RESUMO
BALB/cJ mice housed under normal vivarium lighting conditions can exhibit profound retinal abnormalities, including retinal infoldings, autofluorescent inflammatory cells, and photoreceptor degeneration. To explore the sensitivity of the outer retina to cyclic lighting during aging, a cohort of BALB/cJ mice was evaluated with Scanning Laser Ophthalmoscopy (SLO), Spectral-Domain Optical Coherence Tomography (OCT) and conventional histopathology. Mice were bred and reared in a low-illuminance (extracage/intracage: 13 lx/1 lx) vivarium under cyclic light (14 h light: 10 h dark). Retinal imaging (around postnatal day 70) was performed to screen for any pre-existing abnormalities and to establish a baseline. Mice with normal retinas were separated into groups (A, B, C) and placed on bottom (Groups A & B) or top (Group C) of the cage racks where cage illumination was <10 & 150 lx respectively. Experimental groups B & C were imaged multiple times over a 17 month period. Mice from group A (controls) were imaged only once post-baseline at various times for comparison to groups B & C. Mice were assessed by histology at 8, 15, 20, 36, and 56 weeks and immunohistochemistry at 15 weeks post-baseline. SLO and OCT retinal images were measured and the resulting trends displayed as a function of age and light exposure. Retinal lesions (RL) and autofluorescent foci (AFF) were identified with histology as photoreceptor layer infoldings (IF) and localized microglia/macrophages (MM), respectively. Few RL and AFF were evident at baseline. Retinal infoldings were the earliest changes followed by subjacent punctate autofluorescent MM. The colocalization of IF and MM suggests a causal relationship. The incidence of these pathological features increased in all groups relative to baseline. OCT imaging revealed thinning of the outer nuclear layer (ONL) in all groups at 1 year relative to baseline. ONL thinning followed an exponential rate of change but the decay constant varied depending on intensity of illumination of the groups. Advanced age and top row illuminance conditions resulted in significant photoreceptor cell loss as judged by decreased thickness of the ONL. Photoreceptor loss was preceded by both retinal infoldings and the presence of autofluorescent inflammatory cells in the outer retina, suggesting that these changes are early indicators of light toxicity in the BALB/cJ mouse.
Assuntos
Envelhecimento/efeitos da radiação , Luz/efeitos adversos , Camundongos Endogâmicos BALB C/fisiologia , Lesões Experimentais por Radiação/patologia , Retina/efeitos da radiação , Degeneração Retiniana/etiologia , Envelhecimento/fisiologia , Animais , Camundongos , Microscopia de Fluorescência , Retina/patologia , Degeneração Retiniana/patologia , Tomografia de Coerência ÓpticaRESUMO
DJ-1/PARK7 mutations or deletions cause autosomal recessive early onset Parkinson's disease (PD). Thus, DJ-1 protein has been extensively studied in brain and neurons. PD patients display visual symptoms; however, the visual symptoms specifically attributed to PD patients carrying DJ-1/PARK7 mutations are not known. In this study, we analyzed the structure and physiology of retinas of 3- and 6-month-old DJ-1 knockout (KO) mice to determine how loss of function of DJ-1 specifically contributes to the phenotypes observed in PD patients. As compared to controls, the DJ-1 KO mice displayed an increase in the amplitude of the scotopic ERG b-wave and cone ERG, while the amplitude of a subset of the dc-ERG components was decreased. The main structural changes in the DJ-1 KO retinas were found in the outer plexiform layer (OPL), photoreceptors and retinal pigment epithelium (RPE), which were observed at 3 months and progressively increased at 6 months. RPE thinning and structural changes within the OPL were observed in the retinas in DJ-1 KO mice. DJ-1 KO retinas also exhibited disorganized outer segments, central decrease in red/green cone opsin staining, decreased labeling of ezrin, broader distribution of ribeye labeling, decreased tyrosine hydroxylase in dopaminergic neurons, and increased 7,8-dihydro-8-oxoguanine-labeled DNA oxidation. Accelerated outer retinal atrophy was observed in DJ-1 KO mice after selective oxidative damage induced by a single tail vein injection of NaIO3, exposing increased susceptibility to oxidative stress. Our data indicate that DJ-1-deficient retinas exhibit signs of morphological abnormalities and physiological dysfunction in association with increased oxidative stress. Degeneration of RPE cells in association with oxidative stress is a key hallmark of age-related macular degeneration (AMD). Therefore, in addition to detailing the visual defects that occur as a result of the absence of DJ-1, our data is also relevant to AMD pathogenesis.
