RESUMO
BACKGROUND: Dopamine neurotransmission plays a critical role in reward in drug abuse and drug addiction. However, the role of dopamine in the recognition of drug-associated environmental stimuli, retrieval of drug-associated memory, and drug-seeking behaviors is not fully understood. METHODS: Roles of dopamine neurotransmission in the prefrontal cortex (PFC) and nucleus accumbens (NAc) in the cocaine-conditioned place preference (CPP) paradigm were evaluated using in vivo microdialysis. RESULTS: In mice that had acquired cocaine CPP, dopamine levels in the PFC, but not in the NAc, increased in response to cocaine-associated cues when mice were placed in the cocaine chamber of an apparatus with 2 separated chambers. The induction of the dopamine response and the development of cocaine CPP were mediated through activation of glutamate NMDA (N-methyl-D-aspartate)/AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptor signaling in the PFC during conditioning. Activation of dopamine D1 or D2 receptor signaling in the PFC was required for cocaine-induced locomotion, but not for the induction of the dopamine response or the development of cocaine CPP. Interestingly, dopamine levels in the NAc increased in response to cocaine-associated cues when mice were placed at the center of an apparatus with 2 connected chambers, which requires motivated exploration associated with cocaine reward. CONCLUSIONS: Dopamine neurotransmission in the PFC is activated by the exposure to the cocaine-associated cues, whereas dopamine neurotransmission in the NAc is activated in a process of motivated exploration of cues associated with cocaine reward. Furthermore, the glutamate signaling cascade in the PFC is suggested to be a potential therapeutic target to prevent the progression of drug addiction.
Assuntos
Cocaína/farmacologia , Dopamina/metabolismo , Comportamento de Procura de Droga , Núcleo Accumbens/metabolismo , Córtex Pré-Frontal/metabolismo , Animais , Condicionamento Clássico , Sinais (Psicologia) , Inibidores da Captação de Dopamina/farmacologia , Masculino , Camundongos , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2 , Receptores de N-Metil-D-Aspartato , Recompensa , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiônicoRESUMO
We reported a rare case of intrascrotal lymphangioma in an adult. A 31-year-old man visited a urological clinic with a chief complaint of left scrotal swelling since a few days ago, and was pointed out to have a left intrascrotal cystic mass. The patient was sent to our hospital for further examination in 23 April 2008. The left scrotal mass was palpated elastic hard below the left testis and its surface was irregular. Light transillumination test showed positive. Ultrasonography revealed a cystic mass 7.0 x 4.4 x 4.5 cm with multiseptate accumulation at the lower pole of the left testis. Magnetic resonance imaging showed low intensity by T1WI and high intensity by T2WI, suggesting a protein-rich component. We suspected left intrascrotal lymphangioma and extirpated the scrotal mass under lumbar anesthesia. Pathological examination demonstrated lymphangioma. The patient had no evidence of recurrence after 1 year.
Assuntos
Neoplasias dos Genitais Masculinos/patologia , Linfangioma/patologia , Escroto , Adulto , Neoplasias dos Genitais Masculinos/cirurgia , Humanos , Linfangioma/cirurgia , MasculinoRESUMO
The transcription factor DeltaFosB is accumulated in the addiction circuitry, including the orbitofrontal and medial prefrontal cortices of rodents chronically exposed to ethanol or other drugs of abuse, and has been suggested to play a direct role in addiction maintenance. To address this hypothesis in the context of substance dependence in humans, we compared the immunoreactivities of FOSB proteins in the orbitofrontal and dorsolateral prefrontal cortices (OFC and DLPFC respectively) between controls and alcoholics using semiquantitative immunoblotting. In both structures, we detected three forms of FOSB, one of which was DeltaFOSB, but in neither case did their immunoreactivities differ between the groups. Our results indicate that the DeltaFOSB immunoreactivity in the human brain is very low, and that it is not accumulated in the OFC and DLPFC of human alcoholics, suggesting that it may not be directly involved in addiction maintenance, at least not in ethanol dependence.
