There is currently limited data available pertaining to the global characterization of the cell surface proteome. We have implemented a strategy for the comprehensive profiling and identification of surface membrane proteins. This strategy has been applied to cancer cells, including the SH-SY5Y neuroblastoma, the A549 lung adenocarcinoma, the LoVo colon adenocarcinoma, and the Sup-B15 acute lymphoblastic leukemia (B cell) cell lines and ovarian tumor cells. Surface membrane proteins of viable, intact cells were subjected to biotinylation then affinity-captured and purified on monomeric avidin columns. The biotinylated proteins were eluted from the monomeric avidin columns as intact proteins and were subsequently separated by two-dimensional PAGE, transferred to polyvinylidene difluoride membranes, and visualized by hybridization with streptavidin-horseradish peroxidase. Highly reproducible, but distinct, two-dimensional patterns consisting of several hundred biotinylated proteins were obtained for the different cell populations analyzed. Identification of a subset of biotinylated proteins among the different cell populations analyzed using matrix-assisted laser desorption ionization and tandem mass spectrometry uncovered proteins with a restricted expression pattern in some cell line(s), such as CD87 and the activin receptor type IIB. We also identified more widely expressed proteins, such as CD98, and a sushi repeat-containing protein, a member of the selectin family. Remarkably, a set of proteins identified as chaperone proteins were found to be highly abundant on the cell surface, including GRP78, GRP75, HSP70, HSP60, HSP54, HSP27, and protein disulfide isomerase. Comprehensive profiling of the cell surface proteome provides an effective approach for the identification of commonly occurring proteins as well as proteins with restricted expression patterns in this compartment.

Previously we observed that capsaicin treatment in rats inhibited sensory neuropeptide signaling, with a concurrent reduction in trabecular bone formation and bone volume, and an increase in osteoclast numbers and bone resorption. Calcitonin gene-related peptide (CGRP) is a neuropeptide richly distributed in sensory neurons innervating the skeleton and we postulated that CGRP signaling regulates bone integrity. In this study we examined CGRP effects on stromal and bone cell differentiation and activity in vitro. CGRP receptors were detected by immunocytochemical staining and real time PCR assays in mouse bone marrow stromal cells (BMSCs) and bone marrow macrophages (BMMs). CGRP effects on BMSC proliferation and osteoblastic differentiation were studied using BrdU incorporation, PCR products, alkaline phosphatase (ALP) activity, and mineralization assays. CGRP effects on BMM osteoclastic differentiation and activity were determined by quantifying tartrate-resistant acid phosphatase positive (TRAP + ) multinucleated cells, Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptBone. Author manuscript; available in PMC 2011 May 1. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript pit erosion area, mRNA levels of TRAP and cathepsin K, and nuclear factor-κB (NF-κB) nuclear localization. BMSCs, osteoblasts, BMMs, and osteoclasts all expressed CGRP receptors. CGRP (10 -10 -10 -8 M) stimulated BMSC proliferation, up-regulated the expression of osteoblastic genes, and increased ALP activity and mineralization in the BMSCs. In BMM cultures CGRP (10 -8 M) inhibited receptor activator of NF-κB ligand (RANKL) activation of NF-κB. CGRP also down-regulated osteoclastic genes like TRAP and cathepsin K, decreased the numbers of TRAP+ cells, and inhibited bone resorption activity in RANKL stimulated BMMs. These results suggest that CGRP signaling maintains bone mass both by directly stimulating stromal cell osteoblastic differentiation and by inhibiting RANKL induced NF-κB activation, osteoclastogenesis, and bone resorption.

For patients up to 80 years who suffered mMCAI, DHC within 48 h of stroke onset not only is a life-saving treatment, but also increases the possibility of surviving without severe disability (mRS = 5).

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