Assuntos
DNA/genética , Mutação , Proteínas Oncogênicas/genética , Peroxirredoxinas/genética , Degeneração Retiniana/genética , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Análise Mutacional de DNA , Modelos Animais de Doenças , Eletrorretinografia , Feminino , Genótipo , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Oncogênicas/biossíntese , Estresse Oxidativo , Peroxirredoxinas/biossíntese , Reação em Cadeia da Polimerase , Proteína Desglicase DJ-1 , Degeneração Retiniana/patologia , Degeneração Retiniana/fisiopatologia , Epitélio Pigmentado da Retina/fisiopatologia , Epitélio Pigmentado da Retina/ultraestrutura , Transdução de SinaisRESUMO
Deimination is a form of protein posttranslational modification carried out by the peptidyl arginine deiminases (PADs) enzymes. PAD2 is the principal deiminase expressed in the retina. Elevated levels of PAD2 and protein deimination are present in a number of human neurological diseases, with or without ocular manifestation. To define the association of deimination with the pathogenesis of age-related macular degeneration (AMD), we studied protein deimination and PAD2 levels in retinas of AMD donor eyes compared to age-matched non-AMD retinas. Eyes from non-AMD and AMD donors were fixed in 4% paraformaldehyde and 0.5% glutaraldehyde in phosphate buffer. Retina and retinal pigment epithelium (RPE) from donor eyes were processed for immunohistochemical detection and western blotting using antibodies to PAD2 and citrulline residues. The ganglion cell, inner plexiform, inner nuclear and outer nuclear layers were labeled by both PAD2 and citrulline antibodies. Changes in the localization of deiminated residues and PAD2 were evident as the retinal layers were remodeled coincident with photoreceptor degeneration in AMD retinas. Immunodetection of either PAD2 or citrulline residues could not be evaluated in the RPE layer due to the high autofluorescence levels in this layer. Interestingly, higher deimination immunoreactivity was detected in AMD retinal lysates. However, no significant changes in PAD2 were detected in the AMD and non-AMD retinas and RPE lysates. Our observations show increased levels of protein deimination but not PAD2 in AMD retinas and RPE, suggesting a reduced rate of turnover of deiminated proteins in these AMD retinas.
Assuntos
Hidrolases/metabolismo , Degeneração Macular/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Retina/enzimologia , Epitélio Pigmentado da Retina/enzimologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Citrulina/metabolismo , Bancos de Olhos , Feminino , Humanos , Imuno-Histoquímica , Degeneração Macular/patologia , Masculino , Pessoa de Meia-Idade , Proteína-Arginina Desiminase do Tipo 2 , Desiminases de Arginina em Proteínas , Retina/patologia , Epitélio Pigmentado da Retina/patologiaRESUMO
A quantitative proteomics analysis of the macular Bruch membrane/choroid complex was pursued for insights into the molecular mechanisms of age-related macular degeneration (AMD). Protein in trephine samples from the macular region of 10 early/mid-stage dry AMD, six advanced dry AMD, eight wet AMD, and 25 normal control post-mortem eyes was analyzed by LC MS/MS iTRAQ (isobaric tags for relative and absolute quantitation) technology. A total of 901 proteins was quantified, including 556 proteins from > or =3 AMD samples. Most proteins differed little in amount between AMD and control samples and therefore reflect the proteome of normal macular tissues of average age 81. A total of 56 proteins were found to be elevated and 43 were found to be reduced in AMD tissues relative to controls. Analysis by category of disease progression revealed up to 16 proteins elevated or decreased in each category. About 60% of the elevated proteins are involved in immune response and host defense, including many complement proteins and damage-associated molecular pattern proteins such as alpha-defensins 1-3, protein S100s, crystallins, histones, and galectin-3. Four retinoid processing proteins were elevated only in early/mid-stage AMD, supporting a role for retinoids in AMD initiation. Proteins uniquely decreased in early/mid-stage AMD implicate hematologic malfunctions and weakened extracellular matrix integrity and cellular interactions. Galectin-3, a receptor for advanced glycation end products, was the most significantly elevated protein in advanced dry AMD, supporting a role for advanced glycation end products in dry AMD progression. The results endorse inflammatory processes in both early and advanced AMD pathology, implicate different pathways of progression to advanced dry and wet AMD, and provide a new database for hypothesis-driven and discovery-based studies of AMD.
