A Bacterial Iron Exporter for Maintenance of Iron Homeostasis

Nutritional iron acquisition by bacteria is well described, but almost nothing is known about bacterial iron export even though it is likely to be an important homeostatic mechanism. Here, we show that Bradyrhizobium japonicum MbfA (Blr7895) is an inner membrane protein expressed in cells specifically under high iron conditions. MbfA contains an N-terminal ferritin-like domain (FLD) and a C-terminal domain homologous to the eukaryotic vacuolar membrane Fe²⁺/Mn²⁺ transporter CCC1. An mbfA deletion mutant is severely defective in iron export activity, contains >2-fold more intracellular iron than the parent strain, and displays an aberrant iron-dependent gene expression phenotype. B. japonicum is highly resistant to iron and H₂O₂ stresses, and MbfA contributes substantially to this as determined by phenotypes of the mbfA mutant strain. The N-terminal FLD was localized to the cytoplasmic side of the inner membrane. Substitution mutations in the putative iron-binding amino acid residues E20A and E107A within the N-terminal FLD abrogate iron export activity and stress response function. Purified soluble FLD oxidizes ferrous iron (Fe²⁺) to incorporate ferric iron (Fe³⁺) in a 2:1 iron:protein ratio, which does not occur in the E20A/E107A mutant. The FLD fragment is a dimer in solution, implying that the MbfA exporter functions as a dimer. MbfA belongs to a protein family found in numerous prokaryotic genera. The findings strongly suggest that iron export plays an important role in bacterial iron homeostasis.     

Characterization of a vacuolar zinc transporter OZT1 in rice (Oryza sativa L.)

The CDF family is a ubiquitous family that has been identified in prokaryotes, eukaryotes, and archaea. Members of this family are important heavy metal transporters that transport metal ions out of the cytoplasm. In this research, a full length cDNA named Oryza sativa Zn Transporter 1 (OZT1) that closely related to rat ZnT-2 (Zn Transporter 2) gene was isolated from rice. The OZT1 encoding a CDF family protein shares 28.2 % ~ 84.3 % of identities and 49.3 % ~ 90.9 % of similarities with other zinc transporters such as RnZnT-2, HsZnT-8, RnZnT-8 and AtMTP1. OZT1 was constitutively expressed in various rice tissues. The OZT1 expression was significantly induced both in the seedlings of japonica rice Nipponbare and indica rice IR26 in response to Zn²⁺ and Cd²⁺ treatments. Besides, OZT1 expression was also increased when exposed to other excess metals, such as Cu²⁺, Fe²⁺ and Mg²⁺. Subcellular localization analysis indicated that OZT1 localized to vacuole. Heterologous expression of OZT1 in yeast increased tolerance to Zn²⁺ and Cd²⁺ stress but not the Mg²⁺ stress. Together, OZT1 is a CDF family vacuolar zinc transporter conferring tolerance to Zn²⁺ and Cd²⁺ stress, which is important to transporting and homeostasis of Zn, Cd or other heavy metals in plants.     

Cadmium uptake kinetics in intact soybean plants

The absorption characteristics of Cd²⁺ by 10- to 12-day-old soybean plants (Glycine max cv Williams) were investigated with respect to influence of Cd concentration on adsorption to root surfaces, root absorption, transport kinetics and interaction with the nutrient cations Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. The fraction of nonexchangeable Cd bound to roots remained relatively constant at 20 to 25% of the absorbed fraction at solution concentration of 0.0025 to 0.5 micromolar, and increased to 45% at solution concentration in excess of 0.5 micromolar. The exchangeable fraction represented 1.4 to 32% of the absorbed fraction, and was concentration dependent. Using dinitrophenol as a metabolic inhibitor, the `metabolically absorbed' fraction was shown to represent 75 to 80% of the absorbed fraction at concentration less than 0.5 micromolar, and decreased to 55% at 5 micromolar. At comparatively low Cd concentrations, 0.0025 to micromolar 0.3, root absorption exhibited two isotherms with K₂ values of 0.08 and 1.2 micromolar. Root absorption and transfer from root to shoot of Cd²⁺ was inhibited by Cu²⁺, Fe²⁺, Mn²⁺, and Zn²⁺. Analyses of kinetic interaction of these nutrient cations with Cd²⁺ indicated that Cu²⁺, Fe²⁺, Zn²⁺, and possibly Mn²⁺ inhibited Cd absorption competitively suggesting an involvement of a common transport site or process.     

