Recent findings suggest that Delta/Serrate/Lag2 (DSL) signals activate Notch by an unprecedented mechanism that requires the ligands to be endocytosed in signalsending cells to activate the receptor in signal-receiving cells. Here, we show that cells devoid of Epsin, a conserved adaptor protein for Clathrin-mediated endocytosis, behave normally except that they cannot send DSL signals. Surprisingly, we find that Epsin is not required for bulk endocytosis of DSL proteins. Instead, Epsin appears to be essential for targeting DSL proteins to a special endocytic pathway that they must enter to acquire signaling activity. We present evidence that DSL proteins must be monoubiquitinated to be targeted by Epsin to this pathway. Furthermore, we show that the requirements for both Epsin and mono-ubiquitination can be bypassed by introducing the internalization signal that mediates endocytosis and recycling of the Low Density Lipoprotein (LDL) receptor. We propose that Epsin is essential for DSL signaling because it targets mono-ubiquitinated DSL proteins to an endocytic recycling compartment that they must enter to be converted into active ligands. Alternatively Epsin may be required to target monoubiquitinated DSL proteins to a particular subclass of coated pits that have special properties essential for Notch activation. Wendland, 2002). Epsins were initially thought to be core components of the endocytic machinery because of the dominant-negative effects of truncated Epsin proteins on endocytosis in mammalian cells (Chen et al., 1998;Ford et al., 2002), their essential role in yeast endocytosis (Wendland et al., 1999), and their inherent capacity to induce membrane curvature (Ford et al., 2002) and bind other core components such as Clathrin and AP-2 (Chen et al., 1998;Owen et al., 1999;Rosenthal et al., 1999;Wendland et al., 1999;Drake et al., 2000). More recently, however, the identification of Ubiquitin-interacting motifs (UIMs) in Epsins, as well as in other proteins involved in membrane trafficking (Hofmann and Falquet, 2001), have led to the suggestion that Epsins belong to a family of cargoselective adapters that link mono-ubiquitinated cell-surface proteins with the endocytic machinery (Wendland, 2002).Here, we report that DSL ligands must normally be endocytosed in signal-sending cells via the action of Lqf to activate Notch on the surface of signal-receiving cells. Surprisingly, however, bulk endocytosis of DSL ligands appears normal in the absence of Lqf. We resolve this apparent paradox by providing evidence that Lqf is unique amongst adapters that target mono-ubiquitinated cargo proteins for internalization, in that it allows them to enter a special endocytic pathway that DSL ligands must enter to acquire signaling activity. We also show that this requirement can be bypassed by introducing the internalization signal that normally mediates internalization and recycling of the Low Density Lipoprotein (LDL) receptor. On the basis of these results, we hypothesize that Epsin-mediated endocytosis might be requi...

Ligands of the Delta/Serrate/Lag2 (DSL) family must normally be endocytosed in signal-sending cells to activate Notch in signal-receiving cells. DSL internalization and signaling are promoted in zebrafish and Drosophila, respectively, by the ubiquitin ligases Mind bomb (Mib)and Neuralized (Neur). DSL signaling activity also depends on Epsin, a conserved endocytic adaptor thought to target mono-ubiquitinated membrane proteins for internalization. Here, we present evidence that the Drosophila ortholog of Mib (Dmib) is required for ubiquitination and signaling activity of DSL ligands in cells that normally do not express Neur,and can be functionally replaced by ectopically expressed Neur. Furthermore,we show that both Dmib and Epsin are required in these cells for some of the endocytic events that internalize DSL ligands, and that the two Drosophila DSL ligands Delta and Serrate differ in their utilization of these Dmib- and Epsin-dependent pathways: most Serrate is endocytosed via the actions of Dmib and Epsin, whereas most Delta enters by other pathways. Nevertheless, only those Serrate and Delta proteins that are internalized via the action of Dmib and Epsin can signal. These results support and extend our previous proposal that mono-ubiquitination of DSL ligands allows them to gain access to a select, Epsin-dependent, endocytic pathway that they must normally enter to activate Notch.

