RESUMO
Vision requires the transport and recycling of the pigment 11-cis retinaldehyde (retinal) between the retinal pigment epithelium (RPE) and photoreceptors. 11-cis retinal is also required for light-mediated photoreceptor death in dark-adapted mouse eye, probably through overstimulation of rod cells adapted for low light. Retbindin is a photoreceptor-specific protein, of unclear function, that is localized between the RPE and the tips of the photoreceptors. Unexpectedly, young Rtbdn-KO mice, with targeted deletion (KO) of retbindin, showed delayed regeneration of retinal function after bleaching and were strongly resistant to light-induced photoreceptor death. Furthermore, bio-layer interferometry binding studies showed recombinant retbindin had significant affinity for retinoids, most notably 11-cis retinal. This suggests that retbindin mediates light damage, probably through a role in transport of 11-cis retinal. In Rtbdn-KO mice, retinal development was normal, as were amplitudes of rod and cone electroretinograms (ERG) up to 4 months, although implicit times and c-waves were affected. However, with aging, both light- and dark-adapted ERG amplitudes declined significantly and photoreceptor outer segments became disordered, However, in contrast to other reports, there was little retinal degeneration or drop in flavin levels. The RPE developed vacuoles and lipid, protein and calcium deposits reminiscent of age-related macular degeneration. Other signs of premature aging included loss of OPN4+ retinal ganglion cells and activation of microglia. Thus, retbindin plays an unexpected role in the mammalian visual cycle, probably as an adaptation for vision in dim light. It mediates light damage in the dark-adapted eye, but also plays a role in light-adapted responses and in long term retinal homeostasis.
Assuntos
Senilidade Prematura/genética , Proteínas do Olho/genética , Regulação da Expressão Gênica , RNA/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Doenças Retinianas/genética , Epitélio Pigmentado da Retina/metabolismo , Senilidade Prematura/metabolismo , Animais , Adaptação à Escuridão/fisiologia , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/biossíntese , Camundongos , Microscopia Eletrônica de Transmissão , Células Fotorreceptoras Retinianas Cones/ultraestrutura , Doenças Retinianas/metabolismo , Doenças Retinianas/patologia , Epitélio Pigmentado da Retina/ultraestruturaRESUMO
The polycistronic miR-183/96/182 cluster is preferentially and abundantly expressed in terminally differentiating sensory epithelia. To clarify its roles in the terminal differentiation of sensory receptors in vivo, we deleted the entire gene cluster in mouse germline through homologous recombination. The miR-183/96/182 null mice display impairment of the visual, auditory, vestibular, and olfactory systems, attributable to profound defects in sensory receptor terminal differentiation. Maturation of sensory receptor precursors is delayed, and they never attain a fully differentiated state. In the retina, delay in up-regulation of key photoreceptor genes underlies delayed outer segment elongation and possibly mispositioning of cone nuclei in the retina. Incomplete maturation of photoreceptors is followed shortly afterward by early-onset degeneration. Cell biologic and transcriptome analyses implicate dysregulation of ciliogenesis, nuclear translocation, and an epigenetic mechanism that may control timing of terminal differentiation in developing photoreceptors. In both the organ of Corti and the vestibular organ, impaired terminal differentiation manifests as immature stereocilia and kinocilia on the apical surface of hair cells. Our study thus establishes a dedicated role of the miR-183/96/182 cluster in driving the terminal differentiation of multiple sensory receptor cells.
