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1.
PLoS One ; 6(9): e25158, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21949876

RESUMO

Synaptosomal-associated protein of 25 kDa (SNAP-25) is a presynaptic protein essential for neurotransmitter release. Previously, we demonstrate that protein kinase C (PKC) phosphorylates Ser(187) of SNAP-25, and enhances neurotransmitter release by recruiting secretory vesicles near to the plasma membrane. As PKC is abundant in the brain and SNAP-25 is essential for synaptic transmission, SNAP-25 phosphorylation is likely to play a crucial role in the central nervous system. We therefore generated a mutant mouse, substituting Ser(187) of SNAP-25 with Ala using "knock-in" technology. The most striking effect of the mutation was observed in their behavior. The homozygous mutant mice froze readily in response to environmental change, and showed strong anxiety-related behavior in general activity and light and dark preference tests. In addition, the mutant mice sometimes exhibited spontaneously occurring convulsive seizures. Microdialysis measurements revealed that serotonin and dopamine release were markedly reduced in amygdala. These results clearly indicate that PKC-dependent SNAP-25 phosphorylation plays a critical role in the regulation of emotional behavior as well as the suppression of epileptic seizures, and the lack of enhancement of monoamine release is one of the possible mechanisms underlying these defects.


Assuntos
Ansiedade/etiologia , Mutação/genética , Proteína Quinase C/metabolismo , Proteína 25 Associada a Sinaptossoma/fisiologia , Substituição de Aminoácidos , Animais , Ansiedade/psicologia , Comportamento Animal , Northern Blotting , Western Blotting , Membrana Celular/metabolismo , Dopamina/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação , Vesículas Secretórias/metabolismo
2.
Development ; 138(9): 1771-82, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21447557

RESUMO

In general, cell proliferation and differentiation show an inverse relationship, and are regulated in a coordinated manner during development. Embryonic cardiomyocytes must support embryonic life by functional differentiation such as beating, and proliferate actively to increase the size of the heart. Therefore, progression of both proliferation and differentiation is indispensable. It remains unknown whether proliferation and differentiation are related in these embryonic cardiomyocytes. We focused on abnormal phenotypes, such as hyperproliferation, inhibition of differentiation and enhanced expression of cyclin D1 in cardiomyocytes of mice with mutant jumonji (Jmj, Jarid2), which encodes the repressor of cyclin D1. Analysis of Jmj/cyclin D1 double mutant mice showed that Jmj was required for normal differentiation and normal expression of GATA4 protein through cyclin D1. Analysis of transgenic mice revealed that enhanced expression of cyclin D1 decreased GATA4 protein expression and inhibited the differentiation of cardiomyocytes in a CDK4/6-dependent manner, and that exogenous expression of GATA4 rescued the abnormal differentiation. Finally, CDK4 phosphorylated GATA4 directly, which promoted the degradation of GATA4 in cultured cells. These results suggest that CDK4 activated by cyclin D1 inhibits differentiation of cardiomyocytes by degradation of GATA4, and that initiation of Jmj expression unleashes the inhibition by repression of cyclin D1 expression and allows progression of differentiation, as well as repression of proliferation. Thus, a Jmj-cyclin D1 pathway coordinately regulates proliferation and differentiation of cardiomyocytes.


Assuntos
Diferenciação Celular/genética , Proliferação de Células , Ciclina D1/fisiologia , Coração/embriologia , Miócitos Cardíacos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Animais , Ciclina D1/genética , Embrião de Mamíferos , Fator de Transcrição GATA4/genética , Fator de Transcrição GATA4/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Coração/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos C3H , Camundongos Knockout , Miócitos Cardíacos/metabolismo , Proteínas do Tecido Nervoso/genética , Complexo Repressor Polycomb 2 , Transdução de Sinais , Fatores de Tempo
3.
Immunobiology ; 216(3): 374-8, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20832138

RESUMO

We have previously shown that over-expression of the invariant Vα19-Jα33 TCR α transgene (Tg) using a natural TCR α promoter in mice induces the development of NK1.1(+) T cells (Vα19 NKT cells) in lymphoid organs, including the liver and intestine. These cells produce different spectra of immunoregulatory cytokines such as IL-4, IL-10, IL-17, and IFN-γ depending on the duration and intensity of the invariant TCR stimulation. In this study, we examined the effects of over-expression of invariant Vα19-Jα33 TCR-bearing cells on disease progress in the models of immunological disorders. The introduction of invariant Vα19 TCR Tg into non-obese diabetic mice delayed the onset of the disease. In addition, delayed-type hypersensitivity (DTH) to sheep erythrocytes was suppressed in the Vα19 Tg mice. DTH was also suppressed in the wild type mice previously transferred with Vα19 Tg(+) but not non-Tg cells. Thus, invariant Vα19 TCR-bearing cells are suggested to participate in the homeostasis of immunity to suppress disease progression resulting from Th1-immunity excess.


