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1.
Gene ; 806: 145920, 2022 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-34455026

RESUMO

Depression is deemed a mood disorder characterized by a high rate of relapse. Therefore, overcoming of the recurrent depression is globally expecting. Kososan, a traditional Japanese herbal medicine, has been clinically used for mild depressive mood, and our previous studies have shown some evidence for its antidepressive-like efficacy in experimental animal models of depression. However, it remains unclear whether kososan has beneficial effects on recurrent depression. Here, we examined its effect using a mouse model of modified repeated social defeat stress (SDS) paradigm. Male BALB/c mice were exposed to a 5-min SDS from unfamiliar aggressive CD-1 mice for 5 days. Kososan extract (1.0 kg/kg/day) or an antidepressant milnacipran (60 mg/kg/day) was administered orally for 26 days (days 7-32) to depression-like mice with social avoidant behaviors on day 6. Single 5 min of SDS was subjected to mice recovered from the social avoidance on day 31, and then the recurrence of depression-like behaviors was evaluated on day 32. Hippocampal gene expression patterns were also assayed by DNA microarray analysis. Water- or milnacipran-administered mice resulted in a recurrence of depression-like behaviors by re-exposure of single SDS, whereas kososan-administered mice did not recur depression-like behaviors. Distinct gene expression patterns were also found for treating kososan and milnacipran. Collectively, this finding suggests that kososan exerts a preventive effect on recurrent depression-like behaviors in mice. Pretreatment of kososan is more useful for recurrent depression than that of milnacipran.


Assuntos
Antidepressivos/farmacologia , Depressão/prevenção & controle , Medicamentos de Ervas Chinesas/farmacologia , Proteínas do Tecido Nervoso/genética , Derrota Social , Estresse Psicológico/tratamento farmacológico , Administração Oral , Animais , Depressão/genética , Depressão/fisiopatologia , Depressão/psicologia , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Japão , Masculino , Medicina Kampo/métodos , Camundongos , Camundongos Endogâmicos BALB C , Milnaciprano/farmacologia , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/classificação , Proteínas do Tecido Nervoso/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Recidiva , Estresse Psicológico/genética , Estresse Psicológico/fisiopatologia
2.
Genes Cells ; 24(2): 151-161, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30570184

RESUMO

Small Ras-like GTPases act as molecular switches for various signal transduction pathways. RagA, RagB/RagC and RagD are small Ras-like GTPases that play regulatory roles in mTORC1. Lack of proper activation of mTORC1 can lead to diseases, such as cancer and diabetes. In this study, we found an interaction between RagA and WDR35. Mutations of WDR35 may cause genetic diseases including Sensenbrenner syndrome. WDR35 seems to be a hedgehog signaling protein with a possible ciliary function and a possible upstream regulator of RagA. RagB is a homologue of RagA and is also associated with WDR35. WDR35 is present in the endoplasmic reticulum, but usually not in lysosomes, where Rag family proteins act as an mTORC1 switch. Over-expression of WDR35 results in decreased phosphorylation of ribosome S6 protein in a RagA-, RagB- and RagC-dependent manner. Thus, WDR35 is associated with RagA, RagB and RagC and might negatively influence mTORC1 activity.


Assuntos
Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas/metabolismo , Proteínas do Citoesqueleto , Células HEK293 , Proteínas Hedgehog , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Lisossomos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/genética , Proteínas Monoméricas de Ligação ao GTP/genética , Complexos Multiproteicos/genética , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/genética , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido
3.
J Neuroinflammation ; 14(1): 98, 2017 05 03.
Artigo em Inglês | MEDLINE | ID: mdl-28468634

