RESUMO
The Zfp296 gene encodes a zinc finger-type protein. Its expression is high in mouse embryonic stem cells (ESCs) but rapidly decreases following differentiation. Zfp296-knockout (KO) ESCs grew as flat colonies, which were reverted to rounded colonies by exogenous expression of Zfp296. KO ESCs could not form teratomas when transplanted into mice but could efficiently contribute to germline-competent chimeric mice following blastocyst injection. Transcriptome analysis revealed that Zfp296 deficiency up- and down-regulates a distinct group of genes, among which Dppa3, Otx2, and Pou3f1 were markedly downregulated. Chromatin immunoprecipitation sequencing demonstrated that ZFP296 binding is predominantly seen in the vicinity of the transcription start sites (TSSs) of a number of genes, and ZFP296 was suggested to negatively regulate transcription. Consistently, chromatin accessibility assay clearly showed that ZFP296 binding reduces the accessibility of the TSS regions of target genes. Zfp296-KO ESCs showed increased histone H3K9 di- and trimethylation. Co-immunoprecipitation analyses revealed interaction of ZFP296 with G9a and GLP. These results show that ZFP296 plays essential roles in maintaining the global epigenetic state of ESCs through multiple mechanisms including activation of Dppa3, attenuation of chromatin accessibility, and repression of H3K9 methylation, but that Zfp296-KO ESCs retain a unique state of pluripotency while lacking the teratoma-forming ability.
Assuntos
Cromatina , Teratoma , Animais , Camundongos , Células-Tronco Embrionárias , Histonas , Células-Tronco Embrionárias Murinas , Bioensaio , Proteínas Cromossômicas não Histona , Fator 6 de Transcrição de OctâmeroRESUMO
Free fatty acid receptor 1 (FFAR1 or GPR40) has attracted attention for the treatment of type 2 diabetes mellitus, and various small-molecule agonists have been developed. However, most FFAR1 agonists as well as endogenous ligands, such as linoleic acids, have high lipophilicity, and their high lipophilicity is related to off-target toxicity. Therefore, we need to focus on new ligand candidates with less toxicity. In this study, we screened peptides with FFAR1 agonist activity as new ligand candidates. First, we used phage display to identify peptides with high affinity to FFAR1. Next, the agonist activities of peptides determined by the phage display were evaluated by the TGF-α shedding assay. Finally, to improve the FFAR1 agonist activity of the peptide, we performed an inclusive single amino acid substitution and sequence analysis. Logistic regression (LR) analysis using 120 physiochemical properties was performed to predict peptides with high FFAR1 agonist activity. STTGTQY determined by phage display promoted glucose-stimulated insulin secretion in pancreatic MIN6 cells. Furthermore, STKGTF predicted by the LR analysis showed high insulin secretion at low concentrations compared to STTGTQY. The results of this study suggest that peptides could be new candidates as FFAR1 agonists.
Assuntos
Substituição de Aminoácidos , Avaliação Pré-Clínica de Medicamentos/métodos , Aprendizado de Máquina , Biblioteca de Peptídeos , Peptídeos/química , Peptídeos/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Glucose/farmacologia , Células HEK293 , Humanos , Insulina/metabolismo , Peptídeos/efeitos adversos , Peptídeos/genética , Ligação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Análise de Regressão , Fator de Crescimento Transformador alfa/metabolismoRESUMO
A pancreatic ß-cell line MIN6 was previously established in our lab from an insulinoma developed in an IT6 transgenic mouse expressing the SV40 T antigen in ß-cells. This cell line has been widely used for in vitro analysis of ß-cell function, but tends to lose the mature ß-cell features, including glucose-stimulated insulin secretion (GSIS), in long-term culture. The aim of this study was to develop a stable ß-cell line that retains the characteristics of mature ß-cells. Considering that mice derived from a cross between C3H and C57BL/6 strains are known to exhibit higher insulin secretory capacity than C57BL/6 mice, an IT6 male mouse of this hybrid background was used to isolate insulinomas, which were independently cultured. After 7 months of continuous culturing, we obtained the MIN6-CB4 ß-cell line, which stably maintains its GSIS. It has been noted that ß-cell lines express the glucagon (Gcg) gene at certain levels. MIN6-CB4 cells were utilized to assess the effects of differential Gcg expression on ß-cell function. Our data show the functional importance of Gcg expression and resulting basal activation of the GLP-1 receptor in ß-cells. MIN6-CB4 cells can serve as an invaluable tool for studying the regulatory mechanisms of insulin secretion, such as the GLP-1/cAMP signaling, in ß-cells.
