BioMetals (v.30, #4)

Metal homeostasis in bacteria: the role of ArsR–SmtB family of transcriptional repressors in combating varying metal concentrations in the environment by Rudra P. Saha; Saikat Samanta; Surajit Patra; Diganta Sarkar; Abinit Saha; Manoj Kumar Singh (459-503).
Bacterial infections cause severe medical problems worldwide, resulting in considerable death and loss of capital. With the ever-increasing rise of antibiotic-resistant bacteria and the lack of development of new antibiotics, research on metal-based antimicrobial therapy has now gained pace. Metal ions are essential for survival, but can be highly toxic to organisms if their concentrations are not strictly controlled. Through evolution, bacteria have acquired complex metal-management systems that allow them to acquire metals that they need for survival in different challenging environments while evading metal toxicity. Metalloproteins that controls these elaborate systems in the cell, and linked to key virulence factors, are promising targets for the anti-bacterial drug development. Among several metal-sensory transcriptional regulators, the ArsR–SmtB family displays greatest diversity with several distinct metal-binding and nonmetal-binding motifs that have been characterized. These prokaryotic metolloregulatory transcriptional repressors represses the expression of operons linked to stress-inducing concentrations of metal ions by directly binding to the regulatory regions of DNA, while derepression results from direct binding of metal ions by these homodimeric proteins. Many bacteria, e.g., Mycobacterium tuberculosis, Bacillus anthracis, etc., have evolved to acquire multiple metal-sensory motifs which clearly demonstrate the importance of regulating concentrations of multiple metal ions. Here, we discussed the mechanisms of how ArsR–SmtB family regulates the intracellular bioavailability of metal ions both inside and outside of the host. Knowledge of the metal-challenges faced by bacterial pathogens and their survival strategies will enable us to develop the next generation drugs.
Keywords: ArsR–SmtB family; Metalloprotein; Transcriptional repressor; Allostery; Redox switch; Antibiotic-resistant bacteria

Arsenic toxicity and epimutagenecity: the new LINEage by Somnath Paul; Pritha Bhattacharjee; Ashok K. Giri; Pritha Bhattacharjee (505-515).
Global methylation pattern regulates the normal functioning of a cell. Research have shown arsenic alter these methylation landscapes within the genome leading to aberrant gene expression and inducts various pathophysiological outcomes. Long interspersed nuclear elements (LINE-1) normally remains inert due to heavy methylation of it’s promoters, time and various environmental insults, they lose these methylation signatures and begin retro-transposition that has been associated with genomic instability and cancerous outcomes. Of the various high throughput technologies available to detect global methylation profile, development of LINE-1 methylation index shall provide a cost effect-screening tool to detect epimutagenic events in the wake of toxic exposure in a large number of individuals. In the present review, we tried to discuss the state of research and whether LINE-1 methylation can be considered as a potent epigenetic signature for arsenic toxicity.
Keywords: Arsenic; Epigenetic signature; Genomic instability; LINE-1 methylation; Human health

The interactions of metal cations and oxyanions with protein tyrosine phosphatase 1B by Kshetrimayum Birla Singh; Wolfgang Maret (517-527).
Protein tyrosine phosphatases are not considered to be metalloenzymes. Yet, they are inhibited by zinc cations and metal and non-metal oxyanions that are chemical analogues of phosphate, e.g. vanadate. Metal inhibition is generally not recognized as these enzymes are purified, supplied, and assayed with buffers containing chelating and reducing agents. We screened a series of cations and anions for their capacity to inhibit protein tyrosine phosphatase 1B and discuss the ensuing general issues with inhibition constants reported in the scientific literature. In contrast to zinc, which binds to the phosphocysteine intermediate in the closed conformation of protein tyrosine phosphatase 1B when the catalytic aspartate has moved into the active site, other divalent cations such as cadmium and copper may also bind to the enzyme in the open conformation. Inhibition by both anions and cations, conditions such as pH, the presence of metal ligands such as glutathione, and the existence of multiple conformational states of protein tyrosine phosphatases in the reaction cycle establish a complex pattern of inhibition of these important regulatory enzymes with implications for the physiology, pharmacology and toxicology of metal ions.
Keywords: Metal cations; Oxyanions; Inhibition; Protein tyrosine phosphatase