RESUMO

BACKGROUND: Kososan, a Kampo (traditional Japanese herbal) medicine, has been used for the therapy of depressive mood in humans. However, evidence for the antidepressant efficacy of kososan and potential mechanisms are lacking. Recently, it has been recognized that stress triggers neuroinflammation and suppresses adult neurogenesis, leading to depression and anxiety. Here, we examined whether kososan extract affected social behavior in mice exposed to chronic social defeat stress (CSDS), an animal model of prolonged psychosocial stress, and neuroinflammation induced by CSDS. METHODS: In the CSDS paradigm, C57BL/6J mice were exposed to 10 min of social defeat stress from an aggressive CD-1 mouse for 10 consecutive days (days 1-10). Kososan extract (1.0 g/kg) was administered orally once daily for 12 days (days 1-12). On day 11, the social avoidance test was performed to examine depressive- and anxious-like behaviors. To characterize the impacts of kososan on neuroinflammation and adult neurogenesis, immunochemical analyses and ex vivo microglial stimulation assay with lipopolysaccharide (LPS) were performed on days 13-15. RESULTS: Oral administration of kososan extract alleviated social avoidance, depression- and anxiety-like behaviors, caused by CSDS exposure. CSDS exposure resulted in neuroinflammation, as indicated by the increased accumulation of microglia, the resident immune cells of the brain, and their activation in the hippocampus, which was reversed to normal levels by treatment with kososan extract. Additionally, in ex vivo studies, CSDS exposure potentiated the microglial pro-inflammatory response to a subsequent LPS challenge, an effect that was also blunted by kososan extract treatment. Indeed, the modulatory effect of kososan extract on neuroinflammation appears to be due to a hippocampal increase in an anti-inflammatory phenotype of microglia while sparing an increased pro-inflammatory phenotype of microglia caused by CSDS. Moreover, reduced adult hippocampal neurogenesis in defeated mice was recovered by kososan extract treatment. CONCLUSIONS: Our findings suggest that kososan extract prevents a social avoidant behavior in socially defeated mice that is partially mediated by the downregulation of hippocampal neuroinflammation, presumably by the relative increased anti-inflammatory microglia and regulation of adult hippocampal neurogenesis. Our present study also provides novel evidence for the beneficial effects of kososan on depression/anxiety and the possible underlying mechanisms.


Assuntos
Aprendizagem da Esquiva/efeitos dos fármacos , Medicamentos de Ervas Chinesas/farmacologia , Mediadores da Inflamação/antagonistas & inibidores , Medicina Kampo , Extratos Vegetais/farmacologia , Comportamento Social , Animais , Aprendizagem da Esquiva/fisiologia , Medicamentos de Ervas Chinesas/isolamento & purificação , Medicamentos de Ervas Chinesas/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/uso terapêutico , Estresse Psicológico/tratamento farmacológico , Estresse Psicológico/metabolismo , Estresse Psicológico/patologia
4.
Cell J ; 17(4): 692-700, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26862528

RESUMO

OBJECTIVE: Neutrophils have an important role in the rapid innate immune response, and the release or active secretion of elastase from neutrophils is linked to various inflammatory responses. Purpose of this study was to determine how the human neutrophil elastase affects the interleukin-10 (IL-10) response in peripheral blood mononuclear cells (PBMC). MATERIALS AND METHODS: In this prospective study, changes in IL-10 messenger RNA (mRNA) and protein expression levels in monocytes derived from human PBMCs were investigated after stimulation with human neutrophil elastase (HNE). A set of inhibitors was used for examining the pathways for IL-10 production induced by HNE. RESULTS: Reverse transcription polymerase chain reaction (RT-PCR) showed that stimulation with HNE upregulated IL-10 mRNA expression by monocytes, while the enzyme-linked immunosorbent assay (ELISA) revealed an increase of IL-10 protein level in the culture medium. A phospholipase C inhibitor (U73122) partially blunt- ed the induction of IL-10 mRNA expression by HNE, while IL-10 mRNA expression was significantly reduced by a protein kinase C (PKC) inhibitor (Rottlerin). A calcium chelator (3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester: TMB-8) inhibited the response of IL-10 mRNA to stimulation by HNE. In addition, pretreatment with a broad-spectrum PKC inhibitor (Ro-318425) partly blocked the response to HNE. Finally, an inhibitor of PKC theta/delta abolished the increased level of IL-10 mRNA expression. CONCLUSION: These results indicate that HNE mainly upregulates IL-10 mRNA ex- pression and protein production in moncytes via a novel PKC theta/delta, although partially via the conventional PKC pathway.

