BioMetals (v.28, #1)

Chronic copper toxicity has been long known to cause hepatotoxicity and liver cirrhosis as observed in Wilson’s disease; however, substantial evidence accrued over the time have shown considerable increase in animal studies demonstrating Alzheimer’s disease like pathology due to chronic copper-intoxication under certain conditions. This review integrates the contemporary mammalian studies in which the effect of chronic copper intoxication was assessed on the central nervous system and cognition of animals.
Keywords: Copper toxicity; Neurodegeneration; Central nervous system; Cognition: Alzheimer’s disease

Estimation of trace metal elements in oral mucosa specimens by using SR-XRF, PIXE, and XAFS by Tomoko Sugiyama; Motohiro Uo; Takahiro Wada; Daisuke Omagari; Kazuo Komiyama; Tadahide Noguchi; Yoshinori Jinbu; Mikio Kusama (11-20).
The effects of dissolved elements from metal dental restorations are a major concern in lesions of the oral mucosa, and the evaluation of accumulated metal elements, especially their distribution and chemical state, is essential for determining the precise effects of trace metals. In this study, X-ray fluorescence with synchrotron radiation (SR-XRF) and particle-induced X-ray emission (PIXE) were applied for distribution analysis of the trace metal elements contained in the oral mucosa, and the chemical states of the elements were estimated using X-ray absorption fine structure (XAFS) analysis. Appropriate combination of these analysis techniques, particularly SR-XRF and PIXE, to visualize the distributions of the elements in the oral mucosa allowed for the observation and evaluation of accumulated metal ions and debris. Importantly, the analyses in this study could be carried out using conventional histopathological specimens without damaging the specimens. Therefore, this method would be applicable for the detection of accumulated trace metal elements in biopsy specimens from the oral mucosa.
Keywords: Synchrotron radiation X-ray fluorescence analysis; Particle-induced X-ray emission analysis; X-ray absorption fine structure analysis; Trace elemental analysis; Histopathological specimen

Effect of Cr(VI) and Ni(II) metal ions on human adipose derived stem cells by R. Indra; K. Purna Sai; A. Rajaram; Rama Rajaram (21-33).
Environmental exposure of Cr(VI) and Ni(II) due to rapid industrialization causes adverse effects in living tissues. Small quantities of these ions also find their way into tissues when metal alloys are used as implants. Even though considerable research has been done on the effects due to their exposure in animal cells, there are only very few reports on how they can affect stem cells which have been shown to be found in adult tissues as well, albeit in small quantities. Hence this study was aimed at understanding how Cr(VI) and Ni(II) affect human adipose derived stem cells (hADSCs) in a cell culture environment. Our results indicate that both ions induce apoptosis in a concentration and time dependent manner with loss of mitochondrial membrane potential (MMP) and corresponding increase in caspase-3 activity. With regard to Ni(II), apoptosis seems to occur only in a small percentage of cells while necrosis is predominant. It can be inferred that the long term exposure of these metals may cause adverse effects in stem cell proliferation and differentiation.
Keywords: hADSCs; Apoptosis; Necrosis; Cr(VI); Ni(II); Caspase-3 activity

Iron sucrose and ferric carboxymaltose: no correlation between physicochemical stability and biological activity by Monika Praschberger; Kathrin Haider; Carolin Cornelius; Markus Schitegg; Brigitte Sturm; Hans Goldenberg; Barbara Scheiber-Mojdehkar (35-50).
Intravenous iron preparations, like iron sucrose (IS) and ferric carboxymaltose (FCM) differ in their physicochemical stability. Thus differences in storage and utilization can be expected and were investigated in a non-clinical study in liver parenchyma HepG2-cells and THP-1 macrophages as models for toxicological and pharmacological target cells. HepG2-cells incorporated significant amounts of IS, elevated the labile iron pool (LIP) and ferritin and stimulated iron release. HepG2-cells had lower basal cellular iron and ferritin content than THP-1 macrophages, which showed only marginal accumulation of IS and FCM. However, FCM increased the LIP up to twofold and significantly elevated ferritin within 24 h in HepG2-cells. IS and FCM were non-toxic for HepG2-cells and THP-1 macrophages were more sensitive to FCM compared to IS at all concentrations tested. In a cell-free environment redox-active iron was higher with IS than FCM. Biostability testing via assessment of direct transfer to serum transferrin did not reflect the chemical stability of the complexes (i.e., FCM > IS). Effect of vitamin C on mobilisation to transferrin was an increase with IS and interestingly a decrease with FCM. In conclusion, FCM has low bioavailability for liver parenchyma cells, therefore liver iron deposition is unlikely. Ascorbic acid reduces transferrin-chelatable iron from ferric carboxymaltose, thus effects on hepcidin expression should be investigated in clinical studies.
Keywords: Intravenous iron; Iron sucrose; Ferric carboxymaltose; Liver iron overload; Macrophages; Ascorbic acid

Transcriptional regulation of copper metabolism genes in the liver of fetal and neonatal control and iron-deficient rats by Malgorzata Lenartowicz; Christine Kennedy; Helen Hayes; Harry J. McArdle (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.
Keywords: Copper-iron interactions; Perinatal development; Metallochaperones; ATP7A; ATP7B; Copper metabolism; Hooded Lister rats