Assuntos
Receptor do Peptídeo Semelhante ao Glucagon 1/metabolismo , Glucagon/fisiologia , Secreção de Insulina , Células Secretoras de Insulina/metabolismo , Insulinoma/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Feminino , Células Secretoras de Insulina/citologia , Insulinoma/patologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neoplasias Pancreáticas/patologiaRESUMO
A common variant in the RAB27A gene in adults was recently found to be associated with the fractional exhaled nitric oxide level, a marker of eosinophilic airway inflammation. The small GTPase Rab27 is known to regulate intracellular vesicle traffic, although its role in allergic responses is unclear. We demonstrated that exophilin-5, a Rab27-binding protein, was predominantly expressed in both of the major IL-33 producers, lung epithelial cells, and the specialized IL-5 and IL-13 producers in the CD44hiCD62LloCXCR3lo pathogenic Th2 cell population in mice. Exophilin-5 deficiency increased stimulant-dependent damage and IL-33 secretion by lung epithelial cells. Moreover, it enhanced IL-5 and IL-13 production in response to TCR and IL-33 stimulation from a specific subset of pathogenic Th2 cells that expresses a high level of IL-33 receptor, which exacerbated allergic airway inflammation in a mouse model of asthma. Mechanistically, exophilin-5 regulates extracellular superoxide release, intracellular ROS production, and phosphoinositide 3-kinase activity by controlling intracellular trafficking of Nox2-containing vesicles, which seems to prevent the overactivation of pathogenic Th2 cells mediated by IL-33. This is the first report to our knowledge to establish the significance of the Rab27-related protein exophilin-5 in the development of allergic airway inflammation, and provides insights into the pathophysiology of asthma.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/imunologia , Asma/imunologia , Proteína 1 Semelhante a Receptor de Interleucina-1/imunologia , Interleucina-33/imunologia , Células Th2/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Asma/genética , Asma/patologia , Modelos Animais de Doenças , Proteína 1 Semelhante a Receptor de Interleucina-1/genética , Interleucina-33/genética , Camundongos , Camundongos Knockout , NADPH Oxidase 2/genética , NADPH Oxidase 2/imunologia , Espécies Reativas de Oxigênio/imunologia , Células Th2/patologiaRESUMO
Lymphangiogenesis plays important roles in normal fetal development and postnatal growth. However, its molecular regulation remains unclear. Here, we have examined the function of forkhead box protein O1 (FOXO1) transcription factor, a known angiogenic factor, in developmental dermal lymphangiogenesis using endothelial cell-specific FOXO1-deficient mice. FOXO1-deficient mice showed disconnected and dilated lymphatic vessels accompanied with increased proliferation and decreased apoptosis in the lymphatic capillaries. Comprehensive DNA microarray analysis of the causes of in vivo phenotypes in FOXO1-deficient mice revealed that the gene encoding C-X-C chemokine receptor 4 (CXCR4) was the most drastically downregulated in FOXO1-deficient primary lymphatic endothelial cells (LECs). CXCR4 was expressed in developing dermal lymphatic capillaries in wild-type mice but not in FOXO1-deficient dermal lymphatic capillaries. Furthermore, FOXO1 suppression impaired migration toward the exogenous CXCR4 ligand, C-X-C chemokine ligand 12 (CXCL12), and coordinated proliferation in LECs. These results suggest that FOXO1 serves an essential role in normal developmental lymphangiogenesis by promoting LEC migration toward CXCL12 and by regulating their proliferative activity. This study provides valuable insights into the molecular mechanisms underlying developmental lymphangiogenesis.