Derivatives of the purple phosphatase from red kidney bean: Replacement of zinc with other divalent metal ions

Apoenzyme, containing ⩽0.1 zinc atoms and ⩽0.2 Fe atoms per subunit and with ⩽3% of the phosphatase activity, has been prepared from native red kidney bean purple phosphatase. Treatment of this apoenzyme with Fe³⁺ or Zn²⁺ separately gave very little recovery of activity, whereas treatment with both Fe³⁺ and Zn²⁺ resulted in complete restoration of activity, indicating that both metal ions are essential. ZnFe enzyme with close to one iron and one zinc atom per subunit has been reconstituted by this procedure. Essentially full reactivation was also achieved by addition of Fe³⁺ together with Fe²⁺ or Co²⁺ to the apoenzyme; Fe³⁺ and Cd²⁺ gave 27% restoration of activity, whereas Fe³⁺ with Mn²⁺, Cu²⁺, Ni²⁺ or Hg²⁺ gave little or no increase in activity. Kinetic parameters for the hydrolysis of p-nitrophenyl phosphate and ATP by the FeFe derivative are reported.     

Metal binding to the tetrathiolate motif of desulforedoxin and related polypeptides

 Desulforedoxin and the N-terminus of desulfoferrodoxin share a 36 amino acid domain containing a (Cys-S)₄ metal binding site. Recombinant forms of desulforedoxin, an N-terminal fragment of desulfoferrodoxin, and two desulforedoxin mutant proteins were reconstituted with Fe³⁺, Cd²⁺, and Zn²⁺ and relative metal ion affinities assessed by proton titrations. Protons compete with metal for protein ligands, a process that can be followed by monitoring the optical spectrum of the metal-protein complex as a function of pH. For all polypeptides, Fe³⁺ bound with the highest affinity, whereas the affinity of Zn²⁺ was greater than Cd²⁺ in desulforedoxin and the N-terminal fragment of desulfoferrodoxin, but this order was reversed in desulforedoxin mutant proteins. Metal binding in both mutants was significantly impaired. Furthermore, the Fe³⁺ complex of both mutants underwent a time-dependent bleaching process which coincided with increased reactivity of cysteine residues to Ellman's reagent and concomitant metal dissociation. It is hypothesized that this results from an autoredox reaction in which Fe³⁺ is reduced to Fe²⁺ with attendant oxidation of ligand thiols. Received: 17 June 1998 / Accepted: 3 September 1998     

ZIP8 Is an Iron and Zinc Transporter Whose Cell-surface Expression Is Up-regulated by Cellular Iron Loading

ZIP8 (SLC39A8) belongs to the ZIP family of metal-ion transporters. Among the ZIP proteins, ZIP8 is most closely related to ZIP14, which can transport iron, zinc, manganese, and cadmium. Here we investigated the iron transport ability of ZIP8, its subcellular localization, pH dependence, and regulation by iron. Transfection of HEK 293T cells with ZIP8 cDNA enhanced the uptake of ⁵⁹Fe and ⁶⁵Zn by 200 and 40%, respectively, compared with controls. Excess iron inhibited the uptake of zinc and vice versa. In RNA-injected Xenopus oocytes, ZIP8-mediated ⁵⁵Fe²⁺ transport was saturable (K_0.5 of ∼0.7 μm) and inhibited by zinc. ZIP8 also mediated the uptake of ¹⁰⁹Cd²⁺, ⁵⁷Co²⁺, ⁶⁵Zn²⁺ > ⁵⁴Mn²⁺, but not ⁶⁴Cu (I or II). By using immunofluorescence analysis, we found that ZIP8 expressed in HEK 293T cells localized to the plasma membrane and partially in early endosomes. Iron loading increased total and cell-surface levels of ZIP8 in H4IIE rat hepatoma cells. We also determined by using site-directed mutagenesis that asparagine residues 40, 88, and 96 of rat ZIP8 are glycosylated and that N-glycosylation is not required for iron or zinc transport. Analysis of 20 different human tissues revealed abundant ZIP8 expression in lung and placenta and showed that its expression profile differs markedly from ZIP14, suggesting nonredundant functions. Suppression of endogenous ZIP8 expression in BeWo cells, a placental cell line, reduced iron uptake by ∼40%, suggesting that ZIP8 participates in placental iron transport. Collectively, these data identify ZIP8 as an iron transport protein that may function in iron metabolism.     

