BioMetals (v.27, #6)

Zinc and autophagy by Juan P. Liuzzi; Liang Guo; Changwon Yoo; Tiffanie S. Stewart (1087-1096).
Autophagy is a highly conserved degradative process through which cells overcome stressful conditions. Inasmuch as faulty autophagy has been associated with aging, neuronal degeneration disorders, diabetes, and fatty liver, autophagy is regarded as a potential therapeutic target. This review summarizes the present state of knowledge concerning the role of zinc in the regulation of autophagy, the role of autophagy in zinc metabolism, and the potential role of autophagy as a mediator of the protective effects of zinc. Data from in vitro studies consistently support the notion that zinc is critical for early and late autophagy. Studies have shown inhibition of early and late autophagy in cells cultured in medium treated with zinc chelators. Conversely, excess zinc added to the medium has shown to potentiate the stimulation of autophagy by tamoxifen, H2O2, ethanol and dopamine. The potential role of autophagy in zinc homeostasis has just begun to be investigated. Increasing evidence indicates that autophagy dysregulation causes significant changes in cellular zinc homeostasis. Autophagy may mediate the protective effect of zinc against lipid accumulation, apoptosis and inflammation by promoting degradation of lipid droplets, inflammasomes, p62/SQSTM1 and damaged mitochondria. Studies with humans and animal models are necessary to determine whether autophagy is influenced by zinc intake.
Keywords: Autophagy; Zinc; Inflammation; Lipophagy; Alcohol; Apoptosis

There is increasing evidence that toxic metals play a role in diseases of unknown etiology. Their action is often mediated by membrane proteins, and in particular neurotransmitter receptors. This brief review will describe recent findings on the direct interaction of metal ions with ionotropic γ-aminobutyric acid (GABAA) and glutamate receptors, the main inhibitory and excitatory neurotransmitter receptors in the mammalian central nervous system, respectively. Both hyper and hypo function of these receptors are involved in neurological and psychotic syndromes and modulation by metal ions is an important pharmacological issue. The focus will be on three xenobiotic metals, lead (Pb), cadmium (Cd) and nickel (Ni) that have no biological function and whose presence in living organisms is only detrimental, and two trace metals, zinc (Zn) and copper (Cu), which are essential for several enzymatic functions, but can mediate toxic actions if deregulated. Despite limited access to the brain and tight control by metalloproteins, exogenous metals interfere with receptor performances by mimicking physiological ions and occupying one or more modulatory sites on the protein. These interactions will be discussed as a potential cause of neuronal dysfunction.
Keywords: GABAA receptor; NMDA receptor; Lead; Cadmium; Nickel; Zinc; Copper

Synthesis, spectroscopic characterization, electrochemical behavior and computational analysis of mixed diamine ligand gold(III) complexes: antiproliferative and in vitro cytotoxic evaluations against human cancer cell lines by Said S. Al-Jaroudi; M. Monim-ul-Mehboob; Muhammad Altaf; Abdulaziz A. Al-Saadi; Mohammed I. M. Wazeer; Saleh Altuwaijri; Anvarhusein A. Isab (1115-1136).
The gold(III) complexes of the type [(DACH)Au(en)]Cl3, 1,2-Diaminocyclohexane ethylenediamine gold(III) chloride [where 1,2-DACH = cis-, trans-1,2- and S,S-1,2diaminocyclohexane and en = ethylenediamine] have been synthesized and characterized using various analytical and spectroscopic techniques including elemental analysis, UV–Vis and FTIR spectra; and solution as well as solid-state NMR measurements. The solid-state 13C NMR shows that 1,2-diaminocyclohexane (1,2-DACH) and ethylenediamine (en) are strongly bound to the gold(III) center via N donor atoms. The stability of the mixed diamine ligand gold(III) was determined by 1H and 13C NMR spectra. Their electrochemical behavior was studied by cyclic voltammetry. The structural details and relative stabilities of the four possible isomers of the complexes were also reported at the B3LYP/LANL2DZ level of theory. The coordination sphere of these complexes around gold(III) center adopts distorted square planar geometry. The computational study also demonstrates that trans- conformations is slightly more stable than the cis-conformations. The antiproliferative effects and cytotoxic properties of the mixed diamine ligand gold(III) complexes were evaluated in vitro on human gastric SGC7901 and prostate PC3 cancer cells using MTT assay. The antiproliferative study of the gold(III) complexes on PC3 and SGC7901 cells indicate that complex 1 is the most effective antiproliferative agent among mixed ligand based gold(III) complexes 13. The IC50 data reveal that the in vitro cytotoxicity of complexes 1 and 3 against SGC7901 cancer cells are fairly better than that of cisplatin.
Keywords: Gold(III) complex; 1,2-Diaminocyclohexane (1,2-DACH); Ethylenediamine (en); Antiproliferative effect; In vitro cytototoxicity; Gastric cancer (SGC7901); Prostate cancer (PC3)