The interaction between two chromates [sodium chromate (Na2CrO4) and potassium chromate K2CrO4)] and bovine serum albumin (BSA) in physiological buffer (pH 7.4) was investigated by the fluorescence quenching technique. The results of fluorescence titration revealed that two chromates could strongly quench the intrinsic fluorescence of BSA through a static quenching procedure. The apparent binding constants K and number of binding sites n of chromate with BSA were obtained by the fluorescence quenching method. The thermodynamic parameters enthalpy change (ΔH), entropy change (ΔS) were negative, indicating that the interaction of two chromates with BSA was driven mainly by van der Waals forces and hydrogen bonds. The process of binding was a spontaneous process in which Gibbs free energy change was negative. The distance r between donor (BSA) and acceptor (chromate) was calculated based on Forster’s non-radiative energy transfer theory. The results of UV–Vis absorption, synchronous fluorescence, three-dimensional fluorescence and circular dichroism (CD) spectra showed that two chromates induced conformational changes of BSA.
Keywords: Bovine serum albumin; Chromate; Fluorescence quenching; Interaction

The most accepted hypothesis of magnetoreception for social insects is the ferromagnetic hypothesis which assumes the presence of magnetic material as a sensor coupled to sensitive structures that transmit the geomagnetic field information to the nervous system. As magnetite is the most common magnetic material observed in living beings, it has been suggested as basic constituent of the magnetoreception system. Antennae and head have been pointed as possible magnetosensor organs in social insects as ants, bees and termites. Samples of three antenna joints: head-scape, scape-pedicel and pedicel-third segment joints were embedded in epoxi resin, ultrathin sectioned and analyzed by transmission electron microscopy. Selected area electron diffraction patterns and X-ray energy dispersive spectroscopy were obtained to identify the nanoparticle compound. Besides iron oxides, for the first time, nanoparticles containing titanium have been identified surrounded by tissue in the antennae of ants. Given their dimension and related magnetic characteristics, these nanoparticles are discussed as being part of the magnetosensor system.
Keywords: Magnetic nanoparticles; Magnetosensor; Social insects; Electron microscopy

Fructose-1,6-bisphosphate reverts iron-induced phenotype of hepatic stellate cells by chelating ferrous ions by Henrique Bregolin Dias; Gabriele Catyana Krause; Eamin Daidrê Squizani; Kelly Goulart Lima; Aline Daniele Schuster; Leonardo Pedrazza; Bruno de Souza Basso; Bianca Andrade Martha; Fernanda Cristina de Mesquita; Fernanda Bordignon Nunes; Márcio Vinicius Fagundes Donadio; Jarbas Rodrigues de Oliveira (549-558).
An erratum to this article has been published.Hepatic fibrosis is an extracellular matrix deposition by hepatic stellate cells (HSC). Fibrosis can be caused by iron, which will lead to hydroxyl radical production and cell damage. Fructose-1,6-bisphosphate (FBP) has been shown to deliver therapeutic effects in many pathological situations. In this work, we aimed to test the effects of FBP in HSC cell line, GRX, exposed to an excess of iron (Fe). The Fe-treatment increased cell proliferation and FBP reversed this effect, which was not due to increased necrosis, apoptosis or changes in cell cycle. Oil Red-O staining showed that FBP successfully increased lipid content and lead GRX cells to present characteristics of quiescent HSC. Fe-treatment decreased PPAR-γ expression and increased Col-1 expression. Both effects were reversed by FBP which also decreased TGF-β1 levels in comparison to both control and Fe groups. FBP, also, did not present scavenger activity in the DPPH assay. The treatment with FBP resulted in decreased proliferation rate, Col-1 expression and TGF-β1 release by HSC cells. Furthermore, activated PPAR-γ and increased lipid droplets induce cells to become quiescent, which is a key event to reversion of hepatic fibrosis. FBP also chelates iron showing potential to improve Cell redox state
Keywords: Hepatic fibrosis; Fructose-1,6-bisphosphate; Hepatic stellate cell; Iron

