Molecular mechanisms of non‐transferrin‐bound and transferring‐bound iron uptake in primary hippocampal neurons

The molecular mechanisms of iron trafficking in neurons have not been elucidated. In this study, we characterized the expression and localization of ferrous iron transporters Zip8, Zip14 and divalent metal transporter 1 (DMT1), and ferrireductases Steap2 and stromal cell‐derived receptor 2 in primary rat hippocampal neurons. Steap2 and Zip8 partially co‐localize, indicating these two proteins may function in Fe³⁺ reduction prior to Fe²⁺ permeation. Zip8, DMT1, and Steap2 co‐localize with the transferrin receptor/transferrin complex, suggesting they may be involved in transferrin receptor/transferrin‐mediated iron assimilation. In brain interstitial fluid, transferring‐bound iron (TBI) and non‐transferrin‐bound iron (NTBI) exist as potential iron sources. Primary hippocampal neurons exhibit significant iron uptake from TBI (Transferrin‐⁵⁹Fe³⁺) and NTBI, whether presented as ⁵⁹Fe²⁺‐citrate or ⁵⁹Fe³⁺‐citrate; reductase‐independent ⁵⁹Fe²⁺ uptake was the most efficient uptake pathway of the three. Kinetic analysis of Zn²⁺ inhibition of Fe²⁺ uptake indicated that DMT1 plays only a minor role in the uptake of NTBI. In contrast, localization and knockdown data indicate that Zip8 makes a major contribution. Data suggest also that cell accumulation of ⁵⁹Fe from TBI relies at least in part on an endocytosis‐independent pathway. These data suggest that Zip8 and Steap2 play a major role in iron accumulation from NTBI and TBI by hippocampal neurons.

     

FTIR, Raman, and UV-Vis spectroscopic and DFT investigations of the structure of iron-lead-tellurate glasses

In this work, the effects of iron ion intercalations on lead–tellurate glasses were investigated via FTIR, Raman and UV-Vis spectroscopies. This homogeneous glass system has compositions xFe₂O₃·(100−x)[4TeO₂·PbO₂], where x = 0–60 mol%. The presented observations in these mechanisms show that the lead ions have a pronounced affinity towards [TeO₃] structural units, resulting in the deformation of the Te–O–Te linkages, and leading to the intercalation of [PbO _n ] (n = 3, 4) and [FeO _n ] (n = 4, 6) entities in the [TeO₄] chain network. The formation of negatively charged [FeO₄]¹⁻ structural units implies the attraction of Pb²⁺ ions in order to compensate for this electrical charge. Upon increasing the Fe₂O₃ content to 60 mol%, the network can accommodate an excess of oxygen through the formation of [FeO₆] structural units and the conversion of [TeO₄] into [TeO₃] structural units. For even higher Fe₂O₃ contents, Raman spectra indicate a greater degree of depolymerization of the vitreous network than FTIR spectra do. The bands due to the Pb–O bond vibrations are very strongly polarized and the [TeO₄] structural units convert into [TeO₃] units via an intermediate coordination stage termed “[TeO₃₊₁]” structural units. Our UV-Vis spectroscopic data show two mechanisms: (i) the conversion of the Fe³⁺ to Fe²⁺ at the same time as the oxidation of Pb²⁺ to Pb⁺⁴ ions for samples with low Fe₂O₃ contents; (ii) when the Fe₂O₃ content is high (x ≥ 50 mol%), the Fe²⁺ ions capture positive holes and are transferred to Fe³⁺ ions through a photochemical reaction, while the Pb²⁺ ions are formed by the reduction of Pb⁴⁺ ions. DFT calculations show that the addition of Fe₂O₃ to lead–tellurate glasses seems to break the axial Te–O bonds, and the [TeO₄] structural units are gradually transformed into [TeO₃₊₁]- and [TeO₃]-type polyhedra. Analyzing these data further indicates a gradual conversion of the lead ions from covalent to ionic environment. There is then a charge transfer between the tri- and tetracoordinated tellurium atoms due to the capacity of the lead–tellurate network to form the appropriate coordination environments containing structural units of opposite charge, such as iron ions, [FeO₄]¹⁻.     