Assuntos
Diabetes Mellitus Tipo 1/imunologia , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Hipersensibilidade Tardia/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Antígenos Ly/imunologia , Citocinas/biossíntese , Ensaio de Imunoadsorção Enzimática , Eritrócitos/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo
4.
Learn Mem ; 17(4): 176-85, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20332189

RESUMO

A recent study has revealed that fear memory may be vulnerable following retrieval, and is then reconsolidated in a protein synthesis-dependent manner. However, little is known about the molecular mechanisms of these processes. Activin betaA, a member of the TGF-beta superfamily, is increased in activated neuronal circuits and regulates dendritic spine morphology. To clarify the role of activin in the synaptic plasticity of the adult brain, we examined the effect of inhibiting or enhancing activin function on hippocampal long-term potentiation (LTP). We found that follistatin, a specific inhibitor of activin, blocked the maintenance of late LTP (L-LTP) in the hippocampus. In contrast, administration of activin facilitated the maintenance of early LTP (E-LTP). We generated forebrain-specific activin- or follistatin-transgenic mice in which transgene expression is under the control of the Tet-OFF system. Maintenance of hippocampal L-LTP was blocked in the follistatin-transgenic mice. In the contextual fear-conditioning test, we found that follistatin blocked the formation of long-term memory (LTM) without affecting short-term memory (STM). Furthermore, consolidated memory was selectively weakened by the expression of follistatin during retrieval, but not during the maintenance phase. On the other hand, the maintenance of memory was also influenced by activin overexpression during the retrieval phase. Thus, the level of activin in the brain during the retrieval phase plays a key role in the maintenance of long-term memory.


Assuntos
Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Animais , Comportamento Animal , Biofísica , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/genética , Condicionamento Psicológico/efeitos dos fármacos , Condicionamento Psicológico/fisiologia , Giro Denteado/efeitos dos fármacos , Giro Denteado/fisiologia , Doxiciclina/administração & dosagem , Estimulação Elétrica/métodos , Inibidores Enzimáticos/farmacologia , Ensaio de Imunoadsorção Enzimática/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/genética , Medo , Folistatina/genética , Folistatina/farmacologia , Lateralidade Funcional , Técnicas In Vitro , Subunidades beta de Inibinas/genética , Subunidades beta de Inibinas/metabolismo , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Prosencéfalo/metabolismo , Ratos , Ratos Wistar
5.
Hum Mol Genet ; 18(19): 3708-24, 2009 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-19605412

RESUMO

ARX (the aristaless-related homeobox gene) is a transcription factor that participates in the development of GABAergic and cholinergic neurons in the forebrain. Many ARX mutations have been identified in X-linked lissencephaly and mental retardation with epilepsy, and thus ARX is considered to be a causal gene for the two syndromes although the neurobiological functions of each mutation remain unclear. We attempted to elucidate the causal relationships between individual ARX mutations and disease phenotypes by generating a series of mutant mice. We generated three types of mice with knocked-in ARX mutations associated with X-linked lissencephaly (P353R) and mental retardation [P353L and 333ins(GCG)7]. Mice with the P355R mutation (equivalent to the human 353 position) that died after birth were significantly different in Arx transcript/protein amounts, GABAergic and cholinergic neuronal development, brain morphology and lifespan from mice with P355L and 330ins(GCG)7 but considerably similar to Arx-deficient mice with truncated ARX mutation in lissencephaly. Mice with the 330ins(GCG)7 mutation showed severe seizures and impaired learning performance, whereas mice with the P355L mutation exhibited mild seizures and only slightly impaired learning performance. Both types of mutant mice exhibited the mutation-specific lesser presence of GABAergic and cholinergic neurons in the striatum, medial septum and ventral forebrain nuclei when compared with wild-type mice. Present findings that reveal a causal relationship between ARX mutations and the pleiotropic phenotype in mice, suggest that the ARX-related syndrome, including lissencephaly or mental retardation, is caused by only the concerned ARX mutations without the involvement of other genetic factors.