Most fungi are known to synthesize siderophores under iron limitation. However, arbuscular mycorrhizal fungi (AM fungi) have so far not been reported to produce siderophores, although their metabolism is iron-dependent. In an approach to isolate siderophores from AM fungi, we have grown plants of Tagetes patula nana in the presence of spores from AM fungi of the genus Glomus (G. etunicatum, G. mossae & unidentified Glomus sp.) symbiotically under iron limitation and sterile conditions. A siderophore was isolated from infected roots after 2–3 weeks of growth in pots containing low-iron sand with Hoagland solution. HPLC analysis of the root cell lysate revealed a peak at a retention time of 6.7 min which showed iron-binding properties in a chrome azurol S test. The compound was isolated by preparative HPLC and the structure was determined by high resolution electrospray FTICR-MS and GC/MS analysis of the hydrolysis products. From an observed absolute mass to charge ratio (m/z) of 401.11925 [M+H]+ with a relative mass error of ∆ = 0.47 ppm an elemental composition of C16H21N2O10 [M+H]+ was derived, suggesting a molecular weight of 400 Da for glomuferrin. Corresponnding ion masses of m/z 423.10 and m/z 439.06 were asigned to the Na-adduct and K-adduct respectively. A mass of 455.03836 confirmed an Fe- complex with an elemental composition of C16H19N2O10Fe (∆ = 0.15 ppm). GC/MS analysis of the HCl lysate (6 N HCL, 12 h) revealed 1,4 butanediamine. Thus the proposed structure of the isolated siderophore from Glomus species consisted of 1,4 butanediamine amidically linked to two dehydrated citrate residues, similar to the previously identified bis-amidorhizoferrin. Thus, the isolated siderophore (glomuferrin) is a member of the rhizoferrin family previously isolated from fungi of the Mucorales (Zygomycetes).
Keywords: Siderophores; Iron; Glomuferrin; VAM fungi; Mycorrhiza

Chromium(III) and iron(III) inhibits replication of DNA and RNA viruses by Sylwia Terpiłowska; Andrzej Krzysztof Siwicki (565-574).
The aim of this study was to examine the effect of treating of chromium(III) and iron(III) and their combinations on Herpes Simplex Virus type 1 (HSV-1) and Bovine Viral Diarrhoea virus (BVDV) replication. The antiviral efficacies of chromium(III) and iron(III) on HSV-1 and BVDV were evaluated using Real Time PCR method. Moreover, the cytotoxicity of these microelements was examined using the MTT reduction assay. The IC50 (50% inhibiotory concentration) for the chromium chloride was 1100 μM for Hep-2 cells and 1400 μM for BT cells. The IC50 for the iron chloride was 1200 μM for Hep-2 cells and more than1400 μM for BT cells. The concentration-dependent antiviral activity of chromium chloride and iron chloride against HSV-1 and BVDV viruses was observed. In cultures simultaneously treated with (1) 200 μM of CrCl3 and 1000 μM of FeCl3, (2) 1000 μM of CrCl3 and 200 μM of FeCl3, (3) 400 μM of CrCl3 and 800 μM of FeCl3, (4) 800 μM of CrCl3 and 400 μM of FeCl3 a decrease in number of DNA or RNA copies was observed compared with control cells and cells incubated with chromium(III) and iron(III) used separately. The synergistic antiviral effects were observed for chromium(III) and iron(III) against HSV-1 and BVDV.
Keywords: Chromium(III); Iron(III); BVDV; HSV-1