Metabolic changes associated with selenium deficiency in mice by Beata Mickiewicz; Michelle L. Villemaire; Linda E. Sandercock; Frank R. Jirik; Hans J. Vogel (1137-1147).
Selenium (Se), which is a central component for the biosynthesis and functionality of selenoproteins, plays an important role in the anti-oxidative response, reproduction, thyroid hormone metabolism and the protection from infection and inflammation. However, dietary Se effects have not well been established to date and the available studies often present contradictory results. To obtain a better understanding of Se intake and its influence on the metabolism of living systems, we have utilized a metabolomics approach to gain insight into the specific metabolic alterations caused by Se deficiency in mice. Serum samples were collected from two groups of C57BL/6 mice: an experimental group which was fed a Se-deficient diet and controls consuming normal chow. The samples were analyzed by 1H nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry. The resulting metabolite data were examined separately for both analytical methods and in a combined manner. By applying multivariate statistical analysis we were able to distinguish the two groups and detect a metabolite pattern associated with Se deficiency. We found that the concentrations of 15 metabolites significantly changed in serum samples collected from Se-deficient mice when compared to the controls. Many of the perturbed biological pathways pointed towards compensatory mechanisms during Se deficiency and were associated with amino acid metabolism. Our findings show that a metabolomics approach may be applied to identify the metabolic impact of Se and reveal the most impaired biological pathways as well as induced regulatory mechanisms during Se deficiency.
Keywords: Selenium; Biomarkers; Metabolomics; Multivariate data analysis

We have previously reported that nickel acetate (Ni2+), a well-known human carcinogenic agents, differentially affected apoptosis in two different airway epithelial cell lines derived from the human respiratory tract (A549 and Beas-2B, respectively), suggesting a potential involvement of epidermal growth factor receptor (EGFR)/Neu receptors in mediating this effect. Since ErbBs are closely associated to Mucin 1 (MUC1), a glycoprotein component of airway mucus that is overexpressed in lung tumors, we have investigated the role of this signaling system in the survival response of airway epithelial cells against Ni2+-induced cell death. We found that A549 cells exposed to Ni2+ do not show any significant increase of MUC1 levels. Conversely, Beas-2B cells exposed to equivalent concentrations of Ni2+ showed increased expression of MUC1 levels and this correlated with increased phosphorylation of both EGFR and of the extracellular-regulated kinase 1/2 (ERK1/2) and increase resistance to apoptosis, as indicated by cell viability assessments and DNA damage. Interestingly, suppression of MUC1 by small interfering RNA inhibited the EGFR activation in Beas-2B cells, leading to a significant decrease of survival and enhancement of DNA fragmentation and cleaved Caspase-3 expression. These results strongly suggest a role for MUC1 in Ni2+-induced neoplastic transformation, which likely involves the activation of the EGFR-mediated cell survival pathway, highlighting new avenues in the molecular approach to lung cancer prevention.
Keywords: Nickel; Lung cancer; Apoptosis; Mucin-1; EGFR

