Inducing Ni Sensitivity in the Ni Hyperaccumulator Plant Alyssum inflatum Nyárády (Brassicaceae) by Transforming with CAX1, a Vacuolar Membrane Calcium Transporter

The importance of calcium in nickel tolerance was studied in the nickel hyperaccumulator plant Alyssum inflatum by gene transformation of CAX1, a vacuolar membrane transporter that reduces cytosolic calcium. CAX1 from Arabidopsis thaliana with a CaMV35S promoter accompanying a kanamycin resistance gene was transferred into A. inflatum using Agrobacterium tumefaciens. Transformed calli were subcultured three times on kanamycin-rich media and transformation was confirmed by PCR using a specific primer for CAX1. At least 10 callus lines were used as a pool of transformed material. Both transformed and untransformed calli were treated with varying concentrations of either calcium (1–15 mM) or nickel (0– 500 lM) to compare their responses to those ions. Increased external calcium generally led to increased callus biomass, however, the increase was greater for untransformed callus. Further, increased external calcium led to increased callus calcium concentrations. Transformed callus was less nickel tolerant than untransformed callus: under increasing nickel concentrations callus relative growth rate was significantly less for transformed callus. Transformed callus also contained significantly less nickel than untransformed callus when exposed to the highest external nickel concentration (200 lM). We suggest that transformation with CAX1 decreased cytosolic calcium and resulted in decreased nickel tolerance. This in turn suggests that, at low cytosolic calcium concentrations, other nickel tolerance mechanisms (e.g., complexation and vacuolar sequestration) are insufficient for nickel tolerance. We propose that high cytosolic calcium is an important mechanism that results in nickel tolerance by nickel hyperaccumulator plants

Ebselen is a potent non-competitive inhibitor of extracellular nucleoside diphosphokinase

Nucleoside di- and triphosphates and adenosine regulate several components of the mucocilairy clearance process (MCC) that protects the lung against infections, via activation of epithelial purinergic receptors. However, assessing the contribution of individual nucleotides to MCC functions remains difficult due to the complexity of the mechanisms of nucleotide release and metabolism. Enzymatic activities involved in the metabolism of extracellular nucleotides include ecto-ATPases and secreted nucleoside diphosphokinase (NDPK) and adenyl kinase, but potent and selective inhibitors of these activities are sparse. In the present study, we discovered that ebselen markedly reduced NDPK activity while having negligible effect on ecto-ATPase and adenyl kinase activities. Addition of radiotracer [γ32P]ATP to human bronchial epithelial (HBE) cells resulted in rapid and robust accumulation of [32P]-inorganic phosphate (32Pi). Inclusion of UDP in the incubation medium resulted in conversion of [γ32P]ATP to [32P]UTP, while inclusion of AMP resulted in conversion of [γ32P]ATP to [32P]ADP. Ebselen markedly reduced [32P]UTP formation but displayed negligible effect on 32Pi or [32P]ADP accumulations. Incubation of HBE cells with unlabeled UTP and ADP resulted in robust ebselen-sensitive formation of ATP (IC50 = 6.9 ± 2 μM). This NDPK activity was largely recovered in HBE cell secretions and supernatants from lung epithelial A549 cells. Kinetic analysis of NDPK activity indicated that ebselen reduced the Vmax of the reaction (Ki = 7.6 ± 3 μM), having negligible effect on KM values. Our study demonstrates that ebselen is a potent non-competitive inhibitor of extracellular NDPK