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Miura D.,Teijin Institute for Bio Medical Research
Genes and Environment | Year: 2014

The phosphatidylinositol glycan anchor biosynthesis, Class A gene (Pig-ain rodents, PIG-Ain humans) codes for a catalytic subunit of the N-acetylglucosamine transferase complex that is involved in an early step of glycosyl-phosphatidyl inositol (GPI) anchor synthesis, and GPI anchors tether specific protein markers to the surface of various types of cells. Two distinct strategies for Pig-a gene mutation assay used peripheral blood have been developed, one using flow cytometry and the other using limiting-dilution cloning. The limiting-dilution cloning assay using bacterial protoxin, proaerolysin, as a selective agent is resource intensive. For routine analysis of mutant frequency, the flow cytometric procedures employing fluorescently labeled antibodies against GPI-anchored markers (e.g., anti-CD59 for rat red blood cells, anti-CD24 for mouse red blood cells) are preferred for routine analysis of mutant frequency. The advantage of the cloning assay, however, is in-depth analyses of the mutant genotype (mutational spectra analysis). The characteristics of the flow cytometric Pig-a gene mutation assays, i.e., induced Pig-a mutant frequencies were persistence and the effect of split doses of chemicals were additive, make the assay on reticulocytes and total red blood cells an attractive possibility for developing detailed mutagenicity data in vivo. Although a large amount of information must be gathered on assay performance, progress toward the goal in the last few years has been rapid, and multi-laboratory trial has been initiated and ongoing. © 2014 The Japanese Environmental Mutagen Society. Source

Takano Y.,Teijin Institute for Bio Medical Research | Takano Y.,Chiba University | Mitsuhashi H.,Teijin Institute for Bio Medical Research | Mitsuhashi H.,Teijin Pharma Ltd | Ueno K.,Chiba University
Steroids | Year: 2011

The chemokine interleukin-8 (IL-8) is involved in the pathogenesis of acute lung injury (ALI). Although several studies have reported that 1α,25-dihydroxyvitamin D 3 (1α,25(OH) 2D 3) suppresses IL-8 production in vitro and in vivo, 1α,25(OH) 2D 3 has not been demonstrated to be effective in an animal model of ALI. Here, we determined its effects of 1α,25(OH) 2D 3 in a hamster model where ALI was induced by lipopolysaccharide (LPS) inhalation. 1α,25(OH) 2D 3 inhibited neutrophil recruitment in the lung by approximately 40% without increasing plasma calcium concentration, while it did not inhibit monocyte recruitment. Our findings show that vitamin D 3 analogues may be suitable as novel anti-inflammatory agents for ALI. © 2011 Elsevier Inc. All rights reserved. Source

Kakuda S.,Teijin Institute for Bio Medical Research | Ishizuka S.,Teijin Institute for Bio Medical Research | Ishizuka S.,University of Pittsburgh | Eguchi H.,Teijin Institute for Bio Medical Research | And 3 more authors.
Acta Crystallographica Section D: Biological Crystallography | Year: 2010

TEI-9647 antagonizes vitamin D receptor (VDR) mediated genomic actions of 1α,25(OH)2D3 in human cells but is agonistic in rodent cells. The presence of Cys403, Cys410 or of both residues in the C-terminal region of human VDR (hVDR) results in antagonistic action of this compound. In the complexes of TEI-9647 with wild-type hVDR (hVDRwt) and H397F hVDR, TEI-9647 functions as an antagonist and forms a covalent adduct with hVDR according to MALDI-TOF MS. The crystal structures of complexes of TEI-9647 with rat VDR (rVDR), H305F hVDR and H305F/H397F hVDR showed that the agonistic activity of TEI-9647 is caused by a hydrogen-bond inter-action with His397 or Phe397 located in helix 11. Both biological activity assays and the crystal structure of H305F hVDR complexed with TEI-9647 showed that the interaction between His305 and TEI-9647 is crucial for antagonist activity. This study indicates the following stepwise mechanism for TEI-9647 antagonism. Firstly, TEI-9647 forms hydrogen bonds to His305, which promote conformational changes in hVDR and draw Cys403 or Cys410 towards the ligand. This is followed by the formation of a 1,4-Michael addition adduct between the thiol (-SH) group of Cys403 or Cys410 and the exo-methylene group of TEI-9647. © 2010 International Unin of Crystallography Printed in Singapore-all rights reserved. Source

Saitoh H.,Teijin Institute for Bio Medical Research | Chida T.,Teijin Institute for Bio Medical Research | Takagi K.,Teijin Institute for Bio Medical Research | Horie K.,Teijin Institute for Bio Medical Research | And 5 more authors.
Organic and Biomolecular Chemistry | Year: 2011

In order to obtain vitamin D derivatives, which have strong activity for enhancing bone growth, we designed vitamin D derivatives with various substitutions at the C-2 position. Novel 2 α-substituted vitamin D derivatives were synthesized starting from d-glucose as a chiral template of the A-ring with a CD-ring bromoolefin unit using the Trost coupling method. We evaluated these compounds by two in vitro assays, affinity to VDR and transactivation assays, using human osteosarcoma (Hos) cells, and demonstrated the SAR of the C-2 position of VD3. Furthermore, by using the OVX model, we found that compound 5c, which has a hydroxypropoxy side chain at C-2 and 2,2-dimethyl cyclopentanone in the CD-ring side chain, has a strong activity for enhancing bone growth, same as the reported compound, 2α-(3- hydroxypropoxy)-1α,25-dihydroxyvitamin D31d, and this derivative shows a possibility that calcemic activity is less than 1din vivo. © 2011 The Royal Society of Chemistry. Source

Takano Y.,Teijin Institute for Bio Medical Research | Takano Y.,Chiba University | Mitsuhashi H.,Teijin Institute for Bio Medical Research | Ishizuka S.,Teijin Institute for Bio Medical Research | And 10 more authors.
Steroids | Year: 2012

While searching for vitamin D3 analogues which inhibit neutrophil recruitment in the lung without elevating plasma calcium level, we found that (5Z,7E)-(1S,3R)-20(R)-[(5E)-(2S)-2-hydroxy-2-methyl-cyclopentanone-5- ylidene]methyl-9,10-secopregna-5,7,10(19)-triene-1,3-diol (TEI-A00114) had the best efficacy and calcemic action. TEI-A00114 has a vitamin D receptor affinity 2.5-fold weaker and a vitamin D binding protein affinity 330.9-fold weaker than those of 1α,25(OH)2D3. The estimated effective doses for 40% inhibition (ED40) via peroral and intratracheal administration are 7.6 and 0.4 μg/kg, respectively. TEI-A00114 was also tested by inhaled administration, and its ED40 was calculated as 0.2 μg/kg. The estimated 40% inhibitory concentration (IC40) of TEI-A00114 on interleukin (IL)-8 production induced by lipopolysaccharide and on IL-1β in human whole blood cells in vitro were 9.8 × 10-8 or 1.8 × 10-9 M, respectively. These levels of TEI-A00114's activities are equal to those of 1α,25(OH)2D3. On the other hand, the calcemic action of TEI-A00114, which was evaluated at day 14 after sequential peroral quaque die administration, was 89-fold weaker (molar ratio) than that of 1α,25(OH)2D3. These results indicate that TEI-A00114 has a dissociated profile between inhibition of neutrophil recruitment in the lung and calcemic action, suggesting its suitability over 1α,25(OH)2D3 as a candidate for the treatment of acute lung injury. © 2012 Elsevier Inc. All rights reserved. Source

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