Purpose: Various protein contents such as enzymes, growth factors, and structural components are responsible for biological activities in organs. We have created a map of vitreous proteins and developed a proteome analysis of human vitreous samples to understand the underlying molecular mechanism and to provide clues to new therapeutic approaches in eyes with proliferative diabetic retinopathy (PDR). Methods: Vitreous and serum samples were obtained from subjects with idiopathic macular hole (MH, 26 cases) and PDR (33 cases). The expressed proteins in the samples were separated by two-dimensional (2-D) polyacrylamide gel electrophoresis. Protein spots were visualized by silver staining, and their expression patterns were analyzed. Some protein spots of concern were excised from the 2-D gels, digested in situ with trypsin, and analyzed by mass spectrometry. Results: More than 400 spots were detected on 2-D gels of MH cases, of which 78 spots were successfully analyzed. The spots corresponded to peptide fragments of 18 proteins, including pigment epithelium-derived factor, prostaglandin-D2 synthase, and interphotoreceptor retinoidbinding protein. These were not identified in the corresponding serum samples. These proteins were also expressed in PDR samples, with no distinct tendency to increase or decrease compared with the MH samples. More than 600 spots were detected on 2-D gels of PDR cases, of which 141 spots were successfully analyzed. The spots corresponded to peptide fragments of 38 proteins. Enolase and catalase were identified among four detected spots. Neither was found in MH vitreous or in PDR serum samples. Conclusion: A map of protein expression was made in human vitreous from eyes with MH and PDR. In the PDR eyes, the increased protein expression observed was due to barrier dysfunction and/or production in the eye. Proteome analysis was useful in systematic screening of various protein expression in human Hyperglycemia induces many abnormal changes, which are observed as diabetic retinopathy, in vascular and retinal cells in eyes with diabetes mellitus. The breakdown of the bloodretina barrier and new vessel formation are caused by hyperglycemia. In these processes, many hyperglycemia-induced biochemical changes occur, which cause vascular dysfunction. According to previous reports, various factors, including many kinds of proteins, are involved in the pathological processes of diabetic retinopathy. A breakdown of the bloodretina barrier is caused by an intraocular increase of vascular endothelial growth factor (VEGF) 1 (1-7), interleukin-6, angiotensin II, and many other cytokines and/or growth factors. New vessel formation is a very complex multistep process and is regulated by many proteins including cytokines and/or growth factors. In addition to the factors mentioned, basic fibroblast growth factor (bFGF) (8, 9), insulin-like growth factor-1 (4), hepatocyte growth factor (HGF) (6, 7), and others are known to be involved during the destructive processes of endogenous ocular tissue. Changes in the...

Glycerophospholipids, the structural components of cell membranes, have not been considered to be spatial cues for intercellular signaling because of their ubiquitous distribution. We identified lyso-phosphatidyl-β-D-glucoside (LysoPtdGlc), a hydrophilic glycerophospholipid, and demonstrated its role in modality-specific repulsive guidance of spinal cord sensory axons. LysoPtdGlc is locally synthesized and released by radial glia in a patterned spatial distribution to regulate the targeting of nociceptive but not proprioceptive central axon projections. Library screening identified the G protein-coupled receptor GPR55 as a high-affinity receptor for LysoPtdGlc, and GPR55 deletion or LysoPtdGlc loss of function in vivo caused the misallocation of nociceptive axons into proprioceptive zones. These findings show that LysoPtdGlc/GPR55 is a lipid-based signaling system in glia-neuron communication for neural development.

