on the fact that the re-aggregation of mouse renal progenitor cells can be used for nephron formation, even when merged into single cells. First, we examined the conditions under which nephron formation is likely to occur in mice during re-aggregation. Next, to improve the differentiation potential of human NPCs derived from pluripotent stem cells, NPCs were sorted using Integrin subunit alpha 8 (ITGA8). Finally, we demonstrated chimera formation between different species by mixing mouse cells with purified, selectively-induced human NPCs under optimum conditions. We observed these chimeric organoids at different time points to learn about these human-mouse chimeric structures at various stages of renal development. We found that the rate of chimera formation was affected by the purity of the human NPCs and the cell ratios used. We demonstrated that chimeric nephrons can be generated using a simple model, even between distant species. We believe that this admixture of human and mouse renal progenitor cells is a promising technology with potential application for the evaluation of the chimera formation abilities of NPCs.


Assuntos
Rim , Néfrons , Humanos , Camundongos , Animais , Células-Tronco Embrionárias , Diferenciação Celular , Camundongos Transgênicos , Organoides
2.
Stem Cell Res ; 39: 101514, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31376722

RESUMO

Functional decline and loss of the retinal pigment epithelium (RPE) cause retinal diseases. Clinical studies using human embryonic stem cell (hESC)- or induced pluripotent stem cell (hiPSC)-derived RPE cells have shown the safety and potential efficacy of hESC/iPSC-RPE cell transplantation. However, the production of RPE cells remains somewhat problematic. hESCs/iPSCs co-cultured with mouse feeder cells carry the risk of xeno-transmitted infections and immune reactions. Moreover, increasing the rate of cell division to ensure the quantity and purity of cells with low differentiation efficiency elevates the risk of gene mutations and chromosomal abnormalities. Here, we show that the transient inhibition of the FGF/MAPK signaling pathway during the hiPSC maintenance period markedly promotes RPE differentiation efficiency under feeder-free culture conditions. Blockage of FGF/MAPK signal induces neural differentiation and generates RPE cells without subsequent inhibition of Wnt and Nodal signals, which is known to be effective for retinal specification. We also found that additional inhibition of the PKC or BMP signaling pathway together with FGF/MAPK signal inhibition further elevates RPE differentiation efficiency. Our study will be helpful for producing clinical-grade RPE cells and will facilitate the development of therapies using hESC/hiPSC-RPE cells.


Assuntos
Fatores de Crescimento de Fibroblastos/metabolismo , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Epitélio Pigmentado da Retina/citologia , Diferenciação Celular/fisiologia , Linhagem Celular , Terapia Baseada em Transplante de Células e Tecidos , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo
3.
Cell Rep ; 10(8): 1310-23, 2015 Mar 03.
Artigo em Inglês | MEDLINE | ID: mdl-25732822

RESUMO

The 5S ribonucleoprotein particle (RNP) complex, consisting of RPL11, RPL5, and 5S rRNA, is implicated in p53 regulation under ribotoxic stress. Here, we show that the 5S RNP contributes to p53 activation and promotes cellular senescence in response to oncogenic or replicative stress. Oncogenic stress accelerates rRNA transcription and replicative stress delays rRNA processing, resulting in RPL11 and RPL5 accumulation in the ribosome-free fraction, where they bind MDM2. Experimental upregulation of rRNA transcription or downregulation of rRNA processing, mimicking the nucleolus under oncogenic or replicative stress, respectively, also induces RPL11-mediated p53 activation and cellular senescence. We demonstrate that exogenous expression of certain rRNA-processing factors rescues the processing defect, attenuates p53 accumulation, and increases replicative lifespan. To summarize, the nucleolar-5S RNP-p53 pathway functions as a senescence inducer in response to oncogenic and replicative stresses.