Effects of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac function and Ca2+ regulation in iron-overloaded thalassemic mice

Although disturbance of cardiac Ca²⁺ regulation is involved in the pathophysiology of iron-overload cardiomyopathy, the obvious mechanisms involved in the dysregulation of iron-induced cardiac Ca²⁺ are unclear. Moreover, the roles of the iron chelator deferiprone and the T-type calcium channel blocker efonidipine on cardiac intracellular Ca²⁺ transients and Ca²⁺ regulatory proteins in thalassemic mice are still unknown. We tested the hypothesis that treatment with either deferiprone or efonidipine attenuated cardiac Ca²⁺ dysregulation and led to improved left ventricular (LV) function in iron-overloaded thalassemic mice. Wild-type (WT) mice and β-thalassemic (HT) mice were fed with either a normal diet (ND) or a high iron-diet (FE) for 90 days. Then, the FE-fed mice were treated with either deferiprone (75 mg/kg/day) or efonidipine (4 mg/kg/day) for 30 days. ND-fed HT mice had an increase in T-type calcium channels (TTCC) and an increased level of sarcoplasmic reticulum Ca²⁺-ATPase (SERCA), compared with ND-fed WT mice. Chronic iron feeding led to an increase in TTCC and expression of SERCA proteins in FE-fed WT mice. Moreover, chronic iron overload led to increased plasma non-transferrin bound iron (NTBI) and cardiac iron deposition, impaired cardiac intracellular Ca²⁺ transients including decreased intracellular Ca²⁺ transient amplitude, rising rate and decay rate, as well as impaired LV function as indicated by a decreased %LV ejection fraction (%LVEF) in both WT and HT mice. Our findings showed that treatment with either deferiprone (DFP) or efonidipine (EFO) showed similar benefits in reducing plasma NTBI and cardiac iron deposition, and improving %LVEF from 84.3 (WT) to 89.3 (DFP) and 89.2 (EFO) treatment; and from 84.2 (HT) to 88.8 (DFP) and 89.5 (EFO) treatment, however there was no improvement in the regulation of cardiac Ca²⁺ in iron-overloaded thalassemic mice. These findings provide the understanding of the effects of these drugs on the iron-overloaded heart in thalassemic mice and suggest that an alternative intervention that could improve calcium regulation under this condition is needed to improve the therapeutic outcome. Moreover, whether the benefits of the TTCC blocker is via its inhibition of the TTCC alone or together with its ability to chelate iron are still unclear and need further investigation.     

A hypothesis for the basis of the pro-oxidant nature of calcium ions

A new hypothesis describing the role of the redox inactive Ca²⁺ ion in the expression of physiological oxidative damage is described. The hypothesis is based on the optimization of the chelation characteristics of iron complexes for pro-oxidant activity. In a previous investigation it was found that an excess of ligand kinetically hindered the Fenton reaction activity of the Fe^II/IIIEDTA complex (Bobier et al. 2003). EDTA, citrate, NTA, and glutamate were selected as models for the coordination sites likely encountered by mobile iron, i.e. proteins. The optimal [EDTA]:[Fe^III] ratio for Fenton reaction activity as measured by electrocatalytic voltammetry in a solution was found to be 1:1. An excess of EDTA in the amount of 10:1 [ligand]:[metal] suppresses the Fenton reaction activity to nearly the control. It is expected that the physiological coordination characteristics of mobile Fe would have a very large excess of [ligand]:[metal] and thus not be optimized for the Fenton reaction. Introduction of Ca²⁺ in to a ratio of 10:10:1 [Ca²⁺]:[EDTA]:[Fe^III] to the system reinvigorated the Fenton reaction activity to nearly the value of the optimal 1:1 [EDTA]:[Fe^III] complex. The pH distribution diagrams of Ca²⁺ in the presence of EDTA and Fe^II/III indicate that Ca²⁺ has the ability to uptake excess EDTA without displacing either Fe^II of Fe^III from their respective complexed forms. The similarity in the presence for hard ligand sites albeit with a lower binding constant for Ca²⁺ accounts for this action.     

Penicillin V production by Penicillium chrysogenum in the presence of Fe3+ and in low-iron culture medium

Late-exponential-phasePenicillium chrysogenum mycelia grown in a complex medium possessed an intracellular iron concentration of 650 μmol/L (2.2±0.6 μmol per g mycelial dry mass). This iron reserve was sufficient to ensure growth and antibiotic production after transferring mycelia into a defined low-iron minimal medium. Although the addition of Fe³⁺ to the Fe-limited cultures increased significantly the intracellular iron levels the surplus iron did not influence the production of penicillin V. Supplements of purified majorP. chrysogenum siderophores (coprogen and ferrichrome) into the fermentation media did not affect the β-lactam production and intracellular iron level. Neither 150 nor 300 μmol/L extracellular Fe³⁺ concentrations disturbed the glutathione metabolism of the fungus, and increased the oxidative stress caused by 700 mmol/L H₂O₂. Nevertheless, when iron was applied in the Fe^II oxidation state the oxidative cell injuries caused by the peroxide were significantly enhanced.     

