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1.
Anticancer Res ; 39(7): 3311-3315, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31262851

RESUMO

Pigment epithelium-derived factor (PEDF) is an important antiangiogenic and antitumorigenic factor in a variety of cancer forms, including pancreatic cancer. PEDF is mainly secreted as a soluble monomeric glycoprotein. In human pancreatic cancer PEDF levels are decreased, both in the tissue and serum. The decrease is associated with increased tumor angiogenesis, fibrosis, inflammation, autophagy, occurrence of liver metastasis and worse prognosis. In murine models, loss of PEDF is sufficient to induce invasive carcinoma and this phenotype is associated with large lesions characterized by poor differentiation. Lentiviral gene transfer of PEDF has resulted in decreased microvessel density and has inhibited tumor growth. Herein we review the multifunctional role of PEDF in pancreatic cancer and its therapeutic potential.


Assuntos
Proteínas do Olho/metabolismo , Fatores de Crescimento Neural/metabolismo , Neoplasias Pancreáticas/metabolismo , Serpinas/metabolismo , Animais , Humanos
2.
Nat Commun ; 10(1): 2477, 2019 06 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171770

RESUMO

Blood vessels in the central nervous system (CNS) develop unique features, but the contribution of CNS neurons to regulating those features is not fully understood. We report that inhibiting spontaneous cholinergic activity or reducing starburst amacrine cell numbers prevents invasion of endothelial cells into the deep layers of the retina and causes blood-retinal-barrier (BRB) dysfunction in mice. Vascular endothelial growth factor (VEGF), which drives angiogenesis, and Norrin, a Wnt ligand that induces BRB properties, are decreased after activity blockade. Exogenous VEGF restores vessel growth but not BRB function, whereas stabilizing beta-catenin in endothelial cells rescues BRB dysfunction but not vessel formation. We further identify that inhibiting cholinergic activity reduces angiogenesis during oxygen-induced retinopathy. Our findings demonstrate that neural activity lies upstream of VEGF and Norrin, coordinating angiogenesis and BRB formation. Neural activity originating from specific neural circuits may be a general mechanism for driving regional angiogenesis and barrier formation across CNS development.


Assuntos
Células Amácrinas/fisiologia , Barreira Hematorretiniana/crescimento & desenvolvimento , Neurônios Colinérgicos/fisiologia , Células Endoteliais/fisiologia , Neovascularização Fisiológica/fisiologia , Células Ganglionares da Retina/fisiologia , Animais , Barreira Hematorretiniana/efeitos dos fármacos , Barreira Hematorretiniana/inervação , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Neurônios Colinérgicos/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Proteínas do Olho/metabolismo , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Agonistas Nicotínicos/farmacologia , Oxigênio/efeitos adversos , Piridinas/farmacologia , Doenças Retinianas , Células Ganglionares da Retina/metabolismo , Neovascularização Retiniana/etiologia , Tetrodotoxina/farmacologia , Fator A de Crescimento do Endotélio Vascular/metabolismo , beta Catenina/metabolismo
3.
Indian J Ophthalmol ; 67(7): 1226-1229, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31238476

RESUMO

A 11-year-old boy presented with complaints of blurred vision and on evaluation was found to have X-linked retinoschisis (XLRS) with angle-closure glaucoma. Clinical and genetic evaluation of first-degree family members was done. His brother had a milder form of XLRS with shallow anterior chamber. Topical dorzolamide 2% and timolol 0.5% were used to control intraocular pressure. Genetic analysis revealed a novel three base pair deleterious mutation (c. 375_377 del AGA) in exon-5 of the RS1 gene in three members of the family.


Assuntos
DNA/genética , Proteínas do Olho/genética , Glaucoma de Ângulo Fechado/diagnóstico , Mutação , Retinosquise/diagnóstico , Criança , Análise Mutacional de DNA , Proteínas do Olho/metabolismo , Testes Genéticos , Glaucoma de Ângulo Fechado/complicações , Glaucoma de Ângulo Fechado/genética , Humanos , Masculino , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Retinosquise/complicações , Retinosquise/genética , Tomografia de Coerência Óptica , Acuidade Visual
4.
J Photochem Photobiol B ; 195: 51-57, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31082734