Assuntos
Células Ciliadas Auditivas/citologia , Células Ciliadas Vestibulares/citologia , MicroRNAs/genética , Mucosa Olfatória/citologia , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Animais , Regulação da Expressão Gênica no Desenvolvimento/genética , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Vestibulares/metabolismo , Transtornos da Audição/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Família Multigênica , Transtornos do Olfato/genética , Mucosa Olfatória/metabolismo , Equilíbrio Postural/genética , Células Fotorreceptoras Retinianas Cones/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Transtornos de Sensação/genética , Transtornos da Visão/genéticaRESUMO
Photoreceptor degeneration is a cause of irreversible vision loss in incurable blinding retinal diseases including retinitis pigmentosa (RP) and atrophic age-related macular degeneration. We found in two separate mouse models of photoreceptor degeneration that tamoxifen, a selective estrogen receptor modulator and a drug previously linked with retinal toxicity, paradoxically provided potent neuroprotective effects. In a light-induced degeneration model, tamoxifen prevented onset of photoreceptor apoptosis and atrophy and maintained near-normal levels of electroretinographic responses. Rescue effects were correlated with decreased microglial activation and inflammatory cytokine production in the retina in vivo and a reduction of microglia-mediated toxicity to photoreceptors in vitro, indicating a microglia-mediated mechanism of rescue. Tamoxifen also rescued degeneration in a genetic (Pde6brd10) model of RP, significantly improving retinal structure, electrophysiological responses, and visual behavior. These prominent neuroprotective effects warrant the consideration of tamoxifen as a drug suitable for being repurposed to treat photoreceptor degenerative disease.SIGNIFICANCE STATEMENT Photoreceptor degeneration is a cause of irreversible blindness in a number of retinal diseases such as retinitis pigmentosa (RP) and atrophic age-related macular degeneration. Tamoxifen, a selective estrogen receptor modulator approved for the treatment of breast cancer and previously linked to a low incidence of retinal toxicity, was unexpectedly found to exert marked protective effects against photoreceptor degeneration. Structural and functional protective effects were found for an acute model of light-induced photoreceptor injury and for a genetic model for RP. The mechanism of protection involved the modulation of microglial activation and the production of inflammatory cytokines, highlighting the role of inflammatory mechanisms in photoreceptor degeneration. Tamoxifen may be suitable for clinical study as a potential treatment for diseases involving photoreceptor degeneration.
Assuntos
Regeneração Nervosa/fisiologia , Células Fotorreceptoras de Vertebrados/efeitos dos fármacos , Células Fotorreceptoras de Vertebrados/patologia , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/fisiopatologia , Tamoxifeno/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Regeneração Nervosa/efeitos da radiação , Fármacos Neuroprotetores/administração & dosagem , Células Fotorreceptoras de Vertebrados/fisiologia , Recuperação de Função Fisiológica/efeitos dos fármacos , Degeneração Retiniana/patologia , Resultado do TratamentoRESUMO
KLPH/lctl belongs to the Klotho family of proteins. Expressed sequence tag analyses unexpectedly revealed that KLPH is highly expressed in the eye lens while northern blots showed that expression is much higher in the eye than in other tissues. In situ hybridization in mouse localized mRNA to the lens, particularly in the equatorial epithelium. Immunofluorescence detected KLPH in lens epithelial cells with highest levels in the germinative/differentiation zone. The gene for KLPH in mouse was deleted by homologous recombination. Littermate knockout (KO) and wild type (WT) mice were compared in a wide panel of pathology examinations and were all grossly normal, showing no systemic effects of the deletion. However, the lens, while superficially normal at young ages, had focusing defects and exhibited age-related cortical cataract by slit lamp examination. Whole-lens imaging showed that KO mice had disorganized lens sutures, forming a loose double-y or x instead of the tight y formation of WT. RNA-seq profiles for KO and WT littermates confirmed the absence of KLPH mRNA in KO lens and also showed complete absence of transcripts for Clic5, a protein associated with cilium/basal body related auditory defects in a mouse model. Immunofluorescence of lens epithelial flat mounts showed that Clic5 localized to cilia/centrosomes. Mice mutant for Clic5 (jitterbug) also had defective sutures. These results suggest that KLPH is required for lens-specific expression of Clic5 and that Clic5 has an important role in the machinery that controls lens fiber cell extension and organization.