Understanding the interaction of an antitumoral platinum(II) 7-azaindolate complex with proteins and DNA by Katia G. Samper; Venancio Rodríguez; Elisabeth Ortega-Carrasco; Sílvia Atrian; Jean Didier Maréchal; Natalia Cutillas; Ana Zamora; Concepción de Haro; Mercè Capdevila; José Ruiz; Òscar Palacios (1159-1177).
The reactivity of the [Pt(dmba)(aza-N1)(dmso)] complex 1, (a potential antitumoral drug with lower IC50 than cisplatin in several tumoral cell lines) with different proteins and oligonucleotides is investigated by means of mass spectrometry (ESI-TOF MS). The results obtained show a particular binding behaviour of this platinum(II) complex. The interaction of 1 with the assayed proteins apparently takes place by Pt-binding to the most accessible coordinating amino acids, presumably at the surface of the protein -this avoiding protein denaturation or degradation- with the subsequent release of one or two ligands of 1. The specific reactivity of 1 with distinct proteins allows to conclude that the substituted initial ligand (dmso or azaindolate) is indicative of the nature of the protein donor atom finally bound to the platinum(II) centre, i.e. N- or S-donor amino acid. Molecular modeling calculations suggest that the release of the azaindolate ligand is promoted by a proton transfer to the non-coordinating N present in the azaindolate ring, while the release of the dmso ligand is mainly favoured by the binding of a deprotonated Cys. The interaction of complex 1 with DNA takes always place through the release of the azaindolate ligand. Interestingly, the interaction of 1 with DNA only proceeds when the oligonucleotides are annealed forming a double strand. Complex 1 is also capable to displace ethidium bromide from DNA and it also weakly binds to DNA at the minor groove, as shown by Hoechst 33258 displacement experiments. Furthermore, complex 1 is also a good inhibitor of cathepsin B (an enzyme implicated in a number of cancer related events). Therefore, although compound 1 is definitely able to bind proteins that can hamper its arrival to the nuclear target, it should be taken into consideration as a putative anticancer drug due to its strong interaction with oligonucleotides and its effective inhibition of cat B. Reactivity of a Pt complex: The interaction of a potential antitumoral drug, [Pt(dmba)(aza-N1)(dmso)], with distinct proteins and oligonucleotides has been studied by means of ESI-TOF MS. The particular reactivity of this complex in front of the assayed biomolecules required the use of spectroscopic techniques and theoretical calculations to elucidate its mechanism. This information will contribute to its consideration as a putative anticancer drug.
Keywords: Antitumoral compound; Platinum; DNA interaction; Protein interaction; Mass spectrometry

Inactivation of bacterial and viral biothreat agents on metallic copper surfaces by Pauline Bleichert; Christophe Espírito Santo; Matthias Hanczaruk; Hermann Meyer; Gregor Grass (1179-1189).
In recent years several studies in laboratory settings and in hospital environments have demonstrated that surfaces of massive metallic copper have intrinsic antibacterial and antiviral properties. Microbes are rapidly inactivated by a quick, sharp shock known as contact killing. The underlying mechanism is not yet fully understood; however, in this process the cytoplasmic membrane is severely damaged. Pathogenic bacterial and viral high-consequence species able to evade the host immune system are among the most serious lethal microbial challenges to human health. Here, we investigated contact-killing mediated by copper surfaces of Gram-negative bacteria (Brucella melitensis, Burkholderia mallei, Burkholderia pseudomallei, Francisella tularensis tularensis and Yersinia pestis) and of Gram-positive endospore-forming Bacillus anthracis. Additionally, we also tested inactivation of monkeypox virus and vaccinia virus on copper. This group of pathogens comprises biothreat species (or their close relatives) classified by the Center for Disease and Control and Prevention (CDC) as microbial select agents posing severe threats to public health and having the potential to be deliberately released. All agents were rapidly inactivated on copper between 30 s and 5 min with the exception of B. anthracis endospores. For vegetative bacterial cells prolonged contact to metallic copper resulted in the destruction of cell structure.
Keywords: Metallic copper surfaces; Antimicrobial; Biothreat agent; Contact-killing

Silver nanoparticles are known to have antimicrobial properties and have been used extensively in medicine, although the mechanism(s) of action have not yet been clearly established. In the present study, the findings suggest a novel mechanism for the antibacterial effect of silver nanoparticles on Escherichia coli, namely, the induction of a bacterial apoptosis-like response. We propose a possible mechanism for the bacterial apoptosis-like response that includes the following: accumulation of reactive oxygen species (ROS) (detected with H2DCFDA staining), increased intracellular calcium levels (detected with Fura-2 AM), phosphatidylserine exposure in the outer membrane (detected with Annexin V) which is the hallmarks of early apoptosis, disruption of the membrane potential [detected with DiBAC4(3)], activation of a bacterial caspase-like protein (detected by FITC-VAD-FMK staining) and DNA degradation (detected with TUNEL assay) which is the hallmarks of late apoptosis in bacterial cells treated with silver nanoparticles. We also performed RecA expression assay with western blotting and observed activation of SOS response to repair the damaged DNA. To summarize, silver nanoparticles are involved in the apoptosis-like response in E. coli and the novel mechanisms which were identified in this study, suggest that silver nanoparticles may be an effective antimicrobial agent with far lower propensity for inducing microbial resistance than antibiotics.
Keywords: Silver nanoparticle; Antibacterial effect; Bacterial cell death; Apoptosis-like response; Esherichia coli