Assuntos
Lâmina Basilar da Corioide/metabolismo , Lâmina Basilar da Corioide/patologia , Proteínas do Olho/metabolismo , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Proteômica/métodos , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Lâmina Basilar da Corioide/citologia , Progressão da Doença , Feminino , Humanos , Degeneração Macular/classificação , Masculino , Proteoma/metabolismoRESUMO
PURPOSE: To quantify and evaluate the distribution of angiotensin II (Ang II) and its receptors in the human retina. METHODS: Donor eyes were obtained within 12 hours postmortem and classified as hypertensive or normotensive and diabetic or nondiabetic, based on the donors' medical histories. Ang II in retina and vitreous was quantified by RIA. Ang II receptors were characterized and quantified by competitive membrane-binding assays. Ang II, its heptapeptide metabolite Ang-(1-7), and AT1 and AT2 receptors were localized by immunohistochemistry and confocal imaging. RESULTS: Levels of Ang II in the retina were significantly higher than in vitreous (P < 0.05). Ang II in the diabetic retina had a higher median compared with that in the nondiabetic retina. Ang II and Ang-(1-7) colocalized in retinal Müller cells. The retina had the highest levels of Ang II receptors that were significantly higher than the optic nerve, retinal pigment epithelium-choroid complex, and ciliary body-iris complex (P < 0.05). AT1 receptors were more abundant than AT2 receptors in the retina. Immunoreactivity for AT1 was detected in Müller cells and on blood vessels. AT2 receptors were localized throughout the Müller cells and nuclei of ganglion cells and neurons in the inner nuclear layer. CONCLUSIONS: In the human retina, identification of Ang II and its bioactive metabolite Ang-(1-7) in Müller cells suggests that these glial cells are able to produce and process Ang II. Ang receptors were localized in the blood vessels and neural cells. Local Ang II signaling may thus allow for autoregulation of neurovascular activity. Such an autonomous system could modulate the onset and severity of retinovascular disease.
Assuntos
Angiotensina II/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Receptor Tipo 2 de Angiotensina/metabolismo , Retina/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Angiotensina I/metabolismo , Enzima de Conversão de Angiotensina 2 , Ligação Competitiva , Western Blotting , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Masculino , Microscopia Confocal , Pessoa de Meia-Idade , Fragmentos de Peptídeos/metabolismo , Peptidil Dipeptidase A/metabolismo , Radioimunoensaio , Doadores de TecidosRESUMO
[This corrects the article DOI: 10.1371/journal.pone.0067983.].
RESUMO
BACKGROUND: DJ-1 is found in many tissues, including the brain, where it has been extensively studied due to its association with Parkinson's disease. DJ-1 functions as a redox-sensitive molecular chaperone and transcription regulator that robustly protects cells from oxidative stress. METHODOLOGY: Retinal pigment epithelial (RPE) cultures were treated with H2O2 for various times followed by biochemical and immunohistological analysis. Cells were transfected with adenoviruses carrying the full-length human DJ-1 cDNA and a mutant construct, which has the cysteine residues at amino acid 46, 53 and 106 mutated to serine (C to S) prior to stress experiments. DJ-1 localization, levels of expression and reactive oxygen species (ROS) generation were also analyzed in cells expressing exogenous DJ-1 under baseline and oxidative stress conditions. The presence of DJ-1 and oxidized DJ-1 was evaluated in human RPE total lysates. The distribution of DJ-1 was assessed in AMD and non-AMD cryosectionss and in isolated human Bruch's membrane (BM)/choroid from AMD eyes. PRINCIPAL FINDINGS: DJ-1 in RPE cells under baseline conditions, displays a diffuse cytoplasmic and nuclear staining. After oxidative challenge, more DJ-1 was associated with mitochondria. Increasing concentrations of H2O2 resulted in a dose-dependent increase in DJ-1. Overexpression of DJ-1 but not the C to S mutant prior to exposure to oxidative stress led to significant decrease in the generation of ROS. DJ-1 and oxDJ-1 intensity of immunoreactivity was significantly higher in the RPE lysates from AMD eyes. More DJ-1 was localized to RPE cells from AMD donors with geographic atrophy and DJ-1 was also present in isolated human BM/choroid from AMD eyes. CONCLUSIONS/SIGNIFICANCE: DJ-1 regulates RPE responses to oxidative stress. Most importantly, increased DJ-1 expression prior to oxidative stress leads to decreased generation of ROS, which will be relevant for future studies of AMD since oxidative stress is a known factor affecting this disease.