Assuntos
Alcoolismo/patologia , Lobo Frontal/patologia , Córtex Pré-Frontal/patologia , Proteínas Proto-Oncogênicas c-fos/análise , Células HeLa , Humanos , Immunoblotting , Peso Molecular , Rede Nervosa/patologia , Valores de ReferênciaRESUMO
ΔFosB is a stable transcription factor which accumulates in the nucleus accumbens (NAc), a key part of the brain's reward circuitry, in response to chronic exposure to cocaine or other drugs of abuse. While ΔFosB is known to heterodimerize with a Jun family member to form an active transcription factor complex, there has not to date been an open-ended exploration of other possible binding partners for ΔFosB in the brain. Here, by use of yeast two-hybrid assays, we identify PSMC5-also known as SUG1, an ATPase-containing subunit of the 19S proteasomal complex-as a novel interacting protein with ΔFosB. We verify such interactions between endogenous ΔFosB and PSMC5 in the NAc and demonstrate that both proteins also form complexes with other chromatin regulatory proteins associated with gene activation. We go on to show that chronic cocaine increases nuclear, but not cytoplasmic, levels of PSMC5 in the NAc and that overexpression of PSMC5 in this brain region promotes the locomotor responses to cocaine. Together, these findings describe a novel mechanism that contributes to the actions of ΔFosB and, for the first time, implicates PSMC5 in cocaine-induced molecular and behavioral plasticity.
Assuntos
Transtornos Relacionados ao Uso de Cocaína/fisiopatologia , Núcleo Accumbens/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Animais , Encéfalo/metabolismo , Linhagem Celular Tumoral , Cocaína/administração & dosagem , DNA Helicases/metabolismo , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/metabolismo , Núcleo Accumbens/fisiopatologia , Fosfoproteínas/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
We have previously demonstrated that endothelin-1 (ET-1)-induced extracellular signal-regulated kinase (Erk) activity via the ETB receptor (EDNRB) is mediated through two independent pathways, a protein kinase C-dependent pathway and a pertussis toxin (PTX)-sensitive pathway, in astrocytes. In this study, we showed that the molar potency of ET-1 to induce Erk activation was two orders of magnitude higher in dibutyryl cAMP (DBcAMP)-treated astrocytes than in quiescent astrocytes. This DBcAMP-enhanced molar potency of ET-1 in Erk activation was selectively inhibited by pretreatment of astrocytes with PTX. The expression level of EDNRB in astrocytes was markedly upregulated by DBcAMP-induced cytodifferentiation. However, this up-regulation was simply attributed to the high expression of low-affinity sites. The molar potency of ET-1 to induce both stimulation of inositol trisphosphate production and activation of protein kinase C in DBcAMP-treated astrocytes was similar to that in quiescent astrocytes. On the contrary, the molar potency of ET-1 to induce accumulation of Ras-GTP was two orders of magnitude higher in DBcAMP-treated astrocytes than in quiescent astrocytes, which was consistent with the case of ET-1-induced Erk activation. Moreover, the ET-1-induced Ras activation was PTX sensitive. These results suggest that cytodifferentiation selectively enhances the PTXsensitive Ras/Erk pathway induced by ET-1 in astrocytes, and that cytodifferentiation-induced EDNRB up-regulation might not contribute to this selective potentiation of ET-1 signaling.
