BioMetals (v.28, #2)

Mercury and metabolic syndrome: a review of experimental and clinical observations by Alexey A. Tinkov; Olga P. Ajsuvakova; Margarita G. Skalnaya; Elizaveta V. Popova; Anton I. Sinitskii; Olga N. Nemereshina; Evgenia R. Gatiatulina; Alexandr A. Nikonorov; Anatoly V. Skalny (231-254).
A significant interrelation between heavy metal exposure and metabolic syndrome (MetS) development has been demonstrated earlier. Despite the presence of a number of works aimed at the investigation of the role of Hg in MetS development, the existing data remain contradictory. Therefore, the primary objective of the current work is to review the existing data regarding the influence of mercury on universal mechanisms involved in the pathogenesis of the development of MetS and its components. The brief chemical characterization of mercury is provided. The role of mercury in induction of oxidative stress has been discussed. In particular, Hg-induced oxidative stress may occur due to both prooxidant action of the metal and decrease in antioxidant enzymes. Despite the absence of direct indications, it can be proposed that mercury may induce endoplasmic reticulum stress. As it is seen from both in vivo and in vitro studies, mercury is capable of inducing inflammation. The reviewed data demonstrate that mercury affects universal pathogenetic mechanisms of MetS development. Moreover, multiple investigations have indicated the role of mercury in pathogenesis of MetS components: dyslipidemia, hypertension, insulin resistance, and obesity to a lesser extent. The present state of data regarding the interrelation between mercury and MetS denotes the following perspectives: (1) Further clinic-epidemiologic and experimental studies are required to estimate the association between mercury exposure and the development of MetS components, especially obesity; (2) Additional investigations of the possible effect of organism’s mercury content modulation on MetS pathogenesis should be undertaken.
Keywords: Mercury; Toxicity; Obesity; Insulin resistance; Hypertension; Dyslipidemia; Atherosclerosis

Roles of oxidative stress and endoplasmic reticulum stress in selenium deficiency-induced apoptosis in chicken liver by Linlin Yao; Qiang Du; Haidong Yao; Xi Chen; Ziwei Zhang; Shiwen Xu (255-265).
Oxidative stress and endoplasmic reticulum (ER) stress are involved in different types of stress-induced injuries. The aim of the present study was to evaluate the effect of Se deficiency on oxidative stress, ER stress and apoptosis in chicken livers. Chickens (1 day old, n = 180) were randomly divided into two groups: the L group [fed with a Se-deficient (Se 0.033 mg/kg) diet] and the control group [fed with a normal (Se 0.2 mg/kg) diet]. Factor-associated oxidative stress, catalase (CAT) activity, H2O2 production and the inhibition of hydroxyl radicals (·OH) in the chicken liver were determined on days 15, 25, 35, 45, 55 and 65, respectively. In addition, ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) and apoptosis-related genes (caspase3 and Bcl-2) were examined by fluorescence quantitative PCR or western blot analysis. Apoptosis levels were also measured using ultrastructural observations and the TdT-mediated dUTP nick end labeling assay. The results showed that CAT activity and ·OH inhibition were decreased and that H2O2 production was increased in the low-Se group, which demonstrated that oxidative stress occurred in the chicken liver. The ER stress-related genes (GRP78, GRP94, ATF4, ATF6 and IRE) and the apoptosis-related gene caspase3 were increased (p < 0.05), while Bcl-2 was decreased (p < 0.05) by Se deficiency. In addition, apoptosis and ER lesions were observed by ultrastructural observations of the chicken liver in the low-Se group. The level of apoptosis and the number of apoptotic cells increased with time. These results indicated that the oxidative-ER stress pathway participates in Se deficiency-induced apoptosis in the chicken liver.
Keywords: Selenium deficiency; Oxidative stress; Endoplasmic reticulum stress; Apoptosis; Chicken liver