RESUMO

This study includes the fabrication of gold nanoparticles (AuNPs) with the help of a plant polyphenol called Resveratrol through an ecofriendly synthetic process without any use of harmful reductants. In the fabrication of AuNPs, Resveratrol acts as both stabilizing and reducing agent. The prepared AuNPs is tested on streptozotocin (STZ) induced diabetic rats for their amelioration consequence. The images of TEM displayed the development of spherical nanoparticles (NPs) with a median of 20 nm particle size. The STZ injected diabetic rats were administrated orally with calcium dobesilate (CD; 500 mg/kg/day) or AuNPs (200, 300 mg/kg/day) for a period of 3 months. The characteristics displayed by AuNPs were found to be similar with CD in decreasing permeability of blood-retinal barrier in STZ injected diabetic rats. The retinal vessels in the AuNPs administrated diabetic rats were observed to be decreased through the retinal histopathological examination. In the AuNPs administrated diabetic rats, the retinal expression of renal Pigment Epithelium-Derived Factor (PEDF) was observed to be increased and the Vascular Endothelial Growth Factor (VEGF-1), which was increased in diabetic rats was declined on treating with AuNPs. On treating the STZ injected diabetic rats with AuNPs, all the retinal mRNA expressions of VEGF-1, Tumor Necrosis Factor (TNFα), Monocyte Chemotactic Proteins-1 (MCP-1), Intercellular Adhesion Molecule-1 (ICAM-1), and Interleukin (IL)-6, IL-1ß were observed to be reduced. Furthermore, AuNPs can reduce phosphorylation of Nuclear Factor Kappa B (NF-κB) p65 and Extracellular signal Regulated Kinase (ERK) 1/2 along with a growth in nuclear translocation of pNF-κB p65 produced by STZ. To conclude, the protective effect of AuNPs on STZ injected diabetic rats could help in redeveloping the balance among the inhibitors and stimulators of angiogenesis. Furthermore, on treating with AuNPs results in inhibiting the signaling pathway of ERK1/2 as well as with amelioration of retinal inflammation through trans repression of NF-κB.


Assuntos
Retinopatia Diabética/tratamento farmacológico , Ouro/química , Nanopartículas Metálicas/química , Resveratrol/química , Animais , Glicemia/análise , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Retinopatia Diabética/etiologia , Proteínas do Olho/metabolismo , Molécula 1 de Adesão Intercelular/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Masculino , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , NF-kappa B/metabolismo , Fatores de Crescimento Neural/metabolismo , Ratos , Ratos Wistar , Resveratrol/uso terapêutico , Retina/metabolismo , Retina/patologia , Serpinas/metabolismo , Transdução de Sinais , Estreptozocina/toxicidade , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Genes Cells ; 24(7): 496-510, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31124270

RESUMO

In the Drosophila brain, neurons form genetically specified synaptic connections with defined neuronal targets. It is proposed that each central nervous system neuron expresses specific cell surface proteins, which act as identification tags. Through an RNAi screen of cell surface molecules in the Drosophila visual system, we found that the cell adhesion molecule Klingon (Klg) plays an important role in repressing the ectopic formation of extended axons, preventing the formation of excessive synapses. Cell-specific manipulation of klg showed that Klg is required in both photoreceptors and the glia, suggesting that the balanced homophilic interaction between photoreceptor axons and the glia is required for normal synapse formation. Previous studies suggested that Klg binds to cDIP and our genetic analyses indicate that cDIP is required in glia for ectopic synaptic repression. These data suggest that Klg play a critical role together with cDIP in refining synaptic specificity and preventing unnecessary connections in the brain.


Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/fisiologia , Proteínas do Olho/metabolismo , Neuroglia/fisiologia , Células Fotorreceptoras de Invertebrados/fisiologia , Sinapses/fisiologia , Vias Visuais , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/fisiologia , Axônios/fisiologia , Moléculas de Adesão Celular/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas do Olho/genética , Feminino
6.
Mol Vis ; 25: 222-236, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31057322

RESUMO

Purpose: Glaucoma is characterized by optic nerve damage and retinal ganglion cell loss. The glycoprotein neuromedin B-associated (Gpnmb) gene is well-known to be involved in the glaucoma disease process. The purpose of this study is to identify a downstream gene through which Gpnmb affects the glaucoma phenotypes using a systems genetics approach. Methods: Retinal gene expression data for the BXD recombinant inbred (RI) strains (n=75) have previously been generated in our laboratory for a glaucoma study, and these data were used for genetic and bioinformatics analysis. Expression quantitative trait locus (eQTL) mapping and genetic correlation methods were used to identify a gene downstream of Gpnmb. Gene-set enrichment analysis was used to evaluate gene function and to construct coexpression networks. Results: The level of Gpnmb expression is associated with a highly statistically significant cis-eQTL. Stanniocalcin 1 (Stc1) has a significant trans-eQTL mapping to the Gpnmb locus. The expression of Gpnmb and Stc1 is highly correlated in the retina and other tissues, as well as with glaucoma-related phenotypes. Gene Ontology and pathway analysis showed that Stc1 and its covariates are highly associated with apoptosis, oxidative stress, and mitochondrial activity. A generated gene network indicated that Gpnmb and Stc1 are directly connected to and interact with other genes with similar biologic functions. Conclusions: These results suggest that Stc1 may be a downstream candidate of Gpnmb, and that both genes interact with other genes in a network to develop glaucoma pathogenesis through mechanisms such as apoptosis and oxidative stress.


