BioMetals (v.24, #2)

Heme oxygenase is involved in cobalt chloride-induced lateral root development in tomato by Sheng Xu; Bo Zhang; Ze-Yu Cao; Teng-Fang Ling; Wen-Biao Shen (181-191).
In animals, heme oxygenase (HO), a rate-limiting enzyme responsible for carbon monoxide (CO) production, was regarded as a protective system maintaining cellular homeostasis. It was also established that metal ions are powerful HO-inducing agents and cobalt chloride (CoCl2) was the first metal ion identified with an inducing property. Previous study suggests that CoCl2 stimulates adventitious root formation in tomato and cucumber cuttings. In this test, we discover that both CoCl2 and an inducer of HO-1, hemin, could lead to the promotion of lateral root development, as well as the induction of HO-1 protein expression, HO activity, or LeHO-1/2 transcripts, in lateral root initiation zone of tomato seedlings. The effect is specific for HO since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) blocked the above actions of CoCl2, and the inhibitory effect was reversed partially when 50% CO aqueous solution was added. However, the addition of ascorbic acid (AsA), a well-known antioxidant, exhibited no obvious effect on lateral root formation. Molecular evidence further showed that CoCl2-induced the up-regulation of target genes responsible for lateral root formation, including LeCDKA1, LeCYCA2;1, and LeCYCA3;1, was suppressed differentially by ZnPPIX. And these decreases were reversed further by the addition of CO. All together, these results suggest a novel role for HO in the CoCl2-induced tomato lateral root formation.
Keywords: Cell cycle regulatory gene; Cobalt chloride; Heme oxygenase; Lateral root development; Lycopersicon esculentum

TonB-dependent outer-membrane proteins and siderophore utilization in Pseudomonas fluorescens Pf-5 by Sierra L. Hartney; Sylvie Mazurier; Teresa A. Kidarsa; Maria Carolina Quecine; Philippe Lemanceau; Joyce E. Loper (193-213).
The soil bacterium Pseudomonas fluorescens Pf-5 produces two siderophores, a pyoverdine and enantio-pyochelin, and its proteome includes 45 TonB-dependent outer-membrane proteins, which commonly function in uptake of siderophores and other substrates from the environment. The 45 proteins share the conserved β-barrel and plug domains of TonB-dependent proteins but only 18 of them have an N-terminal signaling domain characteristic of TonB-dependent transducers (TBDTs), which participate in cell-surface signaling systems. Phylogenetic analyses of the 18 TBDTs and 27 TonB-dependent receptors (TBDRs), which lack the N-terminal signaling domain, suggest a complex evolutionary history including horizontal transfer among different microbial lineages. Putative functions were assigned to certain TBDRs and TBDTs in clades including well-characterized orthologs from other Pseudomonas spp. A mutant of Pf-5 with deletions in pyoverdine and enantio-pyochelin biosynthesis genes was constructed and characterized for iron-limited growth and utilization of a spectrum of siderophores. The mutant could utilize as iron sources a large number of pyoverdines with diverse structures as well as ferric citrate, heme, and the siderophores ferrichrome, ferrioxamine B, enterobactin, and aerobactin. The diversity and complexity of the TBDTs and TBDRs with roles in iron uptake clearly indicate the importance of iron in the fitness and survival of Pf-5 in the environment.
Keywords: Pyoverdine; Iron-acquisition; TonB-dependent receptors; Pseudomonas fluorescens

Previous studies noted specific changes in urinary porphyrin excretion patterns associated with exposure to mercury (Hg) in animals and humans. In our study, urinary porphyrin concentrations were examined in normal children 8–18 years-old from a reanalysis of data provided from a randomized, prospective clinical trial that was designed to evaluate the potential health consequences of prolonged exposure to Hg from dental amalgam fillings (the parent study). Our analysis examined dose-dependent correlations between increasing Hg exposure from dental amalgams and urinary porphyrins utilizing statistical models with adjustments for the baseline level (i.e. study year 1) of the following variables: urinary Hg, each urinary porphyrin measure, gender, race, and the level of lead (Pb) in each subject’s blood. Significant dose-dependent correlations between cumulative exposure to Hg from dental amalgams and urinary porphyrins associated with Hg body-burden (pentacarboxyporphyrin, precoproporphyrin, and coproporphyrin) were observed. Overall, 5–10% increases in Hg-associated porphyrins for subjects receiving an average number of dental amalgam fillings in comparison to subjects receiving only composite fillings were observed over the 8-year course of the study. In contrast, no significant correlations were observed between cumulative exposure to Hg from dental amalgams and urinary porphyrins not associated with Hg body-burden (uroporphyrin, heptacarboxyporphyrin, and hexacarboxyporphyrin). In conclusion, our study, in contrast to the no-effect results published from the parent study, further establishes the sensitivity and specificity of specific urinary porphyrins as a biomarker for low-level Hg body-burden, and also reveals that dental amalgams are a significant chronic contributor to Hg body-burden.
Keywords: Body burden; Dental amalgam; Mercury; Porphyrin