Assuntos
Derme/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Linfangiogênese/genética , Receptores CXCR4/genética , Cauda/metabolismo , Regulação para Cima/genética , Animais , Animais Recém-Nascidos , Antígenos CD/metabolismo , Apoptose , Sequência de Bases , Caderinas/metabolismo , Morte Celular , Proliferação de Células , Quimiocina CXCL12/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Elementos Facilitadores Genéticos/genética , Deleção de Genes , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Regiões Promotoras Genéticas/genética , Ligação Proteica , Receptores CXCR4/metabolismoRESUMO
In mammalian pancreatic ß cells, the IRE1α-XBP1 pathway is constitutively and highly activated under physiological conditions. To elucidate the precise role of this pathway, we constructed ß cell-specific Ire1α conditional knockout (CKO) mice and established insulinoma cell lines in which Ire1α was deleted using the Cre-loxP system. Ire1α CKO mice showed the typical diabetic phenotype including impaired glycemic control and defects in insulin biosynthesis postnatally at 4-20 weeks. Ire1α deletion in pancreatic ß cells in mice and insulinoma cells resulted in decreased insulin secretion, decreased insulin and proinsulin contents in cells, and decreased oxidative folding of proinsulin along with decreased expression of five protein disulfide isomerases (PDIs): PDI, PDIR, P5, ERp44, and ERp46. Reconstitution of the IRE1α-XBP1 pathway restored the proinsulin and insulin contents, insulin secretion, and expression of the five PDIs, indicating that IRE1α functions as a key regulator of the induction of catalysts for the oxidative folding of proinsulin in pancreatic ß cells.
Assuntos
Endorribonucleases/metabolismo , Células Secretoras de Insulina/enzimologia , Insulina/metabolismo , Proinsulina/metabolismo , Dobramento de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Proteína 1 de Ligação a X-Box/metabolismo , Fator 6 Ativador da Transcrição/metabolismo , Animais , Sítios de Ligação , Glicemia/metabolismo , Linhagem Celular Tumoral , Diabetes Mellitus/sangue , Diabetes Mellitus/enzimologia , Diabetes Mellitus/genética , Endorribonucleases/deficiência , Endorribonucleases/genética , Insulina/genética , Insulinoma/enzimologia , Insulinoma/genética , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos Knockout , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Oxirredução , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/genética , Fosforilação , Proinsulina/química , Proinsulina/genética , Regiões Promotoras Genéticas , Isomerases de Dissulfetos de Proteínas/genética , Isomerases de Dissulfetos de Proteínas/metabolismo , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Transdução de Sinais , Tiorredoxinas/genética , Tiorredoxinas/metabolismo , Proteína 1 de Ligação a X-Box/genética , eIF-2 Quinase/metabolismoRESUMO
Forkhead box protein O1 (FoxO1) is a transcription factor and a critical regulator of angiogenesis. Various environmental stimuli, including growth factors, nutrients, shear stress, oxidative stress and hypoxia, affect FoxO1 subcellular localization and strongly influence its transcriptional activity; however, FoxO1-localization patterns in endothelial cells (ECs) during development have not been clarified in vivo. Here, we reported that FoxO1 expression was observed in three layers of angiogenic vessels in developing mouse retinas and that among these layers, the front layer showed high levels of FoxO1 expression in the nuclei of most tip ECs. Because tip ECs migrate toward the avascular hypoxic area, we focused on hypoxia as a major stimulus regulating FoxO1 subcellular localization in tip cells. In cultured ECs, FoxO1 accumulated into the nucleus under hypoxic conditions, with hypoxia also inducing expression of tip-cell-specific genes, including endothelial-specific molecule 1 (ESM1), which was suppressed by FoxO1 knockdown. Additionally, in murine models, EC-specific FoxO1 deletion resulted in reduced ESM1 expression and suppressed tip-cell migration during angiogenesis. These findings indicated roles for FoxO1 in tip-cell migration and that its transcriptional activity is regulated by hypoxia.