5.
Blood Cells Mol Dis ; 55(2): 127-33, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26142328

RESUMO

Neutrophil extracellular traps (NETs) have an important role in antimicrobial innate immunity and release substances that may modulate the immune response. We investigated the effects of soluble factors from NETs and neutrophil granule proteins on human monocyte function by using the Transwell system to prevent cell-cell contact. NET formation was induced by exposing human neutrophils to phorbol myristate acetate (PMA). When monocytes were incubated with PMA alone, expression of interleukin (IL)-4, IL-6, IL-8, and tumor necrosis factor (TNF)-alpha mRNA was upregulated, but IL-10, IL-12, and interferon (IFN)-gamma mRNA were not detected. Incubation of monocytes with NETs enhanced the expression of IL-10 and IFN-gamma mRNA, but not IL-12 mRNA. Myeloperoxidase stimulated IFN-gamma production by monocytes in a dose-dependent manner. Both a nuclear factor-kappaB inhibitor (PDTC) and an intracellular calcium antagonist (TMB-8) prevented upregulation of IFN-gamma production. Neither a combined p38alpha and p38beta inhibitor (SB203580) nor an extracellular signal-regulated kinase inhibitor (PD98059) suppressed IFN-gamma production. Interestingly, a combined p38gamma and p38delta inhibitor (BIRB796) significantly decreased IFN-gamma production. These findings suggest that myeloperoxidase induces IFN-gamma production by monocytes via p38gamma/delta mitogen-activated protein kinase.


Assuntos
Armadilhas Extracelulares/metabolismo , Interferon gama/biossíntese , Monócitos/metabolismo , Neutrófilos/metabolismo , Peroxidase/metabolismo , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Inibidores Enzimáticos/farmacologia , Armadilhas Extracelulares/imunologia , Expressão Gênica , Humanos , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Neutrófilos/imunologia
6.
Blood Cells Mol Dis ; 55(1): 21-6, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25976462

RESUMO

BACKGROUND: Granulocyte-macrophage colony-stimulating factor (GM-CSF) promotes classically activated M1 macrophages. GM-CSF upregulates protease-activated receptor-2 (PAR-2) protein expression and activation of PAR-2 by human neutrophil elastase (HNE) regulates cytokine production. AIM: This study investigated the mechanism of PAR-2-mediated interleukin (IL)-13 production by GM-CSF-dependent macrophages stimulated with HNE. METHODS: Adherent macrophages were obtained from primary cultures of human mononuclear cells. After stimulation with HNE to activate the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway, IL-13 mRNA and protein levels were assessed by the reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: PAR-2 protein was detected in GM-CSF-dependent macrophages by Western blotting. Unexpectedly, PD98059 (an ERK1 inhibitor) increased IL-13 production, even at higher concentrations. Interestingly, U0126 (an ERK1/2 inhibitor) reduced IL-13 production in a concentration-dependent manner. Neither SB203580 (a p38alpha/p38beta inhibitor) nor BIRB796 (a p38gamma/p38delta inhibitor) affected IL-13 production, while TMB-8 (a calcium chelator) diminished IL-13 production. DISCUSSION: Stimulation with HNE promoted the production of IL-13 (a Th2 cytokine) by GM-CSF-dependent M1 macrophages. PAR-2-mediated IL-13 production may be dependent on the Ca(2+)/ERK2 signaling pathway.


Assuntos
Cálcio/metabolismo , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-13/genética , Macrófagos/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/imunologia , Receptor PAR-2/genética , Butadienos/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Adesão Celular , Diferenciação Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Flavonoides/farmacologia , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Interleucina-13/imunologia , Elastase de Leucócito/farmacologia , Macrófagos/citologia , Macrófagos/imunologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/imunologia , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/imunologia , Naftalenos/farmacologia , Nitrilas/farmacologia , Cultura Primária de Células , Inibidores de Proteínas Quinases/farmacologia , Pirazóis/farmacologia , Piridinas/farmacologia , Receptor PAR-2/imunologia , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno
7.
Blood Cells Mol Dis ; 54(4): 353-9, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25633855

