Showing papers in "Biometals in 2003"

DMT1: A mammalian transporter for multiple metals

Abstract: DMT1 has four names, transports as many as eight metals, may have four or more isoforms and carries out its transport for multiple purposes. This review is a start at sorting out these multiplicities. A G185R mutation results in diminished gastrointestinal iron uptake and decreased endosomal iron exit in microcytic mice and Belgrade rats. Comparison of mutant to normal rodents is one analytical tool. Ectopic expression is another. Antibodies that distinguish the isoforms are also useful. Two mRNA isoforms differ in the 3' UTR: +IRE DMT1 has an IRE (Iron Responsive Element) but -IRE DMT1 lacks this feature. The +/-IRE proteins differ in the distal 18 or 25 amino acid residues after shared identity for the proximal 543 residues. A major function is serving as the apical iron transporter in the lumen of the gut. The +IRE isoform appears to have that role. Another role is endosomal exit of iron. Some evidence indicts the -IRE isoform for this function. In our ectopic expression assay for metal uptake, four metals--Fe2+, Mn2+, Ni2+ and Co2+--respond to the normal DMT1 cDNA but not the G185R mutant. Two metals did not--Cd2+ and Zn2+--and two--Cu2+ and Pb2+--remain to be tested. In competition experiments in the same assay, Cd2+, Cu2+ and Pb2+ inhibit Mn2+ uptake but Zn2+ did not. In rodent mutants, Fe and Mn appear more dependent on DMT1 than Cu and Zn. Experiments based on ectopic expression, specific antibodies that inhibit metal uptake and labeling data indicate that Fe3+ uptake depends on a different pathway in multiple cells. Two isoforms localize differently in a number of cell types. Unexpectedly, the -IRE isoform is in the nuclei of cells with neuronal properties. While the function of -IRE DMT1 in the nucleus is speculative, one may safely infer that this localization identifies new role(s) for this multifunctional transporter. Management of toxic challenges is another function related to metal homeostasis. Airways represent a gateway tissue for metal entry. Preliminary evidence using specific PCR primers and antibodies specific to the two isoforms indicates that -IRE mRNA and protein increase in response to exposure to metal in lungs and in a cell culture model; the +IRE form is unresponsive. Thus the -IRE form could be part of a detoxification system in which +IRE DMT1 does not participate. How does iron status affect other metals' toxicity? In the case of Mn, iron deficiency may enhance cellular responses.

Brain iron uptake and homeostatic mechanisms: an overview

Abstract: Timely and adequate iron acquisition by the brain is essential to normal neurological function. Despite the numerous cognitive and neurological impairments that are associated with disruptions in brain iron acquisition, including both too much and too little iron, the mechanism and regulation of the mechanisms by which the brain acquires iron are poorly understood. In this article, we review the current state of knowledge regarding expression of iron transport proteins in the brain, brain iron uptake and discuss why a model for brain iron uptake must take into consideration the potentially competing influences on the endothelial cell between the status of iron in the brain versus the systemic iron status.

The copper-iron chronicles: The story of an intimate relationship

Abstract: During the last decade there has been a surge of interest and activity in exploring the metabolic links between copper and iron. This review describes more than a century and a half of effort that has led to our current understanding. Particular attention is given to the early events since these are less well-known and appreciated. The landmark 1928 paper of Hart, Elvehjem and coworkers is generally given credit for the start of the copper/iron field, and specifically for the discovery of the role of copper in forming hemoglobin and in overcoming anemia. However, some credit for the ideas, observations, and experiments should be shared with several investigators of the previous century. These scientists and physicians were primarily motivated to find the causes and cures of chlorosis, a common form of anemia at the time. From his chemical determination of copper in red blood cells in 1848, Millon proposed a form of chlorosis due to copper deficiency. Likewise, Pecholier and Saint-Pierre, observing the robust health of young women working in copper factories, concluded that copper was helpful in preventing and overcoming chlorosis. The first direct experimental evidence for the theory was provided by the Italian physician Mendini, who in 1862 reported that supplementation of the diet with copper salts overcame chlorosis in young women. In the 1890s Cervello and his students in Italy, using semi-quantitative hematological measurements, confirmed the beneficial effects of copper on anemia both in patients and in animal models. There was nearly a 30-year period of inactivity, but the decade of the 1930s saw renewed interest and exciting developments in the field. The Elvehjem report of 1928 was quickly verified and extended by multiple laboratories on four continents. In the 1950s and 1960s Wintrobe and Cartwright and their colleagues localized, and started to systematically evaluate, the potential sites at which copper was likely to effect iron for hemoglobin synthesis, namely, intestinal absorption, release from storage, and cellular utilization during synthesis. The copper/iron connection also has a 'flip-side', i.e., iron status can influence copper metabolism as first described by Warburg and Krebs in 1927. Thus, there are opportunities for feedback mechanisms at the cellular and physiological level that are not yet understood. The evaluation of these processes continues to this day, aided by modern molecular and genetic approaches. Studies of two copper proteins, ceruloplasmin and its recently discovered homologue hephaestin, have provided two molecular links connecting the pathways of copper and iron metabolism. The recent identification of other proteins of iron and copper metabolism, for example, copper ATPases and the membrane iron transporters DCT1/DMT1/Nramp2 and IREG1/MTP1/ferroportinl, are likely to fill crucial pathway gaps. The ongoing discovery of genes and gene mutations involved in the metabolism of copper and iron provides an important key to a deeper understanding of the connections between the pathways, and their physiological and pathological consequences. It is hoped that this historical review, by illuminating the complex paths that have led to the current state of knowledge, will contribute to our appreciation, our understanding, and perhaps our continued discovery of the connections between copper and iron.

