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1.
Am J Pathol ; 180(5): 1879-96, 2012 May.
Artigo em Inglês | MEDLINE | ID: mdl-22440255

RESUMO

An imbalance between free radical generation and radical scavenging antioxidant systems results in oxidative stress, which has been associated with cell injury observed in many age-related diseases. The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase-1 (Sod1) in mice leads to many different phenotypes that resemble accelerated aging. In this study we examined the morphologic features and the secretory functions of the lacrimal glands in Sod1(-/-) mice. Lacrimal glands showed atrophy of acinar units; fibrosis; infiltration with CD4(+) T cells, monocytes, and neutrophils; increased staining with both 4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine; increases in apoptotic cells; and the presence of the epithelial-mesenchymal transition in senescent Sod1(-/-) mice. Electron microscopy findings revealed evidence of epithelial-mesenchymal transition, presence of swollen and degenerated mitochondria, and the presence of apoptotic cell death in the lacrimal glands of senescent Sod1(-/-) mice. These alterations were also associated with the accumulation of secretory vesicles in acinar epithelial cells, decreased production of both stimulated and nonstimulated tears, and a decline in total protein secretion from the lacrimal glands. Our results suggest that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated lacrimal gland alterations.


Assuntos
Envelhecimento/fisiologia , Aparelho Lacrimal/fisiopatologia , Estresse Oxidativo/fisiologia , Superóxido Dismutase/fisiologia , 8-Hidroxi-2'-Desoxiguanosina , Envelhecimento/patologia , Animais , Apoptose/fisiologia , Citocinas/metabolismo , Dano ao DNA , Desoxiguanosina/análogos & derivados , Desoxiguanosina/sangue , Modelos Animais de Doenças , Síndromes do Olho Seco/patologia , Síndromes do Olho Seco/fisiopatologia , Transição Epitelial-Mesenquimal/fisiologia , Fibrose , Aparelho Lacrimal/patologia , Aparelho Lacrimal/ultraestrutura , Peroxidação de Lipídeos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica , Mitocôndrias/ultraestrutura , Superóxido Dismutase/deficiência , Lágrimas/metabolismo
2.
Hum Mol Genet ; 19(13): 2606-15, 2010 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-20388642

RESUMO

Glaucoma is one of the leading causes of bilateral blindness affecting nearly 8 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells (RGCs) and is often associated with elevated intraocular pressure (IOP). However, patients with normal tension glaucoma (NTG), a subtype of primary open-angle glaucoma (POAG), develop the disease without IOP elevation. The molecular pathways leading to the pathology of NTG and POAG are still unclear. Here, we describe the phenotypic characteristics of transgenic mice overexpressing wild-type (Wt) or mutated optineurin (Optn). Mutations E50K, H486R and Optn with a deletion of the first (amino acids 153-174) or second (amino acids 426-461) leucine zipper were used for overexpression. After 16 months, histological abnormalities were exclusively observed in the retina of E50K mutant mice with loss of RGCs and connecting synapses in the peripheral retina leading to a thinning of the nerve fiber layer at the optic nerve head at normal IOP. E50K mice also showed massive apoptosis and degeneration of entire retina, leading to approximately a 28% reduction of the retina thickness. At the molecular level, introduction of the E50K mutation disrupts the interaction between Optn and Rab8 GTPase, a protein involved in the regulation of vesicle transport from Golgi to plasma membrane. Wt Optn and an active GTP-bound form of Rab8 complex were localized at the Golgi complex. These data suggest that alternation of the Optn sequence can initiate significant retinal degeneration in mice.


Assuntos
Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Glaucoma/genética , Degeneração Retiniana/genética , Proteínas rab de Ligação ao GTP/metabolismo , Substituição de Aminoácidos , Animais , Apoptose , Proteínas de Ciclo Celular , Proteínas de Membrana Transportadoras , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nervo Óptico/patologia , Ligação Proteica , Células Ganglionares da Retina/patologia
3.
Mol Vis ; 17: 1222-30, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21617757

