Myosin heavy chains from two different adult fast-twitch muscles have different peptide maps but identical mRNAs

Myosin heavy chains prepared from the pectoralis major and from the posterior latissimus dorsi of the same adult chicken exhibit different peptide maps when cleaved with Staphylococcus aureus V8 protease. These differences were observed at five different enzyme concentrations and in chickens of various strains. The cleavage pattern of pectoralis major myosin heavy chain from different adult chickens was always identical, as was that of posterior latissimus dorsi myosin heavy chain, demonstrating the reproducibility of the technique. However, when RNAs extracted from the pectoralis major and from the posterior latissimus dorsi were translated in a cell-free reticulocyte lysate, the myosin heavy chain encoded by pectoralis major RNA and the myosin heavy chain encoded by posterior latissimus dorsi RNA exhibited identical peptide maps. These results suggest that the different peptide maps of myosin heavy chains from the pectoralis major and posterior latissimus dorsi may arise from posttranslational modifications.     

Expression of 150-kDa oxygen-regulated protein (ORP150) stimulates bleomycin-induced pulmonary fibrosis and dysfunction in mice

Idiopathic pulmonary fibrosis (IPF) involves pulmonary injury associated with inflammatory responses, fibrosis and dysfunction. Myofibroblasts and transforming growth factor (TGF)-β1 play major roles in the pathogenesis of this disease. Endoplasmic reticulum (ER) stress response is induced in the lungs of IPF patients. One of ER chaperones, the 150-kDa oxygen-regulated protein (ORP150), is essential for the maintenance of cellular viability under stress conditions. In this study, we used heterozygous ORP150-deficient mice (ORP150(+/-) mice) to examine the role of ORP150 in bleomycin-induced pulmonary fibrosis. Treatment of mice with bleomycin induced the expression of ORP150 in the lung. Bleomycin-induced inflammatory responses were slightly exacerbated in ORP150(+/-) mice compared to wild-type mice. On the other hand, bleomycin-induced pulmonary fibrosis, alteration of lung mechanics and respiratory dysfunction was clearly ameliorated in the ORP150(+/-) mice. Bleomycin-induced increases in pulmonary levels of both active TGF-β1 and myofibroblasts were suppressed in ORP150(+/-) mice. These results suggest that although ORP150 is protective against bleomycin-induced lung injury, this protein could stimulate bleomycin-induced pulmonary fibrosis by increasing pulmonary levels of TGF-β1 and myofibroblasts.     

C-terminal region of GADD34 regulates eIF2α dephosphorylation and cell proliferation in CHO-K1 cells

GADD34 is a member of a growth arrest and DNA damage (GADD)-inducible gene family. Here, we established a novel Chinese hamster ovary (CHO)-K1-derived cell line, CHO-K1-G34M, which carries a nonsense mutation (termed the Q525X mutation) in the GADD34 gene. The Q525X mutant protein lacks the C-terminal 66 amino acids required for GADD34 to bind to and activate protein phosphatase 1 (PP1). We investigated the effects of GADD34 with or without the Q525X mutation on the phosphorylation status of PP1 target proteins, including the α subunit of eukaryotic initiation factor 2 (eIF2α) and glycogen synthase kinase 3β (GSK3β). CHO-K1-G34M cells had higher levels of eIF2α phosphorylation compared to the control CHO-K1-normal cells both in the presence and absence of endoplasmic reticulum stress. Overexpression of the wild-type GADD34 protein in CHO-K1-normal cells largely reduced eIF2α phosphorylation, while overexpression of the Q525X mutant did not produce similar reductions. Meanwhile, neither wild type nor Q525X mutation of GADD34 affected the GSK3β phosphorylation status. GADD34 also did not affect the canonical Wnt signaling pathway downstream of GSK3β. Cell proliferation rates were higher, while expression levels of the cyclin-dependent kinase inhibitor p21 were lower in CHO-K1-G34M cells compared to the CHO-K1-normal cells. The GADD34 Q525X mutant had a reduced ability to inhibit cell proliferation and enhance p21 expression of the CHO-K1-normal cells compared to the wild-type GADD34 protein. These results suggest that the GADD34 protein C-terminal plays important roles in regulating not only eIF2α dephosphorylation but also cell proliferation in CHO-K1 cells.     

