Alterations in manganese, copper, and zinc contents, and intracellular status of the metal-containing superoxide dismutase in human mesothelioma cells

BACKGROUND AND AIM: Molecular diagnostics and therapeutics of human mesothelioma using disease-related markers present major challenges in clinical practice. To identify biochemical alternations that would be markers of human mesothelioma, we measured the intracellular steady-state levels of biologically important trace metals such as manganese (Mn), copper (Cu), and zinc (Zn) in a human mesothelial cell line, MeT-5A, and in five human mesothelioma cell lines (MSTO-211H, NCI-H226, NCI-H2052, NCI-H2452, ACC-MESO-1) by inductively coupled plasma-mass spectrometry (ICP-MS). We also aimed to investigate whether the alterations were related to the intracellular status of metal-containing superoxide dismutase (SOD). RESULTS: There were no significant differences in the contents of the trace metals among MeT-5A, MSTO-211H, and ACC-MESO-1 cells. However, each of the other three mesothelioma cell lines had a unique characteristic in terms of the intracellular amounts of the metals; NCI-H226 contained an extremely high level of Mn, an amount 7.3-fold higher than that in MeT-5A. NCI-H2052 had significantly higher amounts of Cu (3.4-fold) and Zn (1.3-fold) compared with MeT-5A. NCI-H2452 contained about 5.8-fold the amount of Cu and 2.5-fold that of Mn compared with MeT-5A. As for the intracellular levels of copper/zinc-SOD (Cu/Zn-SOD) and manganese-SOD (Mn-SOD), those of Cu/Zn-SOD were relatively unchanged among the cells tested, and no notable correlation with Cu or Zn contents was observed. On the other hand, all mesothelioma cells highly expressed Mn-SOD compared with MeT-5A, and a very high expression of the enzyme with a robust activity was observed in the two mesothelioma cells (NCI-H226, NCI-H2452) containing a large amount of Mn. CONCLUSIONS: In comparison with MeT-5A human mesothelial cells, some human mesothelioma cells had significantly higher amounts of Mn or Cu and one mesothelioma cell had a significantly higher amount of Zn. Interestingly, all mesothelioma cells overexpressed Mn-SOD compared with MeT-5A, and the cells whose Mn-SOD activity was increased contained higher amounts of Mn. It seemed that intracellular Mn content was positively correlated with Mn-SOD, suggesting that the intracellular Mn level is associated with Mn-SOD activity. These biochemical signatures could be potential disease-related markers of mesothelioma.     

IKKε regulates cell elongation through recycling endosome shuttling

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Highlights? Recycling endosomes shuttle along the proximal-distal axis of growing bristles ? IKK? locally antagonizes Rab11 effector Nuf at the bristle tip by phosphorylation ? Nuf-endosome pathway is independent of actin cytoskeleton or DIAP1-caspase pathway ? Mammalian IKK-related kinases antagonize Rab11-FIP3 through phosphorylation     

The propeptide in the precursor form of carboxypeptidase Y ensures cooperative unfolding and the carbohydrate moiety exerts a protective effect against heat and pressure.

The heat- and pressure-induced unfolding of the glycosylated and unglycosylated forms of mature carboxypeptidase Y and the precursor procarboxypeptidase Y were analysed by differential scanning calorimetry and/or by their intrinsic fluorescence in the temperature range of 20-75 degrees C or the pressure range of 0.1-700 MPa. Under all conditions, the precursor form showed a clear two-state transition from a folded to an unfolded state, regardless of the presence of the carbohydrate moiety. In contrast, the mature form, which lacks the propeptide composed of 91 amino acid residues, showed more complex behaviour: differential scanning calorimetry and pressure-induced changes in fluorescence were consistent with a three-step transition. These results show that carboxypeptidase Y is composed of two structural domains, which unfold independently but that procarboxypeptidase Y behaves as a single domain, thus ensuring cooperative unfolding. The carbohydrate moiety has a slightly protective role in heat-induced unfolding and a highly protective role in pressure-induced unfolding.     