The epidemic of obesity and diabetes is causing an increased incidence of dyslipidemia-related heart failure. While the primary etiology of lipotoxic cardiomyopathy is an elevation of lipid levels resulting from an imbalance in energy availability and expenditure, increasing evidence suggests a relationship between dysregulation of membrane phospholipid homeostasis and lipid-induced cardiomyopathy. In the present study, we report that the Drosophila easily shocked (eas) mutants that harbor a disturbance in phosphatidylethanolamine (PE) synthesis display tachycardia and defects in cardiac relaxation and are prone to developing cardiac arrest and fibrillation under stress. The eas mutant hearts exhibit elevated concentrations of triglycerides, suggestive of a metabolic, diabetic-like heart phenotype. Moreover, the low PE levels in eas flies mimic the effects of cholesterol deficiency in vertebrates by stimulating the Drosophila sterol regulatory element-binding protein (dSREBP) pathway. Significantly, cardiac-specific elevation of dSREBP signaling adversely affects heart function, reflecting the cardiac eas phenotype, whereas suppressing dSREBP or lipogenic target gene function in eas hearts rescues the cardiac hyperlipidemia and heart function disorders. These findings suggest that dysregulated phospholipid signaling that alters SREBP activity contributes to the progression of impaired heart function in flies and identifies a potential link to lipotoxic cardiac diseases in humans.

The identification of factors that promote β cell proliferation could ultimately move type 1 diabetes treatment away from insulin injection therapy and toward a cure. We have performed high-throughput, cell-based screens using rodent β cell lines to identify molecules that induce proliferation of β cells. Herein we report the discovery and characterization of WS6, a novel small molecule that promotes β cell proliferation in rodent and human primary islets. In the RIP-DTA mouse model of β cell ablation, WS6 normalized blood glucose and induced concomitant increases in β cell proliferation and β cell number. Affinity pulldown and kinase profiling studies implicate Erb3 binding protein-1 and the IκB kinase pathway in the mechanism of action of WS6.

To identify small molecules that can induce β-cell replication, a large chemical library was screened for proliferation of growth-arrested, reversibly immortalized mouse β cells by using an automated high-throughput screening platform. A number of structurally diverse, active compounds were identified, including phorbol esters, which likely act through protein kinase C, and a group of thiophene-pyrimidines that stimulate β-cell proliferation by activating the Wnt signaling pathway. A group of dihydropyridine (DHP) derivatives was also shown to reversibly induce β-cell replication in vitro by activating L-type calcium channels (LTCCs). Our data suggest that the LTCC agonist 2a affects the expression of genes involved in cell cycle progression and cellular proliferation. Furthermore, treatment of β cells with both LTCC agonist 2a and the Glp-1 receptor agonist Exendin-4 showed an additive effect on β-cell replication. The identification of small molecules that induce β-cell proliferation suggests that it may be possible to reversibly expand other quiescent cells to overcome deficits associated with degenerative and/or autoimmune diseases.

SUMMARY Reactive oxygen species (ROS) can act cell autonomously and in a paracrine manner by diffusing into nearby cells. Here, we reveal a ROS-mediated paracrine signaling mechanism that does not require entry of ROS into target cells. We found that under physiological conditions, nonmyocytic pericardial cells (PCs) of the Drosophila heart contain elevated levels of ROS compared to the neighboring cardiomyocytes (CMs). We show that ROS in PCs act in a paracrine manner to regulate normal cardiac function, not by diffusing into the CMs to exert their function, but by eliciting a downstream D-MKK3-D-p38 MAPK signaling cascade in PCs that acts on the CMs to regulate their function. We find that ROS-D-p38 signaling in PCs during development is also important for establishing normal adult cardiac function. Our results provide evidence for a previously unrecognized role of ROS in mediating PC/CM interactions that significantly modulates heart function.

