Goat anti-Human DPM1 polyclonal antibody

Dolichyl phosphate mannose synthetase (GDP-mannose: dolichyl-phosphate O-beta-D-mannosyltransferase; EC 2.4.1.83) is an enzyme that is involved in glycoconjugate biosynthesis and possesses a putatively conserved dolichol binding site. In order to probe the interaction between the enzyme and the dolichol chain., lipid phosphates varying in length and extent of branching have been tested as substrates in crude microsomal preparations from Saccharomyces cerevisiae. It was found that phytanyl (3,7,11,15-tetramethylhexadecanyl) phosphate was utilized at 60-70% of the efficiency of the natural dolichyl lipid in transfer of [3,4,-H-3]mannose from GDP-Man to organic soluble material, whereas addition of S-3-methyloctadecanyl phosphate, which is of similar length to the phytanyl analogue but with only one branch, resulted in approximately 25 % of the incorporation of the natural substrate. Incubations with the unbranched tetradecanyl phosphate and with the short, doubly branched R- and S-dihydrocitronellyl (3,7-dimethyloctanyl) phosphates exhibited levels of activity similar to incubations with no exogenous acceptor. These results were qualitatively confirmed with experiments on Escherichia coli harbouring the S. cerevisiae DPM1 gene. The [H-3]mannosylated lipid-linked material from microsomal incubations was purified by anion-exchange chromatography. The major saccharide component recovered after hydrolysis was determined to be mannose, but a mannose-containing disaccharide was also present. It is concluded that branching of lipid phosphates is essential for substrates of dolichyl phosphate mannose synthetase and that significant transfer of mannose occurs even if only branching at C-3 is present.

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