Production of infectious hepatitis C virus in tissue culture from a cloned viral genome

Hepatitis C virus (HCV) infection causes chronic liver diseases and is a global public health problem. Detailed analyses of HCV have been hampered by the lack of viral culture systems. Subgenomic replicons of the JFH1 genotype 2a strain cloned from an individual with fulminant hepatitis replicate efficiently in cell culture. Here we show that the JFH1 genome replicates efficiently and supports secretion of viral particles after transfection into a human hepatoma cell line (Huh7). Particles have a density of about 1.15-1.17 g/ml and a spherical morphology with an average diameter of about 55 nm. Secreted virus is infectious for Huh7 cells and infectivity can be neutralized by CD81-specific antibodies and by immunoglobulins from chronically infected individuals. The cell culture-generated HCV is infectious for chimpanzee. This system provides a powerful tool for studying the viral life cycle and developing antiviral strategies.     

Involvement of heme regulatory motif in heme-mediated ubiquitination and degradation of IRP2

Iron regulatory protein 2 (IRP2), a regulator of iron metabolism, is modulated by ubiquitination and degradation. We have shown that IRP2 degradation is triggered by heme-mediated oxidation. We report here that not only Cys201, an invariant residue in the heme regulatory motif (HRM), but also His204 is critical for IRP2 degradation. Spectroscopic studies revealed that Cys201 binds ferric heme, whereas His204 is a ferrous heme binding site, indicating the involvement of these residues in sensing the redox state of the heme iron and in generating the oxidative modification. Moreover, the HRM in IRP2 has been suggested to play a critical role in its recognition by the HOIL-1 ubiquitin ligase. Although HRMs are known to sense heme concentration by simply binding to heme, the HRM in IRP2 specifically contributes to its oxidative modification, its recognition by the ligase, and its sensing of iron concentration after iron is integrated into heme.     

Evidence for Cd101 but not Fcgr1 as candidate for type 1 diabetes locus, Idd10

Among polygenes conferring susceptibility to type 1 diabetes in the NOD mouse, Idd10 on distal chromosome 3 has been shown to be important for disease susceptibility. In this study, we investigated the candidacy of Fcgr1 and Cd101 for Idd10, by congenic mapping and candidate gene sequencing. Among seven NOD-related strains studied, the IIS mouse was found to possess a recombinant Idd10 interval with the same sequence at Fcgr1 as the NOD mouse, but a different sequence at Cd101 from that in the NOD mouse with 10 amino acid substitutions. The frequency of type 1 diabetes in NOD mice congenic for IIS Idd10 (NOD.IISIdd10) was significantly reduced as compared to that in the NOD mouse, despite the presence of the identical Fcgr1 sequence. These data indicate that IIS mice possess a resistant allele at Idd10, and suggest that Cd101, but not Fcgr1, is responsible for the Idd10 effect.     

Genomewide high-density SNP linkage analysis of 236 Japanese families supports the existence of schizophrenia susceptibility loci on chromosomes 1p, 14q, and 20p

The Japanese Schizophrenia Sib-Pair Linkage Group (JSSLG) is a multisite collaborative study group that was organized to create a national resource for affected sib pair (ASP) studies of schizophrenia in Japan. We used a high-density single-nucleotide–polymorphism (SNP) genotyping assay, the Illumina BeadArray linkage mapping panel (version 4) comprising 5,861 SNPs, to perform a genomewide linkage analysis of JSSLG samples comprising 236 Japanese families with 268 nonindependent ASPs with schizophrenia. All subjects were Japanese. Among these families, 122 families comprised the same subjects analyzed with short tandem repeat markers. All the probands and their siblings, with the exception of seven siblings with schizoaffective disorder, had schizophrenia. After excluding SNPs with high linkage disequilibrium, we found significant evidence of linkage of schizophrenia to chromosome 1p21.2-1p13.2 (LOD=3.39) and suggestive evidence of linkage to 14q11.2 (LOD=2.87), 14q11.2-q13.2 (LOD=2.33), and 20p12.1-p11.2 (LOD=2.33). Although linkage to these regions has received little attention, these regions are included in or partially overlap the 10 regions reported by Lewis et al. that passed the two aggregate criteria of a meta-analysis. Results of the present study—which, to our knowledge, is the first genomewide analysis of schizophrenia in ASPs of a single Asian ethnicity that is comparable to the analyses done of ASPs of European descent—indicate the existence of schizophrenia susceptibility loci that are common to different ethnic groups but that likely have different ethnicity-specific effects.