Assuntos
Senescência Celular , Ribossomos/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Nucléolo Celular/metabolismo , Células Cultivadas , Humanos , Células MCF-7 , Camundongos , Proteínas Nucleares/antagonistas & inibidores , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-mdm2/genética , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Interferência de RNA , RNA Ribossômico 5S/metabolismo , RNA Interferente Pequeno/metabolismo , Proteínas de Ligação a RNA , Proteínas Ribossômicas/antagonistas & inibidores , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Ativação Transcricional , Regulação para Cima
4.
Sci Rep ; 5: 8903, 2015 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-25754892

RESUMO

Various cellular stresses activate autophagy, which is involved in lysosomal degradation of cytoplasmic materials for maintaining nutrient homeostasis and eliminating harmful components. Here, we show that RNA polymerase I (Pol I) transcription inhibition induces nucleolar disruption and autophagy. Treatment with autophagy inhibitors or siRNA specific for autophagy-related (ATG) proteins inhibited autophagy but not nucleolar disruption induced by Pol I transcription inhibition, which suggested that nucleolar disruption was upstream of autophagy. Furthermore, treatment with siRNA specific for nucleolar protein nucleophosmin (NPM) inhibited this type of autophagy. This showed that NPM was involved in autophagy when the nucleolus was disrupted by Pol I inhibition. In contrast, NPM was not required for canonical autophagy induced by nutrient starvation, as it was not accompanied by nucleolar disruption. Thus, our results revealed that, in addition to canonical autophagy, there may be NPM-dependent autophagy associated with nucleolar disruption.


Assuntos
Autofagia/genética , Proteínas Nucleares/genética , RNA Polimerase I/genética , Transcrição Gênica , Nucléolo Celular/genética , Humanos , Células MCF-7 , Proteínas Nucleares/antagonistas & inibidores , Região Organizadora do Nucléolo/genética , Nucleofosmina , RNA Polimerase I/antagonistas & inibidores , RNA Interferente Pequeno
5.
Dev Biol ; 392(2): 182-92, 2014 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-24929238

RESUMO

Nanog, a core pluripotency factor, is required for stabilizing pluripotency of inner cell mass (ICM) and embryonic stem cells (ESCs), and survival of primordial germ cells in mice. Here, we have addressed function and regulation of Nanog in epiblasts of postimplantation mouse embryos by conditional knockdown (KD), chromatin immunoprecipitation (ChIP) using in vivo epiblasts, and protein interaction with the Nanog promoter in vitro. Differentiation of Nanog-KD epiblasts demonstrated requirement for Nanog in stabilization of pluripotency. Nanog expression in epiblast is directly regulated by Nodal/Smad2 pathway in a visceral endoderm-dependent manner. Notably, Nanog promoters switch from Oct4/Esrrb in ICM/ESCs to Oct4/Smad2 in epiblasts. Smad2 directly associates with Oct4 to form Nanog promoting protein complex. Collectively, these data demonstrate that Nanog plays a key role in stabilizing Epiblast pluripotency mediated by Nodal/Smad2 signaling, which is involved in Nanog promoter switching in early developing embryos.


Assuntos
Camadas Germinativas/embriologia , Proteínas de Homeodomínio/metabolismo , Modelos Biológicos , Células-Tronco Pluripotentes/fisiologia , Animais , Linhagem Celular , Imunoprecipitação da Cromatina , Técnicas de Silenciamento de Genes , Camadas Germinativas/metabolismo , Proteínas de Homeodomínio/genética , Imuno-Histoquímica , Luciferases , Camundongos , Proteína Homeobox Nanog , Proteína Nodal/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Células-Tronco Pluripotentes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína Smad2/genética , Proteína Smad2/metabolismo
6.
PLoS One ; 9(5): e98586, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24879416

