Characterizing the normal proteome of human ciliary body

BACKGROUND: The ciliary body is the circumferential muscular tissue located just behind the iris in the anterior chamber of the eye. It plays a pivotal role in the production of aqueous humor, maintenance of the lens zonules and accommodation by changing the shape of the crystalline lens. The ciliary body is the major target of drugs against glaucoma as its inhibition leads to a drop in intraocular pressure. A molecular study of the ciliary body could provide a better understanding about the pathophysiological processes that occur in glaucoma. Thus far, no large-scale proteomic investigation has been reported for the human ciliary body. RESULTS: In this study, we have carried out an in-depth LC-MS/MS-based proteomic analysis of normal human ciliary body and have identified 2,815 proteins. We identified a number of proteins that were previously not described in the ciliary body including importin 5 (IPO5), atlastin-2 (ATL2), B-cell receptor associated protein 29 (BCAP29), basigin (BSG), calpain-1 (CAPN1), copine 6 (CPNE6), fibulin 1 (FBLN1) and galectin 1 (LGALS1). We compared the plasma proteome with the ciliary body proteome and found that the large majority of proteins in the ciliary body were also detectable in the plasma while 896 proteins were unique to the ciliary body. We also classified proteins using pathway enrichment analysis and found most of proteins associated with ubiquitin pathway, EIF2 signaling, glycolysis and gluconeogenesis. CONCLUSIONS: More than 95% of the identified proteins have not been previously described in the ciliary body proteome. This is the largest catalogue of proteins reported thus far in the ciliary body that should provide new insights into our understanding of the factors involved in maintaining the secretion of aqueous humor. The identification of these proteins will aid in understanding various eye diseases of the anterior segment such as glaucoma and presbyopia

A Bioinformatics Resource for TWEAK-Fn14 Signaling Pathway

TNF-related weak inducer of apoptosis (TWEAK) is a new member of the TNF superfamily. It signals through TNFRSF12A, commonly known as Fn14. The TWEAK-Fn14 interaction regulates cellular activities including proliferation, migration, differentiation, apoptosis, angiogenesis, tissue remodeling and inflammation. Although TWEAK has been reported to be associated with autoimmune diseases, cancers, stroke, and kidney-related disorders, the downstream molecular events of TWEAK-Fn14 signaling are yet not available in any signaling pathway repository. In this paper, we manually compiled from the literature, in particular those reported in human systems, the downstream reactions stimulated by TWEAK-Fn14 interactions. Our manual amassment of the TWEAK-Fn14 pathway has resulted in cataloging of 46 proteins involved in various biochemical reactions and TWEAK-Fn14 induced expression of 28 genes. We have enabled the availability of data in various standard exchange formats from NetPath, a repository for signaling pathways. We believe that this composite molecular interaction pathway will enable identification of new signaling components in TWEAK signaling pathway. This in turn may lead to the identification of potential therapeutic targets in TWEAK-associated disorders

5. 慢性円板状エリテマトーデスに続発せる棘細胞癌の1例について(第434(B)回千葉医学会例会 第15回千葉皮膚科臨床談話会)

Additional file 3: Table S3. Details of proteins identified in the study with reports on whether or not reported in PPD and gene ontology-based classification

Berberine Chloride Mediates Its Anti-Leishmanial Activity via Differential Regulation of the Mitogen Activated Protein Kinase Pathway in Macrophages

BACKGROUND: A complex interplay between Leishmania and macrophages influences parasite survival and necessitates disruption of signaling molecules, eventually resulting in impairment of macrophage function. In this study, we demonstrate the immunomodulatory activity of Berberine chloride in Leishmania infected macrophages. PRINCIPAL FINDINGS: The IC(50) of Berberine chloride, a quaternary isoquinoline alkaloid was tested in an amastigote macrophage model and its safety index measured by a cell viability assay. It eliminated intracellular amastigotes, the IC(50) being 2.8 fold lower than its IC(50) in promastigotes (7.10 µM vs. 2.54 µM) and showed a safety index >16. Levels of intracellular and extracellular nitric oxide (NO) as measured by flow cytometry and Griess assay respectively showed that Berberine chloride in Leishmania infected macrophages increased production of NO. Measurement of the mRNA expression of iNOS, IL-12 and IL-10 by RT-PCR along with levels of IL-12p40 and IL-10 by ELISA showed that in infected macrophages, Berberine chloride enhanced expression of iNOS and IL-12p40, concomitant with a downregulation of IL-10. The phosphorylation status of extracellular signal related kinase (ERK1/2) and p38 mitogen activated protein kinase (p38 MAPK) was studied by western blotting. In infected macrophages, Berberine chloride caused a time dependent activation of p38 MAPK along with deactivation of ERK1/2; addition of a p38 MAPK inhibitor SB203580 inhibited the increased generation of NO and IL-12p40 by Berberine chloride as also prevented its decrease of IL-10. CONCLUSIONS: Berberine chloride modulated macrophage effector responses via the mitogen activated protein kinase (MAPK) pathway, highlighting the importance of MAPKs as an antiparasite target

