M-tropic human immunodeficiency virus (HIV-1) strains enter the cell after interaction with their receptors, CD4 and the G-protein-coupled chemokine receptor CCR5. The number of cell surface CCR5 molecules is thought to be important in determining the infection rate for HIV. Cell surface CCR5 is dependent on the rate of receptor internalization and recycling. Internalization of G-protein-coupled receptors after agonist activation is thought to occur either through clathrin-coated pits or through caveolae. In this study, the role of these different pathways was investigated in Chinese hamster ovary cells expressing CCR5 using specific inhibitors. Internalization of CCR5 after chemokine treatment was inhibited by sucrose, indicating a role for the clathrin-coated pit pathway. Activation of CCR5 leads to arrestin-2 movement in the cells, providing further evidence for the involvement of clathrin-coated pits. Nystatin and filipin also affected the rate of internalization of CCR5, indicating a role for caveolae. Using inhibitors of vesicle transport in the cell, it was found that the CCR5 recycling pathway is independent of the Golgi apparatus and late endosomes. Protein synthesis is not involved in receptor recovery. It seems likely that after internalization, CCR5 is directed to early endosomes and subsequently recycled to the cell surface. (Blood. 2002;99:785-791)

s u m m a r yThe skill of a forecast can be assessed by comparing the relative proximity of both the forecast and a benchmark to the observations. Example benchmarks include climatology or a naïve forecast. Hydrological ensemble prediction systems (HEPS) are currently transforming the hydrological forecasting environment but in this new field there is little information to guide researchers and operational forecasters on how benchmarks can be best used to evaluate their probabilistic forecasts. In this study, it is identified that the forecast skill calculated can vary depending on the benchmark selected and that the selection of a benchmark for determining forecasting system skill is sensitive to a number of hydrological and system factors. A benchmark intercomparison experiment is then undertaken using the continuous ranked probability score (CRPS), a reference forecasting system and a suite of 23 different methods to derive benchmarks. The benchmarks are assessed within the operational set-up of the European Flood Awareness System (EFAS) to determine those that are 'toughest to beat' and so give the most robust discrimination of forecast skill, particularly for the spatial average fields that EFAS relies upon.Evaluating against an observed discharge proxy the benchmark that has most utility for EFAS and avoids the most naïve skill across different hydrological situations is found to be meteorological persistency. This benchmark uses the latest meteorological observations of precipitation and temperature to drive the hydrological model. Hydrological long term average benchmarks, which are currently used in EFAS, are very easily beaten by the forecasting system and the use of these produces much naïve skill. When decomposed into seasons, the advanced meteorological benchmarks, which make use of meteorological observations from the past 20 years at the same calendar date, have the most skill discrimination. They are also good at discriminating skill in low flows and for all catchment sizes. Simpler meteorological benchmarks are particularly useful for high flows. Recommendations for EFAS are to move to routine use of meteorological persistency, an advanced meteorological benchmark and a simple meteorological benchmark in order to provide a robust evaluation of forecast skill. This work provides the first comprehensive evidence on how benchmarks can be used in evaluation of skill in probabilistic hydrological forecasts and which benchmarks are most useful for skill discrimination and avoidance of naïve skill in a large scale HEPS. It is recommended that all HEPS use the evidence and methodology provided here to evaluate which benchmarks to employ; so forecasters can have trust in their skill evaluation and will have confidence that their forecasts are indeed better.

The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T- lymphocytes. We describe here a study of the endocytosis of CXCR3 using T-lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 downregulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was dependent upon mRNA transcription, de novo protein synthesis and transport through the ER and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C-terminus revealed that internalization and degradation were independent of phophorylation, ubiquitination or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C-terminus. Although freshly isolated T-lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having significant activity on activated T-lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Since CXCR3+ cells are themselves a source of IFN-γ, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T-lymphocyte recruitment.

We have recently identified Np9 as a novel nuclear protein produced by the human endogenous retrovirus K and were able to document the exclusive presence of np9 transcript in tumors and transformed cells. With the aim of studying whether Np9 has a role in tumorigenesis, a systematic search for interacting proteins was performed. Here, we identify the RING-type E3 ubiquitin ligase LNX (ligand of Numb protein X) as an Np9-interacting partner. We furthermore show that the interaction involves N-and C-terminal domains of both proteins and can affect the subcellular localization of LNX. LNX has been reported to target the cell fate determinant and Notch antagonist Numb for proteasome-dependent degradation, thereby causing an increase in transactivational activity of Notch. We document that LNX-interacting Np9, like Numb, is unstable and degraded via the proteasome pathway and that ectopic Numb can stabilize recombinant Np9. Combined, these findings point to the possibility that Np9 affects tumorigenesis through the LNX/Numb/Notch pathway.Up to 8% of the human genome consists of retrovirusderived sequences (19). They are the result of numerous infections and subsequent integration of proviral elements into the germ line of human ancestors during the past 40 million years (2). Most of the recent human endogenous retrovirus (HERV) elements are defective due to the accumulation of mutations. Nonetheless, retroviral genes are frequently expressed in normal and transformed human tissues. The function of these genes in normal human cell physiology is still unclear (32). So far, only the expression of the env gene of HERV-W is known to have a crucial function in normal organismal physiology, namely human trophoblast cell fusion and differentiation (5,12,23). In contrast, retroviral gene expression has been linked to several human diseases, including tumorigenesis. In particular, expression of HERV-K seems to be strongly associated with transformation. Early hints pointing in this direction came from the observation that expression of the HERV-K proteins Gag and Env, as well as antibodies directed against these proteins, could be specifically detected in patients suffering from germ cell tumors (17,24,30,31). Other studies implicate a contribution of HERV-K10 gag RNA expression to the development of leukemia (9) or show that env as well as the expression of subgenomic env transcripts correlates with human breast cancer (35,36).It is unclear, at the present, how these proteins may support tumorigenesis. The Rev-like regulatory protein Rec (20,21)