RESUMO

Responding to various stimuli is indispensable for the maintenance of homeostasis. The downregulation of ribosomal RNA (rRNA) transcription is one of the mechanisms involved in the response to stimuli by various cellular processes, such as cell cycle arrest and apoptosis. Cell differentiation is caused by intra- and extracellular stimuli and is associated with the downregulation of rRNA transcription as well as reduced cell growth. The downregulation of rRNA transcription during differentiation is considered to contribute to reduced cell growth. However, the downregulation of rRNA transcription can induce various cellular processes; therefore, it may positively regulate cell differentiation. To test this possibility, we specifically downregulated rRNA transcription using actinomycin D or a siRNA for Pol I-specific transcription factor IA (TIF-IA) in HL-60 and THP-1 cells, both of which have differentiation potential. The inhibition of rRNA transcription induced cell differentiation in both cell lines, which was demonstrated by the expression of the common differentiation marker CD11b. Furthermore, TIF-IA knockdown in an ex vivo culture of mouse hematopoietic stem cells increased the percentage of myeloid cells and reduced the percentage of immature cells. We also evaluated whether differentiation was induced via the inhibition of cell cycle progression because rRNA transcription is tightly coupled to cell growth. We found that cell cycle arrest without affecting rRNA transcription did not induce differentiation. To the best of our knowledge, our results demonstrate the first time that the downregulation of rRNA levels could be a trigger for the induction of differentiation in mammalian cells. Furthermore, this phenomenon was not simply a reflection of cell cycle arrest. Our results provide a novel insight into the relationship between rRNA transcription and cell differentiation.


Assuntos
Diferenciação Celular/genética , Regulação para Baixo/genética , RNA Ribossômico/genética , Transcrição Gênica/genética , Animais , Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Células HL-60 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/genética , RNA Polimerase I/genética , RNA Interferente Pequeno/genética , Fatores de Transcrição
7.
PLoS One ; 9(1): e86356, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24489718

RESUMO

Here, we found that the PR domain protein Prdm8 serves as a key regulator of the length of the multipolar phase by controlling the timing of morphological transition. We used a mouse line with expression of Prdm8-mVenus reporter and found that Prdm8 is predominantly expressed in the middle and upper intermediate zone during both the late and terminal multipolar phases. Prdm8 expression was almost coincident with Unc5D expression, a marker for the late multipolar phase, although the expression of Unc5D was found to be gradually down-regulated to the point at which mVenus expression was gradually up-regulated. This expression pattern suggests the possible involvement of Prdm8 in the control of the late and terminal multipolar phases, which controls the timing for morphological transition. To test this hypothesis, we performed gain- and loss-of-function analysis of neocortical development by using in utero electroporation. We found that the knockdown of Prdm8 results in premature change from multipolar to bipolar morphology, whereas the overexpression of Prdm8 maintained the multipolar morphology. Additionally, the postnatal analysis showed that the Prdm8 knockdown stimulated the number of early born neurons, and differentiated neurons located more deeply in the neocortex, however, majority of those cells could not acquire molecular features consistent with laminar location. Furthermore, we found the candidate genes that were predominantly utilized in both the late and terminal multipolar phases, and these candidate genes included those encoding for guidance molecules. In addition, we also found that the expression level of these guidance molecules was inhibited by the introduction of the Prdm8 expression vector. These results indicate that the Prdm8-mediated regulation of morphological changes that normally occur during the late and terminal multipolar phases plays an important role in neocortical development.


Assuntos
Córtex Cerebral/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histona-Lisina N-Metiltransferase/genética , Neurogênese/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Córtex Cerebral/crescimento & desenvolvimento , Proteínas de Ligação a DNA , Eletroporação , Embrião de Mamíferos , Feminino , Técnicas de Silenciamento de Genes , Genes Reporter , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/antagonistas & inibidores , Histona-Lisina N-Metiltransferase/metabolismo , Injeções Intraventriculares , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Plasmídeos , Gravidez , Útero , Proteína Vermelha Fluorescente
8.
J Biol Chem ; 289(8): 4928-40, 2014 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-24375404