Promotion of Iron Oxide Reduction and Extracellular Electron Transfer in Shewanella oneidensis by DMSO

The dissimilatory metal reducing bacterium Shewanella oneidensis MR-1, known for its capacity of reducing iron and manganese oxides, has great environmental impacts. The iron oxides reducing process is affected by the coexistence of alternative electron acceptors in the environment, while investigation into it is limited so far. In this work, the impact of dimethyl sulphoxide (DMSO), a ubiquitous chemical in marine environment, on the reduction of hydrous ferric oxide (HFO) by S. oneidensis MR-1 was investigated. Results show that DMSO promoted HFO reduction by both wild type and ΔdmsE, but had no effect on the HFO reduction by ΔdmsB, indicating that such a promotion was dependent on the DMSO respiration. With the DMSO dosing, the levels of extracellular flavins and omcA expression were significantly increased in WT and further increased in ΔdmsE. Bioelectrochemical analysis show that DMSO also promoted the extracellular electron transfer of WT and ΔdmsE. These results demonstrate that DMSO could stimulate the HFO reduction through metabolic and genetic regulation in S. oneidensis MR-1, rather than compete for electrons with HFO. This may provide a potential respiratory pathway to enhance the microbial electron flows for environmental and engineering applications.     

黄瓜MTP基因家族分析及重金属胁迫下表达特征

该研究以模式物种(拟南芥、水稻、玉米)的MTP序列作为种子序列,对黄瓜MTP基因家族(CsMTP)成员进行了系统鉴定和分析;并以Zn~(2+)、Cd~(2+)、Mn~(2+)、Cu~(2+)、Fe~(2+)和Mo~(2+)处理后的黄瓜叶片和根系为材料,对CsMTP响应金属离子的表达模式进行了研究,为探究该基因家族对黄瓜重金属胁迫调控机制提供理论依据。结果表明:(1)黄瓜含有10个CsMTP,可进一步分为Fe/Zn-CDF(2个)、Mn-CDF(3个)和Zn-CDF(5个)三个亚家族,Mn-CDF成员含有保守基序DxxxD,Zn-CDF和Fe/Zn-CDFs成员含有保守基序HxxxD,大部分CsMTP成员含有6个跨膜结构域。(2)RNA-seq数据分析表明,CsMTP具备组织特异性表达和响应不同处理的特征,CsMTP11、CsMTP3和CsMTP7在不同组织和处理下普遍高水平表达,且外源化学试剂处理可诱导同一基因的不同表达模式。(3)RT-PCR分析表明,CsMTPs可被不同重金属离子诱导差异表达,包括不是MTP家族潜在底物的重金属。     

Cadmium ion stimulation of growth and versicolorin synthesis in a mutant strain ofAspergillus parasiticus

Cultures ofAspergillus parasiticus produce the polyketide versicolorin A in response to elevation of the Zn²⁺ content of the growth medium. With suboptimal Zn²⁺ (0.8 μM) mycelial growth is about half maximal, and versicolorin synthesis is essentially zero. Inclusion of Cd²⁺ (1–100 μM) in the Zn²⁺-limiting growth medium allows optimal growth and stimulates full versicolorin synthesis. Cd²⁺, like Zn²⁺, will stimulate versicolorin sysnthesis only when added within the first 30 h after conidial inoculation. The transport system for Cd²⁺ uptake may be the same as that for Zn²⁺, as judged byin vivo competition studies. Cd²⁺ is a competitive inhibitor of Zn²⁺ uptake, with K_i = 20 μM.     