Assuntos
Epilepsia/genética , Proteínas de Homeodomínio/genética , Deficiência Intelectual/genética , Lisencefalia/genética , Mutação , Fatores de Transcrição/genética , Animais , Modelos Animais de Doenças , Proteína Duplacortina , Epilepsia/metabolismo , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Deficiência Intelectual/metabolismo , Lisencefalia/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fenótipo , Fatores de Transcrição/metabolismo
6.
J Reprod Dev ; 55(4): 386-92, 2009 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-19367085

RESUMO

Cryopreservation of mouse sperm is useful for maintaining various strains. However, fertility generally decreases after freezing. In particular, the fertility of cryopreserved C57BL/6J sperm is very low. To improve the fertility of frozen sperm, we examined the efficiencies of various media used for sperm preincubation (SP) and in vitro fertilization (IVF) in frozen C57BL/6J sperm. In this study, SP medium was examined for efficiency of fertility with respect to content, especially calcium (Ca(2+)), phosphate (PO(4)(3-)) and lactate. In all media containing no Ca(2+), including medium lacking Ca(2+), lacking Ca(2+) and PO(4)(3-), lacking Ca(2+) and lactate and lacking Ca(2+), PO(4)(3-) and lactate, high IVF rates were obtained (79, 69, 76 and 71%, respectively). On the other hand, the rates for media containing Ca(2+) were significantly lower (30-38%, P<0.05). After transfer, 41-50% of newborns were obtained in all media containing no Ca(2+). In conclusion, preincubation of thawed sperm in medium containing no Ca(2+) markedly improved the fertility of cryopreserved C57BL/6J sperm. These results indicate that the present method of IVF using medium with no Ca(2+) is practical for use in cryopreserved C57BL/6J sperm.


Assuntos
Cálcio/metabolismo , Criopreservação/métodos , Crioprotetores/farmacologia , Meios de Cultura/farmacologia , Espermatozoides/metabolismo , Animais , Criopreservação/instrumentação , Feminino , Fertilidade , Fertilização in vitro , Lactatos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfatos/metabolismo , Motilidade dos Espermatozoides
7.
Mol Brain ; 2: 7, 2009 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-19265511

RESUMO

BACKGROUND: The formation of long-term memory (LTM) and the late phase of long-term potentiation (L-LTP) depend on macromolecule synthesis, translation, and transcription in neurons. vesl-1S (VASP/Ena-related gene upregulated during seizure and LTP, also known as homer-1a) is an LTP-induced immediate early gene. The short form of Vesl (Vesl-1S) is an alternatively spliced isoform of the vesl-1 gene, which also encodes the long form of the Vesl protein (Vesl-1L). Vesl-1L is a postsynaptic scaffolding protein that binds to and modulates the metabotropic glutamate receptor 1/5 (mGluR1/5), the IP3 receptor, and the ryanodine receptor. Vesl-1 null mutant mice show abnormal behavior, which includes anxiety- and depression-related behaviors, and an increase in cocaine-induced locomotion; however, the function of the short form of Vesl in behavior is poorly understood because of the lack of short-form-specific knockout mice. RESULTS: In this study, we generated short-form-specific gene targeting (KO) mice by knocking in part of vesl-1L/homer-1c cDNA. Homozygous KO mice exhibited normal spine number and morphology. Using the contextual fear conditioning test, we demonstrated that memory acquisition and short-term memory were normal in homozygous KO mice. In contrast, these mice showed impairment in fear memory consolidation. Furthermore, the process from recent to remote memory was affected in homozygous KO mice. Interestingly, reactivation of previously consolidated fear memory attenuated the conditioning-induced freezing response in homozygous KO mice, which suggests that the short form plays a role in fear memory reconsolidation. General activity, emotional performance, and sensitivity to electrofootshock were normal in homozygous KO mice. CONCLUSION: These results indicate that the short form of the Vesl family of proteins plays a role in multiple steps of long-term, but not short-term, fear memory formation.