RESUMO

Tetramerization of p53 is crucial to exert its biological activity, and nucleolar disruption is sufficient to activate p53. We previously demonstrated that nucleolar stress induces translocation of the nucleolar protein MYBBP1A from the nucleolus to the nucleoplasm and enhances p53 activity. However, whether and how MYBBP1A regulates p53 tetramerization in response to nucleolar stress remain unclear. In this study, we demonstrated that MYBBP1A enhances p53 tetramerization, followed by acetylation under nucleolar stress. We found that MYBBP1A has two regions that directly bind to lysine residues of the p53 C-terminal regulatory domain. MYBBP1A formed a self-assembled complex that provided a molecular platform for p53 tetramerization and enhanced p300-mediated acetylation of the p53 tetramer. Moreover, our results show that MYBBP1A functions to enhance p53 tetramerization that is necessary for p53 activation, followed by cell death with actinomycin D treatment. Thus, we suggest that MYBBP1A plays a pivotal role in the cellular stress response.


Assuntos
Nucléolo Celular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Multimerização Proteica , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sítios de Ligação , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Proteína p300 Associada a E1A/metabolismo , Humanos , Modelos Biológicos , Proteínas Nucleares/química , Proteínas de Transporte Nucleocitoplasmático/química , Regiões Promotoras Genéticas/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Transcrição , Proteína Supressora de Tumor p53/genética
9.
Biochem Biophys Res Commun ; 434(3): 659-63, 2013 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-23583237

RESUMO

Nucleolar dynamics are important for cellular stress response. We previously demonstrated that nucleolar stress induces nucleolar protein Myb-binding protein 1A (MYBBP1A) translocation from the nucleolus to the nucleoplasm and enhances p53 activity. However, the underlying molecular mechanism is understood to a lesser extent. Here we demonstrate that MYBBP1A interacts with lysine residues in the C-terminal regulatory domain region of p53. MYBBP1A specifically interacts with nonacetylated p53 and induces p53 acetylation. We propose that MYBBP1A dissociates from acetylated p53 because MYBBP1A did not interact with acetylated p53 and because MYBBP1A was not recruited to the p53 target promoter. Therefore, once p53 is acetylated, MYBBP1A dissociates from p53 and interacts with nonacetylated p53, which enables another cycle of p53 activation. Based on our observations, this MYBBP1A-p53 binding property can account for efficient p53-activation by MYBBP1A under nucleolar stress. Our results support the idea that MYBBP1A plays catalytic roles in p53 acetylation and activation.


Assuntos
Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Ativação Transcricional , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Primers do DNA , Proteínas de Ligação a DNA , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA , Fatores de Transcrição , Proteína Supressora de Tumor p53/química
10.
J Biol Chem ; 286(23): 20861-9, 2011 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-21471221

RESUMO

In response to a shortage of intracellular energy, mammalian cells reduce energy consumption and induce cell cycle arrest, both of which contribute to cell survival. Here we report that a novel nucleolar pathway involving the energy-dependent nucleolar silencing complex (eNoSC) and Myb-binding protein 1a (MYBBP1A) is implicated in these processes. Namely, in response to glucose starvation, eNoSC suppresses rRNA transcription, which results in a reduction in nucleolar RNA content. As a consequence, MYBBP1A, which is anchored to the nucleolus via RNA, translocates from the nucleolus to the nucleoplasm. The translocated MYBBP1A induces acetylation and accumulation of p53 by enhancing the interaction between p300 and p53, which eventually leads to the cell cycle arrest (or apoptosis). Taken together, our results indicate that the nucleolus works as a sensor that transduces the intracellular energy status into the cell cycle machinery.