A Combined Zinc/Cadmium Sensor and Zinc/Cadmium Export Regulator in a Heavy Metal Pump

Heavy metal pumps (P1B-ATPases) are important for cellular heavy metal homeostasis. AtHMA4, an Arabidopsis thaliana heavy metal pump of importance for plant Zn²⁺ nutrition, has an extended C-terminal domain containing 13 cysteine pairs and a terminal stretch of 11 histidines. Using a novel size-exclusion chromatography, inductively coupled plasma mass spectrometry approach we report that the C-terminal domain of AtHMA4 is a high affinity Zn²⁺ and Cd²⁺ chelator with capacity to bind 10 Zn²⁺ ions per C terminus. When AtHMA4 is expressed in a Zn²⁺-sensitive zrc1 cot1 yeast strain, sequential removal of the histidine stretch and the cysteine pairs confers a gradual increase in Zn²⁺ and Cd²⁺ tolerance and lowered Zn²⁺ and Cd²⁺ content of transformed yeast cells. We conclude that the C-terminal domain of AtHMA4 serves a dual role as Zn²⁺ and Cd²⁺ chelator (sensor) and as a regulator of the efficiency of Zn²⁺ and Cd²⁺ export. The identification of a post-translational handle on Zn²⁺ and Cd²⁺ transport efficiency opens new perspectives for regulation of Zn²⁺ nutrition and tolerance in eukaryotes.     

Resistance acquisition of Thiobacillus thiooxidans upon cadmium and zinc ion addition and formation of cadmium ion-binding and zinc ion-binding proteins exhibiting metallothionein-like properties

Through subcultivations of Thiobacillus thiooxidans WU-79A in autotrophic media in which the concentrations of Cd²⁺ and Zn²⁺ were increased successively, Cd²⁺-resistant (CDR) and Zn²⁺-resistant strains (ZNR) were obtained. The growth of WU-79A was inhibited by the addition of 25 mM Cd²⁺ as well as Zn²⁺. However, CDR and ZNR could grow without any lag phase in media containing 200 mM Cd²⁺ and 250 mM Zn²⁺, respectively. CDR and ZNR were able to grow even in media containing up to 400 mM Cd²⁺ and 600 mM Zn²⁺, respectively, although they exhibited lag phases. CDR could grow in medium containing up to 250 mM Zn²⁺, as could ZNR in medium containing up to 200 mM Cd²⁺. Cd²⁺-binding and Zn²⁺-binding proteins were isolated from CDR and ZNR, respectively, by gel filtration and ion exchange chromatography. The molecular weights of both proteins were estimated to be approximately 13,000 by gel filtration. The fact that there was no strong absorption at 280 nm of the proteins suggested that they had few aromatic amino acids. Broad absorption bands which are typical of mercaptide (metal thiolate) complexes were detected. The properties of the proteins were spectrophotometrically similar to those of metallothionein.     

Iron promotes cadmium binding to citrate

Ironcadmium interactions are important in cadmium toxicity. Dietary iron supplements may decrease cadmium retention after oral cadmium exposure but the underlying mechanism is not known. Using a CdS/AgS ion selective electrode to measure [Cd²⁺] in physiological saline solution at pH 7.4, we show that Fe²⁺ promotes Cd²⁺ binding to citrate thereby decreasing the availability of free Cd²⁺. This suggests the formation of high molecular weight Cd²⁺Fe²⁺citrate complexes. We confirm this suggestion by showing that ¹⁰⁹Cd²⁺ is retained by 1 kDa cut off filters when present with total 50 M Fe²⁺ plus 1 mM citrate but not when present with citrate alone. The formation of high molecular weight complexes may prevent Cd²⁺ absorption. As citrate is part of the diet, we suggest that these ironcadmium interactions may contribute to the protective effect of iron against cadmium toxicity.     