Basic fibroblast growth factor increases intracellular magnesium concentration through the specific signaling pathways

Basic fibroblast growth factor (bFGF) plays an important role in angiogenesis. However, the underlying mechanisms are not clear. Mg²⁺ is the most abundant intracellular divalent cation in the body and plays critical roles in many cell functions. We investigated the effect of bFGF on the intracellular Mg²⁺ concentration ([Mg²⁺]_i) in human umbilical vein endothelial cells (HUVECs). bFGF increased [Mg²⁺]_i in a dose-dependent manner, independent of extracellular Mg²⁺. This bFGF-induced [Mg²⁺]_i increase was blocked by tyrosine kinase inhibitors (tyrphostin A-23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and a phospholipase Cγ (PLCγ) inhibitor (U73122). In contrast, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) did not affect the bFGF-induced [Mg²⁺]_i increase. These results suggest that bFGF increases the [Mg²⁺]_i from the intracellular Mg²⁺ stores through the tyrosine kinase/PI3K/PLCγ-dependent signaling pathways.     

Crystal and molecular structure of 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and its iron(III) complex: an iron chelator with anti-tumour activity

 Previous studies have demonstrated that 2-hydroxy-1-naphthaldehyde isonicotinoyl hydrazone (NIH) and several other aroylhydrazone chelators possess anti-neoplastic activity due to their ability to bind intracellular iron. In this study we have examined the structure and properties of NIH and its Fe^III complex in order to obtain further insight into its anti-tumour activity. Two tridentate NIH ligands deprotonate upon coordination to Fe^III in a meridional fashion to form a distorted octahedral, high-spin complex. Solution electrochemistry of [Fe(NIH–H)₂]⁺ shows that the trivalent oxidation state is dominant over a wide potential range and that the Fe^II analogue is not a stable form of this complex. The fact that [Fe(NIH–H)₂]⁺ cannot cycle between the Fe^II and Fe^III states suggests that the production of toxic free-radical species, e.g. OH ^. or O₂ ^{. –}, is not part of this ligand's cytotoxic action. This suggestion is supported by cell culture experiments demonstrating that the addition of Fe^III to NIH prevents its anti-proliferative effect. The chemistry of this chelator and its Fe^III complex are discussed in the context of understanding its anti-tumour activity. Received: 12 November 1998 / Accepted: 9 February 1999     

Non-transferrin bound iron,cytokine activation and intracellular reactive oxygen species generation in hemodialysis patients receiving intravenous iron dextran or iron sucrose

Intravenous (IV) iron supplementation is widely used to support erythropoeisis in hemodialysis patients. IV iron products are associated with oxidative stress that has been measured principally by circulating biomarkers such as products of lipid peroxidation. The pro-oxidant effects of IV iron are presumed to be due at least in part, by free or non-transferrin bound iron (NTBI). However, the effects of IV iron on intracellular redox status and downstream effectors is not known. This prospective, crossover study compared cytokine activation, reactive oxygen species generation and oxidative stress after single IV doses of iron sucrose and iron dextran. This was a prospective, open-label, crossover study. Ten patients with end-stage renal disease (ESRD) on hemodialysis and four age and sex-matched healthy were assigned to receive 100 mg of each IV iron product over 5 min in random sequence with a 2 week washout between products. Subjects were fasted and fed a low iron diet in the General Clinical Research Center at the University of New Mexico. Serum and plasma samples for IL-1, IL-6, TNF-α and IL-10 and NTBI were obtained at baseline, 60 and 240 min after iron infusion. Peripheral blood mononuclear cells (PBMC) were isolated at the same time points and stained with fluorescent probes to identify intracellular reactive oxygen species and mitochondrial membrane potential (Δψm) by flow cytometry. Lipid peroxidation was assessed by plasma F₂ isoprostane concentration. Mean ± SEM maximum serum NTBI values were significantly higher among patients receiving IS compared to ID (2.59 ± 0.31 and 1.0 ± 0.36 μM, respectively, P = 0.005 IS vs. ID) Mean ± SEM NTBI area under the serum concentration–time curve (AUC) was 3-fold higher after IS versus ID (202 ± 53 vs. 74 ± 23 μM*min/l, P = 0.04) in ESRD patients, indicating increased exposure to NTBI. IV iron administration was associated with increased pro-inflammatory cytokines. Serum IL-6 concentrations increased most profoundly, with a 2.6 and 2.1 fold increase from baseline in ESRD patients given IS and ID, respectively (P < 0.05 compared to baseline). In healthy controls, serum IL-6 was undetectable at baseline and after IV iron administration. Most ESRD patients had increased intracellular ROS generation, however, there was no difference between ID and IS. Only one healthy control had increased ROS generation post IV iron. All healthy controls experienced a loss of Δψm (100% with IS and 50% with ID). ESRD patients also had loss of Δψm with a nadir at 240 min. IS administration was associated with higher maximum serum NTBI concentrations compared to ID, however, the both compounds produced similar ROS generation and cytokine activation that was more pronounced among ESRD patients. The effect of IV iron-induced ROS production on pivotal signaling pathways needs to be explored.     