RESUMO

PURPOSE: To investigate the transcriptional factors associated with epithelial-mesenchymal transition (EMT) in choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). METHODS: Paraffin sections of CNV obtained from patients with AMD (n = 12) were stained for transcriptional factors related to EMT, i.e., Snail, Slug, SIP1, and Twist. As a control, postmortem sections of ocular normal tissue were used. Furthermore, using a human retinal pigment epithelial (RPE) cell line (ARPE-19), reverse transcription-polymerase chain reaction (RT-PCR) and immunofluorescence microscopy were performed to explore the cellular localization and expression levels of EMT-associated transcriptional factors upon cytokine stimulation. RESULTS: Of 12 specimens, 11 CNV tissues (91.6%) showed staining for Snail localized in cellular nuclei, particularly in those of RPE cells. Snail was strongly co-localized with α-smooth muscle antigen (SMA) in RPE cells. In contrast, postmortem human retina showed no Snail staining in RPE cells. Other transcriptional factors, Slug, Twist and SIP1 were not detected in CNV or normal human retina. In ARPE-19 cells, RT-PCR and immunofluorescence microscopy showed that Snail mRNA was upregulated by transforming growth factor (TGF)-ß and VEGF stimulation. Furthermore, TGF-ß induced relocalization of Snail to the nucleus in RPE cells. CONCLUSIONS: The current data indicate that Snail is a major transcriptional factor for EMT changes of RPE cells in human CNV.


Assuntos
Neovascularização de Coroide/genética , Transição Epitelial-Mesenquimal/genética , Degeneração Macular/genética , Fatores de Transcrição , Idoso , Linhagem Celular , Neovascularização de Coroide/etiologia , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Imuno-Histoquímica , Degeneração Macular/complicações , Masculino , Microtomia , Pessoa de Meia-Idade , Inclusão em Parafina , Retina , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição da Família Snail , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia
4.
Genomics ; 96(2): 102-11, 2010 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-20580816

RESUMO

YPEL5 is a member of the YPEL gene family that is highly conserved in the eukaryotic species and apparently involved in a certain cell division-related function. In this study, we examined the functional and phylogenetic aspects of YPEL5 protein in more detail. During cell cycle, YPEL5 protein was detected at different subcellular localizations; at interphase, it was located in the nucleus and centrosome, then it changed location sequentially to spindle poles, mitotic spindle, and spindle midzone during mitosis, and finally transferred to midbody at cytokinesis. Knockdown of YPEL5 function by siRNA or anti-sense morpholino oligonucleotide inhibited the growth of cultured COS-7 cells and early development of medaka fish embryos, indicating its involvement in cell cycle progression. Interestingly, RanBPM (Ran Binding Protein in the Microtubule organizing center, encoded by RANBP9) was identified as a YPEL5-binding protein by yeast two-hybrid method. A paralog of RanBPM, namely RanBP10 (encoded by RANBP10), was found to be another YPEL5-binding protein, and these two protein genes are highly conserved each other. Comparative genomic analysis allowed us to define a new gene family consisting of RanBPM and RanBP10, named Scorpin, providing a basis to better understand how they interact with YPEL5.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Família Multigênica/genética , Proteínas Nucleares/metabolismo , Filogenia , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células COS , Proteínas de Ciclo Celular/genética , Chlorocebus aethiops , Clonagem Molecular , Análise por Conglomerados , DNA Complementar/genética , Bases de Dados Genéticas , Embrião não Mamífero/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Imunoprecipitação , Dados de Sequência Molecular , Oligonucleotídeos/genética , Oryzias , RNA Interferente Pequeno/genética , Análise de Sequência de DNA , Técnicas do Sistema de Duplo-Híbrido
5.
Am J Pathol ; 172(5): 1325-31, 2008 May.
Artigo em Inglês | MEDLINE | ID: mdl-18372426

RESUMO

The superoxide dismutase (SOD) family is a major antioxidant system, and deficiency of Cu,Zn-superoxide dismutase (SOD1) in mice leads to many different phenotypes that resemble accelerated aging. The purpose of this study was to examine the morphology and physiology of the sensory retina in Sod1(-/-) mice. The amplitudes of the a- and b-waves of electroretinograms elicited by stimuli of different intensity were reduced in senescent Sod1(-/-) mice, and this reduction in amplitude was more pronounced with increasing age. Retinal morphometric analyses showed a reduced number of nuclei in both the inner nuclear cell layer and outer nuclear cell layer. Electron microscopy revealed swollen cells and degenerated mitochondria in the inner nuclear cell and outer nuclear cell layer of senescent Sod1(-/-) mice indicating necrotic cell death. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling revealed no significant differences in the number of apoptotic cells between Sod1(-/-) and wild-type mice, and activated caspase-3 could not be detected in the retina of Sod1(-/-) mice. In addition to the age-related macular degeneration-like phenotypes previously reported, Sod1(-/-) mice also present progressive retinal degeneration. Our results indicate that Sod1(-/-) mice may be a good model system in which to study the mechanism of reactive oxygen species-mediated retinal degeneration.


