NDRG1 is important to maintain the integrity of airway epithelial barrier through claudin‐9 expression

Impairment of epithelial barrier integrity caused by environmental triggers is associated with the pathogenesis of airway inflammation. Using human airway epithelial cells, we attempted to identify molecule(s) that promote airway epithelial barrier integrity. Microarray analyses were conducted using the Affimetrix human whole genome gene chip, and we identified the N‐myc downstream‐regulated gene 1 (NDRG1) gene, which was induced during the development of the epithelial cell barrier. Immunohistochemical analysis revealed strong NDRG1 expression in ciliated epithelial cells in nasal tissues sampled from patients with chronic rhinosinusitis (CRS), and the low expression of NDRG1 was observed in goblet cells or damaged epithelial cells. NDRG1 gene knockdown with its specific siRNA decreased the transepithelial electrical resistance and increased the dextran permeability. Immunocytochemistry revealed that NDRG1 knockdown disrupted tight junctions of airway epithelial cells. Next, we analyzed the effects of NDRG1 knockdown on the expression of tight and adhesion junction molecules. NDRG1 knockdown significantly decreased only claudin‐9 expression, but did not decrease other claudin family molecules, such as E‐cadherin, and ZO‐1, ‐2, or ‐3. Knockdown of claudin‐9 markedly impaired the barrier function in airway epithelial cells. These results suggest that NDRG1 is important for the barrier integrity in airway epithelial cells.     

Development of molecular markers for breeding of double flowers in Japanese gentian

Double flowers are valuable floral traits in most floricultural plants. We recently revealed that a double-flowered mutant of Gentiana scabra was caused by an insertion of a retrotransposable element (Tgs1) into GsAG1, one of the C-class MADS-box genes in gentian. In this study, we developed a PCR-based molecular DNA marker to distinguish double- and single-flower phenotypes at the young seedling stage in Japanese gentian plants. To test the validity of the markers, 17 F₂ populations were produced by selfing F₁ plants crossed between the double-flower mutant and seven breeding lines. Multiplex PCR demonstrated that the Tgs1 insertion in GsAG1 cosegregated with the double-flower phenotype in two F₂ populations, indicating that the PCR-based DNA marker was useful to discriminate between double- and single-flower phenotypes in advance of flowering in Japanese gentian. Given that Japanese gentians lack variation in flower shape and require a long breeding period, the DNA marker developed here will be helpful for efficient breeding of double-flowered cultivars in the future.     

Hemin reduces cellular sensitivity to imatinib and anthracyclins via Nrf2

Heme plays an important biomodulating role in various cell functions. In this study, we examined the effects of hemin on cellular sensitivity to imatinib and other anti-leukemia reagents. Hemin treatment of human BCR/ABL-positive KCL22 leukemia cells increased IC(50) values of imatinib, that is, the drug resistance, in a dose-dependent manner without any change in the BCR/ABL kinase activity. Imatinib-induced apoptosis was also suppressed by hemin treatment in KCL22 cells. Hemin treatment increased the activity of gamma-glutamylcysteine synthetase (gamma-GCS) light subunit gene promoter, which contains a Maf recognition element (MARE). Protein levels of gamma-GCS and heme oxygenase-1 (HO-1), two MARE-containing genes, were also increased after hemin treatment. Knockdown of Nrf2 expression by RNA interference largely abolished the effect of hemin on imatinib-treated cells, suggesting that Nrf2 recognition of MARE is essential for the hemin-mediated protective effect. Similar to hemin, treatment of cells with delta-aminolevulinic acid (delta-ALA), the obligatory heme precursor, also increased IC(50) values of imatinib. In contrast, inhibition of cellular heme synthesis by succinylacetone increased the sensitivity of cells to imatinib in two imatinib-resistant cell lines, KCL22/SR and KU812/SR. Hemin treatment also decreased the sensitivity of cells to four anthracyclins, daunorubicin, idarubicin, doxorubicin, and mitoxantrone, in BCR/ABL-negative leukemia U937 and THP-1 cells, as well as in KCL22 cells. These findings thus indicate that cellular heme level plays an important role in determining the sensitivity of cells to imatinib and certain other anti-leukemia drugs and that the effect of heme may be mediated via its ability to upregulate Nrf2 activity.     

Compact optical cell system for vacuum ultraviolet absorption and circular dichroism spectroscopy and its application to aqueous solution sample

We have designed a compact optical cell for studying the absorption and circular dichroism (CD) of a solution sample in the vacuum ultraviolet (VUV) region using a temperature control unit. The cell size was 34 mm in diameter and 14 mm in length. Such compactness was obtained by coating the VUV scintillator onto the outside of the back window. Because this scintillator converts the transmitted VUV light to visible light, the outside of this cell is operated under atmospheric pressure. The temperature of the sample solution was maintained in the range of 5 degrees C to 80 degrees C using a temperature control unit with a Peltier thermoelectric element. Changes in the sample temperature were observed by monitoring the absorption intensity of water. Through the study of VUV-CD spectra of ammonium camphor-10-sulfonate aqueous solutions and the transmitted spectrum of an empty cell, it was concluded that this cell unit has sufficient performance for use in VUV spectroscopy.     

