Nitric oxide decreases cell surface expression of aquaporin-5 and membrane water permeability in lung epithelial cells

Nitric oxide (NO) is implicated in the pathogenesis of lung inflammation and edema. In this study, the effects of nitric oxide (NO)-donors on membrane water permeability and cell surface expression of aquaporin-5 (AQP5) in mouse lung epithelial cells were examined. NO-donors, GSNO and NOC-18 decreased cell surface expression of AQP5, concentration- and time-dependently, whereas they did not affect the amount of AQP5 in whole cell lysates. The membrane water permeability of cells was also decreased by treatment with NO-donors. The decrease in cell surface AQP5 by NO was abolished by simultaneous treatment with methyl-beta-cyclodextrin, but not with ODQ, an inhibitor of the cGMP-dependent pathway. In addition, immunocytochemistry with anti-AQP5 indicated that NO changed AQP5 localization from the plasma membrane to the intracellular fraction. These data indicate that NO stimulates AQP5 internalization from the plasma membrane through a cGMP-independent mechanism, and decreases membrane water permeability.     

Isolation of a class IV chitin synthase gene from a zygomycete fungus, Rhizopus oligosporus

We found the presence of DNA sequence which shows sequence similarity to the class IV chitin synthase gene (CHS3) of Saccharomyces cerevisiae in the genome of 14 Rhizopus species which belong to zygomycetes. We cloned a gene (chs3), which might correspond to one of these homologous sequences, from Rhizopus oligosporus by low stringency plaque hybridization probed with CHS3. The deduced amino acid sequence of this gene showed highest similarity to the class IV chitin synthase of Neurospora crassa (46.7% identity over 1087 amino acids), showing that this gene encodes a class IV chitin synthase. Northern analysis revealed the differential expression pattern of this gene in the asexual life cycle with highest expression in the early stage of asexual spore formation. This is the first report of the isolation and analysis of a class IV chitin synthase gene from zygomycete fungi.     

Dynamics of calcium regulation of chloride currents in Xenopus oocytes

Ca-activated Cl currents are widely expressed in many cell typesand play diverse and important physiological roles. TheXenopus oocyte is a good model systemfor studying the regulation of these currents. We previously showedthat inositol 1,4,5-trisphosphate (IP₃) injection intoXenopus oocytes rapidly elicits anoninactivating outward Cl current(I_Cl1-S)followed several minutes later by the development of slow inward(I_Cl2) andtransient outward(I_Cl1-T) Clcurrents. In this paper, we investigate whether these three currentsare mediated by the same or different Cl channels. Outward Cl currentswere more sensitive to Ca than inward Cl currents, as shown byinjection of different amounts of Ca or by Ca influx through aheterologously expressed ligand-gated Ca channel, the ionotropicglutamate receptor iGluR3. These data could be explained by twochannels with different Ca affinities or one channel with a higher Caaffinity at depolarized potentials. To distinguish between thesepossibilities, we determined the anion selectivity of the threecurrents. The anion selectivity sequences for the three currents werethe same (I > Br > Cl), butI_Cl1-Shad an I-to-Cl permeability ratio more than twofold smaller than the other two currents. The different anion selectivities and instantaneous current-voltage relationships were consistent with at least two different channels mediating these currents. However, afterconsideration of possible errors, the hypothesis that a single type ofCl channel underlies the complex waveforms of the three differentmacroscopic Ca-activated Cl currents inXenopus oocytes remains a viable alternative.

     

Crosstalk between oral squamous cell carcinoma cells and cancer-associated fibroblasts via the TGF-β/SOX9 axis in cancer progression

Cancer-associated fibroblasts (CAFs) have important roles in promoting cancer development and progression. We previously reported that high expression of sex-determining region Y (SRY)-box9 (SOX9) in oral squamous cell carcinoma (OSCC) cells was positively correlated with poor prognosis. This study developed three-dimensional (3D) in vitro models co-cultured with OSCC cells and CAFs to examine CAF-mediated cancer migration and invasion in vitro and in vivo. Moreover, we performed an immunohistochemical analysis of alpha-smooth muscle actin and SOX9 expression in surgical specimens from 65 OSCC patients. The results indicated that CAFs promote cancer migration and invasion in migration assays and 3D in vitro models. The invading OSCC cells exhibited significant SOX9 expression and changes in the expression of epithelial–mesenchymal transition (EMT) markers, suggesting that SOX9 promotes EMT. TGF-β1 signalling inhibition reduced SOX9 expression and cancer invasion in vitro and in vivo, indicating that TGF-β1-mediated invasion is dependent on SOX9. In surgical specimens, the presence of CAFs was correlated with SOX9 expression in the invasive cancer nests and had a significant impact on regional recurrence. These findings demonstrate that CAFs promote cancer migration and invasion via the TGF-β/SOX9 axis.     

Alleviation of lipopolysaccharide/d-galactosamine-induced liver injury in leukocyte cell-derived chemotaxin 2 deficient mice

Leukocyte cell-derived chemotaxin 2 (LECT2) is a secreted pleiotropic protein that is mainly produced by the liver. We have previously shown that LECT2 plays an important role in the pathogenesis of inflammatory liver diseases. Lipopolysaccharide/d-galactosamine (LPS/d-GalN)-induced acute liver injury is a known animal model of fulminant hepatic failure. Here we found that this hepatic injury was alleviated in LECT2-deficient mice. The levels of TNF-α and IFN-γ, which mediate this hepatitis, had significantly decreased in these mice, with the decrease in IFN-γ production notably greater than that in TNF-α. We therefore analyzed IFN-γ-producing cells in liver mononuclear cells. Flow cytometric analysis showed significantly reduced IFN-γ production in hepatic NK and NKT cells in LECT2-deficient mice compared with in wild-type mice. We also demonstrated a decrease in IFN-γ production in LECT2-deficient mice after systemic administration of recombinant IL-12, which is known to induce IFN-γ in NK and NKT cells. These results indicate that a decrease of IFN-γ production in NK and NKT cells was involved in the alleviation of LPS/d-GalN-induced liver injury in LECT2-deficient mice.     