Assuntos
Proteínas do Olho/genética , Redes Reguladoras de Genes , Glaucoma/genética , Glicoproteínas/genética , Glicoproteínas de Membrana/genética , Animais , Apoptose , Biologia Computacional/métodos , Modelos Animais de Doenças , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Ontologia Genética , Glaucoma/metabolismo , Glaucoma/patologia , Glicoproteínas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Anotação de Sequência Molecular , Estresse Oxidativo , Fenótipo , Mapeamento de Interação de Proteínas , Locos de Características Quantitativas , Transdução de Sinais
7.
Jpn J Ophthalmol ; 63(4): 317-321, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31104165

RESUMO

PURPOSE: To investigate the perioperative clinical factors, which contribute to the postsurgical aqueous flare intensity (AFI) following 27-gauge pars plana vitrectomy (27GPPV) for primary rhegmatogenous retinal detachment (RRD). STUDY DESIGN: Retrospective clinical study. METHODS: We performed retrospective analyses of the medical records of 47 eyes of 47 patients with primary RRD who had undergone 27GPPV with a wide-angle viewing system. AFI was measured preoperatively and 1 week, 1 month, 3 months, 6 months, and 12 months after the surgery. RESULTS: AFI was significantly increased 1 week after the surgery (p<0.01) and then decreased overtime. At 6 months after surgery it was still statistically significantly higher than preoperative AFI (p=0.03). There was no statistical difference between preoperative AFI and that at 12 months following surgery. Multiple regression analyses revealed that the number of retinal photocoagulations and the performance of scleral indentation had significant positive correlation with AFI at 1 week, 1 month, 3 months, and 6 months, and at 1 month and 3 months after the surgery, respectively. CONCLUSION: Intraoperative retinal photocoagulation and scleral indentation are probable causes of increased AFI after 27G PPV for RRD.


Assuntos
Humor Aquoso/diagnóstico por imagem , Proteínas do Olho/metabolismo , Descolamento Retiniano/cirurgia , Acuidade Visual , Vitrectomia/métodos , Humor Aquoso/metabolismo , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Período Pós-Operatório , Descolamento Retiniano/diagnóstico , Descolamento Retiniano/fisiopatologia , Estudos Retrospectivos , Fatores de Tempo , Resultado do Tratamento
8.
Invest Ophthalmol Vis Sci ; 60(6): 2034-2037, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31067323

RESUMO

Antibodies are key reagents used in vision research, indeed across biomedical research, but they often do not reveal the whole story about a sample. It is important for researchers to be aware of aspects of antibodies that may affect or limit data interpretation. Federal agencies now require funded grants to demonstrate how they will authenticate reagents used. There is also a push for recombinant antibodies, enabled by phage display technology awarded the 2018 Nobel Prize in Chemistry, which allow for thorough validation and a fixed DNA sequence. Here, we discuss how issues surrounding antibodies are pertinent to detecting myocilin, a protein found in trabecular meshwork and associated with a portion of hereditary glaucoma. Confirmation of myocilin expression in tissues and cell culture has been adopted as validation standard in trabecular meshwork research; thus, a discussion of antibody characteristics and fidelity is critical. Further, based on our basic structural understanding of myocilin architecture and its biophysical aggregation properties, we provide a wish list for the characteristics of next-generation antibody reagents for vision researchers. In the long term, well-characterized antibodies targeting myocilin will enable new insights into its function and involvement in glaucoma pathogenesis.


Assuntos
Anticorpos/imunologia , Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glaucoma/imunologia , Glicoproteínas/metabolismo , Malha Trabecular/metabolismo , Proteínas do Citoesqueleto/imunologia , Proteínas do Olho/imunologia , Glaucoma/diagnóstico , Glaucoma/metabolismo , Glicoproteínas/imunologia , Humanos
9.
Invest Ophthalmol Vis Sci ; 60(6): 2049-2063, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31074760

RESUMO

Purpose: To assess the phenotypic variability and natural course of inherited retinal diseases (IRDs) caused by EYS mutations. Methods: Multiethnic cohort study (N = 30) with biallelic EYS variants from a clinical IRD database (retinitis pigmentosa [RP], N = 27; cone-rod dystrophy [CRD], N = 1; and macular dystrophy, N = 2). In vitro minigene splice assay was performed to determine the effect on EYS pre-mRNA splicing of the c.1299+5_1299+8del variant in macular dystrophy patients. Results: We found 27 different EYS variants in RP patients and 7 were novel. The rate of visual field loss of the V4e isopter area was -0.84 ± 0.44 ln(deg2) per year, and the rate of visual acuity loss was 0.75 Early Treatment Diabetic Retinopathy Study letters per year. Ellipsoid zone width was correlated with area of the hyperautofluorescent ring, with rs = 0.78 and P < 0.001. Rate of decline in ellipsoid zone width was -57 ± 17 µm per year (P < 0.01) (n = 14) or -3.69% ± 0.51% from baseline per year (P < 0.001). An isolated CRD patient carried a homozygous EYS variant (c.9405T>A), previously identified in RP patients. Two siblings with macular dystrophy carried compound heterozygous EYS variants: c.1299+5_1299+8del and c.6050G>T. The former was novel and shown to result in skipping of exon 8, and the latter was a known RP variant. Conclusions: We report on EYS-associated macular dystrophy, extending the spectrum of EYS-associated IRDs. We observed heterogeneity between RP patients in age of onset and disease progression. Identical EYS variants were found in cases with RP, CRD, and macular dystrophy. Screening for EYS variants in CRD and macular dystrophy patients might increase the diagnostic yield in previously unsolved cases.