Antiproliferative effects of copper(II)–polypyridyl complexes in breast cancer cells through inducing apoptosis by Mona Salimi; Khatereh Abdi; Hirsa Mostafapour Kandelous; Hassan Hadadzadeh; Kayhan Azadmanesh; Amir Amanzadeh; Hassan Sanati (267-278).
Although cisplatin has been used for decades to treat human cancer, some toxic side effects and resistance are observed. Previous investigations have suggested copper complexes as a novel class of tumor-cell apoptosis inducers. The present study aimed to evaluate the anti-breast cancer activities of two polypyridyl-based copper(II) complexes, [Cu(tpy)(dppz)](NO3)2 (1) and [Cu(tptz)2](NO3)2 (2) (tpy = 2,2′:6′,2″-terpyridine, dppz = dipyrido[3,2-a:2′,3′-c]phenazine, tptz = 2,4,6-tris(2-pyridyl)-1,3,5-triazine), using human breast adenocarcinoma cell line (MCF-7). The ability of the complexes to cleave supercoiled DNA in the presence and absence of external agents was also examined. The apoptotic activities of the complexes were assessed using flow cytometry, fluorescence microscope and western blotting analysis. Our results indicated the high DNA affinity and nuclease activity of complexes 1 and 2. The cleavage mechanisms between the complexes and plasmid DNA are likely to involve a singlet oxygen or singlet oxygen-like entity as the reactive oxygen species. Complexes 1 and 2 also significantly inhibited the proliferation of MCF-7 cells in a dose-dependent manner (IC50 values = 4.57 and 1.98 μM at 24 h, respectively). Complex 2 remarkably induced MCF-7 cells to undergo apoptosis, which was demonstrated by cell morphology, annexin-V and propidium iodide staining. The caspase cascade was activated as shown by the proteolytic cleavage of caspase-3 after treatment of MCF-7 cells with complex 2. Additionally, complex 2 significantly increased the expression of the Bax-to-Bcl-2 ratio to induce apoptosis. In conclusion, these results revealed that complex 2 may be a potential and promising chemotherapeutic agent to treat breast cancer.
Keywords: Cancer; Apoptosis; DNA cleavage; Cytotoxicity

Iron can both induce and inhibit nitrosative stress. Intracellular iron levels play an important role in nitric oxide (NO) signaling mechanisms. Depending on various factors, such as the cell’s redox state and transition metal levels, NO generation may lead to lipid peroxidation and DNA damage as well as both anti- and pro-apoptotic effects. Administration of intravenous iron sucrose originator (ISORIG) has been shown not to cause significant tyrosine nitration or significantly increased caspase 3 levels in non-anemic rats. In this study, the potential of several marketed iron sucrose similars (ISSs) to induce tyrosine nitration and caspase 3 expression in non-anemic rats was assessed. Although the physico-chemical properties of most of the analyzed ISSs complied with the United States Pharmacopeia for iron sucrose injection, all ISSs resulted in higher levels of tyrosine nitration and increased the expression of caspase 3 versus ISORIG. Moreover, significant differences were detected in tissue iron distribution between ISORIG- and ISS-treated animals. In general, ISORIG resulted in higher levels of ferritin deposits versus ISSs whereas ISSs showed higher Prussian blue-stainable iron(III) deposits than ISORIG. This result suggests that some iron from ISSs bypassed the tightly regulated pathway through resident macrophages of the liver, spleen and bone marrow thus, ending up in the cellular compartment that favors oxidative and or nitrosative stress as well as apoptosis. The results also confirm that polynuclear iron(III)-oxyhydroxide carbohydrates, such as iron sucrose, cannot be fully characterized by physico-chemical methods alone.
Keywords: Apoptosis; Intravenous iron; Iron sucrose; Nitrosative stress; Non-clinical