Assuntos
Células Endoteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Regulação da Expressão Gênica , Hipóxia/metabolismo , Retina/crescimento & desenvolvimento , Neovascularização Retiniana/metabolismo , Animais , Células Endoteliais/patologia , Proteína Forkhead Box O1/genética , Técnicas de Silenciamento de Genes , Humanos , Hipóxia/genética , Hipóxia/patologia , Camundongos , Camundongos Transgênicos , Retina/patologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/patologiaRESUMO
The Cys2/His2-type zinc finger protein Zfp296 has been implicated in stem cell pluripotency and tumor pathogenesis. However, its mechanisms remain elusive. Here, we demonstrated that a Zfp296 deficiency in mice impairs germ-cell development and embryonic growth. Zfp296 was intracellularly localized to heterochromatin in embryos. A GST-Zfp296 pull-down experiment using ES cell nuclear extract followed by LC-MS/MS showed that Zfp296 interacts with component proteins of heterochromatin (such as HP1, Dnmt1, Dnmt3b, and ATRX) and the NuRD complex. We focused on H3K9 methylation as a hallmark of heterochromatin, and found that Zfp296 overexpression in cultured cells reduces the Suv39h1-mediated H3K9 methylation. Consistent with this finding, in Zfp296 -/- mouse embryos, we observed a global increase in H3K9 methylation in a developmental stage-dependent manner, and showed, by ChIP-qPCR, that the H3K9me3 levels at major satellite repeats were elevated in Zfp296 -/- embryos. Our results demonstrate that Zfp296 is a component of heterochromatin that affects embryonic development by negatively regulating H3K9 methylation.
Assuntos
Proteínas de Ligação a DNA/genética , Desenvolvimento Embrionário/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Células-Tronco Embrionárias Murinas/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Diferenciação Celular , Homólogo 5 da Proteína Cromobox , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas de Ligação a DNA/deficiência , Embrião de Mamíferos , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/química , Histonas/genética , Masculino , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Camundongos , Camundongos Knockout , Células-Tronco Embrionárias Murinas/citologia , Ovário/anormalidades , Ovário/crescimento & desenvolvimento , Ovário/metabolismo , Cultura Primária de Células , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Testículo/anormalidades , Testículo/crescimento & desenvolvimento , Testículo/metabolismo , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , DNA Metiltransferase 3BRESUMO
Recent studies have revealed that newly emerging transformed cells are often apically extruded from epithelial tissues. During this process, normal epithelial cells can recognize and actively eliminate transformed cells, a process called epithelial defence against cancer (EDAC). Here, we show that mitochondrial membrane potential is diminished in RasV12-transformed cells when they are surrounded by normal cells. In addition, glucose uptake is elevated, leading to higher lactate production. The mitochondrial dysfunction is driven by upregulation of pyruvate dehydrogenase kinase 4 (PDK4), which positively regulates elimination of RasV12-transformed cells. Furthermore, EDAC from the surrounding normal cells, involving filamin, drives the Warburg-effect-like metabolic alteration. Moreover, using a cell-competition mouse model, we demonstrate that PDK-mediated metabolic changes promote the elimination of RasV12-transformed cells from intestinal epithelia. These data indicate that non-cell-autonomous metabolic modulation is a crucial regulator for cell competition, shedding light on the unexplored events at the initial stage of carcinogenesis.
Assuntos
Comunicação Celular , Transformação Celular Neoplásica/metabolismo , Metabolismo Energético , Células Epiteliais/metabolismo , Animais , Linhagem Celular Transformada , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Técnicas de Cocultura , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Cães , Feminino , Genes ras , Glucose/metabolismo , Glicólise , Ácido Láctico/metabolismo , Células Madin Darby de Rim Canino , Masculino , Potencial da Membrana Mitocondrial , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Interferência de RNA , Transdução de Sinais , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
A promising approach to new diabetes therapies is to generate ß cells from other differentiated pancreatic cells in vivo. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into ß cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into ß cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of in vivo reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes.