Synthesis, X-ray crystal structure, DNA/BSA binding, DNA cleavage and cytotoxicity studies of phenanthroline based copper(II)/zinc(II) complexes by Qingming Wang; Han Mao; Wenling Wang; Huimin Zhu; Lihui Dai; Yanli Chen; Xinhui Tang (575-587).
Research on copperII 1,10-phenanththroline (phen) derivatives continues to attract interest in the context of structure and biological properties. In this paper, two metal complexes [Cu2(phen)2(μ-Cl)2]Cl2 (1), [Zn(phen)2(H2O)Cl]Cl·4H2O (2) were synthesized and characterized. The crystal structures of 1 and 2 were determined by X-ray diffraction. In order to investigate the biological properties of the prepared complexes, spectroscopic and biological studies were performed. Results of X-ray diffraction showed that 1 and 2 form two types of crystal structures in a given system: dinuclear and mono-nuclear complex. The preliminary study on the DNA cleavage activity has shown that 1 under study behaved as the chemical nucleases. The DNA binding interaction of 1 & 2 with CT-DNA has been investigated by UV–Visible and fluorescence emission spectrometry and the apparent binding constant (K app) values are 5.1 × 104 and 1.2 × 104 M−1, respectively. In addition, fluorescence spectrometry of bovine serum albumin (BSA) with 1 & 2 showed that the quenching mechanism might be a static quenching procedure with one binding sites for BSA. In addition, the cytotoxicity of 1 in vitro on tumor cells lines (MCF-7, HepG2 and HT29) was examined by MTT and showed better antitumor effect on the tested cells.
Keywords: Potential anticancer agents; DNA binding; Bull serum albumin (BSA) binding; DNA cleavage; Binding modes; Metals complexes

Structural characterization of pyoverdines produced by Pseudomonas putida KT2440 and Pseudomonas taiwanensis VLB120 by Matthias Baune; Yulin Qi; Karen Scholz; Dietrich A. Volmer; Heiko Hayen (589-597).
The previously unknown sequences of several pyoverdines (PVD) produced by a biotechnologically-relevant bacterium, namely, Pseudomonas taiwanensis VLB120, were characterized by high performance liquid chromatography (HPLC)–high resolution mass spectrometry (HRMS). The same structural characterization scheme was checked before by analysis of Pseudomonas sp. putida KT2440 samples with known PVDs. A new sample preparation strategy based on solid-phase extraction was developed, requiring significantly reduced sample material as compared to existing methods. Chromatographic separation was performed using hydrophilic interaction liquid chromatography with gradient elution. Interestingly, no signals for apoPVDs were detected in these analyses, only the corresponding aluminum(III) and iron(III) complexes were seen. The chromatographic separation readily enabled separation of PVD complexes according to their individual structures. HPLC-HRMS and complementary fragmentation data from collision-induced dissociation and electron capture dissociation enabled the structural characterization of the investigated pyoverdines. In Pseudomonas sp. putida KT2240 samples, the known pyoverdines G4R and G4R A were readily confirmed. No PVDs have been previously described for Pseudomonas sp. taiwanensis VLB120. In our study, we identified three new PVDs, which only differed in their acyl side chains (succinic acid, succinic amide and malic acid). Peptide sequencing by MS/MS provided the sequence Orn-Asp-OHAsn-Thr-AcOHOrn-Ser-cOHOrn. Of particular interest is the presence of OHAsn, which has not been reported as PVD constituent before.
Keywords: Pseudomonas taiwanensis ; Pyoverdine; Siderophore; Structure characterization; Hydrophilic interaction liquid chromatography (HILIC); Solid phase extraction

Iron modulates the activity of monoamine oxidase B in SH-SY5Y cells by Huiru Lu; Jun Chen; Hui Huang; Mengxue Zhou; Qing Zhu; Shao Q. Yao; Zhifang Chai; Yi Hu (599-607).
Both monoamine oxidase B (MAO-B) and iron accumulation are associated with neurologic diseases including Parkinson’s disease. However, the association of iron with MAO-B activity was poorly understood. Here we took advantage of highly sensitive and specific fluorescence probes to examine the change in MAO-B activity in human dopaminergic neuroblastoma (SH-SY5Y) cells upon iron exposure. Both ferric and ferrous ions could significantly enhance the activity of MAO-B, instead of MAO-A, in SH-SY5Y cells. In addition, iron-induced increase in MAO-B probe fluorescence could be prevented by pargyline and other newly developed MAO-B inhibitors, suggesting that it was MAO-B activity-dependent. These findings may suggest MAO-B is an important sensor in iron-stressed neuronal cells.
Keywords: Iron; MAO-B; Fluorescence probe