The assembly of progenitor cells is a crucial step for organ formation during vertebrate development. Kupffer's vesicle (KV), a key organ required for the left-right asymmetric body plan in zebrafish, is generated from a cluster of ∼20 dorsal forerunner cells (DFCs). Although several genes are known to be involved in KV formation, how DFC clustering is regulated and how cluster formation then contributes to KV formation remain unclear. Here we show that positive feedback regulation of FGF signaling by Canopy1 (Cnpy1) controls DFC clustering. Cnpy1 positively regulates FGF signals within DFCs, which in turn promote Cadherin1-mediated cell adhesion between adjacent DFCs to sustain cell cluster formation. When this FGF positive feedback loop is disrupted, the DFC cluster fails to form, eventually leading to KV malformation and defects in the establishment of laterality. Our results therefore uncover both a previously unidentified role of FGF signaling during vertebrate organogenesis and a regulatory mechanism underlying cell cluster formation, which is an indispensable step for formation of a functional KV and establishment of the left-right asymmetric body plan.left-right patterning | ciliogenesis F ibroblast growth factor (FGF) signaling plays crucial roles in multiple morphogenetic processes of vertebrate development, including gastrulation movement, mesoderm formation, and leftright (LR) patterning (1-3). Because gain or loss of function of FGF signaling results in morphological changes in the embryo, some mechanism must ensure appropriate FGF signal levels in space and time for proper morphogenesis throughout development. FGF effectors acting as positive or negative regulators show a wide range of expression patterns and activities, contributing to the precise regulation of FGF signal activity (1, 4). Although most effectors identified to date act as negative regulators of FGF signaling, a few that positively regulate FGF activity have been reported (1, 4).We recently identified in zebrafish a positive regulator of FGF signaling named canopy1 (cnpy1), which is required for maintenance of the midbrain-hindbrain boundary (MHB) (5). Expression of cnpy1 was restricted to the MHB at late-somitogenesis stages, whereas cnpy1 was broadly distributed in earlier embryos (5) (SI Appendix, Fig. S1A), suggesting an additional role(s) for Cnpy1-mediated FGF signaling beyond the regulation of MHB formation. In this study, we characterize cnpy1 in detail during early zebrafish development and show that a Cnpy1-mediated positive feedback loop of FGF signaling promotes cell cluster formation between dorsal forerunner cells (DFCs) during gastrulation. We also demonstrate that the failure of DFCs to cluster when this FGF positive loop is disrupted eventually leads to Kupffer's vesicle (KV) malformation and randomization of LR asymmetric patterning. Results Positive Feedback Loop of FGF Signaling Mediated by Cnpy1 IsActivated Specifically in DFCs During Zebrafish Gastrulation. To reveal the role of Cnpy1-mediated FGF signaling in ...

To contribute to physiology and pathophysiology of the glomerulus of human kidney, we have launched a proteomic study of human glomerulus, and compiled a profile of proteins expressed in the glomerulus of normal human kidney by two-dimensional gel electrophoresis (2-DE) and identification with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) and/or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Kidney cortices with normal appearance were obtained from patients under surgical nephrectomy due to renal tumor, and glomeruli were highly purified by a standard sieving method followed by picking-up under a phase-contrast microscope. The glomerular proteins were separated by 2-DE with 24 cm immobilized pH gradient strips in the 3-10 range in the first dimension and 26 x 20 cm sodium dodecyl sulfate polyacrylamide electrophoresis gels of 12.5% in the second dimension. Gels were silver-stained, and valid spots were processed for identification through an integrated robotic system that consisted of a spot picker, an in-gel digester, and a MALDI-TOF MS and / or a LC-MS/MS. From 2-DE gel images of glomeruli of four subjects with no apparent pathologic manifestations, a synthetic gel image of normal glomerular proteins was created. The synthetic gel image contained 1713 valid spots, of which 1559 spots were commonly observed in the respective 2-DE gels. Among the 1559 spots, 347 protein spots, representing 212 proteins, have so far been identified, and used for the construction of an extensible markup language (XML)-based database. The database is deposited on a web site (http://www.sw.nec.co.jp/bio/rd/hgldb/index.html) in a form accessible to researchers to contribute to proteomic studies of human glomerulus in health and disease.

A genome-wide association study identified a strong correlation between body mass index and the presence of a 21-kb copy number variation upstream of the human GPRC5B gene; however, the functional role of GPRC5B in obesity remains unknown. We report that GPRC5B-deficient mice were protected from diet-induced obesity and insulin resistance because of reduced inflammation in their white adipose tissue. GPRC5B is a lipid raft-associated transmembrane protein that contains multiple phosphorylated residues in its carboxyl terminus. Phosphorylation of GPRC5B by the tyrosine kinase Fyn and the subsequent direct interaction with Fyn through the Fyn Src homology 2 (SH2) domain were critical for the initiation and progression of inflammatory signaling in adipose tissue. We demonstrated that a GPRC5B mutant lacking the direct binding site for Fyn failed to activate a positive feedback loop of nuclear factor kB-inhibitor of kB kinase e signaling. These findings suggest that GPRC5B may be a major node in adipose signaling systems linking diet-induced obesity to type 2 diabetes and may open new avenues for therapeutic approaches to diabetic progression.