Characterization of Iron Uptake from Ferrioxamine B by Chlorella vulgaris

Iron uptake from two Fe³⁺-hydroxamate siderophores, ferrioxamine B and Fe³⁺-rhodotorulate, by iron-stressed Chlorella vulgaris (ATCC strain 11468) was evaluated with some comparison to iron uptake from synthetic and organic acid ferric chelates. Iron-stress induced iron uptake from ferrioxamine B. Dissipation of the electrochemical gradient, via uncouplers, inhibited iron uptake. Respiratory inhibitors gave variable results, an indication that a direct link to respiration was not apparent. Vanadate inhibition of iron uptake indicated that an ATPase or phosphate intermediate could be involved in the uptake mechanism. Divalent cations manifested variable effects dependent on the cation and chelator used. These data confirm that C. vulgaris has an inducible iron-uptake system for Fe³⁺-hydroxamic acid siderophores which may involve a different mechanism than that observed for other chelates.     

BIOLOGICAL AVAILABILITY OF IRON TO THE FRESHWATER CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition from various ferric chelates and colloids was studied using iron‐limited cells of Anabaena flos‐aquae (Lyng.) Brèb UTEX 1444, a cyanobacterial strain that produces high levels of siderophores under iron limitation. Various chelators of greatly varying affinity for Fe³⁺ (HEDTA, EDDHA, desferrioxamine mesylate, HBED, 8‐hydroxyquinoline) were assayed for the degree of iron acquisition by iron‐limited cyanobacterial cells. Iron uptake rates (measured by graphite furnace atomic absorption spectrometry) varied approximately inversely with calculated [Fe³⁺] (calculated as pFe) and decreased with increasing chelator‐to‐iron ratio. No iron uptake was observed when Fe³⁺ was chelated with HBED, the strongest of the tested chelators. Iron‐limited Anabaena cells were able to take up iron from 8‐hydroxyquinoline (oxine or 8HQ), a compound sometimes used to quantify aquatic iron bioavailability. Iron bound to purified humic acid was poorly available but did support some growth at high humic acid concentrations. These results suggest that for cyanobacteria, even tightly bound iron is biologically available, including to a limited extent iron bound to humic acids. However, iron bound to some extremely strong chelators (e.g. HBED) is likely to be biologically unavailable.     

The yeast mitochondrial carrier proteins Mrs3p/Mrs4p mediate iron transport across the inner mitochondrial membrane

The yeast proteins Mrs3p and Mrs4p are two closely related members of the mitochondrial carrier family (MCF), which had previously been implicated in mitochondrial Fe²⁺ homeostasis. A vertebrate Mrs3/4 homologue named mitoferrin was shown to be essential for erythroid iron utilization and proposed to function as an essential mitochondrial iron importer. Indirect reporter assays in isolated yeast mitochondria indicated that the Mrs3/4 proteins are involved in mitochondrial Fe²⁺ utilization or transport under iron-limiting conditions. To have a more direct test for Mrs3/4p mediated iron uptake into mitochondria we studied iron (II) transport across yeast inner mitochondrial membrane vesicles (SMPs) using the iron-sensitive fluorophore PhenGreen SK (PGSK). Wild-type SMPs showed rapid uptake of Fe²⁺ which was driven by the external Fe²⁺ concentration and stimulated by acidic pH. SMPs from the double deletion strain mrs3/4Δ failed to show this rapid Fe²⁺ uptake, while SMPs from cells overproducing Mrs3/4p exhibited increased Fe²⁺ uptake rates. Cu²⁺ was transported at similar rates as Fe²⁺, while other divalent cations, such as Zn²⁺ and Cd²⁺ apparently did not serve as substrates for the Mrs3/4p transporters. We conclude that the carrier proteins Mrs3p and Mrs4p transport Fe²⁺ across the inner mitochondrial membrane. Their activity is dependent on the pH gradient and it is stimulated by iron shortage.     