ESI–MS studies of the reactions of novel platinum(II) complexes containing O,O′-chelated acetylacetonate and sulfur ligands with selected model proteins by Tiziano Marzo; Sandra A. De Pascali; Chiara Gabbiani; Francesco P. Fanizzi; Luigi Messori; Alessandro Pratesi (609-614).
A group of mixed-ligand Pt(II) complexes bearing acetylacetonate and sulphur ligands were recently developed in the University of Lecce as a new class of prospective anticancer agents that manifested promising pharma-cological properties in preliminary in vitro and in vivo tests. Though modelled on the basis of cisplatin, these Pt(II) complexes turned out to exhibit a profoundly distinct mode of action as they were found to act mainly on non-genomic targets rather than on DNA. Accordingly, we have explored here their reactions with two representative model proteins through an established ESI–MS procedure with the aim to describe their general interaction mechanism with protein targets. A pronounced reactivity with the tested proteins was indeed documented; the nature of the resulting metallodrug-protein interactions could be characterised in depth in the various cases. Preferential binding to protein targets compared to DNA is supported by independent ICP-OES measurements. The implications of these findings are discussed.
Keywords: Anticancer drugs; Platinum compounds; ESI–MS; Protein interaction

Bovine apo-transferrin (Tf) dose-dependently inhibited zinc (Zn) measurement if apo-Tf was added to a Zn standard solution followed by Zn measurement using a commercial Zn assay kit. Incubation of apo-Tf with zinc sulfate results in loss of Tf to inhibit Zn measurement, probably due to the binding of Tf with Zn. After treatment of Zn-binding Tf with ethylenediaminetetraacetic acid (EDTA) to generate apo-Tf, Zn measurement was even more strongly inhibited. However, when Zn standard solution was added to individual serum samples obtained from four dairy cows, the added Zn was almost recovered quantitatively. Apo-Tf had no effect on serum Zn measurement following its addition to serum samples. Apo-Tf and Zn standard solution was added to serum sample added Zn standard solution and apo-Tf, respectively, beforehand. The last added apo-Tf to the mixed solution showed higher Zn recovery (76–96%) as compared with the last added Zn standard solution (33–61%). Bovine serum albumin (BSA) did not affect the Zn recovery test, but apo-Tf inhibited Zn recovery even in the presence of BSA. These results suggested that, although Tf does not always inhibit serum Zn measurement, the Zn content of Zn-bound Tf could not be measured using the present Zn assay. Bovine serum contains Zn-binding protein with higher affinity to Zn than that of Apo-Tf. In addition, BSA does not inhibit the binding of apo-Tf with Zn, suggesting that BSA has lower affinity to Zn than that of apo-Tf.
Keywords: Albumin; Bovine; Apotransferrin; Serum; Zinc

Iron-induced generation of mitochondrial ROS depends on AMPK activity by Hui Huang; Jun Chen; Huiru Lu; Mengxue Zhou; Zhifang Chai; Yi Hu (623-628).
Deregulated iron homeostasis is generally believed to be implicated in neurodegenerative diseases, including Parkinson’s disease. Nevertheless, it is not fully understood how iron overload can elicit neuronal cell damage. Here we examined mitochondrial reactive oxygen species (ROS) levels in human dopaminergic neuroblastoma SH-SY5Y cells upon iron exposure. A relatively high concentration of iron could significantly increase mitochondrial ROS levels in SH-SY5Y cells. Pharmacological activation of AMP-activated protein kinase (AMPK) almost completely inhibited the effect of iron on mitochondrial ROS. By contrast, AMPK inhibition aggravated the neurotoxicity of iron and enhanced the production of mitochondrial ROS. Collectively, these findings suggested that excess iron may be able to perturb mitochondrial function, and AMPK activity is important for the association of iron and mitochondria.
Keywords: Fe(II); AMPK; Neurotoxicity; Mitochondria; ROS