GNL3L depletion destabilizes MDM2 and induces p53-dependent G2/M arrest.

Guanine nucleotide binding protein-like 3-like (GNL3L) is a nucleolar protein and the vertebrate paralogue of nucleostemin (NS). We previously reported that nucleoplasmic mobilization of NS stabilizes MDM2 (mouse double minute 2). Here, we investigated the role of GNL3L as a novel MDM2 regulator. We found that GNL3L binds MDM2 in vivo and displays the same function as NS in stabilizing MDM2 protein and preventing its ubiquitylation. The interaction between GNL3L and MDM2 also takes place in the nucleoplasm. However, the MDM2 regulatory activity of GNL3L occurs constitutively and does not so much depend on the nucleolar release mechanism as NS does. GNL3L depletion triggers G2/M arrest in the p53-wild-type HCT116 cells more than in the p53-null cells, and upregulates specific p53 targets (that is, Bax, 14-3-3σ and p21) without affecting the ubiquitylation or stability of p53 proteins. The inhibitory activity of GNL3L on p53-mediated transcription correlates with the increased expression of GNL3L and reduced expression of 14-3-3σ and p21 in human gastrointestinal tumors. This work shows that in contrast to most nucleolar proteins that negatively control MDM2, GNL3L and NS are the only two that are designed to stabilize MDM2 protein under basal or induced condition, respectively, and may act as tumor-promoting genes.

Changes in corneal endothelial apical junctional protein organization after corneal cold storage.

PURPOSE: Understanding the mechanisms regulating corneal endothelial permeability during storage and recovery is of critical importance both to improving Eye Banking practices and preventing corneal transplant failure. The goal of this study was to determine the effects of cold storage on the organization of apical junctional complex (AJC) proteins and their relationship to F-actin in corneal endothelium. METHODS: Immunostaining using antibodies to the AJC proteins, ZO-1, cadherin, and alpha- and beta-catenin was performed on 16 eye bank corneas and four cat corneas after 2-8 days of storage at 4 degrees C in Optisol-GS, and compared with fresh corneas. The 3-D in situ localization of the AJC proteins was then determined by using laser confocal microscopy. AJC organization also was assessed after stored human corneas were further incubated at 37 degrees C in Optisol-GS or in serum-free media. RESULTS: In normal human and cat corneas, F-actin was organized into dense peripheral bands (DPBs) along the apical cell border. The tight-junction protein, ZO-1, and the adherens junction proteins, cadherin and alpha- and beta-catenin, each formed a uniquely discontinuous hexagonal apical band with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. In stored eye bank and cat corneas, cells lost their normal hexagonal F-actin staining pattern and appeared rounded and distorted, with increased cytoplasmic staining and incomplete and condensed DPBs. Similar distortions were observed in the apical bands of cadherin, catenin, and ZO-1 staining between endothelial cells. Gaps in staining at the endothelial Y-junctions were significantly enlarged; corresponding gaps also were observed with phalloidin staining. These changes were reversed after overnight incubation at 37 degrees C in either serum-free media or Optisol-GS. Quantitative analysis demonstrated a significant increase in the size of the Y-junctional gaps (p < 0.0001) after cold storage of cat corneas as compared with fresh corneas. CONCLUSION: These results suggest that disruption of the F-actin cytoskeleton and AJC may explain, in part, the loss of function (corneal swelling) after prolonged cold storage.

The spatial organization of apical junctional complex-associated proteins in feline and human corneal endothelium.

PURPOSE: Previous studies suggest that proteins associated with the apical junctional complex (AJC) play essential roles in the development, maintenance and regulation of barrier function in transport epithelium and vascular endothelium. The goal of this study is to identify and determine the spatial organization of several major AJC-associated proteins in normal human and feline corneal endothelium. METHODS: Fresh corneal tissue was obtained from 4 recipient buttons removed during penetrating keratoplasty (two from keratoconus patients, and two from patients with post-traumatic stromal scarring) as well as from 16 cat eyes. En bloc double- and triple-labeling of corneas was performed using phalloidin, and mouse, rat or rabbit antibodies to ZO-1, occludin, pan-cadherin, alpha-catenin, beta-catenin and plakoglobin (gamma-catenin). The 3-D localization of the proteins was then determined in situ using laser confocal microscopy. RESULTS: Similar staining patterns were obtained for the corneal endothelium of normal cat corneas and fresh human buttons. Apically, f-actin was arranged into dense peripheral bands (DPB) in individual cells that were separated from those in adjacent cells. Diffuse phalloidin staining also extended from the DPB into the cytoplasm apically. Although weaker, phalloidin staining also appeared to be associated with the basolateral cell borders. The adherens junction protein, cadherin, formed a thin pericellular band at the apical cell junctions between the DPB. In addition, cadherin staining also appeared to extend along the basolateral cell borders in a convoluted pattern. Staining for alpha-catenin, beta-catenin and plakoglobin each showed a nearly identical organization as cadherin. ZO-1 formed a single apical band that was localized between the DPB; however, no basolateral ZO-1 staining was detected. Interestingly, the distribution of ZO-1 was discontinuous around the cell, with the largest gaps occurring at the Y-junctions between adjacent endothelial cells. Positive staining for occludin was not detected in either human or feline corneal endothelium. CONCLUSIONS: The composition and organization of the AJC of corneal endothelium appears to be different from that of classical transport epithelia; these findings may be related to functional differences between these two cell types.