Assuntos
Apoptose/fisiologia , Nucléolo Celular/metabolismo , Metabolismo Energético/fisiologia , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular Tumoral , Nucléolo Celular/genética , Proteínas de Ligação a DNA , Humanos , Complexos Multiproteicos/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/genética , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteínas de Ligação a RNA , Fatores de Transcrição , Proteína Supressora de Tumor p53/genética , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismo
11.
Biochem Biophys Res Commun ; 407(2): 378-82, 2011 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-21396915

RESUMO

Cells eventually exit from mitosis during sustained arrest at the spindle checkpoint, without sister chromatid separation and cytokinesis. The resulting tetraploid cells are arrested in the subsequent G1 phase in a p53-dependent manner by the regulatory function of the postmitotic G1 checkpoint. Here we report how the nucleolus plays a critical role in activation of the postmitotic G1 checkpoint. During mitosis, the nucleolus is disrupted and many nucleolar proteins are translocated from the nucleolus into the cytoplasm. Among the nucleolar factors, Myb-binding protein 1a (MYBBP1A) induces the acetylation and accumulation of p53 by enhancing the interaction between p300 and p53 during prolonged mitosis. MYBBP1A-dependent p53 activation is essential for the postmitotic G1 checkpoint. Thus, our results demonstrate a novel nucleolar function that monitors the prolongation of mitosis and converts its signal into activation of the checkpoint machinery.


Assuntos
Nucléolo Celular/metabolismo , Fase G1 , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proteínas de Ligação a DNA , Técnicas de Silenciamento de Genes , Humanos , Mitose , Nocodazol/farmacologia , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Poliploidia , Proteínas de Ligação a RNA , Fatores de Transcrição , Proteína Supressora de Tumor p53/genética , Fatores de Transcrição de p300-CBP/metabolismo
12.
EMBO J ; 30(6): 1054-66, 2011 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-21297583

RESUMO

A number of external and internal insults disrupt nucleolar structure, and the resulting nucleolar stress stabilizes and activates p53. We show here that nucleolar disruption induces acetylation and accumulation of p53 without phosphorylation. We identified three nucleolar proteins, MYBBP1A, RPL5, and RPL11, involved in p53 acetylation and accumulation. MYBBP1A was tethered to the nucleolus through nucleolar RNA. When rRNA transcription was suppressed by nucleolar stress, MYBBP1A translocated to the nucleoplasm and facilitated p53-p300 interaction to enhance p53 acetylation. We also found that RPL5 and RPL11 were required for rRNA export from the nucleolus. Depletion of RPL5 or RPL11 blocked rRNA export and counteracted reduction of nucleolar RNA levels caused by inhibition of rRNA transcription. As a result, RPL5 or RPL11 depletion inhibited MYBBP1A translocation and p53 activation. Our observations indicated that a dynamic equilibrium between RNA generation and export regulated nucleolar RNA content. Perturbation of this balance by nucleolar stress altered the nucleolar RNA content and modulated p53 activity.


Assuntos
Nucléolo Celular/química , Proteínas Nucleares/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , RNA Ribossômico/análise , Proteína Supressora de Tumor p53/metabolismo , Acetilação , Linhagem Celular , Proteínas de Ligação a DNA , Humanos , Proteínas de Ligação a RNA , Proteínas Ribossômicas/metabolismo , Fatores de Transcrição
13.
Cell ; 133(4): 627-39, 2008 May 16.
Artigo em Inglês | MEDLINE | ID: mdl-18485871

RESUMO

Intracellular energy balance is important for cell survival. In eukaryotic cells, the most energy-consuming process is ribosome biosynthesis, which adapts to changes in intracellular energy status. However, the mechanism that links energy status and ribosome biosynthesis is largely unknown. Here, we describe eNoSC, a protein complex that senses energy status and controls rRNA transcription. eNoSC contains Nucleomethylin, which binds histone H3 dimethylated Lys9 in the rDNA locus, in a complex with SIRT1 and SUV39H1. Both SIRT1 and SUV39H1 are required for energy-dependent transcriptional repression, suggesting that a change in the NAD(+)/NADH ratio induced by reduction of energy status could activate SIRT1, leading to deacetylation of histone H3 and dimethylation at Lys9 by SUV39H1, thus establishing silent chromatin in the rDNA locus. Furthermore, eNoSC promotes restoration of energy balance by limiting rRNA transcription, thus protecting cells from energy deprivation-dependent apoptosis. These findings provide key insight into the mechanisms of energy homeostasis in cells.


