A proteomic map for human urine on two-dimensional (2-D) gels has been developed. Initial studies demonstrated that the urine proteins prepared by conventional methods showed interference and poor reproducibility in 2-D electrophoresis (2-DE). To address this issue, urine samples were dialyzed to remove any interfering molecules. The dialysis of urine proteins and the concentration by lyophilization without fractionation significantly improved the reproducibility and resolution and likely represents the total urine proteins on a 2-D gel. In addition, removing albumin from urine using Affi-Gel Blue helped to identify the low-abundant proteins. Using the developed method, we prepared proteins from urine collected from healthy females and males. The large inter- and intra-subject variation in protein profiles on 2-D gels made it difficult to establish a normal human urine proteomic 2-D map. To resolve this problem, urinary proteins were prepared from the pooled urine collected from 20 healthy females and males, respectively. The established male and female urine proteomes separated on 2-D gels were almost identical except for some potential sex-dependent protein spots. We have annotated 113 different proteins on the 2-D gel by peptide mass fingerprinting (PMF). We propose that the established total urine proteome can be used for 2-DE analysis, liquid chromatography-tandem mass spectrometry (LC-MS/MS), and identification of novel disease-specific biomarkers.

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The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, have been implicated in regulating cell adhesion and migration during development by mediating cell-to-cell signaling events. Genetic evidence suggests that ephrins may transduce signals and become tyrosine phosphorylated during embryogenesis. However, the induction and functional significance of ephrin phosphorylation is not yet clear. Here, we report that when we used ectopically expressed proteins, we found that an activated fibroblast growth factor (FGF) receptor associated with and induced the phosphorylation of ephrin B1 on tyrosine. Moreover, this phosphorylation reduced the ability of overexpressed ephrin B1 to reduce cell adhesion. In addition, we identified a region in the cytoplasmic tail of ephrin B1 that is critical for interaction with the FGF receptor; we also report FGF-induced phosphorylation of ephrins in a neural tissue. This is the first demonstration of communication between the FGF receptor family and the Eph ligand family and implicates cross talk between these two cell surface molecules in regulating cell adhesion.Cell adhesion events, coordinated both spatially and temporally, are critical to the development and maintenance of the tissue structures of an organism. Interactions with other cells and extracellular matrix components govern the growth, differentiation, migration, and ultimate location and function of a given cell. An increasing number of proteins expressed at the cell surface have been implicated in regulating cell adhesion and movement within specific developmental contexts. For example, considerable progress has been made in identifying cell surface proteins which regulate neurite outgrowth and pathfinding during nervous system development (9). These proteins include classic cell adhesion molecules such as the neural cell adhesion molecules of the immunoglobulin superfamily (58, 69), members of the cadherin superfamily (20), and integrins (47), as well as a growing number of other protein families including collapsins/semaphorins, neuropilins, netrin receptors (10, 11, 57), various peptide growth factor receptors (49), and receptor-type tyrosine phosphatases (13, 42). These cell surface proteins respond to molecular guidance cues which can arise locally or diffuse from distant sources. In one such case, control of growth cone physiology is achieved at the most intimate level of cell-to-cell contact, when neuronal cell surface proteins interact with attractive or repulsive cues present on the surface of a neighboring cell.The Eph family of receptor tyrosine kinases and their membrane-associated activating ligands, the ephrins, are recent additions to this growing repertoire of cell surface proteins which mediate cell-to-cell interactions. They are highly expressed in the developing and adult nervous system and have been implicated in regulating cell adhesion events in both neural and nonneural tissues including neural crest cell migration (43, 62, 71), hindbrain segmentation (4, 5, 23,...