Assuntos
Proteínas do Olho/genética , Degeneração Macular/genética , Mutação , RNA Mensageiro/genética , Retina/patologia , Retinite Pigmentosa/genética , Acuidade Visual , Adulto , Idoso , Idoso de 80 Anos ou mais , Análise Mutacional de DNA , Eletrorretinografia , Proteínas do Olho/metabolismo , Feminino , Homozigoto , Humanos , Degeneração Macular/diagnóstico , Degeneração Macular/metabolismo , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Retina/metabolismo , Retina/fisiopatologia , Retinite Pigmentosa/diagnóstico , Retinite Pigmentosa/metabolismo , Tomografia de Coerência Óptica , Adulto Jovem
10.
Int J Mol Sci ; 20(8)2019 Apr 19.
Artigo em Inglês | MEDLINE | ID: mdl-31010136

RESUMO

Sjögren syndrome (SS) or dry eye disease (DED) is one of the most complicated ocular surface diseases. The goal of this study is to elucidate the relationship of the changes in clinical indices of tear film (TF) homeostasis with respect to tear components to allow for SS-DED monitoring and avoid stably controlled SS-DED patients from re-entering a vicious cycle. This prospective case-control study compared stable SS-DED patients with non-SS-DED control from several aspects, including clinical indices for TF homeostasis, 2 DED diagnostic biomarkers (MMP-9 and lactoferrin), and the proteome of flush tears. Compared with non-SS-DED controls, stably controlled SS-DED subjects had less tear secretion and higher ocular surface inflammation, a higher concentration ratio of tear MMP-9/lactoferrin, a more diverse tear proteome, and lower spectral intensities of lipocalin-1, lacritin, and prolactin-inducible protein among the abundant tear proteins. For stable SS-DED patients, the concentration ratio of tear MMP-9/lactoferrin and the corrected lipocalin-1 signal was positively correlated with ocular inflammation and TF stability, respectively. MMP-9 released from stressed ocular surface epithelium and lipocalin-1 secreted from the energetic lacrimal gland are two tear biomarkers responding well to TF homeostasis. The tear proteomics approach through flush tears is a promising method for monitoring SS-DED patients with a standardized sampling procedure and lactoferrin-corrected analysis.


Assuntos
Síndromes do Olho Seco/metabolismo , Proteômica/métodos , Síndrome de Sjogren/metabolismo , Lágrimas/metabolismo , Biomarcadores/metabolismo , Estudos de Casos e Controles , Proteínas do Olho/metabolismo , Feminino , Humanos , Lactoferrina/metabolismo , Masculino , Pessoa de Meia-Idade
11.
Prog Retin Eye Res ; 69: 137-158, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30982505

RESUMO

ELOngation of Very Long chain fatty acids-4 (ELOVL4) is an elongase responsible for the biosynthesis of very long chain (VLC, ≥C28) saturated (VLC-SFA) and polyunsaturated (VLC-PUFA) fatty acids in brain, retina, skin, Meibomian glands, and testes. Fascinatingly, different mutations in this gene have been reported to cause vastly different phenotypes in humans. Heterozygous inheritance of seven different mutations in the coding sequence and 5' untranslated region of ELOVL4 causes autosomal dominant Stargardt-like macular dystrophy (STGD3), while homozygous inheritance of three more mutant variants causes severe seizures with ichthyosis, hypertonia, and even death. Some recent studies have described heterozygous inheritance in yet another three mutant ELOVL4 variants, two that cause spinocerebellar ataxia-34 (SCA34) with erythrokeratodermia (EKV) and one that causes SCA34 without EKV. We identified the specific enzymatic reactions catalyzed by ELOVL4 and, using a variety of genetically engineered mouse models, have actively searched for the mechanisms by which ELOVL4 impacts neural function and health. In this review, we critically compare and contrast the various animal model and case studies involving ELOVL4 deficiency via either mutation or deletion, and the resulting consequences on neuronal health and function in both the retina and central nervous system.