Yeast, a model organism for iron and copper metabolism studies.

Abstract: Virtually all organisms on earth depend on transition metals for survival. Iron and copper are particularly important because they participate in vital electron transfer reactions, and are thus cofactors of many metabolic enzymes. Their ability to transfer electrons also render them toxic when present in excess. Disturbances of iron and copper steady-state levels can have profound effects on cellular metabolism, growth and development. It is critical to maintain these metals in a narrow range between utility and toxicity. Organisms ranging from bacteria and plants to mammals have developed sophisticated mechanisms to control metal homeostasis. In this review, we will present an overview of the current understanding of iron and copper metabolism in yeast, and the utility of yeast as a model organism to investigate iron and copper metabolism in mammals and plants.

Oxidative stress-mediated hepatotoxicity of iron and copper: role of Kupffer cells.

Abstract: Iron- or copper-mediated catalysis leads to the generation of reactive oxygen species (ROS) that can attack biomolecules directly, with the consequent enhancement in membrane lipid peroxidation, DNA damage, and protein oxidation Reactive nitrogen species (RNS) can also be formed, leading to nitration of aromatic structures in addition to the oxidative deterioration of cellular components Kupffer cells, the resident macrophages of the liver, play significant roles in immunomodulation, phagocytosis, and biochemical attack Upon stimulation, liver macrophages release biologically active products related to cell injury, namely, ROS, RNS, and both immunomodulatory and fibrogenic cytokines, with production of chemokines and adhesion molecules by other cells of the liver sinusoid Iron and copper alter the functional status of Kupffer cells by enhancing their respiratory burst activity without modifying particle phagocytosis This effect is probably due to extra O2 equivalents used in the oxidation of biomolecules and/or in the activating action of iron/copper on nitric oxide synthase, in addition to those employed by NADPH oxidase activity Changes in gene expression of Kupffer cells may also be accomplished by iron and copper through modulation of the activity of transcription factors such as NF-κB, which signals the production of cytotoxic, proinflammatory, or fibrogenic mediators Thus, iron/copper-induced hepatotoxicity is a multifactorial phenomenon underlying actions due to the generation of ROS and RNS that may alter essential biomolecules with loss of their biological functions, modulate gene expression of Kupffer cells with production of cytotoxic mediators, or both

Platinum(II) and palladium(II) complexes with 2-acetyl pyridine 4N-ethyl thiosemicarbazone able to overcome the cis-platin resistance. Structure, antibacterial activity and DNA strand breakage

Abstract: The reactions of Pd(II) and Pt(II) with 2-Acetyl Pyridine N(4)-Ethyl-Thiosemicarbazones, HAc4Et and 2-Acetyl Pyridine N(4)-1-(2-pyridyl)-piperazinyl Thiosemicarbazone, HAc4PiPiz and 2-Formyl Pyridine N(4)-1-(2-pyridyl)-piperazinyl Thiosemicarbazone, HFo4PiPiz afforded the complexes, [Pd(Ac4Et)], 1, [Pd(HAc4Et)2]Cl2, 2 and [Pd(Ac4Et)2], 3 [Pt(Ac4Et)], 4, [Pt(HAc4Et)2]Cl2, 5, [Pt(Ac4Et)2], 6 and [Pd(Fo4PipePiz)Cl], 7, [Pd(Fo4PipePiz)2], 8, [Pd(Ac4PipePiz)Cl], 9 and [Pd(Ac4PipePiz)2], 10. The crystal structure of the complex [Pt(Ac4Et)2], 6 has been solved. The platinum(II) atom is in a square planar environment surrounded by two cis nitrogen atoms and two cis sulfur atoms. The ligands are not equivalent, one being tridentate with (N,N,S) donation, the other being monodentate using only the sulfur atom to coordinate to the metal. The tridentate ligand shows a Z, E, Z configuration while the monodentate ligand shows an E, E, Z. Inter-molecular hydrogen bonds stabilize the structure, while the crystal packing is determined by pi-pi, and Pt-C interactions. The antibacterial effect of Pd(II) and Pt(II) complexes were studied in vitro. The complexes were found to have effect on Gram(+) bacteria, while the same complexes showed no bactericidal effect on Gram(-) bacteria. The effect of the Pd(II) and Pt(II) complexes on the in vitro DNA strand breakage was studied by agarose gel electrophoresis. The complexes 1-6 were found to exhibit a cytotoxic potency in a very low micromolar range and to be able to overcome the cisplatin resistance of A2780/Cp8 cells.

Thiol-induced reduction of antimony(V) into antimony(III): a comparative study with trypanothione, cysteinyl-glycine, cysteine and glutathione.