Fluorescence detection of intracellular cadmium with Leadmium Green

Leadmium Green is a commercially available, small molecule, fluorescent probe advertised as a detector of free intracellular cadmium (Cd²⁺) and lead (Pb²⁺). Leadmium Green has been used in various paradigms, such as tracking Cd²⁺ sequestration in plant cells, heavy metal export in protozoa, and Pb²⁺ absorption by vascular endothelial cells. However very little information is available regarding its affinity and selectivity for Cd²⁺, Pb²⁺, and other metals. We evaluated the in vitro selectivity of Leadmium Green using spectrofluorimetry. Consistent with manufacturer’s claims, Leadmium Green was sensitive to Cd²⁺ (K_D ~600 nM) and also Pb²⁺ (K_D ~9.0 nM) in a concentration-dependent manner, and furthermore proved insensitive to Ca²⁺, Co²⁺, Mn²⁺ and Ni²⁺. Leadmium Green also responded to Zn²⁺ with a K_D of ~82 nM. Using fluorescence microscopy, we evaluated Leadmium Green in live mouse hippocampal HT22 cells. We demonstrated that Leadmium Green detected ionophore-mediated acute elevations of Cd²⁺ or Zn²⁺ in a concentration-dependent manner. However, the maximum fluorescence produced by ionophore-delivered Zn²⁺ was much less than that produced by Cd²⁺. When tested in a model of oxidant-induced liberation of endogenous Zn²⁺, Leadmium Green responded weakly. We conclude that Leadmium Green is an effective probe for monitoring intracellular Cd²⁺, particularly in models where Cd²⁺ accumulates rapidly, and when concomitant fluctuations of intracellular Zn²⁺ are minimal.     

不同浓度Fe2+与Zn2+对羊草幼苗生长和生理特性的影响

以羊草幼苗为研究对象,通过调整全营养培养基(CK,0.05 mmol/L Fe²⁺、0.015 mmol/L Zn²⁺)中铁或者锌含量设置0、10倍、20倍Fe²⁺(Zn²⁺)浓度处理Fe₀(Zn₀)、Fe₁₀(Zn₁₀)、Fe₂₀(Zn₂₀),以及在高铁培养基中单独添加0.15 mmol/L Zn²⁺或同时添加10 mmol/L Ca²⁺、5 mmol/L Mg²⁺、20 mmol/L K⁺处理,测定培养6 d后幼苗生长指标和矿质元素含量、以及高铁(Fe₂₀)处理下幼苗根中抗氧化指标和相关基因表达量,探究不同浓度Fe²⁺、Zn²⁺对羊草幼苗生长、矿质元素吸收积累及抗氧化指标、基因表达的影响。结果表明：(1)缺锌(Zn₀)显著抑制羊草幼苗鲜重的增加和Zn元素的积累,但促进Fe、Mg元素的积累;高浓度锌(Zn₁₀、Zn₂₀)显著促进幼苗叶片生长和Zn元素的积累;缺铁(Fe₀)显著抑制幼苗的根长、鲜重和Fe元素的积累,促进Mg、Zn元素的积累;高浓度铁(Fe₁₀、Fe₂₀)显著抑制羊草幼苗根叶生长、根毛发育和Ca、Zn、Mg、K元素的积累。(2)增加Zn²⁺和Ca²⁺、Mg²⁺、K⁺浓度无法恢复高铁胁迫对幼苗生长的抑制作用。(3)高浓度铁(Fe₂₀)处理羊草幼苗48 h后,根部过氧化物酶、超氧化物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶、谷胱甘肽还原酶活性和丙二醛、抗坏血酸、还原型谷胱甘肽含量显著升高;烟酰胺合成酶基因、过氧化物酶基因表达量显著下调,植物类萌发素蛋白基因表达量显著上调。研究发现,羊草幼苗生长发育和矿质元素积累对环境中Zn²⁺浓度变化不敏感,却受到环境中高浓度Fe²⁺的显著抑制,并造成严重的氧化胁迫伤害,这种伤害无法在添加Zn²⁺或同时添加Ca²⁺、Mg²⁺、K⁺的条件下恢复。     

The interaction of zinc, nickel and cadmium with serum albumin and histidine-rich glycoprotein assessed by equilibrium dialysis and immunoadsorbent chromatography