A role for ferritin in the antioxidant system in coffee cell cultures by Alexandra Bottcher; Paula Macedo Nobile; Paula Fabiane Martins; Fábio Frangiotti Conte; Ricardo Antunes Azevedo; Paulo Mazzafera (225-237).
Iron (Fe) is an essential nutrient for plants, but it can generate oxidative stress at high concentrations. In this study, Coffea arabica L. cell suspension cultures were exposed to excess Fe (60 and 240 μM) to investigate changes in the gene expression of ferritin and antioxidant enzymes. Iron content accumulated during cell growth, and Western blot analysis showed an increase of ferritin in cells treated with Fe. The expression of two ferritin genes retrieved from the Brazilian coffee EST database was studied. CaFER1, but not CaFER2, transcripts were induced by Fe exposure. Phylogenetic analysis revealed that CaFER1 is not similar to CaFER2 or to any ferritin that has been characterised in detail. The increase in ferritin gene expression was accompanied by an increase in the activity of antioxidant enzymes. Superoxide dismutase, guaiacol peroxidase, catalase, and glutathione reductase activities increased in cells grown in the presence of excess Fe, especially at 60 μM, while the activity of glutathione S-transferase decreased. These data suggest that Fe induces oxidative stress in coffee cell suspension cultures and that ferritin participates in the antioxidant system to protect cells against oxidative damage. Thus, cellular Fe concentrations must be finely regulated to avoid cellular damage most likely caused by increased oxidative stress induced by Fe. However, transcriptional analyses indicate that ferritin genes are differentially controlled, as only CaFER1 expression was responsive to Fe treatment.
Keywords: Coffea arabica ; Ferritin; Oxidative stress; Iron toxicity; Antioxidant enzymes

Desferrithiocin analogue iron chelators: iron clearing efficiency, tissue distribution, and renal toxicity by Raymond J. Bergeron; Jan Wiegand; Neelam Bharti; James S. McManis; Shailendra Singh (239-258).
The current solution to iron-mediated damage in transfusional iron overload disorders is decorporation of excess unmanaged metal, chelation therapy. The clinical development of the tridentate chelator deferitrin (1, Table 1) was halted due to nephrotoxicity. It was then shown by replacing the 4′-(HO) of 1 with a 3,6,9-trioxadecyloxy group, the nephrotoxicity could be ameliorated. Further structure–activity relationship studies have established that the length and the position of the polyether backbone controlled: (1) the ligand’s iron clearing efficiency (ICE), (2) chelator tissue distribution, (3) biliary ferrokinetics, and (4) tissue iron reduction. The current investigation compares the ICE and tissue distribution of a series of (S)-4,5-dihydro-2-[2-hydroxy-4-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table 1, 35) and the (S)-4,5-dihydro-2-[2-hydroxy-3-(polyether)phenyl]-4-methyl-4-thiazolecarboxylic acids (Table 1, 810). The three most effective polyether analogues, in terms of performance ratio (PR), defined as mean ICEprimate/ICErodent, are 3 (PR 1.1), 8, (PR 1.5), and 9, now in human trials, (PR 2.2). At the onset of the clinical trial on 9, no data were available for ligand 3 or 8. This is unfortunate, as 3 has many advantages over 9, e.g., the ICE of 3 in rats is 2.5-fold greater than that of 9 and analogue 3 achieves very high levels in the liver, pancreas, and heart, the organs most affected by iron overload. Finally, the impact of 3 on the urinary excretion of kidney injury molecule-1 (Kim-1), an early diagnostic biomarker for monitoring acute kidney toxicity, has been carried out in rats; no evidence of nephrotoxicity was found. Overall, the results suggest that 3 would be a far superior clinical candidate to 9.
Keywords: Iron chelator; Desazadesferrithiocin polyether analogues; Tissue distribution; Nephrotoxicity; Urinary biomarker; Kidney injury molecule-1 (Kim-1)

