Original Research Report: Structure-Function Analysis of the ArsA ATPase: Contribution of Histidine Residues

The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance. Either antimonite or arsenite stimulates ArsA ATPase activity. In this study, the role of histidine residues in ArsA activity was investigated. Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity. The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues. ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site. ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558. Each histidine was individually altered to alanine by site-directed mutagenesis. Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite. The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group.     

Arsenite inhibits Ras-dependent activation of ERK but activates ERK in the presence of oncogenic Ras in baboon vascular smooth muscle cells

Exposure to arsenical compounds enhances the risk of atherosclerosis. The reason is unknown but it might be because an effect of arsenite (As3+) on plaque smooth muscle cells (SMCs) activation of extracellular signal-regulated kinase (ERK), a crucial mediator of SMC function. We found that arsenite inhibits the activation of ERK by platelet-derived growth factor-BB (PDGF-BB). This inhibitory effect depends on the time of arsenite exposure, is reversible, and is attenuated by preincubation of SMCs with the antioxidant N-acetyl-cysteine. These observations are consistent with the assumption that oxidative stress is involved. The blockade of ERK by arsenite may be mediated by an inhibition of Ras as arsenite prevents GTP-loading of Ras in response to PDGF-BB. Moreover, the Ras blockade by arsenite is not specific for PDGF-BB because it was also observed following stimulation of SMCs with EGF. To address the role of Ras, we expressed constitutively active, GTP-bound Ha-Ras (V12Ras). Unexpectedly, in V12Ras expressing-SMCs, arsenite stimulates ERK, but still decreases ERK activity in the presence of PDGF-BB. Our data suggest that arsenite inhibits the Ras/ERK pathway in SMCs, and that arsenite may activate ERK in Ras-transformed cells by mechanisms different from those employed by growth factors.     

Arsenite as an Inducer of Lipid Peroxidation in Saccharomyces cerevisiae Cells

The ability of sodium arsenite at concentrations of 10^–2, 10^–4, and 10^–6 M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10^–2 and 10^–4 M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10^–6 M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of -ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Bioadsorption of arsenic from aqueous solution by the extremophilic bacterium Acidithiobacillus ferrooxidans DLC-5

The bioadsorption of heavy metal ions to process industrial and solid wastes is an attractive technology from an economical and environmental point of view. This study investigated the equilibrium, thermodynamics and bioadsorption characteristics of arsenite (iAs^III) and dimethylarsinate (DMA^V) by Acidithiobacillus ferrooxidans (A. ferrooxidans) DLC-5 in aqueous solution. Optimum bioadsorption conditions were determined by identifying the optimum temperature, pH, biomass dosage, initial arsenic concentration and contact time. The equilibrium data were then applied to Langmuir and Freundlich isotherm models. The results indicated that the bioadsorption processes for both iAs^III and DMA^V involved pseudo-second-order kinetics. Additionally, the bioadsorption of iAs^III and DMA^V by A. ferrooxidans DLC-5 was feasible, spontaneous and endothermic under the tested conditions. Fourier transform infrared spectroscopy (FT-IR) showed that –OH and –NH groups were involved in the bioadsorption process. A. ferrooxidans DLC-5 demonstrates potential for use in removing arsenic from aqueous solutions, especially those with very low arsenic concentrations.     

PI-3K/Akt signal pathway plays a crucial role in arsenite-induced cell proliferation of human keratinocytes through induction of cyclin D1

Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite-induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI-3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI-3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI-3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI-3K/Akt pathway is implicated in arsenite-induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI-3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI-3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI-3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite.     

Phosphoenolpyruvate metabolism in Jerusalem artichoke mitochondria

We report here initial studies on phosphoenolpyruvate metabolism in coupled mitochondria isolated from Jerusalem artichoke tubers. It was found that:

(1)

phosphoenolpyruvate can be metabolized by Jerusalem artichoke mitochondria by virtue of the presence of the mitochondrial pyruvate kinase, shown both immunologically and functionally, located in the inner mitochondrial compartments and distinct from the cytosolic pyruvate kinase as shown by the different pH and inhibition profiles.