In the present study, the expression of p53, mouse double minute 2 homolog (MDM2), eukaryotic translation initiation factor 4E (eIF4E), and epidermal growth factor receptor (EGFR) were investigated in nasopharyngeal carcinoma (NPC), and the correlation between their expression and clinicopathological characteristics and prognosis was analyzed. The medical records of 96 NPC patients who had undergone biopsy prior to radical radiotherapy and chemotherapy between 2005 and 2009 were reviewed, retrospectively. All patients received intensity-modulated radiotherapy with concurrent platinum-based chemotherapy. Patients were followed-up for three years. Streptavidin-peroxidase immunohistochemistry was used to evaluate the expression of p53, MDM2, eIF4E and EGFR in NPC biopsy specimens, and the association between their expression and clinical parameters and survival was analyzed. The p53, MDM2, eIF4E and EGFR expression rates were 65.6% (63/96), 79.16% (76/96), 77.08% (74/96) and 89.5% (86/96), respectively. p53 (χ2,20.322; P=0.001) and EGFR (χ2,8.337; P=0.005) expression were found to correlate with T stage, whereas MDM2 (χ2,16.361; P=0.001) expression was found to correlate with lymph node metastasis. p53 expression was found to inversely correlate with MDM2 expression (r, −3.24; P<0.05). Three-year survival rates were lower in p53-positive (76.2%) patients when compared with p53-negative (93.9%) patients. In addition, three-year survival rates were lower in EGFR-positive (75.8%) patients than in EGFR-negative patients (91.2%). The Cox proportional-hazards regression model revealed that p53 (β,−0.455; χ2,5.491; P=0.019) and EGFR (β, 3.93; χ2, 11.95; P=0.001) expression were independent prognostic factors. Thus, it was hypothesized that p53 and EGFR expression present potential unfavorable prognostic markers for patients with NPC.

Purpose: Repair of radiation-induced DNA damage plays a critical role for both the susceptibility of patients to side effects after radiotherapy and their subsequent cancer risk.The study objective was to evaluate whether DNA repair data determined in vitro are correlated with the occurrence of acute side effects during radiotherapy. Experimental Design: Nasopharyngeal cancer patients receiving radiation therapy were recruited in a prospective epidemiologic study. As an indicator for clinical radiosensitivity, adverse reactions of the skin were recorded. Cryopreserved lymphocytes from 100 study participants were g-irradiated with 5 Gy in vitro and analyzed using the alkaline comet assay. Reproducibility of the assay was determined by repeated analysis (n = 22) of cells from a healthy donor. A coefficient of variation of 0.24 was calculated. Results: The various parameters determined to characterize the individual DNA repair capacity showed large differences between patients. Twenty-one patients were identified with considerably enhanced DNA damage induction, and 19 patients exhibited severely reduced DNA repair capacity after 15 and 30 minutes. Eight patients were considered as clinically radiosensitive, indicated by moist desquamation of the skin after a total radiation dose of 70 Gy. Conclusions: Using the alkaline comet assay as described here, nasopharyngeal cancer patients were identified showing abnormal cellular radiation effects, but this repair deficiency corresponded only at a very limited extent to the acute radiation sensitivity of the skin.Despite various therapeutic improvements, efficient radiation therapy of malignant cancers is limited by the adverse side effects occurring in the normal tissue when exposed to radiation. In general, >15% of nasopharyngeal cancer patients develop acute or late symptoms of enhanced radiosensitivity (1, 2). Several patient-and treatment-related factors are known to influence the variability of side effects; however, up to 70% of the cases remain unexplained (1). Therefore, there is much interest among clinicians for in vitro detection of cellular radiosensitivity as an indicator of the extent of a patient's normal tissue reaction (3). The use of such predictive assays would enable clinicians to adjust radiation therapy for both sensitive and resistant patients (4) with consequent improvement in the therapeutic ratio (5). For example, a strategy based on testing human normal tissue radiosensitivity to identify the patients with a high risk of developing unacceptable severe reactions after radiotherapy might also permit the individualization of treatment (5) by dose escalation in resistant patients without increasing normal tissue complications (6 -8).Three biological parameters have been used mainly to determine radiosensitivity in vitro. Data from assays based on clonogenic survival (9 -12) and chromosomal aberrations (11,12) showed, at least in several studies, a good correlation of in vitro data with late radiation effects, but these assays are time consu...