RESUMO
MicroRNAs (miRNAs) belonging to the same family have similar sequences and are difficult to identify. Herein, we report the reverse transcription-hairpin-probe-polymerase chain reaction (RT-Hpro-PCR) technique, which utilises a reverse transcription (RT) primer containing a 5'-end deoxyribonucleic acid (DNA) tag, to detect miRNAs with similar sequences. This strategy follows a two-step RT-PCR method using 6-7-mer RT-primers with a ~ 10-mer tag sequence at the 5'-end and a probe with a hairpin structure (Hpro), including two C-bulges, attached. The findings demonstrate that the specificity of RT could be increased by shortening the complementary part of the RT primer containing a different base, wherein the PCR could successfully progress with the use of 5'-end DNA tag because of an increase in the length of the hybridised tagged primer. This study shows the potential of RT-Hpro-PCR to precisely detect miRNAs with similar sequences, which could help explore the roles of miRNAs in several biological processes.
Assuntos
MicroRNAs/genética , Polimorfismo de Nucleotídeo Único/genética , Alelos , Humanos , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Loss of oligodendrocytes, the myelin-forming cells of the central nervous system, and subsequent failure of myelin development result in serious neurological disorders such as multiple sclerosis. Using primary mouse embryonic neural stem cells (NSCs), we previously demonstrated that donepezil, an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates the differentiation of NSCs into oligodendrocytes and neurons, albeit at the expense of astrogenesis. However, the precise mechanisms underlying donepezil-induced differentiation remain unclear. In this study, we aimed at elucidating the molecular pathways contributing to donepezil-induced differentiation of mouse-induced pluripotent stem cell-derived neural stem cells (miPSC-NSCs). We used cell-based reporter gene arrays to investigate effects of donepezil on differentiation of miPSC-NSCs. Subsequently, we assessed the molecular pathway underlying donepezil action on differentiation of miPSC-NSCs into mature oligodendrocytes. Donepezil increased the transcriptional activity of estrogen response element under differentiating conditions. Moreover, estrogen receptors α (ERα) and ß (ERß) were highly expressed in MBP-positive mature oligodendrocytes. The ER antagonist ICI 182,780 abrogated the number of MBP-positive oligodendrocytes induced by donepezil, but showed no effect on the differentiation of miPSC-NSCs into Tuj1-positive neurons and GFAP-positive astrocytes. Furthermore, the donepezil-induced generation of mature oligodendrocytes from miPSC-NSC was significantly attenuated by antagonists and siRNA targeting ERα and ERß. In conclusion, we demonstrated, for the first time, that donepezil-induced oligodendrogenesis is mediated through both ER subtypes, ERα and ERß. Cover Image for this issue: https://doi.org/10.1111/jnc.14771.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Donepezila/farmacologia , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Receptores de Estrogênio/metabolismo , Animais , Diferenciação Celular/fisiologia , Células Cultivadas , Inibidores da Colinesterase/farmacologia , Antagonistas do Receptor de Estrogênio/farmacologia , Fulvestranto/farmacologia , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Oligodendroglia/fisiologia , RNA Interferente Pequeno/administração & dosagem , Receptores de Estrogênio/antagonistas & inibidoresRESUMO
Overactivation of N-methyl-d-aspartate glutamate receptors (NMDARs) after traumatic brain injury (TBI) contributes to excitotoxic cell death. The hyperactivation of NMDARs results in toxic levels of intracellular Ca2+ and in the activation of p53-mediated apoptosis pathway. Neuronal Ca2+ -dependent activator protein 1 (NCDAP1) was identified as an epileptogenic gene of unknown function in our laboratory. In this study, we investigated the expression and cellular localization of NCDAP1 in rat models of fluid percussion-induced TBI. NCDAP1 expression increased in the ipsilateral cortex and hippocampus adjacent to the lesion of the TBI rats compared with that in the sham-operated controls. In addition, NCDAP1 was co-expressed with neuronal marker (NeuN), and the results of terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining suggest that NCDAP1 is involved in neuronal apoptosis that occurs after brain injury. In addition, the expression levels of p53, Bax, and active caspase-3 correlated with those of NCDAP1. To further investigate the function of NCDAP1, primary cultured neurons were employed to establish an apoptosis model. The expression of NCDAP1 was induced by NMDA-induced Ca2+ influx, and the knockdown of NCDAP1 by siRNA decreased apoptosis caused by treatment with NMDA. Silencing of NCDAP1 also reduced p53 expression, whereas the over-expression of NCDAP1 induced cell death and up-regulated the expression of p53. The inhibition of p53 with pifithrin alpha or siRNA counteracted the effects of NCDAP1. Based on our data, we suggest that NCDAP1 plays an important role in p53-mediated neuronal apoptosis following TBI.
