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1.  Pentavalent methylated arsenicals are substrates of human AQP9 
Liver aquaglyceroporin AQP9 facilitates movement of trivalent inorganic arsenite (AsIII) and organic monomethylarsonous acid (MAsIII). However, the transport pathway for the two major pentavalent arsenic cellular metabolites, MAsV and DMAsV, remains unknown in mammals. These products of arsenic metabolism, in particular DMAsV, are the major arsenicals excreted in the urine of mammals. In this study, we examined the uptake of the two pentavalent organic arsenicals by human AQP9 in Xenopus laevis oocytes. Xenopus laevis oocytes microinjected with AQP9 cRNA exhibited uptake of both MAsV and DMAsV in a pH-dependent manner. The rate of transport was much higher at acidic pH (pH5.5) than at neutral pH. Hg(II), an aquaporin inhibitor, inhibited transport of AsIII, MAsIII, MAsV and DMAsV via AQP9. However, phloretin, which inhibits water and glycerol permeation via AQP9, can only inhibit transport of pentavalent MAsV and DMAsV but not trivalent AsIII and MAsIII, indicating the translocation mechanisms of these arsenic species are not exactly the same. Reagents such as FCCP, valinomycin and nigericin that dissipate transmembrane proton potential or change the transmemebrane pH gradient did not significantly inhibit all arsenic transport via AQP9, suggesting the transport of pentavalent arsenic is not proton coupled. The results suggest that in addition to the initial uptake of trivalent inorganic AsIII inside cells, AQP9 plays a dual role in the detoxification of arsenic metabolites by facilitating efflux from cells.
PMCID: PMC4266138  PMID: 19802720
AQP9; Liver; Urine; Methylation; Arsenite; Arsenate; Monomethylarsonous acid; Monomethylarsonate; Dimethylarsinate
2.  Boric acid induces cytoplasmic stress granule formation, eIF2α phosphorylation, and ATF4 in prostate DU-145 cells 
Biometals  2014;28:133-141.
Dietary boron intake is associated with reduced prostate and lung cancer risk and increased bone mass. Boron is absorbed and circulated as boric acid (BA) and at physiological concentrations is a reversible competitive inhibitor of cyclic ADP ribose, the endogenous agonist of the ryanodine receptor calcium (Ca+2) channel, and lowers endoplasmic reticulum (ER) [Ca2+]. Low ER [Ca2+] has been reported to induce ER stress and activate the eIF2α/ATF4 pathway. Here we report that treatment of DU-145 prostate cells with physiological levels of BA induces ER stress with the formation of stress granules and mild activation of eIF2α, GRP78/BiP, and ATF4. Mild activation of eIF2α and its downstream transcription factor, ATF4, enables cells to reconfigure gene expression to manage stress conditions and mild activation of ATF4 is also required for the differentiation of osteoblast cells. Our results using physiological levels of boric acid identify the eIF2α/ATF pathway as a plausible mode of action that underpins the reported health effects of dietary boron.
PMCID: PMC4300416  PMID: 25425213
Boron; Boric acid; eIF2α; ATF4; DU-145 cells
3.  Transcriptional regulation of copper metabolism genes in the liver of fetal and neonatal control and iron-deficient rats 
Biometals  2014;28:51-59.
Copper and iron metabolism have been known to interact for many years. We have previously shown, during pregnancy, that copper levels in the maternal liver rise as a consequence of iron deficiency, but that levels in the fetal liver decrease. In this paper, we measure expression of genes involved in copper metabolism in fetal and postnatal liver, to test whether alterations can explain this observation. Additionally, we study the extent to which gene expression changes in the latter stages of pregnancy and in the perinatal period. Ctr1 expression levels dropped to term, rising again thereafter. There was no difference in gene expression between control and iron deficient animals. Atox1 expression remained approximately stable until term, and then there was a rise to a maximum at about Day 8. Atp7a expression levels remained constant, except for a brief drop at term. Atp7b levels, in contrast, decreased from a maximum early in gestation to low levels in the term and post-natal livers. Ceruloplasmin expression appeared to be diametrically opposite to Atp7b. The other two metallochaperones showed the same pattern of expression as Atox1, with a decrease to term, a rise at Day 1, or a rise after birth followed by a brief decrease at about Day 3. None of the genes were significantly affected by iron deficiency, suggesting that changes in expression cannot explain the altered copper levels in the fetal and neonatal liver.