(2)

Jerusalem artichoke mitochondria can take up externally added phosphoenolpyruvate in a proton compensated manner, in a carrier-mediated process which was investigated by measuring fluorimetrically the oxidation of intramitochondrial pyridine nucleotide which occurs as a result of phosphoenolpyruvate uptake and alternative oxidase activation.

(3)

The addition of phosphoenolpyruvate causes pyruvate and ATP production, as monitored via HPLC, with their efflux into the extramitochondrial phase investigated fluorimetrically. Such an efflux occurs via the putative phosphoenolpyruvate/pyruvate and phosphoenolpyruvate/ATP antiporters, which differ from each other and from the pyruvate and the adenine nucleotide carriers, in the light of the different sensitivity to non-penetrant compounds. These carriers were shown to regulate the rate of efflux of both pyruvate and ATP. The appearance of citrate and oxaloacetate outside mitochondria was also found as a result of phosphoenolpyruvate addition.

     

Arsenite oxidation in batch reactors with alginate-immobilized ULPAs1 strain

Arsenic is one of the major groundwater contaminants worldwide. It was previously demonstrated that the beta-proteobacterium Cenibacterium arsenoxidans has an efficient As[III] oxidation ability. The present study was conducted to evaluate the performance of alginate-immobilized ULPAs1 in the oxidation of As[III] to As[V] in batch reactors. A two-level full factorial experimental design was applied to investigate the influence of main parameters involved in the oxidation process, i.e., pH (7-8), temperature (4 degrees C-25 degrees C), kind of nutrient media (2%-20% sauerkraut brine), and arsenic concentration (10-100 mg/L). One hundred milligram per liter of As[III] was fully oxidized by calcium-alginate immobilized cells in 1 h. It was found that the temperature as well as the kind of nutrient media used were significant parameters at a 95% confidence interval whereas only temperature was a significant parameter at a 99% confidence interval. The immobilization of the As[III] oxidizing strain in alginate beads offers a promising way to implement new treatment processes in the remediation of arsenic contaminated waters.     

Removal of selenium and arsenic by animal biopolymers

The animal biopolymers prepared from hen eggshell membrane and broiler chicken feathers, which are byproducts of the poultry-processing industry, were evaluated for the removal of the oxyanions selenium [Se(IV) and Se(VI)] and arsenic [As(III) and As(V)] from aqueous solutions. The biopolymers were found to be effective at removing Se(VI) from solution. Optimal Se(IV) and Se(VI) removal was achieved at pH 2.5–3.5. At an initial Se concentration of 100 mg/L (1.3 m M), the eggshell membrane removed approx 90% Se(VI) from the solution. Arsenic was removed less effectively than Se, but the chemical modification of biopolymer carboxyl groups dramatically enhanced the As(V) sorption capacity. Se(VI) and As(V) sorption isotherms were developed at optimal conditions and sorption equilibrium data fitted the Langmuir isotherm model. The maximum uptakes by the Langmuir model were about 37.0 mg/g and 20.7 mg/g of Se(VI) and 24.2 mg/g and 21.7 mg/g of As(V) for eggshell membrane and chicken feathers, respectively.     

外源As(Ⅲ)在不同母质发育土壤中的老化过程

应用模拟试验方法,研究了外源As(Ⅲ)在第四纪红土、紫色砂页岩和花岗岩3种母质发育土壤中的老化过程,并分析了该过程中砷的有效性、结合态等的变化.结果表明: 经过120 d老化后,3种土壤中均只能检测到As(Ⅴ)的存在;伴随老化过程的进行,土壤中有效态砷的含量均呈下降趋势,且下降幅度为:紫色砂页岩发育土壤(RS₂)>花岗岩发育土壤(RS₃)>第四纪红土发育土壤(RS₁).应用准二级动力学方程可以较好地模拟3种土壤中有效态砷含量的变化(P＜0.05),土壤pH、有机质及铁铝锰氧化物是影响砷老化的主要因素,且锰氧化物的影响大于铁铝氧化物(P＜0.05).相关分析表明,非专性吸附态砷和专性吸附态砷是构成土壤有效砷的主要形态.