ABSTRACT
The aqueous humor is a colorless, transparent fluid that fills the anterior chamber of the eye. It plays an important role in maintaining the intraocular pressure and providing nourishment to the lens and cornea. The constitution of the aqueous humor is controlled by the blood-aqueous barrier. Though this ocular fluid has been extensively studied, its role in ocular physiology is still not completely understood. In this study, aqueous humor samples were collected from 250 patients undergoing cataract surgery, subjected to multiple fractionation strategies and analyzed on a Fourier transform LTQ-Orbitrap Velos mass spectrometer. In all, we identified 763 proteins, of which 386 have been identified for the first time in this study. Sorbitol dehydrogenase (SORD), filensin (BFSP1), and phakinin (BFSP2) are some of the proteins that have not been previously reported in the aqueous humor. Gene Ontology analysis revealed 35% of the identified proteins to be extracellular, with a majority of them involved in cell communication and signal transduction. This study comprehensively reports 386 novel proteins that have important potential as biomarker candidates for future research into personalized medicine and diagnostics aimed towards improving visual health.
Subject(s)
Aqueous Humor/chemistry , Proteomics/methods , Chromatography, Liquid , Eye Proteins/analysis , Humans , Intermediate Filament Proteins/analysis , L-Iditol 2-Dehydrogenase/analysis , Tandem Mass SpectrometryABSTRACT
The PIK3CA gene is frequently mutated in human cancers. Here we carry out a SILAC-based quantitative phosphoproteomic analysis using isogenic knockin cell lines containing 'driver' oncogenic mutations of PIK3CA to dissect the signalling mechanisms responsible for oncogenic phenotypes induced by mutant PIK3CA. From 8,075 unique phosphopeptides identified, we observe that aberrant activation of PI3K pathway leads to increased phosphorylation of a surprisingly wide variety of kinases and downstream signalling networks. Here, by integrating phosphoproteomic data with human protein microarray-based AKT1 kinase assays, we discover and validate six novel AKT1 substrates, including cortactin. Through mutagenesis studies, we demonstrate that phosphorylation of cortactin by AKT1 is important for mutant PI3K-enhanced cell migration and invasion. Our study describes a quantitative and global approach for identifying mutation-specific signalling events and for discovering novel signalling molecules as readouts of pathway activation or potential therapeutic targets.
Subject(s)
Cortactin/metabolism , Phosphatidylinositol 3-Kinases/genetics , Phosphoproteins/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology , Chromatography, Liquid , Class I Phosphatidylinositol 3-Kinases , DNA Primers/genetics , Fluorescent Antibody Technique , Gene Knock-In Techniques , Humans , Immunoblotting , Immunoprecipitation , Mutagenesis, Site-Directed , Mutation/genetics , Proteomics , RNA, Small Interfering/genetics , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction/genetics , Tandem Mass SpectrometryABSTRACT
OBJECTIVES: To develop a kinetic, inexpensive, automatable assay for ceruloplasmin as ferroxidase. DESIGN AND METHODS: Dithiothreitol has been used to stabilize the substrate and quinolones to complex the ferric. RESULTS: Mean ferroxidase activity in healthy adults, Wilson's disease and pregnancy was 787.29, 178.5 and 1828.09 IU/L, respectively. Correlation coefficient of ferroxidase vis-à-vis copper, ferroxidase by ferrozine and ceruloplasmin were 0.90, 0.94 and 0.92, respectively. CONCLUSIONS: Norfloxacin method is a simple, inexpensive and automatable kinetic assay.
Subject(s)
Ceruloplasmin/analysis , Ceruloplasmin/metabolism , Clinical Chemistry Tests/methods , Adult , Clinical Chemistry Tests/economics , Dithiothreitol/chemistry , Female , Ferrozine/chemistry , Hepatolenticular Degeneration/enzymology , Humans , Hydrogen-Ion Concentration , Kinetics , Male , Norfloxacin/chemistry , Pregnancy , Quinolones/chemistry , Reproducibility of ResultsABSTRACT
Serum ferroxidase and albumin levels were determined in 98 patients of tubercuiosis, of whom 49 were freshly diagnosed, sputum positive (group-I) & 49 were completely treated patients (group-II). Forty nine age and sex matched healthy individuals were taken as controls. Mean±SD of serum ferroxidase and albumin levels in controls, group-I and group-II was found to be 864.35±106.35 IU/L & 3.91±0.234 g/dL, 1603.76±222.65 IU/L & 3.24±0.518 g/dL and 1001.78±201.63 IU/L & 3.82±0.43 g/dL, respectively. Serum ferroxidase in group I was significantly higher as compared to controls and group-II (p<0.01). The decreased levels of serum albumin in group I, as compared to control and group-II was statistically significant (p<0.01). Serum ferroxidase: albumin ratio (Ferroxidase in International Unit per gram of albumin) in group I (50.47±10.36 IU/g) was significantly higher than controls (22.22±3.3 IU/g), (p<0.001) while in group II it was significantly lower (26.72±7.18 IU/g, p<0.001) than group-I and close to control values. Serum ferroxidase: albumin ratio (IU/g) can therefore be incorporated as a surrogate marker to assist in diagnosis and prognosis of pulmonary tuberculosis.
