Nel positively regulates the genesis of retinal ganglion cells by promoting their differentiation and survival during development

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Experimental rat bladder urothelial cell carcinoma models

Bladder cancer is a major public health problem. Currently available therapeutic options seem to be unable to prevent bladder cancer recurrence and progression. To enable preclinical testing of new intravesical therapeutic agents, a suitable bladder tumor model that resembles human disease is highly desirable. The aim of this topic paper was to discuss the problems associated with current in vivo animal bladder tumor models, focusing on the orthotopic syngeneic rat bladder tumor model. In the second part of the paper the development of a potential new orthotopic rat bladder tumor model is described

The Neuronal EGF-Related Gene Nell2 Interacts with Macf1 and Supports Survival of Retinal Ganglion Cells after Optic Nerve Injury

Nell2 is a neuron-specific protein containing six epidermal growth factor-like domains. We have identified Nell2 as a retinal ganglion cell (RGC)-expressed gene by comparing mRNA profiles of control and RGC-deficient rat retinas. The aim of this study was to analyze Nell2 expression in wild-type and optic nerve axotomized retinas and evaluate its potential role in RGCs. Nell2-positive in situ and immunohistochemical signals were localized to irregularly shaped cells in the ganglion cell layer (GCL) and colocalized with retrogradely-labeled RGCs. No Nell2-positive cells were detected in 2 weeks optic nerve transected (ONT) retinas characterized with approximately 90% RGC loss. RT-PCR analysis showed a dramatic decrease in the Nell2 mRNA level after ONT compared to the controls. Immunoblot analysis of the Nell2 expression in the retina revealed the presence of two proteins with approximate MW of 140 and 90 kDa representing glycosylated and non-glycosylated Nell2, respectively. Both products were almost undetectable in retinal protein extracts two weeks after ONT. Proteome analysis of Nell2-interacting proteins carried out with MALDI-TOF MS (MS) identified microtubule-actin crosslinking factor 1 (Macf1), known to be critical in CNS development. Strong Macf1 expression was observed in the inner plexiform layer and GCL where it was colocalizied with Thy-1 staining. Since Nell2 has been reported to increase neuronal survival of the hippocampus and cerebral cortex, we evaluated the effect of Nell2 overexpression on RGC survival. RGCs in the nasal retina were consistently more efficiently transfected than in other areas (49% vs. 13%; n = 5, p<0.05). In non-transfected or pEGFP-transfected ONT retinas, the loss of RGCs was approximately 90% compared to the untreated control. In the nasal region, Nell2 transfection led to the preservation of approximately 58% more cells damaged by axotomy compared to non-transfected (n = 5, p<0.01) or pEGFP-transfected controls (n = 5, p<0.01)

Dynamic Ligand Modulation of EPO Receptor Pools, and Dysregulation by Polycythemia-Associated EPOR Alleles

Erythropoietin (EPO) and its cell surface receptor (EPOR) are essential for erythropoiesis; can modulate non-erythroid target tissues; and have been reported to affect the progression of certain cancers. Basic studies of EPOR expression and trafficking, however, have been hindered by low-level EPOR occurrence, and the limited specificity of anti-EPOR antibodies. Consequently, these aspects of EPOR biology are not well defined, nor are actions of polycythemia- associated mutated EPOR alleles. Using novel rabbit monoclonal antibodies to intracellular, PY- activated and extracellular EPOR domains, the following properties of the endogenous hEPOR in erythroid progenitors first are unambiguously defined. 1) High- Mr EPOR forms become obviously expressed only when EPO is limited. 2) EPOR-68K plus-70K species sequentially accumulate, and EPOR-70K comprises an apparent cell surface EPOR population. 3) Brefeldin A, N-glycanase and associated analyses point to EPOR-68K as a core-glycosylated intracellular EPOR pool (of modest size). 4) In contrast to recent reports, EPOR inward trafficking is shown (in UT7epo cells, and primary proerythroblasts) to be sharply ligand-dependent. Beyond this, when C-terminal truncated hEPOR-T mutant alleles as harbored by polycythemia patients are co-expressed with the wild-type EPOR in EPO-dependent erythroid progenitors, several specific events become altered. First, EPOR-T alleles are persistently activated upon EPO- challenge, yet are also subject to apparent turn-over (to low-Mr EPOR products). Furthermore, during exponential cell growth EPOR-T species become both over-represented, and hyper-activated. Interestingly, EPOR-

Upon EPO ligation, the polycythemia-associated truncated EPOR allele EPOR-T-392 exhibit sustained activation.

<p><b>A, B</b>] UT7-wtEPOR and UT7-EPOR-T-392 cells were cultured for 20 hours in the absence of EPO. Cells were then exposed to EPO at 0.8 U/mL (left panel), or 3.2 U/mL (right panel). At 0, 10, 30 and 90 minutes, lysates were prepared and analyzed by western blotting for levels of PY344- EPOR (panel A). Levels of activated JAK2 also were determined (panel B). PY-EPOR and PY-JAK2 levels also were quantitatively estimated (panel B, lower sub-panels).</p

In exponentially growing erythroid progenitors, EPOR-T-392 and EPOR-T-374 accumulate, and decrease endogenous wt-EPOR expression.

<p><b>A</b>] UT7-wtEPOR, UT7-EPOR-T392 and UT7-EPOR-T374 cells were cultured in EPO at either 0.3 or 3 U/mL. Levels of EPOR expression in directly prepared lysates then were assessed via western blotting. <b>B</b>] EPO dose-dependent increases in truncated EPOR-T-392 expression, <i>vs</i> EPO dose-dependent decreases in wild-type EPOR expression– For the above western blot analyses, quantitative imaging illustrates converse effects of EPO on expression levels of WT-EPOR <i>vs</i> EPOR-T-392 receptors. <b>C</b>] In erythroid progenitor cells expressing truncated EPOR forms, heightened EPO levels lead to marked decreases in wild-type EPOR levels– For the above western blot analyses, quantitative imaging illustrates clear inhibitory effects of EPOR-T392, and EPOR-T-374 expression on wild-type EPOR levels. <b>D</b>] Truncated alleles EPOR-T-392 and EPOR-T-374 each exhibit clear EPO dose-dependent turnover to low- Mr EPOR-T species (*,**).</p