BioMetals (v.25, #1)

ZnT3: a zinc transporter active in several organs by Kamille Smidt; Jørgen Rungby (1-8).
The review collects the emerging information about zinc transporter 3 (ZnT3). ZnT3 has been associated with Alzheimer’s disease, airway diseases and diabetes. ZnT3 was discovered and cloned in 1996. Since then, the major interest in the protein has been in its ability to transport zinc into pre-synaptic vesicles of glutamatergic neurones and its role during the development of amyloid β plaques in Alzheimer’s disease. Increasing evidence suggests that ZnT3 is present in various cell types like different cell types in the brain, cells from adipose tissue, beta-cells from pancreatic islets, epithelial cells, cells from testis, prostate cancer cells and cells from retina. The expression of ZnT3 is regulated by age, hormones, fatty acids, zinc chelation, and glucose.
Keywords: Slc30A3; ZnT3; Zinc transport; Apoptosis; Alzheimer’s disease; Diabetes

Perspective: the potential of pyrazole-based compounds in medicine by Frankline K. Keter; James Darkwa (9-21).
Pyrazoles are widely used as core motifs for a large number of compounds for various applications such as catalysis, agro-chemicals, building blocks of other compounds and in medicine. The attractiveness of pyrazole and its derivatives is their versatility that allows for synthesis of a series of analogues with different moieties in them, thus affecting the electronics and by extension the properties of the resultant compounds. In medicine pyrazole is found as a pharmacophore in some of the active biological molecules. While pyrazole derivatives have been extensively studied for many applications including anticancer, antimicrobial, anti-inflammatory, antiglycemic, anti-allergy and antiviral, much less has been reported on their metal counterparts in spite of the fact that metals have been shown to impart activity to ligands. Thus this perspective is intended to demonstrate the potential of pyrazole and pyrazolyl metal complexes in the areas of drug discovery and development. Several examples, that include palladium, platinum, copper, gold, zinc, cobalt, nickel, iron, copper, silver and gallium complexes, are used to bolster the above point. For the purposes of this review three areas are discussed, that is pyrazole metal complexes as: (i) anticancer, (ii) antibacterial/parasitic and (iii) antiviral agents.
Keywords: Pyrazole; Metal complexes; Anticancer; Antibacterial; Antiparasitic; Antiviral

In this study, the effects of cadmium (Cd) on cell morphology and antioxidant enzyme activities as well as the distribution of the metal in different cell compartments in Bradyrhizobium sp. strains were investigated. These strains were previously classified as sensitive (Bradyrhizobium sp. SEMIA 6144) and tolerant (Bradyrhizobium sp. NLH25) to Cd. Transmission electron micrographs showed large electron-translucent inclusions in the sensitive strain and electron-dense bodies in the tolerant strain, when exposed to Cd. Analysis of Cd distribution revealed that it was mainly bounded to cell wall in both strains. Antioxidant enzyme activities were significantly different in each strain. Only the tolerant strain was able to maintain a glutathione/oxidized glutathione (GSH/GSSG) ratio by an increase of GSH reductase (GR) and GSH peroxidase (GPX) enzyme activities. GSH S-transferase (GST) and catalase (CAT) activities were drastically inhibited in both strains while superoxide dismutase (SOD) showed a significant decrease only in the sensitive strain. In conclusion, our findings suggest that GSH content and its related enzymes are involved in the Bradyrhizobium sp. tolerance to Cd contributing to the cellular redox balance.
Keywords: Antioxidant enzymes; Bradyrhizobium sp.; Cadmium; Glutathione; Tolerance