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Oncogênicas/metabolismo , Estresse Oxidativo , Epitélio Pigmentado da Retina/metabolismo , Animais , Western Blotting , Linhagem Celular , Núcleo Celular/metabolismo , Células Cultivadas , Citoplasma/metabolismo , Células HEK293 , Humanos , Peróxido de Hidrogênio/farmacologia , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular/genética , Degeneração Macular/metabolismo , Degeneração Macular/patologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Confocal , Mitocôndrias/metabolismo , Mutação , Proteínas Oncogênicas/genética , Oxidantes/farmacologia , Oxirredução , Proteína Desglicase DJ-1 , Transporte Proteico/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/efeitos dos fármacosAssuntos
Anexinas/biossíntese , Anexinas/química , Lâmina Basilar da Corioide/metabolismo , Regulação da Expressão Gênica , Drusas Retinianas/metabolismo , Idoso , Idoso de 80 Anos ou mais , Olho/metabolismo , Humanos , Imuno-Histoquímica/métodos , Peptídeos/química , Ligação Proteica , Proteômica/métodos , Tripsina/farmacologiaRESUMO
The two cellular targets of interest in age-related macular degeneration (AMD) are the photoreceptors and the RPE. However, the mechanisms involved in AMD pathology are not yet fully understood. In the present report, we extend our previous studies on semenogelin proteins (Sgs) in normal human retina and compare these with the distribution in retinas from AMD donor eyes. Semenogelins I (SgI) and II (SgII) are the major structural protein components of semen coagulum, but have been recently found in non-genital tissues as well. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with a specific antibody. The presence of SgI was analyzed in retina and RPE total lysates and SgI was detected by western blot in human retina and RPE. The intensity of immunoreactivity was significantly reduced in the AMD eyes. SgI is expressed in the normal human retina and in the retina of AMD donor eyes, where localization was detected in the photoreceptors and in a few ganglion cells. We find the distribution of SgI in the AMD retinas substantially lower than observed in normal retina. SgI localization to photoreceptors and the RPE suggests a possible function related to the ability of these cells to sequester zinc.
Assuntos
Proteínas do Olho/análise , Degeneração Macular/metabolismo , Retina/química , Proteínas Secretadas pela Vesícula Seminal/análise , Western Blotting/métodos , Humanos , Células Fotorreceptoras de Vertebrados/química , Epitélio Pigmentado Ocular/química , Drusas Retinianas/metabolismoRESUMO
Oxidative damage and inflammation are postulated to be involved in age-related macular degeneration (AMD). However, the molecular signal(s) linking oxidation to inflammation in this late-onset disease is unknown. Here we describe AMD-like lesions in mice after immunization with mouse serum albumin adducted with carboxyethylpyrrole, a unique oxidation fragment of docosahexaenoic acid that has previously been found adducting proteins in drusen from AMD donor eye tissues and in plasma samples from individuals with AMD. Immunized mice develop antibodies to this hapten, fix complement component-3 in Bruch's membrane, accumulate drusen below the retinal pigment epithelium during aging, and develop lesions in the retinal pigment epithelium mimicking geographic atrophy, the blinding end-stage condition characteristic of the dry form of AMD. We hypothesize that these mice are sensitized to the generation of carboxyethylpyrrole adducts in the outer retina, where docosahexaenoic acid is abundant and conditions for oxidative damage are permissive. This new model provides a platform for dissecting the molecular pathology of oxidative damage in the outer retina and the immune response contributing to AMD.
