BioMetals (v.22, #3)

Imaging atoms in medicine by M. Teresa Albelda; Enrique García-España; Juan Carlos Frías (393-399).
The innovations in science and technology have allowed researchers to look inside the human body. In some cases, like MRI, the protons present in the body generate enough signal for an image. However, the employ of certain atoms, metallic or non-metallic, enable detection through different imaging techniques (computed tomography, nuclear imaging, ultrasound or optical imaging), and improve the quality of the images. Here we discuss the different imaging atoms used depending on the imaging technique and the new possible imaging atoms for medical applications.
Keywords: Atoms; MRI; CT; Nuclear imaging; US; OI

The ability of Abortiporus biennis to tolerate and solubilize toxic metal oxides (Cu2O, Al2O3, ZnO, CuFe2O4Zn, CdO, and MnO2) incorporated into agar media was investigated and the growth rate, oxalic acid secretion, and mycelial morphology were monitored. Among the tested metal oxides, formation of clear zones underneath the mycelium growing on Cu2O- and ZnO-amended plates was observed. ZnO, CdO and Cu2O caused the highest rate of fungal growth inhibition. An increased level of oxalic acid concentration was detected as a response of A. biennis to the presence of Cu2O, MnO2, ZnO and CuFe2O4Zn in growth medium. The oxalate oxidase (OXO) was found to be responsible for oxalic acid degradation in A. biennis cultivated in metal-amended media. An increased level of OXO was observed in media amended with Cu2O, ZnO and MnO2. Confocal microscopy used in this study revealed changes in mycelial morphology which appeared as increased hyphal branching, increased septation and increased spore number.
Keywords: Metal oxides; Oxalic acid; Oxalate oxidase; Wood-rotting fungi; Confocal microscopy

Functional characterization of tzn1 and tzn2-zinc transporter genes in Neurospora crassa by Patnala Kiranmayi; Anand Tiwari; Korripally Prem Sagar; Adhikarla Haritha; Pamarthi Maruthi Mohan (411-420).
Previous work from our laboratory involved the description of the Neurospora metal transportome, which included seven hypothetical zinc transporters belonging to the ZIP family. The aim of the present study was to make a comparative functional evaluation of two hypothetical zinc transporters named tzn1 (NCU07621.3) and tzn2 (NCU11414.3). Phenotypic analysis of ∆tzn1 and ∆tzn2 mutants and a double mutant (∆tzn1tzn2) revealed that the deletion of tzn1 causes aconidiation and a greater defect in growth than the single deletion of tzn2. Supplementation with zinc restores growth but not conidiation in ∆tzn1 and ∆tzn1tzn2. TZN1 complemented a zinc-uptake-deficient Saccharomyces cerevisiae mutant (∆zrt1zrt2) in zinc-deficient conditions, while tzn2 restored growth upon supplementation with zinc (0.05 mM). Furthermore, the Δtzn1 mutant was found to have severely reduced zinc content indicating that tzn1 functions as a key regulator of intracellular zinc levels in Neurospora crassa. Zinc uptake studies indicate tzn1 is a specific transporter of zinc, while tzn2 transports both zinc and cadmium. Quantitative RT-PCR showed up-regulation of tzn1 (128-fold) under zinc-depleted conditions and down-regulation (>1,000-fold) in zinc-replete conditions. The present study indicates that the zinc transport proteins encoded by tzn1 and tzn2 are members of the zinc uptake system regulated by zinc status in N. crassa.
Keywords: Zinc; Metal ion transporters; ZIP family; Aconidiation; Neurospora crassa