The pathway of copper entry into Escherichia coli is still unknown. In an attempt to shed light on this process, a lux-based biosensor was utilized to monitor intracellular copper levels in situ. From a transposon-mutagenized library, strains were selected in which copper entry into cells was reduced, apparent as clones with reduced luminescence when grown in the presence of copper (low-glowers). One low-glower had a transposon insertion in the comR gene, which encodes a TetR-like transcriptional regulator. The mutant strain could be complemented by the comR gene on a plasmid, restoring luminescence to wild-type levels. ComR did not regulate its own expression, but was required for copper-induction of the neighboring, divergently transcribed comC gene, as shown by real-time quantitative PCR and with a promoter-lux fusion. The purified ComR regulator bound to the promoter region of the comC gene in vitro and was released by copper. By membrane fractionation, ComC was shown to be localized in the outer membrane. When grown in the presence of copper, ∆comC cells had higher periplasmic and cytoplasmic copper levels, compared to the wild-type, as assessed by the activation of the periplasmic CusRS sensor and the cytoplasmic CueR sensor, respectively. Thus, ComC is an outer membrane protein which lowers the permeability of the outer membrane to copper. The expression of ComC is controlled by ComR, a novel, TetR-like copper-responsive repressor.
Keywords: Copper homeostasis; Outer membrane protein; Copper regulator; Biosensor

Role of reactive oxygen species in the antibacterial mechanism of silver nanoparticles on Escherichia coli O157:H7 by Hengyi Xu; Feng Qu; Hong Xu; Weihua Lai; Y. Andrew Wang; Zoraida P. Aguilar; Hua Wei (45-53).
In this study, the conditions and mechanism of antibacterial activity of hydrophilic polymer coated silver nanoparticles (AgNPs) against E. coli O157:H7 (CMCC44828) as model pathogen was studied. The AgNPs were coated with amphiphilic polymer that introduced carboxyl groups on the surface to make it water-soluble. The AgNPs were exposed to various treatment conditions of pH and temperature before these were combined with the E. coli. The mechanism of the antibacterial activity was studied through the formation of reactive oxygen species (ROS) that was later suppressed with antioxidant to establish correlation with the AgNPs antimicrobial activity. Studies were carried out at both anaerobic and aerobic conditions. The results indicated that 5 mg/L AgNPs inhibited ~50% of the growth of 106 colony forming units per milliliter (cfu/mL) E. coli cells in liquid Luria–Bertani (LB) medium. This dose-dependent antimicrobial activity was higher at increased temperature (37°C) but was lower when the AgNPs were treated with acid at pH 2 before exposure to the bacteria. It was also established that the conditions of higher antimicrobial effect generated more ROS that was dependent on the presence of oxygen. The antibacterial activity was suppressed in the presence of an antioxidant.
Keywords: Silver; Nanoparticle; Antibacterial mechanism; Reactive oxygen species; Escherichia coli

Coordination of lapachol to bismuth(III) improves its anti-inflammatory and anti-angiogenic activities by Gabrieli L. Parrilha; Rafael P. Vieira; Paula P. Campos; Grácia Divina F. Silva; Lucienir P. Duarte; Silvia P. Andrade; Heloisa Beraldo (55-62).
Complex [Bi(Lp)2]Cl was obtained with 4-hydroxy-3-(3-methylbut-2-enyl)naphthalene-1,2-dione, “lapachol” (HLp). Lapachol, [Bi(Lp)2]Cl and BiCl3 were evaluated in a murine model of inflammatory angiogenesis induced by subcutaneous implantation of polyether polyurethane sponge discs. Intraperitoneal (i.p.) administration of lapachol or [Bi(Lp)2]Cl reduced the hemoglobin content in the implants suggesting that reduction of neo-vascularization was caused by lapachol. In the per os treatment only [Bi(Lp)2]Cl decreased the hemoglobin content in the implants. Likewise, N-acetylglucosaminidase (NAG) activity decreased in the implants of the groups i.p. treated with lapachol and [Bi(Lp)2]Cl while in the per os treatment inhibition was observed only for [Bi(Lp)2]Cl. Histological analysis showed that the components of the fibro-vascular tissue (vascularization and inflammatory cell population) were decreased in lapachol- and complex-treated groups. Our results suggest that both lapachol and [Bi(Lp)2]Cl exhibit anti-angiogenic and anti-inflammatory activities which have been attributed to the presence of the lapachol ligand. However, coordination to bismuth(III) could be an interesting strategy for improvement of lapachol’s therapeutic properties.
Keywords: Lapachol; Bismuth(III) complex; Sponge implants; Angiogenesis; Inflammation