Assuntos
Retina/patologia , Retina/fisiopatologia , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Superóxido Dismutase/fisiologia , Envelhecimento , Animais , Apoptose , Caspase 3/fisiologia , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Cruzamentos Genéticos , Eletrorretinografia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Necrose , Retina/metabolismo , Degeneração Retiniana/metabolismo , Superóxido Dismutase/genética , Superóxido Dismutase-1
6.
J Radiat Res ; 50(1): 73-83, 2009 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19218782

RESUMO

Much attention has been focused on the mitochondrial superoxide anion (O2(-)), which is also a critical free radial produced by ionizing radiation. The specific role of the mitochondrial O2(-) on physiological aging in mammals is still unclear despite wide-spread evidence that oxidative stress is involved in aging and age-related diseases. The major endogenous source of O2(-) is generated as a byproduct of energy metabolism from mitochondria. In order to better understand how O2(-)relates to metazoan aging, we have comprehensively examined age-related changes in the levels of oxidative damage, mitochondrial O2(-) production, mitochondrial antioxidant enzyme activity and apoptosis induction in key organs of an inbred mouse strain (C57BL/6J). Oxidative damage accumulated and excess apoptosis occurred in the brain, oculus and kidney with aging, but comparatively little occurred in the heart and muscle. These rates are correlated with O2(-) levels. Mitochondrial O2(-) production levels increased with aging in the brain, oculus and kidney, and did not significantly increased in the heart and muscle. In contrast to O2(-) production, mitochondrial SOD activities increased in heart and muscle, and remained unchanged in the brain, oculus and kidney with aging. These results suggest that O2(-) production has high organ specificity, and oxidative damage by O2(-) from mitochondria mediated apoptosis can lead to organ atrophy and physiological dysfunction. In addition, O2(-) from mitochondria plays a core role in physiological aging.


Assuntos
Envelhecimento/fisiologia , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Estresse Oxidativo/fisiologia , Superóxidos/metabolismo , Animais , Células Cultivadas , Camundongos , Camundongos Endogâmicos C57BL , Especificidade de Órgãos , Distribuição Tecidual
7.
Eur J Pharmacol ; 548(1-3): 74-6, 2006 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-16973158

RESUMO

Real-time quantitative PCR, Western blot and in situ hybridization techniques were employed to clarify the presence of serine racemase in the primary cultures of rat neurons. We have detected both serine racemase mRNA and protein in the cultured neurons. Both the mRNA and the protein levels in the neurons are higher than those in the astrocytes. Sequential detection of serine racemase mRNA and MAP2 immunoreactivity also revealed that serine racemase and MAP2 are co-localized in the cultured neurons. These data are the first to demonstrate that a substantial amount of serine racemase exists in the cultured neurons.


Assuntos
Neurônios/metabolismo , Racemases e Epimerases/metabolismo , Animais , Astrócitos/metabolismo , Células Cultivadas , Expressão Gênica , RNA Mensageiro/metabolismo , Racemases e Epimerases/genética , Ratos
8.
Gene ; 318: 45-53, 2003 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-14585497

RESUMO

We have previously cloned a human, retina-specific, amine oxidase gene (RAO, gene symbol: AOC2), a member of the copper-binding amine oxidase super family. AOC2 shares sequence identity with the human kidney amine oxidase gene (KAO, gene symbol: AOC1) and the vascular adhesion protein-1 gene (VAP-1, gene symbol: AOC3). For further analysis of AOC2, the sequences surrounding the human AOC2 and the complete mouse and partial rat homologue of AOC2 were cloned for characterization. Real-time quantitative PCR, in situ hybridization, and immunohistochemistry were performed to determine the specific expression of AOC2 in the mouse retina and especially in the retinal ganglion cells. Our results demonstrated that the copper-binding motif and the enzyme active site of AOC1 and AOC3 were both conserved in mouse AOC2. The human and mouse AOC2 was flanked by two genes, the Psme3 gene for PA-28 gamma subunit and, surprisingly, the AOC3 gene. Rat AOC2 contained a stop codon that terminated the peptide length to 127 amino acids. The presence of human and rat AOC pseudogene in this region, in addition to the tandemly positioned two AOC genes, indicates the possibility of successful AOC3 replication to retina-specific AOC2 for human and mouse but unsuccessful for rat.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Retina/enzimologia , Amina Oxidase (contendo Cobre)/genética , Amina Oxidase (contendo Cobre)/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , Cobre/metabolismo , DNA/química , DNA/genética , DNA/isolamento & purificação , DNA Complementar/química , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Éxons , Expressão Gênica , Genes/genética , Humanos , Imuno-Histoquímica , Hibridização In Situ , Íntrons , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Dados de Sequência Molecular , Mutagênese Insercional , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Ratos , Retina/metabolismo , Análise de Sequência de DNA , Deleção de Sequência
9.
Invest Ophthalmol Vis Sci ; 45(8): 2652-9, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15277488