Beryllium is an inhibitor of cellular GSK-3β that is 1,000-fold more potent than lithium by Swapna R. Mudireddy; Ataur Rahman Mohammed Abdul; Priyatham Gorjala; Ronald K. Gary (1203-1216).
Glycogen synthase kinase 3β (GSK-3β) is a key regulator in signaling networks that control cell proliferation, metabolism, development, and other processes. Lithium chloride is a GSK-3 family inhibitor that has been a mainstay of in vitro and in vivo studies for many years. Beryllium salt has the potential to act as a lithium-like inhibitor of GSK-3, but it is not known whether this agent is effective under physiologically relevant conditions. Here we show that BeSO4 inhibits endogenous GSK-3β in cultured human cells. Exposure to 10 µM Be2+ produced a decrease in GSK-3β kinase activity that was comparable to that produced by 10 mM Li+, indicating that beryllium is about 1,000-fold more potent than the classical inhibitor when treating intact cells. There was a statistically significant dose-dependent reduction in specific activity of GSK-3β immunoprecipitated from cells that had been treated with either agent. Lithium inhibited GSK-3β kinase activity directly, and it also caused GSK-3β in cells to become phosphorylated at serine-9 (Ser-9), a post-translational modification that occurs as part of a well-known positive feedback loop that suppresses the kinase activity. Beryllium also inhibited the kinase directly, but unlike lithium it had little effect on Ser-9 phosphorylation in the cell types tested, suggesting that alternative modes of feedback inhibition may be elicited by this agent. These results indicate that beryllium, like lithium, can induce perturbations in the GSK-3β signaling network of treated cells.
Keywords: Beryllium sulfate; Lithium chloride; Glycogen synthase kinase; Phosphorylation

In vivo and in vitro evaluation of effects of Mg-6Zn alloy on apoptosis of common bile duct epithelial cell by Yigang Chen; Jun Yan; Xiaohu Wang; Song Yu; Zigang Wang; Xiaonong Zhang; Shaoxiang Zhang; Yufeng Zheng; Changli Zhao; Qi Zheng (1217-1230).
Biodegradable magnesium alloy implants have attracted much attention because of their excellent biocompatibility and good mechanical properties. However, effects of Mg alloy on cell apoptosis remain unclear. The aim of this study was to investigate the effects of the Mg-6Zn alloy on the apoptosis and necrosis of common bile duct (CBD) epithelial cells. In the in vitro experiments, primary mouse extrahepatic bile epithelial cells (MEBECs) were exposed to Mg-6Zn alloy extracts with different concentrations (0, 40, 80, and 100 %). Flow cytometry analysis indicated that low concentration Mg-6Zn extract can induce apoptosis of MEBECs, and high concentration Mg-6Zn extracts may relate to necrosis and/or ‘apoptotic necrosis’. Real-time PCR results showed that when MEBECs were treated with 40 % extracts for 3 days, the relative apoptotic genes including Bax, Bax/Bcl-2 ratio, NF-κB and caspase-3 were higher than those in the control group. In the in vivo experiments, Mg-6Zn alloy stents were implanted into rabbits’ CBD for 1, 2, 3 weeks, respectively. Based on the hematoxylin and eosin (H&E) staining of peri-implant CBD tissue, no apoptotic bodies and necrotic cells were observed. Results of immunohistochemical staining also showed Mg-6Zn stents did not increase expression levels of apoptosis related gene such as Bax, Bcl-2, Bax/Bcl-2 ratio, TNF-α, NF-κB and caspase-3 in CBD, which indicating Mg-6Zn did not induce significant apoptosis in the in vivo experiments. The different results of in vitro and in vivo experiment may result from the low corrosion rate of Mg-6Zn alloy stents in vivo and local Mg2+ ion concentration in CBD.
Keywords: Biodegradable stent; Mg-6Zn alloy; Common bile duct; Apoptosis