Cadmium induces alterations in the human spinal cord morphogenesis by Erica Sarchielli; Stefania Pacini; Gabriele Morucci; Tiziana Punzi; Mirca Marini; Gabriella B. Vannelli; Massimo Gulisano (63-74).
The effects of cadmium on the central nervous system are still relatively poorly understood and its role in neurodegenerative diseases has been debated. In our research, cultured explants from 25 human foetal spinal cords (10–11 weeks gestational age) were incubated with 10 and 100 μM cadmium chloride (CdCl2) for 24 h. After treatment, an immunohistochemical study [for Sglial fibrillary acidic protein (GFAP) and choline acetyltransferase (ChAT)], a Western blot analysis (for GFAP, β-Tubulin III, nerve growth factor receptor, Caspase 8 and poly (ADP-ribose) polymerase), and a terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) assay (for detection of apoptotic bodies) were performed. The treatment with CdCl2 induced a significant and dose-dependent change in the ratio motor neurons/glial cells in the ventral horns of human foetal spinal cord. The decrease of the choline acetyltransferase-positive cells (motor neurons) and the reduction of β Tubulin III indicate that CdCl2 specifically affects motor neurons of the ventral horns. While the number of motor neurons decreased for the activation of apoptotic pathways (as shown by the increased expression of Caspase 8, nerve growth factor receptor, and poly (ADP-ribose) polymerase), glial cells, both in the subependymal zone and in the gray matter of the ventral horns, increased (as shown by the increase of GFAP expression). These results provide the evidence that during human spinal cord development, CdCl2 may affect the fate of neural and glial cells thus, being potentially involved in the etiopathogenesis of neurodegenerative diseases.
Keywords: Neurodegenerative diseases; Human spinal cord; Cadmium

Genome wide identification of Acidithiobacillus ferrooxidans (ATCC 23270) transcription factors and comparative analysis of ArsR and MerR metal regulators by Christian Hödar; Pablo Moreno; Alex di Genova; Mauricio Latorre; Angélica Reyes-Jara; Alejandro Maass; Mauricio González; Verónica Cambiazo (75-93).
Acidithiobacillus ferrooxidans is a chemolithoautotrophic acidophilic bacterium that obtains its energy from the oxidation of ferrous iron, elemental sulfur, or reduced sulfur minerals. This capability makes it of great industrial importance due to its applications in biomining. During the industrial processes, A. ferrooxidans survives to stressing circumstances in its environment, such as an extremely acidic pH and high concentration of transition metals. In order to gain insight into the organization of A. ferrooxidans regulatory networks and to provide a framework for further studies in bacterial growth under extreme conditions, we applied a genome-wide annotation procedure to identify 87 A. ferrooxidans transcription factors. We classified them into 19 families that were conserved among diverse prokaryotic phyla. Our annotation procedure revealed that A. ferrooxidans genome contains several members of the ArsR and MerR families, which are involved in metal resistance and detoxification. Analysis of their sequences revealed known and potentially new mechanism to coordinate gene-expression in response to metal availability. A. ferrooxidans inhabit some of the most metal-rich environments known, thus transcription factors identified here seem to be good candidates for functional studies in order to determine their physiological roles and to place them into A. ferrooxidans transcriptional regulatory networks.
Keywords: Acidithiobacillus ferrooxidans ; Transcriptional regulators; Metal resistance; ArsR family; MerR family