Assuntos
Fenômenos Fisiológicos Celulares/fisiologia , Doenças do Sistema Nervoso Central/fisiopatologia , Proteínas do Olho/fisiologia , Mamíferos/fisiologia , Proteínas de Membrana/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Doenças Retinianas/fisiopatologia , Animais , Doenças do Sistema Nervoso Central/genética , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mutação , Doenças Retinianas/genética , Doenças Retinianas/metabolismo
12.
Invest Ophthalmol Vis Sci ; 60(5): 1461-1469, 2019 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-30951588

RESUMO

Purpose: To determine the effect of molecular weight (MW) on the concentration of plasma-derived proteins in aqueous humor and to estimate the plasma-derived and eye-derived fractions for each protein. Methods: Aqueous humor and plasma samples were obtained during cataract surgery on an institutional review board-approved protocol. Protein concentrations were determined by ELISA and quantitative antibody microarrays. A total of 93 proteins were studied, with most proteins analyzed using 27 to 116 aqueous and 6 to 30 plasma samples. Results: Plasma proteins without evidence of intraocular expression by sequence tags were used to fit a logarithmic model relating aqueous-plasma ratio (AH:PL) to MW. The log(AH:PL) appears to be well predicted by the log(MW) (P < 0.0001), with smaller proteins such as cystatin C (13 kDa) having a higher AH:PL (1:6) than larger proteins such as albumin (66 kDa, 1:300) and complement component 5 (188 kDa, 1:2500). The logarithmic model was used to calculate the eye-derived intraocular fraction (IOF) for each protein. Based on the IOF, 66 proteins could be categorized as plasma-derived (IOF<20), whereas 10 proteins were primarily derived from eye tissue (IOF >80), and 17 proteins had contribution from both plasma and eye tissue (IOF 20-80). Conclusions: Protein concentration of plasma-derived proteins in aqueous is nonlinearly dependent on MW in favor of smaller proteins. Our study demonstrates that for proper interpretation of results, proteomic studies evaluating changes in aqueous humor protein levels should take into account the plasma and eye-derived fractions.


Assuntos
Barreira Hematoaquosa/metabolismo , Catarata/metabolismo , Proteínas do Olho/metabolismo , Idoso , Idoso de 80 Anos ou mais , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Peso Molecular , Proteômica/métodos
13.
Nat Neurosci ; 22(5): 741-752, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30936556

RESUMO

Despite expanding knowledge regarding the role of astroglia in regulating neuronal function, little is known about regional or functional subgroups of brain astroglia and how they may interact with neurons. We use an astroglia-specific promoter fragment in transgenic mice to identify an anatomically defined subset of adult gray matter astroglia. Using transcriptomic and histological analyses, we generate a combinatorial profile for the in vivo identification and characterization of this astroglia subpopulation. These astroglia are enriched in mouse cortical layer V; express distinct molecular markers, including Norrin and leucine-rich repeat-containing G-protein-coupled receptor 6 (LGR6), with corresponding layer-specific neuronal ligands; are found in the human cortex; and modulate neuronal activity. Astrocytic Norrin appears to regulate dendrites and spines; its loss, as occurring in Norrie disease, contributes to cortical dendritic spine loss. These studies provide evidence that human and rodent astroglia subtypes are regionally and functionally distinct, can regulate local neuronal dendrite and synaptic spine development, and contribute to disease.


Assuntos
Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Proteínas do Olho/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Animais , Células Cultivadas , Espinhas Dendríticas/fisiologia , Substância Cinzenta/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Córtex Motor/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Transcriptoma
14.
eNeuro ; 6(1)2019 Jan-Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30815534

RESUMO

Increasing evidence suggests that necroptosis, a form of programmed cell death (PCD), contributes to neurodegeneration in several disorders, including ALS. Supporting this view, investigations in both in vitro and in vivo models of ALS have implicated key molecular determinants of necroptosis in the death of spinal motor neurons (MNs). Consistent with a pathogenic role of necroptosis in ALS, we showed increased mRNA levels for the three main necroptosis effectors Ripk1, Ripk3, and Mlkl in the spinal cord of mutant superoxide dismutase-1 (SOD1G93A) transgenic mice (Tg), an established model of ALS. In addition, protein levels of receptor-interacting protein kinase 1 (RIPK1; but not of RIPK3, MLKL or activated MLKL) were elevated in spinal cord extracts from these Tg SOD1G93A mice. In postmortem motor cortex samples from sporadic and familial ALS patients, no change in protein levels of RIPK1 were detected. Silencing of Ripk3 in cultured MNs protected them from toxicity associated with SOD1G93A astrocytes. However, constitutive deletion of Ripk3 in Tg SOD1G93A mice failed to provide behavioral or neuropathological improvement, demonstrating no similar benefit of Ripk3 silencing in vivo. Lastly, we detected no genotype-specific myelin decompaction, proposed to be a proxy of necroptosis in ALS, in either Tg SOD1G93A or Optineurin knock-out mice, another ALS mouse model. These findings argue against a role for RIPK3 in Tg SOD1G93A-induced neurodegeneration and call for further preclinical investigations to determine if necroptosis plays a critical role in the pathogenesis of ALS.