Abstract: Gluthathione (GSH) has been previously shown to promote the reduction of pentavalent antimony (Sb(V)) into the more toxic trivalent antimony (Sb(III)) in the antimonial drug, meglumine antimonate However, this reaction occurred at acidic pH (pH 5) but not at the pH of the cytosol (pH 72) in which GSH is encountered The aim of the present study was to further characterize the reaction between thiols and antimonial drugs, addressing the following aspects: (i) the reducing activity of cysteine (Cys) and cysteinyl-glycine (Cys-Gly), expected to be the predominant thiols in the acidic compartiments of mammalian cells; (ii) the reducing activity of trypanothione (T(SH)2), the main intracelular thiol in Leishmania parasites; (iii) the influence of the state of complexation of Sb(V) on the rate of Sb(V) reduction We report here that Cys, Cys-Gly and T(SH)2 did promote the reduction of Sb(V) into Sb(III) at 37 °C Strikingly, the initial rates of reduction of Sb(V) were much greater in the presence of Cys-Gly, Cys and T(SH)2 than in the presence of GSH These reactions occurred at both pH 5 and pH 7 but were favored at acidic pH Moreover, our data shows that Sb(V) is reduced more slowly in the form of meglumine antimonate than in its non-complexed form, indicating that the complexation of Sb(V) tends to slow down the rate of its reduction In conclusion, our data supports the hypothesis that Sb(V) is reduced in vivo by T(SH)2 within Leishmania parasites and by Cys or Cys-Gly within the acidic compartments of mammalian cells

Aceruloplasminemia, an inherited disorder of iron metabolism.

Abstract: Ceruloplasmin, a multi-copper ferroxidase that affects the distribution of tissue iron, has antioxidant effects through the oxidation of ferrous iron to ferric iron Aceruloplasminemia is an inherited disorder of iron metabolism due to the complete lack of ceruloplasmin ferroxidase activity caused by mutations in the ceruloplasmin gene It is characterized by iron accumulation in the brain as well as visceral organs Clinically, the disease consists of the triad of retinal degeneration, diabetes mellitus, and neurological disease, which include ataxia, involuntary movements, and dementia These symptoms reflect the sites of iron deposition The unique involvement of the central nervous system distinguishes aceruloplasminemia from other inherited and acquired iron storage disorders Twenty-one mutations in the ceruloplasmin gene have been reported in 24 families worldwide In Japan, the incidence was estimated to be approximately one per 2,000,000 in the case of non-consanguineous marriages Excess iron functions as a potent catalyst of biologic oxidation Previously we showed that an increased iron concentration is associated with increased levels of lipid peroxidation in the serum, cerebrospinal fluid, and erythrocyte membranes The levels of malondialdehyde and 4-hydroxynonenals, indicators of lipid peroxidation, were also elevated in the basal ganglia and cerebral cortex Positron emission tomography showed diminished brain metabolism of glucose and oxygen Enzyme activities in the mitochondrial respiratory chain of the basal ganglia were reduced to approximate 45% and 42%, respectively, for complexes I and IV These findings suggest that iron-mediated free radicals causes neuronal cell damage through lipid peroxidation and mitochondrial dysfunction in aceruloplasminemia brains

Mode of anti-fungal activity of 1,10-phenanthroline and its Cu(II), Mn(II) and Ag(I) complexes.

Abstract: The mode of action of the anti-fungal compounds, 1,10-phenanthroline (phen), [Cu(phen)2(mal)] ⋅ 2H2O, [Mn(phen)2(mal)] ⋅ 2H2O and [Ag2(phen)3(mal)] ⋅ 2H2O (malH2 = malonic acid), was examined using the pathogenic yeast Candida albicans. The compounds have minimum inhibitory concentrations (MIC's) in the range 1.25–5.0 μg cm−3 and at a concentration of 10 μg cm−3 display some fungicidal activity. Yeast cells exposed to these drugs show a diminished ability to reduce 2,3,5-triphenyltetrazolium chloride (TTC), indicating a reduction in respiratory function. Treating exponential and stationary phase yeast cells with phen and the Cu(II) and Mn(II) complexes causes a dramatic increase in oxygen consumption. All of the drugs promote reductions in the levels of cytochromes b and c in the cells, whilst the Ag(I) complex also lowers the amount of cytochrome aa3. Cells treated with phen and the Cu(II) and Ag(I) species show reduced levels of ergosterol whilst the Mn(II) complex induces an increase in the sterol concentration. The general conclusion is that the drugs damage mitochondrial function and uncouple respiration. That the drugs are not uniformly active suggests their bioactivity has a degree of metal-ion dependency. Phen and metal-phen complexes represent a novel set of highly active anti-fungal agents whose mode of action is significantly different to that of the polyene and azole prescription drugs.