PMCID: PMC4300417  PMID: 25349135
Copper-iron interactions; Perinatal development; Metallochaperones; ATP7A; ATP7B; Copper metabolism; Hooded Lister rats
4.  Impairment of antioxidant defenses as a contributor to arsenite-induced cell transformation 
Arsenite (As) causes transformation of human osteogenic sarcoma cells (HOS) when applied continuously at low doses (0.1–0.5 μM) during 8-weeks of exposure. However, the mechanisms by which As transforms human cells are not known. We investigated whether alterations occurred in gene expression and protein levels of antioxidant defense proteins, such as superoxide dismutase 1 (SOD1) and ferritin. In comparison to control HOS cells, 0.1 μM As induced greater cell proliferation and decreased anti-oxidant defenses. The tumor suppressor protein p53 was also decreased at both mRNA and protein levels. Further, pig3 (p53-induced-gene 3), a homolog of NQO1 (NADPH quinone oxidoreductase 1), was also down-regulated after 8 weeks of As challenge. The treatment of HOS cells with dicumarol, a NQO1 inhibitor, caused a dose-dependent decline in p53 protein levels, proving the effect of an antioxidant enzyme on p53 expression and, potentially, downstream processes. Caffeic acid phenethyl ester, an antioxidant, prevented the As-induced decreases in SOD1, p53, and ferritin mRNA and protein levels. SOD1, p53 and ferritin levels were inversely related to As-induced cell proliferation. Cumulatively, these results strongly suggest that impairment in antioxidant defenses contributes to As-induced human cell transformation and that the p53 pathway is involved in the process.
PMCID: PMC4208698  PMID: 22692362
Arsenite; CAPE; Ferritin; NQO1; p53; SOD1
5.  Conserved metallomics in two insect families evolving separately for a hundred million years 
Biometals  2014;27(6):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.
PMCID: PMC4223573  PMID: 25298233
Physiology; Evolution; Genetics; Nutrition; Fruit flies of economic importance; Agriculture; Mediterranean fruit fly
6.  Zinc pyrithione impairs zinc homeostasis and upregulates stress response gene expression in reconstructed human epidermis 
Zinc ion homeostasis plays an important role in human cutaneous biology where it is involved in epidermal differentiation and barrier function, inflammatory and antimicrobial regulation, and wound healing. Zinc-based compounds designed for topical delivery therefore represent an important class of cutaneous therapeutics. Zinc pyrithione (ZnPT) is an FDA-approved microbicidal agent used worldwide in over-the-counter topical antimicrobials, and has also been examined as an investigational therapeutic targeting psoriasis and UVB-induced epidermal hyperplasia. Recently, we have demonstrated that cultured primary human skin keratinocytes display an exquisite sensitivity to nanomolar ZnPT concentrations causing induction of heat shock response gene expression and poly(ADP-ribose) polymerase (PARP)-dependent cell death (Cell Stress Chaperones 15:309–322, 2010). Here we demonstrate that ZnPT causes rapid accumulation of intracellular zinc in primary keratinocytes as observed by quantitative fluorescence microscopy and inductively coupled plasma mass spectrometry (ICP-MS), and that PARP activation, energy crisis, and genomic impairment are all antagonized by zinc chelation. In epidermal reconstructs (EpiDerm™) exposed to topical ZnPT (0.1–2% in Vanicream™), ICP-MS demonstrated rapid zinc accumulation, and expression array analysis demonstrated upregulation of stress response genes encoding metallothionein-2A (MT2A), heat shock proteins (HSPA6, HSPA1A, HSPB5, HSPA1L, DNAJA1, HSPH1, HSPD1, HSPE1), antioxidants (SOD2, GSTM3, HMOX1), and the cell cycle inhibitor p21 (CDKN1A). IHC analysis of ZnPT-treated EpiDerm™ confirmed upregulation of Hsp70 and TUNEL-positivity. Taken together our data demonstrate that ZnPT impairs zinc ion homeostasis and upregulates stress response gene expression in primary keratinocytes and reconstructed human epidermis, activities that may underlie therapeutic and toxicological effects of this topical drug.