Conformational study of bovine lactoferricin in membrane-micking conditions by molecular dynamics simulation and circular dichroism by Isabella Daidone; Alessandro Magliano; Alfredo Di Nola; Giuseppina Mignogna; Matilda Manuela Clarkson; Anna Rita Lizzi; Arduino Oratore; Fernando Mazza (259-268).
Lactoferricins are potent antimicrobial peptides released by pepsin cleavage of Lactoferrins. Bovine Lactoferricin (LfcinB) has higher activity than the intact bovine Lactoferrin, and is the most active among the other Lactoferricins of human, murine and caprine origin. In the intact protein the fragment corresponding to LfcinB is in an helical conformation, while in water LfcinB adopts an amphipathic β-hairpin structure. However, whether any of these structural motifs is the antibacterial active conformation, i.e., the one interacting with bacterial membrane components, remains to be seen. Here we present Circular Dichroism (CD) spectra and Molecular Dynamics (MD) simulations indicating that in membrane-mimicking solvents the LfcinB adopts an amphipathic β-hairpin structure similar to that observed in water, but differing in the dynamic behavior of the side-chains of the two tryptophan residues. In the membrane-mimicking solvent these side-chains show a high propensity to point towards the hydrophobic environment, rather than being in the hydrophobic core as seen in water, while the backbone preserves the hairpin conformation as found in water. These results suggest that the tryptophans might act as anchors pulling the stable, solvent-invariant hairpin structure into the membrane.
Keywords: Antimicrobial peptides; Molecular dynamics simulation; Circular dichroism; Amphipathic; β-hairpin

Copper depletion increases the mitochondrial-associated SOD1 in neuronal cells by Mario Arciello; Concetta Rosa Capo; Sara D’Annibale; Mauro Cozzolino; Alberto Ferri; Maria Teresa Carrì; Luisa Rossi (269-278).
The role of copper in the toxicity of mutant copper-dependent enzyme superoxide dismutase (SOD1) found in patients affected with the familial form of amyotrophic lateral sclerosis (fALS) is widely debated. Here we report that treatment of human neuroblastoma cells SH-SY5Y with a specific copper chelator, triethylene tetramine (Trien) induces the decrease of intracellular copper level, paralleled by decreased activity of SOD1. A comparable effect is observed in mouse NSC-34-derived cells, a motoneuronal model, transfected for the inducible expression of either wild-type or G93A mutant human SOD1, one of the mutations associated with fALS. In both cell types, the drop of SOD1 activity is not paralleled by the same extent of decrease in SOD1 protein content. This discrepancy can be explained by the occurrence of a fraction of copper-free SOD1 upon copper depletion, which is demonstrated by the partial recovery of the enzyme activity after the addition of copper sulphate to homogenates of SH-SY5Y cells. Furthermore, copper depletion produces the enrichment of the physiological mitochondrial fraction of SOD1 protein, in both cells models. However, increasing the fraction of mitochondrial, possibly copper-free, mutant human SOD1 does not further alter mitochondrial morphology in NSC-34-derived cells. Thus, copper deficiency is not a factor which may worsen mitochondrial damage, which is one of the earliest events in fALS associated with mutant SOD1.
Keywords: Copper; Mitochondria; SOD1; SH-SY5Y; NSC-34; ALS

