Fructosyl-amino acid oxidases of Aspergillus oryzae are induced by the reaction product, glucosone

Aspergillus oryzae has two fructosyl-amino acid oxidase (FAOD) isozymes (AoFao1 and AoFao2), which are different in the substrate specificities. Northern blot analysis showed both FAO genes were induced by autoclave-browned medium containing l-lysine or l-valine. Studies with a mutant, that had a disrupted AoFAO2 gene, revealed that the expression of AoFAO1 by fructosyl l-valine depended on the expression of AoFAO2. Both genes were also induced by one of the FAOD-reaction products, glucosone. In contrast, other alpha-dicarbonyl compounds, which display a similar structure to that of glucosone were not able to induce the genes expression. These results imply that glucosone may contribute to the expression of FAO genes.     

Defects of protein phosphatase 2A causes corticosteroid insensitivity in severe asthma

Background

Corticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma.

Methodology/Principal Findings

Corticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser²²⁶, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2A_C-Tyr³⁰⁷ phosphorylation compared with those in healthy volunteers. As the results, GR-Ser²²⁶ and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser²²⁶. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells.

Conclusions/Significance

PP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser²²⁶ via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma.     

G-Protein-Coupled Estrogen Receptor Agonist Suppresses Airway Inflammation in a Mouse Model of Asthma through IL-10

Estrogen influences the disease severity and sexual dimorphism in asthma, which is caused by complex mechanisms. Besides classical nuclear estrogen receptors (ERαβ), G-protein-coupled estrogen receptor (GPER) was recently established as an estrogen receptor on the cell membrane. Although GPER is associated with immunoregulatory functions of estrogen, the pathophysiological role of GPER in allergic inflammatory lung disease has not been examined. We investigated the effect of GPER-specific agonist G-1 in asthmatic mice. GPER expression in asthmatic lung was confirmed by immunofluorescent staining. OVA-sensitized BALB/c and C57BL/6 mice were treated with G-1 by daily subcutaneous injections during an airway challenge phase, followed by histological and biochemical examination. Strikingly, administration of G-1 attenuated airway hyperresponsiveness, accumulation of inflammatory cells, and levels of Th2 cytokines (IL-5 and IL-13) in BAL fluid. G-1 treatment also decreased serum levels of anti-OVA IgE antibodies. The frequency of splenic Foxp3⁺CD4⁺ regulatory T cells and IL-10-producing GPER⁺CD4⁺ T cells was significantly increased in G-1-treated mice. Additionally, splenocytes isolated from G-1-treated mice showed greater IL-10 production. G-1-induced amelioration of airway inflammation and IgE production were abolished in IL-10-deficient mice. Taken together, these results indicate that extended GPER activation negatively regulates the acute asthmatic condition by altering the IL-10-producing lymphocyte population. The current results have potential importance for understanding the mechanistic aspects of function of estrogen in allergic inflammatory response.     

Effects of narrow roads on the movement of carabid beetles (Coleoptera,Carabidae) in Nopporo Forest Park,Hokkaido

Forest-dwelling carabid beetles that have no flight ability were studied using mark-recapture methods in late-June to mid-October 2007. This study was done to determine the effects of narrow roads in Nopporo Forest Park, Hokkaido on carabid beetle movement and habitat use. The investigation was conducted at four sites: one site was an abandoned grassy road with a width of 3.5 m, two sites were gravel roads with widths of 3.5 and 4.5 m, and another site was an asphalt-paved road with a width of 4.5 m. A total of 3,580 individuals from six species of carabid beetles were collected using dry pit-fall traps, and recapture rates ranged from 6.1 to 36.2%. All examined roads acted as barriers against the movement of Leptocarabus arboreus ishikarinus. All roads, except the abandoned grassy road, acted as a barrier against Carabus granulatus yezoensis movement. Forest–roadside verge comparisons demonstrated that some carabid beetles avoid even narrow roadside verges. Harmful effects increase with increasing road width and both paved roads and narrow roads negatively affect the movement of carabid beetles inhabiting the bordering forest. Therefore, forest specialist beetles are influenced by a barrier effect that starts at the forest road verge, and this barrier effect may be exacerbated by vehicular traffic. Therefore, these barrier effects on carabid beetles should be considered when planning and implementing road construction and maintenance in forests.     