Assuntos
Canais de Cloreto/metabolismo , Regulação da Expressão Gênica/fisiologia , Glicosídeo Hidrolases/genética , Cristalino/metabolismo , Proteínas de Membrana/genética , Animais , Northern Blotting , Western Blotting , Catarata/metabolismo , Catarata/patologia , Diferenciação Celular , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas do Olho/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Deleção de Genes , Imunoquímica , Hibridização In Situ , Cristalino/citologia , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genéticaRESUMO
PURPOSE: Having observed that confluent ARPE-19 cells (derived from human RPE) survive well in high-glucose serum-free medium (SFM) without further feeding for several days, we investigated the expression profile of RPE cells under the same conditions. METHODS: Expression profiles were examined with microarray and quantitative PCR (qPCR) analyses, followed by western blot analysis of key regulated proteins. The effects of low-density lipoprotein (LDL) and zinc supplementation were examined with qPCR. Immunofluorescence was used to localize the LDL receptor and to examine LDL uptake. Cellular cholesterol levels were measured with filipin binding. Expression patterns in primary fetal RPE cells were compared using qPCR. RESULTS: Microarray analyses of gene expression in ARPE-19, confirmed with qPCR, showed upregulation of lipid and cholesterol biosynthesis pathways in SFM. At the protein level, the cholesterol synthesis control factor SRBEF2 was activated, and other key lipid synthesis proteins increased. Supplementation of SFM with LDL reversed the upregulation of lipid and cholesterol synthesis genes, but not of cholesterol transport genes. The LDL receptor relocated to the plasma membrane, and LDL uptake was activated by day 5-7 in SFM, suggesting increased demand for cholesterol. Confluent ARPE-19 cells in SFM accumulated intracellular cholesterol, compared with cells supplemented with serum, over 7 days. Over the same time course in SFM, the expression of metallothioneins decreased while the major zinc transporter was upregulated, consistent with a parallel increase in demand for zinc. Supplementation with zinc reversed expression changes for metallothionein genes, but not for other zinc-related genes. Similar patterns of regulation were also seen in primary fetal human RPE cells in SFM. CONCLUSIONS: ARPE-19 cells respond to serum deprivation and starvation with upregulation of the lipid and cholesterol pathways, accumulation of intracellular cholesterol, and increased demand for zinc. Similar trends are seen in primary fetal RPE cells. Cholesterol accumulation basal to RPE is a prominent feature of age-related macular degeneration (AMD), while dietary zinc is protective. It is conceivable that accumulating defects in Bruch's membrane and dysfunction of the choriocapillaris could impede transport between RPE and vasculature in AMD. Thus, this pattern of response to serum deprivation in RPE-derived cells may have relevance for some aspects of the progression of AMD.
Assuntos
Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Meios de Cultura Livres de Soro , Epitélio Pigmentado da Retina/metabolismo , Zinco/metabolismo , Células Cultivadas , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Immunoblotting , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMO
Viscoelastic braking theories developed by Shanahan and de Gennes and by others predict deformation of a solid surface at the solid-liquid-air contact line. This phenomenon has only been observed for soft smooth surfaces and results in a protrusion of the solid surface at the three-phase contact line, in agreement with the theoretical predictions. Despite the large (enough to break chemical bonds) forces associated with it, this deformation was not confirmed experimentally for hard surfaces, especially for hydrophobic ones. In this study we use superhydrophobic surfaces composed of an array of silicon nanostructures whose Young modulus is 4 orders of magnitude higher than that of surfaces in earlier recorded viscoelastic braking experiments. We distinguish between two cases: when a water drop forms an adhesive contact, albeit small, with the apparent contact angle θ < 180° and when the drop-surface adhesion is such that the conditions for placing a resting drop on the surface cannot be reached (i.e., θ = 180°). In the first case we show that there is a surface deformation at the three-phase contact line which is associated with a reduction in the hydrophobicity of the surface. For the second case, however, there cannot be a three-phase contact line associated with a drop in contact with the surface, and indeed, if we force-place a drop on the surface by holding it with a needle, no deformation is detected, nor is there a reduction in the hydrophobic properties of the surface. Yet, if we create a long horizontal three-phase contact line by partially immersing the superhydrophobic substrate in a water bath, we see a localized reduction in the hydrophobic properties of the surface in the region where the three-phase contact line used to be. The SEM scan of that region shows a narrow horizontal stripe where the nanorods are no longer there, and instead there is only a shallow structure that is lower than the nanorods height and resembles fused or removed nanorods. Away from that region, either on the part of the surface which was exposed to bulk water or the part which was exposed to air, no change in the hydrophobic properties of the surface is observed, and the SEM scan confirms that the nanorods seem intact in both regions.
