BioMetals (v.21, #5)

Phytosiderophores (PS) are strong iron chelators, produced by graminaceous plants under iron deficiency. The ability of released PS to chelate iron(III), and subsequent uptake of this chelate into roots by YS1-type transport proteins, are well-known. The mechanism of iron release from the stable chelate inside the plant cell, however, is unclear. One possibility involves the reduction of ferric PS in the presence of an iron(II) chelator via ternary complex formation. Here, the conversion of ferric PS species by ascorbate in the presence of the intracellular ligand nicotianamine (NA) has been investigated at cytosolic pH (pH 7.3), leading to the formation of a ferrous NA chelate. This reaction takes place when supplying Fe(III) as a chelate with 2′-deoxymugineic acid (DMA), mugineic acid (MA), and 3-epi-hydroxymugineic acid (epi-HMA), with the reaction rate decreasing in this order. The progress of the conversion of ferric DMA to ferrous NA was monitored in real-time by high resolution mass spectrometry (FTICR-MS), and the results are complemented by electrochemical measurements (cyclic voltammetry), which allows detecting reactive intermediates and their change with time at high sensitivity. Hence, the combined results of electrochemistry and mass spectrometry indicate an ascorbate-mediated mechanism for the iron release from ferric PS, which highlights the role of ascorbate as a simple, but effective plant reductant.
Keywords: Phytosiderophores; Iron; Reduction; Cyclic voltammetry; Mass spectrometry

East Kolkata Wetlands is a conserved wetland utilizing sewage and garbage, generated by Kolkata Municipal Corporation area for cultivation purpose. Cyanobacteria are the photosynthetic prokaryotes having bioremedial capacity. We have isolated a cyanobacterium from the sewage recycling fish-pond of East Kolkata Wetlands. Partial sequence of 16S rDNA gene of the isolated strain showed 100% similarity with that of genus Synechocystis. Isolated strain and Synechocystis sp. PCC6803 survived up to 300 μg ml−1 Pb2+ and growth was completely inhibited at 400 μg ml−1 Pb2+. All experiments were carried out with 100 μg ml−1 Pb2+ in which growth was the maximum. 91.67% of the total Pb2+ got adsorbed to the outer surface of the cell and 1% of the total Pb2+ entered the cell of the isolated strain as estimated by atomic absorption spectrometry, but in Synechocystis sp. PCC6803 72.72% adsorbed and 0.96% penetrated. Intracellular and periplasmic depositions of Pb2+ were observed in both the strain. A filamentous structure developed outside the cell wall of the isolated cyanobacterium, but very little change was observed in Synechocystis sp. PCC6803. ZiaR—SmtB like regulator gene was expressed in both the strains after Pb2+ induction. The cDNA sequence of ZiaR of the isolated cyanobacterium shows 100% homology with that of Synechocystis sp. PCC6803. Upon Pb2+ induction, expression of SOD gene increased. cDNA sequence of the SOD gene from the isolated strain showed 98% homology with that of Synechocystis sp. PCC6803. Enzymatic activity of catalase and SOD was also increased. No DNA damage was monitored upon induction with Pb2+.
Keywords: Synechocystis sp. PCC6803; East Kolkata Wetlands; Pb2+ uptake; 16S rDNA; Ramsar site

Copper binding components of blood plasma and organs, and their responses to influx of large doses of 65Cu, in the mouse by Anthony Cabrera; Erin Alonzo; Eric Sauble; Yu Ling Chu; Dionne Nguyen; Maria C. Linder; Dee S. Sato; Andrew Z. Mason (525-543).
To establish for the first time how mice might differ from rats and humans in terms of copper transport, excretion, and copper binding proteins, plasma and organ cytosols from adult female C57CL6 mice were fractionated and analyzed by directly coupled size exclusion HPLC-ICP-MS, before and after i.p. injection of large doses of 65Cu. Plasma from untreated mice had different proportions of Cu associated with transcuprein/macroglobulin, ceruloplasmin and albumin than in humans and rats, and two previously undetected copper peaks (Mr 700 k and 15 k) were observed. Cytosols had Cu peaks seen previously in rat liver (Mr > 1000 k, 45 k and 11 k) plus one of 110 kDa. 65Cu (141 μg) administered over 14 h, initially loaded plasma albumin and mainly entered liver and kidney (especially 28 kDa and 11 kDa components). Components of other organs were less (but still significantly) enriched. 63Cu/65Cu ratios returned almost to normal by 14 days, indicating a robust system for excreting excess copper. We conclude that there are significant differences but also strong similarities in copper metabolism between mice, rats and humans; that the liver is able to buffer enormous changes in copper status; and that a large number of mammalian copper proteins remain to be identified.
Keywords: Copper binding proteins; Mouse plasma; Cytosol; Heavy isotope; Excretion