Screen for alterations of iron related parameters in N-ethyl-N-nitrosourea-treated mice identified mutant lines with increased plasma ferritin levels by Birgit Rathkolb; Martina Klempt; Sibylle Sabrautzki; Dian Michel; Matthias Klaften; Jürgen Laufs; Reinhard Sedlmeier; Wolfgang Hans; Helmut Fuchs; Martina U. Muckenthaler; Marion Horsch; Dean R. Campagna; Mark Fleming; Martin Hrabé de Angelis; Eckhard Wolf; Bernhard Aigner (293-306).
Iron is essential for numerous cellular processes. For diagnostic purposes iron-related parameters in patients are assessed by clinical chemical blood analysis including the analysis of ferritin, transferrin and iron levels. Here, we retrospectively evaluated the use of these parameters in the phenotype-driven Munich N-ethyl-N-nitrosourea mouse mutagenesis project for the generation of novel animal models for human diseases. The clinical chemical blood analysis was carried out on more than 10,700 G1 and G3 offspring of chemically mutagenized inbred C3H mice to detect dominant and recessive mutations leading to deviations in the plasma levels of iron-related plasma parameters. We identified animals consistently exhibiting altered plasma ferritin or transferrin values. Transmission of the phenotypic deviations to the subsequent generations led to the successful establishment of three mutant lines with increased plasma ferritin levels. For two of these lines the causative mutations were identified in the Fth1gene and the Ireb2 gene, respectively. Thus, novel mouse models for the functional analysis of iron homeostasis were established by a phenotype-driven screen for mutant mice.
Keywords: Iron; Ferritin; Transferrin; Mouse; ENU; Mutagenesis; Hyperferritinemia

Physicochemical properties of ternary oxovanadium(IV) complexes with oxydiacetate and 1,10-phenanthroline or 2,2′-bipyridine. Cytoprotective activity in hippocampal neuronal HT22 cells by Dariusz Wyrzykowski; Iwona Inkielewicz-Stępniak; Joanna Pranczk; Krzysztof Żamojć; Patrycja Zięba; Aleksandra Tesmar; Dagmara Jacewicz; Tadeusz Ossowski; Lech Chmurzyński (307-320).
The aim of this work was to find a relationship between physicochemical properties of the oxovanadium(IV) complexes, namely [VO(ODA)(H2O)2], [VO(ODA)(phen)]·1.5H2O and [VO(ODA)(bipy)]·2H2O (ODA = oxydiacetate) as well as [VO(H2O)5]2+, and their biological activity. A potentiometric titration method has been used to characterize the stability of the complexes in aqueous solutions. Furthermore, the reactivity of the complexes towards superoxide free radicals was assessed by employing the NBT assay as well as a cyclic voltammetry (CV) technique. Additionally, the investigations of the antioxidant properties of the complexes were complemented by studying their reactivity towards organic radicals (the ABTS and DPPH tests). Finally, the biological properties of the complexes were investigated in relation to their cytoprotective activity against the oxidative damage generated exogenously by using hydrogen peroxide in the Hippocampal neuronal cell line HT22 (the MTT and LDH tests). The obtained results showed that all the compounds under study display antioxidant properties but a concentration-depended protective effect against the oxidative damage was found for [VO(ODA)(bipy)]·2H2O only.
Keywords: Oxydiacetate complexes; Potentiometric titration; Cyclic voltammetry; Free radicals; Cytoprotective activity; Hippocampal HT22 cells