Assuntos
Células Acinares/citologia , Transdiferenciação Celular/genética , Pâncreas/citologia , Transativadores/genética , Células Acinares/metabolismo , Animais , Glicemia/metabolismo , Reprogramação Celular , Glândulas Exócrinas/citologia , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Insulina/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
BACKGROUND: Keratin 5 (K5) is a cytoskeletal tissue-specific protein expressed in the epithelial cells of skin and esophagus and ectopic K5 expression in lymphocytes has never been reported. OBJECTIVE: Here we demonstrate an ectopic epidermal self-protein expression in B-1 B cell by fate mapping of K5-expressing cells. METHODS: K5-Cre×CAG-CAT-loxP-EGFP double Tg (K5×GFP) mice that express enhanced GFP under the control of the K5 promoter were employed. RESULTS: Unexpectedly, B220(+)GFP(+) cells were found in LN, spleen, peripheral blood and peritoneal cavity. These cells were IgM(+)IgD(low)CD23(-)CD43(+)CD19(+)CD93(-), indicating that they were B-1 B cells. The number of B220(+)GFP(+) cells was significantly larger in spleen than in the other tissues tested. Although GFP(+) B-1 cells did not express K5 in the periphery, Lin(-)CD93(+)B220(low-neg)CD19(+) B-1 B cell progenitors expressed GFP and B220(+)CD93(+) progenitor cells expressed K5 and MHC-class II in BM, indicating that GFP(+) B-1 cells transiently expressed K5 and the progenitor cells were potential APC. GFP(+) B-1 cells in the periphery continued expressing MHC class II and had exogenous antigen-presenting capacity comparable to non-follicular B cells. GFP(+) B-1 cells spontaneously secreted more IgM than GFP(-) B-1 cells in vitro. CONCLUSION: These results indicate that B-1 B cells transiently and partially express K5 in BM and are potent for both natural antibody production and antigen presentation.
Assuntos
Células da Medula Óssea/metabolismo , Diferenciação Celular , Queratina-15/metabolismo , Células Precursoras de Linfócitos B/metabolismo , Animais , Formação de Anticorpos , Apresentação de Antígeno , Biomarcadores/metabolismo , Células da Medula Óssea/efeitos dos fármacos , Células da Medula Óssea/imunologia , Linhagem da Célula , Células Cultivadas , Genótipo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Imunoglobulina M/biossíntese , Queratina-15/genética , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Células Precursoras de Linfócitos B/efeitos dos fármacos , Células Precursoras de Linfócitos B/imunologia , Regiões Promotoras Genéticas , Fatores de TempoRESUMO
OBJECTIVES: Although the somatostatin analog octreotide (OCT) has been used for uncontrollable insulinoma, the mechanism involved is still unknown. The aim of this study was to elucidate the therapeutic effect of OCT for insulinoma. METHODS: Mouse insulinoma cell line MIN6 cells were cultured with OCT to clarify its antiproliferative effects, the expression of somatostatin receptor subtypes, cell cycle, p27 expression, and cdc2 kinase activity. The changes of the messenger RNA expression profiles were examined by microarray analysis. Intraperitoneal OCT treatment was given to insulinoma model IT6 mice for 4 weeks. RESULTS: MIN6 cells expressed somatostatin receptor 2A, 3, and 5 under the OCT treatment. Octreotide showed a dose-dependent antiproliferative effect on MIN6 cells but not on the other cell lines. p27 expression and cdc2 kinase activity in MIN6 cells became prominent with OCT treatment. At the messenger RNA level, several molecules in the mitogen-activated protein kinase signaling pathway were downregulated. The sizes of the individual tumors tended to be smaller in the OCT-treated group. p27 expression was seen in the tumor tissue, but no apoptotic marker was detected. CONCLUSION: Octreotide acted through a cytostatic mechanism and could be an effective therapy for insulinoma.