Assuntos
Inflamação/metabolismo , Inflamação/patologia , Degeneração Macular/patologia , Estresse Oxidativo , Envelhecimento , Animais , Anticorpos/imunologia , Antígenos/imunologia , Lâmina Basilar da Corioide/patologia , Lâmina Basilar da Corioide/ultraestrutura , Complemento C3d/metabolismo , Imunização , Degeneração Macular/imunologia , Degeneração Macular/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Epitélio Pigmentado Ocular/patologia , Epitélio Pigmentado Ocular/ultraestrutura , Transporte Proteico , Pirróis/metabolismo , Fatores de Tempo , TitulometriaRESUMO
Clathrin was identified in a recent proteomic analysis of Bruch's membrane from age-related macular degeneration (AMD) donor eyes. The present study was conducted to determine the localization of clathrin in AMD tissues and to compare this distribution and relative content with that in non-AMD control tissues. The distribution of adaptin, which is functionally linked to clathrin, was also evaluated. Human eyes were from donors between 66 and 94 years of age; 13 eyes were from donors with AMD and 13 from non-AMD donors. Bruch's membrane and choroid from the macula of each donor eye were prepared for immunohistochemistry and Western blotting. Differences in immunoreactivity were quantitated. Drusen, Bruch's membrane and choroid from AMD tissues showed greater immunoreactivity for clathrin and adaptin than did non-AMD tissues. Western blots also showed more intense clathrin and adaptin immunoreactivity in AMD tissues than were present in non-AMD samples. This study suggests that accumulation of clathrin and adaptin in drusen, Bruch's membrane and choroid may reflect a higher rate of clathrin mediated endocytosis in AMD tissues. Alternatively, the accumulation of these proteins in these extracellular compartments may reflect a higher susceptibility to oxidative damage.
Assuntos
Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Corioide/metabolismo , Clatrina/metabolismo , Degeneração Macular/metabolismo , Drusas do Disco Óptico/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting/métodos , Lâmina Basilar da Corioide/metabolismo , Feminino , Humanos , Técnicas Imunoenzimáticas/métodos , Masculino , Epitélio Pigmentado Ocular/metabolismoRESUMO
Semenogelin I and II are the major proteins present in semen coagulum. In the present study, semenogelin I and II were detected in human RPE lysates by proteomic analysis. We further analyzed the expression of these proteins in the retinal cells in vivo and in vitro. Western blots detected semenogelin I and II in both RPE and neural retina while the vitreous contained only SgII. Cryo and paraffin sections of human retina were processed for both immunofluorescence and DAB reaction with an antibody that recognizes both forms of semenogelin proteins. Retina and RPE total lysates were evaluated for the presence of these proteins and in a human RPE cell line (D407). Both proteins were detected by western blot in human RPE and in D407 cell lysates. Immunoreactivity was detected in the ganglion cell and photoreceptor layer of the retina. Our data support the expression of semenogelin I and II in the human retina in several different compartments. Further studies towards addressing the function of these proteins in the retina are in progress.
Assuntos
Proteínas do Olho/análise , Retina/química , Proteínas Secretadas pela Vesícula Seminal/análise , Idoso , Idoso de 80 Anos ou mais , Corioide/química , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Células Fotorreceptoras de Vertebrados/química , Epitélio Pigmentado Ocular/química , Proteômica/métodos , Retina/citologia , Células Ganglionares da Retina/química , Corpo Vítreo/químicaRESUMO
While the production of nitric oxide by human corneas in storage has recently been demonstrated, protein nitration as a result of this production has not been demonstrated. In this study, nitrated protein accumulation in the epithelium of stored human corneas was assessed. One half of five donor corneas maintained in storage media for 3 days were prepared for immunohistochemical studies. The other halves remained in storage media for 7 additional days and were also processed for immunohistochemistry. Mouse monoclonal antibody to nitrotyrosine adducts was used to define the localisation of these epitopes. The density of antibody staining was observed and quantified on a digital camera system and statistically analysed. Immunostaining in the epithelium was greater in tissues recovered after 10 days in storage compared to the intensity of staining after 3 days of storage (p<0.0001). No staining was evident in the epithelium in sections exposed to non-immune mouse IgG. Western blot analysis was performed on epithelial cells scraped from corneal surfaces of one-half of four donor corneas in storage for 3 days and from the other half at 10 days of storage. Nitrated BSA was used as a positive control. After extraction and homogenisation, identical protein concentrations of each sample were loaded per lane on 10% gels and subjected to SDS-PAGE. Proteins were blotted and probed with the anti-nitrotyrosine antibody. Western blot immunoreactivity was detected in epithelial samples at the 3 and 10 day recovery times with the latter samples showing greater staining intensity. Nitrated protein, thought to indicate toxic peroxynitrite formation, accumulates in the human corneal epithelium with time of storage. Our study shows that there is an association between increased nitrated protein and storage time.