Transcriptional activation of glutathione pathways and role of glucose homeostasis during copper imbalance by Natalia Quiroz; Nicole Rivas; Talía del Pozo; Jason Burkhead; Miriam Suazo; Mauricio González; Mauricio Latorre (321-328).
Copper is an essential micronutrient for organism health. Dietary changes or pathologies linked to this metal induce changes in intracellular glutathione concentrations. Here, we studied the transcriptional activation of glutathione pathways in Jurkat cell lines, analyzing the effect of change in glucose homeostasis during a physiological and supra-physiological copper exposure. An immortalized line of human T lymphocyte cell line (Jurkat) was exposed to different copper and glucose conditions to mimic concentrations present in human blood. We applied treatments for 6 (acute) and 24 h (sustained) to 2 µM (physiological) or 20 µM (supra-physiological, Wilson disease scenario) of CuSO4 in combination with 25 mg/dL (hypoglycemia), 100 mg/dL (normal) and 200 mg/dL (hyperglycemia, diabetes scenario) of glucose. The results indicate that a physiological concentration of copper exposure does not induce transcriptional changes in the glutathione synthesis pathway after 6 or 24 h. The G6PDH gene (regeneration pathway), however, is induced during a supra-physiological copper condition. This data was correlated with the viability assays, where fluctuation in both glucose conditions (hypo and hyperglycemia scenario) affected Jurkat proliferation when 20 µM of CuSO4 was added to the culture media. Under a copper overload condition, the transcription of a component of glutathione regeneration pathway (G6PDH gene) is activated in cells chronically exposed to a hyperglycemia scenario, indicating that fluctuations in glucose concentration impact the resistance against the metal. Our findings illustrate the importance of glucose homeostasis during copper excess.
Keywords: Copper; Glutathione; Glucose; Metabolism; Nutrigenomic

Legionella pneumophila is a waterborne pathogen that can cause Legionnaires’ disease, a fatal pneumonia, or Pontiac fever, a mild form of disease. Copper is an antimicrobial material used for thousands of years. Its incorporation in several surface materials to control the transmission of pathogens has been gaining importance in the past decade. In this work, the ability of copper to control the survival of L. pneumophila in biofilms was studied. For that, the incorporation of L. pneumophila in polymicrobial drinking water biofilms formed on copper, PVC and PEX, and L. pneumophila mono-species biofilms formed on copper and uPVC were studied by comparing cultivable and total numbers (quantified by peptide nucleic acid (PNA) hybridisation). L. pneumophila was never recovered by culture from heterotrophic biofilms; however, PNA-positive numbers were slightly higher in biofilms formed on copper (5.9 × 105 cells cm−2) than on PVC (2.8 × 105 cells cm−2) and PEX (1.7 × 105 cells cm−2). L. pneumophila mono-species biofilms grown on copper gave 6.9 × 105 cells cm−2 for PNA-positive cells and 4.8 × 105 CFU cm−2 for cultivable numbers, showing that copper is not directly effective in killing L. pneumophila. Therefore previous published studies showing inactivation of L. pneumophila by copper surfaces in potable water polymicrobial species biofilms must be carefully interpreted.
Keywords: Legionella pneumophila ; Drinking water biofilms; PNA hybridisation; Copper

Shotgun metabolomic approach based on mass spectrometry for hepatic mitochondria of mice under arsenic exposure by M. A. García-Sevillano; T. García-Barrera; F. Navarro; Z. Montero-Lobato; J. L. Gómez-Ariza (341-351).
Mass spectrometry (MS)-based toxicometabolomics requires analytical approaches for obtaining unbiased metabolic profiles. The present work explores the general application of direct infusion MS using a high mass resolution analyzer (a hybrid systems triple quadrupole–time-of-flight) and a complementary gas chromatography–MS analysis to mitochondria extracts from mouse hepatic cells, emphasizing on mitochondria isolation from hepatic cells with a commercial kit, sample treatment after cell lysis, comprehensive metabolomic analysis and pattern recognition from metabolic profiles. Finally, the metabolomic platform was successfully checked on a case-study based on the exposure experiment of mice Mus musculus to inorganic arsenic during 12 days. Endogenous metabolites alterations were recognized by partial least squares-discriminant analysis. Subsequently, metabolites were identified by combining MS/MS analysis and metabolomics databases. This work reports for the first time the effects of As-exposure on hepatic mitochondria metabolic pathways based on MS, and reveals disturbances in Krebs cycle, β-oxidation pathway, amino acids degradation and perturbations in creatine levels. This non-target analysis provides extensive metabolic information from mitochondrial organelle, which could be applied to toxicology, pharmacology and clinical studies.
Keywords: Metabolomics; Mass spectrometry; Mouse mitochondria; Arsenic; Toxicometabolomics; Non-target analysis