PMCID: PMC4169675  PMID: 21424779
Zinc pyrithione; Keratinocyte; Reconstructed epidermis; Heat shock response; HSPA1A; ICP-MS
7.  Zinc transporter expression profiles in the rat prostate following alterations in dietary zinc 
Zinc plays important roles in numerous cellular activities and physiological functions. Intracellular zinc levels are strictly maintained by zinc homeostatic mechanisms. Zinc concentrations in the prostate are the highest of all soft tissues and could be important for prostate health. However, the mechanisms by which the prostate maintains high zinc levels are still unclear. In addition, the response of the prostate to alterations in dietary zinc is unknown. The current study explored cellular zinc levels and zinc transporter expression profiles in the lobes of the prostate during dietary marginal zinc depletion. Rats were given either zinc-adequate (ZA, 30 mg Zn/kg) or marginal zinc-deficient (MZD, 5 mg Zn/kg) diet for 9 weeks. In addition, a subgroup of the MZD rats was supplemented with phytase (1,500 unit/kg diet) to improve zinc bioavailability. We found that both zinc concentrations and ZnT2 expression in the prostate dorsolateral lobes were substantially higher than in the ventral lobes (P<0.05). Marginal zinc depletion significantly decreased ZnT2 expression in the dorsolateral lobes (P<0.05), and phytase supplementation had a trend to increase ZnT2 expression. In addition, of all measured zinc transporters, only ZnT2 mRNA abundance was significantly correlated to the zinc concentrations in the dorsolateral lobe. No correlations were found between zinc transporter expression and zinc concentrations in the ventral lobes. These results indicate that ZnT2 may play a significant role in the maintenance of zinc homeostasis in the prostate.
PMCID: PMC4152227  PMID: 19760107
Zinc transporter; ZnT2; Prostate; Marginal zinc deficiency
8.  Dietary zinc depletion and repletion affects plasma proteins: an analysis of the plasma proteome 
Zinc (Zn) deficiency is a problem worldwide. Current methods for assessing Zn status are limited to measuring plasma or serum Zn within populations suspected of deficiency. Despite the high prevalence of Zn deficiency in the human population there are no methods currently available for sensitively assessing Zn status among individuals. The purpose of this research was to utilize a proteomic approach using two-dimensional gel electrophoresis (2DE) and mass spectrometry to identify protein biomarkers that were sensitive to changes in dietary Zn levels in humans. Proteomic analysis was performed in human plasma samples (n = 6) obtained from healthy adult male subjects that completed a dietary Zn depletion/repletion protocol, current dietary zinc intake has a greater effect on fractional zinc absorption than does longer term zinc consumption in healthy adult men. Chung et al. (Am J Clin Nutr 87 (5):1224–1229, 2008). After a 13 day Zn acclimatization period where subjects consumed a Zn-adequate diet, the male subjects consumed a marginal Zn-depleted diet for 42 days followed by consumption of a Zn-repleted diet for 28 days. The samples at baseline, end of depletion and end of repletion were pre-fractionated through immuno-affinity columns to remove 14 highly abundant proteins, and each fraction separated by 2DE. Following staining by colloidal Coomassie blue and densitometric analysis, three proteins were identified by mass spectrometry as affected by changes in dietary Zn. Fibrin β and chain E, fragment double D were observed in the plasma protein fraction that remained bound to the immuno-affinity column. An unnamed protein that was related to immunoglobulins was observed in the immunode-pleted plasma fraction. Fibrin β increased two-fold following the Zn depletion period and decreased to baseline values following the Zn repletion period; this protein may serve as a viable biomarker for Zn status in the future.
PMCID: PMC4152233  PMID: 23255060
Fibrin β; Zn biomarker; Marginal Zn deficiency; Dietary Zn depletion and repletion; Two-dimensional gel electrophoresis; Hemostasis
9.  Plasmid- and chromosome-encoded siderophore anguibactin systems found in marine vibrios: biosynthesis, transport and evolution 
Vibrio anguillarum is a marine pathogen that causes vibriosis, a hemorrhagic septicemia in aquatic invertebrate as well as vertebrate animals. The siderophore anguibactin system is one of the most important virulence factors of this bacterium. Most of the anguibactin biosynthesis and transport genes are located in the 65-kb pJM1 virulence plasmid although some of them are found in the chromosome of this fish pathogen. Over 30 years of research unveiled the role numerous chromosomal and pJM1 genes play in the synthesis of anguibactin and the transport of cognate ferric complexes into the bacterial cell. Furthermore, these studies showed that pJM1-carrying strains might be originated from pJM1-less strains producing the chromosome-mediated siderophore vanchrobactin. Additionally, we recently identified a chromosome-mediated anguibactin system in V. harveyi suggesting the possible evolutional origin of the V. anguillarum anguibactin system. In this review, we present our current understanding of the mechanisms and evolution hypothesis of the anguibactin system that might have occurred in these pathogenic vibrios.
