ABSTRACT
BACKGROUND: Testing for 25-hydroxyvitamin D (25(OH)D) has increased dramatically over the past decade and several automated immunoassays exist to measure serum 25(OH)D. Here we assess the performance of the recently released automated Bio-Rad BioPlex® 2200 25-OH vitamin D immunoassay, claimed to equally detect 25(OH)D2 and 25(OH)D3, and compare its results against a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method and the well-established DiaSorin LIAISON® 25-OH vitamin D total immunoassay. METHODS: Imprecision was determined using third party controls over 20days. Linearity over the claimed measuring range was assessed using admixtures of a high and a low patient pool. Correlation between the BioPlex and LC-MS/MS (n=137) or the LIAISON (n=56) was assessed using patient samples with varying amounts of 25(OH)D3 and/or 25(OH)D2. RESULTS: The total imprecision was 9.4%, 6.9% and 4.5% at concentrations of 39.4nmol/L, 70.6nmol/L and 242.8nmol/L, respectively. The assay was linear from 33.1-375.0nmol/L with a R(2) of 0.993. Method comparison revealed a strong correlation between the BioPlex assay and LC-MS/MS for samples containing 25(OH)D2 alone (n=5; R(2)=0.999), 25(OH)D3 alone (n=119; R(2)=0.935) and both (n=13; R(2)=0.919). In samples tested by all three methods (n=56), the correlation between the BioPlex and the LIAISON (R(2)=0.853) was poorer than that of the BioPlex and LC-MS/MS (R(2)=0.942). CONCLUSION: The BioPlex assay is suitable for the measurement of total serum 25(OH)D. The strong correlation between the BioPlex assay and LC-MS/MS in detecting 25(OH)D2 and 25(OH)D3 provides evidence that the BioPlex assay is capable of the equivalent detection of both forms.
Subject(s)
25-Hydroxyvitamin D 2/blood , Biomarkers/blood , Calcifediol/blood , Chromatography, Liquid/methods , Immunoassay/methods , Polymers/chemistry , Tandem Mass Spectrometry/methods , Humans , Reproducibility of ResultsABSTRACT
Mutations in p73 are rare in cancer. Emerging evidence suggests that the relative expression of various p73 isoforms may contribute to tumorigenesis. Alternative promoters and N-terminal splicing result in the transcription and processing of either full-length (TA) or N-terminally truncated (deltaN) p73 isoforms. TAp73 possesses pro-apoptotic functions, while deltaNp73 has anti-apoptotic properties via functional inhibition of TAp73 and p53. Here, we report that TAp73, but not deltaNp73, is covalently modified by NEDD8 under physiologic conditions in an Mdm2-dependent manner. Co-expression of NEDP1, a cysteine protease that specifically cleaves NEDD8 conjugates, was shown to deneddylate TAp73. In addition, blockage of the endogenous NEDD8 pathway increased TAp73-mediated transactivation of p53- and p73-responsive promoter-driven reporter activity, and in conjunction, neddylated TAp73 species were found preferentially in the cytoplasm. These results suggest that Mdm2 attenuates TAp73 transactivation function, at least in part, by promoting NEDD8-dependent TAp73 cytoplasmic localization and provide the first evidence of a covalent post-translational modification exclusively targeting the TA isoforms of p73.
Subject(s)
DNA-Binding Proteins/metabolism , Gene Expression Regulation , Nuclear Proteins/metabolism , Proto-Oncogene Proteins c-mdm2/metabolism , Transcriptional Activation , Tumor Suppressor Proteins/metabolism , Ubiquitins/metabolism , Cells, Cultured , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , Endopeptidases/metabolism , Genes, Dominant , Humans , Immunoblotting , Immunoprecipitation , Kidney/metabolism , Kidney/pathology , Luciferases/metabolism , NEDD8 Protein , Nuclear Proteins/genetics , Osteosarcoma/genetics , Osteosarcoma/metabolism , Osteosarcoma/pathology , Plasmids , Promoter Regions, Genetic/genetics , Protein Processing, Post-Translational , Proto-Oncogene Proteins c-mdm2/genetics , Subcellular Fractions , Transcription, Genetic , Tumor Protein p73 , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Tumor Suppressor Proteins/genetics , Ubiquitins/geneticsABSTRACT
The evolutionarily conserved proteins Par-6, atypical protein kinase C (aPKC), Cdc42 and Par-3 associate to regulate cell polarity and asymmetric cell division, but the downstream targets of this complex are largely unknown. Here we identify direct physiological interactions between mammalian aPKC, murine Par-6C (mPar-6C) and Mlgl, the mammalian orthologue of the Drosophila melanogaster tumour suppressor Lethal (2) giant larvae. In cultured cell lines and in mouse brain, aPKC, mPar-6C and Mlgl form a multiprotein complex in which Mlgl is targeted for phosphorylation on conserved serine residues. These phosphorylation sites are important for embryonic fibroblasts to polarize correctly in response to wounding and may regulate the ability of Mlgl to direct protein trafficking. Our data provide a direct physical and regulatory link between proteins of distinct polarity complexes, identify Mlgl as a functional substrate for aPKC in cell polarization and indicate that aPKC is directed to cell polarity substrates through a network of protein-protein interactions.