Chromate induces adventitious root formation via auxin signalling and SOLITARY-ROOT/IAA14 gene function in Arabidopsis thaliana by José López-Bucio; Randy Ortiz-Castro; León Francisco Ruíz-Herrera; Consuelo Vargas Juárez; Fátima Hernández-Madrigal; Yazmín Carreón-Abud; Miguel Martínez-Trujillo (353-365).
Morphological root plasticity optimizes nutrient and water uptake by plants and is a promising target to improve tolerance to metal toxicity. Exposure to sublethal chromate [Cr(VI)] concentrations inhibits root growth, decreases photosynthesis and compromises plant development and productivity. Despite the increasing environmental problem that Cr(VI) represents, to date, the Cr tolerance mechanisms of plants are not well understood, and it remains to be investigated whether root architecture remodelling is important for plant adaptation to Cr(VI) stress. In this report, we analysed the growth response of Arabidopsis thaliana seedlings to concentrations of Cr(VI) that strongly repress primary and lateral root growth. Interestingly, adventitious roots started developing, branched and allowed seedlings to grow under highly growth-repressing Cr(VI) concentrations. Cr(VI) negatively regulates auxin transport and response gene expression in the primary root tip, as evidenced by decreased expression of auxin-related reporters DR5::GFP, DR5::uidA and PIN1::PIN1::GFP, and then, another auxin maximum is established at the site of adventitious root initiation that drives adventitious root organogenesis. Both primary root growth inhibition and adventitious root formation induced by high Cr(VI) levels are blocked by a gain-of-function mutation in the SOLITARY-ROOT/IAA14 gene of Arabidopsis. These data provide evidence that suggests a critical role for auxin transport and signalling via IAA14/SLR1 in the developmental program linking Cr(VI) to root architecture remodelling.
Keywords: Adventitious roots; Chromate; Auxin; Arabidopsis thaliana ; Heavy metals

Investigation of the multifaceted iron acquisition strategies of Burkholderia cenocepacia by J. Tyrrell; N. Whelan; C. Wright; I. Sá-Correia; S. McClean; M. Thomas; Máire Callaghan (367-380).
Burkholderia cenocepacia is a bacterial pathogen which causes severe respiratory infections in cystic fibrosis (CF). These studies were aimed at gaining an insight into the iron acquisition strategies of B. cenocepacia. In iron restricted conditions, genes associated with the synthesis and utilisation of ornibactin (pvdA, orbA, orb F) were significantly upregulated compared to the expression of pyochelin associated genes (pchD, fptA). In the absence of alternative iron sources, B. cenocepacia J2315 and 715j utilised ferritin and haemin, but not transferrin or lactoferrin for growth. Significantly, mutants unable to produce ornibactin, (715j-orbI) or ornibactin and pyochelin, (715j-pobA), utilised haemin and ferritin more efficiently than the wild-type. Moreover, both mutants were also able to utilise lactoferrin for growth (P ≤ 0.01) and additionally 715j-pobA utilised transferrin (P ≤ 0.01), potentially facilitating adaptation to the host environment. Furthermore, B. cenocepacia increased ornibactin gene expression in response to pyoverdine from Pseudomonas aeruginosa (P ≤ 0.01), demonstrating the capacity to compete for iron in co-colonised niches. Pyoverdine also significantly diminished the growth of B. cenocepacia (P < 0.001) which was related to its iron chelating activity. In a study of three B. cenocepacia sequential clonal isolates obtained from a CF patient over a 3.5 year period, ornibactin upregulation in response to pyoverdine was less pronounced in the last isolate compared to the earlier isolates, as was growth in the presence of haemin and ferritin, indicating alternative iron acquisition mechanism(s) may dominate as chronic infection progresses. These data demonstrate the multifaceted iron acquisition strategies of B. cenocepacia and their capacity to be differentially activated in the presence of P. aeruginosa and during chronic infection.
Keywords: Burkholderia cenocepacia ; Ornibactin; Siderophore; Iron acquisition; Bacterial adaptation; Cystic fibrosis

