Improvement of Agrobacterium-mediated transformation of embryogenic calluses from maize elite inbred lines

Summary This study was carried out to evaluate the effects of purine synthesis inhibitor mizoribine, purine and pyrimidine synthesis inhibitors azaserine and acivicin, and surfactant Silwet L-77 on Agrobacterium-mediated transformation efficiency of embryogenic calluses from maize elite inbred lines Qi 319 and Ye 515. After transformation and three rounds of selection on 2.8 μM chlorsulfuron, resistant calluses were obtained subsequently, and morphologically normal plantlets were regenerated from 80 to 90% of the resistant calluses treated with the compounds. There were no obvious discrepancies between the frequencies of plantlet regeneration and the ratio of PCR positive plantlets of calluses treated with different compounds. Results of PCR assay with primers for betA showed that 40.2% (103/256) of the regenerated plantlets were positive. The percentage of resistant calluses was 2–3-fold higher than the control after being treated with 0.19–0.27 mM mizoribine. The most suitable concentration of azaserin was 0.36 mM, which gave a 4-fold increase in the percentage of resistant calluses. Acivicin at 0.28–0.84 mM yielded a 3–5-fold increase in the percentage of resistant calluses, which is significantly better than the control. When the calluses were treated with 0.01 or 0.02% Silwet L-77, the percentages of resistant calluses were 34.89 and 25.60%, respectively. We concluded that purine synthesis inhibitors, purine and pyrimidine synthesis inhibitor and the surfactant Silwet L-77 at optimal concentrations significantly improved the Agrobacterium-mediated transformation of maize calluses.     

Antipyrimidine effects of five different pyrimidine de novo synthesis inhibitors in three head and neck cancer cell lines

The pyrimidine de novo nucleotide synthesis consists of 6 sequential steps. Various inhibitors against these enzymes have been developed and evaluated in the clinic for their potential anticancer activity: acivicin inhibits carbamoyl-phosphate-synthase-II, N-(phosphonacetyl)-L- aspartate (PALA) inhibits aspartate-transcarbamylase, Brequinar sodium and dichloroallyl-lawsone (DCL) inhibit dihydroorotate-dehydrogenase, and pyrazofurin (PF) inhibits orotate-phosphoribosyltransferase. We compared their growth inhibition against 3 cell lines from head-and-neck-cancer (HEP-2, UMSCC-14B and UMSCC-14C) and related the sensitivity to their effects on nucleotide pools. In all cell lines Brequinar and PF were the most active compounds with IC50 (50% growth inhibition) values between 0.06–0.37 µM, Acivicin was as potent (IC50s 0.26-1 µM), but DCL was 20-31-fold less active. PALA was most inactive (24–128 µM). At equitoxic concentrations, all pure antipyrimidine de novo inhibitors depleted UTP and CTP after 24 hr exposure, which was most pronounced for Brequinar (between 6–10% of UTP left, and 12–36% CTP), followed by DCL and PF, which were almost similar (6–16% UTP and 12–27% CTP), while PALA was the least active compound (10–70% UTP and 13–68% CTP). Acivicin is a multi-target inhibitor of more glutamine requiring enzymes (including GMP synthetase) and no decrease of UTP was found, but a pronounced decrease in GTP (31–72% left). In conclusion, these 5 inhibitors of the pyrimidine de novo nucleotide synthesis varied considerably in their efficacy and effect on pyrimidine nucleotide pools. Inhibitors of DHO-DH were most effective suggesting a primary role of this enzyme in controlling pyrimidine nucleotide pools     

Mechanisms of γ-glutamylcysteine ligase regulation

The principal objective of this study was to investigate the mechanisms regulating the activity of γ-glutamylcysteine ligase (GCL; EC 6.3.2.2), the rate limiting enzyme in glutathione biosynthesis. Two phylogenetically divergent species, mouse and the fruitfly, Drosophila melanogaster were used to test the hypothesis that reversible protein phosphorylation and pyridine dinucleotide phosphate dependent allostery regulate GCL activity. GCL was almost completely inhibited under phosphorylating conditions, involving preincubations with MgATP and endogenous protein kinases. Maximal GCL inhibitions of 94%, 77%, 85%, 87%, 83%, 95% and 89% occurred, respectively, in mouse cerebellum, hippocampus, brainstem, striatum, cortex and heart, and Drosophila. These changes in GCL activity were detected using saturating levels of substrates, suggesting that V_max was dramatically affected, whereas K_m values showed no differences. In vitro activation of GCL, presumably due to dephosphorylation, was blocked by inhibitors of protein phosphatases, suggesting that GCL exists in vivo as a mixture of phosphorylated and dephosphorylated forms. The reversibility of the dephosphorylation-dependent activation was indicated by the time-dependent inactivation of the in vitro activated Drosophila GCL, by preincubation with MgATP. NADPH increased maximal GCL activity by up to 93%, whereas several other nucleotide analogues did not, thereby demonstrating specificity. Kinetic analysis using Hanes–Woolf replots of initial velocity data suggested that the NADPH-dependent stimulation of GCL activity is brought about by a change in the maximal activity, V_max, rather than changes in substrate affinity. Results of this study suggest that mechanisms of modulation of eukaryotic GCL enzymes may include specific binding of ligands such as pyridine dinucleotide phosphates and reversible protein phosphorylation.