Receptor tyrosine kinase inhibitor profiling using bead-based multiplex sandwich immunoassays.

Receptor tyrosine kinases (RTK) are important targets in drug discovery processes. Studying the phosphorylation pattern of RTKs enables the determination of their activation and inactivation states. Multiplex bead-based sandwich immunoassays are powerful tools for measuring the phosphorylation state of key regulators within cellular signalling networks. Here, we describe the analysis of the phosphorylation state of receptor tyrosine kinases using the epidermal growth factor receptor (EGFR) as an example. We provide a protocol for a bead-based sandwich immunoassay that enables a relative quantification of the EGFR and its generic tyrosine phosphorylation. We also present data from a kinase inhibitor experiment using 96-well cell-culture plates and a commercially available kit for the analysis of seven receptor tyrosine kinases.

Evaluation of a urinary kidney biomarker panel in rat models of acute and subchronic nephrotoxicity.

Several novel urinary kidney biomarkers were recently approved by the US-FDA and EMA for improved detection of nephrotoxicity, but few data regarding their performance are publicly available so far. In this study, we investigated the potential of some of the newly accepted makers (Kim-1, ß-2-microglobulin, cystatin C, clusterin) along with six additional urinary key proteins of kidney injury (GST-α, Timp-1, VEGF, calbindin, NGAL/lipocalin-2, osteopontin) to detect proximal tubule damage in the rat model studying either acute drug-induced kidney injury or subchronic nephrotoxicity. Candidate proteins were measured in urine samples obtained from rats treated with gentamicin (0, 60 and 120 mg/kg bw for 7 days), BI-3 [3-pyrrolidineacetic acid, 5-[[[4'-[imino[(methoxycarbonyl) amino]methyl] [1,1'-biphenyl]-4-yl]oxy]methyl]-2-oxo-, methyl ester,(3S-trans)] (0, 100, and 1000 mg/kg bw for up to 14 days) or with the mycotoxin ochratoxin A (OTA) (0, 21, 70 and 210 µg/kg bw for up to 90 days) using a Luminex(®) xMAP(®) platform. Cystatin C and NGAL appeared to be the most sensitive indicators of gentamicin nephrotoxicity, with significant changes occurring as early as day 1, and importantly before alterations in serum creatinine or blood urea nitrogen (BUN). Altered urinary excretion of KIM-1, clusterin, calbindin and Timp-1 accompanied by a rise in BUN was observed in rats with BI-3 at 1000 mg/kg bw for 14 days. In contrast, histopathological alterations induced by OTA, which preceded effects on traditional clinical parameters, were best reflected by changes in urinary Kim-1. Overall, our data confirm increased sensitivity of new markers as compared to traditional clinical chemistry parameters.

Sequential multiplex analyte capturing for phosphoprotein profiling.

Microarray-based sandwich immunoassays can simultaneously detect dozens of proteins. However, their use in quantifying large numbers of proteins is hampered by cross-reactivity and incompatibilities caused by the immunoassays themselves. Sequential multiplex analyte capturing addresses these problems by repeatedly probing the same sample with different sets of antibody-coated, magnetic suspension bead arrays. As a miniaturized immunoassay format, suspension bead array-based assays fulfill the criteria of the ambient analyte theory, and our experiments reveal that the analyte concentrations are not significantly changed. The value of sequential multiplex analyte capturing was demonstrated by probing tumor cell line lysates for the abundance of seven different receptor tyrosine kinases and their degree of phosphorylation and by measuring the complex phosphorylation pattern of the epidermal growth factor receptor in the same sample from the same cavity.

Fully functional, naturally occurring and C-terminally truncated variant human immunodeficiency virus (HIV) Vif does not bind to HIV Gag but influences intermediate filament structure.

A variant human immunodeficiency virus type 1 (HIV-1) vif gene, vifA45-2, which encodes a protein lacking 19 amino acids at the C terminus but which is fully functional in supporting HIV replication in non-permissive cells has been described previously. By employing newly generated anti-VifA45 serum, further properties of VifA45 and its full-length counterpart, VifA45open, in comparison to Vif from HIV strain BH10 are reported in permissive HeLa and COS-7 cells. The results obtained using confocal microscopic localization studies and in vitro binding assays do not support a requirement for the direct interaction of HIV Gag with Vif. Furthermore and in contrast to previous conclusions, detergent solubility analyses do not demonstrate a role for the C terminus of Vif in mediating localization to the fraction containing cellular membrane proteins. Localization of Vif from HIV strain BH10 to perinuclear aggregates in a small fraction (about 10%) of transfected HeLa cells has been previously reported. The intermediate filament protein vimentin colocalizes to these structures. In contrast, VifA45 and VifA45open form perinuclear aggregates in nearly all transfected HeLa cells; vimentin as well as the cytoskeletal-bridging protein plectin, but not the microtubular protein tubulin, become relocalized to these structures. Interestingly, in COS-7 cells, all of the functional Vif proteins tested (Vif from strain BH10, VifA45 and VifA45open) predominantly localize in the cytoplasm but still induce dramatic aggregation of vimentin and plectin, i.e. in these cells the respective Vif proteins are influencing intermediate filament structure in the absence of colocalization.