Enhancement of hippocampal mossy fiber activity in zinc deficiency and its influence on behavior by Atsushi Takeda; Hiromasa Itoh; Kohei Yamada; Haruna Tamano; Naoto Oku (545-552).
The extracellular concentration of glutamate in the hippocampus is increased by hippocampal perfusion with CaEDTA, a membrane-impermeable zinc chelator, suggesting that the activity of glutamatergic neurons in the hippocampus are influenced by the extracellular concentrations of zinc. In the present study, the relationship between the extracellular concentrations of zinc and mossy fiber activity in the hippocampus was examined in mice and rats fed a zinc-deficient diet for 4 weeks. Timm’s stain, by which histochemically reactive zinc in the presynaptic vesicles is detected, was attenuated in the hippocampus in zinc deficiency. The extracellular signal of ZnAF-2, a membrane-impermeable zinc indicator, was also lower in the hippocampal CA3, suggesting that the basal extracellular concentrations of zinc are lower maintained in zinc deficiency. To check mossy fiber activity after 4-week zinc deprivation, the decrease in the signal of FM4-64, an indicator of presynaptic activity (exocytosis), at mossy fiber synapses was measured under the condition of spontaneous depolarization. The decrease was significantly facilitated by zinc deficiency, suggesting that the basal exocytosis at mossy fiber synapses is enhanced by zinc deficiency. On the other hand, the increase in anxiety-like behavior was observed in the open-field test after 4-week zinc deprivation. The present study demonstrates that the decrease in the basal extracellular concentrations of zinc may be linked to the enhancement of the basal mossy fiber activity in zinc deficiency. This decrease seems to be also involved in neuropsychological behavior in zinc deficiency.
Keywords: Synaptic zinc; Mossy fiber; Exocytosis; Hippocampus; Zinc deficiency; Neuropsychological behavior

Influences of calcium deficiency and cerium on the conversion efficiency of light energy of spinach by Hao Huang; Xiaoqing Liu; Chunxiang Qu; Chao Liu; Liang Chen; Fashui Hong (553-561).
Chloroplast absorbs light energy and transforms it into electron energy, and then converts it into active chemical energy and stable chemical energy. In the present paper, we investigated the effects of Ce3+, which has the most significant catalytic effects and similar characteristics with Ca2+, on light energy conversion of spinach chloroplasts under Ca2+-deficient stress. The results illuminated that the Hill reaction activity, electron flow both photosystems and photophosphorylation rate of spinach chloroplasts reduced significantly under Ca2+-deficient condition, and activities of Mg2+-ATPase and Ca2+-ATPase on the thylakoid membrane were severely inhibited. Meanwhile, the activity of Rubisco, which is the key enzyme of photosynthetic carbon assimilation, was also prohibited. However, Ce3+ decreased the inhibition of calcium deprivation the electron transport rate, the oxygen evolution rate, the cyclic and noncyclic photophosphorylation, the activities of Mg2+-ATPase, Ca2+-ATPase and Rubisco of spinach chloroplasts. All above implied that Ca2+-depletion could disturb light energy conversion of chloroplasts strongly, which could be reversed by Ce3+.
Keywords: Ce3+ ; Ca2+-deficiency; Spinach; Chloroplast; Light energy conversion

Vanadium-enriched chickpea sprout ameliorated hyperglycemia and impaired memory in streptozotocin-induced diabetes rats by Xueqin Mao; Ling Zhang; Qing Xia; Zhaofeng Sun; Xiaomin Zhao; Hongxin Cai; Xiaoda Yang; Zuoli Xia; Yujing Tang (563-570).
Vanadium compounds have been recognized for their hypoglycemic effects; however, potential short and long-term vanadium toxicity has slowed the acceptance for therapeutic use. In the present work, three batches of vanadium-enriched chickpea sprout (VCS) were prepared by incubating chickpea seeds in presence of 200, 100, and 50 μg/ml of sodium orthovanadate (SOV). The effects of oral administration of chickpea sprout (CS) and VCS food for 8 weeks on streptozotocin-induced (STZ) diabetic rats were investigated. Both CS and VCS food was found to ameliorate some hyperglycemic symptoms of the diabetic rats, i.e. improve lipid metabolism, decrease blood glucose level, prevent body weight loss, and reduce impairment of diabetic related spatial learning and memory. Serum insulin was substantially elevated in treated diabetic rats, which is probably one important reason for the hypoglycemic effect. Compared with CS alone, VCS100 food exhibited remarkably enhanced effectiveness in alleviating diabetes induced hyperglycemia and memory loss. Moreover, vanadium-enriched chickpeas appeared to abolish the vanadium induced toxicity associated with administration of this metal for diabetes during the 8-week study period. This study suggested further work of the vanadium speciation in CS and novel hypoglycemic mechanism for the antidiabetic activity of vanadium agents. Vanadium containing (VCS) food could be a dietary supplement for the diabetic status.
Keywords: Chickpea sprout; Enriching; Streptozotocin-induced diabetes; Rats; SOV