Interaction of calcium with the human divalent metal-ion transporter-1

Iron deficiency is the most prevalent micronutrient deficiency worldwide. Whereas dietary calcium is known to reduce the bioavailability of iron, the molecular basis of this interaction is not understood. We tested the hypothesis that divalent metal-ion transporter-1 (DMT1)—the principal or only mechanism by which nonheme iron is taken up at the intestinal brush border—is shared also by calcium. We expressed human DMT1 in RNA-injected Xenopus oocytes and examined its activity using radiotracer assays and the voltage clamp. DMT1 did not mediate ⁴⁵Ca²⁺ uptake. Instead, we found that Ca²⁺ blocked the Fe²⁺-evoked currents and inhibited ⁵⁵Fe²⁺ uptake in a noncompetitive manner (K_i ≈ 20 mM). The mechanism of inhibition was independent of voltage and did not involve intracellular Ca²⁺ signaling. The alkaline-earth metal ions Ba²⁺, Sr²⁺, and Mg²⁺ also inhibited DMT1-mediated iron-transport activity. We conclude that Ca²⁺ is a low-affinity noncompetitive inhibitor—but not a transported substrate—of DMT1, explaining in part the effect of high dietary calcium on iron bioavailability.     

Mechanism of iron uptake by peanut plants : I. Fe reduction, chelate splitting, and release of phenolics

Iron deficiency in peanuts (Arachis hypogeae L.) caused an increase in release of caffeic acid, a higher rate of Fe^III reduction, and increased rates of both Fe^III chelate splitting and iron uptake.

Experiments on Fe^III reduction by phenolics (in vitro experiments) and by roots of Fe-deficient peanuts exclude the direct involvement of released phenolics in Fe^III reduction by roots: Fe^III reduction by phenolics had a pH optimum higher than 8.0 and was strongly dependent on the concentration and the stability of the supplied Fe^III chelates. In contrast, Fe^III reduction by roots of Fe-deficient peanuts had a pH optimum of about 5.0 and was less dependent on the stability of the supplied Fe^III chelates. Furthermore, the observed release of phenolics into nutrient solution would have to be at least 200 times higher to attain the reduction rates of roots of Fe-deficient peanuts. The results of these experiments support the idea of an enzymic reduction of Fe^III on the plasmalemma of cortical cells of roots.

     

Iron and phosphate uptake explains the calcifuge–calcicole behavior of the terricolous lichens Cladonia furcata subsp. furcata and C. rangiformis

Mechanisms causing the calcifuge–calcicole behavior of lichens are largely unexplored. Studying the case examples of two closely related terricolous lichens, the calcifuge Cladonia furcata subsp. furcata and the calcicole C. rangiformis, we found that preference for acidic or calcareous soils in these lichens is related to iron and phosphate uptake as in vascular plants. In laboratory studies, the calcicole species was more efficient in the intracellular uptake of Fe³⁺ and phosphate at pH 8 than the calcifuge species. At pH 3, intracellular uptake of Fe²⁺ in the calcicole species significantly exceeded that in the calcifuge species suggesting that calcicole lichens suffer from toxicity symptoms by excess Fe²⁺ at acidic sites. Though these observations parallel findings from calcifuge and calcicole vascular plants, mechanisms leading to the different iron and phosphate uptake characteristics in the studied calcifuge and calcicole lichens may differ from those in vascular plants and should be the topic of future research. A role of the depside atranorin in facilitating iron uptake by reducing Fe³⁺ in the apoplast is hypothesized.     

Mitoferrin-2-dependent Mitochondrial Iron Uptake Sensitizes Human Head and Neck Squamous Carcinoma Cells to Photodynamic Therapy

Photodynamic therapy (PDT) is a promising approach to treat head and neck cancer cells. Here, we investigated whether mitochondrial iron uptake through mitoferrin-2 (Mfrn2) enhanced PDT-induced cell killing. Three human head and neck squamous carcinoma cell lines (UMSCC1, UMSCC14A, and UMSCC22A) were exposed to light and Pc 4, a mitochondria-targeted photosensitizer. The three cell lines responded differently: UMSCC1 and UMSCC14A cells were more resistant, whereas UMSCC22A cells were more sensitive to Pc 4-PDT-induced cell death. In non-erythroid cells, Mfrn2 is an iron transporter in the mitochondrial inner membrane. PDT-sensitive cells expressed higher Mfrn2 mRNA and protein levels compared with PDT-resistant cells. High Mfrn2-expressing cells showed higher rates of mitochondrial Fe²⁺ uptake compared with low Mfrn2-expressing cells. Bafilomycin, an inhibitor of the vacuolar proton pump of lysosomes and endosomes that causes lysosomal iron release to the cytosol, enhanced PDT-induced cell killing of both resistant and sensitive cells. Iron chelators and the inhibitor of the mitochondrial Ca²⁺ (and Fe²⁺) uniporter, Ru360, protected against PDT plus bafilomycin toxicity. Knockdown of Mfrn2 in UMSCC22A cells decreased the rate of mitochondrial Fe²⁺ uptake and delayed PDT plus bafilomycin-induced mitochondrial depolarization and cell killing. Taken together, the data suggest that lysosomal iron release and Mfrn2-dependent mitochondrial iron uptake act synergistically to induce PDT-mediated and iron-dependent mitochondrial dysfunction and subsequent cell killing. Furthermore, Mfrn2 represents a possible biomarker of sensitivity of head and neck cancers to cell killing after PDT.     