Helicobacter pylori causes various gastric diseases, such as gastritis, peptic ulcerations and gastric cancer. Triple therapy combining bismuth compounds with two antibiotics is the cornerstone of the treatment of H. pylori infections. Up to now, the molecular mechanisms by which bismuth inhibits the growth of H. pylori are far from clear. In the bacterial tricarboxylic acid (TCA) cycle, fumarase catalyses the reversible hydration of fumarate to malic acid. Our previous proteomic work indicated that fumarase was capable of bismuth-binding. The interactions as well as the inhibitory effects of bismuth to fumarase have been characterized in this study. The titration of bismuth showed that each fumarase monomer binds one mol equiv of Bi3+, with negligible secondary structural change. Bismuth-binding results in a near stoichiometric inactivation of the enzyme, leading to an apparent non-competitive mechanism as reflected by the Lineweaver–Burk plots. Our collective data indicate that the TCA cycle is a potential molecular target of bismuth drugs in H. pylori.
Keywords: Helicobacter pylori ; Fumarase; Bismuth; Tricarboxylic acid cycle

As2O3 oxidation by vitamin C: cell culture studies by Andreja Zelenik Pevec; Zdenka Šlejkovec; Johannes T. van Elteren; Ingrid Falnoga (103-113).
The ability of As2O3 to induce apoptosis in various malignant cell lines has made it a potential treatment agent for several malignancies. In this study the chemical stability of As2O3 (As(III)) in cell-free growth media with various compositions was studied (MEM with different amount of amino acids and DMEM). Special attention was given to evaluate the influence of serum (FBS; fetal bovine serum) absence and vitamin C addition on the oxidation of As(III) to As(V) in cell-free growth media. FBS is an important source of antioxidants and vitamin C (ascorbic acid) is acting as a prooxidant in millimolar concentrations. Media were incubated with As(III) (0.6, 2 and 7 μmol l−1) up to 72 h. Experiments were performed at 37°C in light or/and in the dark, with or without added serum (10%) or vitamin C (1.4, 0.14 mM). Metabolites were followed with high-performance liquid chromatography directly coupled to a hydride generation-atomic fluorescence spectrometry system. After 72 h up to 30% of As(III) was transformed into As(V) in MEMs and up to 35% in DMEM when exposed in dark. Light had no influence on transformations in MEMs, but changed the situation dramatically in DMEM where almost all As(III) was oxidized to As(V) after 72 h when exposed to light. Except for some faster oxidation rate the absence of FBS had little effect on the transformation rate in all media. The most visible impact on As(III) oxidation was observed by addition of vitamin C. Addition of vitamin C (1.4 mM) transformed almost all As(III) to As(V) within 72 h. In lower concentrations (0.14 mM) a pro-oxidative effect was still observed reaching approximately 60% oxidation of As(III) during 72 h. All oxidation processes could be explained by pseudo first order reaction kinetics, yielding reaction rates increasing with initial As(III) concentration and vitamin C concentration whereas the FBS content additionally increased the As(III) oxidation rate in the DMEM (light). The temporal oxidation of As(III) to As(V) in various cell-free growth media necessitates routine checking of the valence state of arsenic during cell culture experiments and the results of biological effects attributed to As(III) should be interpreted with caution. Special attention is needed particularly in cases with vitamin C which was acting pro-oxidatively in all conditions examined.
Keywords: Arsenic trioxide; Vitamin C; Serum; Oxidation; Cell culture