Assuntos
Lesões Encefálicas Traumáticas/metabolismo , Calmodulina/biossíntese , Neurônios/metabolismo , Transdução de Sinais/fisiologia , Proteína Supressora de Tumor p53/biossíntese , Animais , Lesões Encefálicas Traumáticas/genética , Lesões Encefálicas Traumáticas/patologia , Calmodulina/genética , Morte Celular/fisiologia , Masculino , Neurônios/patologia , Ratos , Ratos Sprague-Dawley , Proteína Supressora de Tumor p53/genéticaRESUMO
Oligodendrocytes are the myelin-forming cells of the central nervous system. Oligodendrocyte loss and failure of myelin development result in serious human disorders, including multiple sclerosis. Previously, using oligodendrocyte progenitor cells, we have shown that donepezil, which is an acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease, stimulates myelin gene expression and oligodendrocyte differentiation. Here, we aimed to analyze the effects of donepezil on primary mouse embryonic neural stem cells (NSCs). Donepezil treatment led to impaired self-renewal ability and increased apoptosis. These effects appeared to be mediated through the Akt/Bad signaling pathway. Using neurosphere differentiation analysis, we observed that donepezil leads to reduced numbers of astrocytes and increased numbers of oligodendrocytes and neurons. Consistent with this finding, mRNA and protein levels for the oligodendrocyte markers myelin-associated glycoprotein, 2', 3'-cyclic-nucleotide 3'-phosphodiesterase (CNPase), and myelin basic protein, as well as the neuronal marker ß-tubulin type III (Tuj1) were up-regulated. In contrast, the expression of the astrocyte marker glial fibrillary acidic protein (GFAP) was down-regulated by donepezil in a dose- and time-dependent manner. Moreover, donepezil increased oligodendrocyte differentiation, resulting in a reduction in the differentiation of NSCs into astrocytes, by suppressing the activation of signal transducer and activator of transcription 3 (STAT3), SMAD1/5/9, and the downstream target gene GFAP, even under astrocyte-inducing conditions. These results suggest that efficient differentiation of NSCs into oligodendrocytes by donepezil may indicate a novel therapeutic role for this drug in promoting repair in demyelinated lesions in addition to its role in preventing astrogenesis.
Assuntos
Astrócitos/efeitos dos fármacos , Indanos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurogênese/efeitos dos fármacos , Oligodendroglia/efeitos dos fármacos , Piperidinas/farmacologia , Animais , Astrócitos/metabolismo , Células Cultivadas , Donepezila , Proteína Glial Fibrilar Ácida/metabolismo , Proteína Básica da Mielina/metabolismo , Células-Tronco Neurais/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Photodynamic control of gene delivery is a new technology with growing applications in gene therapy and basic cell research. Main approaches of light-selective gene delivery rely on the light-dependent enhancement of transfection efficiency. Studies focused on light-stimulated inhibitory regulation of transfection have rarely been reported. Here, we tried to establish a novel procedure of light-dependent inhibition of transfection. Our experiments, conducted with several types of commercial transfection reagents, revealed that jetPRIME-mediated transfection was strongly inhibited by blue light. Although the uptake of reagent-DNA complex was drastically reduced, preliminary exposure of cells or reagent-DNA complex to blue light had no inhibitory effect on the transfection efficiency. The inhibitory effect was wavelength-dependent and mediated by reactive oxygen species. Partial exposure of a culture vessel to blue light resulted in selective gene delivery into cells grown on the unexposed area of the vessel. By using this approach, different types of plasmid DNA were delivered into different areas in the culture vessel. This novel approach to the inhibitory control of transfection provides practical options for research and therapeutics. Biotechnol. Bioeng. 2016;113: 1560-1567. © 2015 Wiley Periodicals, Inc.