Assuntos
Proteínas de Transporte/genética , Medo/fisiologia , Genes Precoces , Memória/fisiologia , Animais , Proteínas de Transporte/metabolismo , Condicionamento Psicológico , Dendritos/metabolismo , Dendritos/patologia , Eletrochoque , Congelamento , Marcação de Genes , Proteínas de Arcabouço Homer , Imuno-Histoquímica , Memória de Longo Prazo/fisiologia , Memória de Curto Prazo/fisiologia , Camundongos , Camundongos Knockout , Atividade Motora/fisiologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo
8.
Hum Mol Genet ; 18(8): 1424-38, 2009 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-19174477

RESUMO

Mice with maternal duplication of proximal Chromosome 11 (MatDp(prox11)), where Meg1/Grb10 is located, exhibit pre- and postnatal growth retardation. To elucidate the responsible imprinted gene for the growth abnormality, we examined the precise structure and regulatory mechanism of this imprinted region and generated novel model mice mimicking the pattern of imprinted gene expression observed in the MatDp(prox11) by deleting differentially methylated region of Meg1/Grb10 (Meg1-DMR). It was found that Cobl and Ddc, the neighboring genes of Meg1/Grb10, also comprise the imprinted region. We also found that the mouse-specific repeat sequence consisting of several CTCF-binding motifs in the Meg1-DMR functions as a silencer, suggesting that the Meg1/Grb10 imprinted region adopted a different regulatory mechanism from the H19/Igf2 region. Paternal deletion of the Meg1-DMR (+/DeltaDMR) caused both upregulation of the maternally expressed Meg1/Grb10 Type I in the whole body and Cobl in the yolk sac and loss of paternally expressed Meg1/Grb10 Type II and Ddc in the neonatal brain and heart, respectively, demonstrating maternalization of the entire Meg1/Grb10 imprinted region. We confirmed that the +/DeltaDMR mice exhibited the same growth abnormalities as the MatDp(prox11) mice. Fetal and neonatal growth was very sensitive to the expression level of Meg1/Grb10 Type I, indicating that the 2-fold increment of the Meg1/Grb10 Type I is one of the major causes of the growth retardation observed in the MatDp(prox11) and +/DeltaDMR mice. This suggests that the corresponding human GRB10 Type I plays an important role in the etiology of Silver-Russell syndrome caused by partial trisomy of 7p11-p13.


Assuntos
Cromossomos Humanos Par 11/metabolismo , Proteína Adaptadora GRB10/genética , Impressão Genômica , Transtornos do Crescimento/genética , Animais , Feminino , Inativação Gênica , Humanos , Masculino , Camundongos
9.
Immunol Lett ; 121(1): 38-44, 2008 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-18789972

RESUMO

Previously, we found that more than a half of the NK1.1(+) T cell lines prepared from CD1(-/-) livers expressed invariant Valpha19-Jalpha33 TCR alpha chains. Over-expression of the invariant Valpha19-Jalpha33 TCR alpha transgene (Tg) with a natural TCR alpha promoter and an enhancer in mice induced the development of NK1.1(+) T cells (Valpha19 NKT cells) in the lymphoid organs, especially in the liver. Preferential usage of the Valpha19 Tg by NKT cells in the transgenic mouse livers was indirectly indicated by the observation that few NK1.1(+) TCRalphabeta(+) cells of the Valpha19 Tg livers were stained with a cocktail of anti-TCR Valpha antibodies in the FACS analysis. Upon invariant TCR engagement in vivo following injection of mice with anti-CD3 antibody, NKT cells of the Tg mouse livers as well as spleens promptly produced immunoregulatory cytokines such as IL-4 and IFN-gamma and altered surface receptor expression. Collectively, localization of Valpha19 NKT cells in the liver is suggested that are ready to immediately response against antigen stimulation.


Assuntos
Antígenos Ly/imunologia , Fígado/imunologia , Subfamília B de Receptores Semelhantes a Lectina de Células NK/imunologia , Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Anticorpos/imunologia , Antígenos CD1/imunologia , Complexo CD3/imunologia , Citocinas/imunologia , Fígado/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células T Matadoras Naturais/efeitos dos fármacos , Receptores de Antígenos de Linfócitos T alfa-beta/agonistas , Receptores de Antígenos de Linfócitos T alfa-beta/genética
10.
PLoS One ; 3(4): e1869, 2008 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-18382659