RESUMO

PURPOSE: To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions. METHODS: Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O(2)) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR. RESULTS: The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin. CONCLUSIONS: The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.


Assuntos
Astrócitos/metabolismo , Proteínas do Olho/genética , Regulação da Expressão Gênica , Glicoproteínas/genética , Malha Trabecular/metabolismo , Sequência de Aminoácidos , Animais , Astrócitos/efeitos dos fármacos , Sequência de Bases , Hipóxia Celular , Clonagem Molecular , Proteínas do Citoesqueleto , DNA Complementar/análise , Dexametasona/farmacologia , Proteínas do Olho/metabolismo , Glucocorticoides/farmacologia , Glicoproteínas/metabolismo , Pressão Hidrostática , Dados de Sequência Molecular , Disco Óptico , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência de Aminoácidos , Estresse Mecânico , Suínos , Malha Trabecular/efeitos dos fármacos
10.
Dev Growth Differ ; 30(3): 271-282, 1988 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-37281562

RESUMO

Microscopic studies were made on the localizations of three different cytoskeletal proteins, actin, vinculin and fibronectin, in the duodenum of developing chick embryos and chicks by an indirect immuno-fluorescent staining method with specific antibodies. Topographical changes in the distributions of these three proteins seemed to be related to stages in morphogenesis of the duodenum. In early stages of embryonic development, findings suggested interaction between actin and vinculin in the apical region of epithelial cells and between actin and fibronectin in the basal region of these cells. From this stage, vinculin and fibronectin seemed to be of importance in determination and continuity of the polarity of the duodenal epithelium, and in control of the intracellular arrangement of actin. This relation between actin and vinculin seemed to continue throughout embryogenesis. The main role of actin in epithelial cells seemed to change on day 12 from that of forming constricting bundles for morphogenesis of previllous ridges to that of microfilaments in the microvilli and the terminal web.

11.
Arch Histol Cytol ; 71(2): 123-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18974604

RESUMO

D-Serine, an endogenous and obligatory coagonist for the glycine site of the N-methyl-D-aspartate receptor in mammals, is synthesized from L-serine by serine racemase. Serine racemase and D-serine have long been believed to occur predominantly in astrocytes, according to immunohistochemical studies. Recent studies have demonstrated, however, that both the mRNA and protein levels of serine racemase are considerably higher in neurons than in astrocytes in primary cultures of the rat brain and that the mRNA level of serine racemase predominates in neurons of the adult rat brain. Here we report the application of in situ hybridization based on tyramide signal amplification for the detection of serine racemase mRNA in sections of the adult rat retina and optic nerve head. The localization of serine racemase mRNA could be demonstrated in ganglion cells, amacrine cells, bipolar cells, horizontal cells, and Müller cells of the retina as well as in the astrocytes of the optic nerve head and the lamina cribrosa. This is the first study to demonstrate the exact localization of serine racemase mRNA at the cellular or tissue level in the retina and the optic nerve head. These results suggest that both the neuron- and glia-derived D-serine could modulate neurotransmission via the glycine site of the N-methyl-D-aspartate receptors in the retina.


Assuntos
Neuroglia/metabolismo , Neurônios/metabolismo , RNA Mensageiro/metabolismo , Racemases e Epimerases/metabolismo , Retina/metabolismo , Animais , Expressão Gênica , Hibridização In Situ , Masculino , Neuroglia/citologia , Neurônios/citologia , Racemases e Epimerases/genética , Ratos , Ratos Wistar , Retina/citologia
12.
Exp Cell Res ; 313(20): 4196-207, 2007 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-17765891