Assuntos
Astrócitos/enzimologia , Diferenciação Celular , Endotelina-1/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Transdução de Sinais , Animais , Astrócitos/efeitos dos fármacos , Bucladesina/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Inositol 1,4,5-Trifosfato/metabolismo , Proteína Básica da Mielina/metabolismo , Toxina Pertussis/farmacologia , Fosforilação , Proteína Quinase C/metabolismo , Ratos , Receptor de Endotelina B/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas ras/metabolismoRESUMO
Patients with epilepsy are at high risk for major depression relative to the general population, and both disorders are associated with changes in adult hippocampal neurogenesis, although the mechanisms underlying disease onset remain unknown. The expression of fosB, an immediate early gene encoding FosB and ΔFosB/Δ2ΔFosB by alternative splicing and translation initiation, is known to be induced in neural progenitor cells within the subventricular zone of the lateral ventricles and subgranular zone of the hippocampus, following transient forebrain ischemia in the rat brain. Moreover, adenovirus-mediated expression of fosB gene products can promote neural stem cell proliferation. We recently found that fosB-null mice show increased depressive behavior, suggesting impaired neurogenesis in fosB-null mice. In the current study, we analyzed neurogenesis in the hippocampal dentate gyrus of fosB-null and fosB(d/d) mice that express ΔFosB/Δ2ΔFosB but not FosB, in comparison with wild-type mice, alongside neuropathology, behaviors, and gene expression profiles. fosB-null but not fosB(d/d) mice displayed impaired neurogenesis in the adult hippocampus and spontaneous epilepsy. Microarray analysis revealed that genes related to neurogenesis, depression, and epilepsy were altered in the hippocampus of fosB-null mice. Thus, we conclude that the fosB-null mouse is the first animal model to provide a genetic and molecular basis for the comorbidity between depression and epilepsy with abnormal neurogenesis, all of which are caused by loss of a single gene, fosB.
Assuntos
Depressão/genética , Epilepsia/genética , Hipocampo/patologia , Mutação/genética , Neurogênese/genética , Proteínas Proto-Oncogênicas c-fos/deficiência , Animais , Bromodesoxiuridina/metabolismo , Proliferação de Células/efeitos dos fármacos , Depressão/complicações , Modelos Animais de Doenças , Proteínas do Domínio Duplacortina , Eletroencefalografia , Epilepsia/complicações , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Ácido Caínico/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Análise em Microsséries , Proteínas Associadas aos Microtúbulos/metabolismo , Neuropeptídeos/metabolismo , Fosfopiruvato Hidratase/metabolismo , Proteínas Proto-Oncogênicas c-fos/genéticaRESUMO
ΔFosB protein accumulates in the striatum in response to chronic administration of drugs of abuse, L-DOPA, or stress, triggering long lasting neural and behavioral changes that underlie aspects of drug addiction, abnormal involuntary movements (dyskinesia), and depression. ΔFosB binds AP-1 DNA consensus sequences found in promoters of many genes and can both repress or activate gene transcription. In the striatum, ΔFosB is thought to dimerize with JunD to form a functional transcription factor, though strikingly JunD does not accumulate in parallel. One explanation is that ΔFosB can recruit different partners, including itself, depending on the neuron type in which it is induced and the chronic stimulus, generating protein complexes with different effects on gene transcription. To develop chemical probes to study ΔFosB, a high-throughput screen was carried out to identify small molecules that modulate ΔFosB function. Two compounds with low micromolar activity, termed C2 and C6, disrupt the binding of ΔFosB to DNA via different mechanisms, and in in vitro assays stimulate ΔFosB-mediated transcription. In cocaine-treated mice, C2 significantly elevates mRNA levels of the AMPA glutamate receptor GluR2 subunit with specificity, a known target gene of ΔFosB that plays a role in drug addiction and endogenous resilience mechanisms. C2 and C6 show different activities against ΔFosB homodimers compared to ΔFosB/JunD heterodimers, suggesting that these compounds can be used as probes to study the contribution of different ΔFosB-containing complexes on the regulation of gene transcription in biological systems and to assess the utility of ΔFosB as a therapeutic target.