Assuntos
Morte Celular/fisiologia , Neurônios Motores/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/deficiência , Adulto , Idoso , Idoso de 80 Anos ou mais , Esclerose Amiotrófica Lateral/metabolismo , Animais , Astrócitos/metabolismo , Astrócitos/patologia , Linhagem Celular , Técnicas de Cocultura , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Feminino , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Córtex Motor/metabolismo , Córtex Motor/patologia , Neurônios Motores/patologia , Cultura Primária de Células , Proteínas Quinases/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/genética , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Medula Espinal/metabolismo , Medula Espinal/patologia , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo
15.
Mol Vis ; 25: 60-69, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820142

RESUMO

Purpose: To identify novel mutations in FZD4 and to investigate their pathogenicity in a cohort of Chinese patients with familial exudative vitreoretinopathy (FEVR). Methods: Next-generation sequencing was performed in patients with a clinical diagnosis of FEVR. Wide-field angiography was performed in probands and family members if available. Clinical data were collected from patient charts. The effect of the mutations in FZD4 on its biologic activity in the Norrin/ß-catenin signaling pathway was analyzed with the luciferase reporter assay. Results: Four novel mutations in FZD4 (c.1188_1192del/p.F396fs, c.1220delC/p.A407Vfs*24, c.905G>A/p.C302Y, c.1325T>A/p.V442E) were identified in four unrelated families. The mutations were not detected in 200 healthy individuals. The variability of the ocular phenotypes was not only observed in the probands and parents harboring the same mutation but also between two eyes in one individual. All four novel mutations introduced reduction in luciferase activity. Compared with the wild-type, the FZD4 level of the four mutants also decreased variably. Conclusions: Four novel mutations in FZD4 were identified in Chinese patients with FEVR. No correlation in the reduced luciferase activity and the ocular phenotype was observed in this study. This study further emphasized the complexity of the FEVR-causing machinery.


Assuntos
Oftalmopatias Hereditárias/genética , Proteínas do Olho/genética , Receptores Frizzled/genética , Mutação , Proteínas do Tecido Nervoso/genética , Doenças Retinianas/genética , beta Catenina/genética , Adulto , Grupo com Ancestrais do Continente Asiático , Sequência de Bases , Estudos de Casos e Controles , Pré-Escolar , Oftalmopatias Hereditárias/diagnóstico por imagem , Oftalmopatias Hereditárias/etnologia , Oftalmopatias Hereditárias/patologia , Proteínas do Olho/metabolismo , Feminino , Angiofluoresceinografia , Receptores Frizzled/metabolismo , Regulação da Expressão Gênica , Genes Reporter , Humanos , Luciferases/genética , Luciferases/metabolismo , Masculino , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Fenótipo , Doenças Retinianas/diagnóstico por imagem , Doenças Retinianas/etnologia , Doenças Retinianas/patologia , Transdução de Sinais , beta Catenina/metabolismo
16.
Mol Vis ; 25: 93-105, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820145

RESUMO

Purpose: To investigate the genetic basis of primary closed angle glaucoma (PCAG) in European Basset Hounds using genome-wide association and RNA sequencing strategies. Methods: DNA samples from 119 European Basset Hounds were genotyped on the 170 K SNP CanineHD BeadChip array (Illumina) comprising 37 with normal iridocorneal angles (controls), 57 with pectinate ligament abnormality (PLA cases), and 25 with PCAG (PCAG cases). Genome-wide association studies (GWASs) of the PLA and PCAG cases were conducted. Whole transcriptome sequences of iridocorneal angle tissues from five Basset Hounds with PCAG were compared with those from four dogs with normal eyes to investigate differences in gene expression between the affected and unaffected eyes in GWAS-associated loci. A variant in NEB, previously reported to be associated with PCAG in American Basset Hounds, was genotyped in cohorts of European Basset Hounds and non-Basset Hounds. Results: The GWASs revealed 1.4 and 0.2 Mb regions, on chromosomes 24 and 37, respectively, that are statistically associated with PCAG. The former locus has previously been associated with glaucoma in humans. Whole transcriptome analysis revealed differential gene expression of eight genes within these two loci. The NEB variant was not associated with PLA or PCAG in this set of European Basset Hounds. Conclusions: We identified two novel loci for canine PCAG. Further investigation is required to elucidate candidate variants that underlie canine PCAG.


Assuntos
Doenças do Cão/genética , Proteínas do Olho/genética , Predisposição Genética para Doença , Genoma , Glaucoma de Ângulo Fechado/veterinária , Transcriptoma , Animais , Estudos de Casos e Controles , Doenças do Cão/patologia , Cães , Europa (Continente) , Proteínas do Olho/metabolismo , Feminino , Ontologia Genética , Loci Gênicos , Estudo de Associação Genômica Ampla , Glaucoma de Ângulo Fechado/genética , Glaucoma de Ângulo Fechado/patologia , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Anotação de Sequência Molecular , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Polimorfismo de Nucleotídeo Único , Análise de Sequência de RNA , Estados Unidos
17.
Mol Vis ; 25: 106-117, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820146

RESUMO

Purpose: Inherited retinal diseases (IRDs) are clinically and genetically heterogeneous showing progressive retinal cell death which results in vision loss. IRDs include a wide spectrum of disorders, such as retinitis pigmentosa (RP), Leber congenital amaurosis (LCA), cone-rod dystrophy (CRD), and Stargardt disease (STGD1). Methods: In this study, we performed targeted next-generation sequencing based on molecular inversion probes (MIPs) that allowed the sequence analysis of 108 IRD-associated genes in 50 Iranian IRD probands. Results: The sequencing and variant filtering led to the identification of putative pathogenic variants in 36 out of 50 (72%) probands. Among 36 unique variants, we identified 20 novel variants in 15 genes. Four out of 36 probands carry compound heterozygous variants, and 32 probands carry homozygous variants. Conclusions: Employing a cost-effective targeted next-generation sequencing procedure, we identified the genetic causes of different retinal disorders in the majority of Iranian families in this study.


