High-affinity uptake of kynurenine and nitric oxide-mediated inhibition of indoleamine 2,3-dioxygenase in bone marrow-derived myeloid dendritic cells

Indoleamine 2,3-dioxygenase (IDO)-initiated tryptophan metabolism along the kynurenine (Kyn) pathway in some dendritic cells (DC) such as plasmacytoid DC regulates T-cell responses. It is unclear whether bone marrow-derived myeloid DC (BMDC) express functional IDO. The IDO expression was examined in CD11c+CD11b+ BMDC differentiated from mouse bone marrow cells using GM-CSF. CpG oligodeoxynucleotides (CpG) induced the expression of IDO protein with the production of nitric oxide (NO) in BMDC in cultures for 24 hr. In the enzyme assay using cellular extracts of BMDC, the IDO activity of BMDC stimulated with CpG was enhanced by the addition of a NO synthase (NOS) inhibitor, suggesting that IDO activity was suppressed by NO production. On the other hand, the concentration of Kyn in the culture supernatant of BMDC was not increased by stimulation with CpG. Exogenously added Kyn was taken up by BMDC independently of CpG stimulation and NO production, and the uptake of Kyn was inhibited by a transport system L-specific inhibitor or high concentrations of tryptophan. The uptake of tryptophan by BMDC was markedly lower than that of Kyn. In conclusion, IDO activity in BMDC is down-regulated by NO production, whereas BMDC strongly take up exogenous Kyn.journal articl

Upper Bound on the Decay τ→μγ from the Belle Detector

journal articl

Seismic characterisation of recent north Kagoshima Province earthquakes, Kyushu Island, Japan

Several earthquakes with moderate magnitudes have shaken an area in the northem part of Kagoshima Province, Kyushu Island, Japan in the period of March 26 to May 20, 1997. Earthquakes of March 26 and May 13 were the two main shocks with magnitudes of 6.5 and 6.3 respectively, in JMA scale. The seismicity of the area was very low and has not experienced a large earthquake of more than 6.5. This study aimed to clarify their special seismic characteristics from several point of views and some suggestions concerning further investigation of attenuation characteristics for the region are given

hClC-6 is glycosylated upon overexpression.

<p>(A) Western blots showing the effect of tunicamycin and (a) PNGaseF and (b) EndoH on hClC-6. For tunicamycin COS-1 cells were incubated with tunicamycin (0.05 and 0.1 µg/ml) for 36 hours; for PNGaseF and EndoH membrane fractions of hClC-6 expressing COS-1 cells were treated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000474#s2" target="_blank">materials and methods</a>. ‘WT’ refers to untreated hClC-6. The small difference in molecular mass between PNGaseF and tunicamycin-treated hClC-6 might be due to the presence of oligosaccharides carrying fucose-linked α1–3 to the GlcNac attached directly to asparagines, which are PNGaseF resistant as described by Dwek <i>et al. </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000474#pone.0000474-Dwek1" target="_blank">[53]</a>. (B)(a), Model of the hClC multimeric structure of 2 homologous subunits with the possible glycosylation sites marked as spheres. (b), Partial sequence alignment (ClustalW) of all members of the CLC family reveals that Asn residues that possibly participate in glycosylation (marked in red) in hClC-6 are situated in a region that is poorly conserved among the other members of the CLC family and located between predicted helices K and M. (C) Western blots of membrane fractions of COS-1 cells expressing WT or mutant hClC-6. (a), Glycosylation pattern of the triple (AAA: N410A/N422A/N432A) and quadruple (AAAA: N137A/N410A/N422A/N432A) mutant compared to WT and WT with tunicamycin. (b), Glycosylation pattern of the double mutants (AAN: N410A/N422A; ANA: N10A/N432A; NAA: N422A/N432A) compared to glycosylated WT and the triple mutant (AAA). (c), Glycosylation pattern of the single mutants (ANN: N410A, NAN: N422A, NNA: N432A) compared to the glycosylated WT and deglycosylated WT treated with tunicamycin. (d), Effect of PNGaseF treatment on a single (ANN: N410A) and double (AAN: N410A/N422A) mutant compared to WT and triple (AAA) mutant. (e), Effect of EndoH treatment on a single (ANN: N410A) and double (AAN: N410A/N422A) mutant compared to WT and triple (AAA) mutant. Bands marked with an asterisk, occasionally observed in the WT protein and frequently observed in single mutants after sustained exposure represent possible intermediary biosynthetic products, which are PNGaseF-sensitive and EndoH-insensitive as shown in panels (d) and (e).</p

Immunolocalization of overexpressed hClC-6 in SH-SY5Y cells.