Isolation and structure determination of new siderophore albachelin from Amycolatopsis alba by Shinya Kodani; Hisayuki Komaki; Masahiro Suzuki; Hikaru Hemmi; Mayumi Ohnishi-Kameyama (381-389).
A new siderophore named albachelin was isolated from iron deficient culture of Amycolatopsis alba. The planar structure of albachelin was elucidated by the combination of ESI–MS/MS experiment and NMR spectroscopic analyses of the gallium (III) complex. The structure of albachelin was determined to be a linear peptide consisting of 6 mol of amino acids including 3 mol of serine, one mol each of N-α-acethyl-N-δ-hydroxy-N-δ-formylornithine, N-α-methyl-N-δ-hydroxyornithine, and cyclic N-hydroxyornithine. The stereochemistries of amino acids constituting albachelin were analyzed by applying modified Marfey method to the hydrolysate of albachelin. Based on bioinformatics, we deduced and discussed the possible biosynthetic gene cluster involved in albachelin biosynthesis from the genome sequence of A. alba. By prediction of substrates for adenylation domains, a non-ribosomal peptide biosynthetase gene (AMYAL_RS0130210) was proposed to be the main biosynthetic gene for albachelin biosynthesis. The related genes including transporter for siderophore were found near the NRPS gene as a gene cluster.
Keywords: Siderophore; Amycolatopsis alba ; Peptide; Biosynthesis

Accumulation of alkaline earth metals by the green macroalga Bryopsis maxima by Shigekazu Takahashi; Kyoko Aizawa; Saki Nakamura; Katsumi Nakayama; Shingo Fujisaki; Soichiro Watanabe; Hiroyuki Satoh (391-400).
Twenty-five days after the disaster at the Fukushima Daiichi nuclear power plant in 2011, we collected samples of the green macroalga Bryopsis maxima from the Pacific coast of Japan. Bryopsis maxima is a unicellular, multinuclear, siphonous green macroalga. Radiation analysis revealed that B. maxima emitted remarkably high gamma radiation of 131I, 134Cs, 137Cs, and 140Ba as fission products of 235U. Interestingly, B. maxima contained naturally occurring radionuclides derived from 226Ra and 228Ra. Analysis of element content revealed that B. maxima accumulates many ocean elements, especially high quantities of the alkaline earth metals Sr (15.9 g per dry-kg) and Ba (3.79 g per dry-kg), whereas Ca content (12.5 g per dry-kg) was lower than that of Sr and only 61 % of the mean content of 70 Japanese seaweed species. Time-course analysis determined the rate of radioactive 85Sr incorporation into thalli to be approximately 0.13 g Sr per dry-kg of thallus per day. Subcellular fractionation of B. maxima cells showed that most of the 85Sr was localized in the soluble fraction, predominantly in the vacuole or cytosol. Given that 85Sr radioactivity was permeable through a dialysis membrane, the 85Sr was considered to be a form of inorganic ion and/or bound with a small molecule. Precipitation analysis with sodium sulfate showed that more than 70 % of the Sr did not precipitate as SrSO4, indicating that a proportion of the Sr may bind with small molecules in B. maxima.
Keywords: Alkaline earth metal; Strontium; Bryopsis maxima ; Nuclear accident; Phytoaccumulation; Ulva arasakii