Assuntos
DNA Ribossômico/genética , Metabolismo Energético , Inativação Gênica , Transcrição Gênica , Morte Celular , Linhagem Celular , Nucléolo Celular/metabolismo , Glucose/metabolismo , Histona Metiltransferases , Histona-Lisina N-Metiltransferase/química , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/metabolismo , Humanos , Metiltransferases/química , Metiltransferases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , NAD/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Metiltransferases , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA , Proteínas Repressoras/metabolismo , Sirtuína 1 , Sirtuínas/metabolismo
14.
Biochem Biophys Res Commun ; 370(1): 145-8, 2008 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-18355447

RESUMO

PPARgamma is a nuclear hormone receptor that plays a key role in the induction of peroxisome proliferation. A number of studies showed that PPARgamma ligands suppress cell cycle progression; however, the mechanism remains to be determined. Here, we showed that PPARgamma ligand troglitazone inhibited G1/S transition in colon cancer cells, LS174T. Troglitazone did not affect on either expression of CDK inhibitor (p18) or Wnt signaling pathway, indicating that these pathways were not involved in the troglitazone-dependent cell cycle arrest. GeneChip and RT-PCR analyses revealed that troglitazone decreased mRNA levels of cell cycle regulatory factors E2F2 and cyclin-E1 whose expression is activated by E2F2. Down-regulation of E2F2 by troglitazone results in decrease of cyclin-E1 transcription, which could inhibit phosphorylation of Rb protein, and consequently evoke the suppression of E2F2 transcriptional activity. Thus, we propose that troglitazone suppresses the feedback loop containing E2F2, cyclin-E1, and Rb protein.


Assuntos
Antineoplásicos/farmacologia , Cromanos/farmacologia , Neoplasias do Colo/metabolismo , Ciclina E/antagonistas & inibidores , Fator de Transcrição E2F2/antagonistas & inibidores , Proteínas Oncogênicas/antagonistas & inibidores , PPAR gama/agonistas , Tiazolidinedionas/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Ciclina E/genética , Ciclina E/metabolismo , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Humanos , Ligantes , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , PPAR gama/metabolismo , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/metabolismo , Proteína do Retinoblastoma/antagonistas & inibidores , Proteína do Retinoblastoma/genética , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Troglitazona , Proteínas Wnt/metabolismo
15.
Am J Orthod Dentofacial Orthop ; 129(4): 562-70, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16627186

RESUMO

INTRODUCTION: Dolichofacial skeletal patterns are a challenge for the orthodontist. Even when treatment for a long-face patient begins before the adolescent growth spurt, excellent compliance is generally needed. The patient whose care is presented here started treatment at age 14. RESULTS: The extraction of 4 premolars, rapid palatal expansion, and excellent compliance wearing a combination occipital and vertical-pull chincup over a 2-year period led to good results at age 16, with minimal dental or skeletal relapse at age 18 years 5 months.


Assuntos
Aparelhos de Tração Extrabucal , Anormalidades Maxilomandibulares/complicações , Má Oclusão Classe I de Angle/complicações , Mandíbula/anormalidades , Ortodontia Corretiva/instrumentação , Adolescente , Cefalometria , Queixo , Face/anatomia & histologia , Feminino , Humanos , Anormalidades Maxilomandibulares/terapia , Má Oclusão Classe I de Angle/terapia , Contenções Ortodônticas , Prevenção Secundária
17.
Mol Cell Biol ; 25(6): 2475-85, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15743839

