RESUMO
Tektins are a group of microtubule-stabilizing proteins necessary for cilia and flagella assembly. TEKTIN1 (TEKT1) is used as a sperm marker for monitoring germ cell differentiation in embryonic stem (ES) and induced pluripotent stem (iPS) cells. Although upregulation of TEKT1 has been reported during spontaneous differentiation of ES and iPS cells, it is unclear which cells express TEKT1. To identify TEKT1-expressing cells, we established an ES cell line derived from cynomolgus monkeys (Macaca fascicularis), which expresses Venus controlled by the TEKT1 promoter. Venus expression was detected at 5 weeks of differentiation on the surface of the embryoid body (EB), and it gradually increased with the concomitant formation of a leash-like structure at the EB periphery. Motile cilia were observed on the surface of the Venus-positive leash-like structure after 8 weeks of differentiation. The expression of cilia markers as well as TEKT1-5 and 9 + 2 microtubule structures, which are characteristic of motile cilia, were detected in Venus-positive cells. These results demonstrated that TEKT1-expressing cells are multiciliated epithelial-like cells that form a leash-like structure during the spontaneous differentiation of ES and iPS cells. These findings will provide a new research strategy for studying cilia biology, including ciliogenesis and ciliopathies.
Assuntos
Primatas , Sêmen , Animais , Masculino , Diferenciação Celular , Células Germinativas , Células-Tronco Embrionárias/metabolismoRESUMO
Fishes expressing a fluorescent protein in germ cells are useful to perform germ cell transfer experiments for conservation study. Nonetheless, no such fish has been generated in endangered endemic fishes. In this study, we tried to produce a fish expressing Venus fluorescent protein in germ cells using Honmoroko (Gnathopogon caerulescens), which is one of the threatened small cyprinid endemic to the ancient Lake Biwa in Japan. To achieve germ cell-specific expression of Venus, we used piwil1 (formally known as ziwi) promoter and Tol2 transposon system. Following the co-injection of the piwil1-Venus expression vector and the Tol2 transposase mRNA into fertilized eggs, presumptive transgenic fish were reared. At 7 months of post-fertilization, about 19% (10/52) of the examined larvae showed Venus fluorescence in their gonad specifically. Immunohistological staining and in vitro spermatogenesis using gonads of the juvenile founder fish revealed that Venus expression was detected in spermatogonia and spermatocyte in male, and oogonia and stage I and II oocytes in female. These results indicate that the Tol2 transposon and zebrafish piwil1 promoter enabled gene transfer and germ cell-specific expression of Venus in G. caerulescens. In addition, in vitro culture of juvenile spermatogonia enables the rapid validation of temporal expression of transgene during spermatogenesis.
Assuntos
Cyprinidae , Animais , Cyprinidae/genética , Feminino , Técnicas de Transferência de Genes , Masculino , Espermatogônias , Peixe-Zebra/genéticaRESUMO
Measles virus (MV) C protein has been known a multifunctional protein involved in anti-IFN response, viral RNA synthesis, and so on. Recent studies have clarified that double-stranded RNA (dsRNA) is derived from viral genomic RNA (vRNA), and the amount of dsRNA was increased in an MV lacking C protein (MV(C-))-infected cells, suggesting that C protein blocks viral RNA synthesis. However, detailed roles of C protein in viral RNA synthesis remain unknown. Here, we have confirmed through time course experiments using Vero/hSLAM cells that as reported previously, the amount of mRNA is increased in MV(C-)-infected cells at 36 h post infection (hpi). In contrast, we found that the transcription level is lower in MV(C-)-infected cells than wild-type MV-infected cells in early phases of infection. Immunoprecipitation assays were performed to find an interactor(s) of C protein, revealed that C protein interacts with N protein in the absence of vRNA and P protein. RNA immunoprecipitation (RIP) assays showed that the interaction between N protein and vRNA was increased in MV(C-)-infected cells. These results suggest that in early phases of infection C protein facilitates viral transcription to control the formation of nascent nucleocapsid composed of N protein and vRNA.