RESUMO

Chronic inflammation is often linked to the presence of type 2-polarized macrophages, which are induced by the T helper type 2 cytokines interleukin-4 and interleukin-13 (IL-13). IL-13 is a key mediator of tissue fibrosis caused by T helper type 2-based inflammation. Human neutrophil elastase (HNE) plays a pivotal role in the pathogenesis of pulmonary fibrosis. This study investigated the priming effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on IL-13 expression by macrophages stimulated with HNE. Adherent macrophages were obtained from primary cultures of human mononuclear cells. Expression of IL-13 mRNA and protein by GM-CSF-dependent macrophages was investigated after stimulation with HNE, using the polymerase chain reaction and enzyme-linked immunosorbent assay. GM-CSF had a priming effect on IL-13 mRNA and protein expression by macrophages stimulated with HNE, while this effect was not observed for various other cytokines. GM-CSF-dependent macrophages showed a significant increase in the expression of protease activated receptor-2 (PAR-2) mRNA and protein. The response of IL-13 mRNA to HNE was significantly decreased by pretreatment with alpha1-antitrypsin, a PAR-2 antibody (SAM11), or a PAR-2 antagonist (ENMD-1068). These findings suggest that stimulation with HNE can induce IL-13 production by macrophages, especially GM-CSF-dependent macrophages. Accordingly, neutrophil elastase may have a key role in fibrosis associated with chronic inflammation.


Assuntos
Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Interleucina-13/genética , Elastase de Leucócito/farmacologia , Macrófagos/efeitos dos fármacos , RNA Mensageiro/genética , Receptor PAR-2/genética , Anticorpos Monoclonais/farmacologia , Regulação da Expressão Gênica , Humanos , Interleucina-13/antagonistas & inibidores , Interleucina-13/biossíntese , Interleucina-13/imunologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Piperazinas/farmacologia , Cultura Primária de Células , Proteínas Secretadas Inibidoras de Proteinases/farmacologia , RNA Mensageiro/antagonistas & inibidores , RNA Mensageiro/imunologia , Receptor PAR-2/antagonistas & inibidores , Receptor PAR-2/imunologia , Transdução de Sinais , alfa 1-Antitripsina/farmacologia
8.
Blood Cells Mol Dis ; 54(2): 206-9, 2015 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-25465717

RESUMO

BACKGROUND: Monocytes and neutrophils are activated during disseminated intravascular coagulation. Tissue factor, the main initiator of coagulation, is expressed by monocytes, while elastase is released by neutrophils. AIMS: This study investigated tissue factor production by peripheral monocytes after stimulation with human neutrophil elastase. METHODS: Tissue factor mRNA levels were investigated by the reverse transcriptase-polymerase chain reaction and tissue factor protein production was assessed by western blotting when monocytes were exposed to neutrophil elastase with or without preincubation using various inhibitors. RESULTS: Neutrophil elastase upregulated tissue factor mRNA and protein levels in monocytes. Both U73122 (phospholipase C inhibitor) and TMB-8 (intracellular calcium antagonist) prevented the upregulation of tissue factor mRNA. SB203580 (p38 mitogen-activated protein kinase inhibitor) suppressed this response, but PD98059 (extracellular signal-regulated kinase inhibitor) did not. Ro-318425 (ATP-competitive and selective protein kinase C (PKC) inhibitor) and Go 6976 (inhibitor of conventional PKCs and PKCµ) blocked the upregulation of tissue factor mRNA expression. Go 6983 (broad-spectrum PKC inhibitor) and CGP 4125 (staurosporine analog) partially attenuated it, as did a PKC theta/delta inhibitor. CONCLUSIONS: Neutrophil elastase mainly enhances tissue factor production by monocytes via the phospholipase C/conventional PKC/p38 MAPK pathway, although a novel PKC is also involved.


Assuntos
Elastase de Leucócito/farmacologia , Monócitos/efeitos dos fármacos , Proteína Quinase C/genética , RNA Mensageiro/metabolismo , Tromboplastina/genética , Fosfolipases Tipo C/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genética , Bloqueadores dos Canais de Cálcio/farmacologia , Carbazóis/farmacologia , Inibidores Enzimáticos/farmacologia , Estrenos/farmacologia , Flavonoides/farmacologia , Ácido Gálico/análogos & derivados , Ácido Gálico/farmacologia , Regulação da Expressão Gênica , Humanos , Imidazóis/farmacologia , Indóis/farmacologia , Maleimidas/farmacologia , Monócitos/citologia , Monócitos/metabolismo , Cultura Primária de Células , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Piridinas/farmacologia , Pirrolidinonas/farmacologia , RNA Mensageiro/genética , Transdução de Sinais , Tromboplastina/metabolismo , Fosfolipases Tipo C/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
9.
J Neurochem ; 128(4): 507-22, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24117785