PMCID: PMC3747315  PMID: 23660776
Iron transport; Siderophore; Evolution; Anguibactin; Vibrio anguillarum; Vibrio harveyi
10.  Structural Mechanisms of heavy-metal extrusion by the Cus efflux system 
Resistance-nodulation-cell division (RND) superfamily efflux systems are responsible for the active transport of toxic compounds from the Gram-negative bacterial cell. These pumps typically assemble as tripartite complexes, spanning the inner and outer membranes of the cell envelope. In Escherichia coli, the CusC(F)BA complex, which exports copper(I) and silver(I) and mediates resistance to these two metal ions, is the only known RND transporter with a specificity for heavy metals. We have determined the crystal structures of both the inner membrane pump CusA and membrane fusion protein CusB, as well as the adaptor-transporter CusBA complex formed by these two efflux proteins. In addition, the crystal structures of the outer membrane channel CusC and the periplasmic metallochaperone CusF have been resolved. Based on these structures, the entire assembled model of the tripartite efflux system has been developed, and this efflux complex should be in the form of CusC3-CusB6-CusA3. It has been shown that CusA utilizes methionine clusters to bind and export Cu(I) and Ag(I). This pump is likely to undergo a conformational change, and utilize a relay network of methionine clusters as well as conserved charged residues to extrude the metal ions from the bacterial cell.
PMCID: PMC3732547  PMID: 23657864
11.  Ferritin-like family proteins in the anaerobe Bacteroides fragilis: when an oxygen storm is coming, take your iron to the shelter 
Bacteroides are gram-negative anaerobes and one of the most abundant members the lower GI tract microflora where they play an important role in normal intestinal physiology. Disruption of this commensal relationship has a great impact on human health and disease. Bacteroides spp. are significant opportunistic pathogens causing infections when the mucosal barrier integrity is disrupted following predisposing conditions such as GI surgery, perforated or gangrenous appendicitis, perforated ulcer, diverticulitis, trauma and inflammatory bowel diseases. B. fragilis accounts for 60–90 % of all anaerobic infections despite being a minor component of the genus (<1 % of the flora). Clinical strains of B. fragilis are among the most aerotolerant anaerobes. When shifted from anaerobic to aerobic conditions B. fragilis responds to oxidative stress by inducing the expression of an extensive set of genes involved in protection against oxygen derived radicals and iron homeostasis. In Bacteroides, little is known about the metal/oxidative stress interactions and the mobilization of intra-cellular non-heme iron during the oxidative stress response has been largely overlooked. Here we present an overview of the work carried out to demonstrate that both oxygen-detoxifying enzymes and iron-storage proteins are essential for B. fragilis to survive an adverse oxygen-rich environment. Some species of Bacteroides have acquired multiple homologues of the iron storage and detoxifying ferritin-like proteins but some species contain none. The proteins found in Bacteroides are classical mammalian H-type non-heme ferritin (FtnA), non-specific DNA binding and starvation protein (Dps) and the newly characterized bacterial Dps-Like miniferritin protein. The full contribution of ferritin-like proteins to pathophysiology of commensal and opportunistic pathogen Bacteroides spp. still remains to be elucidated.
PMCID: PMC3815548  PMID: 23842847
Bacteroides fragilis ferritin superfamily; Iron-storage and detoxification in anaerobes; Ferritin; Dps; Dps-Like
12.  Utilization of microbial iron assimilation processes for the development of new antibiotics and inspiration for the design of new anticancer agents 
Pathogenic microbes rapidly develop resistance to antibiotics. To keep ahead in the “microbial war”, extensive interdisciplinary research is needed. A primary cause of drug resistance is the overuse of antibiotics that can result in alteration of microbial permeability, alteration of drug target binding sites, induction of enzymes that destroy antibiotics (ie., beta-lactamase) and even induction of efflux mechanisms. A combination of chemical syntheses, microbiological and biochemical studies demonstrate that the known critical dependence of iron assimilation by microbes for growth and virulence can be exploited for the development of new approaches to antibiotic therapy. Iron recognition and active transport relies on the biosyntheses and use of microbe-selective iron-chelating compounds called siderophores. Our studies, and those of others, demonstrate that siderophores and analogs can be used for iron transport-mediated drug delivery (“Trojan Horse” antibiotics) and induction of iron limitation/starvation (Development of new agents to block iron assimilation). Recent extensions of the use of siderophores for the development of novel potent and selective anticancer agents are also described.
