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  • Effect of quiescence on integrin alpha5beta1 expression in human retinal pigment epithelium. 14551535

    PURPOSE: The retinal pigment epithelium (RPE) is differentiated and mitotically inactive in the normal eye, but several pathologies such as proliferative vitreoretinopathy (PVR) cause RPE cells to dedifferentiate and resume proliferation. Integrins, a family of cell surface glycoproteins that mediate cell proliferation and differentiation, are thought to play fundamental roles in PVR. The aim of this study was to evaluate protein expression and gene regulation of the integrin alpha5 subunit in proliferating and quiescent RPE cells. METHODS: Protein expression was studied in situ by immunohistochemistry and in vitro at different cell confluences by immunoprecipitation. Semi-quantitative RT-PCR and transient transfections were used to determine whether increasing cell confluence also affected alpha5 subunit mRNA levels and promoter activity, respectively. RESULTS: We demonstrated that the integrin alpha5 subunit is present at the RPE cell surface both in situ and in vitro, and that alpha5 protein level is influenced by confluence. Levels of integrin alpha5 transcripts are similar for sub-confluent and confluent cells, and a small increase in the promoter activity was observed between sub-confluent and confluent cells. However, both the integrin alpha5 subunit transcript and the alpha5 promoter activity decreased when cells reached post-confluence. CONCLUSIONS: We demonstrated that cell confluence affected protein and gene expression of the integrin alpha5 subunit. Proliferating RPE cells expressed high levels of both the alpha5 protein and mRNA transcripts and showed a high promoter activity. However, when cells reached quiescence, alpha5 gene expression was substantially reduced and RPE cells expressed little alpha5 protein at their cell surface.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Visual cycle protein RPE65 persists in new retinal cells during retinal regeneration of adult newt. 16485283

    Adult newts can regenerate their entire retina through transdifferentiation of the retinal pigment epithelium (RPE). The objective of this study was to redescribe the retina regeneration process by means of modern biological techniques. We report two different antibodies (RPE-No.112 and MAB5428) that recognize the newt homolog of RPE65, which is involved in the visual cycle and exclusively label the RPE cell-layer in the adult newt eye. We analyzed the process of retinal regeneration by immunohistochemistry and immunoblotting and propose that this process should be divided into nine stages. We found that the RPE65 protein is present in the RPE-derived new retinal rudiment at 14 days postoperative (po) and in the regenerating retinas at the 3-4 cell stage (19 days po). These observations suggest that certain characteristics of RPE cells overlap with those of retinal stem/progenitor cells during the period of transdifferentiation. However, RPE65 protein was not detected in either retinal stem/progenitor cells in the ciliary marginal zone (CMZ) of adult eyes or in neuroepithelium present during retina development, where it was first detected in differentiated RPE. Moreover, the gene expression of RPE65 was drastically downregulated in the early phase of transdifferentiation (by 10 days po), while those of Connexin43 and Pax-6, both expressed in regenerating retinas, were differently upregulated. These observations suggest that the RPE65 protein in the RPE-derived retinal rudiment may represent the remainder after protein degradation or discharge rather than newly synthesized protein.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5428
    Nombre del producto:
    Anti-Retinal Pigment Epithelium 65 Antibody

  • Histopathology and functional correlations in a patient with a mutation in RPE65, the gene for retinol isomerase. 21931134

    Here the authors describe the structural features of the retina and retinal pigment epithelium (RPE) in postmortem donor eyes of a 56-year-old patient with a homozygous missense RPE65 mutation (Ala132Thr) and correlate the pathology with the patient's visual function last measured at age 51.Eyes were enucleated within 13.5 hours after death. Representative areas from the macula and periphery were processed for light and electron microscopy. Immunofluorescence was used to localize the distribution of RPE65, rhodopsin, and cone arrestin. The autofluorescence in the RPE was compared with that of two normal eyes from age-similar donors.Histologic examination revealed the loss of rods and cones across most areas of the retina, attenuated retinal vessels, and RPE thinning in both eyes. A small number of highly disorganized cones were present in the macula that showed simultaneous labeling with cone arrestin and red/green or blue opsin. RPE65 immunoreactivity and RPE autofluorescence were reduced compared with control eyes in all areas studied. Rhodopsin labeling was observed in rods in the far periphery. The optic nerve showed a reduced number of axons.The clinical findings of reduced visual acuity, constricted fields, and reduced electroretinograms (ERGs) 5 years before death correlated with the small number of cones present in the macula and the extensive loss of photoreceptors in the periphery. The absence of autofluorescence in the RPE suggests that photoreceptor cells were probably missing across the retina for extended periods of time. Possible mechanisms that could lead to photoreceptor cell death are discussed.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Structural and functional characterization of recombinant human cellular retinaldehyde-binding protein. 9541407