Iron accumulation in bronchial epithelial cells is dependent on concurrent sodium transport by Jennifer L. Turi; Claude A. Piantadosi; Jackie D. Stonehuerner; Andrew J. Ghio (571-580).
Airway epithelial cells prevent damaging effects of extracellular iron by taking up the metal and sequestering it within intracellular ferritin. Epithelial iron transport is associated with transcellular movement of other cations including changes in the expression or activity of Na, K-ATPase and epithelial Na+ channel (ENaC). Given this relationship between iron and Na+, we hypothesized that iron uptake by airway epithelial cells requires concurrent Na+ transport. In preliminary studies, we found that Na+-free buffer blocked iron uptake by human airway epithelial cell. Na+ channels inhibitors, including furosemide, bumetanide, and ethylisopropyl amiloride (EIPA) significantly decreased epithelial cell concentrations of non-heme iron suggesting that Na+-dependent iron accumulation involves generalized Na+ flux into the cells rather than participation of one or more specific Na+ channels. In addition, efflux of K+ was detected during iron uptake, as was the influx of phosphate to balance the inward movement of cations. Together, these data demonstrate that intracellular iron accumulation by airway epithelium requires concurrent Na+/K+exchange.
Keywords: Iron; Sodium; Cation exchange

Petrobactin is produced by both pathogenic and non-pathogenic isolates of the Bacillus cereus group of bacteria by Andrew T. Koppisch; Suraj Dhungana; Karen K. Hill; Hakim Boukhalfa; Henry S. Heine; Leslie A. Colip; Raymond B. Romero; Yulin Shou; Lawrence O. Ticknor; Babetta L. Marrone; Larry E. Hersman; Srinivas Iyer; Christy E. Ruggiero (581-589).
Petrobactin is the primary siderophore synthesized by Bacillus anthracis str Sterne and is required for virulence of this organism in a mouse model. The siderophore’s biosynthetic machinery was recently defined and gene homologues of this operon exist in several other Bacillus strains known to be mammalian pathogens, but are absent in several known to be harmless such as B. subtilis and B. lichenformis. Thus, a common hypothesis regarding siderophore production in Bacillus species is that petrobactin production is exclusive to pathogenic isolates. In order to test this hypothesis, siderophores produced by 106 strains of an in-house library of the Bacillus cereus sensu lato group were isolated and identified using a MALDI-TOF-MS assay. Strains were selected from a previously defined phylogenetic tree of this group in order to include both known pathogens and innocuous strains. Petrobactin is produced by pathogenic strains and innocuous isolates alike, and thus is not itself indicative of virulence.
Keywords: Siderophores; Iron; Bacillus cereus ; Bacillus anthracis ; Bacillus thuringiensis

Chromate removal by yeasts isolated from sediments of a tanning factory and a mine site in Argentina by Liliana B. Villegas; Pablo M. Fernández; María J. Amoroso; Lucía I. C. de Figueroa (591-600).
Twenty-one yeast-like microorganisms were isolated from tannery effluents and from a nickel–copper mine in Argentina. They were tested for their Cu(II), Ni(II), Cd(II) and Cr(VI) tolerance in qualitative assays on solid medium. Three isolates were selected for their multiple tolerance to the different heavy metals and highest tolerance to Cr(VI). According to morphological and physiological analysis and 26S rDNA D1/D2 domain sequences the isolates were characterized as: Lecythophora sp. NGV-1, Candida sp. NGV-9 and Aureobasidium pullulans VR-8. Resistance of the three strains to high Cr(VI) concentrations and their ability to remove Cr(VI) were assessed using YNB-glucose medium supplemented with 0.5 and 1 mM Cr(VI). Chromate removal activity was estimated by measuring remaining Cr(VI) concentration in the supernatant using the colorimetric 1,5-diphenylcarbazide method and total chromium was determined by flame atomic absorption spectroscopy. The results indicate that the initial Cr(VI) concentration negatively influenced growth and the specific growth rate but stimulated the metabolic activity of the three strains; resistance to Cr(VI) by these strains was mainly due to reduction of Cr(VI) rather than chromium bioaccumulation. This study showed the potential ability of these strains as tools for bioremediation of Cr(VI) from contaminated sites.
Keywords: Bioremediation; Cr(VI) removal; Cr(VI) tolerant yeasts