Assuntos
Biotecnologia/métodos , Técnicas de Transferência de Genes , Células HEK293 , Células HeLa , Humanos , LuzRESUMO
Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS). Failure of myelin development and oligodendrocyte loss results in serious human disorders, including multiple sclerosis. Here, we show that donepezil, an acetlycholinesterase inhibitor developed for the treatment of Alzheimer's disease, can stimulate oligodendrocyte differentiation and maturation of neural stem cell-derived oligodendrocyte progenitor cells without affecting proliferation or cell viability. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase, and MOG, in addition to transcription factors that regulate oligodendrocyte differentiation and myelination, were rapidly increased after treatment with donepezil. Furthermore, luciferase assays confirmed that both MAG and MBP promoters display increased activity upon donepezil-induced oligodendrocytes differentiation, suggesting that donepezil increases myelin gene expression mainly through enhanced transcription. We also found that the increase in the number of oligodendrocytes observed following donepezil treatment was significantly inhibited by the nicotinic acetylcholine receptor (nAChR) antagonist mecamylamine, but not by the muscarinic acetylcholine receptor antagonist scopolamine. Moreover, donepezil-induced myelin-related gene expression was suppressed by mecamylamine at both the mRNA and protein level. These results suggest that donepezil stimulates oligodendrocyte differentiation and myelin-related gene expression via nAChRs in neural stem cell-derived oligodendrocyte progenitor cells. We show that donepezil, a drug for the treatment of Alzheimer disease, can stimulate oligodendrocyte differentiation and maturation of oligodendrocyte progenitor cells. Transcripts for essential myelin-associated genes, such as PLP, MAG, MBP, CNPase and MOG in addition to transcripton factors that regulate oligodendrocyte differentiation and myelination were rapidly increased after treatment with donepezil. These effects were partly dependent on nicotinic acetylcholine receptor (nAChR).
Assuntos
Diferenciação Celular/efeitos dos fármacos , Indanos/farmacologia , Neurogênese/efeitos dos fármacos , Oligodendroglia/citologia , Oligodendroglia/efeitos dos fármacos , Piperidinas/farmacologia , Receptores Nicotínicos/metabolismo , Animais , Células Cultivadas , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Donepezila , Feminino , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Camundongos , Esclerose Múltipla/tratamento farmacológico , Esclerose Múltipla/metabolismo , Proteína Básica da Mielina/metabolismo , Receptores Nicotínicos/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/efeitos dos fármacosRESUMO
We previously demonstrated that indoxyl sulfate induces senescence and dysfunction of proximal tubular cells by activating p53 expression. However, little is known about the role of nuclear factor (NF)-κB in these processes. The present study examines whether activation (phosphorylation) of NF-κB by indoxyl sulfate promotes senescence and dysfunction in human proximal tubular cells (HK-2 cells). Indoxyl sulfate induced phosphorylation of NF-κB p65 on Ser-276, which was suppressed by N-acetylcysteine, an antioxidant. Furthermore, indoxyl sulfate induced NF-κB p65 expression. Inhibitors of NF-κB (pyrrolidine dithiocarbamate and isohelenin) and NF-κB p65 small interfering RNA (siRNA) suppressed indoxyl sulfate-induced senescence-associated ß-galactosidase activity and expression of p53, transforming growth factor (TGF)-ß1, and α-smoothe muscle actin (SMA). The induction of p53 expression and p53 promoter activity by indoxyl sulfate were inhibited by pifithrin-α, p-nitro, an inhibitor of p53, whereas p53-transfected cells showed enhanced p53 promoter activity. NF-κB inhibitors suppressed indoxyl sulfate-induced p21 expression, whereas NF-κB p65 siRNA enhanced its expression. NF-κB inhibitors partially alleviated indoxyl sulfate-induced inhibition of cellular proliferation. NF-κB p65 siRNA-transfected cells showed less proliferation in the presence of indoxyl sulfate than control cells. Phosphorylated NF-κB p65 was expressed and colocalized with p53, p21, ß-galactosidase, TGF-ß1, and α-SMA in the kidneys of chronic renal failure (CRF) rats. AST-120, which reduces serum indoxyl sulfate level, suppressed their expression in the CRF rat kidneys. Taken together, NF-κB plays an important role in indoxyl sulfate-induced cellular senescence, fibrotic gene expression, and inhibition of proliferation in proximal tubular cells. More notably, indoxyl sulfate accelerates proximal tubular cell senescence with progression of CRF through reactive oxygen species-NF-κB-p53 pathway.