RESUMO

Dendritic spines are small, actin-rich protrusions on dendrites, the development of which is fundamental for the formation of neural circuits. The actin cytoskeleton is central to dendritic spine morphogenesis. Drebrin is an actin-binding protein that is thought to initiate spine formation through a unique drebrin-actin complex at postsynaptic sites. However drebrin overexpression in neurons does not increase the final density of dendritic spines. In this study, we have identified and characterized a novel drebrin-binding protein, spikar. Spikar is localized in cell nuclei and dendritic spines, and accumulation of spikar in dendritic spines directly correlates with spine density. A reporter gene assay demonstrated that spikar acts as a transcriptional co-activator for nuclear receptors. We found that dendritic spine, but not nuclear, localization of spikar requires drebrin. RNA-interference knockdown and overexpression experiments demonstrated that extranuclear spikar regulates dendritic spine density by modulating de novo spine formation and retraction of existing spines. Unlike drebrin, spikar does not affect either the morphology or function of dendritic spines. These findings indicate that drebrin-mediated postsynaptic accumulation of spikar regulates spine density, but is not involved in regulation of spine morphology.


Assuntos
Espinhas Dendríticas/fisiologia , Neuropeptídeos/metabolismo , Transativadores/fisiologia , Animais , Western Blotting , Células Cultivadas , Clonagem Molecular , DNA Complementar/biossíntese , DNA Complementar/genética , Fenômenos Eletrofisiológicos , Feminino , Genes Reporter/genética , Vetores Genéticos , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase , Gravidez , Interferência de RNA , Ratos , Saccharomyces cerevisiae , Frações Subcelulares/metabolismo , Sinapses/fisiologia , Transfecção
10.
Eur J Cell Biol ; 89(7): 547-56, 2010 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-20188437

RESUMO

Claudins constitute tight junction (TJ) strands and regulate paracellular permeability, which varies in the epithelial cells of various organs. Heterotypic claudin compatibility and/or the association of TJ particles to either the protoplasmic (P) or exoplasmic (E) face may be related to paracellular permeability. This study examined the relationship between the TJ morphology, heterotypic claudin compatibility and paracellular permeability using claudin-10b- or claudin-15-expressing HEK293 cells and MDCK I cells. Claudin-10b or -15 expressed in TJ-free HEK293 cells formed E-face- or P-face-associated TJ particles, respectively. The coculture of claudin-1-expressing HEK293 cells and either claudin-10b- or claudin-15-expressing HEK293 cells showed that claudin-10b and -15 were not compatible with claudin-1. The expression of claudin-10b or -15 in high-resistance MDCK I cells did not alter the expression of endogenous claudins except for claudin-3 and dramatically reduced transepithelial electrical resistance by increasing the permeability of Na(+) but it did not change that of Cl(-). The expression of claudin-10b or -15 in MDCK I cells either decreased or increased the flux of 4 kDa dextran, respectively. The coculture of MDCK I cells and either claudin-10b- or claudin-15-expressing MDCK I cells showed claudin-10b to be partly compatible, while claudin-15 was incompatible with the endogenous claudins in MDCK I cells. These results indicate that the TJ morphology cannot predict the properties of either paracellular permeability or heterotypic claudin compatibility.


Assuntos
Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Animais , Linhagem Celular , Claudina-1 , Claudinas , Cães , Eletroforese , Técnica de Fratura por Congelamento , Humanos , Immunoblotting , Camundongos , Microscopia Confocal
11.
Histochem Cell Biol ; 131(6): 681-90, 2009 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19234713

RESUMO

Claudins constitute tight junction (TJ) strands. In order to examine the function of the second extracellular loop (ECL2), we constructed 1CLDeltaFY and 1CLDeltaPL in which highly conserved amino acids, FY or PL, in the ECL2 of mouse claudin-1 were deleted. They were then tagged with either EGFP at the NH(2)-terminus (EGFP1CLDeltaFY and EGFP1CLDeltaPL) or the myc-epitope at the COOH-terminus (1CLDeltaFYmyc and 1CLDeltaPLmyc). The expression of EGFP1CLDeltaFY and EGFP1CLDeltaPL in TJ-free HEK293 cells formed TJ strands resembling those formed by wild-type claudin-1. The expression of 1CLDeltaPLmyc in TJ-bearing MDCK II cells induced aberrant TJ strands in the lateral plasma membranes whose intramembranous particles were almost equally distributed in the P- and E-face. In contrast, 1CLDeltaFYmyc formed aggregates of short continuous strands which were frequently associated with vesicle-like structures. Coculture experiments with MDCK II cells showed that 1CLDeltaPLmyc was localized at heterotypic cell-cell junctions but 1CLDeltaFYmyc was not. These results suggest that changes in the TJ morphology due to the expression of either 1CLDeltaFYmyc or 1CLDeltaPLmyc may be caused by some factors specific to epithelial MDCK II cells including endogenous claudins.


