DNA−mediated transport of the intermediate filament protein vimentin into the nucleus of cultured cells

A number of characteristic properties of intermediate filament (IF) proteins, such as nucleic acid−binding activity, affinity for histones and structural relatedness to transcription factors and nuclear matrix proteins, in conjunction with the tight association of IFs with the nucleus, suggest that these proteins might also fulfill nuclear functions in addition to their structure−organizing and −stabilizing activities in the cytoplasm. Yet, cytoplasmic IF proteins do not possess nuclear localization signals. In a search for carriers capable of transporting the IF protein vimentin into the nucleus, complexes of FITC−vimentin with various DNAs were microinjected into the cytoplasm of cultured cells and the intracellular distribution of the protein was followed by confocal laser scanning microscopy. The single−stranded oligodeoxyribonucleotides oligo(dG)25, oligo[d(GT)12G] and oligo[d(G3T2A)4G] proved to be excellent nuclear carriers for vimentin. However, in fibroblasts, fluorescence−labeled vimentin taken up by the nuclei remained undetectable with affinity−purified, polyclonal anti−vimentin antibody, whereas it was readily identifiable in the nuclei of microinjected epithelial cells in this way. Moreover, when FITC−vimentin was preinjected into fibroblasts and allowed to assemble into the endogenous vimentin filament system, it was still transferred into the nucleus by post−injected oligo(dG)25, although to a lesser extent. Superhelical circular DNAs, like pBR322, SV40 and mitochondrial DNA, were also characterized by considerable capacities for nuclear vimentin transport; these transport potentials were totally destroyed by relaxation or linearization of the DNA molecules. Nevertheless, certain linear double−stranded DNA molecules with a high affinity for vimentin IFs, such as repetitive telomere and centromere or mobile long interspersed repeat (LINE) DNA, could carry FITC−vimentin into the nucleus. This was also true for a 375 bp extrachromosomal linear DNA fragment which occurs in the cytoplasm of mouse tumor cells and which is capable of immortalizing human lymphocytes. On the basis of these results, it appears very likely that cellular and viral products of reverse transcription as well as other extrachromosomal DNAs, which are circular, superhelical and apparently shuttling between the cytoplasm and the nucleus (eccDNA), are constantly loaded with vimentin in vimentin−positive cells. Since such DNAs are considered as markers of genomic instability, it is conceivable that vimentin directly participates as an architectural, chromatin−modifying protein in recombinatorial processes set off by these DNAs in the nucleu

A novel monoclonal antibody immunoassay for the detection of human serum hepcidin

BACKGROUND: Hepcidin is a liver-derived peptide hormone regulating iron metabolism. Changes in the expression of hepcidin are known to be the key pathogenic factors in hereditary hemochromatosis and are associated with infection and inflammation. To better understand the hormone's function in human disease, we aimed to establish an immunoassay to determine hepcidin concentrations in serum. METHODS: Monoclonal antibodies mHK(8) and mHK(9) were generated and characterized by dot blot, Western blot, and immunofluorescence. A competitive enzyme-linked immunosorbent assay (ELISA) was established with mHK(9). RESULTS: Both antibodies recognized hepcidin, by dot blot and Western blot, respectively. In human liver, mHK(8)/(9) showed an immunofluorescence staining pattern in hepatocytes identical to that of established prohepcidin antibodies. The developed immunoassay with mHK(9), reliably detecting mature hepcidin in serum over a large concentration range (0.9-140 ng ml(-1)), showed high sensitivity and precision (intra-/interassay coefficients of variation: 4-5 and 7-11%; mean linearity: 85-112%; mean recovery: 87-114%). To test the clinical functionality of the developed assay we measured hepcidin serum concentrations in healthy volunteers, hepatitis C virus (HCV) patients, and two groups of hemochromatotic patients undergoing phlebotomy. The assay distinguished low hepcidin level in HCV and homozygous hemochromatosis patients from normal-range controls and compound heterozygous hemochromatosis patients. In healthy subjects and HCV patients, hepcidin levels were correlated with iron and transferrin saturation; no correlation was observed in the hemochromatotic patients. CONCLUSION: We developed a monoclonal antibody ELISA that quantifies serum hepcidin levels with high sensitivity, robustness, and reliability of detection. The hepcidin ELISA should help to enhance our understanding of hepcidin-related human disorders