Assuntos
Água/química , Adesivos/química , Elasticidade , Interações Hidrofóbicas e Hidrofílicas , Nanoestruturas/química , Silício/química , Propriedades de Superfície , ViscosidadeRESUMO
OBJECTIVE: To preliminarily explore death risk factors in primary melanoma patients. METHOD: Competing risk model analysis was used using a large sample public cohort and Cox proportional hazard model was compared. RESULT: In the competing risk model analysis, age, gender, ethnicity, stage, site, TMN stage and metastases were the independent risk factors of single primary melanoma (SPM) death. T stage had a particularly important impact on SPM death. T2 stage had a 3.212 times greater risk of interest event than T1 stage [hazard ratio (HR)=3.212, 95%CI: 2.994-3.446], T3 stage was 5.747 times greater than that T1 stage (HR=5.747, 95%CI: 5.337-6.187) and T4 stage had a 7.086 times than T1 stage (HR=7.086, 95%CI: 6.514-7.708). Gender, ethnicity, stage, site, T stage and brain and liver metastases were the independent risk factors of multiple primary melanoma (MPM) death. When some groups had a very high death rate or the reference group had a very low death rate in competing events, the results of Cox proportional hazard model may not be as accurate as the results obtained by fine-Gray regression model. CONCLUSION: Early diagnosis and therapy, and prevention of tumor progression and metastases of primary melanoma patients are important measures to improve its prognosis and survival.
Assuntos
Melanoma/mortalidade , Adulto , Fatores Etários , Idoso , Neoplasias Ósseas/mortalidade , Neoplasias Ósseas/secundário , Neoplasias Encefálicas/mortalidade , Neoplasias Encefálicas/secundário , Estudos de Coortes , Intervalos de Confiança , Feminino , Humanos , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/secundário , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/secundário , Masculino , Melanoma/etnologia , Melanoma/patologia , Melanoma/secundário , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Modelos de Riscos Proporcionais , Análise de Regressão , Fatores de Risco , Fatores SexuaisRESUMO
By coating a fluorocarbon monolayer on a bundled Si nanorod array substrate, a superhydrophobic surface with contact angle approximately 167 degrees and sliding angle approximately 2 degrees is created due to the nanocarpet effect. Without forming the nanocarpet, we can only obtain a moderately hydrophobic surface with contact angle <151 degrees and sliding angle >17 degrees. Comparison between nanocarpets formed from nanorods with low and high densities confirms that the main reason for the superhydrophobicity is the formation of sharp pyramidal bundles, which effectively reduces the area of solid-liquid contact. Video recording and analysis of millimeter-sized water droplets bouncing on the solid surface are used to ascertain the superhydrophobicity, and the energy dissipation during a low speed impact is estimated to be several nanojoules.
RESUMO
Deposition of hydroxyapatite (HAP) basal to the retinal pigment epithelium (RPE) is linked to the progression of age-related macular degeneration (AMD). Serum-deprivation of RPE cells in culture mimics some features of AMD. We now show that serum-deprivation also leads to the induction of amelotin (AMTN), a protein involved in hydroxyapatite mineralization in enamel. HAP is formed in our culture model and is blocked by siRNA inhibition of AMTN expression. In situ hybridization and immunofluorescence imaging of human eye tissue show that AMTN is expressed in RPE of donor eyes with geographic atrophy ("dry" AMD) in regions with soft drusen containing HAP spherules or nodules. AMTN is not found in hard drusen, normal RPE, or donor eyes diagnosed with wet AMD. These findings suggest that AMTN is involved in formation of HAP spherules or nodules in AMD, and as such provides a new therapeutic target for slowing disease progression.
Assuntos
Proteínas do Esmalte Dentário/metabolismo , Durapatita/metabolismo , Atrofia Geográfica/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Idoso , Meios de Cultura Livres de Soro , HumanosRESUMO
PURPOSE: To investigate the functional significance of MIP/AQP0 phosphorylation at serine(235). METHODS: MIP/AQP0 expression and cellular localization was studied in rat lens epithelia explants induced to differentiate by FGF-2. MIP wild type (WT) and MIP (S235A) mutant expression plasmids were constructed and transiently expressed in RK13 cells. Subcellular localization of endogenous MIP in differentiating lens epithelia explants or of transfected MIP expression vectors in RK13 cells was analyzed by immunofluorescence confocal microscopy. RESULTS: MIP/AQP0 expressed in lens epithelia explants induced to differentiate by FGF-2 localizes to the plasma membrane of elongating cells. However, MIP/AQP0 translocation to the plasma membrane was prevented by inhibiting PKC activity with Go6976, resulting in retention in the cytoplasmic compartment. This effect was specific to MIP/AQP0; localization of AQP1 to the cell membrane was not affected by Go6976. When the consensus PKC phosphorylation site at MIP Ser(235) was mutated to alanine and transiently expressed in transfected RK13 cells, the mutant MIP was retained in the cytoplasmic compartment in contrast to WT MIP that localized to the plasma membrane of the transfected RK13 cells. Colocalization studies indicated that the mutant MIP was retained in the trans-Golgi network. CONCLUSIONS: Our results indicate that serine(235) is required for proper intracellular transport of MIP/AQP0 from the trans-Golgi network to the plasma membrane. A PKC dependent phosphorylation event involving MIP at serine(235) is most likely involved in this process.