Ovarian follicular differentiation with prepubertal gonadotropin surges and gonadotropin priming in mice

Preantral follicles were mechanically isolated from the ovaries of 1.5 to 8 week old mice and cultured in vitro for 4 days in the presence or absence of either activin A or FSH. Plasma gonadotropin, estradiol and immunoreactive (IR) inhibin levels were measured. Cultured follicles showed stepwise changes in response to recombinant human (rh) FSH, with no response until 11 days, a gradual increase from 2 weeks, culminating in a strong response to rhFSH at 8 weeks. The response to activin A was vice versa. It enhanced the effect of rhFSH on preantral follicular growth of up to 4-week-old mice, but inhibited the effect of rhFSH in 8-week-old mice. The peak of the prepubertal gonadotropin surge was observed on day 11. Seven-day-old mice were treated with either luteinizing hormone releasing hormone (LHRH) or rhFSH or human chorionic gonadotropin (hCG) for 3 consecutive days from day 7, and follicles were collected on day 11. Those follicles showed enhanced response to rhFSH, no response to activin A, and an enhanced response to the combination of rhFSH and activin A, suggesting that the chronological changes in follicular response are a result of the prepubertal gonadotropin surge.     

Diet-induced insulin resistance in mice lacking adiponectin/ACRP30

Here we investigated the biological functions of adiponectin/ACRP30, a fat-derived hormone, by disrupting the gene that encodes it in mice. Adiponectin/ACRP30-knockout (KO) mice showed delayed clearance of free fatty acid in plasma, low levels of fatty-acid transport protein 1 (FATP-1) mRNA in muscle, high levels of tumor necrosis factor-alpha (TNF-alpha) mRNA in adipose tissue and high plasma TNF-alpha concentrations. The KO mice exhibited severe diet-induced insulin resistance with reduced insulin-receptor substrate 1 (IRS-1)-associated phosphatidylinositol 3 kinase (PI3-kinase) activity in muscle. Viral mediated adiponectin/ACRP30 expression in KO mice reversed the reduction of FATP-1 mRNA, the increase of adipose TNF-alpha mRNA and the diet-induced insulin resistance. In cultured myocytes, TNF-alpha decreased FATP-1 mRNA, IRS-1-associated PI3-kinase activity and glucose uptake, whereas adiponectin increased these parameters. Our results indicate that adiponectin/ACRP30 deficiency and high TNF-alpha levels in KO mice reduced muscle FATP-1 mRNA and IRS-1-mediated insulin signaling, resulting in severe diet-induced insulin resistance.     

Mouse semaphorin H induces PC12 cell neurite outgrowth activating Ras-mitogen-activated protein kinase signaling pathway via Ca(2+) influx.

We recently showed that mouse semaphorin H (MSH), a secreted semaphorin molecule, acts as a chemorepulsive factor on sensory neurites. In this study, we found for the first time that MSH induces neurite outgrowth in PC12 cells in a dose-dependent manner. Comparison of Ras-mitogen-activated protein kinase (MAPK) signaling pathways between MSH and nerve growth factor (NGF) revealed that these pathways are crucial for MSH action as well as NGF. K-252a, an inhibitor of tyrosine autophosphorylation of tyrosine kinase receptors (Trks), did not inhibit the action of MSH, suggesting that MSH action occurs via a different receptor than NGF. L- and N-types of voltage-dependent Ca(2+) channel blockers, diltiazem and omega-conotoxin, inhibited MSH-induced neurite outgrowth and MAPK phosphorylation in a Ca(2+)-dependent manner. A transient elevation in intracellular Ca(2+) level was observed upon MSH stimulation. These findings suggest that extracellular Ca(2+) influx, followed by activation of the Ras-MAPK signaling pathway, is required for MSH induced PC12 cell neurite outgrowth.     

Immobilization of bioactive fibroblast growth factor-2 into cubic proteinous microcrystals (Bombyx mori cypovirus polyhedra) that are insoluble in a physiological cellular environment

The supramolecular architecture of the extracellular matrix and the disposition of its specific accessory molecules give rise to variable heterotopic signaling cues for single cells. Here we have described the successful occlusion of human fibroblast growth factor-2 (FGF-2) into the cubic inclusion bodies (FGF-2 polyhedra) of the Bombyx mori cytoplasmic polyhedrosis virus (BmCPV). The polyhedra are proteinous cubic crystals of several microns in size that are insoluble in the extracellular milieu. Purified FGF-2 polyhedra were found to stimulate proliferation and phosphorylation of p44/p42 mitogen-activated protein kinase in cultured fibroblasts. Moreover, cellular responses were blocked by a synthetic inhibitor of the FGF signaling pathway, SU5402, suggesting that FGF-2 polyhedra indeed act through FGF receptors. Furthermore, FGF-2 polyhedra retained potent growth stimulatory properties even after desiccation. We have demonstrated that BmCPV polyhedra microcrystals that occlude extracellular signaling proteins are a novel and versatile tool that can be employed to analyze cellular behavior at the single cell level.     

Crystallographic study of lead-substituted hydroxyapatite synthesized by high-temperature mixing method under hydrothermal conditions

The solid solutions in the system of Ca and Pb hydroxyapatite, Ca_10−χPb_χHA (χ = 0-10), were successfully synthesized by high-temperature mixing method (HTMM) at 200 °C for 12 h under hydrothermal conditions. The samples were characterized by X-ray diffraction, chemical analysis and electron microscopic observation, and the site of the metal ions in the solid solutions was analyzed with the Rietveld method. The lattice parameters of the solid solutions prepared by both of the methods didn’t vary linearly with Pb contents, and they have two turning points of 0.4 and 0.6 of Pb content. It was found that Pb ions in the solid solutions preferentially occupied the M (2) site in the apatite structure. HTMM gives Ca-Pb HA solid solutions much better crystallization. However, due to the formation of intermediate compound of Pb₃O₂(OH)₂ in the Pb(NO₃)₂·4H₂O solution before mixing with (NH₄)₂HPO₄ solution at 200 °C, HTMM causes the decrease of crystallization of the samples with high Pb content.