ZAT12 a C2H2-zinc-finger protein is an abiotic stress-responsive transcription factor in plants having less information about their structure. Transcription analysis proved that ZAT12 transcripts over-expressed during drought, heat and salt stress conditions which led to an interest in 3-D structural studies of ZAT12in Brassica carinata. Over-expression of BcZAT12 in transformed tomato plants under abiotic stresses, suggest role of ZAT12 in conferring stress-tolerance in tomato. Sequence analysis of ZAT12 protein (Accession No. ABB55254.1) from B. carinata revealed it as a 161 amino acid long protein with short conserved motif 140LDLXL144 in C-terminal, a leucine rich L-Box with—14EXXAXCLXXL23 motif in N-terminal region and presence of two conserved Zinc-Finger motifs “CXXCXXXXXXXQALGGHXXXH” between positions 42–62 and 85–105. The two zinc finger motifs have presence of two conserved glutamic acid (Glu) and phenylalanine (Phe) residues. Two methionine (Met) residues at position 94 and 102 present in ZF-motif-2 were absent in ZF-motif-1. The 94Met and 97Ala in ZF-motif-2 were found to be replaced by serine (Ser) in ZF-motif-1. Homology and ab initio structural modeling of ZAT12 encoded BcZAT12 protein of B. carinata resulted in robust 3-D models and were evaluated for structural motifs, associated GO terms and protein-DNA interactions. The BcZAT12 protein model, was of good quality, reliable, stable and is deposited in PMDB database (PMDB ID: PM0078213). BcZAT12 is annotated as an intracellular protein having molecular function in Zn-binding which in turn regulates signal transduction/translation processes in response to abiotic stresses in plants. Results suggest BcZAT12 protein to interact directly with one strand of dsDNA via electrostatic and H-bonds.
Keywords: Brassica carinata ; Oxidative stress; Modeling; ZAT12; Zinc finger protein

Oral zinc aspartate treats experimental autoimmune encephalomyelitis by Claudia Schubert; Karina Guttek; Kurt Grüngreiff; Anja Thielitz; Frank Bühling; Annegret Reinhold; Stefan Brocke; Dirk Reinhold (1249-1262).
The essential trace element zinc plays a critical role in the regulation of immune homeostasis. Zinc deficiency or excess can cause severe impairment of the immune response, which points to the importance of the physiological and dietary control of zinc levels for a functioning immune system. We previously reported that injection of zinc aspartate suppresses experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS), as well as effector T cell functions in vitro. Among the preferred characteristics of novel therapeutics for the treatment of autoimmune diseases such as MS are oral availability and a tolerable effective dose to minimize side effects. In this study, we investigated whether oral administration of zinc aspartate, an approved drug to treat zinc deficiency in humans, is effective in controlling EAE at clinically approved doses. We show that oral administration of 6 µg/day [0.3 mg/kg body weight (BW)] or 12 µg/day [0.6 mg/kg BW] of zinc aspartate reduces clinical and histopathological signs during the relapsing remitting phase of the disease in SJL mice. The clinical effect in mice was accompanied by suppression of IFN-γ, TNF-α, GM-CSF and IL-5 production in stimulated human T cells and mouse splenocytes in a dose-dependent manner. Furthermore, a large array of proinflammatory cytokines was modulated by zinc aspartate exposure in vitro. These data suggest that administration of oral zinc aspartate may have beneficial effects on autoimmune diseases like MS.
Keywords: Zinc aspartate; Experimental autoimmune encephalomyelitis (EAE); IFN-γ; TNF-α; GM-CSF; IL-5; T cell activation

Mutations in the ArsA ATPase that restore interaction with the ArsD metallochaperone by Jitesh K. Pillai; Sarkarai Venkadesh; A. Abdul Ajees; Barry P. Rosen; Hiranmoy Bhattacharjee (1263-1275).
The ArsA ATPase is the catalytic subunit of the ArsAB As(III) efflux pump. It receives trivalent As(III) from the intracellular metallochaperone ArsD. The interaction of ArsA and ArsD allows for resistance to As(III) at environmental concentrations. A quadruple mutant in the arsD gene encoding a K2A/K37A/K62A/K104A ArsD is unable to interact with ArsA. An error-prone mutagenesis approach was used to generate random mutations in the arsA gene that restored interaction with the quadruple arsD mutant in yeast two-hybrid assays. A number of arsA genes with multiple mutations were isolated. These were analyzed in more detail by separation into single arsA mutants. Three such mutants encoding Q56R, F120I and D137V ArsA were able to restore interaction with the quadruple ArsD mutant in yeast two-hybrid assays. Each of the three single ArsA mutants also interacted with wild type ArsD. Only the Q56R ArsA derivative exhibited significant metalloid-stimulated ATPase activity in vitro. Purified Q56R ArsA was stimulated by wild type ArsD and to a lesser degree by the quadruple ArsD derivative. The F120I and D137V ArsAs did not show metalloid-stimulated ATPase activity. Structural models generated by in silico docking suggest that an electrostatic interface favors reversible interaction between ArsA and ArsD. We predict that mutations in ArsA propagate changes in hydrogen bonding and salt bridges to the ArsA–ArsD interface that affect their interactions.
Keywords: Arsenite; ArsA; ArsD; Metallochaperone; Resistance; Transport ATPase