Evaluation of Reductive Release as a Mechanism for Iron Uptake from Ferrioxamine B by Chlorella vulgaris

The involvement of ferric reduction in the iron uptake mechanism of iron-stressed Chlorella vulgaris from ferrioxamine B was investigated. Some comparative data for ferric-citrate was also obtained. EPR and a spectrophotometric assay were used to measure Fe³⁺ reduction. These two methods differed in the absolute quantity but not in effectors of ferric reduction. The mechanism governing ferric reduction was investigated by use of respiratory inhibitors, uncouplers, alternative electron acceptors, and ATPase inhibitors. Reduction appears to play a role in iron uptake from both Fe³⁺-deferrioxamine B and Fe³⁺-citrate; however, the involvement of photoreduction in Fe³⁺-citrate uptake implies multiple reductive mechanisms could be involved.     

Assessment of iron toxicity using a luminescent bacterial test with an Escherichia coli recombinant strain

The toxic effect of the Fe²⁺ and Fe³⁺ ions on the luminescent recombinant Escherichia coli strain with the luxCDABE operon was studied in short- and long-term experiments. At 30-min exposure of bacteria to the iron ions, the effective concentrations of Fe²⁺ and Fe³⁺ resulting in acute toxicity (EC₅₀) were 8.5 and 1.3 mg/L, respectively. In the long-term (24 h) experiment, during active bacterial growth, the toxicity index for Fe²⁺ and Fe³⁺ was 65.5 and 62.8, respectively. Addition of the iron ions into the medium did not suppress growth, although it inhibited luminescence. Comparative analysis of the short- and long-term experiments made it possible to assess iron toxicity at the concentrations from 0.5 to 20 mg/L (as calculated for the Fe²⁺ and Fe³⁺ ions). Iron ions were found to affect only the reactions that were not vitally important for the cell. At the same time, they had no negative effect on the genetic mechanisms and protein synthesis, thus indicating non-specific toxicity of Fe²⁺ and Fe³⁺.     

The role of iron uptake in pathogenicity and symbiosis in <Emphasis Type="Italic">Photorhabdus luminescens</Emphasis> TT01

Background  

Photorhabdus are Gram negative bacteria that are pathogenic to insect larvae whilst also having a mutualistic interaction with nematodes from the family Heterorhabditis. Iron is an essential nutrient and bacteria have different mechanisms for obtaining both the ferrous (Fe²⁺) and ferric (Fe³⁺) forms of this metal from their environments. In this study we were interested in analyzing the role of Fe³⁺ and Fe²⁺ iron uptake systems in the ability of Photorhabdus to interact with its invertebrate hosts.     

Zusammenhänge zwischen der Eisenaufnahme und dem Eisentransport in der Pflanze

Zusammenfassung Junge Sonnenblumenpflanzen nahmen 8 Std. lang nicht markiertes und danach 15 Std. lang mit Fe⁵⁹ markiertes Eisen in Form von Fe-Chlorid, Fe-EDTA oder Fe-Citrat auf. 1. Bei allen drei Eisenformen absorbierten die Pflanzenwurzeln mehr Eisen aus der N?hrl?sung mit 0,1 ppm Fe als aus der mit 1,0 ppm Fe. 2. Die Wurzeln der mit Fe-Chlorid ern?hrten Pflanzen enthalten den h?chsten Gehalt an Fe⁵⁹. 3. Das Stengelexsudat der 0,1 ppm Fe-Reihe enthielt weniger Fe⁵⁹ als das Exsudat der 1,0 ppm Fe-Reihe. 4. Eine sichere Beziehung zwischen der Ern?hrung mit den verschiedenen drei Eisenformen und der ausgeschiedenen Exsudatmenge konnte nicht festgestellt werden. Ebenfalls bestand kein Zusammenhang zwischen der ausgeschiedenen Exsudatmenge und ihrem Fe⁵⁹-Gehalt. 5. Der Gehalt des Stengelexsudats an Fe⁵⁹ war bei der Ern?hrung mit Fe-EDTA und Fe-Citrat h?her als bei Fe-Chlorid.