RESUMO

The pluripotential cell-specific gene Nanog encodes a homeodomain-bearing transcription factor required for maintaining the undifferentiated state of stem cells. However, the molecular mechanisms that regulate Nanog gene expression are largely unknown. To address this important issue, we used luciferase assays to monitor the relative activities of deletion fragments from the 5'-flanking region of the gene. An adjacent pair of highly conserved Octamer- and Sox-binding sites was found to be essential for activating pluripotential state-specific gene expression. Furthermore, the 5'-end fragment encompassing the Octamer/Sox element was sufficient for inducing the proper expression of a green fluorescent protein reporter gene even in human embryonic stem (ES) cells. The potential of OCT4 and SOX2 to bind to this element was verified by electrophoretic mobility shift assays with extracts from F9 embryonal carcinoma cells and embryonic germ cells derived from embryonic day 12.5 embryos. However, in ES cell extracts, a complex of OCT4 with an undefined factor preferentially bound to the Octamer/Sox element. Thus, Nanog transcription may be regulated through an interaction between Oct4 and Sox2 or a novel pluripotential cell-specific Sox element-binding factor which is prominent in ES cells.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Regulação da Expressão Gênica , Proteínas de Homeodomínio/genética , Proteínas Nucleares/fisiologia , Elementos de Resposta/genética , Células-Tronco/metabolismo , Fatores de Transcrição/fisiologia , Região 5'-Flanqueadora/genética , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Ligação a DNA/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Embrião de Mamíferos/citologia , Genes Reporter/genética , Proteínas de Fluorescência Verde/análise , Proteínas de Fluorescência Verde/genética , Proteínas HMGB , Humanos , Luciferases/análise , Luciferases/genética , Camundongos , Dados de Sequência Molecular , Mutação/genética , Proteína Homeobox Nanog , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero , Fatores de Transcrição SOXB1 , Deleção de Sequência/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica
18.
Biol Reprod ; 70(1): 154-9, 2004 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-12954723

RESUMO

The function of cyclin B1 (CB1) and cyclin B2 (CB2) during porcine oocyte maturation was investigated by injecting oocytes with their antisense RNAs (asRNAs). At first, protein levels of both cyclin Bs were examined by immunoblotting, revealing that immature oocytes had only CB2, at a level comparable to 1/20 to 1/40 of that detected in first metaphase oocytes. Both cyclin B syntheses were started around germinal vesicle breakdown (GVBD); CB1 and CB2 peaked at the second metaphase and first metaphase, respectively. We obtained a porcine CB2 cDNA fragment, which was 88% homologous with human CB2, by reverse-transcriptase polymerase chain reaction (RT-PCR) using total RNAs of immature porcine oocytes and a primer set of human CB2. Specific asRNAs of CB1 and CB2 were prepared in vitro. Then one, the other, or both were injected into the cytoplasm of immature oocytes. CB1 asRNA inhibited CB1 synthesis specifically; the injected oocytes underwent first meiosis normally but could not arrest at the second meiotic metaphase. CB2 asRNA inhibited CB2 synthesis specifically, but had almost no effect on the maturation of injected oocytes. When both CB1 and CB2 asRNAs were injected, synthesis of both cyclin Bs was inhibited, and GVBD was significantly suppressed but occurred slowly. These results suggest that CB1 is the principal molecule for regulation in mammalian oocyte maturation, whereas CB2 has only an accessory role. They also show that in porcine oocytes, cyclin B synthesis is not necessary for GVBD induction itself, but synthesis of at least one cyclin B, CB1 or CB2, is necessary for GVBD induction in a normal time course.