Comparative proteomic analysis of two tobacco (Nicotiana tabacum) genotypes differing in Cd tolerance by Lupeng Xie; Xiaoyan He; Shenghua Shang; Weite Zheng; Wenxing Liu; Guoping Zhang; Feibo Wu (1277-1289).
Tobacco can easily accumulate cadmium (Cd) in leaves and thus poses a potential threat to human health. Cd-stress-hydroponic-experiments were performed, and the proteomic and transcriptional features of two contrasting tobacco genotypes Yun-yan2 (Cd-tolerant) and Guiyan1 (Cd-sensitive) were compared. We identified 18 Cd-tolerance-associated proteins in leaves, using 2-dimensional gel electrophoresis coupled with mass spectrometry, whose expression were significantly induced in Yunyan2 leaves but down-regulated/unchanged in Guiyan1, or unchanged in Yunyan2 but down-regulated in Guiyan1 under 50 µM Cd stress. They are including epoxide hydrolase, enoyl-acyl-carrier-protein reductase, NPALDP1, chlorophyll ab binding protein 25, heat shock protein 70 and 14-3-3 proteins. They categorized as 8 groups of their functions: metabolism, photosynthesis, stress response, signal transduction, protein synthesis, protein processing, transport and cell structure. Furthermore, the expression patterns of three Cd-responsive proteins were validated by quantitative real-time PCR. Our findings provide an insight into proteomic basis for Cd-detoxification in tobacco which offers molecular resource for Cd-tolerance.
Keywords: Cadmium (Cd); Proteomic analysis; qRT-PCR; Tobacco (Nicotiana tabacum); 2-DE (2-dimensional gel electrophoresis)

Abnormal metal levels in the primary visual pathway of the DBA/2J mouse model of glaucoma by Alaina S. DeToma; Christine M. Dengler-Crish; Aniruddha Deb; Joseph J. Braymer; James E. Penner-Hahn; Cornelis J. van der Schyf; Mi Hee Lim; Samuel D. Crish (1291-1301).
The purpose of this study was to determine metal ion levels in central visual system structures of the DBA/2J mouse model of glaucoma. We used inductively coupled plasma mass spectrometry (ICP-MS) to measure levels of iron (Fe), copper (Cu), zinc (Zn), magnesium (Mg), manganese (Mn), and calcium (Ca) in the retina and retinal projection of 5-month (pre-glaucomatous) and 10-month (glaucomatous) old DBA/2J mice and age-matched C57BL/6J controls. We used microbeam X-ray fluorescence (μ-XRF) spectrometry to determine the spatial distribution of Fe, Zn, and Cu in the superior colliculus (SC), which is the major retinal target in rodents and one of the earliest sites of pathology in the DBA/2J mouse. Our ICP-MS experiments showed that glaucomatous DBA/2J had lower retinal Fe concentrations than pre-glaucomatous DBA/2J and age-matched C57BL/6J mice. Pre-glaucomatous DBA/2J retina had greater Mg, Ca, and Zn concentrations than glaucomatous DBA/2J and greater Mg and Ca than age-matched controls. Retinal Mn levels were significantly deficient in glaucomatous DBA/2J mice compared to aged-matched C57BL/6J and pre-glaucomatous DBA/2J mice. Regardless of age, the SC of C57BL/6J mice contained greater Fe, Mg, Mn, and Zn concentrations than the SC of DBA/2J mice. Greater Fe concentrations were measured by μ-XRF in both the superficial and deep SC of C57BL/6J mice than in DBA/2J mice. For the first time, we show direct measurement of metal concentrations in central visual system structures affected in glaucoma and present evidence for strain-related differences in metal content that may be specific to glaucomatous pathology.
Keywords: Superior colliculus; Retina; ICP-MS; Neurodegeneration; Glaucoma