Ferrous iron is found in mesenteric lymph bound to TIMP-2 following hemorrhage/resuscitation by James L. Atkins; Nikolai V. Gorbunov; Valerie Trabosh; Rachel Van Duyne; Fatah Kashanchi; Andrei M. Komarov (279-289).
Extracellular iron has been implicated in the pathogenesis of post-injury organ failure. However, the source(s) and biochemical species of this iron have not been identified. Based upon evidence that distant organ injury results from an increase in intestinal permeability, we looked for ferrous iron in mesenteric lymph in anesthetized rats undergoing hemorrhage and fluid resuscitation (H/R). Ferrous iron increased in lymph from 4.7 nmol/mg of protein prior to hemorrhage to 86.6 nmol/mg during resuscitation. Utilizing immuno-spin trapping in protein fractions that were rich in iron, we tentatively indentified protein carrier(s) of ferrous iron by MALDI-TOF MS. One of the identified proteins was the metalloproteinase (MMP) inhibitor, TIMP-2. Antibody to TIMP-2 immunoprecipitated 74% of the ferrozine detectable iron in its protein fraction. TIMP-2 binds iron in vitro at pH 6.3, which is typical of conditions in the mesentery during hemorrhage, but it retains the ability to inhibit the metalloproteases MMP-2 and MMP-9. In summary, there is a large increase in extracellular ferrous iron in the gut in H/R demonstrating dysregulation of iron homeostasis. We have identified, for the first time, the binding of extracellular iron to TIMP-2.
Keywords: Ferrous iron; TIMP-2; Hemorrhage; Extracellular

Selenoprotein W gene expression in the gastrointestinal tract of chicken is affected by dietary selenium by Jin-Long Li; Hui-Xin Li; Shu Li; Zhi-Hui Jiang; Shi-Wen Xu; Zhao-Xin Tang (291-299).
Selenoprotein W (SelW) and selenium (Se) plays important roles in gastrointestinal function and that SelW expression in the gastrointestinal system of mammals is sensitive to Se levels. However, little is known about the pattern of SelW expression in the bird gastrointestinal tract. To investigate the distribution of SelW and effects of dietary Se levels on the SelW mRNA expression in the gastrointestinal tract tissues of birds, 1-day-old male chickens were fed either a commercial diet or a Se-supplemented diet containing 1.0, 2.0, 3.0 or 5.0 mg/kg sodium selenite for 90 days. The gastrointestinal tract tissues (tongue, esophagus, crop, proventriculus, gizzard, duodenum, small intestine, cecum and rectum) were collected and examined for Se content and mRNA levels of SelW. The mRNA expression of SelW was detected in all tissues. The greatest increase in SelW mRNA levels was observed in the gizzard, whereas Se content was highest in the duodenum and small intestine. A significant increase in SelW mRNA levels was observed in the gastrointestinal tract tissues of chickens fed the diets containing 1–3 mg/kg sodium selenite while decreased SelW mRNA levels were observed in the esophagus, crop, proventriculus, gizzard, duodenum and cecum in chickens fed the diet containing 5 mg/kg sodium selenite. These data indicate that SelW is widely expressed in the gastrointestinal tract tissues of birds and the transcription of the SelW gene is very sensitive to dietary Se.
Keywords: Chicken; Selenoprotein W; Selenium; mRNA expression; Gastrointestinal tract tissues

Mercury resistance in Sporosarcina sp. G3 by Amit Bafana (301-309).
Mercuric reductase (MerA) enzyme plays an important role in biogeochemical cycling and detoxification of Hg and recently, has also been shown to be useful in clean up of Hg-contaminated effluents. Present study describes isolation of a heavy metal-resistant isolate of Sporosarcina, which could tolerate up to 40, 525, 210, 2900 and 370 μM of Cd, Co, Zn, Cr and Hg respectively. It was found to reduce and detoxify redox-active metals like Cr and Hg. The chromate reductase and MerA activities in the crude cell extract of the culture were 1.5 and 0.044 units/mg protein respectively. The study also describes designing of a new set of highly degenerate primers based on a dataset of 23 Firmicute merA genes. As the primers encompass the known diversity of merA genes within the phylum Firmicutes, they can be very useful for functional diversity analysis. They were successfully used to amplify a 787 bp merA fragment from the current isolate. A 1174 bp merA fragment was further cloned by designing an additional downstream primer. It was found to show 92% similarity to the putative merA gene from Bacillus cereus AH820. To the best of our knowledge, this is the first report of mercury resistance and merA gene sequence from Sporosarcina.
Keywords: Mercuric reductase; merA ; Firmicutes ; Gram positive; Degenerate primer