Assuntos
Células Epiteliais/ultraestrutura , Fibroblastos/ultraestrutura , Proteínas de Membrana/metabolismo , Junções Íntimas/ultraestrutura , Animais , Linhagem Celular , Claudina-1 , Cães , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Técnica de Fratura por Congelamento , Humanos , Proteínas de Membrana/genética , Camundongos , Microscopia Eletrônica , Mutação , Junções Íntimas/metabolismo , Transfecção
12.
Anat Rec (Hoboken) ; 291(5): 577-85, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18384062

RESUMO

Tight junctions (TJs) create a paracellular permeability barrier to restrict the passage of ions, small solutes, and water. Ameloblasts are enamel-forming cells that sequentially differentiate into preameloblasts, secretory, transition, and ruffle-ended and smooth-ended maturation ameloblasts (RAs and SAs). TJs are located at the proximal and distal ends of ameloblasts. TJs at the distal ends of secretory ameloblasts and RAs are well-developed zonula occludens, but other TJs are moderately developed but incomplete zonula occludens (ZO) or less-developed macula occludens. We herein examined the immunofluorescence localization of TJ proteins, 10 claudin isoforms, occludin, ZO-1, and PAR3, a cell polarity-related protein, in ameloblasts of rat upper incisors. ZO-1 and claudin-1 were detected at both ends of all ameloblasts except for the distal ends of SAs. Claudin-4 and occludin were detected at both ends of transition and maturation ameloblasts except for the distal ends of SAs. PAR3 was detected at the proximal TJs of all ameloblasts and faintly at the distal TJs of early RAs. These results indicate that functional zonula occludens formed at the distal ends of the secretory ameloblasts and RAs consisted of different TJ proteins. Therefore, the distal TJs of secretory ameloblasts and RAs may differentially regulate the paracellular permeability to create a microenvironment suitable for enamel deposition and enamel maturation, respectively. In addition, PAR3 may be principally involved in the formation and maintenance of the proximal, but not distal, TJs.


Assuntos
Ameloblastos/metabolismo , Proteínas de Transporte/metabolismo , Incisivo/metabolismo , Proteínas de Membrana/metabolismo , Junções Íntimas/metabolismo , Animais , Claudina-1 , Claudina-4 , Proteínas do Tecido Nervoso , Ocludina , Fosfoproteínas/metabolismo , Ratos , Ratos Wistar , Proteína da Zônula de Oclusão-1
13.
Histochem Cell Biol ; 129(2): 223-32, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18034259

RESUMO

Recent studies suggest that the morphological and physiological properties of tight junctions (TJs) are determined by the combination and mixing ratios of claudin isoforms. In this study, we tried to characterize mouse cell lines by expression of claudin isoforms to use for studying epithelial TJs by overexpression or suppression of claudin(s) in the cells and found that claudin-2 was expressed in a few mouse rectum carcinoma cells, CMT93 cells. We have isolated CMT93-I and -II cells from CMT93 cells by immunohistochemical screening for the presence or absence of claudin-2 expression. Immunofluorescence and RT-PCR analyses showed that expression of claudin-4, -6, -7 and -12 was detected in both cell lines, but claudin-2 was only expressed in CMT93-II cells. There were no differences in paracellular permeability between CMT93-I and -II cells examined by 4 kDa FITC-dextran and fluorescein sodium, or in the number of TJ strands examined by freeze-fracture electron microscopy. However, the transepithelial electrical resistance (TER) of CMT93-I cells was approximately 6.5 times higher than that of CMT93-II cells, suggesting that expression of claudin-2 may be related to decreased TER. Comparative examinations of CMT93-I and -II cells provide a clue how the combination and mixing ratios of claudin isoforms regulate the paracellular permeability.