Role for copper in the cellular and regulatory effects of heme-hemopexin by Ann Smith; Kimberly R. Rish; Rachel Lovelace; Jennifer F. Hackney; Rachel M. Helston (421-437).
Hemopexin (HPX) binds heme tightly, thus protecting cells from heme toxicity during hemolysis, trauma and ischemia-reperfusion injury. Heme uptake via endocytosis of heme-HPX followed by heme catabolism by heme oxygenase-1 (HMOX1) raises regulatory iron pools, thus linking heme metabolism with that of iron. Normal iron homeostasis requires copper-replete cells. When heme-HPX induces HMOX1, the copper-storing metallothioneins (MTs) are also induced whereas the copper-responsive copper chaperone that delivers copper to Cu, Zn superoxide dismutase, CCS1, is decreased; both are known responses when cellular copper levels rise. Endocytosis of heme-HPX is needed to regulate CCS1 since the signaling ligand cobalt-protoporphyrin (CoPP)-HPX, which does not induce HMOX1 but does co-localize with heme-HPX in endosomes, also decreased CCS1. These observations support that heme-HPX mobilizes copper in cells. The regulation of both hmox1 and mt1 is prevented by the copper-chelator, bathocuproinedisulfonate (BCDS), but not uptake of heme-AlexaFluor-labeled HPX into endosomes. Supporting a role for copper in HMOX1 regulation by heme-HPX, nutritional copper deficiency generated by tetraethylene pentamine or 232 tetraamine prevented HMOX1 induction. Using conditions that mimic maturing endosomes, we found that copper prevents rebinding of heme to apo-HPX. A model is presented in which copper endocytosis together with that of heme-HPX provides a means to facilitate heme export from HPX in the maturing endosomes: heme is needed for hmox1 transcription, while cytosolic copper and CCS1 provide a link for the known simultaneous regulation of hmox1 and mt1 by heme-HPX.
Keywords: Heme; Hemopexin; Copper; Heme oxygenase; Iron; Cobalt protoporphyrin IX; Endocytosis; Endosomes; Metallothionein; CCS1

Kinetic analysis of ligand interaction with the gonococcal transferrin-iron acquisition system by Amanda J. DeRocco; Mary Kate Yost-Daljev; Christopher D. Kenney; Cynthia Nau Cornelissen (439-451).
The transferrin iron acquisition system of Neisseria gonorrhoeae consists of two dissimilar transferrin binding proteins (Tbp) A and B. TbpA is a TonB dependent transporter while TbpB is a lipoprotein that makes iron acquisition from transferrin (Tf) more efficient. In an attempt to further define the individual roles of these receptors in the process of Tf-iron acquisition, the kinetics of the receptor proteins in regards to ligand association and dissociation were evaluated. Tf association with TbpB was rapid as compared to TbpA. Tf dissociation from the wild-type receptor occurred in a biphasic manner; an initial rapid release was followed by a slower dissociation over time. Both TbpA and TbpB demonstrated a two-phase release pattern; however, TbpA required both TonB and TbpB for efficient Tf dissociation from the cell surface. The roles of TbpA and TbpB in Tf dissociation were further examined, utilizing previously created HA fusion proteins. Using a Tf-utilization deficient TbpA-HA mutant, we concluded that the slower rate of ligand dissociation demonstrated by the wild-type transporter was a function of successful iron internalization. Insertion into the C-terminus of TbpB decreased the rate of Tf dissociation, while insertion into the N-terminus had no effect on this process. From these studies, we propose that TbpA and TbpB function synergistically during the process of Tf iron acquisition and that TbpB makes the process of Tf-iron acquisition more efficient at least in part by affecting association and dissociation of Tf from the cell surface.
Keywords: Neisseria ; TonB; Transferrin; Receptor