Lipocalin 2 attenuates iron-related oxidative stress and prolongs the survival of ovarian clear cell carcinoma cells by up-regulating the CD44 variant

Ovarian clear cell carcinoma (CCC) arises from ovarian endometriosis. Intra-cystic fluid contains abundant amounts of free iron, which causes persistent oxidative stress, a factor that has been suggested to induce malignant transformation. However, the mechanisms linking oxidative stress and carcinogenesis in CCC currently remain unclear. Lipocalin 2 (LCN2), a multifunctional secretory protein, functions as an iron transporter as well as an antioxidant. Therefore, we herein examined the roles of LCN2 in the regulation of intracellular iron concentrations, oxidative stress, DNA damage, and antioxidative functions using LCN2-overexpressing (ES2), and LCN2-silenced (RMG-1) CCC cell lines. The results of calcein staining indicated that the up-regulated expression of LCN2 correlated with increases in intracellular iron concentrations. However, a DCFH-DA assay and 8OHdG staining revealed that LCN2 reduced intracellular levels of reactive oxygen species and DNA damage. Furthermore, the expression of LCN2 suppressed hydrogen peroxide-induced apoptosis and prolonged cell survival, suggesting an antioxidative role for LCN2. The expression of mRNAs and proteins for various oxidative stress-catalyzing enzymes, such as heme oxygenase (HO), superoxide dismutase (SOD), and glutathione peroxidase, was not affected by LCN2, whereas the intracellular concentration of the potent antioxidant, glutathione (GSH), was increased by LCN2. Furthermore, the expression of xCT, a cystine transporter protein, and CD44 variant 8-10 (CD44v), a stem cell marker, was up-regulated by LCN2. Although LCN2 increased intracellular iron concentrations, LCN2-induced GSH may catalyze and override oxidative stress via CD44v and xCT, and subsequently enhance the survival of CCC cells in oxidative stress-rich endometriosis.     

A regulated adaptor function of p40phox: distinct p67phox membrane targeting by p40phox and by p47phox

In the phagocytic cell, NADPH oxidase (Nox2) system, cytoplasmic regulators (p47(phox), p67(phox), p40(phox), and Rac) translocate and associate with the membrane-spanning flavocytochrome b(558), leading to activation of superoxide production. We examined membrane targeting of phox proteins and explored conformational changes in p40(phox) that regulate its translocation to membranes upon stimulation. GFP-p40(phox) translocates to early endosomes, whereas GFP-p47(phox) translocates to the plasma membrane in response to arachidonic acid. In contrast, GFP-p67(phox) does not translocate to membranes when expressed alone, but it is dependent on p40(phox) and p47(phox) for its translocation to early endosomes or the plasma membrane, respectively. Translocation of GFP-p40(phox) or GFP-p47(phox) to their respective membrane-targeting sites is abolished by mutations in their phox (PX) domains that disrupt their interactions with their cognate phospholipid ligands. Furthermore, GFP-p67(phox) translocation to either membrane is abolished by mutations that disrupt its interaction with p40(phox) or p47(phox). Finally, we detected a head-to-tail (PX-Phox and Bem1 [PB1] domain) intramolecular interaction within p40(phox) in its resting state by deletion mutagenesis, cell localization, and binding experiments, suggesting that its PX domain is inaccessible to interact with phosphatidylinositol 3-phosphate without cell stimulation. Thus, both p40(phox) and p47(phox) function as diverse p67(phox) "carrier proteins" regulated by the unmasking of membrane-targeting domains in distinct mechanisms.     