Assuntos
Carcinoma/metabolismo , Carcinoma/ultraestrutura , Proteínas de Membrana/biossíntese , Neoplasias Retais/metabolismo , Neoplasias Retais/ultraestrutura , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Animais , Linhagem Celular , Membrana Celular/fisiologia , Claudinas , Microscopia Crioeletrônica , Impedância Elétrica , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Técnica de Fratura por Congelamento , Immunoblotting , Imuno-Histoquímica , Proteínas de Membrana/genética , Camundongos , Isoformas de Proteínas/biossíntese , Isoformas de Proteínas/genética , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Anat Rec (Hoboken) ; 290(11): 1431-8, 2007 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17853415

RESUMO

Claudins are integral membrane proteins at tight junctions (TJs) and form TJ strands. In the present study, we found that claudin-7 was localized along the entire lateral membranes of epididymal epithelium, including the apical junctional region throughout the epididymis, but claudin-8 was restricted to the apical junctional region. This finding raises the possibility that aberrant TJ strands may be formed on lateral membranes. Thus, we focused on examining whether TJ strands exist on lateral membranes of epididymal epithelium. Freeze-fracture electron microscopy showed that aberrant TJ strands were observed in only a few principal cells in all segments of the epididymis except for the initial segment, indicating that the occurrence of aberrant strands is very rare. Aberrant TJ strands were smooth and not subdivided into individual particles in the protoplasmic face, and complementary grooves in the extracellular face were almost free of particles. Aberrant TJ strands in the distal caput and corpus epididymis were accompanied by many vesicle-like structures but those in the proximal caput and cauda epididymis were not. These results suggest that most of claudin-7 in lateral membranes may exist in a nonpolymerized form and may play some different roles other than the formation of TJ strands, for example, in the formation of a pool of claudin proteins or in the reinforcement of cell adhesion.


Assuntos
Membrana Celular/metabolismo , Epididimo/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Animais , Adesão Celular/fisiologia , Membrana Celular/ultraestrutura , Claudinas , Epididimo/citologia , Epididimo/ultraestrutura , Epitélio/metabolismo , Epitélio/ultraestrutura , Técnica de Fratura por Congelamento , Masculino , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica , Ocludina , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestrutura , Ratos , Ratos Wistar , Proteína da Zônula de Oclusão-1
15.
Genes Cells ; 11(1): 29-46, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16371130

RESUMO

RCC1, a conserved chromosomal protein with a seven-bladed propeller is a GDP/GTP nucleotide exchange factor for RanGTPase that mediates various cellular events. We isolated 16 temperature-sensitive (ts) mutants of S. pombeRCC1-homolog, pim1+, by error-prone PCR. Five pim1(ts) mutants had a single mutation. The obtained pim1(ts) mutations and previously reported mutations were localized on similar sites in seven RCC1 repeats. Those mutations resulted in a reduced binding of Pim1 with Spi1. All pim1(ts) mutants showed a defect in nucleocytoplasmic protein transports, whereas the majority of them showed a normal mRNA export. In all pim1(ts) examined, chromosomal DNA replication was completed. However, mitotic spindle formation was abrogated, the septum was formed being uncoupled with nuclear division and abnormally widened, thus resulting in chromosomal DNA mis-segregation and the accumulation of enucleated cells. As a result, a defect of RanGEF/Pim1 abolished the orchestration of sequential mitotic events, spindle formation, septation and cytokinesis that are essential to produce two identical daughter cells.


Assuntos
Fatores de Troca do Nucleotídeo Guanina/genética , Mitose/fisiologia , Proteínas de Schizosaccharomyces pombe/genética , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Proteína ran de Ligação ao GTP/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Animais , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Temperatura Alta , Humanos , Dados de Sequência Molecular , Mutação , Transporte Proteico , RNA Mensageiro/biossíntese , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alinhamento de Sequência
16.
Arch Histol Cytol ; 68(5): 349-60, 2005 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-16505581