Effects of copper overload in P19 neurons: impairment of glutathione redox homeostasis and crosstalk between caspase and calpain protease systems in ROS-induced apoptosis by Maja Jazvinšćak Jembrek; Josipa Vlainić; Vedrana Radovanović; Julija Erhardt; Nada Oršolić (1303-1322).
Copper, a transition metal with essential biological functions, exerts neurotoxic effects when present in excess. The aim of the present study was to better elucidate cellular and molecular mechanisms of CuSO4 toxicity in differentiated P19 neurons. Exposure to 0.5 mM CuSO4 for 24 h provoked moderate decrease in viability, accompanied with barely increased generation of reactive oxygen species (ROS) and caspase-3/7 activity. Glutathione (GSH) and ATP contents were depleted, lactate dehydrogenase inactivated, and glyceraldehyde-3-phosphate dehydrogenase overexpressed. In severely damaged neurons exposed to only two times higher concentration, classical caspase-dependent apoptosis was triggered as evidenced by marked caspase-3/7 activation and chromatin condensation. Multifold increase in ROS, together with very pronounced ATP and GSH loss, strongly suggests impairment of redox homeostasis. At higher copper concentration protease calpains were also activated, and neuronal injury was prevented in the presence of calpain inhibitor leupeptin through the mechanism that affects caspase activation. MK-801 and nifedipine, inhibitors of calcium entry, and H-89 and UO126, inhibitors of PKA and ERK signaling respectively, exacerbated neuronal death only in severely damaged neurons, while ROS-scavenger quercetin and calcium chelator BAPTA attenuated toxicity only at lower concentration. In a dose-dependent manner copper also provoked transcriptional changes of genes involved in intracellular signaling and induction of apoptosis (p53, c-fos, Bcl-2 and Bax). The obtained results emphasize differences in triggered neuronal-death processes in a very narrow range of concentrations and give further insight into the molecular mechanisms of copper toxicity with the potential to improve current therapeutic approaches in curing copper-related neurodegenerative diseases.
Keywords: Copper toxicity; P19 neurons; Glutathione; Lactate dehydrogenase; Apoptosis; Calpain; ERK pathway

Conserved metallomics in two insect families evolving separately for a hundred million years by Polychronis Rempoulakis; Negar Afshar; Beatriz Osorio; Martha Barajas-Aceves; Joanna Szular; Sohel Ahmad; Thilakasiri Dammalage; Ulysses Sto Tomas; Esther Nemny-Lavy; Mor Salomon; Marc J. B. Vreysen; David Nestel; Fanis Missirlis (1323-1335).
Μetal cofactors are required for enzymatic catalysis and structural stability of many proteins. Physiological metal requirements underpin the evolution of cellular and systemic regulatory mechanisms for metal uptake, storage and excretion. Considering the role of metal biology in animal evolution, this paper asks whether metal content is conserved between different fruit flies. A similar metal homeostasis was previously observed in Drosophilidae flies cultivated on the same larval medium. Each species accumulated in the order of 200 µg iron and zinc and approximately ten-fold less manganese and copper per gram dry weight of the adult insect. In this paper, data on the metal content in fourteen species of Tephritidae, which are major agricultural pests worldwide, are presented. These fruit flies can be polyphagous (e.g., Ceratitis capitata) or strictly monophagous (e.g., Bactrocera oleae) or oligophagous (e.g., Anastrepha grandis) and were maintained in the laboratory on five distinct diets based on olive oil, carrot, wheat bran, zucchini and molasses, respectively. The data indicate that overall metal content and distribution between the Tephritidae and Drosophilidae species was similar. Reduced metal concentration was observed in B. oleae. Feeding the polyphagous C. capitata with the diet of B. oleae resulted in a significant quantitative reduction of all metals. Thus, dietary components affect metal content in some Tephritidae. Nevertheless, although the evidence suggests some fruit fly species evolved preferences in the use or storage of particular metals, no metal concentration varied in order of magnitude between these two families of Diptera that evolved independently for over 100 million years.
Keywords: Physiology; Evolution; Genetics; Nutrition; Fruit flies of economic importance; Agriculture; Mediterranean fruit fly

Investigation of metal sorption behavior of Slp1 from Lysinibacillus sphaericus JG-B53: a combined study using QCM-D, ICP-MS and AFM by Matthias Suhr; Nancy Unger; Karen E. Viacava; Tobias J. Günther; Johannes Raff; Katrin Pollmann (1337-1349).
Surface layer proteins (S-layer) of Lysinibacillus sphaericus JG-B53 are biological compounds with several bio-based technical applications such as biosorptive materials for metal removal or rare metals recovery from the environment. Despite their well-described applications, a deeper understanding of their metal sorption behavior still remains challenging. The metal sorption ability of Au3+, Pd2+, Pt2+ and Eu3+ was investigated by ICP-MS, AFM and QCM-D which enables the sorption detection in real-time during in situ experiments. Results indicate a high binding of Pd, followed by Au, Eu and Pt to the proteins. The comparison between different methods allowed a deeper understanding of the metal sorption of isolated S-layer either frees in liquid, adsorbed forming a protein layer or as the bacteria surface.
Keywords: Biosorption; Metals; S-layer; QCM-D; AFM; ICP-MS