Variability of the Fenton reaction characteristics of the EDTA, DTPA, and citrate complexes of iron

Abstract: The common metal chelation agents, DTPA and EDTA are often used as models for physiological low-molecular weight iron complexes in biochemical studies, or for common biochemical protocols In the biochemical literature there are apparent conflicts as to whether EDTA and DTPA are pro-oxidant or antioxidant additives This apparent conflict is puzzling since in chemical systems FeIIEDTA and FeIIDTPA are well known Fenton reaction reagents In this investigation we examined the voltammetric characteristics of the iron complexes of EDTA, DTPA, and citrate and the effect of the ligand:metal ratio (L:M) on the electrocatalytic (EC') waves that result from reduction of H2O2 by this complex At a ratio of 1:1, the cyclic voltammetric waves of the complexes indicate the presence of a reversible species corresponding to the Fe(II/III)L couple, along with a second irreversible reduction peak The second irreversible voltammetric peak decreases at higher L:M ratios for EDTA and citrate The 1:1 iron complexes of EDTA, DTPA, and citrate clearly induce the catalytic reduction of H2O2 In the presence of a greater than 100 fold excess of H2O2 relative to iron, higher L:M ratios greatly reduced the catalytic EC' wave compared to the 1:1 ratios At H2O2:Fe ratios less than 50, the L:M ratio has very little effect of the EC' current These observations may explain the apparent discrepancies in the biochemical literature Addition of EDTA or DTPA may enhance oxidative processes if the L:M is low (less than unity), whereas rates of on-going oxidative processes may decrease if that ratio, along with the relative amount of H2O2, are both high (excess ligand) The impact of this study is of particular importance given the widespread use of these ligands in biochemical studies

Iron-induced oxidative stress modify tau phosphorylation patterns in hippocampal cell cultures.

Abstract: Oxidative stress phenomena have been related with the onset of neurodegenerative diseases. Particularly in Alzheimer Disease (AD), oxygen reactive species (ROS) and its derivatives can be found in brain samples of postmortem AD patients. However, the mechanisms by which oxygen reactive species can alter neuronal function are still not elucidated. There is a growing amount of evidence pointing to a role for mitochondrial damage as the source of free radicals involved in oxidative stress. Among the species that participate in the production of oxygen reactive radicals, transition metals are one of the most important. Several reports have implicated the involvement of redox-active metals with the onset of different neurodegenerative diseases such as Alzheimer's Disease (AD), Progressive Supranuclear Palsy (PSP), Amyotrophic Lateral Sclerosis (ALS) and Parkinson's Disease (PD). On the other hand, our previous studies have indicated that Aβ-induced deregulation of the protein kinase Cdk5 associated with tau protein hyperphosphorylation constitute a critical pathway toward neurodegeneration. In the current paper we have shown that iron induces an imbalance in the function of Cdk5/p25 system of hippocampal neurons, resulting in a marked decrease in tau phosphorylation at the typical Alzheimer's epitopes. The loss of phosphorylated tau epitopes correlated with an increase in 4-hydroxy-nonenal (HNE) adducts revealing damage by oxidative stress. This effects on tau phosphorylation patterns seems to be a consequence of a decrease in the Cdk5/p25 complex activity that appears to result from a depletion of the activator p25, a mechanism in which calcium transients could be implicated.

Are cadmium effects on plasma gonadotropins, prolactin, ACTH, GH and TSH levels, dose-dependent?

Abstract: It is well established that cadmium affects plasma levels of the pituitary hormones studied. However, whether the effects of the metal are dose dependent needs to be clarify. This work was designed to evaluate the possible changes in plasma levels of gonadotropins, prolactin, ACTH, GH and TSH after oral cadmium exposure in adult male rats. Plasma levels of these hormones were measured in adult male rats exposed to cadmium chloride (CdCl2) in the drinking water at the doses of 5, 10, 25, 50 or 100 ppm for one month. The lower dose of cadmium increased plasma prolactin levels and higher doses of the metal (25 or 50 ppm) decreased them. There was a continuous increase of plasma ACTH levels from the lower to 25 ppm dose of CdCl2 and decreased them after to basal values with the highest dose. Plasma GH levels were increased with the dose of cadmium of 10 ppm, although the doses of 5, 25 and 50 ppm decreased them. Plasma LH levels were only reduced with the dose of 50 ppm of CdCl2, whereas those of FSH increased. Plasma TSH levels were increased with the doses of 5, 25 and 100 ppm of CdCl2. Cadmium concentration increased in pituitary with the doses of 125, 50 and 100 ppm of CdCl2. These data suggest that cadmium differentially affects the secretory mechanisms of the pituitary hormones studied depending on the dose used. The effects of the metal on prolactin and ACTH are dose-dependent.

Adventiously-bound redox active iron and copper are at the center of oxidative damage in Alzheimer disease

Abstract: Central to oxidative damage in Alzheimer disease is the production of metal-catalyzed hydroxyl radicals that damage every category of macromolecule. Studies on redox-competent copper and iron indicate that redox activity in Alzheimer disease resides exclusively within the cytosol of vulnerable neurons and that chelation with deferoxamine or DTPA removes this activity. We have also found that while proteins that accumulate in Alzheimer disease such as tau, amyloid beta, and apolipoprotein E possess metal-binding sites, metal-associated cellular redox activity is more dependent on metal-nucleic acid binding. Consistent with this finding is the large amount of cytoplasmic RNA in pyramidal neurons. Still, the source of metal-catalyzed redox activity is controversial. Heme oxygenase-1, an enzyme that catalyzes the conversion of heme to iron and biliverdin, is increased in Alzheimer disease suggesting increased heme turnover as a source of redox-active iron. Additionally, the role of mitochondria as a potential source of redox-active metals and oxygen radical production is assuming more prominence. In recent studies, we have found that while mitochondrial DNA and cytochrome C oxidase activity are increased in Alzheimer disease, the number of mitochondria is decreased, indicating accelerated mitochondria turnover. This finding, as well as preliminary studies demonstrating a reduction in microtubule density in neurons in Alzheimer disease suggests mitochondrial dysfunction as a potentially inseparable component of the initiation and progression of Alzheimer disease.