PMCID: PMC4066965  PMID: 19130268
Siderophores; Drug conjugates; Antibiotics; Mycobactins; Antituberculosis agents; Anticancer agents
13.  Inhibition of intestinal polyp growth by oral ingestion of bovine lactoferrin and immune cells in the large intestine 
Biometals  2014;27(5):1017-1029.
Studies using animal models have demonstrated that ingestion of bovine lactoferrin (bLF) inhibits carcinogenesis in the colon and other organs of experimental animals. As a result of these studies, a blinded, randomized, controlled clinical trial was conducted in the National Cancer Center Hospital, Tokyo, Japan to determine whether ingestion of bLF had an effect on the growth of colorectal polyps in humans. Patients with colorectal polyps ≤5 mm diameter and likely to be adenomas ingested 0, 1.5, or 3.0 g bLF daily for 1 year. Ingestion of 3.0 g bLF suppressed the growth of colorectal polyps and increased the level of serum human lactoferrin in trial participants 63 years old or younger. The purpose of the present study was to investigate correlations between immune parameters and changes in polyp size. Trial participants with regressing polyps had increased NK cell activity, increased serum hLF levels (indicating increased neutrophil activity), and increased numbers of CD4+ cells in the polyps. These findings are consistent with a correlation between higher immune activity and suppression of colorectal polyps. In addition, participants with regressing polyps had lower numbers of PMNs and increased numbers of S100A8+ cells in the polyps, consistent with a correlation between lower inflammatory potential in the colon and suppression of colorectal polyps. Trial participants ingesting bLF had increased serum hLF levels, a possible increase in systemic NK cell activity, and increased numbers of CD4+ and CD161+ cells in the polyps. Taken together, our findings suggest that bLF suppressed colorectal polyps by enhancing immune responsiveness.
PMCID: PMC4155176  PMID: 24867408
Ingestion of bovine lactoferrin; Immune function; Human intestine; Ancillary study of a human clinical trial
14.  Elevated Transferrin Saturation, Health-Related Quality of Life and Telomere Length 
We sought to examine the relationship between elevated transferrin saturation (TS) and measures of health status (telomere length and patient-reported health-related quality of life) to assess whether elevated TS is associated with negative patient outcomes beyond increased risk for morbidity and mortality, using a cross-sectional analysis of the Hemochromatosis and Iron Overload Screening Study supplemented with assays for leukocyte telomere length in adults ≥25 years old (n=669). Among individuals with elevated TS (≥45% for women and ≥50% for men), who also had a usual source of care, only 5.2% reported ever being told by a doctor that they had an elevated iron condition. In a fully adjusted general linear regression model controlling for demographic characteristics as well as health conditions associated with iron overload, elevated TS versus non-elevated TS was associated with worse general health status (60.4 vs. 63.8, p<0.05), mental health status (76.5 vs. 82.2, p<0.0001) and shorter telomere length (241.4 vs. 261.3, p<0.05). Increased surveillance of elevated TS may be in order as elevated TS is associated with decreased health status and very few patients with elevated TS are aware of their condition.
PMCID: PMC4034347  PMID: 24337410
Iron; telomere length; quality of life; functional status; health status
15.  Novel antisense oligonucleotides containing hydroxamate linkages: targeted iron-triggered chemical nucleases 
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.
PMCID: PMC4030709  PMID: 19184459
Hydroxamic acid; Iron binding; Fenton chemistry; Antisense oligonucleotides; Chemical nucleases
16.  Killing of melanoma cells and their metastases by human lactoferricin derivatives requires interaction with the cancer marker phosphatidylserine 
Biometals  2014;27(5):981-997.
Despite favorable advancements in therapy cancer is still not curative in many cases, which is often due to inadequate specificity for tumor cells. In this study derivatives of a short cationic peptide derived from the human host defense peptide lactoferricin were optimized in their selective toxicity towards cancer cells. We proved that the target of these peptides is the negatively charged membrane lipid phosphatidylserine (PS), specifically exposed on the surface of cancer cells. We have studied the membrane interaction of three peptides namely LF11-322, its N-acyl derivative 6-methyloctanoyl-LF11-322 and its retro repeat derivative R(etro)-DIM-P-LF11-322 with liposomes mimicking cancerous and non-cancerous cell membranes composed of PS and phosphatidylcholine (PC), respectively. Calorimetric and permeability studies showed that N-acylation and even more the repeat derivative of LF11-322 leads to strongly improved interaction with the cancer mimic PS, whereas only the N-acyl derivative also slightly affects PC. Tryptophan fluorescence of selective peptide R-DIM-P-LF11-322 revealed specific peptide penetration into the PS membrane interface and circular dichroism showed change of its secondary structure by increase of proportion of β-sheets just in the presence of the cancer mimic. Data correlated with in vitro studies with cell lines of human melanomas, their metastases and melanocytes, revealing R-DIM-P-LF11-322 to exhibit strongly increased specificity for cancer cells. This indicates the need of high affinity to the target PS, a minimum length and net positive charge, an adequate but moderate hydrophobicity, and capability of adoption of a defined structure exclusively in presence of the target membrane for high antitumor activity.