Assuntos
Senescência Celular/fisiologia , Indicã/metabolismo , NF-kappa B/metabolismo , Actinas/metabolismo , Injúria Renal Aguda/tratamento farmacológico , Injúria Renal Aguda/metabolismo , Animais , Benzotiazóis/farmacologia , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Humanos , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , NF-kappa B/antagonistas & inibidores , Regiões Promotoras Genéticas , Pirrolidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Sesquiterpenos/farmacologia , Tiocarbamatos/farmacologia , Tolueno/análogos & derivados , Tolueno/farmacologia , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , beta-Galactosidase/metabolismoRESUMO
The liver has a considerable capacity of regeneration against the damage. The regulatory factors and molecular mechanism for the capacity are not fully appreciated. In developmental processes, hepatocyte nuclear factor 1ß (HNF1ß) is a cooperative factor for HNF6, which is a known stimulatory factor for hepatocyte proliferation after partial hepatectomy. We showed that carbon tetrachloride (CCl4)-induced liver injury up-regulated HNF1ß, whereas the expression of HNF6 was not affected by the chemical stress, indicating unknown physiological roles of HNF1ß against the chemical stress, not in cooperation with HNF6. To determine whether HNF1ß has a novel function in the liver regeneration, we overexpressed HNF1ß in the mouse liver by adenoviral gene delivery. We revealed that overexpression of HNF1ß resulted in accelerated cell proliferation with the protein level up-regulation of plasminogen and plasmin, a converted active form of plasminogen, which play a pivotal role in liver regeneration inducing hepatocyte proliferation. Despite this stimulatory effect for the liver regeneration, HNF1ß overexpression significantly increased genomic instability with decreased protein level of mediator of DNA damage checkpoint 1 (MDC1) and dephosphorylation of SP1 transcription factor. The increased expression of HNF1ß is associated with several types of hepatocyte carcinomas, indicating possible involvement of the factor in carcinogenesis. Our data extend the current understanding of the mechanism underlying liver regeneration against chemical stress, and identified HNF1ß as a novel regulatory factor in this mechanism and as a potential initiator for carcinogenesis.