Copper-induced trafficking of the cU-ATPases: a key mechanism for copper homeostasis.

Abstract: The Menkes protein (MNK) and Wilson protein (WND) are transmembrane, CPX-type Cu-ATPases with six metal binding sites (MBSs) in the N-terminal region containing the motif GMXCXXC. In cells cultured in low copper concentration MNK and WND localize to the transGolgi network but in high copper relocalize either to the plasma membrane (MNK) or a vesicular compartment (WND). In this paper we investigate the role of the MBSs in Cu-transport and trafficking. The copper transport activity of MBS mutants of MNK was determined by their ability to complement a strain of Saccharomyces cerevisiae deficient in CCC2 (Δccc2), the yeast MNK/WND homologue. Mutants (CXXC to SXXS) of MBS1, MBS6, and MBSs1-3 were able to complement Δccc2 while mutants of MBS4-6, MBS5-6 and all six MBS inactivated the protein. Each of the inactive mutants also failed to display Cu-induced trafficking suggesting a correlation between trafficking and transport activity. A similar correlation was found with mutants of MNK in which various MBSs were deleted, but two constructs with deletion of MBS5-6 were unable to traffic despite retaining 25% of copper transport activity. Chimeras in which the N-terminal MBSs of MNK were replaced with the corresponding MBSs of WND were used to investigate the region of the molecules that is responsible for the difference in Cu-trafficking of MNK and WND. The chimera which included the complete WND N-terminus localized to a vesicular compartment, similar to WND in elevated copper. Deletions of various MBSs of the WND N-terminus in the chimera indicate that a targeting signal in the region of MBS6 directs either WND/MNK or WND to a vesicular compartment of the cell.

Copper reduction by copper binding proteins and its relation to neurodegenerative diseases.

Abstract: Increasing evidence supports an important role for metals in neurobiology In fact, copper binding proteins that form bioinorganic complexes are able to display oxidant or anti-oxidant properties, which would impact on neuronal function or in the triggering of neurodegenerative process Two proteins related to neurodegenerative diseases have been described as copper binding proteins: the amyloid precursor protein (APP), a protein related to Alzheimer's disease, and the Prion protein (PrP), related to Creutzfeldt-Jakob disease We used different synthetic peptides from APP and PrP sequences in order to evaluate the ability to reduce copper We observed that APP(135-156), amyloid-beta-peptide (A beta(1-40)), and PrP(59-91) all have copper reducing ability, with the APP(135-156) peptide being more potent than the other fragments Moreover, we identify His, Cys and Trp residues as key amino acids involved in the copper reduction of A beta, APP and PrP, respectively We postulated, that in a cellular context, the interaction of these proteins with copper could be necessary to reduce copper on plasma membrane, possibly presenting Cu(I) to the copper transporter, driving the delivery of this metal to antioxidant enzymes Moreover, protein-metal complexes could be the catalytic centers for the formation of reactive oxygen species involved in the oxidative damage present both in Alzheimer's and Prion disease

The Saccharomyces cerevisiae Arr4p is involved in metal and heat tolerance

Abstract: Homologues of the bacterial ArsA ATPase are found in nearly every organism. While the enzyme is involved in arsenic detoxification in bacteria, the roles of eukaryotic homologues have not been identified. This article reports the function of the Saccharomyces cerevisiae homologue encoded by ARR4 gene (YDL100c ORF). Disruption of ARR4 was not lethal, but the disrupted strain displayed increased sensitivity to As3+, As5+, Co2+, Cr3+, Cu2+ or VO4(3-) salts and temperature. A plasmid-encoded copy of a wild-type ARR4 gene could complement the heat- or metal-related stress responses. Mutation of a codon within the consensus sequence for the nucleotide-binding site resulted in loss of complementation of the disrupted strain and produced a dominant negative phenotype in a wild type strain. Wild type and mutant Arr4p were purified from Escherichia coli. The wild type protein exhibited a low level of ATPase activity, and the mutant was inactive. The purified ATPase eluted as a dimer of 80-kDa species. A fusion of ARR4 and the GFP (green fluorescent protein) gene was constructed. The gene fusion was able to complement stress-related phenotype of the ARR4 disruption. Under non-stress conditions, GFP fluorescence was found diffusely in the cytosol. Under stress conditions GFP was localized in a few punctate bodies resembling late endosomes. It is proposed that under heat or metal stress, the soluble ATPase becomes membrane-associated, perhaps through interaction with a partner protein, and that this complex is involved in stress tolerance.

Bacillus subtilis CPx-type ATPases: characterization of Cd, Zn, Co and Cu efflux systems.