Assuntos
Genoma Viral , Proteínas do Nucleocapsídeo/metabolismo , Transcrição Gênica , Proteínas não Estruturais Virais/fisiologia , Animais , Linhagem Celular , Células Cultivadas , Vírus do Sarampo/genética , Vírus do Sarampo/fisiologia , Ligação ProteicaRESUMO
Cancer cachexia is a complex multifaceted syndrome involving functional impairment and changes in body composition that cannot be reversed by nutritional support. Cancer cachexia is characterized by decreased skeletal muscle mass, increased lipolysis, and decreased food intake. Cancer cachexia decreases chemotherapy tolerance as well as quality of life. However, because no fully effective interventions are available, cancer cachexia remains an unmet need in cancer treatment. In recent years, several discoveries and treatments for cancer cachexia have been studied, and guidelines have been published. We believe that the development of effective strategies for the diagnosis and treatment of cancer cachexia will lead to breakthroughs in cancer treatment.
Assuntos
Caquexia , Neoplasias , Humanos , Caquexia/tratamento farmacológico , Caquexia/etiologia , Qualidade de Vida , Anorexia/etiologia , Neoplasias/complicaçõesRESUMO
Nonylphenol (NP), an endocrine disrupting chemical, is widely used in industrial and agricultural processes, causing NP influx into aquatic environments. NP induces hormonal imbalance, and male feminization, and reduces germ cell production during spermatogenesis; however, the mechanism by which it affects spermatogenesis remains unknown. Here, we investigated the effect of NP on spermatogenesis in honmoroko (Gnathopogon caerulescens), an endangered fish endemic to Lake Biwa, Japan, using an in vitro differentiation system. We collected spermatogonia from the testes of non-spawning G. caerulescens and subjected them to suspension culture. The spermatogonia differentiated into flagellated spermatozoa in 3 weeks, regardless of the presence of NP. NP concentrations as low as 1 nM caused a decrease in the number of germ cells in a dose-dependent manner, whereas the number of somatic cells decreased only at a high concentration of 1 µM. Flow cytometric analysis revealed that the decrease in germ cell number was attributed to haploids (spermatids and spermatozoa); the number of spermatogonia and spermatocytes was not affected by NP treatment. This result is consistent with the hypothesis that NP might repress the second meiosis or induce apoptosis in haploids. This study demonstrated that the combination of in vitro germ cell differentiation and flow cytometric analysis is useful for evaluating the direct effects of NP on germ cell differentiation in endangered endemic fish.
Assuntos
Cyprinidae , Espermatogênese , Animais , Masculino , Haploidia , Espermatozoides , Espermatogônias , TestículoRESUMO
Effects of endocrine disrupting chemicals (EDCs) on reproduction have not been fully explained comprehensively. In this study, we tried to validate the common effect of Bisphenol A (BPA) and Nonylphenol (NP) on the differentiation of embryonic stem (ES) cells and found that they modify the expression of germ cell specific genes. To elucidate functional significance on biological process, we performed Gene Ontology (GO)-based microarray analysis comparing with published GeneChip data of primordial germ cell development in vivo. Cluster analysis of gene expression profile revealed that EDC treatment and primordial germ cell (PGC) development shared characteristic cluster consists of GO terms related to "germ cell development" and "reproduction". In the GO term "reproduction", meiosis related genes showed high expression level by EDC exposure. These results suggest that BPA and NP affect not only some of the germ cell specific genes, but functionally interferes germ cell development and reproduction.