Transferrin: a potential source of iron for oxygen free radical-mediated endothelial cell injury.

The ability of transferrin to potentiate oxygen free radical-mediated endothelial cell injury was assessed. 51Cr-labeled endothelial cells derived from rat pulmonary arteries (RPAECs) were incubated with hydrogen peroxide (H2O2) in the presence and absence of holosaturated human transferrin, and the effect of transferrin on H2O2-mediated endothelial cell toxicity was determined. Addition of holosaturated transferrin potentiated H2O2-mediated RPAEC cytotoxicity at concentrations of H2O2 greater than 10 microM, suggesting that transferrin may provide a source of iron for free radical-mediated endothelial cell injury. Free radical-mediated injury is dependent on non-protein-bound iron. The ability of RPAECs to facilitate the release of iron from transferrin was assessed. We determined that RPAECs facilitate the release of transferrin-derived iron by reduction of transferrin-bound ferric iron (Fe3+) to ferrous iron (Fe2+). The reduction and release of transferrin-derived Fe2+ were inhibited by apotransferrin and chloroquine, indicating a dependence on receptor-specific binding of transferrin to the RPAEC cell surface, with subsequent endocytosis, acidification, and reduction of transferrin-bound Fe3+ to Fe2+. The release of transferrin-derived Fe2+ was potentiated by diethyldithiocarbamate, an inhibitor of intracellular superoxide dismutase (SOD). In contrast, exogenous SOD did not alter iron release, suggesting that intracellular superoxide anion (O2-) may play an important role in mediating the reduction and release of transferrin-derived iron. Results of this study suggest that transferrin may provide a source of iron for oxygen free radical-mediated endothelial cell injury and identify a novel mechanism by which endothelial cells may mediate the reduction and release of transferrin-derived iron.     

Regulatory Volume Decrease by SV40-transformed Rabbit Corneal Epithelial Cells Requires Ryanodine-sensitive Ca2+-induced Ca2+ Release

The relationship between relative cell volume and time-dependent changes in intracellular Ca²⁺ concentration ([Ca²⁺] _i ) during exposure to hypotonicity was characterized in SV-40 transformed rabbit corneal epithelial cells (tRCE) (i). Light scattering measurements revealed rapid initial swelling with subsequent 97% recovery of relative cell volume (characteristic time (τ _vr ) was 5.9 min); (ii). Fura2-fluorescence single-cell imaging showed that [Ca²⁺] _i initially rose by 216% in 30 sec with subsequent return to near baseline level after another 100 sec. Both relative cell volume recovery and [Ca²⁺] _i transients were inhibited by either: (a) Ca²⁺-free medium; (b) 5 mm Ni²⁺ (inhibitor of plasmalemma Ca²⁺ influx); (c) 10 μm cyclopiazonic acid, CPA (which causes depletion of intracellular Ca²⁺ content); or (d) 100 μm ryanodine (inhibitor of Ca²⁺ release from intracellular stores). To determine the temporal relationship between an increased plasmalemma Ca²⁺ influx and the emptying of intracellular Ca²⁺ stores during the [Ca²⁺] _i transients, Mn²⁺ quenching of fura2-fluorescence was quantified. In the presence of CPA, hypotonic challenge increased plasmalemma Mn²⁺ permeability 6-fold. However, Mn²⁺ permeability remained unchanged during exposure to either: 1.100 μm ryanodine; 2.10 μm CPA and 100 μm ryanodine. This report for the first time documents the time dependence of the components of the [Ca²⁺] _i transient required for a regulatory volume decrease (RVD). The results show that ryanodine sensitive Ca²⁺ release from an intracellular store leads to a subsequent increase in plasmalemma Ca²⁺ influx, and that both are required for cells to undergo RVD. Received: 7 November 1996/Revised: 6 January 1997     