RESUMO

Activin, a member of the transforming growth factor-beta superfamily, is an endocrine hormone that regulates differentiation and proliferation of a wide variety of cells. In the brain, activin protects neurons from ischemic damage. In this study, we demonstrate that activin modulates anxiety-related behavior by analyzing ACM4 and FSM transgenic mice in which activin and follistatin (which antagonizes the activin signal), respectively, were overexpressed in a forebrain-specific manner under the control of the alphaCaMKII promoter. Behavioral analyses revealed that FSM mice exhibited enhanced anxiety compared to wild-type littermates, while ACM4 mice showed reduced anxiety. Importantly, survival of newly formed neurons in the subgranular zone of adult hippocampus was significantly decreased in FSM mice, which was partially rescued in ACM4/FSM double transgenic mice. Our findings demonstrate that the level of activin in the adult brain bi-directionally influences anxiety-related behavior. These results further suggest that decreases in postnatal neurogenesis caused by activin inhibition affect an anxiety-related behavior in adulthood. Activin and its signaling pathway may represent novel therapeutic targets for anxiety disorder as well as ischemic brain injury.


Assuntos
Ativinas/genética , Ativinas/fisiologia , Encéfalo/metabolismo , Folistatina/genética , Neurônios/metabolismo , Ativinas/química , Ativinas/metabolismo , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Diferenciação Celular , Folistatina/metabolismo , Hipocampo/metabolismo , Isquemia/metabolismo , Camundongos , Camundongos Transgênicos , Modelos Biológicos , Fenótipo , Regiões Promotoras Genéticas , Transgenes
11.
Nat Genet ; 40(2): 243-8, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18176565

RESUMO

Eutherian placenta, an organ that emerged in the course of mammalian evolution, provides essential architecture, the so-called feto-maternal interface, for fetal development by exchanging nutrition, gas and waste between fetal and maternal blood. Functional defects of the placenta cause several developmental disorders, such as intrauterine growth retardation in humans and mice. A series of new inventions and/or adaptations must have been necessary to form and maintain eutherian chorioallantoic placenta, which consists of capillary endothelial cells and a surrounding trophoblast cell layer(s). Although many placental genes have been identified, it remains unknown how the feto-maternal interface is formed and maintained during development, and how this novel design evolved. Here we demonstrate that retrotransposon-derived Rtl1 (retrotransposon-like 1), also known as Peg11 (paternally expressed 11), is essential for maintenance of the fetal capillaries, and that both its loss and its overproduction cause late-fetal and/or neonatal lethality in mice.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Impressão Genômica , Troca Materno-Fetal/fisiologia , Placenta/fisiologia , Proteínas da Gravidez/fisiologia , Retroelementos/fisiologia , Alelos , Animais , Animais Recém-Nascidos , Sequência de Bases , Biomarcadores/metabolismo , Cromossomos Artificiais Bacterianos/genética , Cromossomos de Mamíferos , Embrião de Mamíferos , Feminino , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , MicroRNAs/metabolismo , Dados de Sequência Molecular , Fases de Leitura Aberta , Linhagem , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Gravidez , Trofoblastos/metabolismo
12.
Neurosci Res ; 59(1): 93-100, 2007 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-17640754

RESUMO

The proper regulation of microtubule (MT) structure is important for dendritic and neural circuit development. However, the relationship between the regulation of the MTs in dendrites and the formation of neural function is still unclear. Stathmin is a MT destabilizer, and we have previously reported that the expression and the activity of stathmin is downregulated during cerebellar Purkinje cell (PC) development. In this study, we generated transgenic mice that specifically overexpress the constitutively active form of stathmin in the PCs. These mutant mice did not show any obvious morphological or excitatory transmission abnormalities in the cerebellum. In contrast, we observed a decline in the expression of MAP2 and KIF5 signal in the PC dendrites and a discoordination of motor function in the mutant mice, although they displayed normal general behavior. These data indicate that the overexpression of stathmin disrupts dendritic MT organization, motor protein distribution, and neural function in PCs.