Eryptosis triggered by bismuth by Manuel Braun; Michael Föller; Erich Gulbins; Florian Lang (453-460).
Bismuth is used for multiple industrial purposes and in the treatment of several gastrointestinal diseases. Untoward effects of bismuth include anemia, which could, in theory, result from suicidal erythrocyte death or eryptosis. Hallmarks of eryptosis are cell shrinkage and cell membrane scrambling with phosphatidylserine exposure at the cell surface. Phosphatidylserine-exposing cells are rapidly cleared from circulating blood. Signaling leading to eryptosis includes increase in cytosolic Ca2+ activity and formation of ceramide. The present experiments explored whether bismuth elicits eryptosis. To this end, phosphatidylserine exposure was estimated from annexin V-binding, cell shrinkage from decrease of forward scatter in FACS analysis, cytosolic Ca2+ activity from Fluo3 fluorescence and ceramide abundance from binding of fluorescent antibodies. A 48 h exposure to bismuth (≥500 μg/l BiCl3) enhanced the percentage of annexin V-binding cells and decreased forward scatter, increased cytosolic Ca2+ activity, and stimulated ceramide formation. In conclusion, bismuth stimulates eryptosis, the suicidal death of erythrocytes. The effect may contribute to or even account for the development of anemia during bismuth treatment. Moreover, ceramide formation in intestinal cells may participate in the therapeutic efficacy of bismuth preparations.
Keywords: Cell volume; Annexin; Eryptosis; Calcium; Phosphatidylserine

Use of Galleria mellonella larvae to evaluate the in vivo anti-fungal activity of [Ag2(mal)(phen)3] by Raymond Rowan; Ciara Moran; Malachy McCann; Kevin Kavanagh (461-467).
Larvae of the insect Galleria mellonella were employed to assess the in vivo antifungal efficacy of ([Ag2(mal)(phen)3]), AgNO3 and 1,10-phenanthroline. Larvae pre-inoculated with these compounds were protected from a subsequent lethal infection by the yeast Candida albicans while larvae inoculated 1 and 4 h post-infection showed significantly increased survival (P < 0.01) compared to control larvae. Administration of these compounds resulted in an increase over 48 h in the density of insect haemocytes (immune cells) but there was no widespread activation of genes for antimicrobial peptides. This work demonstrates that G. mellonella larvae may be employed to ascertain the antifungal efficacy of silver(I) compounds and offers a rapid and effective means of assessing the in vivo activity of inorganic antimicrobial compounds.
Keywords: Antifungal; Candida albicans ; Silver(I); Insect; Galleria

Protein phosphatase 2C (PP2C) family is characterized by requirement of metal cation for phosphatase activity. We previously established that PPM1H is a cancer-associated member of the PP2C family. Here we further characterized the phosphatase activity of PPM1H, focusing on its dependence on metal cation. PPM1H possesses the potential to dephosphorylate p-nitrophenyl phosphate (pNPP), casein and phosphopeptides. Interestingly, PPM1H shows the metal preference that is varied depending on the substrate (substrate-dependent metal preference); PPM1H prefers Mn2+ when pNPP or phosphopeptides is used as a substrate. Meanwhile, a preference for Mg2+ is displayed by PPM1H with casein as a substrate. When both cations are added to the reaction, the degree of the effect is always closer to that with Mn2+ alone, irrespective of the substrate. This preponderance of Mn2+ is explained by its greater affinity for PPM1H than Mg2+. From the literature the substrate-dependent metal preference appears to be shared by other PP2Cs. According to the crystal structure, a binuclear metal center of PP2C plays an important role for coordinating the substrate and nucleophilic waters in the active site. Therefore, the differences in the size, preferred geometry and coordination requirements between two metals, in relation to the substrate, may be responsible for this intriguing property.
Keywords: Catalytic domain; Metal cation cofactor; PPM1H; Protein phosphatase 2C

Red cabbage is a source of health beneficial substances with antioxidant and antigenotoxic properties. HPLC analysis specifying the content of the investigated extract indicated that mainly anthocyanins (ATH) were responsible for its abilities. Cytological research was conducted with two experimental models: plant tissues—meristematic cells of Vicia faba, and animal tissue elements—human lymphocytes. Positive influence of ATH extract on mitotic activity of Vicia cells exposed to Cu2+ stress, and inhibitory effect of ATH on cytotoxic actions of Cu2+ on lymphocytes were demonstrated. In all experimental series with ATH application in combinations with Cu2+, mitotic index (MI) were higher than those obtained for only Cu2+ stressed tissues. Preincubation in ATH before Cu2+ stress had the best effect. Similarly, after ATH applications in all tested series decrease in frequency of micronuclei (MN) appearance was noticed in comparison with only Cu2+ stressed material. In the case of Vicia cells ATH acted effectively even applied after Cu2+ stress. It suggests that this ATH mixture not only prevents and limits but also heals the cytological injury caused by Cu2+ stress.
Keywords: Anthocyanins; Brassica oleracea rubrum ; Copper ions; Cytogenetic tests