Assuntos
Preparações Farmacêuticas/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Avaliação Pré-Clínica de Medicamentos/métodos , Insetos , Camundongos , Preparações Farmacêuticas/metabolismo , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas c-fos/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-fos/fisiologia , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/fisiologiaRESUMO
Brain-derived neurotrophic factor (BDNF) is a key positive regulator of neural plasticity, promoting, for example, the actions of stimulant drugs of abuse such as cocaine. We discovered a surprising opposite role for BDNF in countering responses to chronic morphine exposure. The suppression of BDNF in the ventral tegmental area (VTA) enhanced the ability of morphine to increase dopamine (DA) neuron excitability and promote reward. In contrast, optical stimulation of VTA DA terminals in nucleus accumbens (NAc) completely reversed the suppressive effect of BDNF on morphine reward. Furthermore, we identified numerous genes in the NAc, a major target region of VTA DA neurons, whose regulation by BDNF in the context of chronic morphine exposure mediated this counteractive function. These findings provide insight into the molecular basis of morphine-induced neuroadaptations in the brain's reward circuitry.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/fisiologia , Neurônios Dopaminérgicos/efeitos dos fármacos , Dependência de Morfina/fisiopatologia , Morfina/farmacologia , Área Tegmentar Ventral/efeitos dos fármacos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Dopamina/metabolismo , Neurônios Dopaminérgicos/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dependência de Morfina/genética , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/fisiopatologia , Estimulação Luminosa , Receptor trkB/genética , Receptor trkB/fisiologia , Área Tegmentar Ventral/fisiologiaRESUMO
Repeated cocaine administration increases the dendritic arborization of nucleus accumbens neurons, but the underlying signaling events remain unknown. Here we show that repeated exposure to cocaine negatively regulates the active form of Rac1, a small GTPase that controls actin remodeling in other systems. Further, we show, using viral-mediated gene transfer, that overexpression of a dominant negative mutant of Rac1 or local knockout of Rac1 is sufficient to increase the density of immature dendritic spines on nucleus accumbens neurons, whereas overexpression of a constitutively active Rac1 or light activation of a photoactivatable form of Rac1 blocks the ability of repeated cocaine exposure to produce this effect. Downregulation of Rac1 activity likewise promotes behavioral responses to cocaine exposure, with activation of Rac1 producing the opposite effect. These findings establish that Rac1 signaling mediates structural and behavioral plasticity in response to cocaine exposure.
Assuntos
Cocaína/farmacologia , Espinhas Dendríticas/efeitos dos fármacos , Inibidores da Captação de Dopamina/farmacologia , Plasticidade Neuronal/efeitos dos fármacos , Neuropeptídeos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Western Blotting , Transtornos Relacionados ao Uso de Cocaína , Espinhas Dendríticas/metabolismo , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neuropeptídeos/genética , Núcleo Accumbens/efeitos dos fármacos , Núcleo Accumbens/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais/fisiologia , Proteínas rac de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTPRESUMO
BACKGROUND: Molecular mechanisms underlying stress tolerance and vulnerability are incompletely understood. The fosB gene is an attractive candidate for regulating stress responses, because ΔFosB, an alternative splice product of the fosB gene, accumulates after repeated stress or antidepressant treatments. On the other hand, FosB, the other alternative splice product of the fosB gene, expresses more transiently than ΔFosB but exerts higher transcriptional activity. However, the functional differences of these two fosB products remain unclear. METHODS: We established various mouse lines carrying three different types of fosB allele, wild-type (fosB(+)), fosB-null (fosB(G)), and fosB(d) allele, which encodes ΔFosB but not FosB, and analyzed them in stress-related behavioral tests. RESULTS: Because fosB(+/d) mice show enhanced ΔFosB levels in the presence of FosB and fosB(d/d) mice show more enhanced ΔFosB levels in the absence of FosB, the function of FosB can be inferred from differences observed between these lines. The fosB(+/d) and fosB(d/d) mice showed increased locomotor activity and elevated Akt phosphorylation, whereas only fosB(+/d) mice showed antidepressive-like behaviors and increased E-cadherin expression in striatum compared with wild-type mice. In contrast, fosB-null mice showed increased depression-like behavior and lower E-cadherin expression. CONCLUSIONS: These findings indicate that FosB is essential for stress tolerance mediated by ΔFosB. These data suggest that fosB gene products have a potential to regulate mood disorder-related behaviors.