Assuntos
Distrofias de Cones e Bastonetes/genética , Proteínas do Olho/genética , Amaurose Congênita de Leber/genética , Degeneração Macular/congênito , Mutação , Retinite Pigmentosa/genética , Adolescente , Adulto , Criança , Distrofias de Cones e Bastonetes/metabolismo , Distrofias de Cones e Bastonetes/patologia , Proteínas do Olho/metabolismo , Feminino , Expressão Gênica , Estudos de Associação Genética , Genótipo , Heterozigoto , Sequenciamento de Nucleotídeos em Larga Escala/economia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Homozigoto , Humanos , Irã (Geográfico) , Amaurose Congênita de Leber/metabolismo , Amaurose Congênita de Leber/patologia , Degeneração Macular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Masculino , Linhagem , Fenótipo , Retina/metabolismo , Retina/patologia , Retinite Pigmentosa/congênito , Retinite Pigmentosa/metabolismo , Retinite Pigmentosa/patologia
18.
Mol Vis ; 25: 118-128, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820147

RESUMO

Purpose: As the aging population is increasing, the incidence of age-related cataract is expected to increase globally. The surgical intervention, a treatment for cataract, still has complications and is limited to developed countries. In this study, we investigated whether the polyphenol-enriched fraction of Vaccinium uliginosum L. (FH) prevents cataract formation in Sprague-Dawley (SD) rat pups. Methods: Sixty rat pups were randomly divided into six groups: CTL, Se, FH40, FH80, FH120, and Cur80. The cataract was induced with subcutaneous injection of sodium selenite (18 µmol/kg bodyweight) on postnatal (P) day 10. All groups, except CTL, were injected with sodium selenite, and the FH40, FH80, and FH120 groups were given gastric intubation with FH40 mg/kg, 80 mg/kg, and 120 mg/kg on P9, P10, and P11. The Cur80 group was also given gastric intubation with curcumin 80 mg/kg on P9, P10, and P11. All rat pups were euthanized on P30. Results: Lens morphological analysis showed that FH dose-dependently inhibited cataract formation. In the Se group, soluble proteins were insolubilized, and the gene expression of the α-, ß-, and γ-crystallins was downregulated. However, FH treatment statistically significantly inhibited insolubilization of soluble proteins and downregulation of the gene expression of the α-, ß-, and γ-crystallins. In the Se group, the gene and protein levels of m-calpain were downregulated, which were attenuated with FH treatment. In addition, sodium selenite injection caused reduced antioxidant enzymes (superoxide dismutase (SOD) and glutathione peroxidase (GPx)), glutathione (GSH) depletion, and malondialdehyde (MDA) production in the lens. The administration of FH inhibited sodium selenite-induced oxidative stress in a dose-dependent manner. The mechanism of protection against oxidative stress by FH involves NF-E2-related factor (Nrf-2) and hemoxygenase-1 (HO-1). FH treatment inhibited decrease of Nrf-2 in the nucleus fraction and HO-1 in the cytosol fraction. Finally, the FH treatment protected poly (ADP)-ribose polymerase (PARP) from cleavage, determined with western blotting. Conclusions: FH showed a preventive effect against cataract formation by inhibiting m-calpain-mediated proteolysis and oxidative stress in the lens. These results suggest that FH could be a potential anticataract agent in age-related cataract.


Assuntos
Antioxidantes/farmacologia , Mirtilos Azuis (Planta)/química , Catarata/prevenção & controle , Proteínas do Olho/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Polifenóis/farmacologia , Animais , Animais Recém-Nascidos , Antioxidantes/isolamento & purificação , Calpaína/genética , Calpaína/metabolismo , Catarata/induzido quimicamente , Catarata/genética , Catarata/patologia , Proteínas do Olho/metabolismo , Glutationa/metabolismo , Glutationa Peroxidase/genética , Glutationa Peroxidase/metabolismo , Heme Oxigenase-1/genética , Heme Oxigenase-1/metabolismo , Cristalino/efeitos dos fármacos , Cristalino/metabolismo , Cristalino/patologia , Malondialdeído/metabolismo , Fator 2 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Polifenóis/isolamento & purificação , Ratos , Ratos Sprague-Dawley , Selenito de Sódio/administração & dosagem , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo , alfa-Cristalinas/genética , alfa-Cristalinas/metabolismo , beta-Cristalinas/genética , beta-Cristalinas/metabolismo , gama-Cristalinas/genética , gama-Cristalinas/metabolismo
19.
Mol Vis ; 25: 165-173, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30820152