Abstract: Metal ion homeostasis requires the balanced expression of metal ion uptake systems, when metals are limiting, and corresponding efflux or storage systems, when metals are in excess. CPx-type ATPases are a family of membrane-bound transporters that often function to export toxic metals from cells. The Bacillus subtilis genome encodes three CPx-type ATPases: zosA, yvgW and yvgX. We demonstrate that yvgW and yvgX encode CadA and CopA, respectively, and that these genes function in metal ion resistance. A cadA mutant was sensitive to Cd(II), Zn(II) and Co(II), but not copper. Transcription of cadA initiates from a single, sigmaA-type promoter and was induced by Cd(II), Zn(II), and Co(II). The adjacent copZA operon is expressed as a bicistronic transcript from a sigmaA-type promoter and is selectively induced by copper. Mutation of either copZ, encoding a metallochaperone, or copA sensitizes the cells to copper but not to other metal ions.

Complexation of uranium (VI) by three eco-types of Acidithiobacillus ferrooxidans studied using time-resolved laser-induced fluorescence spectroscopy and infrared spectroscopy

Abstract: Time-resolved laser-induced fluorescence spectroscopy (TRLFS) was used to study the properties of uranium complexes (emission spectra and fluorescence lifetimes) formed by the cells of the three recently described eco-types of Acidithiobacillus ferrooxidans. The results demonstrated that these complexes have different lifetimes which increase in the same order as the capability of the strains to accumulate uranium. The complexes built by the cells of the eco-type II were the strongest, whereas, those of the eco-types I and III were significantly weaker. The emission spectra of all A. ferrooxidans complexes were almost identical to those of the uranyl organic phosphate compounds. The latter finding was confirmed by infrared spectroscopic analysis.

Copper exposure modifies the content and distribution of trace metals in mammalian cultured cells

Abstract: With this work, we have determined the cellular content of Cu, Fe and Zn in different cell lines, by using total reflection X-ray fluorescence spectrometry (TXRF). In addition, we examined whether cellular exposure to 100 μmoles l−1 of Cu-His modifies the intracellular content and distribution of these trace metals. Our results indicate that all the cell lines displayed the same pattern of relative intracellular abundance of trace metals (Cu

Role of nitric oxide in cellular iron metabolism.

Abstract: Iron regulatory proteins (IRP1 and IRP2) control the synthesis of transferrin receptors (TfR) and ferritin by binding to iron-responsive elements (IREs) which are located in the 3′ untranslated region (UTR) and the 5′ UTR of their respective mRNAs. Cellular iron levels affect binding of IRPs to IREs and consequently expression of TfR and ferritin. Moreover, NO•, a redox species of nitric oxide that interacts primarily with iron, can activate IRP1 RNA-binding activity resulting in an increase in TfR mRNA levels. We have shown that treatment of RAW 264.7 cells (a murine macrophage cell line) with NO+ (nitrosonium ion, which causes S-nitrosylation of thiol groups) resulted in a rapid decrease in RNA-binding of IRP2, followed by IRP2 degradation, and these changes were associated with a decrease in TfR mRNA levels. Moreover, we demonstrated that stimulation of RAW 264.7 cells with lipopolysaccharide (LPS) and interferon-γ (IFN-γ) increased IRP1 binding activity, whereas RNA-binding of IRP2 decreased and was followed by a degradation of this protein. Furthermore, the decrease of IRP2 binding/protein levels was associated with a decrease in TfR mRNA levels in LPS/IFN-γ-treated cells, and these changes were prevented by inhibitors of inducible nitric oxide synthase. These results suggest that NO+-mediated degradation of IRP2 plays a major role in iron metabolism during inflammation.

Copper exposure and potential biomarkers of copper metabolism.

Abstract: Relevant biological effects associated with mild to moderate copper deficiency and copper excess are unknown. It is difficult to identify markers of these early changes because limits of the homeostatic range are still undefined and early changes may represent adaptive responses that do not imply necessarily risk of damage. We report here a series of studies carried out to shed light on the responses within the homeostatic range, by assessing classic parameters of copper status in humans at different copper exposure. In adult healthy volunteers that had an estimated daily intake of 0.9 mg Cu/day (approximately 15 microg/kg/d), exposure to additional 50-60 microg of copper/kg/day for three months or up to 150 microg/kg/d for two months resulted in no significant changes of SOD activity in erythrocytes, of copper concentration (in serum, erythrocytes and mononuclear cells) and of serum ceruloplasmin (ANOVA). Neither were found differences by gender or age. As in previous studies in infants, the non-ceruloplasmin copper fraction was positively correlated to serum copper (r = 0.58). Assessing variations on copper absorption, infants supplemented/not supplemented with oral copper (80 ug/kg/14 days), at age 1 and 3 months, showed copper absorption close to 80% at both ages; no effect was observed for age or supplementation, suggesting that either these concentrations do not elicit regulatory mechanisms or that at this age down regulation for copper absorption is not efficient. These studies indicate that in the range of the copper homeostasis area the markers tested are not suitable to detect mild changes (within the homeostatic range) of copper metabolism.

Iron and copper homeostasis and intestinal absorption using the Caco2 cell model.

