Cultured pigment epithelial cells of the fetal human retina secrete a protein, pigment epithelium-derived factor (PEDF), that induces a neuronal phenotype in cultured human retinoblastoma cells. Morphological changes include the induction of an extensive neurite meshwork and the establishment of corona-like cellular aggregates surrounding a central lumen. The differentiated cells also show increases in the expression of neuron-specific enolase and the 200-kDa neurofilament subunit. Amino acid and DNA sequence data demonstrate that PEDF belongs to the serine protease inhibitor (serpin) family. The PEDF gene contains a typical signalpeptide sequence, initiator methionine codon, and polyadenylylation signal and matches the size of other members of the serpin superfamily (e.g., a1-antitrypsin). It lacks homology, however, at the putative serpin reactive center. Thus, PEDF could exert a paracrine effect in the embryonic retina, influencing neuronal differentiation by a mechanism that does not involve classic inhibition of serine protease activity.The retinal pigment epithelium (RPE) develops in advance of and lies adjacent to the neural retina. A closed compartment between the two cell layers contains the interphotoreceptor matrix and many soluble secretory products of RPE and neural retinal cells. In the human, the interphotoreceptor matrix is present by 20 weeks of gestation and contains at least one specific marker, the interphotoreceptor retinoidbinding protein (1). Nutrients, metabolites, or trophic factors exchanged between the RPE and neural retina must pass through the interphotoreceptor matrix. RPE cells, for example, are thought to synthesize and secrete a photoreceptor survival-promoting factor, PSPA (2). Cultured RPE cells also synthesize a number of other, well-known trophic factors including PDGF (3), FGF (4), TGF-a (5), and TGF-13 (6). It is thus possible that these or other, unknown factors derived from the RPE could influence neural retina development.We Cell Culture. The Y79 retinoblastoma cell line (American Type Culture Collection) was maintained in suspension culture (7). Cells were harvested by centrifugation for 5 min at 600 x g, washed in prewarmed serum-free medium, centrifuged again, and suspended at 106 cells per ml in serum-free medium with or without HPLC-purified PEDF (50-500 ng/ ml) (9) for a 7-day incubation period prior to attachment to poly(D-lysine)-coated flasks (8). A number of growth factors were tested under similar conditions: NGF at 10-300 ng/ml, PDGF at 1-5 ng/ml, TGF-a at 5 ng/ml, TGF-f3 at 5 ng/ml, FGF at 25-220 ng/ml, and EGF at 25-100 ng/ml (11).Cell Analyses.

SignificanceDuchenne muscular dystrophy (DMD) is a rare and devastating muscle disease caused by mutations in the X-linked DMD gene (which encodes the dystrophin protein). Serum biomarkers hold significant potential as objective phenotypic measures of DMD disease state, as well as potential measures of pharmacological effects of and response to therapeutic interventions. Here we describe a proteomics approach to determine serum levels of 1,125 proteins in 93 DMD patients and 45 controls. The study identified 44 biomarkers that differed significantly between patients and controls. These data are being made available to DMD researchers and clinicians to accelerate the search for new diagnostic, prognostic, and therapeutic approaches.

Antibodies have been the workhorse reagents of protein capture and quantification since their 1959 debut in the RIAs developed by Yalow and Berson. However, there are technical challenges to the use of antibodies in highly multiplexed arrays aimed at measuring hundreds or even thousands of proteins at one time. We describe here a recently developed class of synthetic protein-binding reagents (slow off-rate modified aptamer). We discuss the chemical makeup and protein binding specifications of slow off-rate modified aptamer reagents, compare them to traditional aptamers and antibodies, briefly describe the novel proteomic assay that takes advantage of their unique properties, and provide several examples of their multiple applications to biomarker discovery and validation across a range of biomedical science questions. that the gene sequence was attached to the affinity reagent. At the same time a new molecular recognition element called the aptamer was introduced [7,8]. Aptamers rely on the natural 3D folding of nucleic acids (RNA and then later singlestranded DNA) to generate specific structures that bind to their respective protein targets. These molecules were selected from a large library of molecules using an iterative in vitro selection method, analogous to that for phage display, resulting in identification of the best binder or set of binders. Aptamers took the technology of affinity reagents one step further: the affinity reagent was the nucleic acid sequence. KeywordsAlthough aptamers are generally very good molecular recognition elements, with affinities comparable to high quality antibodies [9], they have been largely ignored in favor of the more established and studied antibody-based industry for several reasons. First, aptamers were relatively new molecules compared to in vivo-derived antibodies and techniques for their use were not as well established. Second, although aptamers to a large number of proteins were rapidly discovered, the generation of high-affinity aptamers across a wide range of proteins was challenging [10

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