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Relationships between iron uptake and iron transport in plants

Summary The uptake of unlabelled iron for 8 h and of Fe⁵⁹, given as Fe chloride, Fe EDTA and Fe citrate for 15 h, was investigated in young sunflower plants. 1. In all three iron forms the roots absorbed more iron from the nutrient solution with 0.1 ppm Fe than with 1.0 ppm Fe. 2. The roots of those plants, supplied with Fe-chloride contained the highest amount of Fe⁵⁹. 3. The stem exudate of plants, given a nutrient solution with 0.1 ppm Fe contained less Fe⁵⁹ than the exudate of plants, grown in a nutrient solution containing 1.0 ppm Fe. 4. No close relationship between the nutrition with the three iron forms and the produced amount of exudate could be established. Furthermore no correlation between the produced amount of exudate and its Fe⁵⁹ content could be found. 5. The content of Fe⁵⁹ in the stem exudate was higher when Fe EDTA and Fe citrate were given as compared with Fe chloride.

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Auszug aus der Dissertation des Verfassers.     

The photocycle and the structure of iron containing bacteriorhodopsin —a kinetic and Mössbauer spectroscopy investigation

Bacteriorhodopsin (bR), converted by deionization to the blue form was reconstituted to the active purple membrane by the addition of Fe²⁺ or Fe³⁺ ions. ⁵⁷Fe Mossbauer spectra of these samples were measured at different pH values (pH 3.9, pH 5.0 and pH 7.0) and at temperatures ranging from 4 K to 300 K. The hyperfine parameters reveal two iron environments with oxygen atoms in the neighbourhood of iron. Iron type 1 is in the 3⁺ high spin state. It is bound to acid side chains of the protein and/or the phosphate groups of the lipids. Iron type 2 is in the 2⁺ high spin state and is linked to carboxy groups of the protein in a rather unspecific way. Dynamics as measured by Mossbauer spectroscopy show that the purple membrane becomes flexible only above 220 K. At the interface between membrane and bulk water the mobility is comparable to that of proteins with hydrophilic surfaces. The photocycle of Fe ³⁺-bR is slowed down compared to native bR. 3–5 Fe³⁺/bR are sufficient to inhibit the photocycle turnover by one order of magnitude. This specific effect is also found with Cr³⁺, though it is less pronounced. Mössbauer spectra of Fe³⁺-bR at 4 K reveal that iron nuclei are spin-coupled, indicating their close spatial proximity. It is proposed that iron trinuclear clusters interact with the proton uptake site of bR. Offprint requests to: M. Engelhard     

Active Transport of Iron in Bacillus megaterium: Role of Secondary Hydroxamic Acids

Kinetics of radioactive iron transport were examined in three strains of Bacillus megaterium. In strain ATCC 19213, which secretes the ferric-chelating secondary hydroxamic acid schizokinen, ⁵⁹Fe³⁺ uptake from ⁵⁹FeCl₃ or the ferric hydroxamate Desferal-⁵⁹Fe³⁺ was rapid and reached saturation within 3 min. In strain SK11, which does not secrete schizokinen, transport from ⁵⁹FeCl₃ was markedly reduced; the two ferric hydroxamates Desferal-⁵⁹Fe³⁺ or schizokinen-⁵⁹Fe³⁺ increased both total ⁵⁹Fe³⁺ uptake and the ⁵⁹Fe³⁺ appearing in a cellular trichloroacetic acid-insoluble fraction, although 10 min was required to reach saturation. Certain characteristics of transport from both ferric hydroxamates and FeCl₃ suggest that iron uptake was an active process. The growth-inhibitory effect of aluminum on strain SK11 was probably due to the formation of nonutilizable iron-aluminum complexes which blocked uptake from ⁵⁹FeCl₃. Desferal or schizokinen prevented this blockage. A strain (ARD-1) resistant to the ferric hydroxamate antibiotic A22765 was isolated from strain SK11. Strain ARD-1 failed to grow with Desferal-Fe³⁺ as an iron source, and it was unable to incorporate ⁵⁹Fe³⁺ from this source. Growth and iron uptake in strain ARD-1 were similar to strain SK11 with schizokinen-Fe³⁺ or the iron salt as sources. It is suggested that the ferric hydroxamates, or the iron they chelate, may be transported by a special system which might be selective for certain ferric hydroxamates. Strain ARD-1 may be unable to recognize both the antibiotic A22765 and the structurally similar chelate Desferal-Fe³⁺, while retaining its capacity to utilize schizokinen-Fe³⁺.     