Assuntos
Compostos Benzidrílicos/toxicidade , Células-Tronco Embrionárias/efeitos dos fármacos , Perfilação da Expressão Gênica , Fenóis/toxicidade , Animais , Células Cultivadas , Células Germinativas/efeitos dos fármacos , Células Germinativas/crescimento & desenvolvimento , Camundongos , Camundongos Endogâmicos ICR , Análise de Sequência com Séries de Oligonucleotídeos , Reprodução/efeitos dos fármacosRESUMO
Measles virus (MV) C protein is a small and basic non-structural protein, but its function is not well understood. We have found that a FLAG-tagged wild-type MV C protein expressed from cDNA was accumulated exclusively in the nucleus. To analyze the amino acid sequence important for the nuclear localization of C protein, a plasmid expressing C protein fused to the enhanced green fluorescent protein (EGFP) was generated. Mutation analysis revealed that (41)PPARKRRQ(48), belonging to the classical nuclear localization signal was important for nuclear localization. Analysis of the amino acid sequence of C protein revealed that it has a nuclear export signal (NES)-like sequence, (76)LEKAMTTLKL(85). Addition of the putative NES to the EGFP resulted in the translocation of EGFP to the cytoplasm. The Rev(1.4)-EGFP nuclear export assay showed that this putative NES has a CRM1-dependent NES activity. C-EGFP accumulated in HeLa nuclei could be translocated to NIH3T3 nuclei in heterokaryon assays. In MV-infected cells, C-EGFP was accumulated in the nuclei in early phase but in the cytoplasm in late phase. These results indicate that the putative NES is functional and that C protein has the ability to shuttle between the nucleus and the cytoplasm.
Assuntos
Núcleo Celular/metabolismo , Citoplasma/metabolismo , Vírus do Sarampo/metabolismo , Proteínas não Estruturais Virais/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Chlorocebus aethiops , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Vírus do Sarampo/genética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Sinais de Exportação Nuclear/genética , Sinais de Localização Nuclear/química , Células Vero , Proteínas não Estruturais Virais/química , Proteínas não Estruturais Virais/genéticaRESUMO
Angiogenesis is important for normal development as well as for tumour growth. However, the molecular and cellular mechanisms underlying angiogenesis are not fully understood, partly because of the lack of a good animal model for imaging. Here, we report the generation of a novel transgenic (Tg) mouse that expresses a bioluminescent reporter protein, Nano-lantern, under the control of Fetal liver kinase 1 (Flk1). Flk1-Nano-lantern BAC Tg mice recapitulated endogenous Flk1 expression in endothelial cells and lymphatic endothelial cells during development and tumour growth. Importantly, bioluminescence imaging of endothelial cells from the aortic rings of Flk1-Nano-lantern BAC Tg mice enabled us to observe endothelial sprouting for 18 hr without any detectable phototoxicity. Furthermore, Flk1-Nano-lantern BAC Tg mice achieved time-lapse luminescence imaging of tumour angiogenesis in freely moving mice with implanted tumours. Thus, this transgenic mouse line contributes a unique model to study angiogenesis within both physiological and pathological contexts.
Assuntos
Carcinoma Pulmonar de Lewis/diagnóstico por imagem , Células Endoteliais/fisiologia , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Neovascularização Patológica/diagnóstico por imagem , Neovascularização Fisiológica , Proteínas Recombinantes de Fusão/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Animais , Carcinoma Pulmonar de Lewis/irrigação sanguínea , Carcinoma Pulmonar de Lewis/metabolismo , Linhagem Celular Tumoral , Células Endoteliais/metabolismo , Fluorescência , Luciferases/genética , Medições Luminescentes/métodos , Proteínas Luminescentes/genética , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Microscopia Confocal , Neovascularização Patológica/genética , Neovascularização Patológica/metabolismo , Proteínas Recombinantes de Fusão/genética , Imagem com Lapso de Tempo/métodos , Fator A de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Human myxovirus resistance gene A (MxA) is a type I interferon-inducible protein and exhibits the antiviral activity against a variety of RNA viruses, including influenza virus. Previously, we reported that MxA accelerates cell death of influenza virus-infected cells through caspase-dependent and -independent mechanisms. Similar to other viruses, influenza virus infection induces endoplasmic reticulum (ER) stress, which is one of cell death inducers. Here, we have demonstrated that MxA enhances ER stress signaling in cells infected with influenza virus. ER stress-induced events, such as expression of BiP mRNA and processing of XBP1 mRNA, were upregulated in cells expressing MxA by treatment with an ER stress inducer, tunicamycin (TM), as well as influenza virus infection. TM-induced cell death was also accelerated by MxA. Furthermore, we showed that MxA interacts with BiP and overexpression of BiP reduces MxA-promoted ER stress signaling. Because cell death in virus-infected cells is one of ultimate anti-virus mechanisms, we propose that MxA-enhanced ER stress signaling is a part of the antiviral activity of MxA by accelerating cell death.