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Su S.,Harbin Medical University | Liu P.,Harbin Medical University | Zhang H.,Harbin Medical University | Li Z.,Harbin Medical University | And 3 more authors.
Investigative Ophthalmology and Visual Science

Purpose. To identify proteomic differences between age-related nuclear cataracts (ARNCs) and normal lens nuclei. Methods. Total solubilized proteins from ARNC lens nuclei with different grades were compared with normal controls by 2-D differential in-gel electrophoresis (2-D DIGE). Proteins with different abundances were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) analyses determined the compositions of high molecular weight (HMW; >200 kDa) aggregates found in ARNC lens nuclei. Western blot analysis was used to verify the changes in αA-crystallin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels. Results. The 2-D differential in-gel electrophoresis results showed that nine proteins were significantly less abundant in lens nuclei from ARNC patients than in control lens nuclei. Six proteins (αA-, βA3-, βA4-, βB1-, and γD-crystallin and putative uncharacterized protein DKFZp434A0627 from the CRYGS family) tended to decrease as the cataract grade increased, while the other three proteins (αB-crystallin, GAPDH, and retinal dehydrogenase 1) did not show such a tendency. SDS-PAGE showed decreased protein levels at ∼20 kDa in ARNC lenses but significantly increased levels at HMW (>200 kDa). Liquid chromatography tandem mass spectrometry analysis showed that the HMW aggregates derived largely from crystallins also contained filensin, phakinin, and carbonyl reductase 1. Of all the components, αA-crystallin accounted for the highest fraction. αA-, αB-, and γD-crystallin and DKFZp434A0627 were more prone to aggregate than other crystallins. Conclusions. The results show that crystallins, especially αA-crystallin, aggregate irreversibly during ARNC development. Some enzymes (GAPDH, retinal dehydrogenase 1, and carbonyl reductase 1) may be involved in and/or accelerate this process. © 2011 The Association for Research in Vision and Ophthalmology, Inc. Source

Yu X.,Beijing Proteome Research Center | Yu X.,Arizona State University | Petritis B.,Arizona State University | Labaer J.,Arizona State University

Protein microarrays are a high-throughput technology used increasingly in translational research, seeking to apply basic science findings to enhance human health. In addition to assessing protein levels, posttranslational modifications, and signaling pathways in patient samples, protein microarrays have aided in the identification of potential protein biomarkers of disease and infection. In this perspective, the different types of full-length protein microarrays that are used in translational research are reviewed. Specific studies employing these microarrays are presented to highlight their potential in finding solutions to real clinical problems. Finally, the criteria that should be considered when developing next-generation protein microarrays are provided. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Yao X.N.,Beijing Proteome Research Center
Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology

AIM: Preparation of monoclonal antibody (mAb) against GP73 protein. METHODS: The N-terminal peptide (AAAERGAVELK) of GP73 protein was displayed on T7 phage, the recombinant phage was amplified and used as the immunogen to immunize mouse to produce antibody. The titer of the antiserum and the positive hybridoma clones which secreted the mAb against GP73 protein were detected by ELISA. The mAb specificity was assayed by ELISA and Western blot. RESULTS: The high specificity mAb against GP73 protein was selected from the mouse immunized with the recombinant T7 phages displaying the epitope of GP73 by cell fusion and screening. CONCLUSION: The appropriate protein epitope displayed on T7 phage could be used as alternative antigen to immunize animals to make specific antibody against the corresponding native protein. Source

Ding Y.,Zhejiang Chinese Medical University | Lu B.,Zhejiang Chinese Medical University | Chen D.,Academy of Military Medical Science | Meng L.,Zhejiang Chinese Medical University | And 2 more authors.

Irritable bowel syndrome (IBS) is one of the most common functional disorders of the gastrointestinal tract. It is characterized by abdominal pain and changes in bowel habits. Various studies have investigated the pathophysiologic processes underlying IBS, but the mechanism remains poorly understood. In the present study, we established an IBS model and identified differentially expressed proteins in colon tissue of IBS rats compared with healthy controls by 2-D gel electrophoresis, MALDI-TOF-MS, and Western blot analysis. Our results showed that 13 of the 1396 protein spots on 2-D gel were differently expressed between the IBS and control groups. Ontological analysis of these proteins revealed primary roles in catalytic activity (protein disulfide-isomerase A3, glyoxalase I, cathepsin S, α-enolase), structural support (cytokeratin 8), antioxidant activity (peroxiredoxin-6), protein binding (transgelin, serpin peptidase inhibitor B5), and signal transduction (40S ribosomal protein SA). Protein disulfide-isomerase A3 and cytokeratin 8 overexpression in IBS were confirmed by Western blot. The findings indicate that multiple proteins are involved in IBS processes that influence intestinal tract immunity, inflammation, and nerve regulation. Our study provides useful candidate genes and proteins for further investigation. © 2010 WILEY-VCH Verlag GmbH & Co. KGaA. Source

Yan Y.,Hefei University of Technology | Jiang W.,Hefei University of Technology | Liu L.,Hefei University of Technology | Wang X.,Hefei University of Technology | And 5 more authors.

Inflammasomes are involved in diverse inflammatory diseases, so the activation of inflammasomes needs to be tightly controlled to prevent excessive inflammation. However, the endogenous regulatory mechanisms of inflammasome activation are still unclear. Here, we report that the neurotransmitter dopamine (DA) inhibits NLRP3 inflammasome activation via dopamine D1 receptor (DRD1). DRD1 signaling negatively regulates NLRP3 inflammasome via a second messenger cyclic adenosine monophosphate (cAMP), which binds to NLRP3 and promotes its ubiquitination and degradation via the E3 ubiquitin ligase MARCH7. Importantly, in vivo data show that DA and DRD1 signaling prevent NLRP3 inflammasome-dependent inflammation, including neurotoxin-induced neuroinflammation, LPS-induced systemic inflammation, and monosodium urate crystal (MSU)-induced peritoneal inflammation. Taken together, our results reveal an endogenous mechanism of inflammasome regulation and suggest DRD1 as a potential target for the treatment of NLRP3 inflammasome-driven diseases. © 2015 Elsevier Inc. All rights reserved. Source

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