    Cellular retinaldehyde-binding protein (CRALBP) is abundant in the retinal pigment epithelium (RPE) and Müller cells of the retina where it is thought to function in retinoid metabolism and visual pigment regeneration. The protein carries 11-cis-retinal and/or 11-cis-retinol as endogenous ligands in the RPE and retina and mutations in human CRALBP that destroy retinoid binding functionality have been linked to autosomal recessive retinitis pigmentosa. CRALBP is also present in brain without endogenous retinoids, suggesting other ligands and physiological roles exist for the protein. Human recombinant cellular retinaldehyde-binding protein (rCRALBP) has been over expressed as non-fusion and fusion proteins in Escherichia coli from pET3a and pET19b vectors, respectively. The recombinant proteins typically constitute 15-20% of the soluble bacterial lysate protein and after purification, yield about 3-8 mg per liter of bacterial culture. Liquid chromatography electrospray mass spectrometry, amino acid analysis, and Edman degradation were used to demonstrate that rCRALBP exhibits the correct primary structure and mass. Circular dichroism, retinoid HPLC, UV-visible absorption spectroscopy, and solution state 19F-NMR were used to characterize the secondary structure and retinoid binding properties of rCRALBP. Human rCRALBP appears virtually identical to bovine retinal CRALBP in terms of secondary structure, thermal stability, and stereoselective retinoid-binding properties. Ligand-dependent conformational changes appear to influence a newly detected difference in the bathochromic shift exhibited by bovine and human CRALBP when complexed with 9-cis-retinal. These recombinant preparations provide valid models for human CRALBP structure-function studies.
    Tipo de documento:
    Referencia
    Referencia del producto:
    13-110
    Nombre del producto:
    MBP, Dephosphorylated

  • Divergent requirements for fibroblast growth factor signaling in zebrafish maxillary barbel and caudal fin regeneration. 23350700

    The zebrafish maxillary barbel is an integumentary organ containing skin, glands, pigment cells, taste buds, nerves, and endothelial vessels. The maxillary barbel can regenerate (LeClair & Topczewski 2010); however, little is known about its molecular regulation. We have studied fibroblast growth factor (FGF) pathway molecules during barbel regeneration, comparing this system to a well-known regenerating appendage, the zebrafish caudal fin. Multiple FGF ligands (fgf20a, fgf24), receptors (fgfr1-4) and downstream targets (pea3, il17d) are expressed in normal and regenerating barbel tissue, confirming FGF activation. To test if specific FGF pathways were required for barbel regeneration, we performed simultaneous barbel and caudal fin amputations in two temperature-dependent zebrafish lines. Zebrafish homozygous for a point mutation in fgf20a, a factor essential for caudal fin blastema formation, regrew maxillary barbels normally, indicating that the requirement for this ligand is appendage-specific. Global overexpression of a dominant negative FGF receptor, Tg(hsp70l:dn-fgfr1:EGFP)(pd1) completely blocked fin outgrowth but only partially inhibited barbel outgrowth, suggesting reduced requirements for FGFs in barbel tissue. Maxillary barbels expressing dn-fgfr1 regenerated peripheral nerves, dermal connective tissue, endothelial tubes, and a glandular epithelium; in contrast to a recent report in which dn-fgfr1 overexpression blocks pharyngeal taste bud formation in zebrafish larvae (Kapsimali et al. 2011), we observed robust formation of calretinin-positive tastebuds. These are the first experiments to explore the molecular mechanisms of maxillary barbel regeneration. Our results suggest heterogeneous requirements for FGF signaling in the regeneration of different zebrafish appendages (caudal fin versus maxillary barbel) and taste buds of different embryonic origin (pharyngeal endoderm versus barbel ectoderm).
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5054

  • Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling. 24468901

    In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MABC32
    Nombre del producto:
    Anti-p62 (Sequestosome-1) Antibody, clone 11C9.2

  • Localization of pigment epithelium derived factor (PEDF) in developing and adult human ocular tissues. 11438800

    PURPOSE: To localize pigment epithelium-derived factor (PEDF) in developing and adult human ocular tissues. METHODS: PEDF was localized in fetal and adult eyes by immunofluorescence with a polyclonal antibody (pAb) against amino acids 327-343 of PEDF, or a monoclonal antibody (mAb) against the C-terminal 155 amino acids of PEDF. Specificity of the antibodies was documented by Western blotting. PEDF mRNA was localized in adult retina by in situ hybridization. RESULTS: In developing retinas (7.4 to 21.5 fetal weeks, Fwks), pAb anti-PEDF labeled retinal pigment epithelium (RPE) granules, developing cones, some neuroblasts and many cells in the ganglion cell layer (GCL). In adult retinas, pAb anti-PEDF labeled rod and cone cytoplasm and nuclei of rods but not cones. Cells in the INL and GCL, choroid, corneal epithelium and endothelium, and ciliary body were also pAb PEDF-positive. Preadsorption of pAb anti-PEDF with the immunizing peptide blocked specific labeling in retina and other tissues, except for photoreceptor outer segments. In agreement with the immunolocalization with pAb anti-PEDF, in situ hybridization revealed PEDF mRNA in the RPE, photoreceptors, inner nuclear layer cells and ganglion cells in adult retina. In developing retinas 18 Fwks and older, and in adult retinas, mAb anti-PEDF labeled the interphotoreceptor matrix (IPM). Western blots of retina, cornea, and ciliary body/iris with pAb anti-PEDF produced several bands at about 46 kDa. With mAb anti-PEDF, retina produced one band at about 46 kDa; cornea and ciliary body/iris had several bands at about 46 kDa. CONCLUSIONS: PEDF, originally reported as a product of RPE cells, is present in photoreceptors and inner retinal cell types in developing and adult human eyes. Photoreceptors and RPE may secrete PEDF into the IPM.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo

  • Assays for the antiangiogenic and neurotrophic serpin pigment epithelium-derived factor. 21683255

    Pigment epithelium-derived factor (PEDF) is a secreted serpin that exhibits a variety of interesting biological activities. The multifunctional PEDF has neurotrophic and antiangiogenic properties, and acts in retinal differentiation, survival, and maintenance. It is also antitumorigenic and antimetastatic, and has stem cell self-renewal properties. It is widely distributed in the human body and exists in abundance in the eye as a soluble extracellular glycoprotein. Its levels are altered in diseases characterized by retinopathies and angiogenesis. Its mechanisms of neuroprotection and angiogenesis are associated with receptor interactions at cell-surface interfaces and changes in protein expression. This serpin lacks demonstrable serine protease inhibitory activity, but has binding affinity to extracellular matrix components and cell-surface receptors. Here we describe purification protocols, methods to quantify PEDF, and determine interactions with specific molecules, as well as neurotrophic and angiogenesis assays for this multifunctional protein.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1059
    Nombre del producto:
    Anti-Pigment Epithelium Derived Factor Antibody, clone 10F12.2

  • Tauroursodeoxycholic acid (TUDCA) protects photoreceptors from cell death after experimental retinal detachment. 21961034

    Detachment of photoreceptors from the underlying retinal pigment epithelium is seen in various retinal disorders such as retinal detachment and age-related macular degeneration and leads to loss of photoreceptors and vision. Pharmacologic inhibition of photoreceptor cell death may prevent this outcome. This study tests whether systemic administration of tauroursodeoxycholic acid (TUDCA) can protect photoreceptors from cell death after experimental retinal detachment in rodents.Retinal detachment was created in rats by subretinal injection of hyaluronic acid. The animals were treated daily with vehicle or TUDCA (500 mg/kg). TUNEL staining was used to evaluate cell death. Photoreceptor loss was evaluated by measuring the relative thickness of the outer nuclear layer (ONL). Macrophage recruitment, oxidative stress, cytokine levels, and caspase levels were also quantified. Three days after detachment, TUDCA decreased the number of TUNEL-positive cells compared to vehicle (651±68/mm(2) vs. 1314±68/mm(2), P = 0.001) and prevented the reduction of ONL thickness ratio (0.84±0.03 vs. 0.65±0.03, P = 0.002). Similar results were obtained after 5 days of retinal detachment. Macrophage recruitment and expression levels of TNF-a and MCP-1 after retinal detachment were not affected by TUDCA treatment, whereas increases in activity of caspases 3 and 9 as well as carbonyl-protein adducts were almost completely inhibited by TUDCA treatment.Systemic administration of TUDCA preserved photoreceptors after retinal detachment, and was associated with decreased oxidative stress and caspase activity. TUDCA may be used as a novel therapeutic agent for preventing vision loss in diseases that are characterized by photoreceptor detachment.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1435
    Nombre del producto:
    Anti-Macrophages/Monocytes Antibody, clone ED-1

  • Pigment epithelium-derived factor is an intrinsic antifibrosis factor targeting hepatic stellate cells. 20709803

    The liver is the major site of pigment epithelium-derived factor (PEDF) synthesis. Recent evidence suggests a protective role of PEDF in liver cirrhosis. In the present study, immunohistochemical analyses revealed lower PEDF levels in liver tissues of patients with cirrhosis and in animals with chemically induced liver fibrosis. Delivery of the PEDF gene into liver cells produced local PEDF synthesis and ameliorated liver fibrosis in animals treated with either carbon tetrachloride or thioacetamide. In addition, suppression of peroxisome proliferator-activated receptor gamma expression, as well as nuclear translocation of nuclear factor-kappa B was found in hepatic stellate cells (HSCs) from fibrotic livers, and both changes were reversed by PEDF gene delivery. In culture-activated HSCs, PEDF, through the induction of peroxisome proliferator-activated receptor gamma, reduced the activity of nuclear factor-kappa B and prevented the nuclear localization of JunD. In conclusion, our observations that PEDF levels are reduced during liver cirrhosis and that PEDF gene delivery ameliorates cirrhosis suggest that PEDF is an intrinsic protector against liver cirrhosis. Direct inactivation of HSCs and the induction of apoptosis of activated HSCs may be two of the mechanisms by which PEDF suppresses liver cirrhosis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5694