The mammalian ZIP (Zrt-, Irt-like Protein) family of transmembrane transport proteins consists of 14 members that share considerable homology. ZIP proteins have been shown to mediate the cellular uptake of the essential trace elements zinc, iron, and manganese. The aim of the present study was to determine the effect of dietary iron deficiency and overload on the expression of all 14 ZIP transporters in the liver, the main site of iron storage. Weanling male rats (n = 6/group) were fed iron-deficient (FeD), iron-adequate (FeA), or iron-overloaded (FeO) diets in two independent feeding studies. In study 1, diets were based on the TestDiet 5755 formulation and contained iron at 9 ppm (FeD), 215 ppm (FeA), and 27,974 ppm (3% FeO). In study 2, diets were based on the AIN-93G formulation and contained iron at 9 ppm Fe (FeD), 50 ppm Fe (FeA), or 18916 ppm (2% FeO). After 3 weeks, the FeD diets depleted liver non-heme iron stores and induced anemia, whereas FeO diets resulted in hepatic iron overload. Quantitative RT-PCR revealed that ZIP5 mRNA levels were 3- and 8-fold higher in 2% FeO and 3% FeO livers, respectively, compared with FeA controls. In both studies, a consistent downregulation of ZIP6, ZIP7, and ZIP10 was also observed in FeO liver relative to FeA controls. Studies in H4IIE hepatoma cells further documented that iron loading affects the expression of these ZIP transporters. Overall, our data suggest that ZIP5, ZIP6, ZIP7, and ZIP10 are regulated by iron, indicating that they may play a role in hepatic iron/metal homeostasis during iron deficiency and overload.
Keywords: SLC39; Iron deficient; Iron overload; Zinc

In this work we demonstrate the existence in Vibrio anguillarum 775 (pJM1) of two chromosomal genes encoding outer membrane proteins that operate in the transport of ferric enterobactin. One of them is a novel receptor that we named FetA and the other is the already characterized FvtA that functions in the uptake of iron complexes of both enterobactin and vanchrobactin. Ferric enterobactin transport proficiency was resumed in double mutants for these two genes when they were complemented with either fetA or fvtA, whereas only the cloned fvtA could complement for ferric vanchrobactin transport. Quantitative RT-PCR assays demonstrated that transcription of the fetA gene is regulated by FetR, that is encoded upstream and in reverse orientation from fetA. This gene as well as fetA, are up-regulated in iron limiting condition in a Fur-dependent manner. The two divergent promoters are located in the intergenic region between fetR and fetA that has a putative Fur binding site and an IrgB binding site in the overlapping promoters of fetR and fetA. FetA and FetR show high homology to V. cholerae IrgA and IrgB respectively and the intergenic regions fetAfetR and irgAirgB are also highly related suggesting a vertical transmission of the fetA–fetR cluster from V. cholerae to V. anguillarum.
Keywords: Iron; Siderophore; Enterobactin; Outer membrane receptor

Iron transport in the genus Marinobacter by Shady A. Amin; David H. Green; Dhuha Al Waheeb; Astrid Gärdes; Carl J. Carrano (135-147).
Marinobacter belong to the class of Gammaproteobacteria and these motile, halophilic or halotolerent bacteria are widely distributed throughout the world’s oceans having been isolated from a wide variety of marine environments. They have also been identified as members of the bacterial flora associated with other marine organisms. Here, using a combination of natural products chemistry and genomic analysis, we assess the nature of the siderophores produced by this genus and their potential relationship to phylogeny and lifestyle/ecological niche of this diverse group of organisms. Our analysis shows a wide level of diversity in siderophore based iron uptake systems among this genus with three general strategies: (1) production and utilization of native siderophores in addition to utilization of a variety of exogenous ones, (2) production and utilization of native siderophores only, (3) lack of siderophore production but utilization of exogenous ones. They all share the presence of at least one siderophore-independent iron uptake ABC transport systems of the FbpABC iron metal type and lack the ability for direct transport of ferrous iron. Siderophore production and utilization can be correlated with phylogeny and thus it forms a type of chemotaxonomic marker for this genus.
Keywords: Iron; Transport; Siderophores; Marinobacter

Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo by Avishek Ganguly; Paramita Chakraborty; Kaushik Banerjee; Shilpak Chatterjee; Soumya Basu; Avijit Sarkar; Mitali Chatterjee; Soumitra Kumar Choudhuri (149-163).
Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.
Keywords: Multi drug resistance; Glutathione; Iron complex; Resistance modifying agent