Abstract: Whole body homeostasis can be viewed as the balance between absorption and excretion, which can be regulated independently. Present evidence suggests that for iron, intestinal absorption is the main site for homeostatic regulation, while for copper it is biliary excretion. There are connections between iron and copper in intestinal absorption and transport. The blue copper plasma protein, ceruloplasmin, and its intracellular homologue, hephaestin, play a role in cellular iron release. The studies reviewed here compare effects of Fe(II) and Cu(II) on their uptake and overall transport by monolayers of polarized Caco2 cells, which model intestinal mucosa. In the physiological range of concentrations, depletion of cellular iron or copper (by half) increased uptake of both metal ions. Depletion of iron or copper also enhanced overall transport of iron from the apical to the basal chamber. Copper depletion enhanced overall copper transport, but iron depletion did not. Pretreatment with excess copper also stimulated copper absorption. Plasma ceruloplasmin (added to the basal chamber) failed to enhance basolateral iron release, and Zn(II) failed to compete with Cu(II) for uptake. Neither copper nor iron deficiency altered expression of IREG1 or DMT1 (-IRE form) at the mRNA level. Thus, in the low-normal range of iron and copper availability, intestinal absorption of both metals appears to be positively related to the need for these elements by the whole organism. The two metal ions also influenced each other's transport; but with copper excess, other mechanisms come into play.

Calcein as a fluorescent iron chemosensor for the determination of low molecular weight iron in biological fluids.

Abstract: The fluorescence quenching of calcein (CA) is not iron specific and results in a negative calibration curve. In the present study, deferoxamine (DFO), a strong iron chelator, was used to regenerate the fluorescence quenched by iron. Therefore, the differences in fluorescence reading of the same sample with or without addition of DFO are positively and specifically proportional to the amounts of iron. We found that the same iron species but different anions (e.g. ferric sulfate or ferric citrate) differed in CA fluorescence quenching, so did the same anions but different iron (e.g. ferrous or ferric sulfates). Excessive amounts of citrate competed with CA for iron and citrate could be removed by barium precipitation. After optimizing the experimental conditions, the sensitivity of the fluorescent CA assay is 0.02 microM of iron, at least 10 times more sensitive than the colorimetric assays. Sera from 6 healthy subjects were tested for low molecular weight (LMW) chelator bound iron in the filtrates of 10 kDa nominal molecular weight limit (NMWL). The LMW iron was marginally detectable in the normal sera. However, increased levels of LMW iron were obtained at higher transferrin (Tf) saturation (1.64-2.54 microM range at 80% Tf saturation, 2.77-3.15 microM range at 100% Tf saturation and 3.09-3.39 microM range at 120% Tf saturation). The application of the assay was further demonstrated in the filtrates of human liver HepG2 and human lung epithelial A549 cells treated with iron or iron-containing dusts.

Identification and mutational studies of conserved amino acids in the outer membrane receptor protein, FepA, which affect transport but not binding of ferric-enterobactin in Escherichia coli.

Abstract: Many gram-negative bacteria produce and excrete siderophores, which complex iron with high affinity in the environment. The ferric siderophore complexes are transported across the outer membrane by receptor proteins. This process requires energy and is TonB dependent and must involve conformational changes in the receptor proteins to allow the transport of the ferric siderophores from the extracellular binding site to the periplasm. There is a large variety in the structures, molecular weights and charges among the siderophores. It was therefore realized that when the sequences of the many different receptor proteins were compared, simultaneously, all identities and close similarities, found in this manner, could only be due to residues involved in the conformational changes and transport mechanism, common to all the proteins, and not be due to the specificity of ligand recognition. Once the crystal structures of FepA, FhuA and FecA became available, it was immediately clear that the sequence similarities which were found in the simultaneous alignment, were all localized in a few structural domains, which are identical in the three structures and can therefore be expected to be maintained in all the proteins in this family. One of these domains, tentatively named the lock region, consists of 10 residues with a central quadrupole formed by two arginines and two glutamates, from the plug region and the beta barrel. We mutated several of these residues in FepA. All showed normal binding in quantitative binding studies. Some showed normal transport as well, however, the majority showed moderate to severe defective transport with ferric enterobactin. The results therefore show the validity of the hypothesis that the simultaneous sequence alignment will select the residues involved in the transport function of the receptor proteins. In addition the results allow to relate the severity of the transport deficiency to be correlated with the structure of the lock region while it is also possible to propose a function of this region in the conformational changes of the protein during the transport of the ligand from the binding site to the periplasm.

The Enterococcus hirae paradigm of copper homeostasis: copper chaperone turnover, interactions, and transactions.

Abstract: The cop operon is a key element of copper homeostasis in Enterococcus hirae. It encodes two copper ATPases, CopA and CopB, the CopY repressor, and the CopZ metallochaperone. The cop operon is induced by copper, which allows uncompromised growth in up to 5 mM ambient copper. Copper uptake appears to be accomplished by the CopA ATPase, a member of the heavy metal CPx-type ATPases and closely related to the human Menkes and Wilson ATPases. The related CopB ATPase extrudes copper when it reaches toxic levels. Intracellular copper routing is accomplished by the CopZ copper chaperone. Using surface plasmon resonance analysis, it was demonstrated that CopZ interacts with the CopA ATPase where it probably becomes copper loaded. CopZ in turn can donate copper to the copper responsive repressor CopY, thereby releasing it from DNA. In high copper, CopZ is proteolyzed. Cell extracts were found to contain a copper activated proteolytic activity that degrades CopZ in vitro. This post-translational control of CopZ expression presumably serves to avoid the accumulation of detrimental Cu-CopZ levels.