Desferrioxamine-caffeine (DFCAF) as a cell permeant moderator of the oxidative stress caused by iron overload by Elizabeth Carmen Pastrana Alta; Dibakar Goswami; M. Teresa Machini; Daniel Menezes Silvestre; Cassiana Seimi Nomura; Breno Pannia Espósito (1351-1360).
Desferrioxamine (DFO) is a potent iron chelator used in the treatment of iron overload (IO) disorders. However, due to its low cell permeability and fast clearance, DFO administration is usually prolonged and of limited use for the treatment of IO in tissues such as the brain. Caffeine is a safe, rapidly absorbable molecule that can be linked to other compounds to improve their cell permeability. In this work, we successfully prepared and described DFO-caffeine, a conjugate with iron scavenging ability, antioxidant properties and enhanced permeation in the HeLa cell model.
Keywords: Iron; Desferrioxamine; Caffeine; HeLa

Revised model of calcium and magnesium binding to the bacterial cell wall by Kieth J. Thomas III; Charles V. Rice (1361-1370).
Metals bind to the bacterial cell wall, yet the binding mechanisms and affinity constants are not fully understood. The cell wall of gram positive bacteria is characterized by a thick layer of peptidoglycan and anionic teichoic acids anchored in the cytoplasmic membrane as lipoteichoic acid or covalently bound to the cell wall as wall teichoic acid. The polyphosphate groups of teichoic acid provide one-half of the metal binding sites for calcium and magnesium, which contradicts previous reports that calcium binding is 100 % dependent on teichoic acid. The remaining binding sites are formed with the carboxyl units of peptidoglycan. In this work we report equilibrium association constants and total metal binding capacities for the interaction of calcium and magnesium ions with the bacterial cell wall. Metal binding is much stronger than previously reported. Curvature of Scatchard plots from the binding data and the resulting two regions of binding affinity suggest the presence of negative cooperative binding, which means that the binding affinity decreases as more ions become bound to the sample. For Ca2+, Region I has a KA = (1.0 ± 0.2) × 106 M−1 and Region II has a KA = (0.075 ± 0.058) × 106 M−1. For Mg2+, KA1 = (1.5 ± 0.1) × 106 and KA2 = (0.17 ± 0.10) × 106. A binding capacity (η) is reported for both regions. However, since binding is still occurring in Region II, the total binding capacity is denoted by η2, which are 0.70 ± 0.04 and 0.67 ± 0.03 µmol/mg for Ca2+ and Mg2+ respectively. These data contradict the current paradigm of only a single metal affinity value that is constant over a range of concentrations. We also find that measurement of equilibrium binding constants is highly sample dependent. This suggests a role for diffusion of metals through heterogeneous cell wall fragments. As a result, we are able to reconcile many contradictory theories that describe binding affinity and the binding mode of divalent metal cations.
Keywords: Peptidoglycan; Teichoic acid; Magnesium; Calcium; Metals

Lactoferrin (Lf), present in colostrum and milk is a member of the transferrin family of iron-binding glyco-proteins, with stronger binding capacity to ferric iron than hemoglobin, myoglobin or transferrin. Unlike hemoglobin and myoglobin, iron-bound Lf is reasonably stable to gastric and duodenal digestive conditions. Unlike ferrous iron, ferric iron is not directly reactive with oxygen supporting the capacity of Lf capture of heme iron to suppress reactive oxygen species (ROS) production. We therefore hypothesized that bovine Lf could capture and thereby terminate the cycle of ROS production by heme iron. The transfer of heme iron from either intact or digested forms of hemoglobin and myoglobin and from intact ferritin was demonstrated by in vitro methods, monitoring Fe-saturation status of Lf by changes in absorptivity at 465 nm. The results are discussed in the context of new proposed opportunities for orally administered Lf to regulate oxidative damage associated with heme iron. In addition to potentially suppressing oxidative heme–iron-mediated tissue damage in the lumen, Lf is expected to also reverse the overload of ferritin-bound iron, that accompanies chronic inflammation and aging. These new proposed uses of Lf are additional to known host defense functions that include anti-microbial, anti-viral properties, immune and cancer cell growth regulation. The findings and interpretations presented require clinical substantiation and may support important additional protective and therapeutic uses for Lf in the future.
Keywords: Lactoferrin; Iron; Heme; Non-heme; Reactive oxygen species; Anti-oxidant; Digestion; Oxidative stress