Effects of added organic matter on iron and manganese redox systems in sediment

Addition of five types of organic matter to Lake Washington sediments resulted in release of high concentrations of iron, organic carbon, and manganese into the interstitial water, and caused an increase in observed sediment oxygen consumption rates. The depressed electrode potentials (E_h < —150 mV) that should accompany such reduction processes did not occur, indicating that E_h was being poised by redox systems present in the sediment. Iron redox systems [Fe(OH)₃‐Fe²⁺, Fe₃(OH)₈‐Fe²⁺, and Fe(OH)₃‐Fe₃(OH)₈] were shown to be poising the E_h of control sediments throughout 13 weeks of incubation and dominating the potential of several of the organically amended sediments following the first three weeks of incubation. Depression of calculated iron system E_o values relative to that of the control sediment early in the incubation appeared to be due to the decreased pH and non‐equilibrium conditions in the organic matter‐amended sediment during the first weeks of incubation. Manganese redox systems exerted no discernable impact on the E_h of the sediment.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of ⁵⁹Fe²⁺ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH₄Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999. © 1999 Wiley-Liss, Inc.     

Role of labile iron in the toxicity of pharmacological ascorbate

Pharmacological ascorbate has been shown to induce toxicity in a wide range of cancer cell lines. Pharmacological ascorbate in animal models has shown promise for use in cancer treatment. At pharmacological concentrations the oxidation of ascorbate produces a high flux of H₂O₂ via the formation of ascorbate radical (Asc^•-). The rate of oxidation of ascorbate is principally a function of the level of catalytically active metals. Iron in cell culture media contributes significantly to the rate of H₂O₂ generation. We hypothesized that increasing intracellular iron would enhance ascorbate-induced cytotoxicity and that iron chelators could modulate the catalytic efficiency with respect to ascorbate oxidation. Treatment of cells with the iron-chelators deferoxamine (DFO) or dipyridyl (DPD) in the presence of 2 mM ascorbate decreased the flux of H₂O₂ generated by pharmacological ascorbate and reversed ascorbate-induced toxicity. Conversely, increasing the level of intracellular iron by preincubating cells with Fe-hydroxyquinoline (HQ) increased ascorbate toxicity and decreased clonogenic survival. These findings indicate that redox metal metals, e.g., Fe³⁺/Fe²⁺, have an important role in ascorbate-induced cytotoxicity. Approaches that increase catalytic iron could potentially enhance the cytotoxicity of pharmacological ascorbate in vivo.     

1H NMR studies of the Fe7S8 ferredoxin from Bacillus schlegelii: a further attempt to understand Fe3S4 clusters

 The oxidized Fe₇S₈ ferredoxin from Bacillus schlegelii, containing both [Fe₃S₄]⁺ and [Fe₄S₄]²⁺ clusters, has been investigated by ¹H NMR spectroscopy. An extensive sequence-specific assignment of the hyperfine-shifted resonances has been obtained by making use of a computer-generated structural model. The pattern and the temperature dependence of the hyperfine shifts of the β-CH₂ protons of the cysteines coordinating the [Fe₃S₄]⁺ cluster are rationalized in terms of magnetic interactions between the iron ions. The same approach holds for the hyperfine coupling with ⁵⁷Fe. It is shown that the magnetic interactions are more asymmetric in Fe₇S₈ ferredoxins than in Fe₃S₄ ferredoxins. The NMR non-observability of the β-CH₂ protons of coordinated cysteines in the one-electron-reduced form has been discussed. Received: 19 June 1996 / Accepted: 2 August 1996     

Fluorescence assay of non-transferrin-bound iron in thalassemic sera using bacterial siderophore