Synechocystis ferredoxin-NADP+ oxidoreductase is capable of functioning as ferric reductase and of driving the Fenton reaction in the absence or presence of free flavin by Junichi Sato; Kouji Takeda; Rika Nishiyama; Toshihiro Watanabe; Mitsuru Abo; Etsuro Yoshimura; Junichi Nakagawa; Akira Abe; Shinji Kawasaki; Youichi Niimura (311-321).
We purified free flavin-independent NADPH oxidoreductase from Synechocystis sp. PCC6803 based on NADPH oxidation activity elicited during reduction of t-butyl hydroperoxide in the presence of Fe(III)-EDTA. The N-terminal sequencing of the purified enzyme revealed it to be ferredoxin-NADP+ oxidoreductase (FNR S ). The purified enzyme reacted with cytochrome c, ferricyanide and 2,6-dichloroindophenol (DCIP). The substrate specificity of the enzyme was similar to the known FNR. DNA degradation occurring in the presence of NADPH, Fe(III)-EDTA and hydrogen peroxide was potently enhanced by the purified enzyme, indicating that Synechocystis FNR S may drive the Fenton reaction. The Fenton reaction by Synechocystis FNR S in the presence of natural chelate iron compounds tended to be considerably lower than that in the presence of synthetic chelate iron compounds. The Synechocystis FNR S is considered to reduce ferric iron to ferrous iron when it evokes the Fenton reaction. Although Synechocystis FNR S was able to reduce iron compounds in the absence of free flavin, the ferric reduction by the enzyme was enhanced by the addition of free flavin. The enhancement was detected not only in the presence of natural chelate iron compounds but also synthetic chelate iron compounds.
Keywords: Ferredoxin-NADP+ oxidoreductase; Ferric reductase; Fenton reaction

Characterization and expression of chicken selenoprotein W by Bor-Rung Ou; Mei-Jung Jiang; Chao-Hsiang Lin; Yu-Chuan Liang; Kuei-Jen Lee; Jan-Ying Yeh (323-333).
As an essential trace element, selenium (Se) deficiency results in White Muscle Disease in livestock and Keshan disease in humans. The main objectives of this study were to clone and characterize the chicken selenoprotein W (SeW) gene and investigate SeW mRNA expression in chicken tissues. The deduced amino acid (AA) sequence of chicken SeW contains 85 AAs with UAG as the stop codon. Like all SeW genes identified in different species, chicken SeW contains one well-conserved selenocysteine (Sec) at the 13th position encoded by the UGA codon. The proposed glutathione (GSH)-binding site at the Cys37 of SeW is not conserved in the chicken, but Cys9 and Sec13, with possible GSH binding, are conserved in SeWs identified from all species. There are 23–59% and 50–61% homology in cDNA and deduced AA sequences of SeW, respectively, between the chicken and other species. The predicted secondary structure of chicken SeW mRNA indicates that the selenocysteine insertion sequence element is type II with invariant adenosines within the apical bulge. The SeW mRNA expression is high in skeletal muscle followed by brain, but extremely low in other tissues from chickens fed a commercial maize-based diet. The SeW gene is ubiquitously expressed in heart, skeletal muscle, brain, testis, spleen, kidney, lung, liver, stomach and pancreas in chickens fed a commercial diet supplemented with sodium selenite. These results indicate that dietary selenium supplementation regulates SeW gene expression in the chicken and skeletal muscle is the most responsive tissue when dietary Se content is low.
Keywords: Selenoprotein W; mRNA expression; Selenium; cDNA sequence; Chicken

Effect of cobalt on Escherichia coli metabolism and metalloporphyrin formation by Tomas Majtan; Frank E. Frerman; Jan P. Kraus (335-347).
Toxicity in Escherichia coli resulting from high concentrations of cobalt has been explained by competition of cobalt with iron in various metabolic processes including Fe–S cluster assembly, sulfur assimilation, production of free radicals and reduction of free thiol pool. Here we present another aspect of increased cobalt concentrations in the culture medium resulting in the production of cobalt protoporphyrin IX (CoPPIX), which was incorporated into heme proteins including membrane-bound cytochromes and an expressed human cystathionine beta-synthase (CBS). The presence of CoPPIX in cytochromes inhibited their electron transport capacity and resulted in a substantially decreased respiration. Bacterial cells adapted to the increased cobalt concentration by inducing a modified mixed acid fermentative pathway under aerobiosis. We capitalized on the ability of E. coli to insert cobalt into PPIX to carry out an expression of CoPPIX-substituted heme proteins. The level of CoPPIX-substitution increased with the number of passages of cells in a cobalt-containing medium. This approach is an inexpensive method to prepare cobalt-substituted heme proteins compared to in vitro enzyme reconstitution or in vivo replacement using metalloporphyrin heme analogs and seems to be especially suitable for complex heme proteins with an additional coenzyme, such as human CBS.
Keywords: Cobalt toxicity; Cobalt protoporphyrin IX; Protein expression; Heme replacement; Chelatase; Fe–S cluster; Cystathionine beta-synthase; Respiration