Comprehensive analysis and characterization of the TCR α chain sequences in the common marmoset

The common marmoset (Callithrix jacchus) is useful as a nonhuman primate model of human diseases. Although the marmoset model has great potential for studying autoimmune diseases and immune responses against pathogens, little information is available regarding the genes involved in adaptive immunity. Here, we identified one TCR α constant (TRAC), 46 TRAJ (joining), and 35 TRAV (variable) segments from marmoset cDNA. Marmoset TRAC, TRAJ, and TRAV shared 80%, 68–100%, and 79–98% identity with their human counterparts at the amino acid level, respectively. The amino acid sequences were less conserved in TRAC than in TCRβ chain constant (TRBC). Comparative analysis of TRAV between marmosets and humans showed that the rates of synonymous substitutions per site (d _S ) were not significantly different between the framework regions (FRs) and complementarity determining regions (CDRs), whereas the rates of nonsynonymous substitutions per site (d _N ) were significantly lower in the FRs than in CDRs. Interestingly, the d _N values of the CDRs were greater for TRBV than TRAV. These results suggested that after the divergence of Catarrhini from Platyrrhini, amino acid substitutions were decreased in the FRs by purifying selection and occurred more frequently in CDRβ than in CDRα by positive selection, probably depending on structural and functional constraints. This study provides not only useful information facilitating the investigation of adaptive immunity using the marmoset model but also new insight into the molecular evolution of the TCR heterodimer in primate species.     

Nitric oxide-cGMP signaling regulates axonal elongation during optic nerve regeneration in the goldfish in vitro and in vivo

Nitric oxide (NO) signaling results in both neurotoxic and neuroprotective effects in CNS and PNS neurons, respectively, after nerve lesioning. We investigated the role of NO signaling on optic nerve regeneration in the goldfish ( Carassius auratus ). NADPH diaphorase staining revealed that nitric oxide synthase (NOS) activity was up-regulated primarily in the retinal ganglion cells (RGCs) 5–40 days after axotomy. Levels of neuronal NOS (nNOS) mRNA and protein also increased in the RGCs alone during this period. This period (5–40 days) overlapped with the process of axonal elongation during regeneration of the goldfish optic nerve. Therefore, we evaluated the effect of NO signaling molecules upon neurite outgrowth from adult goldfish axotomized RGCs in culture. NO donors and dibutyryl cGMP increased neurite outgrowth dose-dependently. In contrast, a nNOS inhibitor and small interfering RNA, specific for the nNOS gene, suppressed neurite outgrowth from the injured RGCs. Intra-ocular dibutyryl cGMP promoted the axonal regeneration from injured RGCs in vivo . None of these molecules had an effect on cell death/survival in this culture system. This is the first report showing that NO-cGMP signaling pathway through nNOS activation is involved in neuroregeneration in fish CNS neurons after nerve lesioning.     

Histology and tetracycline labeling of a single section of alveolar bone of first molars in the rat

We observed the histology and tetracycline (TC) labeling in a single frontal section of alveolar bone of upper first molars of adolescent rats. A single injection of TC was administered intraperitonealy in adolescent rats. After three weeks, the upper jaws were immersed rapidly in liquid nitrogen and sectioned. Five micrometer unfixed, undecalcified frozen sections were cut and observed by light and fluorescence microscopy. Frontal sections of the upper first molar area revealed that the structural relationships among the roots, the periodontal ligament and the alveolar bone, and also between the cervical enamel and the attachment epithelium were well preserved. The TC labeling lines in the sections were very clear and distinguished new bone from old bone. The brightness of the lines differed among regions. An analysis of the brightness in the same section suggested a difference in the bone forming activity at the time of injection.     

Characterization of Inhibitor-Bound α-Synuclein Dimer: Role of α-Synuclein N-Terminal Region in Dimerization and Inhibitor Binding

α-Synuclein is a major component of filamentous inclusions that are histological hallmarks of Parkinson's disease and other α-synucleinopathies. Previous analyses have revealed that several polyphenols inhibit α-synuclein assembly with low micromolar IC₅₀ values, and that SDS-stable, noncytotoxic soluble α-synuclein oligomers are formed in their presence. Structural elucidation of inhibitor-bound α-synuclein oligomers is obviously required for the better understanding of the inhibitory mechanism. In order to characterize inhibitor-bound α-synucleins in detail, we have prepared α-synuclein dimers in the presence of polyphenol inhibitors, exifone, gossypetin, and dopamine, and purified the products. Peptide mapping and mass spectrometric analysis revealed that exifone-treated α-synuclein monomer and dimer were oxidized at all four methionine residues of α-synuclein. Immunoblot analysis and redox-cycling staining of endoproteinase Asp-N-digested products showed that the N-terminal region (1-60) is involved in the dimerization and exifone binding of α-synuclein. Ultra-high-field NMR analysis of inhibitor-bound α-synuclein dimers showed that the signals derived from the N-terminal region of α-synuclein exhibited line broadening, confirming that the N-terminal region is involved in inhibitor-induced dimerization. The C-terminal portion still predominantly exhibited the random-coil character observed in monomeric α-synuclein. We propose that the N-terminal region of α-synuclein plays a key role in the formation of α-synuclein assemblies.