Assuntos
Proliferação de Células/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27/genética , Pontos de Checagem da Fase G1 do Ciclo Celular/genética , Insulinoma/tratamento farmacológico , Octreotida/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Idoso , Animais , Antineoplásicos Hormonais/administração & dosagem , Antineoplásicos Hormonais/farmacologia , Antineoplásicos Hormonais/uso terapêutico , Western Blotting , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p27/metabolismo , Relação Dose-Resposta a Droga , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Injeções Intraperitoneais , Insulinoma/genética , Insulinoma/patologia , Fígado/efeitos dos fármacos , Fígado/patologia , Fígado/cirurgia , Camundongos , Células NIH 3T3 , Octreotida/administração & dosagem , Octreotida/uso terapêutico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Receptores de Somatostatina/genética , Receptores de Somatostatina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcriptoma/efeitos dos fármacos , Resultado do TratamentoRESUMO
Gobionine species belonging to the genera Pseudorasbora, Pseudopungtungia, and Pungtungia (Teleostei; Cypriniformes; Cyprinidae) have been heavily studied because of problems on taxonomy, threats of extinction, invasion, and human health. Nucleotide sequences of three nuclear genes, that is, recombination activating protein gene 1 (rag1), recombination activating gene 2 (rag2), and early growth response 1 gene (egr1), from Pseudorasbora, Pseudopungtungia, and Pungtungia species residing in China, Japan, and Korea, were analyzed to elucidate their intergeneric and interspecific phylogenetic relationships. In the phylogenetic tree inferred from their multiple gene sequences, Pseudorasbora, Pseudopungtungia and Pungtungia species ramified into three phylogenetically distinct clades; the "tenuicorpa" clade composed of Pseudopungtungia tenuicorpa, the "parva" clade composed of all Pseudorasbora species/subspecies, and the "herzi" clade composed of Pseudopungtungia nigra, and Pungtungia herzi. The genus Pseudorasbora was recovered as monophyletic, while the genus Pseudopungtungia was recovered as polyphyletic. Our phylogenetic result implies the unstable taxonomic status of the genus Pseudopungtungia.
Assuntos
Cipriniformes/genética , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Proteínas de Homeodomínio/genética , Proteínas Nucleares/genética , Filogenia , Animais , Núcleo Celular , Classificação , Cipriniformes/classificação , Evolução Molecular , HumanosRESUMO
Neural stem/progenitor cells (NSPCs) have the ability to migrate into the central nervous system (CNS) to replace damaged cells. In inflammatory CNS disease, cytokine transduced neural stem cells may be used as vehicles to specifically reduce inflammation and promote cell replacement. In this study, we used NSPCs overexpressing IL-10, an immunomodulatory cytokine, in an animal model for CNS inflammation and multiple sclerosis (MS). Intravenous injection of IL-10 transduced neural stem/progenitor cells (NSPC(IL-10)) suppressed myelin oligodendrocyte glycoprotein aa 35-55 (MOG35-55)- induced experimental autoimmune encephalomyelitis (EAE) and, following intravenous injection, NSPC(IL-10) migrated to peripheral lymphoid organs and into the CNS. NSPC(IL-10 )suppressed antigen-specific proliferation and proinflammatory cytokine production of lymph node cells obtained from MOG35-55 peptide immunized mice. In this model, IL-10 producing NSPCs act via a peripheral immunosuppressive effect to attenuate EAE.
Assuntos
Encefalomielite Autoimune Experimental/imunologia , Interleucina-10/imunologia , Células-Tronco Neurais/imunologia , Células-Tronco Neurais/transplante , Transplante de Células-Tronco/métodos , Animais , Ensaio de Imunoadsorção Enzimática , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução GenéticaRESUMO
Adaptive radiations present fascinating opportunities for studying the evolutionary process. Most cases come from isolated lakes or islands, where unoccupied ecological space is filled through novel adaptations. Here, we describe an unusual example of an adaptive radiation: symbiotic mussels that colonized island-like chemosynthetic environments such as hydrothermal vents, cold seeps and sunken organic substrates on the vast deep-sea floor. Our time-calibrated molecular phylogeny suggests that the group originated and acquired sulfur-oxidizing symbionts in the Late Cretaceous, possibly while inhabiting organic substrates and long before its major radiation in the Middle Eocene to Early Oligocene. The first appearance of intracellular and methanotrophic symbionts was detected only after this major radiation. Thus, contrary to expectations, the major radiation may have not been triggered by the evolution of novel types of symbioses. We hypothesize that environmental factors, such as increased habitat availability and/or increased dispersal capabilities, sparked the radiation. Intracellular and methanotrophic symbionts were acquired in several independent lineages and marked the onset of a second wave of diversification at vents and seeps. Changes in habitat type resulted in adaptive trends in shell lengths (related to the availability of space and energy, and physiological trade-offs) and in the successive colonization of greater water depths.