Evidence that the degree and duration of acid suppression are related to Helicobacter pylori eradication by triple therapy

BACKGROUNDS AND AIMS: Eradication rates of Helicobacter pylori by a proton pump inhibitor-based triple therapy depend on CYP2C19 genotype status. We investigated whether gastric acid inhibition during an eradication therapy would influence the eradication rates attained by the triple therapy. METHODS: Thirty-two patients with H. pylori infection underwent the first-line triple therapy with lansoprazole 30 mg, amoxicillin 750 mg, and clarithromycin 400 mg b.i.d. for 1 week. In all 32 patients, the 24-hour intragastric pH monitoring was performed on day 6 during the treatment period. RESULTS: The intention-to-treat-based eradication rate by the first-line therapy was 75.0% (24/32, 95%CI: 56.60-88.54%). In patients with successful eradication, the median 24-hour pH was 6.4 (range; 5.0-7.6), which was significantly higher than that in patients without eradication [5.2 (2.2-6.2), p = .0131]. The median percentage time of pH < 4.0 during 24-hour postdose in patients with eradication [0.5% (0.0-31.6%)] was significantly shorter than that in patients without eradication [26.7% (6.0-72.2%), p = .0017]. These parameters for acid inhibition significantly differed among the different CYP2C19 genotype groups. When the percentage time of pH < 4.0 and 24-hour pH were attained < 10% and > 6.0, respectively, during the eradication treatment, the majority of patients could eradicate H. pylori infection, irrespective of the bacterial susceptibility to clarithromycin. CONCLUSIONS: The sustained intragastric pH > 4.0 for a longer postdose time appears to be required for a successful eradication of H. pylori with lansoprazole and acid-labile antibiotics.     

Fabp7 maps to a quantitative trait locus for a schizophrenia endophenotype

Deficits in prepulse inhibition (PPI) are a biological marker for schizophrenia. To unravel the mechanisms that control PPI, we performed quantitative trait loci (QTL) analysis on 1,010 F2 mice derived by crossing C57BL/6 (B6) animals that show high PPI with C3H/He (C3) animals that show low PPI. We detected six major loci for PPI, six for the acoustic startle response, and four for latency to response peak, some of which were sex-dependent. A promising candidate on the Chromosome 10-QTL was Fabp7 (fatty acid binding protein 7, brain), a gene with functional links to the N-methyl-D-aspartic acid (NMDA) receptor and expression in astrocytes. Fabp7-deficient mice showed decreased PPI and a shortened startle response latency, typical of the QTL's proposed effects. A quantitative complementation test supported Fabp7 as a potential PPI-QTL gene, particularly in male mice. Disruption of Fabp7 attenuated neurogenesis in vivo. Human FABP7 showed altered expression in schizophrenic brains and genetic association with schizophrenia, which were both evident in males when samples were divided by sex. These results suggest that FABP7 plays a novel and crucial role, linking the NMDA, neurodevelopmental, and glial theories of schizophrenia pathology and the PPI endophenotype, with larger or overt effects in males. We also discuss the results from the perspective of fetal programming.     

Arrest of cytoplasmic streaming induces algal proliferation in green paramecia

A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.     

Crystal structure of MqnD (TTHA1568), a menaquinone biosynthetic enzyme from Thermus thermophilus HB8

In many microorganisms, menaquinone is an essential lipid-soluble electron carrier. Recently, an alternative menaquinone biosynthetic pathway was found in some microorganisms [Hiratsuka, T., Furihata, K., Ishikawa, J., Yamashita, H., Itoh, N., Seto, H., Dairi, T., 2008. An alternative menaquinone biosynthetic pathway operating in microorganisms. Science 321, 1670–1673]. Here, we report the 1.55 Å crystal structure of MqnD (TTHA1568) from Thermus thermophilus HB8, an enzyme within the alternative menaquinone biosynthetic pathway. The structure comprises two domains with α/β structures, a large domain and a small domain. L(+)-Tartaric acid was bound to the pocket between the two domains, suggesting that this pocket is a putative active site. The conserved glycine residues at positions 78, 80 and 82 seem to act as hinges, allowing the substrate to access the pocket. Highly conserved residues, such as Asp14, Asp38, Asn43, Ser57, Thr107, Ile144, His145, Glu146, Leu176 and Tyr234, are located at this pocket, suggesting that these residues are involved in substrate binding and/or catalysis, and especially, His145 could function as a catalytic base. Since humans and their commensal intestinal bacteria, including lactobacilli, lack the alternative menaquinone biosynthetic pathway, this enzyme in pathogenic species, such as Helicobacter pylori and Campylobacter jejuni, is an attractive target for the development of chemotherapeutics. This high-resolution structure may contribute toward the development of its inhibitors.