RESUMO

We identified 11 proteins that are associated with DGCR8 by immunoprecipitation assay and mass spectrometry. These proteins included Nucleolin, ILF3 and others, most of which appeared to be involved in the RNA processing or RNA transportation. We detected at least four kinds of protein complex, such as DROSHA/DGCR8, DGCR8/Nucleolin, DGCR8/ILF3 and ILF3/XPO5, by co-immunoprecipitation. The complex formation of DGCR8 with Nucleolin was dependent on RNA. Subcellular localization analysis by the immunofluorescent microscopy and immunoelectron microscopy indicated that DGCR8 locates at the nucleolus and small foci adjacent to splicing speckles in the nucleoplasm. Furthermore, the localization of DGCR8 at the nucleolus was changed by the inhibition of RNA transcription. Thus, our studies provided additional new evidence for the involvement of various protein complexes in the molecular mechanisms of apparently complex innate RNA interference machinery.


Assuntos
Nucléolo Celular/metabolismo , Proteínas/metabolismo , Nucléolo Celular/ultraestrutura , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica , Células HeLa , Humanos , Espectrometria de Massas , Modelos Genéticos , Proteínas do Fator Nuclear 90/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Frações Subcelulares/metabolismo , Transcrição Gênica , Nucleolina
13.
Arch Histol Cytol ; 70(2): 127-34, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17827670

RESUMO

D-serine is an endogenous and obligatory coagonist for the glycine site of the N-methyl-D-aspartate receptor in the mammalian brain. D-serine is synthesized from L-serine by serine racemase; immunohistochemical studies have long been believed to indicate that serine racemase and D-serine occur predominantly in astrocytes. However, we have recently demonstrated in the primary cultures that both the mRNA and protein levels of serine racemase are higher in neurons than in astrocytes. Here we report the application of in situ hybridization based on tyramide signal amplification for the detection of serine racemase mRNA in sections of the adult rat brain. Serine racemase mRNA could be demonstrated in a large number of neurons throughout the brain, especially in the forebrain such as the cerebral cortex, striatum, and hippocampus. This is the first study to demonstrate the exact localization of serine racemase mRNA at the cellular or tissue level. These results suggest that neuron-derived D-serine could modulate neurotransmission via the glycine site of N-methyl-D-aspartate receptors.


Assuntos
Encéfalo/metabolismo , Neurônios/metabolismo , RNA Mensageiro/biossíntese , Racemases e Epimerases/metabolismo , Animais , Northern Blotting , Encéfalo/citologia , Hibridização In Situ , Masculino , Racemases e Epimerases/genética , Ratos , Ratos Wistar
14.
Proc Natl Acad Sci U S A ; 103(30): 11282-7, 2006 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-16844785

RESUMO

Oxidative stress has long been linked to the pathogenesis of neurodegenerative diseases; however, whether it is a cause or merely a consequence of the degenerative process is still unknown. We show that mice deficient in Cu, Zn-superoxide dismutase (SOD1) have features typical of age-related macular degeneration in humans. Investigations of senescent Sod1(-/-) mice of different ages showed that the older animals had drusen, thickened Bruch's membrane, and choroidal neovascularization. The number of drusen increased with age, and exposure of young Sod1(-/-) mice to excess light induced drusen. The retinal pigment epithelial cells of Sod1(-/-) mice showed oxidative damage, and their beta-catenin-mediated cellular integrity was disrupted, suggesting that oxidative stress may affect the junctional proteins necessary for the barrier integrity of the retinal pigment epithelium. These observations strongly suggest that oxidative stress may play a causative role in age-related retinal degeneration, and our findings provide evidence for the free radical theory of aging. In addition, these results demonstrate that the Sod1(-/-) mouse is a valuable animal model to study human age-related macular degeneration.


Assuntos
Doenças da Coroide/patologia , Degeneração Macular/genética , Degeneração Macular/patologia , Neovascularização Patológica , Drusas do Disco Óptico/patologia , Epitélio Pigmentado Ocular/metabolismo , Superóxido Dismutase/genética , Animais , Doenças da Coroide/genética , Modelos Animais de Doenças , Luz , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Drusas do Disco Óptico/genética , Estresse Oxidativo , Superóxido Dismutase-1 , beta Catenina/metabolismo
15.
Biochem Biophys Res Commun ; 328(4): 1232-43, 2005 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-15708008