Laboratory simulation of a mining accident: acute toxicity, hsc/hsp70 response, and recovery from stress in Gammarus fossarum (Crustacea, Amphipoda) exposed to a pulse of cadmium.

Abstract: The rate of survival and stress protein (hsc/hsp70) response were investigated in the freshwater amphipod, Gammarus fossarum Koch, 1835, during a 20-day stress and recovery experiment. Adult females and males, were separately exposed to 9 different cadmium concentrations for 5 days to simulate a short-term pulse of xenobiotics in an aquatic environment, followed by a recovery period of 15 days. In terms of mortality, females were much more sensitive to cadmium than males; 4.28±2.45 μg Cd2+/l resulted in strong effects on the rate of survival of females but not males. In both sexes, mortality occurred predominantly within the first 5 days of the recovery period. At the cellular level, cadmium induced an hsc/hsp70 response. The lower Cd2+ concentrations we used led to an induction of stress proteins while higher Cd2+ concentrations resulted in a proportionately reduced hsc/hsp70 response, most likely due to pathological damage. Surviving individuals retained their capacity to induce stress protein production in the recovery period, even if the stress protein response system was overwhelmed by cadmium during the exposure period.

Visible and near-IR light induced biohydrogen production using the system containing Mg chlorophyll-a from Spirulina and colloidal platinum.

Abstract: Photoinduced hydrogen production with Mg chlorophyll-a from Spirulina as a visible and near-IR light photosensitizer by use of three component system consisting of nicotineamide adenine dinucleotide phosphate, reduced form (NADPH) as an electron donor, methylviologen as electron relay reagent and colloidal platinum as hydrogen production catalyst was investigated After 4 h irradiation, the amount of hydrogen production with Mg chlorophyll-a and MgTPP, which was artificial model compound for chlorophyll, were ca 27 and 18 μmol, respectively When the near-IR light was irradiated, little change of hydrogen production was observed Thus, the effective visible and near IR light induced hydrogen production system with colloidal platinum was established using Mg chlorophyll-a

Parallels and contrasts between iron and copper metabolism.

Abstract: This paper reviews the Second International Workshop on Iron and Copper Homeostasis, held in Pucon, Chile 10–13 November, 2001. We cover the presentations and papers published (this issue) with the intent to point out parallels, contrasts and cutting edge areas rather than to say something about every paper. Iron and copper metabolism have been intertwined for nearly 150 years and the interrelationship is growing with advances in understanding the role of ceruloplasmin as one example and the probable role of hephaestin as another. The transporter DMT1 (divalent metal transporter 1) clearly plays a major part in iron uptake and trafficking. Emerging evidence suggests that it plays a lesser role in manganese, cadmium and copper transport; but it is still being evaluated there. Yet another interaction may come from the IRE/IRP (Iron Responsive Element/Iron Regulatory Protein) story where a paradigmatic role in iron homeostasis is well established, but interaction with copper is only now emerging. Parallels include the nutrient status of both metals based on their utility for redox reactions as well as their toxicity primarily via reactive oxygen species. The workshop also revealed that alternate splicing of pre-mRNAs for iron and copper related proteins and tissue specific responses are additional similarities. Regulation of gene expression and excretion offered contrasts between the two metals. The workshop also considered a series of continuing and emerging issues.

The role of the placenta in iron transfer from mother to fetus and the relationship between iron status and fetal outcome

Abstract: During pregnancy, iron is transferred from the mother to the fetus across the placenta. The mechanism has been extensively studied. Altered iron metabolism changes transfer, but also has other consequences. In this review, we examine how the placenta adapts to altered iron supply, both in terms of changing cytokine expression and in relation to the proteins of iron transfer. Changing iron levels alters the levels of other metals, especially copper, and we review how this is related to changing function. There are also consequences to the placenta itself, to vascularisation and other aspects of the physiology. In turn, this has effects on the fetus and we review how growth and development are modified. Finally, we examine in more detail the efflux process, how it is regulated and, especially, the putative role of the placental Cu oxidase in the efflux process. As appropriate, we draw on data from humans, from animal models and from cell culture systems to illustrate the information.

Nickel removal from nickel plating waste water using a biologically active moving-bed sand filter

Abstract: Efficient removal of dissolved nickel was observed in a biologically active moving-bed `MERESAFIN' sand filter treating rinsing water from an electroless nickel plating plant. Although nickel is fully soluble in this waste water, its passage through the sand filter promoted rapid removal of approximately 1 mg Ni/l. The speciation of Ni in the waste water was modelled; the most probable precipitates forming under the conditions in the filter were predicted using PHREEQC. Analyses of the Ni-containing biosludge using chemical, electron microscopical and X-ray spectroscopic techniques confirmed crystallisation of nickel phosphate as arupite (Ni3(PO4)2.8H2O), together with hydroxyapatite within the bacterial biofilm on the filter sand grains. Biosorption contributed less than 1% of the overall sequestered nickel. Metabolising bacteria are essential for the process; the definitive role of specific components of the mixed population is undefined but the increase in pH promoted by metabolic activity of some microbial components is likely to promote nickel desolubilisation by others.