Transfusional iron overload associated with thalassemia leads to the appearance of non-transferrin-bound iron (NTBI) in blood that is toxic and causes morbidity and mortality via tissue damage. Hence, a highly sensitive and accurate assay of NTBI, with broad clinical application in both diagnosis and validation of treatment regimens for iron overload, is important. An assay based on iron chelation by a high-affinity siderophore, azotobactin, has been developed. The steps consist of blocking of native apotransferrin iron binding sites, mobilization of NTBI, ultrafiltration of all serum proteins, and finally the addition of the probe, which has a chromophore that fluoresces at 490 nm. Binding of Fe³⁺ to azotobactin quenches the fluorescence in a concentration-dependent manner. Measured NTBI levels in 63 sera ranged from 0.07 to 3.24 μM (0.375 ± 0.028 μM [means ± SEM]). It correlated well with serum iron and percentage transferrin saturation but not with serum ferritin. Pearson’s correlation coefficients were found to be 0.6074 (P < 0.0001) and 0.6102 (P < 0.0001) for percentage transferrin saturation and total serum iron, respectively. The low values are due to the patients being under regular chelation therapy even prior to sampling, indicating that the method is sensitive to very low levels of NTBI, allowing a much lower detection limit than the available methods.     

New photosensitizers based upon [Fe(L)2(CN)2] and [FeL3], where L is substituted 2,2′-bipyridine

Ultrafast electron transfer in the dye sensitized solar cell (DSSC) has made it possible to use iron(II) polypyridyl complexes as photosensitizers [J. Am. Chem. Soc. 120 (1998) 843]. Although ruthenium(II) polypyridyl complexes comprise an extensively studied and widely utilized photochemical system, comparatively little is known about the photoproperties of their iron analogues. The syntheses and solution properties of the complexes [Fe^II(L)₂(CN)₂] and [Fe^IIL₃] for a series of L, where L is a 2,2′-bipyridine derivative, are presented here. We compare the solvatochromism of [Fe^II(4,4′-dicarboxylic acid-2,2′-bipyridine)₂(CN)₂] to [Fe^II(4,4′-dimethyl-2,2′-bipyridine)₂(CN)₂] and discuss general trends in the electrochemistry and absorption properties within the series. The solvatochromism of these complexes is discussed in terms of their use in a dye sensitized TiO₂ solar cell.     

Ferroxidase activity of recombinant Desulfovibrio vulgaris rubrerythrin

 Rubrerythrin (Rr) is the trivial name given to a non-heme iron protein of unknown function which has been found in anaerobic sulfate-reducing bacteria. Rr is unique in containing both rubredoxin-type FeS₄ and diiron-oxo sites in the same protein. The results described here demonstrate for the recombinant protein that: (a) Rr catalyzes oxidation of Fe²⁺ to Fe³⁺ by O₂, i.e., Rr has ferroxidase activity, (b) both FeS₄ and diiron domains of the Rr protein are required for ferroxidase activity, (c) with excess Fe²⁺ and O₂ the initial rate of this oxidation appears to be first order in [Rr] and independent of starting [Fe²⁺] above 30 μM, (d) the Fe³⁺ is produced in a form which is capable of rapid incorporation into the iron-binding site of ovotransferrin, and (e) the ferroxidase activity of Rr is comparable to that of published ferroxidase activities of apoferritins on a subunit basis. Ferroxidase activity of Rr was monitored either by the rate of increase in absorbance at 315 nm (which lies near an isosbestic point for oxidized and reduced Rr) or by using apoovotransferrin as Fe³⁺ acceptor, and measuring the rate and extent of diferric transferrin formation at 460 nm. No polyironoxyhydroxide aggregates appeared to associate with Rr after the ferroxidase reaction. A truncated form of Rr containing only the diiron domain had little or no ferroxidase activity. Rr could function as one component of a set of enzymes which channels the reaction products of O₂ and Fe²⁺ onto a non-toxic pathway during transient exposure of the bacteria to air.     