Assuntos
Regulação da Expressão Gênica/genética , Transtornos das Habilidades Motoras/genética , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Células de Purkinje/metabolismo , Animais , Animais Recém-Nascidos , Comportamento Animal , Cerebelo/citologia , Relação Dose-Resposta à Radiação , Estimulação Elétrica/métodos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/fisiologia , Potenciais Pós-Sinápticos Excitadores/efeitos da radiação , Comportamento Exploratório/fisiologia , Hibridização In Situ , Técnicas In Vitro , Cinesinas/metabolismo , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/genética , Transtornos das Habilidades Motoras/patologia , Transtornos das Habilidades Motoras/fisiopatologia , Células de Purkinje/fisiologia , Tubulina (Proteína)/metabolismo
13.
Dev Biol ; 303(2): 549-60, 2007 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-17189626

RESUMO

During development of the mouse central nervous system (CNS), most neural progenitor cells proliferate in the ventricular zone (VZ). In many regions of the CNS, neural progenitor cells give rise to postmitotic neurons that initiate neuronal differentiation and migrate out of the VZ to the mantle zone (MZ). Thereafter, they remain in a quiescent state. Here, we found many ectopic mitotic cells and cell clusters expressing neural progenitor or proneural marker genes in the MZ of the hindbrain of jumonji (jmj) mutant embryos. When we examined the expression of cyclin D1, which is repressed by jmj in the repression of cardiac myocyte proliferation, we found many ectopic clusters expressing both cyclin D1 and Musashi 1 in the MZ of mutant embryos. jmj is mainly expressed in the cyclin D1 negative region in the hindbrain, and cyclin D1 expression in the VZ was upregulated in jmj mutant mice. In jmj and cyclin D1 double mutant mice, the ectopic mitosis and formation of the abnormal clusters in the MZ were rescued. These results suggest that a jmj-cyclin D1 pathway is required for the precise coordination of cell cycle exit and migration during neurogenesis in the mouse hindbrain.


Assuntos
Ciclinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Rombencéfalo/embriologia , Rombencéfalo/metabolismo , Animais , Ciclo Celular/genética , Ciclo Celular/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/fisiologia , Movimento Celular/genética , Movimento Celular/fisiologia , Ciclina D , Ciclinas/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos C3H , Camundongos Mutantes , Mitose/genética , Mitose/fisiologia , Proteínas do Tecido Nervoso/genética , Complexo Repressor Polycomb 2 , Gravidez , Regiões Promotoras Genéticas , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Rombencéfalo/citologia
14.
J Biol Chem ; 282(6): 3413-7, 2007 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-17179160

RESUMO

Ras GTPase-activating proteins (GAP) are negative regulators of Ras that convert active Ras-GTP to inactive Ras-GDP. R-Ras GAP is a membrane-associated molecule with stronger GAP activity for R-Ras, an activator of integrin, than H-Ras. We found that R-Ras GAP is down-regulated during neurite formation in rat pheochromocytoma PC12 cells by nerve growth factor (NGF), which is blocked by the transient expression of R-Ras gap or dominant negative R-ras cDNA. By establishing a PC12 subclone that stably expresses exogenous R-Ras GAP, it was found that NGF reduced endogenous R-Ras GAP but not exogenous R-Ras GAP, suggesting that down-regulation of R-Ras GAP occurs at the transcription level. To clarify the physiological role of R-Ras GAP, we generated mice that express mutant Ras GAP with knocked down activity. While heterozygotes are normal, homozygous mice die at E12.5-13.5 of massive subcutaneous and intraparenchymal bleeding, probably due to underdeveloped adherens junctions between capillary endothelial cells. These results show essential roles of R-Ras GAP in development and differentiation: its expression is needed for embryonic development of blood vessel barriers, whereas its down-regulation facilitates NGF-induced neurite formation of PC12 cells via maintaining activated R-Ras.


Assuntos
Hemorragia/embriologia , Hemorragia/fisiopatologia , Neovascularização Patológica , Neuritos/metabolismo , Proteínas Ativadoras de ras GTPase/fisiologia , Animais , Proliferação de Células , Regulação para Baixo/genética , Hemorragia/genética , Camundongos , Camundongos Knockout , Neovascularização Patológica/genética , Neuritos/fisiologia , Células PC12 , Ratos , Proteínas Ativadoras de ras GTPase/antagonistas & inibidores , Proteínas Ativadoras de ras GTPase/biossíntese , Proteínas Ativadoras de ras GTPase/deficiência
15.
Reprod Med Biol ; 6(4): 229-233, 2007 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29662412