Assuntos
Instabilidade Genômica , Fator 1-beta Nuclear de Hepatócito/metabolismo , Fígado/metabolismo , Fígado/patologia , Estresse Fisiológico , Animais , Tetracloreto de Carbono , Proliferação de Células , Vetores Genéticos , Hepatopatias/metabolismo , Hepatopatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Modelos BiológicosRESUMO
The processes involved in the adaptation of animals to environmental factors remain unclear. We examined the mechanisms underlying the adaptive potential of the mouse against hepatotoxic chemical-induced injury. Microarray analysis revealed that ethylbenzene, a hepatotoxic chemical, upregulated PDK4 (encoding pyruvate dehydrogenase kinase isoenzyme 4) in mouse livers and that the upregulation was enhanced by previous exposure to the chemical. Although PDK4 is an energy resource regulator induced by starvation, expression of other fasting-inducible genes was unaffected. PDK4 induced by chemical stress developed hepatic accumulation of sirtuin 1 by regulating pyruvate concentration and activated the Nbn and ATM, which are critical for DNA repair and checkpoint activation. PDK4 overexpression on carbon tetrachloride (CCl(4))-induced liver injury resulted in delayed necrotic tissue recovery with cell cycle arrest and decreased γH2AX foci and micronucleus formation. PDK4 silencing on CCl(4)-induced liver injury accelerated necrotic tissue recovery and increased γH2AX foci and micronucleus formation, indicating the essential role of PDK4 in DNA repair and checkpoint activation. PDK4 overexpression induced pancreas-specific transcription factor 1a (Ptf1a) upregulation and transcriptional activation of several pancreatic genes in the liver. Ptf1a overexpression by adenoviral gene delivery resulted in accelerated tissue recovery on CCl(4)-induced liver injury. Our data identified PDK4 as a novel pivotal factor in adaptation to chemical stress.
Assuntos
Adaptação Fisiológica/genética , Reparo do DNA/genética , Regulação Enzimológica da Expressão Gênica , Hepatopatias/enzimologia , Hepatopatias/patologia , Fígado/patologia , Proteínas Serina-Treonina Quinases/genética , Animais , Sequência de Bases , Ciclo Celular , Proliferação de Células , Genes de Troca , Células Hep G2 , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Pâncreas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Piruvatos/metabolismo , Sirtuína 1/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismoRESUMO
The application of a magnetic field to enhance the transfection efficiency has been reported to be mainly dependent on the magnetic force generated by a magnetic field gradient to attract paramagnetic bead-conjugated carrier and polynucleotide complexes. This strategy has the advantage of targeting a point or an area on the culture vessel. However, it is difficult to target deeply placed tissues in vivo. Uniform magnetic field-correlated effect is applicable to such a purpose. Here, we attempted to establish a novel procedure for uniform magnetic field-dependent enhancement of transfection efficiency. We examined the effect of a 1.5 mT uniform magnetic field on cellular reactive oxygen species (ROS) level and transfection efficiency mediated by a ROS-sensitive transfection carrier. Our experimental results revealed that a 1.5 mT uniform magnetic field transiently decreased cellular ROS levels and strongly enhanced transfection efficiency mediated by polyethylenimine (PEI). The uniform magnetic field-dependent enhancement of PEI-mediated in vivo transfection was confirmed in the livers of mice. Local intensification of a uniform magnetic field in a culture dish resulted in selective gene delivery into cells on the target area. Although further examination and improvement are necessary for this procedure, our findings provide a novel option for spatial control of gene delivery.
Assuntos
Técnicas de Transferência de Genes , Polietilenoimina , Animais , Terapia Genética , Campos Magnéticos , Camundongos , Plasmídeos , TransfecçãoRESUMO
Various uremic toxins accumulate in patients with chronic renal failure (CRF) and one of them is indoxyl sulfate, which accelerates the progression of CRF through unknown mechanisms. The present study investigates how indoxyl sulfate promotes CRF using the proximal tubular cell line HK-2 and CRF rats. Indoxyl sulfate inhibited serum-induced cell proliferation and promoted the activation of senescence-associated ß-galactosidase, a marker of cellular senescence, and the expression of α-smooth muscle actin (α-SMA), a marker of fibrosis, through inducing p53 expression and phosphorylation. Pifithrin-α, p-nitro, a p53 inhibitor, blocked these effects. Indoxyl sulfate evoked reactive oxygen species (ROS), and the antioxidant N-acetylcysteine inhibited indoxyl sulfate-induced p53 expression and phosphorylation, as well as indoxyl sulfate-induced α-SMA expression. We previously demonstrated that although cellular senescence and fibrosis are detectable in the kidneys of CRF rats, the oral adsorbent AST-120 repressed these effects. Here, we found that ß-galactosidase, p53 and α-SMA were expressed and colocalized in the renal tubules of CRF rats, whereas AST-120 decreased the expression of these genes. Taken together, these findings indicate that indoxyl sulfate induces the expression and phosphorylation of p53 though ROS production, thus inhibiting cell proliferation and promoting cellular senescence and renal fibrosis.