Assuntos
Epitélio Corneano/química , Proteínas do Olho/metabolismo , Ácido Peroxinitroso/metabolismo , Tirosina/análogos & derivados , Adulto , Western Blotting/métodos , Células Epiteliais/química , Proteínas do Olho/análise , Humanos , Imuno-Histoquímica/métodos , Pessoa de Meia-Idade , Óxido Nítrico/metabolismo , Oxidantes/análise , Oxidantes/metabolismo , Ácido Peroxinitroso/análise , Técnicas de Cultura de Tecidos/métodos , Tirosina/imunologiaRESUMO
Photoreceptors project from the outer retinal surface into a specialized glycocalyx, the interphotoreceptor matrix (IPM), which contains hyaluronan (HA) and two novel proteoglycans, Spacr and Spacrcan. This matrix must be stable enough to function in the attachment of the retina to the outer eye wall yet porous enough to allow movement of metabolites between these tissues. How this matrix is organized is not known. HA is a potential candidate in IPM organization since biochemical studies show that these proteoglycans bind HA. RHAMM (receptor for HA-mediated motility)-type HA binding motifs (HABMs) are present in their deduced amino acid sequence and may be the sites of this HA interaction. To test this hypothesis, we subcloned three fragments of mouse Spacrcan that contain the putative HABMs. We found that each recombinant fragment binds HA. Binding decreased when residues in the HABMs were mutated. This provides direct evidence that the RHAMM-type HABMs in Spacrcan are involved in hyaluronan binding. Since chondroitin sulfate and heparan sulfate proteoglycans are important for retinal development and function, we also evaluated the binding of these recombinant proteins to heparin and chondroitin sulfates, the glycosaminoglycan side chain of these proteoglycans. We found that each recombinant protein bound to both heparin and chondroitin sulfates. Binding to chondroitin sulfates involved these HABMs, because mutagenesis reduced binding. Binding to heparin was probably not mediated through these HABMs since heparin binding persisted following their mutagenesis. These studies provide the first evidence defining the sites of protein-carbohydrate interaction of molecules present in the IPM.
Assuntos
Ácido Hialurônico/metabolismo , Proteoglicanas/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação/genética , Proteínas da Matriz Extracelular/química , Proteínas da Matriz Extracelular/metabolismo , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Receptores de Hialuronatos/química , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/química , Camundongos , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Proteoglicanas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
SPACRCAN is a novel proteoglycan present in the interphotoreceptor matrix (IPM) of the rat and human retina that resists aqueous extraction through its binding to hyaluronan. The purpose of this study was: to clone mouse Spacrcan; to characterize the promoter elements; to define the deduced amino acid sequence; to establish the time of Spacrcan expression during retinal development; and to determine the time of appearance and distribution of SPACRCAN protein. Spacrcan cDNA clone was obtained through PCR amplification of a mouse retina cDNA library, and RT-PCR amplification and 5'RACE of mouse retina RNA. The deduced polypeptide sequence of mouse SPACRCAN contains a signal peptide at the N-terminal, seven N-link glycosylation sites, numerous potential O-linked glycosylation sites in a central mucin-like domain, two glycosaminoglycan attachment sites, five potential hyaluronan-binding motifs, two epidermal growth factor-like domains, and a hydrophobic stretch of 23 amino acids near the C-terminal. Comparison of the genomic structure of mouse and human SPACRCAN showed significant structure conservation. Analysis of the promoter region revealed several important putative regulatory elements including a Ret-1/PCE-1 element, an 11 base motif for Crx binding, six copies of PIRE, a Ret-4 element, three copies of AP-1, a CRE element, and five copies of GATA3. Northern blot analysis and immunohistochemistry were used to determine the tissue specificity of Spacrcan mRNA and to localize SPACRCAN in developing retina. Spacrcan mRNA is expressed in both retina and pineal gland and was detectable as early as embryonic day 15. The protein is first detectable in the IPM at postnatal day 8 where it increases in concert with the extension of photoreceptor inner and outer segments from the outer retinal surface. The presence of several unique regulatory elements in the promoter region and characteristic molecular features shared with the orthologue in human and rat suggest an important functional role of SPACRCAN in the IPM. The time of appearance of the SPACRCAN protein during retinal development suggests that this matrix protein may establish the extracellular microenvironment into which photoreceptor outer segments are elaborated.