Assuntos
Aquaporinas/metabolismo , Membrana Celular/enzimologia , Proteínas do Olho/metabolismo , Cristalino/citologia , Cristalino/enzimologia , Glicoproteínas de Membrana/metabolismo , Fosfosserina/metabolismo , Proteína Quinase C/metabolismo , Animais , Aquaporina 1/metabolismo , Aquaporinas/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Proteínas do Olho/genética , Técnicas In Vitro , Cristalino/efeitos dos fármacos , Glicoproteínas de Membrana/genética , Camundongos , Proteínas Mutantes/metabolismo , Mutação/genética , Fosforilação/efeitos dos fármacos , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Coelhos , Ratos , Ratos Sprague-Dawley , Transcrição Gênica/efeitos dos fármacos , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
γ-Crystallins, abundant proteins of vertebrate lenses, were thought to be absent from birds. However, bird genomes contain well-conserved genes for γS- and γN-crystallins. Although expressed sequence tag analysis of chicken eye found no transcripts for these genes, RT-PCR detected spliced transcripts for both genes in chicken lens, with lower levels in cornea and retina/retinal pigment epithelium. The level of mRNA for γS in chicken lens was relatively very low even though the chicken crygs gene promoter had lens-preferred activity similar to that of mouse. Chicken γS was detected by a peptide antibody in lens, but not in other ocular tissues. Low levels of γS and γN proteins were detected in chicken lens by shotgun mass spectroscopy. Water-soluble and water-insoluble lens fractions were analyzed and 1934 proteins (< 1% false discovery rate) were detected, increasing the known chicken lens proteome 30-fold. Although chicken γS is well conserved in protein sequence, it has one notable difference in leucine 16, replacing a surface glutamine conserved in other γ-crystallins, possibly affecting solubility. However, L16 and engineered Q16 versions were both highly soluble and had indistinguishable circular dichroism, tryptophan fluorescence and heat stability (melting temperature Tm ~ 65 °C) profiles. L16 has been present in birds for over 100 million years and may have been adopted for a specific protein interaction in the bird lens. However, evolution has clearly reduced or eliminated expression of ancestral γ-crystallins in bird lenses. The conservation of genes for γS- and γN-crystallins suggests they may have been preserved for reasons unrelated to the bulk properties of the lens.
Assuntos
Galinhas/genética , Olho/metabolismo , Cristalino/metabolismo , Família Multigênica , Vertebrados/genética , gama-Cristalinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Galinhas/metabolismo , Dicroísmo Circular , Cristalino/química , Camundongos , Dados de Sequência Molecular , Filogenia , Regiões Promotoras Genéticas/genética , Proteoma , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Homologia de Sequência do Ácido Nucleico , Espectrometria de Fluorescência , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Vertebrados/metabolismo , gama-Cristalinas/química , gama-Cristalinas/metabolismoRESUMO
PURPOSE: Major Intrinsic Protein (MIP)/Aquaporin 0 is required for lens transparency and is specifically expressed in lens fiber cell membranes. We have demonstrated previously that in the rat lens MIP interacts specifically with gammaE-crystallin, resulting in its recruitment to the plasma membrane. Our goal was to examine the interaction or lack of interaction between MIP and all members of the gamma-crystallin family and to provide evidence for a physiological role these interactions may play in gamma-crystallin or MIP function. METHODS: Full length MIP was expressed as untagged, enhanced green fluorescent protein (EGFP) tagged, or myc tagged proteins. Members of the gamma-crystallin family were expressed as red fluorescent protein (HcRed) tagged proteins in the rabbit kidney epithelial cell line RK13. Co-localization of tagged proteins was analyzed by confocal fluorescence microscopy. RESULTS: Confocal fluorescence microscopy demonstrated that gammaE- and gammaF-crystallin co-localize specifically with full length MIP in mammalian cells while other gamma-crystallins, including gammaA-, gammaB-, gammaC-, gammaD-, and gammaS-crystallin do not. As a result of this interaction, either gammaE- or gammaF-crystallin was recruited to the plasma membrane from the cytoplasm. MIP does not interact with the Elo mutant of gammaE-crystallin, which has been linked to a dominant cataract phenotype in mice. CONCLUSIONS: These experiments demonstrate that MIP interacts selectively with gammaE- and gammaF-crystallin, and not with other gamma-crystallins. This raises the possibility of MIP playing a structural role in the organization of gamma-crystallins in rodent lens fibers and/or that gammaE- and gammaF-crystallin may have a specific role in MIP function in the rodent lens.