Human serum albumin (HSA) has been shown to bind 2–3 mol of Zn²⁺, Ni²⁺, or Cd²⁺ per mole of protein with apparent dissociation constants (K_d) in the range of 10 μm. Rabbit histidine-rich glycoprotein (HRG) binds 13, 9, and 6 mol of Zn²⁺, Ni²⁺, and Cd²⁺ per mole of protein, respectively, with apparent K_ds also near 10 μm. However, the binding of metals by HRG exhibits positive cooperativity, so that the apparent K_ds may underestimate HRGs true affinity for metal ions. The relative affinities of HSA and HRG for metal ions were found to be Zn²⁺ > Ni²⁺ > Cd²⁺. In addition, histidine (a serum metal chelator) affected the binding of Ni²⁺ by both proteins but not that of Zn²⁺ or Cd²⁺. At physiological concentrations of HSA (250 μm), HRG (2.5 μm), and histidine (100 μm), HRG bound 36% of the Zn²⁺, 9% of the Ni²⁺, and 13% of the Cd²⁺ at a total metal concentration of 25 μm. Under the same conditions HSA held 37% of the Zn²⁺, 14% of the Ni²⁺, and 56% of the Cd²⁺. Thus, HSA appears to have a lower intrinsic affinity for the three metals than HRG but would be expected to bind a higher proportion of these metals in serum. A specific immunoadsorbent column was prepared and used to study the metal binding by HRG in serum directly. Both ⁶⁵Zn²⁺ and ⁶³Ni²⁺ were associated with HRG in aliquots of rabbit serum after incubation with the corresponding metal ion. This evidence indicates that HRG must be considered as a metal binding component of serum.     

Production of Siderophores Increases Resistance to Fusaric Acid in Pseudomonas protegens Pf-5

Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe²⁺ and Fe³⁺, but also Zn²⁺, Mn²⁺ and Cu²⁺, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.     

Heavy metal ions affect the activity of DNA glycosylases of the Fpg family

Prokaryotic enzymes formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease VIII (Nei) and their eukaryotic homologs NEIL1, NEIL2, and NEIL3 define the Fpg family of DNA glycosylases, which initiate the process of repair of oxidized DNA bases. The repair of oxidative DNA lesions is known to be impaired in vivo in the presence of ions of some heavy metals. We have studied the effect of salts of several alkaline earth and transition metals on the activity of Fpg-family DNA glycosylases in the reaction of excision of 5,6-dihydrouracil, a typical DNA oxidation product. The reaction catalyzed by NEIL1 was characterized by values K _m = 150 nM and k _cat = 1.2 min⁻¹, which were in the range of these constants for excision of other damaged bases by this enzyme. NEIL1 was inhibited by Al³⁺, Ni²⁺, Co²⁺, Cd²⁺, Cu²⁺, Zn²⁺, and Fe²⁺ in Tris-HCl buffer and by Cd²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ in potassium phosphate buffer. Fpg and Nei, the prokaryotic homologs of NEIL1, were inhibited by the same metal ions as NEIL1. The values of I₅₀ for NEIL1 inhibition were 7 μM for Cd²⁺, 16 μM for Zn²⁺, and 400 μM for Cu²⁺. The inhibition of NEIL1 by Cd²⁺, Zn²⁺, and Cu²⁺ was at least partly due to the formation of metal-DNA complexes. In the case of Cd²⁺ and Cu²⁺, which preferentially bind to DNA bases rather than phosphates, the presence of metal ions caused the enzyme to lose the ability for preferential binding to damaged DNA. Therefore, the inhibition of NEIL1 activity in removal of oxidative lesions by heavy metal ions may be a reason for their comutagenicity under oxidative stress.     

Substrate Profile and Metal-ion Selectivity of Human Divalent Metal-ion Transporter-1