PMCID: PMC4155172  PMID: 24838743
Peptides; Cancer therapy; Liposomes; Melanoma; Membrane biophysics; Lactoferricin derivatives; Cell membrane permeabilization; Phosphatidylserine
17.  The many “faces” of copper in medicine and treatment 
Biometals  2014;27(4):611-621.
Copper (Cu) is an essential microelement found in all living organisms with the unique ability to adopt two different redox states—in the oxidized (Cu2+) and reduced (Cu+). It is required for survival and serves as an important catalytic cofactor in redox chemistry for proteins that carry out fundamental biological functions, important in growth and development. The deficit of copper can result in impaired energy production, abnormal glucose and cholesterol metabolism, increased oxidative damage, increased tissue iron (Fe) accrual, altered structure and function of circulating blood and immune cells, abnormal neuropeptides synthesis and processing, aberrant cardiac electrophysiology, impaired myocardial contractility, and persistent effects on the neurobehavioral and the immune system. Increased copper level has been found in several disorders like e.g.: Wilson’s disease or Menke’s disease. New findings with the great potential for impact in medicine include the use of copper-lowering therapy for antiangiogenesis, antifibrotic and anti-inflammatory purposes. The role of copper in formation of amyloid plaques in Alzheimer’s disease, and successful treatment of this disorder in rodent model by copper chelating are also of interest. In this work we will try to describe essential aspects of copper in chosen diseases. We will represent the evidence available on adverse effect derived from copper deficiency and copper excess. We will try to review also the copper biomarkers (chosen enzymes) that help reflect the level of copper in the body.
PMCID: PMC4113679  PMID: 24748564
Copper; Wilson’s disease; Menke’s disease; Enzymes; Alzheimer’s disease; Metallothionein; Aceruloplasmin; Copper complexes
18.  New science challenges old notion that mercury dental amalgam is safe 
Biometals  2014;27(1):19-24.
Mercury dental amalgam has a long history of ostensibly safe use despite its continuous release of mercury vapor. Two key studies known as the Children’s Amalgam Trials are widely cited as evidence of safety. However, four recent reanalyses of one of these trials now suggest harm, particularly to boys with common genetic variants. These and other studies suggest that susceptibility to mercury toxicity differs among individuals based on multiple genes, not all of which have been identified. These studies further suggest that the levels of exposure to mercury vapor from dental amalgams may be unsafe for certain subpopulations. Moreover, a simple comparison of typical exposures versus regulatory safety standards suggests that many people receive unsafe exposures. Chronic mercury toxicity is especially insidious because symptoms are variable and nonspecific, diagnostic tests are often misunderstood, and treatments are speculative at best. Throughout the world, efforts are underway to phase down or eliminate the use of mercury dental amalgam.
PMCID: PMC3905169  PMID: 24420334
Mercury; Dental amalgam; Porphyrins; Chronic mercury toxicity
19.  Treatment with d-penicillamine or zinc sulphate affects copper metabolism and improves but not normalizes antioxidant capacity parameters in Wilson disease 
Biometals  2013;27(1):207-215.
Copper accumulation in tissues due to a biallelic pathogenic mutation of the gene: ATP7B results in a clinical phenotype known as Wilson disease (WD). Aberrations in copper homeostasis can create favourable conditions for superoxide-yielding redox cycling and oxidative tissue damage. Drugs used in WD treatment aim to remove accumulated copper and normalise the free copper concentration in the blood. In the current study the effect of decoppering treatment on copper metabolism and systemic antioxidant capacity parameters was analyzed. Treatment naïve WD patients (TNWD) (n = 33), those treated with anti-copper drugs (TWD) (n = 99), and healthy controls (n = 99) were studied. Both TNWD and TWD patients characterised with decreased copper metabolism parameters, as well as decreased total antioxidant potential (AOP), glutathione (GSH) level, activity of catalase, glutathione peroxidase (GPx), and S-transferase glutathione, compared to controls. TWD patients had significantly lower copper metabolism parameters, higher total AOP and higher levels of GSH than TWD individuals; however, no difference was observed between these two patient groups with respect to the rest of the antioxidant capacity parameters. Patients who had undergone treatment with d-penicillamine or zinc sulphate did not differ with respect to copper metabolism or antioxidant capacity parameters, with the exception of GPx that was lower in d-penicillamine treated individuals. These data suggest that anti-copper treatment affects copper metabolism as well as improves, but does not normalize, natural antioxidant capacity in patients with WD. We propose to undertake studies aimed to evaluate the usefulness of antioxidants as well as selenium as a supplemental therapy in WD.