Assuntos
Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , gama-Cristalinas/metabolismo , Sequência de Aminoácidos , Animais , Aquaporinas , Linhagem Celular , Células Epiteliais/metabolismo , Expressão Gênica , Vetores Genéticos , Proteínas de Fluorescência Verde/metabolismo , Rim/citologia , Rim/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Dados de Sequência Molecular , Plasmídeos , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/metabolismo , Coelhos , Transfecção , Proteína Vermelha FluorescenteRESUMO
PURPOSE: Major intrinsic protein (MIP), also called aquaporin-0, is essential for lens transparency and is specifically expressed in the lens fiber cell membranes. The goal of the current study was to identify and characterize proteins that interact with MIP and to elucidate the role of these interactions in MIP functions. METHODS: The C-terminal 74-amino-acid fragment of MIP was used as bait to screen a rat lens cDNA yeast two-hybrid library. The full-length MIP was expressed as enhanced green fluorescent protein (EGFP)-tagged or myc-tagged proteins, and gammaE-crystallin was expressed as FLAG-tagged or red fluorescent protein (HcRed)-tagged proteins, respectively, in the RK13 rabbit kidney epithelial cell line. Protein-protein interactions were analyzed by coimmunoprecipitation assays and visualized by confocal fluorescence microscopy. RESULTS: gammaE-Crystallin, a water-soluble protein that is specifically expressed in lens fibers, was identified as a binding protein to the MIP C-terminal peptide. Coimmunoprecipitation assays demonstrated that gammaE-crystallin interacts specifically with full-length MIP in mammalian cells. MIP did not interact with gammaD-crystallin, another member of the highly conserved gamma-crystallin gene family. Confocal fluorescence microscopy demonstrated that MIP interacted with gammaE-crystallin in individual mammalian cells and that this interaction resulted in the recruitment of gammaE-crystallin from the cytoplasm to the plasma membrane. CONCLUSIONS: These experiments provide the first demonstration of MIP interaction with other lens proteins at the molecular level and raise the possibility of a structural role of MIP in the organization of gamma-crystallins in lens fibers.
Assuntos
Antígenos de Superfície/metabolismo , Membrana Celular/metabolismo , Cristalinas/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas de Membrana/metabolismo , Animais , Aquaporinas , Western Blotting , Linhagem Celular , Biblioteca Gênica , Proteínas de Fluorescência Verde , Rim/citologia , Rim/metabolismo , Proteínas Luminescentes/metabolismo , Microscopia Confocal , Plasmídeos , Ligação Proteica , Mapeamento de Interação de Proteínas , Coelhos , Ratos , Técnicas do Sistema de Duplo-Híbrido , gama-CristalinasRESUMO
PURPOSE: This study sought to analyze the changes to clots and retinal tissue following direct infusion of tissue plasminogen activator (tPA) at different rates to the obstructed area of the retinal vein in an experimental animal model of retinal vein occlusion. METHODS: The miniature pigs, which underwent unilateral retinal vein thrombosis generated by a combination of light illumination of the retinal vein and I.V. administration of rose Bengal, were randomly divided into two groups. In the experimental group (n = 45), balanced salt solutions + tPA were infused into the obstructed retinal vein at different rates (30,60, and 80 ml/h) for 20 minutes (15 pigs in each subgroups). In the control group (n = 5), balanced salt solutions without tPA were infused. Retinal tissue and clots were evaluated by fundus photographic and histopathologic analysis. RESULTS: Clot lysis occurred in 2/15, 9/15, and 10/15 pigs respectively following the 30, 60, and 80 ml/h tPA infusion. Exudative retinal detachment appeared in 12/15 pigs after infusion of tPa at a rate of 80 ml/h. Retinal histopathology and transmission electron microscopy showed that endothelial cell tight junctions of retinal vein and capillaries were injured in pigs receiving the 80 ml/h tPA infusion. CONCLUSION: Experiments with our pig model suggests that the optimal infusion rate for the treatment of retinal vein thrombosis by direct tPA infusion is 60 ml/h for 20 minutes.