Assuntos
Adaptação Psicológica/fisiologia , Comportamento Exploratório/fisiologia , Atividade Motora/fisiologia , Proteínas Proto-Oncogênicas c-fos/fisiologia , Estresse Psicológico/fisiopatologia , Animais , Caderinas/biossíntese , Corpo Estriado/metabolismo , Dopamina/fisiologia , Masculino , Aprendizagem em Labirinto/fisiologia , Camundongos , Camundongos Mutantes , Atividade Motora/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Estresse Psicológico/genética , Estresse Psicológico/metabolismoRESUMO
In contrast with the many studies of stress effects on the brain, relatively little is known about the molecular mechanisms of resilience, the ability of some individuals to escape the deleterious effects of stress. We found that the transcription factor DeltaFosB mediates an essential mechanism of resilience in mice. Induction of DeltaFosB in the nucleus accumbens, an important brain reward-associated region, in response to chronic social defeat stress was both necessary and sufficient for resilience. DeltaFosB induction was also required for the standard antidepressant fluoxetine to reverse behavioral pathology induced by social defeat. DeltaFosB produced these effects through induction of the GluR2 AMPA glutamate receptor subunit, which decreased the responsiveness of nucleus accumbens neurons to glutamate, and through other synaptic proteins. Together, these findings establish a previously unknown molecular pathway underlying both resilience and antidepressant action.
Assuntos
Antidepressivos/farmacologia , Núcleo Accumbens/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Resiliência Psicológica , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/metabolismo , Animais , Doença Crônica , Dominação-Subordinação , Fluoxetina/farmacologia , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Núcleo Accumbens/efeitos dos fármacos , Receptores de AMPA/metabolismo , Recompensa , Transdução de Sinais , Resultado do TratamentoRESUMO
Among fos family genes encoding components of activator protein-1 complex, only the fosB gene produces two forms of mature transcripts, namely fosB and DeltafosB mRNAs, by alternative splicing of an exonic intron. The former encodes full-length FosB. The latter encodes DeltaFosB and Delta2DeltaFosB by alternative translation initiation, and both of these lack the C-terminal transactivation domain of FosB. We established two mutant mouse embryonic stem (ES) cell lines carrying homozygous fosB-null alleles and fosB(d) alleles, the latter exclusively encoding DeltaFosB/Delta2DeltaFosB. Comparison of their gene expression profiles with that of the wild type revealed that more than 200 genes were up-regulated, whereas 19 genes were down-regulated in a DeltaFosB/Delta2DeltaFosB-dependent manner. We furthermore found that mRNAs for basement membrane proteins were significantly up-regulated in fosB(d/d) but not fosB-null mutant cells, whereas genes involved in the TGF-beta1 signaling pathway were up-regulated in both mutants. Cell-matrix adhesion was remarkably augmented in fosB(d/d) ES cells and to some extent in fosB-null cells. By analyzing ES cell lines carrying homozygous fosB(FN) alleles, which exclusively encode FosB, we confirmed that FosB negatively regulates cell-matrix adhesion and the TGF-beta1 signaling pathway. We thus concluded that FosB and DeltaFosB/Delta2DeltaFosB use this pathway to antagonistically regulate cell matrix adhesion.
Assuntos
Processamento Alternativo/genética , Junções Célula-Matriz/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , Alelos , Animais , Membrana Basal/metabolismo , Adesão Celular , Linhagem Celular , Células-Tronco Embrionárias/metabolismo , Perfilação da Expressão Gênica , Marcação de Genes , Camundongos , Modelos Biológicos , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-fos/deficiência , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Regulação para CimaRESUMO
The transcription factor DeltaFosB (Delta FosB) accumulates in a region-specific manner in the brain during chronic exposure to stress, drugs of abuse or other chronic stimuli. Once induced, DeltaFosB persists in the brain for at least several weeks following cessation of the chronic stimulus. The biochemical basis of the persistent expression of DeltaFosB has remained unknown. Here, we show that the FosB C-terminus, absent in DeltaFosB as a result of alternative splicing, contains two degron domains. Pulse-chase experiments of C-terminal truncation mutants of full-length FosB indicate that removal of its most C-terminal degron increases its half-life approximately fourfold, and prevents its proteasome-mediated degradation and ubiquitylation, properties similar to DeltaFosB. In addition, removal of a second degron domain, which generates DeltaFosB, further stabilizes FosB approximately twofold, but in a proteasome-independent manner. These data indicate that alternative splicing specifically removes two destabilizing elements from FosB in order to generate a longer-lived transcription factor, DeltaFosB, in response to chronic perturbations to the brain.