PMCID: PMC3905172  PMID: 24368744
Antioxidants; Copper; d-Penicillamine; Glutathione peroxidase; Oxidative stress; Treatment; Wilson disease; Zinc sulphate
20.  Competition of zinc ion for the [2Fe–2S] cluster binding site in the diabetes drug target protein mitoNEET 
Human mitochondrial protein mitoNEET is a novel target of type II diabetes drug pioglitazone, and contains a redox active [2Fe–2S] cluster that is hosted by a unique ligand arrangement of three cysteine and one histidine residues. Here we report that zinc ion can compete for the [2Fe–2S] cluster binding site in human mitoNEET and potentially modulate the physiological function of mitoNEET. When recombinant mitoNEET is expressed in Escherichia coli cells grown in M9 minimal media, purified mitoNEET contains very little or no iron–sulfur clusters. Addition of exogenous iron or zinc ion in the media produces mitoNEET bound with a [2Fe–2S] cluster or zinc, respectively. Mutations of the amino acid residues that hosting the [2Fe–2S] cluster in mitoNEET diminish the zinc binding activity, indicating that zinc ion and the [2Fe–2S] cluster may share the same binding site in mitoNEET. Finally, excess zinc ion effectively inhibits the [2Fe–2S] cluster assembly in mitoNEET in E. coli cells, suggesting that zinc ion may impede the function of mitoNEET by blocking the [2Fe–2S] cluster assembly in the protein.
PMCID: PMC3822609  PMID: 22945239
Human mitoNEET; Type II diabetes drug pioglitazone; Iron–sulfur cluster; Zinc binding
21.  Different iron sources to study the physiology and biochemistry of iron metabolism in marine micro-algae 
Biometals  2013;27(1):75-88.
We compared ferric EDTA, ferric citrate and ferrous ascorbate as iron sources to study iron metabolism in Ostreococcus tauri, Phaeodactlylum tricornutum and Emiliania huxleyi. Ferric EDTA was a better iron source than ferric citrate for growth and chlorophyll levels. Direct and indirect experiments showed that iron was much more available to the cells when provided as ferric citrate as compared to ferric EDTA. As a consequence, growth media with iron concentration in the range 1–100 nM were rapidly iron-depleted when ferric citrate—but not ferric EDTA was the iron source. When cultured together, P. tricornutum cells overgrew the two other species in iron-sufficient conditions, but E. huxleyi was able to compete other species in iron-deficient conditions, and when iron was provided as ferric citrate instead of ferric EDTA, which points out the critical influence of the chemical form of iron on the blooms of some phytoplankton species. The use of ferric citrate and ferrous ascorbate allowed us to unravel a kind of regulation of iron uptake that was dependent on the day/night cycles and to evidence independent uptake systems for ferrous and ferric iron, which can be regulated independently and be copper-dependent or independent. The same iron sources also allowed one to identify molecular components involved in iron uptake and storage in marine micro-algae. Characterizing the mechanisms of iron metabolism in the phytoplankton constitutes a big challenge; we show here that the use of iron sources more readily available to the cells than ferric EDTA is critical for this task.
Electronic supplementary material
The online version of this article (doi:10.1007/s10534-013-9688-1) contains supplementary material, which is available to authorized users.