Assuntos
Ciclina B/metabolismo , Oócitos/citologia , Oócitos/fisiologia , Animais , Sequência de Bases , Ciclina B/genética , Ciclina B1 , Ciclina B2 , Feminino , Meiose/fisiologia , Dados de Sequência Molecular , RNA Antissenso/farmacologia
19.
Glycobiology ; 13(5): 315-26, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12626382

RESUMO

Highly water-soluble glycopolymers with poly(alpha-L-glutamic acid) (PGA) backbones carrying multivalent sialyl oligosaccharides units were chemoenzymatically synthesized as polymeric inhibitors of infection by human influenza viruses. p-Aminophenyl disaccharide glycosides were coupled with gamma-carboxyl groups of PGA side chains and enzymatically converted to Neu5Acalpha2-3Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-6Galbeta1-4GlcNAcbeta-, Neu5Acalpha2-3Galbeta1-3GalNAcalpha-, and Neu5Acalpha2-3Galbeta1-3GalNAcbeta- units, respectively, by alpha2,3- or alpha2,6-sialytransferases. The glycopolymers synthesized were used for neutralization of human influenza A and B virus infection as assessed by measurement of the degree of cytopathic inhibitory effect in virus-infected MDCK cells. Among the glycopolymers tested, alpha2,6-sialo-PGA with a high molecular weight (260 kDa) most significantly inhibited infection by an influenza A virus, strain A/Memphis/1/71 (H3N2), which predominantly binds to alpha2-6 Neu5Ac residue. The alpha2,6-sialo-PGA also inhibited infection by an influenza B virus, B/Lee/40. The binding preference of viruses to terminal sialic acids was affected by core determinants of the sugar chain, Galbeta1-4GlcNAcbeta- or Galbeta1-3GalNAcalpha/beta- units. Inhibition of infection by viruses was remarkably enhanced by increasing the molecular weight and sialic acid content of glycopolymers.


Assuntos
Vírus da Influenza A , Vírus da Influenza B , Oligossacarídeos/síntese química , Infecções por Orthomyxoviridae/prevenção & controle , Ácido Poliglutâmico/química , Polissacarídeos/síntese química , Sialoglicoproteínas/síntese química , Animais , Sequência de Carboidratos , Células Cultivadas , Testes Imunológicos de Citotoxicidade , Cães , Soros Imunes/farmacologia , Vírus da Influenza A/efeitos dos fármacos , Vírus da Influenza A/fisiologia , Vírus da Influenza B/efeitos dos fármacos , Vírus da Influenza B/fisiologia , Camundongos , Dados de Sequência Molecular , Oligossacarídeos/imunologia , Oligossacarídeos/farmacologia , Polissacarídeos/imunologia , Polissacarídeos/farmacologia , Sialoglicoproteínas/imunologia , Relação Estrutura-Atividade
20.
Mol Microbiol ; 44(5): 1367-75, 2002 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12068813

RESUMO

Replication of the Escherichia coli chromosome is initiated synchronously from all origins (oriC) present in a cell at a fixed time in the cell cycle under given steady state culture conditions. A mechanism to ensure the cyclic initiation events operates through the chromosomal site, datA, which titrates exceptionally large amounts of the bacterial initiator protein, DnaA, to prevent overinitiation. Deletion of the datA locus results in extra initiations and altered temporal control of replication. There are many other sites on the E. coli chromosome that can bind DnaA protein, but the contribution of these sites to the control of replication initiation has not been investigated. In the present study, seven major DnaA binding sites other than datA have been examined for their influence on the timing of replication initiation. Disruption of these seven major binding sites, either individually or together, had no effect on the timing of initiation of replication. Thus, datA seems to be a unique site that adjusts the balance between free and bound DnaA to ensure that there is only a single initiation event in each bacterial cell cycle. Mutation either in the second or the third DnaA box (a 9 basepair DnaA-binding sequence) in datA was enough to induce asynchronous and extra initiations of replication to a similar extent as that observed with the datA-deleted strain. These DnaA boxes may act as cores for the cooperative binding of DnaA to the entire datA region.


Assuntos
Proteínas de Bactérias/metabolismo , Cromossomos Bacterianos/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Origem de Replicação/genética , Sequência de Bases , Escherichia coli/metabolismo , Citometria de Fluxo , Dados de Sequência Molecular , Regiões Promotoras Genéticas
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