Effects of dietary methylmercury on the zebrafish brain: histological, mitochondrial, and gene transcription analyses by Sébastien Cambier; Patrice Gonzalez; Nathalie Mesmer-Dudons; Daniel Brèthes; Masatake Fujimura; Jean-Paul Bourdineaud (165-180).
The neurotoxic compound methylmercury (MeHg) is a commonly encountered pollutant in the environment, and constitutes a hazard for wildlife and human health through fish consumption. To study the neurotoxic impact of MeHg on piscivorous fish, we contaminated the model fish species Danio rerio for 25 and 50 days with food containing 13.5 μg/g dry weight (dw) of MeHg (0.6 μg MeHg/fish/day), an environmentally relevant dose leading to brain mercury concentrations of 30 ± 4 μg of Hg g−1 (dw) after 25 days of exposure and 46 ± 7 μg of Hg g−1 (dw) after 50 days. Brain mitochondrial respiration was not modified by exposure to MeHg, contrary to what happens in skeletal muscles. A 6-fold increase in the expression of the sdh gene encoding the succinate dehydrogenase Fe/S protein subunit was detected in the contaminated brain after 50 days of exposure. An up regulation of 3 genes, atp2b3a, atp2b3b, and slc8a2b, encoding for calcium transporters was noticed after 25 days of exposure but the atp2b3a and atp2b3b were repressed and the slc8a2b gene expression returned to its basal level after 50 days, suggesting a perturbation of calcium homeostasis. After 50 days, we detected the up regulation of glial fibrillary acidic protein and glutathione S-transferase genes (gfap and gst), along with a repression of the glutathione peroxidase gene gpx1. These results match well with a MeHg-induced onset of oxidative stress and inflammation. A transmission electron microscopic observation confirmed an impairment of the optical tectum integrity, with a decrease of the nucleal area in contaminated granular cells compared to control cells, and a lower density of cells in the contaminated tissue. A potential functional significance of such changes observed in optical tectum when considering wild fish contaminated in their natural habitat might be an impaired vision and therefore a lowered adaptability to their environment.
Keywords: Methylmercury; Dietary exposure; Zebrafish; Bioenergetics; Brain

Siderophore-mediated iron uptake in two clades of Marinobacter spp. associated with phytoplankton: the role of light by Shady A. Amin; David H. Green; Astrid Gärdes; Ariel Romano; Lyndsay Trimble; Carl J. Carrano (181-192).
Iron is an essential element for oceanic microbial life but its low bioavailability limits microorganisms in large areas of the oceans. To acquire this metal many marine bacteria produce organic chelates that bind and transport iron (siderophores). We have previously shown that algal-associated heterotrophic bacteria belonging to the γ-proteobacterial Marinobacter genus release the siderophore vibrioferrin (VF). The iron-VF complex was shown to be both far more photolabile than all previously examined photolabile siderophores and to generate a photoproduct incapable of re-chelating the released iron. Thus, the photo-generated iron was shown to be highly bioavailable both to the producing bacterium and its algal partner. In exchange, we proposed that algal cells produced dissolved organic matter that helped support bacterial growth and ultimately fueled the biosynthesis of VF through a light-dependent “carbon for iron mutualism”. While our knowledge of the importance of light to phototrophs is vast, there are almost no studies that examine the effects of light on microbial heterotrophs. Here, we characterize iron uptake mechanisms in “algal-associated” VF-producers. Fe uptake by a VF knock-out mutant mimics the wild-type strain and demonstrates the versatility of iron uptake mechanisms in Marinobacter VF-producers. We also show that VF-producers selectively regulate a subset of their siderophore-dependent iron uptake genes in response to light exposure. The regulation of iron uptake and transport genes by light is consistent with the light driven algal–bacterial “carbon for iron mutualism” hypothesis in the marine environment.
Keywords: Iron; Transport; Marinobacter ; Light; Gene expression; Siderophores

