Development of a novel immunoassay specific for mouse intact proinsulin.

The blood concentration of intact proinsulin, but not total proinsulin, has been suggested to be a diagnostic marker for type 2 diabetes mellitus (T2DM), but a sensitive assay specific for rodent intact proinsulin is lacking. Here, a novel enzyme-linked immunosorbent assay (ELISA) for mouse intact proinsulin was developed. The developed ELISA detected mouse intact proinsulin with the working range of 8.3 to 2700pg/ml. Cross-reactivity with mouse split-32,33 proinsulin was approximately 100times lower than the reactivity with mouse intact proinsulin, and no cross-reactivity with mouse insulin was detected. The developed ELISA was sufficiently sensitive to detect low levels of intact proinsulin in normal mouse plasma. The measurement by the developed ELISA revealed that intact proinsulin was elevated in the plasma of type 2 diabetic db/db mice as mice aged, and the ratio of intact proinsulin/insulin in plasma was correlated with levels of glycated hemoglobin A1c as seen in T2DM patients. These results suggest that the plasma level of intact proinsulin, but not total proinsulin, is a sensitive marker for pancreatic dysfunction and the ensuring diabetic disease progression of db/db mice. This ELISA could aid nonclinical evaluation of therapeutic interventions in T2DM.

Optimization of enrichment conditions on TiO2 chromatography using glycerol as an additive reagent for effective phosphoproteomic analysis.

Metal oxide affinity chromatography (MOAC) represented by titanium dioxide (TiO2) chromatography has been used for phosphopeptide enrichment from cell lysate digests prior to mass spectrometry. For in-depth phosphoproteomic analysis, it is important for MOAC to achieve high phosphopeptide enrichment efficiency by optimizing purification conditions. However, there are some differences in phosphopeptide selectivity and specificity enriched by various TiO2 materials and procedures. Here, we report that binding/wash buffers containing polyhydric alcohols, such as glycerol, markedly improve phosphopeptide selectivity from complex peptide mixtures. In addition, the elution conditions combined with secondary amines, such as bis-Tris propane, made it possible to recover phosphopeptides with highly hydrophobic properties and/or longer peptide lengths. To assess the practical applicability of our improved method, we confirmed using PC3 prostate cancer cells. By combining the hydrophilic interaction chromatography (HILIC) with the optimized TiO2 enrichment method prior to LC-MS/MS analysis, over 8300 phosphorylation sites and 2600 phosphoproteins were identified. Additionally, some dephosphorylations of those were identified by treatment with dasatinib for a kinase inhibitor. These results indicate that our method is applicable to understanding the profiling of kinase inhibitors such as anticancer compounds, which will be useful for drug discovery and development.

Gata3/Ruvbl2 complex regulates T helper 2 cell proliferation via repression of Cdkn2c expression.

GATA-binding protein 3 (Gata3) controls the differentiation of naive CD4 T cells into T helper 2 (Th2) cells by induction of chromatin remodeling of the Th2 cytokine gene loci, direct transactivation of Il5 and Il13 genes, and inhibition of Ifng. Gata3 also facilitates Th2 cell proliferation via additional mechanisms that are far less well understood. We herein found that Gata3 associates with RuvB-like protein 2 (Ruvbl2) and represses the expression of a CDK inhibitor, cyclin-dependent kinase inhibitor 2c (Cdkn2c) to facilitate the proliferation of Th2 cells. Gata3 directly bound to the Cdkn2c locus in an Ruvbl2-dependent manner. The defect in the proliferation of Gata3-deficient Th2 cells is rescued by the knockdown of Cdkn2c, indicating that Cdkn2c is a key molecule involved in the Gata3-mediated induction of Th2 cell proliferation. Ruvbl2-knockdown Th2 cells showed decreased antigen-induced expansion and caused less airway inflammation in vivo. We therefore have identified a functional Gata3/Ruvbl2 complex that regulates the proliferation of differentiating Th2 cells through the repression of a CDK inhibitor, Cdkn2c.

Potential plasma biomarkers for progression of knee osteoarthritis using glycoproteomic analysis coupled with a 2D-LC-MALDI system.

BACKGROUND: Although osteoarthritis (OA) is a highly prevalent joint disease, to date, no reliable biomarkers have been found for the disease. In this study, we attempted to identify factors the amounts of which significantly change in association with the progression of knee OA. METHODS: A total of 68 subjects with primary knee OA were enrolled in the study. These subjects were followed up over an 18-month period, and plasma and serum samples were obtained together with knee radiographs every 6 months, i.e., 0, 6, 12 and 18 months after the enrollment. Progressors and non-progressors were determined from the changes on radiographs, and plasma samples from those subjects were subjected to N-glycoproteomic 2D-LC-MALDI analysis. MS peaks were identified, and intensities for respective peaks were compared between the progressors and non-progressors to find the peak intensities of which differed significantly between the two groups of subjects. Proteins represented by the chosen peaks were identified by MS/MS analysis. Expression of the identified proteins was evaluated in synovial tissues from 10 OA knee joints by in situ hybridization, western blotting analysis and ELISA. RESULTS: Among the subjects involved in the study, 3 subjects were determined to be progressors, and 6 plasma and serum samples from these subjects were subjected to the analysis together with another 6 samples from the non-progressors. More than 3000 MS peaks were identified by N-glycoproteomic 2D-LC-MALDI analysis. Among them, 4 peaks were found to have significantly different peak intensities between the progressors and non-progressors. MS/MS analysis revealed that these peaks represented clusterin, hemopexin, alpha-1 acid glycoprotein-2, and macrophage stimulating protein, respectively. The expression of these genes in OA synovium was confirmed by in situ hybridization, and for clusterin and hemopexin, by western blotting analysis and ELISA as well. CONCLUSIONS: In this study, 4 potential biomarkers were identified as potential prognostic markers for knee OA through N-glycoproteomic analysis. To the best of our knowledge, this is the first report for the use of glycoproteomic technology in exploring potential biomarkers for knee OA.