<p>Double immunofluorescence confocal images of SH-SY5Y cells, transiently transfected with pcDNA3.1(−)/hClC-6a expression vector. Overexpression levels were very high, so that transfected cells could easily be distinguished from non-transfected cells. Overexpressed hClC-6 (left column) was detected using the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 (green signal). Markers for different endosomal compartments (middle column) were (A) mouse anti-EEA-1 (an early endosome marker); (B) mouse anti-transferrin receptor (TfR, an early/recycling endosome marker); (C) mouse anti-LAMP-1 (a late endosomal/lysosomal marker). Primary antibodies were visualized using anti-mouse IgG antibodies conjugated to Alexa Fluor 594 (red signal). In the merged pictures (right column) colocalization is indicated by a yellow signal. The scale bars represent 10 µm.</p

Immunolocalization of hClC-6 in transfected COS-1 cells.

<p>Double immunofluorescence confocal images of COS-1 cells transiently transfected with pcDNA3.1(−)/hClC-6a. hClC-6 expression (left column) was detected using the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 488 (green signal). Organelles were stained with the following antibodies or markers (middle column): (A) mouse anti-BIP (an endoplasmic reticulum marker); (B) mouse anti-Golgin-97 (a Golgi marker); (C) mouse anti-LAMP-1 (a late endosomal/lysosomal marker); (D) mouse anti-EEA-1 (an early endosome marker); (E) mouse anti-transferrin receptor (TfR, an early/recycling endosome marker). The marker antibodies were visualized by anti-mouse IgG antibodies conjugated to Alexa Fluor 594 (red signal). The column on the right shows merged pictures of ClC-6 expression and marker staining with yellow indicating colocalization. The scale bars represent 10 µm.</p

Colocalization of overexpressed hClC-6 with different endosomal Rab-proteins.

<p>Confocal images of double transiently transfected HeLa cells, expressing hClC-6 and (A) GFP-Rab4, (B) GFP-Rab5, (C) GFP-Rab7, (D) GFP-Rab11. hClC-6 was detected with the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 594. The Rab signals were visualized by the GFP signal. (E) Represents a confocal image of double transiently transfected COS-1 cells, expressing the glycosylation-deficient AAA-hClC-6 (red signal) and GFP-Rab4 (green signal). The scale bars represent 10 µm.</p

Development of a polyclonal antibody against human ClC-6 (hClC-6).

<p>Multiple sequence alignment (ClustalW) of all human CLC proteins revealed a COOH-terminal region that is unique for ClC-6 (aa 639–740). This region interrupts the first cystathionine-β synthase (CBS) domain in the COOH-terminal cytosolic tail <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000474#pone.0000474-Ignoul1" target="_blank">[52]</a>. Polyclonal antibodies were raised against an epitope (aa 672–686; red box) in this unique region.</p

hClC-6 resides in detergent resistant membrane fractions.

<p>(A) DRM fractions of COS-1 cells overexpressing respectively (a) hClC-6 and (b) KKGRR/AAGAA-hClC-6; were prepared and separated on a sucrose gradient. Upward flotation of the DRM's was checked by distribution of caveolin-1 (Cav-1) which migrated to the top of the gradient (fractions 2/3/4). Transferrin receptor (TfR), a non-raft membrane protein, was used as a negative control (fractions 8/9 at the bottom of the gradient). hClC-6 expression was checked by staining with the polyclonal α-hClC-6. (B) Confocal images of double transiently transfected COS-1 cells, expressing GFP-hClC-7 and wild type hClC-6 (panels a to c) or KKGRR/AAGAA-hClC-6 (panels d to f). Wild type and KKGRR/AAGAA-hClC-6 were detected with the polyclonal α-hClC-6 antibody and visualized with anti-rabbit IgG antibodies conjugated to Alexa Fluor 594 (red signal, panels a and d). ClC-7 expression is visualized by the GFP signal (green signal, panels b and e). A yellow signal indicates colocalization (panels c and f). Scale bars represent 10 µm.</p

研究解説 : 数値気候モデルによる巻頭地方の都市気候解析 : 建物のalbedoの変化による影響

departmental bulletin pape