Ferritin is not only important for iron storage and detoxification in living organisms, but a multifunctional size-constrained nanoplatform for biomimetic nanoparticles. In order to tailor the biomimetic nanoparticles for future applications, it is essential to investigate the effects of external factors such as temperature on the particle size and structure of reconstituted cores in ferritin. In this study, we systematically investigated the mineral composition, crystallinity, and particle size of human H-ferritin (HuHF) reconstituted at four different temperatures (25, 30, 37, and 42°C) by integrated magnetic and transmission electron microscopy analyses. Our results showed that the particle size of reconstituted ferrihydrite cores (~5 nm) in HuHF was temperature-independent. However, the significant changes of the induced magnetization at 5 T field (M5T) and remanent magnetization (Mr) at 5 K clearly showed that the crystallinity of reconstituted cores increased with increasing temperature, indicating that the reaction temperature deeply affects the structural order of reconstituted ferrihydrite cores rather than the particle size, and the reconstituted cores become more ordered at higher reaction temperatures. Our findings provide useful insights into biomineralization of ferritin under in vivo fever condition as well as in biomimetic synthesis of nanomaterials using ferritin. Furthermore, the rock magnetic methods should be very useful approaches for characterizing finite ferritin nanoparticles.
Keywords: HuHF; Biomineralization; Biomimetic synthesis; Temperature; Particle size; Degree of crystallinity

Iron-induced remodeling in cultured rat pulmonary artery endothelial cells by Nikolai V. Gorbunov; James L. Atkins; Narasimman Gurusamy; Bruce R. Pitt (203-217).
Although iron is known to be a component of the pathogenesis and/or maintenance of acute lung injury (ALI) in experimental animals and human subjects, the majority of these studies have focused on disturbances in iron homeostasis in the airways resulting from exposure to noxious gases and particles. Considerably less is known about the effect of increased plasma levels of redox-reactive non-transferrin bound iron (NTBI) and its impact on pulmonary endothelium. Plasma levels of NTBI can increase under various pathophysiological conditions, including those associated with ALI, and multiple mechanisms are in place to affect the [Fe2+]/[Fe3+] redox steady state. It is well accepted, however, that intracellular transport of NTBI occurs after reduction of [Fe3+] to [Fe2+] (and is mediated by divalent metal transporters). Accordingly, as an experimental model to investigate mechanisms mediating vascular effects of redox reactive iron, rat pulmonary artery endothelial cells (RPAECs) were subjected to pulse treatment (10 min) with [Fe2+] nitriloacetate (30 μM) in the presence of pyrithione, an iron ionophore, to acutely increase intracellular labile pool of iron. Cellular iron influx and cell shape profile were monitored with time-lapse imaging techniques. Exposure of RPAECs to [Fe2+] resulted in: (i) an increase in intracellular iron as detected by the iron sensitive fluorophore, PhenGreen; (ii) depletion of cell glutathione; and (iii) nuclear translocation of stress-response transcriptional factors Nrf2 and NFkB (p65). The resulting iron-induced cell alterations were characterized by cell polarization and formation of membrane cuplike and microvilli-like projections abundant with ICAM-1, caveolin-1, and F-actin. The iron-induced re-arrangements in cytoskeleton, alterations in focal cell–cell interactions, and cell buckling were accompanied by decrease in electrical resistance of RPAEC monolayer. These effects were partially eliminated in the presence of N,N′-bis (2-hydroxybenzyl) ethylenediamine-N,N′-diacetic acid, an iron chelator, and Y27632, a Rho—kinase inhibitor. Thus acute increases in labile iron in cultured pulmonary endothelium result in structural remodeling (and a proinflammatory phenotype) that occurs via post-transcriptional mechanisms regulated in a redox sensitive fashion.
Keywords: Iron complexes; Endothelial cells; Remodeling; Inflammation; Redox-stress