Assuntos
Processamento Alternativo/genética , Encéfalo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Estresse Fisiológico/metabolismo , Animais , Encéfalo/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Células PC12 , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína/fisiologia , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/genética , Ratos , Estresse Fisiológico/fisiopatologiaRESUMO
To evaluate the antimutagenic role of a mammalian mutY homolog, namely the Mutyh gene, which encodes adenine DNA glycosylase excising adenine misincorporated opposite 8-oxoguanine in the template DNA, we generated MUTYH-null mouse embryonic stem (ES) cells. In the MUTYH-null cells carrying no adenine DNA glycosylase activity, the spontaneous mutation rate increased 2-fold in comparison with wild type cells. The expression of wild type mMUTYH or mutant mMUTYH protein with amino acid substitutions at the proliferating cell nuclear antigen binding motif restored the increased spontaneous mutation rates of the MUTYH-null ES cells to the wild type level. The expression of a mutant mMUTYH protein with an amino acid substitution (G365D) that corresponds to a germ-line mutation (G382D) found in patients with multiple colorectal adenomas could not suppress the elevated spontaneous mutation rate of the MUTYH-null ES cells. Although the recombinant mMUTYH(G365D) purified from Escherichia coli cells had a substantial level of adenine DNA glycosylase activity as did wild type MUTYH, no adenine DNA glycosylase activity was detected in the MUTYH-null ES cells expressing the mMUTYH(G365D) mutant protein. The germ-line mutation (G382D) of the human MUTYH gene is therefore likely to be responsible for the occurrence of a mutator phenotype in these patients.
Assuntos
DNA Glicosilases , Embrião de Mamíferos/citologia , Mutação , N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/genética , Células-Tronco/citologia , Motivos de Aminoácidos , Animais , Western Blotting , Análise Mutacional de DNA , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Vetores Genéticos , Mutação em Linhagem Germinativa , Camundongos , Modelos Genéticos , Mutagênese Sítio-Dirigida , N-Glicosil Hidrolases/metabolismo , Fenótipo , Antígeno Nuclear de Célula em Proliferação/química , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
The present study was designed to elucidate the role of p38 mitogen-activated protein kinase (p38) in the pathogenesis of inflammation, using a mouse contact hypersensitivity (CHS) model induced by 2,4-dinitro-1-fluorobenzene (DNFB). Ear swelling was induced by challenge with DNFB, accompanied by infiltration of mononuclear cells, neutrophils, and eosinophils and a marked increase in mRNA levels of cytokines such as interleukin (IL)-2, interferon (IFN)-gamma, IL-4, IL-5, IL-1beta, IL-18, and tumor necrosis factor-alpha in the challenged ear skin. Both ear swelling and the number of infiltrated cells in DNFB-challenged ear skin were significantly inhibited by treatment with SB202190, a p38 inhibitor. Furthermore, the DNFB-induced expression of all cytokines except IL-4 was significantly inhibited by treatment with SB202190. Ribonuclease protection assay revealed that the mRNA levels of chemokines such as IP-10 and MCP-1 in ear skin were markedly increased at 24 h after challenge with DNFB. The induction of these chemokines was significantly inhibited by treatment with SB202190. In p38alpha +/- mice, both ear swelling and infiltration of cells induced by DNFB were reduced compared with those in wild-type mice. However, induction of cytokines by DNFB was also observed in p38alpha +/- mice, although the induction of IFN-gamma, IL-5, and IL-18 was typically reduced compared with that in wild-type mice. Challenge with DNFB slightly induced IP-10 and MCP-1 mRNA in p38alpha +/- mice, with weaker signals than those in SB202190-treated wild-type mice. These results suggest that p38 plays a key role in CHS and is an important target for the treatment of CHS.