Antisense oligonucleotides with iron binding hydroxamate linkages are designed to act as sequence-selective cleaving agents of complementary nucleic acids through Fenton chemistry. Oligothymidylate analogs with hydroxamate linkages were efficiently synthesized from coupling of nucleoside intermediates, activated as p-nitrophenyl carbonates, with hydroxylamine derivatized nucleosides. Iron binding studies showed that hydroxamate linked oligonucleotides are effective iron chelators when there are three nonadjacent internucleosidic hydroxamate linkages available in the same oligonucleotide molecule. However, analysis of the CD spectra of an oligothymidylate 16mer, which contained complete substitution of all phosphates with hydroxamates, indicated that the hydroxamate linkage was too rigid to allow the analog to base pair with the complementary DNA d(A16). Syntheses of mix-linked thymidine oligomers with up to three hydroxamate linkages incorporated in the center of the sequence are also reported. Iron binding of the thymidine oligomer with hydroxamate linkages was confirmed by matrix assisted laser desorption mass spectrometry analysis. Nuclease stability assays showed that the modified oligonucleotides have enhanced resistance toward nuclease S1 (endonuclease) compared to natural oligonucleotides. A thymidine 16mer with three hydroxamate linkages incorporated in the center of the sequence was shown to be able to bind with both iron and its complementary polyA strand. A small destablizing effect was observed when the phosphodiester linkage was changed to the hydroxamate linkage. Under Fenton chemistry conditions, this novel iron binding oligothymidylate analog cleaved the complementary DNA strand sequence-selectively.
Keywords: Hydroxamic acid; Iron binding; Fenton chemistry; Antisense oligonucleotides; Chemical nucleases

Gadolinium promoted proliferation and enhanced survival in human cervical carcinoma cells by Ying Zhang; Li-Juan Fu; Jin-Xia Li; Xiao-Gai Yang; Xiao-Da Yang; Kui Wang (511-519).
The effect of gadolinium chloride (Gd) on the proliferation of HeLa cells was investigated at lower concentration. The results obtained by MTT and cell cycle analysis showed that Gd promoted proliferation by inducing S phase entry in HeLa cells at the concentration less than 100 μM. It was further evidenced by both an increase in the levels of phosphorylation of retinoblastoma protein (pRb) and a remarkable increase in cyclin E expression. Moreover, the survival of cells, exposed to Gd up to 3–5 days, was increased compared with control. The attenuation of the serum deprivation-induced cell loss by Gd was associated with the sustained activation of FAK (PY397) and the delayed activation of JNKs pathway. Besides, it appeared that Gd promoted cell proliferation and survival in HeLa cells was not contributed to the ROS generation. Based on the present results, both positive and negative effects of the lanthanides as potential drugs or diagnostic agents are discussed.
Keywords: Gadolinium; Proliferation; Cell cycle; Survival