Assuntos
Evolução Biológica , Especiação Genética , Mytilidae/fisiologia , Simbiose , Adaptação Fisiológica , Animais , Tamanho Corporal , Núcleo Celular/genética , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Ecossistema , Evolução Molecular , Dados de Sequência Molecular , Mytilidae/citologia , Mytilidae/genética , Filogenia , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Tcl1 is highly expressed in embryonic stem (ES) cells, but its expression rapidly decreases following differentiation. To assess Tcl1's roles in ES cells, we generated Tcl1-deficient and -overexpressing mouse ES cell lines. We found that Tcl1 was neither essential nor sufficient for maintaining the undifferentiated state. Tcl1 is reported to activate Akt and to enhance cell proliferation. We found that Tcl1 expression levels correlated positively with the proliferation rate and negatively with the apoptosis of ES cells, but did not affect Akt phosphorylation. On the other hand, the phosphorylation level of ß-catenin decreased in response to Tcl1 overexpression. We measured the ß-catenin activity using the TOPflash reporter assay, and found that wild-type ES cells had low activity, which Tcl1 overexpression enhanced 1.8-fold. When the canonical Wnt signaling is activated by ß-catenin stabilization, it reportedly helps maintain ES cells in the undifferentiated state. We then performed DNA microarray analyses between the Tcl1-deficient and -expressing ES cells. The results revealed that Tcl1 expression downregulated a distinct group of genes, including Ndp52, whose expression is very high in blastocysts but reduced in the primitive ectoderm. Based on these results, we discuss the possible roles of Tcl1 in ES cells.
Assuntos
Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Animais , Apoptose/genética , Diferenciação Celular/genética , Linhagem Celular , Proliferação de Células , Perfilação da Expressão Gênica , Técnicas de Transferência de Genes , Camundongos , Camundongos Knockout , Análise em Microsséries , Via de Sinalização Wnt/genéticaRESUMO
Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic islet ß cells is important for understanding and treating diabetes. MIN6 cells, a transformed ß-cell line derived from a mouse insulinoma, retain GSIS and are a popular in vitro model for insulin secretion. However, in long-term culture, MIN6 cells' GSIS capacity is lost. We previously isolated a subclone, MIN6 clone 4, from the parental MIN6 cells, that shows well-regulated insulin secretion in response to glucose, glybenclamide, and KCl, even after prolonged culture. To investigate the molecular mechanisms responsible for preserving GSIS in this subclone, we compared four groups of MIN6 cells: Pr-LP (parental MIN6, low passage number), Pr-HP (parental MIN6, high passage number), C4-LP (MIN6 clone 4, low passage number), and C4-HP (MIN6 clone 4, high passage number). Based on their capacity for GSIS, we designated the Pr-LP, C4-LP, and C4-HP cells as "responder cells." In a DNA microarray analysis, we identified a group of genes with high expression in responder cells ("responder genes"), but extremely low expression in the Pr-HP cells. Another group of genes ("non-responder genes") was expressed at high levels in the Pr-HP cells, but at extremely low levels in the responder cells. Some of the responder genes were involved in secretory machinery or glucose metabolism, including Chrebp, Scgn, and Syt7. Among the non-responder genes were Car2, Maf, and Gcg, which are not normally expressed in islet ß cells. Interestingly, we found a disproportionate number of known imprinted genes among the responder genes. Our findings suggest that the global expression profiling of GSIS-competent and GSIS-incompetent MIN6 cells will help delineate the gene regulatory networks for insulin secretion.