Assuntos
Camundongos/genética , Células Fotorreceptoras de Vertebrados/metabolismo , Regiões Promotoras Genéticas , Proteoglicanas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Regulação da Expressão Gênica no Desenvolvimento , Genes Reguladores , Genoma , Humanos , Camundongos/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos CBA , Dados de Sequência Molecular , Proteoglicanas/biossíntese , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Especificidade da EspécieRESUMO
The nitrate/nitrite content in storage media was determined after nitric oxide synthase inhibition by adding 400 microl of 100 mm N(G)-monomethyl-l-arginine (LMMA) to four chambers of Optisol GS corneal storage media, each containing one viable human cornea. The companion corneas in storage media without LMMA served as controls. Four hundred microlitre aliquots obtained at baseline (day 0) and at one-day intervals for 20 more days for both groups were analyzed for nitrate and nitrite (breakdown products of nitric oxide) concentration levels using a spectrophotometric method based on the Greiss reaction. Average nitrate/nitrite concentrations, statistically analyzed using a polynomial random coefficients model, showed a statistically significant marked reduction in the levels of nitrate and nitrite accumulation in the study chambers as compared to control chambers for days 1-20(P < 0.001) There was also a reduction in the accumulation rate of nitrate and nitrite concentrations, as compared to controls (P < 0.05) until around day 8 when the differences in rates were no longer statistically significant. The progressive increase in nitrate and nitrite accumulation in corneal storage media can be blunted by the addition of a nitric oxide synthase inhibitor. Given the toxic free radical properties of nitric oxide, corneas in storage awaiting transplantation may benefit from having a nitric oxide synthase inhibitor added to storage media.
Assuntos
Córnea/enzimologia , Óxido Nítrico Sintase/antagonistas & inibidores , Preservação de Tecido , ômega-N-Metilarginina/farmacologia , Meios de Cultura/química , Depressão Química , Humanos , Nitratos/análise , Nitritos/análiseRESUMO
The retinal pigment epithelium (RPE) is a single cell layer adjacent to the rod and cone photoreceptors that plays key roles in retinal physiology and the biochemistry of vision. RPE cells were isolated from normal adult human donor eyes, subcellular fractions were prepared, and proteins were fractionated by electrophoresis. Following in-gel proteolysis, proteins were identified by peptide sequencing using liquid chromatography tandem electrospray mass spectrometry and/or by peptide mass mapping using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Preliminary analyses have identified 278 proteins and provide a starting point for building a database of the human RPE proteome.
Assuntos
Epitélio Pigmentado Ocular/química , Cromatografia Líquida , Citosol/metabolismo , Bases de Dados como Assunto , Humanos , Microssomos/metabolismo , Peptídeos/química , Proteoma , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Frações SubcelularesRESUMO
Drusen are extracellular deposits that accumulate below the retinal pigment epithelium on Bruch's membrane and are risk factors for developing age-related macular degeneration (AMD). The progression of AMD might be slowed or halted if the formation of drusen could be modulated. To work toward a molecular understanding of drusen formation, we have developed a method for isolating microgram quantities of drusen and Bruch's membrane for proteome analysis. Liquid chromatography tandem MS analyses of drusen preparations from 18 normal donors and five AMD donors identified 129 proteins. Immunocytochemical studies have thus far localized approximately 16% of these proteins in drusen. Tissue metalloproteinase inhibitor 3, clusterin, vitronectin, and serum albumin were the most common proteins observed in normal donor drusen whereas crystallin was detected more frequently in AMD donor drusen. Up to 65% of the proteins identified were found in drusen from both AMD and normal donors. However, oxidative protein modifications were also observed, including apparent crosslinked species of tissue metalloproteinase inhibitor 3 and vitronectin, and carboxyethyl pyrrole protein adducts. Carboxyethyl pyrrole adducts are uniquely generated from the oxidation of docosahexaenoate-containing lipids. By Western analysis they were found to be more abundant in AMD than in normal Bruch's membrane and were found associated with drusen proteins. Carboxymethyl lysine, another oxidative modification, was also detected in drusen. These data strongly support the hypothesis that oxidative injury contributes to the pathogenesis of AMD and suggest that oxidative protein modifications may have a critical role in drusen formation.