Assuntos
Linhagem Celular/efeitos dos fármacos , Senescência Celular/efeitos dos fármacos , Indicã/farmacologia , Túbulos Renais Proximais/efeitos dos fármacos , Túbulos Renais Proximais/fisiologia , Túbulos Renais Proximais/fisiopatologia , Proteína Supressora de Tumor p53/metabolismo , Acetilcisteína/farmacologia , Animais , Benzotiazóis/farmacologia , Proliferação de Células/efeitos dos fármacos , Senescência Celular/fisiologia , Fibrose/metabolismo , Humanos , Falência Renal Crônica/metabolismo , Falência Renal Crônica/fisiopatologia , Túbulos Renais Proximais/citologia , Masculino , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Espécies Reativas de Oxigênio/metabolismo , Tolueno/análogos & derivados , Tolueno/farmacologia , Proteína Supressora de Tumor p53/genéticaRESUMO
We used bacterial mutation and yeast genotoxicity tests to evaluate the effects of intermediate frequency (IF; 2 kHz, 20 kHz and 60 kHz) magnetic fields (MFs) on mutagenicity, co-mutagenicity and gene conversion. We constructed a Helmholtz type exposure system that generated vertical and sinusoidal IF MFs, such as 0.91 mT at 2 kHz, 1.1 mT at 20 kHz and 0.11 mT at 60 kHz. Mutagenicity, co-mutagenicity and gene conversion assays were performed for each of the three MF exposure conditions. Mutagenicity testing was performed in four strains of Salmonella typhimurium (TA98, TA100, TA1535 and TA1537) and two strains of Escherichia coli (WP2 uvrA and WP2 uvrA/pKM101) to cover a wide spectrum of point mutations. For co-mutagenicity tests, we used four sensitive test strains (TA98, TA100, WP2 uvrA and WP2 uvrA/pKM101) with five chemical mutagens (t-butyl hydroperoxide (BH, a hydroxyl free radical precursor), 2-(2-furyl)-3-(5-nitro-2-furyl) acrylamide (AF2) and N-ethyl-N'-nitro-N-nitrosoguanidine (ENNG, DNA reactive reagents), benz[a]pyrene (BaP) and 2-aminoanthracene (2AA, DNA reactive promutagens). Gene conversion testing was performed in the yeast test strain, Saccharomyces cerevisiae XD83. We also examined the effects on the repair process of DNA damage by UV irradiation. No statistically significant effects were observed between exposed and control groups in any of the genotoxicity tests, indicating that the IF MFs (0.91 mT at 2 kHz, 1.1 mT at 20 kHz or 0.11 mT at 60 kHz) do not have mutagenic or co-mutagenic potentials for the chemical mutagens tested under these experimental conditions. Our findings also indicate that these IF MFs do not induce gene conversion or affect the repair process of DNA damage in eukaryotic cells.
Assuntos
Conversão Gênica/efeitos da radiação , Magnetismo , Mutação/efeitos da radiação , Escherichia coli/genética , Escherichia coli/efeitos da radiação , Conversão Gênica/genética , Testes de Mutagenicidade/métodos , Mutação Puntual/efeitos dos fármacos , Salmonella typhimurium/genética , Salmonella typhimurium/efeitos da radiação , Leveduras/genética , Leveduras/efeitos da radiaçãoRESUMO
Neurochondrin is a cytoplasmic protein possibly involved in neurite outgrowth and chondrocyte differentiation. In the present study, we have identified 202 bp of the mouse neurochondrin minimal promoter sequences encompassing the transcriptional initiation site, and both of the activating and repressing regions in the first exon. These two regulatory regions in the first exon had a cell type dependent effect on the identified minimal promoter. In the regulatory region, the duplication of potential binding sites for GATA family transcriptional factors was observed. Prospective binding sites for sex determining region Y and c-Ets1 were also found in the minimal promoter region. These factors could be potential regulators for the mouse neurochondrin gene.