“Chelatable iron pool”: inositol 1,2,3-trisphosphate fulfils the conditions required to be a safe cellular iron ligand

Mammalian cells contain a pool of iron that is not strongly bound to proteins, which can be detected with fluorescent chelating probes. The cellular ligands of this biologically important “chelatable”, “labile” or “transit” iron are not known. Proposed ligands are problematic, because they are saturated by magnesium under cellular conditions and/or because they are not “safe”, i.e. they allow iron to catalyse hydroxyl radical formation. Among small cellular molecules, certain inositol phosphates (InsPs) excel at complexing Fe³⁺ in such a “safe” manner in vitro. However, we previously calculated that the most abundant InsP, inositol hexakisphosphate, cannot interact with Fe³⁺ in the presence of cellular concentrations of Mg²⁺. In this work, we study the metal complexation behaviour of inositol 1,2,3-trisphosphate [Ins(1,2,3)P ₃], a cellular constituent of unknown function and the simplest InsP to display high-affinity, “safe”, iron complexation. We report thermodynamic constants for the interaction of Ins(1,2,3)P ₃ with Na⁺, K⁺, Mg²⁺, Ca²⁺, Cu²⁺, Fe²⁺ and Fe³⁺. Our calculations indicate that Ins(1,2,3)P ₃ can be expected to complex all available Fe³⁺ in a quantitative, 1:1 reaction, both in cytosol/nucleus and in acidic compartments, in which an important labile iron subpool is thought to exist. In addition, we calculate that the fluorescent iron probe calcein would strip Fe³⁺ from Ins(1,2,3)P ₃ under cellular conditions, and hence labile iron detected using this probe may include iron bound to Ins(1,2,3)P ₃. Therefore Ins(1,2,3)P ₃ is the first viable proposal for a transit iron ligand. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Different mechanism of LPS-induced calcium increase in human lung epithelial cell and microvascular endothelial cell: a cell culture study in a model for ARDS

Acute respiratory distress syndrome (ARDS) is a contemporary term incorporating the historic ‘acute lung injury’ and the colloquial term ‘shock lung’. ARDS remains a serious and enigmatic human disease, causing significant mortality. The mechanisms involved at the alveolar cell/capillary endothelial interface have been explored but to date we lack clarity on the role of intracellular calcium ([Ca²⁺]i) fluxes across this interface. To explore the mechanisms of Ca²⁺ induced inflammatory reaction in epithelial cells and pulmonary microvascular endothelial cells (HMVEC) located at the two sides of blood-air barrier, lung epithelial A549 and HMVEC cells were treated with LPS. Our results demonstrated that LPS evoked the increase of [Ca²⁺]i, TNF-α and IL-8 in both cells types. The [Ca²⁺]i increases involved intracellular but not extracellular Ca²⁺ sources in A549, but both intracellular and extracellular Ca²⁺ sources in HMVEC cells. The effects of LPS on both cells types were completely inhibited by the combination of LPS and CaSR-targeted siRNA. Furthermore, LPS-inhibited cell proliferations were significantly reversed by the combined treatment. Therefore, LPS induced different mechanisms of [Ca²⁺]i increase during the activation of CaSR in A549 and HMVEC cells, which translates into functional outputs related to ARDS.     

Regulation of Intracellular Ca2+ by CFTR in Chinese Hamster Ovary Cells

In cystic fibrosis, the mutation of the CFTR protein causes reduced transepithelial Cl⁻ secretion. As recently proposed, beside its role of Cl⁻ channel, CFTR may regulate the activity of other channels such as a Ca²⁺-activated Cl⁻ channel. Using a calcium imaging system, we show, in adenovirus-CFTR infected Chinese Hamster Ovary (CHO) cell monolayers, that CFTR can act as a regulator of intracellular [Ca²⁺] _i ([Ca²⁺] _i ), involving purino-receptors. Apical exposure to ATP or UTP produced an increase in ([Ca²⁺] _i in noninfected CHO cell monolayers (CHO-WT), in CHO monolayers infected with an adenovirus-CFTR (CHO-CFTR) or infected with an adenovirus-LacZ (CHO-LacZ). The transient [Ca²⁺] _i increase produced by ATP or UTP could be mimicked by activation of CFTR with forskolin (20 μm) in CHO-CFTR confluent monolayers. However, forskolin had no significant effect on [Ca²⁺] _i in noninfected CHO-WT or in CHO-LacZ cells. Pretreatment with purino-receptor antagonists such as suramin (100 μm) or reactive blue-2. (100 μm), and with hexokinase (0.28 U/mg) inhibited the [Ca²⁺] _i response to forskolin in CHO-CFTR infected cells. Taken together, our experiments provide evidence for purino-receptor activation by ATP released from the cell and regulation of [Ca²⁺] _i by CFTR in CHO epithelial cell membranes. Received: 5 April 1999/Revised: 28 June 1999