RESUMO

Aim: Cryopreservation of mouse sperm commonly uses raffinose, which is a trisaccharide, plus 3% skim milk. Because of the present lack of knowledge of the effectiveness of any other saccharides, we examined the cryoprotective effects of various saccharides on the viability of mouse sperm from various strains to determine which saccharides are the best cryoprotectants for mouse sperm. Methods: Sperm from the caudae epididymides of mature C57BL/6J mice were frozen with monosaccharides (fructose, glucose, rhamnose, xylose), disaccharides (lactose, maltose, sucrose, trehalose) or trisaccharides (melezitose, raffinose) in a range of concentrations (4-33%). After thawing, the optimal concentration was determined to be the concentration in which there was the highest proportion of motile sperm. In addition, sperm of inbred and hybrid mice were frozen with the saccharides at the optimal concentrations and used for in vitro fertilization. Results: The optimal concentration was 12% for the disaccharides and 18% for the trisaccharides. The fertility of all strains, except C57BL/6J, showed the best cryoprotective effects with maltose, melezitose and raffinose when compared with fresh sperm. Conclusion: Maltose, melezitose and raffinose have the best effects when used as a protectant for cryopreservation of mouse sperm. (Reprod Med Biol 2007; 6: 229-233).

16.
Fertil Steril ; 86(4 Suppl): 1080-7, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16978625

RESUMO

OBJECTIVE: To study whether fecundity was recovered in mice into which umbilical cord blood cells (UCBCs) were transfused after lethal-dose radiation, followed by transplantation of frozen-thawed ovaries. DESIGN: Prospective basic research study. SETTING: Academic research laboratory. ANIMAL(S): Female C57BL/6 mice as recipients of UCBCs and ovaries, male B6C3F1 mice for mating, and green fluorescent protein (GFP)-transgenic mice: 18.5-day-old fetuses (-/+) for UCBCs and adult GFP mice (+/+) for ovarian tissues. INTERVENTION(S): The UCBCs were transfused into each irradiated mouse, with GFP+ ovaries transplanted 4 weeks later. The chimeric mice were mated 3 weeks after ovarian transplantation and were examined 14 to 16 weeks after the transfusion of UCBCs. MAIN OUTCOME MEASURE(S): Percentage of chimerism, number of GFP+ pups. RESULT(S): The percentage of chimerism in these mice tends to increase with the radiation dose. The recovery of fecundity was observed in the chimeric mice that were transplanted with fresh and previously vitrified ovaries after exposure to radiation. CONCLUSION(S): Even when the exposure dose of radiation administered as pretreatment is lethal, the fecundity of recipients can be maintained if their ovaries are cryopreserved before they are exposed to radiation.


Assuntos
Criopreservação , Morte , Ovário/transplante , Prenhez , Quimera por Radiação , Animais , Relação Dose-Resposta à Radiação , Feminino , Citometria de Fluxo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos , Folículo Ovariano/anatomia & histologia , Ovário/anatomia & histologia , Ovário/fisiologia , Gravidez
17.
Nature ; 441(7095): 885-9, 2006 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-16625204

RESUMO

Autophagy is an intracellular bulk degradation process through which a portion of the cytoplasm is delivered to lysosomes to be degraded. Although the primary role of autophagy in many organisms is in adaptation to starvation, autophagy is also thought to be important for normal turnover of cytoplasmic contents, particularly in quiescent cells such as neurons. Autophagy may have a protective role against the development of a number of neurodegenerative diseases. Here we report that loss of autophagy causes neurodegeneration even in the absence of any disease-associated mutant proteins. Mice deficient for Atg5 (autophagy-related 5) specifically in neural cells develop progressive deficits in motor function that are accompanied by the accumulation of cytoplasmic inclusion bodies in neurons. In Atg5-/- cells, diffuse, abnormal intracellular proteins accumulate, and then form aggregates and inclusions. These results suggest that the continuous clearance of diffuse cytosolic proteins through basal autophagy is important for preventing the accumulation of abnormal proteins, which can disrupt neural function and ultimately lead to neurodegeneration.


Assuntos
Autofagia/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Doenças Neurodegenerativas/patologia , Doenças Neurodegenerativas/fisiopatologia , Neurônios/patologia , Animais , Proteína 5 Relacionada à Autofagia , Encéfalo/metabolismo , Encéfalo/patologia , Morte Celular , Corpos de Inclusão/metabolismo , Proteínas de Filamentos Intermediários/genética , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas do Tecido Nervoso/genética , Nestina , Neurônios/metabolismo , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Ubiquitina/metabolismo
18.
Nat Genet ; 38(1): 101-6, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16341224

RESUMO

By comparing mammalian genomes, we and others have identified actively transcribed Ty3/gypsy retrotransposon-derived genes with highly conserved DNA sequences and insertion sites. To elucidate the functions of evolutionarily conserved retrotransposon-derived genes in mammalian development, we produced mice that lack one of these genes, Peg10 (paternally expressed 10), which is a paternally expressed imprinted gene on mouse proximal chromosome 6. The Peg10 knockout mice showed early embryonic lethality owing to defects in the placenta. This indicates that Peg10 is critical for mouse parthenogenetic development and provides the first direct evidence of an essential role of an evolutionarily conserved retrotransposon-derived gene in mammalian development.