Role of monovalent and divalent metal cations in human ribokinase catalysis and regulation by Diego Quiroga-Roger; Jorge Babul; Victoria Guixé (401-413).
Human ribokinase (RK) is a member of the ribokinase family, and is the first enzyme responsible for d-ribose metabolism, since d-ribose must first be converted into d-ribose-5-phosphate to be further metabolized and incorporated into ATP or other high energy phosphorylated compounds. Despite its biological importance, RK is poorly characterized in eukaryotes and especially in human. We have conducted a comprehensive study involving catalytic and regulatory features of the human enzyme, focusing on divalent and monovalent metal regulatory effects. Mg2+, Mn2+, and Co2+ support enzyme activity although at different rates, with Mn2+ being the most effective. Analysis of the divalent cation requirement in the wild type enzyme demonstrates that in addition to that chelated by the nucleotide substrate, an activating cation (either Mn2+ or Mg2+) is required to obtain full activity of the enzyme, with the affinity for both divalent cations being almost the same (4 and 8 µM respectively). Besides metal cation activation, inhibition of the enzyme activity by increasing concentrations of Mn2+ but not Mg2+ is observed. Also the role of residues N199 and E202 of the highly conserved NXXE motif present at the active site has been evaluated regarding Mg2+ and phosphate binding. K+ (but not Na+) and PO4 3− activate the wild type enzyme, whereas the N199L and E202L mutants display a dramatic decrease in kcat and require higher free Mg2+ concentrations than the wild type enzyme to reach maximal activity, and the activating effect of PO4 3− is lost. The results demonstrated a complex regulation of the human ribokinase activity where residues Asn199 and Glu202 play an important role.
Keywords: Human ribokinase; Enzyme regulation; Divalent metal cation; Manganese; NXXE motif

Clinical isolates of Candida albicans, Candida tropicalis, and Candida krusei have different susceptibilities to Co(II) and Cu(II) complexes of 1,10-phenanthroline by Amanda E. Hoffman; Leann Miles; Tiffany J. Greenfield; Carolyn Shoen; Michelle DeStefano; Michael Cynamon; Robert P. Doyle (415-423).
The minimal inhibitory concentrations (MICs) of copper and cobalt based dimeric pyrophosphate complexes with capping 1,10-phenanthroline groups on clinical isolates of C. albicans (28 isolates), C. krusei (20 isolates) and C. tropicalis (20 isolates) are reported. C. albicans was inhibited by the cobalt complex better than by the copper complex, while C. krusei demonstrated the opposite results. C. tropicalis showed similar sensitivities to both metals in terms of calculated MIC50 values but was more sensitive to cobalt when MIC90 values were noted. Knockout strains of C. albicans that had the copper efflux protein P-type ATPase (CRP1), the copper binding metallothionein CUP1 or both CRP1/CUP1 removed clearly demonstrate that the origins of copper resistant in C. albicans lies primarily in the P-type ATPase, with the MT playing an important secondary role in the absence of the efflux protein. This study suggests that certain strains of Candida have evolved to protect against particular metal ions and that in the case of C. albicans, a primary invasive fungal species, cobalt may be a good starting-point for new therapeutic development.
Keywords: Candida ; Cobalt; Copper; MIC90 ; MIC50 ; Pyrophosphate

Reactions of cytotoxic metallodrugs with lysozyme in pure DMSO explored through UV–Vis absorption spectroscopy and ESI MS by Tiziano Marzo; Aleksandar Savić; Lara Massai; Elena Michelucci; Tibor J. Sabo; Sanja Grguric-Šipka; Luigi Messori (425-430).
The reactions of four representative metallodrugs with the model protein HEWL were investigated within a non-aqueous environment-i.e. in pure DMSO- through UV–Vis absorption spectroscopy and ESI MS analysis. Notably, formation of a variety of metallodrug-protein adducts was clearly documented. This is the first example for this kind of protein metalation reactions carried out within a pure organic solvent. It is shown that the applied solution conditions greatly affect the nature of the formed adducts, this being well accounted for by the fact that the overall protein conformation is greatly perturbed within pure DMSO; in addition, the activation profiles of the studied metallodrugs are also highly dependent on the nature of the solvent. The implications of these results are discussed.
Keywords: Anticancer complexes; Protein interaction; Lysozyme; ESI-MS; Metallodrugs