An unusual case of elastofibroma oculi.

A 22-year-old female college student from the southwestern United States was first seen with progressive fluffy white growths over both corneas nasally and a history of chronic allergies and extensive sun, wind, and chlorine exposure as a competitive swimmer. The lesions were superficial, elevated, crescent-shaped, vascularized, and extended from the limbal conjunctiva onto the peripheral cornea nasally. The lesions were composed of an unencapsulated thick fibrovascular pannus with numerous fibrocytes, chronic inflammatory cells and mast cells, and abundant linear and wavy elastinophilic profiles, but no adipose tissue. Ultrastructurally, the elastinophilic structures were immature elastic fibers arranged in globules and bundles within a thick collagenous matrix. No mature elastic fibers were found. A few areas of elastotic degeneration were also found. This lesion resembled elastofibromas that have been reported more commonly in the subcapsular region and only once in the ocular region. Previous reports debated whether the elastic material in elastofibromas is derived from excessive production of elastic fibers by activated fibrocytes or from elastotic degeneration of collagen. In our case, both processes occur and are presumed to result from excessive ultraviolet radiation, wind and chlorine exposure, and perhaps, chronic inflammation; features that have been ascribed to the pathogenesis of pingueculae and pterygia.

Clinicopathologic studies of an eye after submacular membranectomy for choroidal neovascularization.

BACKGROUND: Submacular membranectomy has been suggested as an alternative treatment for subfoveal choroidal neovascularization (CNV). Clinicopathologic features of the right eye of a 59-year-old man with recurrent subfoveal CNV who underwent submacular membranectomy after two unsuccessful laser photocoagulation treatments are reported. METHODS: The surgically excised subfoveal membrane was sectioned serially and evaluated by light microscopy. The globes were obtained postmortem and serial sectioned through the macula and optic nerve head for light microscopy. Ultrastructural study of a tissue section in the center of the lesion was performed. RESULTS: Histopathologic study of the surgically excised membrane disclosed a thin two-component fibrovascular membrane with the larger component internal to residual retinal pigment epithelium and basal laminar deposit. Photoreceptor outer segments were present on the internal surface of the membrane near one margin. Light and electron microscopic study of the postmortem globe revealed a very thin subfoveal subretinal pigment epithelial fibrovascular membrane with loss of photoreceptor cell layer in a central 0.5 mm area, loss of outer segments, reduction of inner segments, and thinning of the outer nuclear layer in the remainder of the lesion. There was moderate retinal pigment epithelial attenuation and mild basal laminar and basal linear deposits. CONCLUSION: Submacular membranectomy for recurrent subfoveal CNV secondary to age-related macular degeneration after two unsuccessful laser photocoagulation treatments appeared to be effective with repopulation of two thirds of the area of membranectomy by extension of attenuated retinal pigment epithelium from adjacent areas. There was, however, persistence or recurrence of CNV, moderate atrophy of the overlying retina with total loss of the photoreceptor cells over the central 0.5 mm of the membrane, and moderate loss of the photoreceptor cells over the remaining area.

Secondary epiretinal membrane after blunt trauma.

Electron microscopic study of a surgically removed epiretinal membrane secondary to blunt trauma disclosed the membrane to be hypocellular and lined by internal limiting membrane on the external surface and by a layer of fibrocytes and myofibrocytes on the internal surface. The membrane was composed predominantly of new collagen. Occasional fibrous astrocytes, rare macrophages, and no blood vessels were present.

Anterior stromal puncture. Immunohistochemical studies in human corneas.

OBJECTIVE: To investigate the mechanism of action of corneal anterior stromal puncture (ASP) in humans. DESIGN: Immunocytochemical techniques were used to localize fibronectin, type IV collagen, and laminin in human corneas with bullous keratopathy, some of which had undergone ASP. Corneal specimens were obtained from transplant procedures performed in a related clinical study. SETTING: Outpatients in private practice settings. PATIENTS: Nine patients with recurrent erosion secondary to bullous keratopathy who were judged to be poor candidates for keratoplasty. INTERVENTIONS: Anterior stromal puncture was performed on each patient using a standardized needle, and corneal transplants were performed on patients whose erosions did not resolve after ASP. PRIMARY OUTCOME MEASURES: Subjective comfort and slit-lamp verification of resolution of rupture of bullae and erosions in patients who underwent ASP; Nomarski differential interference contrast photography, immunohistochemical staining, and light microscopy were applied to the corneal specimens. RESULTS: All three matrix glycoproteins were observed in the epithelial basement membrane of normal corneas. In patients with bullous keratopathy who did not undergo ASP, the epithelial basement membrane of the cornea did not stain with antibodies against human fibronectin, type IV collagen, or laminin. In patients with bullous keratopathy who underwent ASP, all three major proteins were present at the puncture sites and in the reactive subepithelial pannus adjacent to the puncture site. Epithelial basement membrane of untreated regions showed little or no staining. CONCLUSIONS: The results suggest that the absence of these extracellular matrix proteins in the epithelial basement membrane of patients with bullous keratopathy may be an important factor in the development of poor epithelial adhesion and secondary erosions. Anterior stromal puncture may promote epithelial reattachment, at least in bullous keratopathy, by stimulating the production of extracellular matrix proteins that are important in the attachment of epithelial cells to the underlying connective tissue. Epithelial-stromal reactions and the development of subepithelial fibrosis may also play a role in reestablishing epithelial attachment.