Effect of lactoferrin on oxidative features of ceruloplasmin by Alexej V. Sokolov; Kira V. Ageeva; Maria O. Pulina; Elena T. Zakharova; Vadim B. Vasilyev (521-529).
In our previous report we first described a complex between lactoferrin (Lf) and ceruloplasmin (Cp) with K d ~ 1.8 μM. The presence of this complex in colostrum that never contains more than 0.3 μM Cp questions the reliability of K d value. We carefully studied Lf binding to Cp and investigated the enzymatic activity of the latter in the presence of Lf, which allowed obtaining a new value for K d of Cp–Lf complex. Lf interacting with Cp changes its oxidizing activity with various substrates, such as Fe2+, o-dianisidine (o-DA), p-phenylenediamine (p-PD) and dihydroxyphenylalanine (DOPA). The presence of at least two binding sites for Lf in Cp molecule is deduced from comparison of substrates’ oxidation kinetics with and without Lf. When Lf binds to the first site affinity of Cp to Fe2+ and to o-DA increases, but it decreases towards DOPA and remains unchanged towards p-PD. Oxidation rate of Fe2+ grows, while that of o-DA, p-PD and DOPA goes down. Subsequent Lf binding to the second center has no effect on iron oxidation, hampers DOPA and o-DA oxidation, and reduces affinity towards p-PD. Scatchard plot for Lf sorbing to Cp-Sepharose allowed estimating K d for Lf binding to high-affinity (~13.4 nM) and low-affinity (~211 nM) sites. The observed effect of Lf on ferroxidase activity of Cp is likely to have physiological implications.
Keywords: Lactoferrin; Ceruloplasmin; Protein–protein interaction; Enzymatic kinetics

Copper egress is induced by PMA in human THP-1 monocytic cell line by Scott E. Afton; Joseph A. Caruso; Bradley E. Britigan; Zhenyu Qin (531-539).
Copper egress is an essential regulator of the kinetics of cellular copper and is primarily regulated by ATP7A, a copper-transporting P-type ATPase. However, little is known under which physiological condition copper egress is induced and its molecular consequence. In current manuscript, using THP-1 cells, a human monocytic cell line, we found that ATP7A expression was increased in cells exposed to phorbol-12-myristate-13-acetate (PMA), a potent inducer of neovascularization and cancer. Inductively coupled plasma mass spectrometry revealed that PMA also induced copper egress. Inhibition of ATP7A expression using small interfering RNA abrogated PMA induced copper egress. PMA treatment in THP-1 cells resulted in increased expression of matrix metalloproteinase (MMP) 9 and vascular endothelial growth factor receptor 1 (VEGFR1), whereas inhibition of ATP7A resulted in suppression of PMA-induced expression of VEGFR1, but not MMP9. Finally, addition of exogenous copper into the conditioned medium did not change VEGFR1 expression in THP-1 cells. Collectively, we demonstrate that PMA induces copper egress in THP-1 cells, which is regulated by ATP7A, and ATP7A regulates VEGFR1 expression. Considering the involvement of copper in neovascularization, our current finding provides the potential evidence to interpret the molecular mechanism.
Keywords: Copper; ATP7A

Cyclodextrins improve the antimicrobial activity of the chloride salt of Ruthenium(II) chloro-phenanthroline-trithiacyclononane by Joana Marques; Teresa M. Braga; Filipe A. Almeida Paz; Teresa M. Santos; Maria de Fátima Silva Lopes; Susana S. Braga (541-556).
The complex [Ru([9]aneS3)phenCl]Cl (phen = 1,10-phenanthroline) and its synthetic precursor [Ru([9]aneS3)dmsoCl2] were immobilized in permethylated β-cyclodextrin (TRIMEB). A new crystalline structure of the precursor, obtained from a batch ethanol solution at low temperature (4°C), is fully described from single-crystal X-ray diffraction data. [Ru([9]aneS3)phenCl]Cl was also encapsulated in native β-cyclodextrin for comparison with the TRIMEB compound. All three compounds were obtained with a 1:1 host:guest stoichiometry and were studied by powder X-ray diffraction (including synchrotron radiation data), thermogravimetric analysis (TGA), 13C{1H} CP/MAS NMR and FTIR spectroscopies. The bacterial growth inhibitory action of the complex [Ru([9]aneS3)phenCl]Cl and its two cyclodextrin compounds was tested on Gram-negative (Salmonella, Escherichia) and Gram-positive strains (Bacillus, Listeria, Enterococcus and Staphilococcus) and results show a positive effect of cyclodextrin immobilization on the antimicrobial properties.
Keywords: Ruthenium(II) complexes; Phenanthroline; β-CD; TRIMEB; Antimicrobial activity