Cooperation of metal-ion fixation and target-site activation for efficient site-selective RNA scission

Iminodiacetate–DNA conjugates and acridine–DNA conjugates were synthesized and combined for site-selective RNA hydrolysis by Lu(III). When these conjugates form a ternary complex with complementary RNA, the Lu(III)–iminodiacetate complex is placed near the target phosphodiester linkage of RNA which is in front of the acridine and is activated by noncovalent interactions. The site-selective hydrolysis by these combinations is several times as fast as that achieved by combining unmodified DNA (without iminodiacetate) and the acridine–DNA conjugate.Electronic Supplementary Material Supplementary material is available for this article at .     

Properties, intracellular localization, and stage-specific expression of membrane-bound beta-glucosidase, BglM1, from Physarum polycephalum

Physarum polycephalum expresses a membrane-bound beta-glucosidase (BglM1) with a molecular mass of 130 kDa. The primary structure of BglM1 consists of a glycosyl hydrolase family 3 domain at an amino-terminal domain and a carboxyl-terminal region without homology to the sequence of known glycosidases. The latter region contains two calx-beta motifs known as Ca(2+)-binding sites; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. The molecular mass calculated from the amino acid sequence is 130 kDa, but that in the crude extract was estimated by SDS-PAGE to be 230 kDa, and decreased to 130 kDa during purification. However, when BglM1 was purified in the presence of calcium ion, the molecular mass remained 230 kDa. The biochemical characteristics of the 130- and 230-kDa BglM1 forms were analyzed: differences were found in the kinetic data for some substrates specific for both these enzymes; however, no difference was found in their intrinsic characteristics such as optimum pH and temperature. In addition, the molecular mass of native BglM1 with a calcium ion was estimated to be 1,000 kDa or larger by gel filtration. These results suggest that the calcium ion influences the conformation of BglM1. The evidence that BglM1 localizes on the plasma membrane of plasmodia was confirmed using immunofluorescence microscopy. Although Physarum BglM1 was expressed in microplasmodia and plasmodia, little expression was detected in other stages. BglM1 may have some function only in multinuclear cells.     

Metalloregulation of triple helix formation by control of the loop conformation

The flexible polypyridine ligand, 2,2':6',2(')-terpyridine (terpy), was built into the backbone of oligonucleotides to form DNA conjugates. The terpy unit functioned as a good loop when the conjugates formed the bimolecular triplexes with complementary oligopurine. The triplex structure was destabilized by the specific interaction with divalent transition metal ions (Cu(2+), Zn(2+), and Fe(2+)), in particular Cu(2+) ions. This ion destabilized one of the triplexes by 4.2 kcalmol(-1) or made the triplex formation constant less than 1/10(3) at 298 K. This result is attributed to the substantial turbulence of the terminal structure of the triplexes.     

Identification of DOCK4 and its splicing variant as PIP3 binding proteins

DOCK4, a member of DOCK180 family proteins, was originally identified as a product of a gene deleted during tumor progression. Although its tumor suppression properties have been reported, the regulation mechanism of this protein has not been fully elucidated. DOCK4 shares two conserved domains called as DHR-1 and DHR-2 domain as other members including DOCK180. Although DHR-1 in DOCK180 is reported to bind to PIP(3), whether that of DOCK4 exhibits similar function has yet not been examined. In a search for novel PIP(3) binding proteins by the PIP(3) analog beads binding assay, we found that DOCK4 and its novel splicing variant, whose exon1 and exon52 are different from the known one, bind to PIP(3). Binding assay using deletion mutants of DOCK4 revealed that the binding region falls into the DHR-1 domain. These results raise the possibility that DOCK4 may be regulated by PIP(3) to exert its function.     

MicroRNA-206 is highly expressed in newly formed muscle fibers: implications regarding potential for muscle regeneration and maturation in muscular dystrophy

miR-1, miR-133a, and miR-206 are muscle-specific microRNAs expressed in skeletal muscles and have been shown to contribute to muscle development. To gain insight into the pathophysiological roles of these three microRNAs in dystrophin-deficient muscular dystrophy, their expression in the tibialis anterior (TA) muscles of mdx mice and CXMD(J) dogs were evaluated by semiquantitative RT-PCR and in situ hybridization. Their temporal and spatial expression patterns were also analyzed in C2C12 cells during muscle differentiation and in cardiotoxin (CTX)-injured TA muscles to examine how muscle degeneration and regeneration affect their expression. In dystrophic TA muscles of mdx mice, miR-206 expression was significantly elevated as compared to that in control TA muscles of age-matched B10 mice, whereas there were no differences in miR-1 or miR-133a expression between B10 and mdx TA muscles. On in situ hybridization analysis, intense signals for miR-206 probes were localized in newly formed myotubes with centralized nuclei, or regenerating muscle fibers, but not in intact pre-degenerated fibers or numerous small mononucleated cells, possibly proliferating myoblasts and inflammatory infiltrates. Similar increased expression of miR-206 was also found in C2C12 differentiation and CTX-induced regeneration, in which differentiated myotubes or regenerating fibers showed abundant expression of miR-206. However, CXMD(J) TA muscles contained smaller amounts of miR-206, miR-1, and miR-133a than controls. They exhibited more severe and more progressive degenerative alterations than mdx TA muscles. Taken together, these observations indicated that newly formed myotubes showed markedly increased expression of miR-206, which might reflect active regeneration and efficient maturation of skeletal muscle fibers.