A novel cDNA, IA-2p, was isolated from a mouse neonatal brain library. The predicted protein sequence revealed an extracellular domain, a transmembrane region, and an intracellular domain. The intracellular domain is 376 amino acids long and 74% identical to the intracellular domain of IA-2, a major autoantigen in insulin-dependent diabetes mellitus (IDDM). A partial sequence of the extracellular domain of IA-2p8 indicates that it differs substantially (only 26% identical) from that of IA-2. Both molecules are expressed in islets and brain tissue. Forty-six percent (23 of 50) of the IDDM sera but none of the sera from normal controls (0 of 50) immunoprecipitated the intracellular domain of IA-2,3. Competitive inhibition experiments showed that IDDM sera have autoantibodies that recognize both common and distinct determinants on IA-2 and IA-213. Many IDDM sera are known to immunoprecipitate 37-kDa and 40-kDa tryptic fragments from islet cells, but the identity of the precursor protein(s) has remained elusive. The current study shows that treatment of recombinant IA-2p and IA-2 with trypsin yields a 37-kDa fragment and a 40-kDa fragment, respectively, and that these fragments can be immunoprecipitated with diabetic sera. Absorption of diabetic sera with unlabeled recombinant IA-2 or IA-2f8, prior to incubation with radiolabeled 37-kDa and 40-kDa tryptic fragments derived from insulinoma or glucagonoma cells, blocks the immunoprecipitation of both of these radiolabeled tryptic fragments. We conclude that IA-2,8 and IA-2 are the precursors of the 37-kDa and 40-kDa islet cell autoantigens, respectively, and that both IA-2 and IA-2p8 are major autoantigens in IDDM.

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Hexavalent chromium (Cr(VI)) is a widespread environmental contaminant and a known human carcinogen, generally causing bronchial cancer. Recent studies have shown that the particulate forms of Cr(VI) are the potent carcinogens. Particulate Cr(VI) is known to induce a spectrum of DNA damage such as DNA single strand breaks, Cr-DNA adducts, DNA-protein crosslinks and chromosomal aberrations. However, particulate Cr(VI)-induced DNA double strand breaks (DSBs) have not been reported. Thus, the aim of this study was to determine if particulate Cr(VI)-induces DSBs in human bronchial cells. Using the single cell gel electrophoresis assay (comet assay), showed that lead chromate-induced concentration dependent increases in DSBs with 0.1, 0.5, 1 and 5 microg/cm2 lead chromate inducing a 20, 50, 67 and 109% relative increase in the tail integrated intensity ratio, respectively. Sodium chromate at concentrations of 1, 2.5 and 5 microM induced 38, 78 and 107% relative increase in the tail integrated intensity ratio, respectively. We also show that genotoxic concentrations of lead chromate activate the ataxia telangiectasia mutated (ATM) protein, which is thought to play a central role in the early stages of DSB detection and controls cellular responses to this damage. The H2A.X protein becomes rapidly phosphorylated on residue serine 139 in cells when DSBs are introduced into the DNA by ionizing radiation. By using immunofluorescence, we found that lead chromate-induced concentration-dependent increases in phosphorylated H2A.X (r-H2A.X) foci formation with 0.1, 0.5, 1, 5 and 10 microg/cm2 lead chromate inducing a relative increase in the number of cells with r-H2A.X foci formation of 43, 51, 115 and 129%, respectively.

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Hexavalent chromium [Cr(VI)] is a well-established human lung carcinogen. Water solubility has proven to be a key factor in the carcinogenicity of Cr(VI), with the water insoluble or "particulate" compounds the more potent carcinogens. Pathology studies indicate that chromates target cells at bronchial bifurcation sites in human lungs. However, it is uncertain what roles particle internalization and dissolution play in the genotoxicity of these compounds to human lung cells. We investigated these mechanisms in a human lung cell line after exposure to particulate lead chromate. We found that lead chromate was clastogenic in a concentration-dependent manner with 0.1, 0.5, and 1 microg/cm(2), while 5 and 10 microg/cm(2) caused complete cell cycle arrest. We also found concentration-dependent increases in intracellular and extracellular chromium ion levels. We investigated particle internalization by using transmission electron microscopy and found an apparent relative increase with concentration but no apparent particle internalization at the lowest concentration (0.1 microg/cm(2)) even after 24 h. Furthermore, we found no lysosomal association with the vacuoles containing particles, further suggesting that intracellular dissolution did not occur. Cotreating the cells with lead chromate and vitamin C eliminated both the uptake of ionic chromium and the clastogenic activity of lead chromate but had no effect on particle internalization. These data indicate that in human bronchial cells lead chromate clastogenesis is mediated by the extracellular dissolution of the particles and not their internalization. These findings have important implications for our understanding of the physicochemical mechanism of particulate chromates as they contradict previous indirect data from human bronchial epithelial cells, which suggest that particles dissolve inside those cells. Thus, these new data suggest that there may be different mechanisms of genotoxicity for epithelial cells and fibroblasts exposed to chromate particles.

In this study, a series of di-O-caffeoylquinic acids (di-COQs) were systematically investigated for their antioxidant and cytoprotective effects towards •OH-damaged bone marrow-derived mesenchymal stem cells (bmMSCs). Five di-COQs were measured using a set of antioxidant assays. The results show that adjacent 4,5-Di-O-caffeoylquinic acid (4,5-COQ) and 3,4-di-O-caffeoylquinic acid (3,4-COQ) always gave lower IC50 values than did non-adjacent di-COQs. In the Fe2+-chelating assay, 4,5-COQ and 3,4-COQ presented greater UV-Vis spectra and darker colors than did non-adjacent di-COQs. In the UPLC-ESI-MS/MS analysis, no corresponding radical adduct formation (RAF) peak was found in the reaction products of di-COQs with PTIO•. In the MTT assay, all di-COQs (especially 1,5-COQ, 1,3-COQ, and 4,5-COQ) dose-dependently increased the cellular viabilities of •OH-damaged bmMSCs. Based on this evidence, we conclude that the five antioxidant di-COQs can protect bmMSCs from •OH-induced damage. Their antioxidant mechanisms may include electron-transfer (ET), H+-transfer, and Fe2+-chelating, except for RAF. Two adjacent di-COQs (4,5-COQ and 3,4-COQ) always possessed a higher antioxidant ability than the non-adjacent di-COQs (1,3-COQ, 1,5-COQ, and 3,5-COQ) in chemical models. However, non-adjacent 1,3-COQ and 1,5-COQ exhibited a higher cytoprotective effect than did adjacent di-COQs. These differences can be attributed to the relative positions of two caffeoyl moieties and, ultimately, to the conformational effect from the cyclohexane skeleton.

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