Divalent metal-ion transporter-1 (DMT1) is a H⁺-coupled metal-ion transporter that plays essential roles in iron homeostasis. DMT1 exhibits reactivity (based on evoked currents) with a broad range of metal ions; however, direct measurement of transport is lacking for many of its potential substrates. We performed a comprehensive substrate-profile analysis for human DMT1 expressed in RNA-injected Xenopus oocytes by using radiotracer assays and the continuous measurement of transport by fluorescence with the metal-sensitive PhenGreen SK fluorophore. We provide validation for the use of PhenGreen SK fluorescence quenching as a reporter of cellular metal-ion uptake. We determined metal-ion selectivity under fixed conditions using the voltage clamp. Radiotracer and continuous measurement of transport by fluorescence assays revealed that DMT1 mediates the transport of several metal ions that were ranked in selectivity by using the ratio I_max/K_0.5 (determined from evoked currents at −70 mV): Cd²⁺ > Fe²⁺ > Co²⁺, Mn²⁺ ≫ Zn²⁺, Ni²⁺, VO²⁺. DMT1 expression did not stimulate the transport of Cr²⁺, Cr³⁺, Cu⁺, Cu²⁺, Fe³⁺, Ga³⁺, Hg²⁺, or VO⁺. ⁵⁵Fe²⁺ transport was competitively inhibited by Co²⁺ and Mn²⁺. Zn²⁺ only weakly inhibited ⁵⁵Fe²⁺ transport. Our data reveal that DMT1 selects Fe²⁺ over its other physiological substrates and provides a basis for predicting the contribution of DMT1 to intestinal, nasal, and pulmonary absorption of metal ions and their cellular uptake in other tissues. Whereas DMT1 is a likely route of entry for the toxic heavy metal cadmium, and may serve the metabolism of cobalt, manganese, and vanadium, we predict that DMT1 should contribute little if at all to the absorption or uptake of zinc. The conclusion in previous reports that copper is a substrate of DMT1 is not supported.     

Cadmium and mineral nutrient accumulation in potato plantlets grown under cadmium stress in two different experimental culture conditions

In order to evaluate the effect of cadmium (Cd²⁺) toxicity on mineral nutrient accumulation in potato (Solanum tuberosum L.), two cultivars named Asterix and Macaca were cultivated both in vitro and in hydroponic experiments under increasing levels of Cd²⁺ (0, 100, 200, 300, 400 and 500 μM in vitro and 0, 50, 100, 150 and 200 μM in hydroponic culture). At 22 and 7 days of exposure to Cd²⁺, for the in vitro and hydroponic experiment, respectively, the plantlets were separated into roots and shoot, which were analyzed for biomass as well as Cd²⁺, and macro (Ca²⁺, K⁺ and Mg²⁺) and micronutrient (Cu²⁺, Fe²⁺, Mn²⁺ and Zn²⁺) contents. In the hydroponic experiment, there was no reduction in shoot and root dry weight for any Cd²⁺ level, regardless of the potato cultivar. In contrast, in the in vitro experiment, there was an increase in biomass at low Cd²⁺ levels, while higher Cd²⁺ levels caused a decrease. In general, Cd²⁺ decreased the macronutrient and micronutrient contents in the in vitro cultured plantlets in both roots and shoot of cultivars. In contrast, the macronutrient and micronutrient contents in the hydroponically grown plantlets were generally not affected by Cd²⁺. Our data suggest that the influence of Cd²⁺ on nutrient content in potato was related to the level of Cd²⁺ in the substrate, potato cultivar, plant organ, essential element, growth medium and exposure time.     

Application of Zero-Valent Iron Nanoparticles for the Removal of Aqueous Zinc Ions under Various Experimental Conditions

Application of zero-valent iron nanoparticles (nZVI) for Zn²⁺ removal and its mechanism were discussed. It demonstrated that the uptake of Zn²⁺ by nZVI was efficient. With the solids concentration of 1 g/L nZVI, more than 85% of Zn²⁺ could be removed within 2 h. The pH value and dissolved oxygen (DO) were the important factors of Zn²⁺ removal by nZVI. The DO enhanced the removal efficiency of Zn²⁺. Under the oxygen-contained condition, oxygen corrosion gave the nZVI surface a shell of iron (oxy)hydroxide, which could show high adsorption affinity. The removal efficiency of Zn²⁺ increased with the increasing of the pH. Acidic condition reduced the removal efficiency of Zn²⁺ by nZVI because the existing H⁺ inhibited the formation of iron (oxy)hydroxide. Adsorption and co-precipitation were the most likely mechanism of Zn²⁺ removal by nZVI. The FeOOH-shell could enhance the adsorption efficiency of nZVI. The removal efficiency and selectivity of nZVI particles for Zn²⁺ were higher than Cd²⁺. Furthermore, a continuous flow reactor for engineering application of nZVI was designed and exhibited high removal efficiency for Zn²⁺.