Effect of Berberine chloride on MAPK pathway in macrophages.

<p><b>A:</b> A representative profile of uninfected macrophages was treated with Berberine chloride (10 µM) for 30 min-6 h. The cells were lysed and subjected to Western blotting with anti-pERK1/2 (a), anti-pp38 MAPK (b) and anti-ERK1/2 (c) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. <b>B:</b> A representative profile of <i>Leishmania</i> infected macrophages were treated with Berberine chloride (10 µM) for 30 min-6 h. The cells were lysed and subjected to western blotting with anti-pERK1/2 (a), anti-pp38 MAPK (b) and anti-ERK1/2 (c) as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>.</p

Effect of Berberine chloride on generation of NO and expression of iNOS.

<p><b>A:</b> A representative dot plot of uninfected (a) and <i>Leishmania</i> infected (d) murine peritoneal macrophages, that were treated with Berberine chloride (10 µM, 48 h, b, e). Cells were gated on the basis of characteristic linear forward and side scatter features of macrophages and subsequently DAF-2T fluorescence was measured on a logarithmic scale in the FL1 channel. A representative histogram of uninfected macrophages (c, ) and <i>L. donovani</i> infected macrophages (f, ) for DAF-2T that were treated with Berberine chloride (…) macrophages as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. <b>B:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 24 h with Berberine chloride 2.5 µM (b) and 10 µM (c), and processed for measurement of DAF-2T fluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. <b>C:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 48 h with Berberine chloride 2.5 µM (b) and 10 µM (c) and processed for measurement of DAF-2T fluorescence as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Data are expressed as the mean GMFC ± SEM of at least 3 experiments in duplicate. <b>D:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 24 h with Berberine chloride 2.5 µM (b) and 10 µM (c) and assayed for levels of extracellular NO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point represents the mean ± SEM of NO₂⁻ (µM) of at least 3 experiments in duplicate. <b>E:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated for 48 h with Berberine chloride 2.5 µM (b) and 10 µM (c) and assayed for levels of extracellular NO as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point represents the mean ± SEM of NO₂⁻ (µM) of at least 3 experiments in duplicate. <b>F:</b> Uninfected macrophages (a) and <i>L. donovani</i> infected macrophages (d) were treated for 18 h with Berberine chloride 2.5 µM (b, e) or 10 µM (c, f). RNA was isolated and subjected to RT-PCR and the products of β-actin and iNOS mRNA were resolved on an agarose gel (1.5%) and quantified densitometrically using Total lab software as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>.</p

Effect of Berberine chloride on IL-12p40 in macrophages.

<p><b>A:</b> Uninfected (a) and <i>L. donovani</i> infected (d) macrophages were treated for 18 h with Berberine chloride 2.5 µM (b, e) or 10 µM (c, f). RNA was isolated, subjected to RT-PCR and the products of β-actin and IL-12 p40 mRNA were resolved on an agarose gel (1.5%) and quantified densitometrically using Total lab software as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. <b>B:</b> Uninfected macrophages (1×10⁶/ml, □, a) or <i>L. donovani</i> infected macrophages (▪, a) were treated with Berberine chloride 2.5 µM (b) and 10 µM (c) for 24 h and assayed for levels of IL-12p40 in culture supernatants by ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018467#s2" target="_blank">Methods</a>. Each point represents the mean ± SEM of IL-12p40 (pg/ml) of at least 3 experiments in duplicate.</p