We reported that (23S)-25-dehydro-1alpha-hydroxyvitamin D(3)-26,23-lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1alpha,25-dihydroxyvitamin D(3) [1alpha,25(OH)(2)D(3)] in human cells but is agonistic in rodent cells. Human and rat VDR ligand-binding domains are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1alpha,25(OH)(2)D(3) transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wild-type human (h)VDR, but agonistic with overexpressed wild-type rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (activation function 2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. hVDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1alpha,25(OH)(2)D(3) transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.

Flow cytometry (FCM) analysis of CD45, CD45R, CD71 and CD90 expression on Crj:CD(SD)IGS rat bone marrow cells was done after 5-fluorouracil (5-FU) administration to examine whether these lineage-specific cell surface antigens could be myelotoxic biomarkers. The expression of CD45 (CD45Low and CD45High: differing in expression intensity), CD45R, CD71 and CD90 on bone marrow cells coincided with previous reports. After repeated administration of 5-FU at 50 mg/kg/day for 1-5 days, a time-dependent decrease in cells expressing CD45Low, CD71 and CD90 was observed, whereas a decrease in the CD45High expressing cells was not observed. Furthermore, the decrease was dose-dependent in CD45Low, CD71 and CD90 expressing cells after administration of 5-FU between 2 and 50 mg/kg/day for 4 days. After 4-day repeated dose of 5-FU at 50 mg/kg/day followed by a recovery period, the change in number of CD45Low, CD45R, CD71 and CD90 cells to the bottom and in recovery showed different kinetics. In contrast, the change in number of CD45High cells was minimal, and relatively stable after 5-FU administration. The results suggest that CD45, CD45R and CD90 could each be potential myelotoxic biomarkers for a total proportion of common leukocytes including T- and B-lymphocytes, for a total proportion of B-lymphocytes, and for a total proportion of T-lymphocytes plus immature B-lymphocytes and common progenitor cells, respectively. CD71 could be a single myelotoxic biomarker for erythroid cells. Further study is required for isolation of each of the myelo-lymphocytic lineages. However, the present study showed that FCM analysis could be available to assess the lineage or differentiation stage-specific response, such as the different extent and time-course or the kinetics (the time to reach the bottom and to recover to the normal level) of myelotoxic effect in rat bone marrow.

ABSTRACT

Reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay we developed detects novel influenza A (H1N1) of swine origin and seasonal influenza A (H1N1 and H3N2) viruses. Individual primer sets targeting the HA gene for novel H1N1, H1N1, and H3N2 were newly designed to specifically detect these subtypes. No cross-reactions occurred among novel H1N1, H1N1, and H3N2, and 7 respiratory viruses-influenza B virus, influenza C virus, adenovirus, respiratory syncytial virus, metapneumovirus, parainfluenza virus, and rhinovirus-had no reaction to 3 RT-LAMP assays. RT-LAMP is assayed at 63 degrees C for 40 min. In our RT-LAMP assay, Eriochrome Black T was added to the reaction mixture as an amplification indicator to detect virus genomes without using real-time turbidimetry. Positive reactions were indicated in blue and negative reactions remained purple. Of 139 samples from suspected novel H1N1 subjects tested by both RT-LAMP and real-time RT-PCR assay, 110 were positive in both assays. Two samples with low copy numbers were positive only in real-time RT-PCR assay. Of 27 novel negative H1N1 samples, 4 were positive for H3N2 on viral isolation and conventional RT-PCR assay. RT-LAMP assay for detecting H3N2 obtained the same findings. Our RT-LAMP assay is thus potentially useful in rapidly detecting influenza A virus such as novel H1N1, H1N1, and H3N2.

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