RESUMO

Tight junctions regulate paracellular permeability, create the luminal fluid microenvironment of blood vessels and the digestive tract, and also form the protective barrier in the stratified epithelium including the epidermis. Claudins are the integral membrane proteins at tight junctions and form a multigene family composed of at least 24 members, but knowledge of the subcellular localization of each claudin is still fragmentary. We performed RT-PCR for fifteen claudin species to examine the mRNA expression in various mouse tissues, and focused on investigating the subcellular localization of claudin-10 and -15 by immunofluorescence microscopy in various rat tissues. Neither claudin-10 nor -15 was detected in vascular endothelial cells in most tissues, and these claudins were restricted to the vasa recta in the kidney medulla. Both claudins were also detected at apical tight junctions in the epithelium of the jejunum with no intensity gradients along the crypt-to-villus axis. However, both claudins were expressed only in the basal half of the crypt epithelium in the colon, showing obvious gradients along crypt-to-surface axis. Moreover, claudin-10 showed the ectopic subcellular localization where tight junction strands do not exist. Claudin-10 was detected along the entire lateral membranes of acinar cells in addition to the apical tight junctions in exocrine glands, and in the cytoplasm of basal cells in the stratified epithelium including the dorsal skin and cutaneous stomach. These heterogeneous distributions of claudin-10 and -15 in tissues may be related to the differences in paracellular permeability among tissues.


Assuntos
Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Junções Íntimas/metabolismo , Animais , Claudinas , Imuno-Histoquímica , Proteínas de Membrana/ultraestrutura , Camundongos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Junções Íntimas/ultraestrutura
17.
J Biol Chem ; 279(9): 8343-50, 2004 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-14660641

RESUMO

RRAG A (Rag A)/Gtr1p is a member of the Ras-like small G protein family that genetically interacts with RCC1, a guanine nucleotide exchange factor for RanGTPase. RRAG A/Gtr1p forms a heterodimer with other G proteins, RRAG C and RRAG D/Gtr2p, in a nucleotide-independent manner. To further elucidate the function of RRAG A/Gtr1p, we isolated a protein that interacts with RRAG A. This protein is a novel nucleolar protein, Nop132. Nop132 is associated with the GTP form, but not the GDP form, of RRAG A, suggesting that RRAG A might regulate Nop132 function. Nop132 is also associated with RRAG C and RRAG D. The Nop132 amino acid sequence is similar to the Saccharomyces cerevisiae nucleolar Nop8p, which is associated with Gtr1p, Gtr2p, and Nip7p. Nop132 also interacts with human Nip7 and is colocalized with RRAG A, RRAG C, and Nip7. RNA interference knockdown of Nop132 inhibited cell growth of HeLa cells.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Divisão Celular , Linhagem Celular , DNA/metabolismo , Expressão Gênica , Vetores Genéticos , Glutationa Transferase/genética , Células HeLa , Humanos , Zíper de Leucina , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Nucleares/química , Proteínas Nucleares/genética , RNA/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes de Fusão , Proteínas Ribossômicas/metabolismo , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Spodoptera/metabolismo , Transfecção , Técnicas do Sistema de Duplo-Híbrido
18.
Mol Cell Biol ; 22(24): 8721-34, 2002 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-12446789

RESUMO

Neurotrophins are key regulators of the fate and shape of neuronal cells and act as guidance cues for growth cones by remodeling the actin cytoskeleton. Actin dynamics is controlled by Rho GTPases. We identified a novel Rho GTPase-activating protein (Grit) for Rho/Rac/Cdc42 small GTPases. Grit was abundant in neuronal cells and directly interacted with TrkA, a high-affinity receptor for nerve growth factor (NGF). Another pool of Grit was recruited to the activated receptor tyrosine kinase through its binding to N-Shc and CrkL/Crk, adapter molecules downstream of activated receptor tyrosine kinases. Overexpression of the TrkA-binding region of Grit inhibited NGF-induced neurite elongation. Further, we found some tendency for neurite promotion in full-length Grit-overexpressing PC12 cells upon NGF stimulation. These results suggest that Grit, a novel TrkA-interacting protein, regulates neurite outgrowth by modulating the Rho family of small GTPases.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Ativadoras de GTPase/metabolismo , Neuritos/metabolismo , Neuropeptídeos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor trkA/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas Ativadoras de GTPase/classificação , Proteínas Ativadoras de GTPase/genética , Genes Reporter , Humanos , Dados de Sequência Molecular , Fator de Crescimento Neural/metabolismo , Neuropeptídeos/genética , Filogenia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-crk , Ratos , Ratos Sprague-Dawley , Receptor trkA/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Proteínas Adaptadoras da Sinalização Shc , Transdução de Sinais/fisiologia , Proteína 3 de Transformação que Contém Domínio 2 de Homologia de Src , Técnicas do Sistema de Duplo-Híbrido , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/classificação , Proteínas rho de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Domínios de Homologia de src
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