PMCID: PMC3905174  PMID: 24281777
Iron; Marine micro-algae; Ferric citrate; Ferric EDTA; Ostreococcus; Phaeodactylum; Emiliania
22.  The ArsD As(III) metallochaperone 
Arsenic, a toxic metalloid widely existing in the environment, causes a variety of health problems. The ars operon encoded by Escherichia coli plasmid R773 has arsD and arsA genes, where ArsA is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump, and ArsD is an arsenic chaperone for ArsA. ArsD transfers As(III) to ArsA and increases the affinity of ArsA for As(III), allowing resistance to environmental concentrations of arsenic. Cys12, Cys13 and Cys18 in ArsD form a three sulfur-coordinated As(III) binding site that is essential for metallochaperone activity. ATP hydrolysis by ArsA is required for transfer of As(III) from ArsD to ArsA, suggesting that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle. The 1.4 Å x-ray crystal structure of ArsD shows a core of four β-strands flanked by four α-helices in a thioredoxin fold. Docking of ArsD with ArsA was modeled in silico. Independently ArsD mutants exhibiting either weaker or stronger interaction with ArsA were selected. The locations of the mutations mapped on the surface of ArsD are consistent with the docking model. The results suggest that the interface with ArsA involves one surface of α1 helix and metalloid binding site of ArsD.
PMCID: PMC3773603  PMID: 21188475
Arsenic; ArsD; Metallochaperone; ArsA; ATP-driven efflux pump
23.  Identification, isolation, and analysis of a gene cluster involved in iron acquisition by Pseudomonas mendocina ymp 
Microbial acquisition of iron from natural sources in aerobic environments is a little-studied process that may lead to mineral instability and trace metal mobilization. Pseudomonas mendocina ymp was isolated from the Yucca Mountain Site for long-term nuclear waste storage. Its ability to solubilize a variety of Fe-containing minerals under aerobic conditions has been previously investigated but its molecular and genetic potential remained uncharacterized. Here, we have shown that the organism produces a hydroxamate and not a catecholate-based siderophore that is synthesized via non-ribosomal peptide synthetases. Gene clustering patterns observed in other Pseudomonads suggested that hybridizing multiple probes to the same library could allow for the identification of one or more clusters of syntenic siderophore-associated genes. Using this approach, two independent clusters were identified. An unfinished draft genome sequence of P. mendocina ymp indicated that these mapped to two independent contigs. The sequenced clusters were investigated informatically and shown to contain respectively a potentially complete set of genes responsible for siderophore biosynthesis, uptake, and regulation, and an incomplete set of genes with low individual homology to siderophore-associated genes. A mutation in the cluster’s pvdA homolog (pmhA) resulted in a siderophore-null phenotype, which could be reversed by complementation. The organism likely produces one siderophore with possibly different isoforms and a peptide backbone structure containing seven residues (predicted sequence: Acyl-Asp-Dab-Ser-fOHOrn-Ser-fOHorn). A similar approach could be applied for discovery of Fe− and siderophore-associated genes in unsequenced or poorly annotated organisms.
PMCID: PMC3755509  PMID: 18058194
Siderophore; Iron; Bacteria; Mineral
24.  Intestinal expression of metal transporters in Wilson’s disease 
Biometals  2013;26(6):925-934.
In Wilson’s disease (WND), biallelic ATP7B gene mutation is responsible for pathological copper accumulation in the liver, brain and other organs. It has been proposed that copper transporter 1 (CTR1) and the divalent metal transporter 1 (DMT1) translocate copper across the human intestinal epithelium, while Cu-ATPases: ATP7A and ATP7B serve as copper efflux pumps. In this study, we investigated the expression of CTR1, DMT1 and ATP7A in the intestines of both WND patients and healthy controls to examine whether any adaptive mechanisms to systemic copper overload function in the enterocytes. Duodenal biopsy samples were taken from 108 patients with Wilson’s disease and from 90 controls. CTR1, DMT1, ATP7A and ATP7B expression was assessed by polymerase chain reaction and Western blot. Duodenal CTR1 mRNA and protein expression was decreased in WND patients in comparison to control subjects, while ATP7A mRNA and protein production was increased. The variable expression of copper transporters may serve as a defense mechanism against systemic copper overload resulting from functional impairment of ATP7B.
PMCID: PMC3825560  PMID: 23963605
Wilson’s disease; Metal transporters; Intestine; Copper; ATP7A; DMT1; CTR1; ATP7B
25.  Iron trafficking as an antimicrobial target 
Iron is essential for the survival of most organisms. Microbial iron acquisition depends on multiple, sometimes complex steps, many of which are not shared by higher eukaryotes. Depriving pathogenic microbes of iron is therefore a potential antimicrobial strategy. The following minireview briefly describes general elements in microbial iron uptake pathways and summarizes some of the current work aiming at their medicinal inhibition.
PMCID: PMC3742301  PMID: 19350396
Iron; Antibiotic; Siderophore

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