RESUMO

Purpose: The evolutionarily conserved retinal homeobox (Rax) transcription factor is essential for normal eye development in all vertebrates. Despite Rax's biologic significance, the molecular mechanisms underlying Rax molecular function as a transcriptional regulator are poorly defined. The rax gene encodes a conserved octapeptide motif (OP) near the N-terminus and several conserved regions in the C-terminus of unknown function, including the orthopedia, aristaless, rax (OAR) domain and the RX domain. The purpose of this study is to investigate the contribution of these conserved domains in Rax function. Methods: N-and C-terminal deletion and point mutations were generated in Xenopus laevis rax.L (previously known as Rx1A) using PCR-based methods. We examined the ability of mutated Rax to transactivate a reporter gene consisting of a portion of a rax target gene promoter (from the Xenopus rhodopsin gene) fused to a firefly luciferase coding region and transfected into human embryonic kidney 293T (HEK293T) cells. Portions of the Rax C-terminal region were also assayed for transactivation activity in the context of a heterologous DNA binding domain with an appropriate reporter gene. Results: Full-length Rax weakly activated the reporter. Deletion of the Rax C-terminus increased Rax activity, suggesting that the C-terminus functions to repress Rax activity. Further deletion eventually resulted in a decrease in activity, suggesting that the C-terminal region also can function to enhance Rax activity. Deletion or mutation of the OP motif resulted in a slight decrease in Rax activity. Mutation or deletion of the N-terminal OP motif resulted in a mild decrease in activity and dampened the activity levels of the C-terminal deletions. Further, fusion of the C-terminus of Rax to a heterologous DNA binding domain enhanced transactivation. Conclusions: The present data indicate that the C-terminus of Rax can function to repress or activate transcription in a context-dependent manner. These data support our hypothesis that the highly conserved OAR domain, in combination with other regulatory elements in the Rax C-terminus, coordinates Rax activity, perhaps through functional interaction with the N-terminal OP motif. Taken together, these data provide insight into the structural features that regulate Rax activity.


Assuntos
Sequência de Bases , Proteínas do Olho/genética , Proteínas Recombinantes de Fusão/genética , Retina/metabolismo , Deleção de Sequência , Ativação Transcricional , Proteínas de Xenopus/genética , Motivos de Aminoácidos , Animais , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Expressão Gênica , Genes Reporter , Células HEK293 , Humanos , Luciferases/genética , Luciferases/metabolismo , Mutação Puntual , Regiões Promotoras Genéticas , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Rodopsina/genética , Rodopsina/metabolismo , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Xenopus laevis
20.
Invest Ophthalmol Vis Sci ; 60(4): 868-876, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30821812

RESUMO

Purpose: To identify protein mediators of corneal haze following presbyopic corneal inlay surgery. Methods: Tears were collected from eyes with corneal haze following surgery with a shape-changing corneal inlay. Samples were subjected to quantitative proteomic analysis using iTRAQ and proteins significantly increased or decreased (1.3-fold or more) in haze eyes relative to fellow eyes were identified. Expression ratios were compared to postoperative eyes without corneal haze to identify proteins selectively increased or decreased in corneal haze eyes. Results: Inlay-associated haze occurred in 35% of eyes (6 of 17). Of 1443 unique tear proteins identified, eight proteins were selectively reduced in tears from postoperative haze eyes and one protein selectively increased. Proteins reduced in haze eyes included complement 4a (level relative to nonhaze eyes 0.464, P = 0.037), complement factor H (0.589, P = 0.048), immunoglobulin kappa variable 2-29 (0.128, P = 0.006), immunoglobulin kappa variable 2D-28 (0.612, P = 0.025), immunoglobulin lambda variable 7-46 (0.482, P = 0.007), S100 calcium binding protein A4 (0.614, P = 0.048), Shootin-1 (0.614, P = 0.048), and tissue inhibitor of metalloproteinase-1 (0.736, P = 0.023). The Xaa-Pro aminopeptidase 1 was increased in haze eyes relative to nonhaze eyes (1.517, P = 0.023). Conclusions: Corneal haze following corneal inlay surgery is associated with reduction in levels of known inflammatory and immune mediators. These findings represent a starting point for elucidation of pathways involved in corneal haze following synthetic inlay implantation and may enable development of targeted therapies that modulate the haze response.


Assuntos
Opacidade da Córnea/etiologia , Substância Própria/cirurgia , Proteínas do Olho/metabolismo , Presbiopia/cirurgia , Implantação de Prótese/efeitos adversos , Lágrimas/metabolismo , Adulto , Idoso , Cromatografia Líquida , Opacidade da Córnea/metabolismo , Topografia da Córnea , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Próteses e Implantes , Proteômica , Espectrometria de Massas em Tandem
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