Anti-DPM1 monoclonal antibody Made to order

Dolichol-phosphate-mannose (DPM) synthase generates mannosyl donors for glycosylphosphatidylinositols, N-glycan and protein O- and C-mannosylation. In Saccharomyces cerevisiae, this enzyme is encoded by DPM1. We reported previously that mammalian DPM synthase contains catalytic DPM1 and regulatory DPM2 subunits, and that DPM1 requires DPM2 for its stable expression in the endoplasmic reticulum. Here we report that human DPM synthase consists of three subunits. The third subunit, DPM3, comprises 92 amino acids associated with DPM1 via its C-terminal domain and with DPM2 via its N-terminal portion. The stability of DPM3 was dependent upon DPM2, However, overexpression of DPM3 in Lec15 cells, a null mutant of DPM2, restored the biosynthesis of DPM with an increase in DPM1, indicating that DPM3 directly stabilized DPM1. Therefore, DPM2 stabilizes DPM3 and DPM3 stabilizes DPM1, DPM synthase activity was 10 times higher in the presence of DPM2 indicating that DPM2 also plays a role in the enzymatic reaction. Schizosaccharomyces pombe has proteins that resemble three human subunits; S. pombe DPM3 restored biosynthesis of DPM in Lec15 cells, indicating its orthologous relationship to human DPM3.

Background and purpose: Systems are being developed to assess radiation exposure based on leukocyte mRNA levels obtained by finger-stick sampling. The goal is to provide accurate detection of dose exposures up to 10 Gy for up to 1 week following exposure. We previously showed that specific mRNA sequences increase expression within an hour of exposure, and some genes continue to show elevated expression for at least 24 h. Full duration and dose-dependence of this persistence remain to be determined. In the present study, real-time quantitative PCR (qPCR) was used to determine changes in gene expression. qPCR can rapidly analyze small blood samples and could be adopted into a field-portable instrument that provides a radiation dose readout within 30 min. Materials and methods: From previous microarray analysis of 21,000 genes expressed in human lymphoblastoid cells 4 h post-irradiation (0-4 Gy), 118 genes were selected for evaluation by qPCR of gene expression in the leukocytes of human blood irradiated in vitro with doses of 0-10 Gy from a Co-60 gamma source at a dose rate of 30 cGy/min. Results: Blood from 20 normal healthy human donors yielded many mRNA sequences that could be used for radiation dosimetry. We observed four genes with large and persistent responses following exposure: ASTN2, CDKN1A, GADD45A, and GDF15. Five genes were identified as reliably non-responsive and were suitable for use as endogenous controls: DPM1, ITFG1, MAP4, PGK1, and SLC25A36: of these, ITFG1 was used for the analyses presented here. A significant dose-responsive increase in expression occurred for CDKN1A that was > 16-fold at 10 Gy and 3-fold at 0.5 Gy compared to pre-irradiation values. Conclusions: These data show large, selective increases in mRNA transcript levels that persist for at least 48 h after single exposures between 0.5 and 10 Gy. Stable, non-responsive mRNA sequences for use as endogenous controls were also identified. These results indicate that following further study to establish the most reproducible gene and dose-response models under a wide range of conditions in vivo, rapid real-time qPCR on blood samples could potentially be used to establish biologically-effective dosimetry from either accidental irradiation or clinical radiotherapy. (C) 2011 Elsevier Ireland Ltd. All rights reserved. Radiotherapy and Oncology 101 (2011) 233-236