RESUMO

Transient receptor potential melastatin 2 (TRPM2) is a calcium-permeable cation channel activated by ADP-ribose or reactive oxygen species. In human, a major transcript of 6.5 kb is expressed in various tissues, whereas a minor transcript of 5.5 kb is detected only in striatum (caudate nucleus and putamen). We found that the 5.5-kb shorter transcript is transcribed from the intron 4 of the TRPM2 gene and encodes the striatum short form protein (SSF-TRPM2) with 1289 amino acid residues as compared to the long form protein (LF-TRPM2), in which the N-terminal 214 amino acid residues are removed. The SSF-TRPM2 protein still maintained H2(O2)-induced Ca2+ influx activity. In addition, we found that the major transcripts in human and mouse start from a novel 5' non-coding exon; however, we could not detect any striatum short transcript in mouse brain. These new findings are invaluable to further study the regulation of TRPM2 gene expression and to examine the possible involvement of the TRPM2 gene in the pathophysiology of bipolar disorder.


Assuntos
Corpo Estriado/metabolismo , Canais Iônicos/química , Canais Iônicos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Sequência Conservada , Humanos , Canais Iônicos/genética , Proteínas de Membrana/genética , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade da Espécie , Canais de Cátion TRPM , Distribuição Tecidual
16.
Biol Cell ; 94(6): 389-99, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12500945

RESUMO

In avian, there are three slow skeletal myosin heavy chain (MHC) isoforms, slow skeletal MHC 1, 2, and 3. While slow skeletal MHC 3 has been characterized, slow skeletal MHC 1 and 2 are not yet fully studied. To determine the complete sequence of slow skeletal MHC 2, we isolated six overlapping cDNA clones, each encoding a portion of chick slow skeletal MHC 2, using the reverse transcription polymerase chain reaction (RT-PCR). The entire slow skeletal MHC 2 cDNA consisted of 5927 nucleotides including a 104 bp 3'-untranslated region and encoded 1941 amino acids. Using one of the cDNA clones, we made a probe for in situ hybridization. We also used immunohistochemistry to localize slow skeletal MHC 2 in skeletal and cardiac tissues. These studies showed that in addition to its expected expression in the adult chicken slow skeletal muscle, slow skeletal MHC 2 was expressed in the subendocardial cluster of cells and around the blood vessels within the ventricle of late embryos and adults. This isoform was not expressed in the myocardium throughout the life of the chicken. Based on morphological criteria as well as rich desmin expression, we concluded that the subendocardial cluster of cells were Purkinje cells. Although the physiological significance of the slow skeletal MHC expression remains elusive at this time, this MHC isoform may be used as a specific marker for Purkinje cells.


Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Cadeias Pesadas de Miosina/genética , Ramos Subendocárdicos/metabolismo , Animais , Sequência de Bases , Embrião de Galinha , Galinhas , Coração/embriologia , Sistema de Condução Cardíaco/química , Sistema de Condução Cardíaco/citologia , Sistema de Condução Cardíaco/embriologia , Dados de Sequência Molecular , Miocárdio/química , Miocárdio/citologia , Cadeias Pesadas de Miosina/biossíntese , Isoformas de Proteínas , Ramos Subendocárdicos/química , RNA Mensageiro/metabolismo , Análise de Sequência de DNA , Distribuição Tecidual
17.
Biochem Biophys Res Commun ; 304(1): 184-90, 2003 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-12705904

RESUMO

We have identified and cloned a novel gene (DGCR8) from the human chromosome 22q11.2. This gene is located in the DiGeorge syndrome chromosomal region (DGCR). It consists of 14 exons spanning over 35kb and produces transcripts with ORF of 2322bp, encoding a protein of 773 amino acids. We also isolated a mouse ortholog Dgcr8 and found it has 95.3% identity with human DGCR8 at the amino acid sequence level. Northern blot analysis of human and mouse tissues from adult and fetus showed rather ubiquitous expression. However, the in situ hybridization of mouse embryos revealed that mouse Dgcr8 transcripts are localized in neuroepithelium of primary brain, limb bud, vessels, thymus, and around the palate during the developmental stages of embryos. The expression profile of Dgcr8 in developing mouse embryos is consistent with the clinical phenotypes including congenital heart defects and palate clefts associated with DiGeorge syndrome (DGS)/conotruncal anomaly face syndrome (CAFS)/velocardiofacial syndrome (VCFS), which are caused by monoallelic microdeletion of chromosome 22q11.2.


Assuntos
Cromossomos Humanos Par 22 , Motivos de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Síndrome de DiGeorge/genética , Embrião de Mamíferos/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , Proteínas , RNA Mensageiro/biossíntese , Proteínas de Ligação a RNA , Distribuição Tecidual , Transcrição Gênica
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