Assuntos
Perda do Embrião/genética , Impressão Genômica , Proteínas Nucleares/genética , Placenta/patologia , Retroelementos , Fatores de Transcrição/genética , Animais , Proteínas Reguladoras de Apoptose , Metilação de DNA , Proteínas de Ligação a DNA , Feminino , Retardo do Crescimento Fetal/genética , Deleção de Genes , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Knockout , Proteínas Nucleares/metabolismo , Partenogênese/genética , Placenta/fisiologia , Gravidez , Proteínas de Ligação a RNA , Fatores de Transcrição/metabolismo
19.
Biochem Biophys Res Commun ; 324(4): 1319-23, 2004 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-15504358

RESUMO

jumonji (jmj) mutant mice, obtained by a gene trap strategy, showed several morphological abnormalities including neural tube and cardiac defects, and died in utero around embryonic day 11.5 (E11.5). It is unknown what causes the embryonic lethality. Here, we demonstrate that exogenous expression of jmj gene in the heart of jmj mutant mice rescued the morphological phenotypes in the heart, and these embryos survived until E13.5. These results suggest that there are at least two lethal periods in jmj mutant mice, and that cardiac abnormalities may cause the earlier lethality. In addition, the rescue of the cardiac abnormalities by the jmj transgene provided solid evidence that the cardiac abnormalities resulted from mutation of the jmj gene.


Assuntos
Cardiopatias Congênitas/etiologia , Proteínas do Tecido Nervoso/genética , Animais , Embrião de Mamíferos/patologia , Genes Letais , Cardiopatias Congênitas/metabolismo , Cardiopatias Congênitas/patologia , Camundongos , Camundongos Mutantes , Camundongos Transgênicos , Miocárdio/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Complexo Repressor Polycomb 2
20.
J Neurosci ; 24(40): 8711-9, 2004 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-15470137

RESUMO

Laminar organization, a fundamental neural architecture in the CNS, is a prominent feature of the neocortex, where the cortical neurons in spatially distinct layers are generated from the common progenitors in a temporally distinct manner during development. Despite many advances in the characterization of the molecular mechanisms of the radial migration of cortical neurons, the way in which the early-late temporal sequence of cortical neuron generation is linked with the deep-superficial spatial sequence of cell body positioning remains obscure. Using in vivo electroporation-mediated gene transfer, we show here that the activities mediated by fibroblast growth factor receptors (FGFRs) in cortical progenitors are critical for conferring proper migratory properties on nascent neuronal progeny. Furthermore, we provide supportive evidence that Pea3 subfamily members of Ets (Pea3-Ets) transcription factors mediate the activities of FGFR at the mid to late phase of neocortical development. In addition, using FGF18 knock-out mice, we demonstrate that FGF18 expressed by early-generated cortical neurons in the cortical plate is critical for the expression of Pea3-Ets transcription factors and that FGF18 is sufficient to induce their expressions. Our results thus imply that a feedback mechanism mediated by FGF signaling is involved in setting up the proper laminar positioning of cortical neurons; FGF18 derived from early-generated cortical neurons acts on the cortical progenitors expressing FGFRs and induces the expression of Pea3-Ets transcription factors that, in turn, confer proper migratory behaviors on nascent cortical progeny during the mid to late stages of neocortical development.


Assuntos
Fatores de Crescimento de Fibroblastos/fisiologia , Neocórtex/embriologia , Neurônios/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Padronização Corporal , Movimento Celular , Fatores de Crescimento de Fibroblastos/genética , Cinética , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , Mutação , Neocórtex/citologia , Neocórtex/metabolismo , Neurônios/citologia , Proteínas Tirosina Quinases/metabolismo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos , Células-Tronco/fisiologia , Fatores de Transcrição/genética
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