An in vitro examination of intestinal iron absorption in a freshwater teleost, rainbow trout (Oncorhynchus mykiss)

This study investigated the physiological characteristics of intestinal iron absorption in a freshwater teleost, rainbow trout (Oncorhynchus mykiss). Using an in vitro gastro-intestinal sac technique, we evaluated the spatial pattern and concentration dependent profile of iron uptake, and also the influence of luminal chemistry (pH and chelation) on iron absorption. We demonstrated that the iron uptake rate in the anterior intestine is significantly higher than that in the mid and posterior intestine. Interestingly, absorption of iron in the anterior intestine occurs likely via simple diffusion, whereas a carrier-mediated pathway is apparent in the mid and posterior intestine. The uptake of ferric and ferrous iron appeared to be linear over the entire range of iron concentration tested (0–20 μM), however the uptake of ferrous iron was significantly higher than that of ferric iron at high iron concentrations (>15 μM). An increase in mucosal pH from 7.4 to 8.2 significantly reduced iron absorption in both mid and posterior intestine, implying the involvement of a Fe²⁺/H⁺ symporter. Iron chelators (nitrilotriacetic acid and desferrioxamine mesylate) had no effects on iron absorption, which suggests that fish are able to acquire chelated iron via intestine.     

Iron requirement and iron uptake from various iron compounds by different plant species

The Fe requirements of four monocotyledonous plant species (Avena sativa L., Triticum aestivum L., Oryza sativa L., Zea mays L.) and of three dicotyledonous species (Lycopersicum esculentum Mill., Cucumis sativus L., Glycine maxima (L.) Merr.) in hydroponic cultures were ascertained. Fe was given as NaFe-EDDHA chelate (Fe ethylenediamine di (O-hydroxyphenylacetate). I found that the monocotyledonous species required a substantially higher Fe concentration in the nutrient solution in order to attain optimum growth than did the dicotyledonous species. Analyses showed that the process of iron uptake was less efficient with the monocotyledonous species. When the results obtained by using chelated Fe were compared with those using ionic Fe, it was shown that the inefficient species were equally inefficient in utilizing Fe³⁺ ions. However, the differences between the efficient and the inefficient species disappeared when Fe²⁺ was used. This confirms the work of others who postulated that Fe³⁺ is reduced before uptake of chelated iron by the root. In addition, it was shown that reduction also takes place when Fe is used in ionic form. The efficiency of Fe uptake seems to depend on the efficiency of the root system of the particular plant species in reducing Fe³⁺. The removal of Fe from the chelate complex after reduction to Fe²⁺ seems to present no difficulties to the various plant species.     

Dynamic equilibria in iron uptake and release by ferritin

The function of ferritins is to store and release ferrous iron. During oxidative iron uptake, ferritin tends to lower Fe²⁺ concentration, thus competing with Fenton reactions and limiting hydroxy radical generation. When ferritin functions as a releasing iron agent, the oxidative damage is stimulated. The antioxidant versus pro-oxidant functions of ferritin are studied here in the presence of Fe²⁺, oxygen and reducing agents. The Fe²⁺-dependent radical damage is measured using supercoiled DNA as a target molecule. The relaxation of supercoiled DNA is quantitatively correlated to the concentration of exogenous Fe²⁺, providing an indirect assay for free Fe²⁺. After addition of ferrous iron to ferritin, Fe²⁺ is actively taken up and asymptotically reaches a stable concentration of 1–5 m. Comparable equilibrium concentrations are found with plant or horse spleen ferritins, or their apoferritins. After addition of ascorbate, iron release is observed using ferrozine as an iron scavenger. Rates of iron release are dependent on ascorbate concentration. They are about 10 times larger with pea ferritin than with horse ferritin. In the absence of ferrozine, the reaction of ascorbate with ferritins produces a wave of radical damage; its amplitude increases with increased ascorbate concentrations with plant ferritin; the damage is weaker with horse ferritin and less dependent on ascorbate concentrations.     