Protective effects of Syzygium cumini seed extract against methylmercury-induced sistemic toxicity in neonatal rats by F. H. Abdalla; L. P. Bellé; P. E. R. Bitencourt; K. S. De Bona; R. A. Zanette; A. A. Boligon; M. L. Athayde; A. S. Pigatto; M. B. Moretto (349-356).
Syzygium cumini (L.) Skeels (Sc) belongs to the medicinal plants with an important source of phenolic compounds. Sc has been shown to possess antioxidant and anti-inflammatory properties. Methylmercury (MeHg), a highly toxic environmental pollutant, induces oxidative stress and dysfunction in many cell types. This study was aimed to evaluate the effect of aqueous seed extract of Sc (ASc) on MeHg-induced toxicity in rats. Two-day-old rats (P2) received a single dose of MeHg (10 mg/kg) and two doses of ASc (0.9 mg/kg) per os. After two days, the effects of the treatment were investigated in the cerebral cortex, hippocampus, kidney, liver and urine samples. Our results demonstrated that N-acetyl-β-d-glucosaminidase (NAG) activity in the kidney and urine, the lipid peroxidation levels in the liver and kidney samples, as well as the adenosine deaminase (ADA) activity in the hippocampus, kidney and liver were higher in MeHg-group when compared to the control group. The administration of ASc reverted the toxic effects of MeHg. It is noteworthy to observe that the main compounds present in the ASc, as gallic acid (the major component), chlorogenic acid and rutin, might be the responsible for such benefit, since they were found to display antioxidant properties.
Keywords: Methylmercury; Syzygium cumini ; N-acetyl-β-d-glucosaminidase; Rat; Adenosine deaminase

Heterogenous distribution of ferroportin-containing neurons in mouse brain by Michael W. Boserup; Jacek Lichota; David Haile; Torben Moos (357-375).
Iron is crucial for a variety of cellular functions in neuronal cells. Neuronal iron uptake is reflected in a robust and consistent expression of transferrin receptors and divalent metal transporter 1 (DMT 1). Conversely, the mechanisms by which neurons neutralize and possibly excrete iron are less clear. Studies indicate that neurons express ferroportin which could reflect a mechanism for iron export. We mapped the distribution of ferroportin in the adult mouse brain using an antibody prepared from a peptide representing amino acid sequences 223–303 of mouse ferroportin. The antibody specifically detected ferroportin in brain homogenates, whereas homogenates of cultured endothelial cells were devoid of immunoreactivity. In brain sections, ferroportin was confined to neuronal cell bodies and peripheral processes of cerebral cortex, hippocampus, thalamus, brain stem, and cerebellum. In brain stem ferroportin-labeling was particularly high in neurons of cranial nerve nuclei and reticular formation. Ferroportin was hardly detectable in striatum, pallidum, or hypothalamus. Among non-neuronal cells, ferroportin was detected in oligodendrocytes and choroid plexus epithelial cells. A comparison with previous studies on the distribution of transferrin receptors in neurons shows that many neuronal pools coincide with those expressing ferroportin. The data therefore indicate that neuronal iron homeostasis consists of a delicate balance between transferrin receptor-mediated uptake of iron-transferrin and ferroportin-related iron excretion. The findings also suggest a particular high turnover of iron in neuronal regions, such as habenula, hippocampus, reticular formation and cerebellum, as several neurons in these regions exhibit a prominent co-expression of transferrin receptors and ferroportin.
Keywords: Blood–brain barrier; Iron; Neurodegeneration; Oxidative stress; qPCR; Transferrin