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Células Secretoras de Insulina/efeitos dos fármacos , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Anidrase Carbônica II/genética , Anidrase Carbônica II/metabolismo , Linhagem Celular , Proliferação de Células , Células Clonais , Redes Reguladoras de Genes , Glucose/metabolismo , Secreção de Insulina , Células Secretoras de Insulina/citologia , Camundongos , Análise em Microsséries , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Proglucagon/genética , Proglucagon/metabolismo , Proteínas Proto-Oncogênicas c-maf/genética , Proteínas Proto-Oncogênicas c-maf/metabolismo , Secretagoginas , Sinaptotagminas/genética , Sinaptotagminas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Tubular complexes (TCs) are aggregates of duct-like monolayered cells in the developing and regenerating pancreas. Recent studies showed that TCs have regenerative potential, including islet neogenesis. We previously delivered adenovirus vector (AdV) into exocrine cells of the pancreas by intra-common bile ductal (ICBD) injection, and found that AdV expressing Pdx1, a pancreas-specific transcription factor, causes TC formation and islet neogenesis. We also established RTF-Pdx1-EGFP mice, which ubiquitously express Pdx1 when tetracycline is removed from the drinking water. However, exogenous Pdx1 expression in adult RTF-Pdx1-EGFP mice did not cause any pathological changes in the pancreas during three weeks of observation after tetracycline withdrawal. To examine whether the host immune response induced by AdV was involved in TC formation, we delivered AdVs expressing pancreas-related transcription factors or an irrelevant protein into the pancreas of RTF-Pdx1-EGFP mice. Histological analyses showed that both AdV injection and Pdx1 expression are required for TC formation. We also analyzed the effects of these ICBD-injected AdVs. AdV expressing Isl1, a proendocrine transcription factor, effectively induced TC formation through acinar-to-ductal metaplasia, and exogenous Pdx1 expression facilitated this process. Considering the regenerative potential of TCs, a strategy that efficiently induces TC formation may lead to novel therapies for diabetes.
Assuntos
Proteínas de Homeodomínio , Proteínas com Homeodomínio LIM , Metaplasia , Pâncreas , Transativadores , Fatores de Transcrição , Adenoviridae , Animais , Diabetes Mellitus/genética , Diabetes Mellitus/terapia , Regulação Neoplásica da Expressão Gênica , Vetores Genéticos , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Metaplasia/genética , Metaplasia/metabolismo , Camundongos , Pâncreas/metabolismo , Pâncreas/patologia , Regeneração/genética , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Several reports have suggested that Foxo1, a key regulator in differentiation, growth and metabolism, is involved in pancreatic ß-cell function. However, detailed analyses have been hampered by a lack of Foxo1-deficient ß cells. To elucidate Foxo1's function in ß cells, we produced a ß-cell line with inducible Foxo1 deletion. We generated a conditional knockout mouse line, in which Cre recombinase deletes the Foxo1 gene. We then established a ß-cell line from an insulinoma induced in this knockout mouse by the ß-cell-specific expression of simian virus 40 T antigen. In this cell line, designated MIN6-Foxo1flox/flox, adenovirus-mediated Cre expression ablates the Foxo1 gene, generating MIN6-Foxo1-KO cells. Using these knockout and floxed cell lines, we found that Foxo1 ablation enhanced the glucose-stimulated insulin secretion (GSIS) at high glucose concentrations and enhanced ß-cell proliferation. We also conducted DNA microarray analyses of MIN6-Foxo1-KO cells infected with either an adenovirus vector expressing a constitutively active FOXO1 or a control vector and identified several Foxo1-regulated genes, including some known to be related to ß-cell function. These cells should be useful for further studies on Foxo1's roles in ß-cells and may lead to novel strategies for treating the impaired insulin secretion in type 2 diabetes mellitus.