Assuntos
Fibrinolíticos/administração & dosagem , Veia Retiniana , Vasos Retinianos/efeitos dos fármacos , Ativador de Plasminogênio Tecidual/administração & dosagem , Trombose Venosa/tratamento farmacológico , Animais , Relação Dose-Resposta a Droga , Fibrinolíticos/uso terapêutico , Microinjeções , Retina/efeitos dos fármacos , Retina/patologia , Suínos , Porco Miniatura , Ativador de Plasminogênio Tecidual/uso terapêutico , Resultado do Tratamento , Trombose Venosa/patologia , Trombose Venosa/fisiopatologiaRESUMO
γS-crystallin (γS) is a highly conserved component of the eye lens. To gain insights into the functional role(s) of this protein, the mouse gene (Crygs) was deleted. Although mutations in γS can cause severe cataracts, loss of function of γS in knockout (KO) mice produced no obvious lens opacity, but was associated with focusing defects. Electron microscopy showed no major differences in lens cell organization, suggesting that the optical defects are primarily cytoplasmic in origin. KO lenses were also grossly normal by light microscopy but showed evidence of incomplete clearance of cellular organelles in maturing fiber cells. Phalloidin labeling showed an unusual distribution of F-actin in a band of mature fiber cells in KO lenses, suggesting a defect in the organization or processing of the actin cytoskeleton. Indeed, in wild-type lenses, γS and F-actin colocalize along the fiber cell plasma membrane. Relative levels of F-actin and G-actin in wild-type and KO lenses were estimated from fluorescent staining profiles and from isolation of actin fractions from whole lenses. Both methods showed a two-fold reduction in the F-actin/G-actin ratio in KO lenses, whereas no difference in tubulin organization was detected. In vitro experiments showed that recombinant mouse γS can directly stabilize F-actin. This suggests that γS may have a functional role related to actin, perhaps in 'shepherding' filaments to maintain the optical properties of the lens cytoplasm and normal fiber cell maturation.
Assuntos
Actinas/metabolismo , Cristalino/citologia , gama-Cristalinas/metabolismo , Animais , Cristalino/fisiologia , Cristalino/ultraestrutura , Camundongos , Camundongos Knockout , gama-Cristalinas/genéticaRESUMO
Three-dimensional Cu-Si and Cu-SiO(2) nanorod arrays containing ~68 at% Cu have been fabricated by a glancing angle co-deposition technique. By selectively etching Cu in 0.05 M KCN methanol solution, porous nanorods with different shapes form, which are promising for applications in sensors, catalysts, and as medical capsules that are able to be loaded with functional materials.
RESUMO
Lens major intrinsic protein (MIP), exclusive to the vertebrate lens, otherwise known as MIP26 and Aquaporin 0, is abundantly expressed as a lens fiber membrane protein. Although relatively less efficient compared with other aquaporins, MIP is suggested to function as a water channel, as an adhesion molecule, and is required for lens transparency. Because MIP is specifically expressed in lens fiber cells, we investigated in this study the activation of Mip expression after triggering differentiation of rat lens epithelia explants by fibroblast growth factor (FGF)-2. Here, we show that Mip expression in the lens cells is regulated by FGF-2. Using Real time PCR we demonstrate that endogenous Mip levels in the explants were up-regulated upon FGF-2 stimulation, in a concentration-dependent manner. Up-regulation of Mip at the transcriptional level was simultaneous with the activation of the FGF down-stream signaling components, ERK1/2 and JNK. Specific inhibitors, UO126 for ERK1/2 and SP600125 for JNK, abrogated Mip expression in response to FGF-2 in the explants. This inhibition pattern was recapitulated in reporter assays for transfection of the rat lens epithelia explants, driven by the Mip promoter (-1648/+44). Our studies show that ERK1/2 and JNK signaling pathways are required for Mip expression in lens epithelia explants induced to differentiate by FGF-2.