Influence of water-borne iron on the liver of Poecilia reticulata (Peters 1859)

The influence of water-borne iron (500 μg × 1^-1 and 2 mg × 1^-1) on the liver of Poecilia reticulata is studied by means of electron microscopy. The uptake and effect of iron is affected by a. the chemical speciation of the metal: Iron complexed with tri-polyphosphates is more deleterious than inorganic Fe³⁺; b. the nutritional status of the fish: In food-deprived Poecilia reticulata iron is absorbed and enhances starvation-induced liver damage. In fed animals, there is no uptake at all. Mild alterations of hepatocyte ultrastructure are attributed to an unspecific stress response.     

Evidence for a specific uptake system for iron phytosiderophores in roots of grasses

Roots of grasses in response to iron deficiency markedly increase the release of chelating substances (`phytosiderophores') which are highly effective in solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds by formation of Fe<sup>III</sup>phytosiderophores. In barley (Hordeum vulgare L.), the rate of iron uptake from Fe<sup>III</sup>phytosiderophores is 100 to 1000 times faster than the rate from synthetic Fe chelates (e.g. Fe ethylenediaminetetraacetate) or microbial Fe siderophores (e.g. ferrichrome). Reduction of Fe<sup>III</sup> is not involved in the preferential iron uptake from Fe<sup>III</sup>phytosiderophores by barley. This is indicated by experiments with varied pH, addition of bicarbonate or of a strong chelator for Fe<sup>II</sup> (e.g. batho-phenanthrolinedisulfonate). The results indicate the existence of a specific uptake system for Fe<sup>III</sup>phytosiderophores in roots of barley and all other graminaceous species. In contrast to grasses, cucumber plants (Cucumis sativus L.) take up iron from Fe<sup>III</sup>phytosiderophores at rates similar to those from synthetic Fe chelates. Furthermore, under Fe deficiency in cucumber, increased rates of uptake of Fe<sup>III</sup>phytosiderophores are based on the same mechanism as for synthetic Fe chelates, namely enhanced Fe<sup>III</sup> reduction and chelate splitting. Two strategies are evident from the experiments for the acquisition of iron by plants under iron deficiency. Strategy I (in most nongraminaceous species) is characterized by an inducible plasma membrane-bound reductase and enhancement of H<sup>+</sup> release. Strategy II (in grasses) is characterized by enhanced release of phytosiderophores and by a highly specific uptake system for Fe<sup>III</sup>phytosiderophores. Strategy II seems to have several ecological advantages over Strategy I such as solubilization of sparingly soluble inorganic Fe<sup>III</sup> compounds in the rhizosphere, and less inhibition by high pH. The principal differences in the two strategies have to be taken into account in screening methods for resistance to `lime chlorosis'.     

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.     

A new technique of plant analysis to resolve iron chlorosis

Summary Iron though indispensable for the biosynthesis of chlorophyll, its total content in the plant was not associated with the occurrence of chlorosis. In order to overcome this inconsistency a new technique of plant iron analysis has been developed. It consists of the determination of Fe²⁺, the fraction of iron involved in the synthesis of chlorophyll.The choice of 1–10 o-phenanthroline (o-Ph) as an extractant for Fe²⁺ was based on its remarkably higher stability constant for Fe²⁺ than Fe³⁺. On this basis, it could preferentially chelate Fe²⁺. The highly specific organce colour of the Fe²⁺-phenanthroline complex made possible the determination of Fe²⁺ by reading the transmittancy at 510 nm.The procedure involves extraction of 2 g of thoroughly washed, chopped, fresh plant by 20 ml of o-phenanthroline extractant (pH 3.0, conc. 1.5%). The plant samples treated with the extractant are allowed to stand for 16 hours and Fe²⁺ is determined in the filtrate by reading the transmittancy at 510 nm.In sharp contrast to total iron the green plants always contained more Fe²⁺ than chlorotic plants. The technique has been developed for rice but is expected to be successful for other crops also.