Assuntos
Proteínas do Tecido Nervoso/genética , Regiões Promotoras Genéticas/genética , Transcrição Gênica/genética , Células 3T3 , Animais , Sequência de Bases , Northern Blotting , Células Cultivadas , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fatores de Ligação de DNA Eritroide Específicos , Fator de Transcrição GATA2 , Regulação da Expressão Gênica , Camundongos , Dados de Sequência Molecular , Osteoblastos/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , TransfecçãoRESUMO
Neurochondrin is a brain and bone specific leucine-rich protein. We previously cloned the two types of mRNAs (neurochondrin-1; 729 amino acids and neurochondrin-2; 712 amino acids) from mouse and human species. As a first step, to better understand the mechanism of the bone and brain specific and developmentally regulated expression of the neruochondrin gene, the genomic organization of murine neurochondrin was determined. It consists of 7 exons and spans about 10 kb; all splice junctions conform to the GT/AG rule. It codes for two alternatively spliced messenger RNAs, neurochondrin-1 containing all 7 exons and neurochondrin-2 lacking exon 1b but containing the other exons. Cap site analysis showed that the major transcription initiation occurs at 765 bp upstream of the ATG start codon of neurochondrin-1. The promoter region has no TATA and CAAT box-like sequence but contains potential AP-1 and SP-1 binding sites. The neurochondrin gene is localized to mouse chromosome 4D1 and rat chromosome 5q36.11.
Assuntos
Proteínas do Tecido Nervoso/genética , Região 5'-Flanqueadora , Processamento Alternativo , Sequência de Aminoácidos , Animais , Bacteriófagos/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos , Códon de Iniciação , DNA Complementar , Éxons , Genoma , Hibridização in Situ Fluorescente , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Transcrição GênicaRESUMO
Neurochondrin is a novel cytoplasmic protein and possibly involved in neurite outgrowth, chondrocyte differentiation, and bone metabolism. Our previous trial in disclosing its role by the loss of function in mice failed because of the lethality in utero. In this study, we eliminated the neurochondrin gene expression preferentially in the nervous system by the conditional knockout strategy. Our results showed that neurochondrin is a negative regulator of Ca(2+)/calmodulin-dependent protein kinase II phosphorylation and essential for the spatial learning process but not for the differentiation or neurite outgrowth of the neuron. In addition, the nervous system-specific homozygous gene disruption resulted in epileptic seizure.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Epilepsia/etiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/fisiologia , Animais , Western Blotting , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Diferenciação Celular , Feminino , Expressão Gênica , Homozigoto , Aprendizagem/fisiologia , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas do Tecido Nervoso/deficiência , Neuritos/fisiologia , Neurônios/fisiologia , Neurônios/ultraestrutura , Fosforilação , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Percepção EspacialRESUMO
Neurochondrin/norbin is a cytoplasmic protein involved in dendrite outgrowth. The expression of the gene has been restricted to neural, bone, and chondral tissues. To identify the functions of the gene in vivo, we have generated mice with a disrupted mutation in the neurochondrin/norbin gene. Histological analysis of heterozygous mutant mice indicates the possibility of specific functions of neurochondrin/norbin in chondrocyte differentiation. We defined the expression patterns of neurochondrin/norbin-lacZ fusion protein in the central nervous system. In the developing olfactory bulb, beta-galactosidase activity was detected in the mantle layer at 12.5 dpc and the strongest activity was detected in the presumptive mitral or tufted cell layer at 15.5 dpc. beta-Galactosidase activity was also detected in the lateral choroid plexus. In homozygous (-/-) mutant mice, the disruption of the neurochondrin/norbin gene leads to early embryonic death between 3.5 and 6.5 dpc. This result indicates that neurochondrin/norbin gene function is essential for the early embryogenesis.