Dendrobium Swarz.(ラン科)の類縁に関する研究 : I. Eugenanthe Schlechter節内での交配親和性

1.ノビルタイプのデンドロビウム品種に.新しい遺伝子を導入する可能性を調べるため, Eugenanthe節内の22種と、D. moniliforme(セッコク), D. nobileとの交配を行なった.2.交配稔性からみて, 本節内にはD. moniliforme, D. nobileとは遠縁と思われる種が含まれていた.3.D. moniliformeは, D. nobileに比べ, 多くの種と交雑可能で, 今後の育種のために有用な種と考えられた.In order to check the possibility of introducing new genes into the modern nobile-type cultivars of Dendrobium, D. nobile Lindl. and D. moniliforme (L.) Swarz. were crossed with selected species of section Eugenanthe Schlechter. D. moniliforme showed a wider range of crossability with Eugenanthe species compared to D. nobile. Eugenanthe species were divided into two groups according to their crossability with D. moniliforme

3b expression increases phosphorylated RUNX1b levels through ERK activation.

<p>A. HEK293 cells were transfected with vector, 3b and RUNX1b expression plasmids. ERK immunoprecipitated from vector and 3b lysates were subjected to kinase assay with RUNX1b beads. Phosphorylated RUNX1b was visualized by autoradiography. Input levels of immunoprecipitated ERK and phospho ERK levels in lysates were probed by western blotting. Graph depicts fold increase in the levels of phosphorylated RUNX1b procured after three independent experiments. Bar represents mean±SD of values obtained by densitometry. #, <i>p</i><0.05. B. Jurkat cells were transfected with WT IL2-Luc in the presence or absence of Myc-3b and treated with DMSO or U0126. Relative luciferase activity was measured and is shown as the mean±SD of three independent experiments performed in triplicates. *, <i>p</i><0.005. Phospho ERK levels in lysates were probed by western blotting.</p

3b partially co-localizes with RUNX1b in the nucleus.

<p>Cellular distribution of 3b and RUNX1b proteins were visualized by subjecting Flag-RUNX1b and HA-3b transfected HEK293 cells to immunofluorescence assay. 3b was visualized using primary α–HA and alexa-488 conjugated secondary antibody. RUNX1b was visualized using primary α–RUNX1 and alexa-594 conjugated secondary antibody. Nuclei were visualized by DAPI (4′6-diamidino-2-phenylindole) staining. Arrows indicate extra nucleolar nucleus area of partial co-localization.</p

3b recruitment on RUNX1 binding elements on the IL2 promoter.

<p>Chromatin immunoprecipitation assays were performed with jurkat cells transfected with vector alone or HA-3b using α-RUNX1 and α-HA antibodies. PCR amplifications were performed using IL2 promoter primers and 3′ distal IL2 promoter primers. Results are representative of three independent experiments.</p

3b and RUNX1b cooperatively increase MIP-1α mRNA levels.

<p>Relative mRNA levels of MIP-1α to actin were estimated in U937 cells expressing indicated proteins, using quantitative RT-PCR. Histogram is the result of three independent experiments. Bar values represent fold increase in the mRNA levels. Asterisk *, <i>p</i><0.005.</p

3b increases RUNX1b transactivation potential on the mouse IL2 promoter.

<p>A. HEK293 cells were transfected with WT IL2-Luc plasmid alone or with Flag-RUNX1b, Flag-CBFβ and indicated amounts of Myc-3b. Relative luciferase activities were calculated 48 h post transfection. B. Jurkat cells were transfected with WT IL2-Luc and indicated amounts of Myc-3b. Relative luciferase activities were calculated 48 h post-transfection. 3b expression was probed using α-myc antibody C. Jurkat cells were transfected with WT IL2-Luc or mutant IL2-Luc plasmid in the presence or absence of Myc-3b. Results in each panel are represented as mean±S.D. of triplicate cultures. Bar values represent fold increase in luciferase activity. *, <i>p</i><0.005.</p

Compounds that reverse the slow growth phenotype.

<p>Yeast grown in media containing 2% galactose with the addition of either 1% DMSO or 50 uM compounds dissolved in 1% DMSO. B. Structures of compounds shown in A. C. Effects of compounds on PLP expression. Western blots were performed with protein extracted from HA tagged PLP expressing yeast grown in the presence of 2% galactose and 50 uM of each compound and visualized with anti-HA antibody.</p

Effect of compounds on PLP enzymatic function.

<p>A. PLP protease activity was assayed using a nsp2/3/GFP reporter plasmid. 293T cells were transfected with either nsp2/3/GFP alone or with HA tagged PLP. Cells were treated with 50 uM of each compound or 1% DMSO alone and protease activity was assayed by reduction in size of the nsp2/3/GFP fusion protein by western blot with anti-GFP antibody. B. PLP deubiquitinase activity was assayed in cells transfected with Flag tagged ubiquitin and HA tagged PLP. 293T cells were transfected with both plasmids. Cells were treated with 50 uM of each compound or 1% DMSO alone and deubiquitinase activity was assayed by reduction in ubiquitinated protein by western blot with anti-Flag antibody. C and D. Effects of the compounds on PLP's IFN antagonism ability were analyzed by poly IC treatment of RIGI transfection of cells with and IFNβ/luciferase reporter plasmid with and without PLP and the compounds. Western blot of transfected HA/PLP shown below each graph. E. Effect of 158362 on IFN induction after infection with SARS-CoV. Vero cells were transfected with IFNβ/luciferase reporter plasmid. RIG-I was transfected in 1 set of wells as a positive control. Cells were then treated with either DMSO or 158362 for 2 hours prior to infection with SARS-CoV at an MOI of 3. No increase in IFNβ induction was seen after infection.</p

Toxicity assays.

<p>VeroE6 (A) or 293T (B) cells were treated with 0, 10 uM or 100 uM of each compound in 1% DMSO for 24 hours. Cells were analyzed for viability with the CellTiter Glo assay (Promega).</p

Effects of compounds on virus growth and RNA production.

<p>VeroE6 (A) or MA104 (B) cells were treated with 50 uM of each compound or 1% DMSO alone and infected with SARS-CoV(GFP) at an MOI of 3. Virus titer was assayed by plaque assay on VeroE6 cells after 12 and 24 hours of growth. C. Fluorescence images of SARS-CoV(GFP) infected Vero and MA104 cells at 24 hours post infection. D. Dose curve of NSC158362 on SARS-CoV growth. Various concentrations of drug were added to Vero cells 2 hours before SARS-CoV was added at an MOI of 3. Aliquots were removed at 12, 18 and 24 hours post infection and titered on Vero cells. E. Vero cells were treated with either DMSO or NSC 158263 at -2 hours, 0 hours, + 2 hours or +6 hours after infection with SARS-CoV. RNA was isolated at 12 hours post infection and analyzed by RT-PCR for SARS-CoV specific transcripts and GAPDH. F. MDCK cells were treated with 50 uM of each compound or 1% DMSO alone and infected with influenza A/PR/8 at an MOI of 0.1. Virus titer was determined by hemagglutination assay.</p