The unique role of siderophore in marine-derived Aureobasidium pullulans HN6.2 by Zhe Chi; Xing-Xing Wang; Zai-Chao Ma; Muhammad Aslam Buzdar; Zhen-Ming Chi (219-230).
The l-ornithine-N 5-monooxygenase structural gene (SidA gene, accession number: FJ769160) was isolated from both the genomic DNA and cDNA of the marine yeast Aureobasidium pullulans HN6.2 by inverse PCR and RT-PCR. An open reading frame of 1,461 bp encoding a 486 amino acid protein (isoelectric point: 7.79) with calculated molecular weight of 55.4 kDa was characterized. The promoter of the gene (intronless) was located from −1 to −824 and had three HGATAR boxes which were putative binding motifs for the respective DNA-binding motifs and one CATA box. The SidA gene in A. pullulans HN6.2 was disrupted by integrating the hygromycin B phosphotransferase (HPT) gene into Open Reading Frame of the SidA gene using homologous recombination. Of all the disruptants obtained, one strain S6 (∆sidA) did not synthesize both intracellular and extracellular fusigen so that it could not inhibit growth of the pathogenic bacteria Vibrio anguillarum and Vibrio parahaemolyticus. The disruptant S6 did not grow in the iron-deplete medium and seawater medium because cell budding was stopped, but could grow in the iron-replete medium with 10 μM Fe3+ and Fe2+. H2O2 in the medium was more toxic to the disruptant S6 than to its wild type HN6.2. Thus, we infer that the fusigen produced by the marine-derived A. pullulans HN6.2 can play a unique role in chelating, uptake and concentration of iron to maintain certain proper physiological functions within the cells and secretion of siderophore may represent an efficient tool to eliminate competitors to compete for limiting nutritional resources in marine environments.
Keywords: Fusigen; Marine-derived yeasts; SidA gene; Gene disruption; Physiological function

Arsenic trioxide versus tetraarsenic oxide in biomedical research: misunderstandings and misinterpretations by Zdenka Šlejkovec; Ingrid Falnoga; Johannes T. van Elteren (231-235).
This work presents an analytical chemist’s view on the sometimes unconscious use of arsenic trioxide in (bio)medical research. Arsenic trioxide is a frequently used chemical in cancer treatment research and its action to various malignant cells has been extensively studied and published. Unfortunately some research articles show trivial errors with regards to background knowledge of the chemical, handling the chemical, experimental design and interpretation of results like e.g. in a range of articles comparing advantages of tetraarsenic oxide over arsenic trioxide (dimeric/monomeric) although the dissolution of both yields the same active compound (HAsO2). To fully understand the implications of these errors we will highlight some of them with the intent to harmonize future work in this field.
Keywords: As2O3 ; As4O6 ; Tetras; Concentration units; Redox conversion; Cell cultures

Enhanced iron availability by protein glycation may explain higher infection rates in diabetics by Theodore J. Zwang; Michael V. Gormally; Malkiat S. Johal; Matthew H. Sazinsky (237-245).
Serum proteins exist in a state of higher glycation among individuals with poor glycemic control, notably diabetics. These non-enzymatic modifications via the Maillard reaction have far reaching effects on metabolism and regulation, and may be responsible for increased infection rates within this population. Here we explore the effects of glycation on iron metabolism and innate immunity by investigating the interaction between siderophores and bovine serum albumin (BSA). Using a quartz crystal microbalance with dissipation monitoring to quantify association rates, glycated BSA exhibited a significantly reduced affinity for apo and holo enterobactin compared to a non-glycated BSA standard. Bacterial growth assays in the presence of BSA and under iron-limited conditions indicated the growth rate of enterobactin-producing E. coli increased significantly when the BSA was in a glycated form. The results, in addition to data in the literature, support the hypothesis that glycation of serum proteins may effectively increase the available free iron pool for bacteria in blood serum and weaken our innate immunity. This phenomenon may be partially responsible for higher infection rates in some diabetics, especially those with poor glycemic control.
Keywords: Siderophores; Iron homeostasis; Albumin; Diabetes; Glycation; QCM