Aluminium (Al), a ubiquitous element in nature is associated with the onset of Alzheimer’s disease. On the other hand, zinc (Zn) is an essential trace element that regulates large number of physiological processes in the human body. The present study was conducted to explore the role of zinc, if any, in regulating apoptotic machinery during Al induced neurodegeneration in rat. Male sprague dawley rats weighing 140–160 g were divided into four different groups viz: Normal control, Al treated (100 mg/kg b.wt./day), Zn treated (227 mg/l) and combined Al and Zn treated. All the treatments were carried out for a total duration of 8 weeks. Al treatment resulted in a significant increase in the protein expressions of cytochrome c, Bax, Apaf-1, caspase 9, caspase 3 (p17), caspase 8, caspase 6, caspase 7 but decreased the Bcl-2 in both the cerebrum and cerebellum. However, Zn supplementation to Al treated rats resulted in a reduction in the protein expressions of cytochrome c, Bax, Apaf-1, caspase 9, caspase 3 (p17), caspase 8, caspase 6 and caspase 7 whereas it elevated the Bcl-2 in both the regions. Further, gene expressions of caspase 3 and caspase 9 were also found to be elevated after Al treatment, which however were reduced following Zn co-treatment. The electron-microscopic analysis of brain revealed that Al intoxication resulted in a number of degenerative signs at ultrastructural level, which were appreciably improved upon Zn supplementation. The present study suggests that Zn provides protection against Al induced neurotoxicity by triggering anti-apoptotic machinery.
Keywords: Aluminium; Zinc; Apoptosis; Ultrastructure; Neurodegeneration; Neurotoxicity

Selenoprotein W (SelW) is mainly understood in terms of its antioxidant effects in the cellular defense system. Inflammation is an important indicator of animal tissue injury, and the inflammatory cells may trigger a sophisticated and well-orchestrated inflammatory cascade, resulting in exaggerated oxidative stress. To investigate the role of SelW in inflammatory injury in chicken immune tissues and cultured splenic lymphocyte, in this report, the effects of selenium (Se) on mRNA expressions of SelW and inflammatory factors (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) in the chicken immune organs (spleen, thymus and bursa of Fabricius) and cultured splenic lymphocyte treated with sodium selenite and H2O2, or knocked down SelW with small interfering RNAs (siRNAs) were examined. The results showed that Se-deficient diets effectively decreased the mRNA expression of SelW (P < 0.05), and induced a significantly up-regulation of COX-2, iNOS, NF-κB, PTGEs and TNF-α mRNA levels (P < 0.05). The histopathological analysis showed that immune tissues were obviously injured in the low-Se groups. In vitro, H2O2 induced a significantly up-regulation of the mRNA levels of inflammation-related genes (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) in cultured splenic lymphocyte (P < 0.05). When lymphocytes were pretreated with Se before treated with H2O2, the inflammation-related genes were significantly decreased (P < 0.05). Silencing of SelW significantly up-regulated the inflammation-related genes (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) in cultured splenic lymphocyte (P < 0.05). The results suggested that the expression levels of inflammatory factors (iNOS, COX-2, NF-κB, PTGEs, and TNF-α) and SelW can be influenced by Se in birds. SelW commonly played an important role in the protection of immune organs of birds from inflammatory injury by the regulations of inflammation-related genes.
Keywords: Selenoprotein W; Small interfering RNAs; Inflammatory injury; Splenic lymphocyte; Chicken

Iron-based granules in body of bumblebees by Petr Jandacka; Barbora Kasparova; Yvonna Jiraskova; Katerina Dedkova; Katerina Mamulova-Kutlakova; Jana Kukutschova (89-99).
The paper deals with the presence of iron-based granules in body parts of bumblebees. Two groups of bumblebees were collected from their natural habitat, industrial landscape, and from a breeding station. Detection of the magnetic particles was performed by a vibratory magnetometer and their morphology and elemental composition was analysed by scanning electron microscopy with EDX microanalysis. By means of the EDX spectra, wild bumblebees were found to have many magnetic and non-magnetic particles on their body, containing Fe, O, Al, Si, Bi, Mg, K, and Ni, likely having origin in the industrial pollution of the environment. In the case of bred bumblebees the presence of iron-rich granules, which occurred more abundantly in subsurface tissues on the head and wings, was observed. Phase analysis based on X-ray diffraction shows that iron-based granules contain magnetite and wuestite and Mössbauer spectroscopy admits a superparamagnetic form of these minerals. Magnetoreception, i.e. the sensory function of these granules, is discussed within the paper.
Keywords: Magnetoreception; Bumblebee; Biomineralisation; Iron granules

The phytotoxicity of ZnO nanoparticles on wheat varies with soil properties by Jean-Luc Watson; Tommy Fang; Christian O. Dimkpa; David W. Britt; Joan E. McLean; Astrid Jacobson; Anne J. Anderson (101-112).
Zn is an essential element for plants yet some soils are Zn-deficient and/or have low Zn-bioavailability. This paper addresses the feasibility of using ZnO nanoparticles (NPs) as soil amendments to improve Zn levels in the plant. The effects of soil properties on phytotoxicity and Zn bioavailability from the NPs were studied by using an acidic and a calcareous alkaline soil. In the acid soil, the ZnO NPs caused dose-dependent phytotoxicity, observed as inhibition of elongation of roots of wheat, Triticum aestivum. Phytotoxicity was mitigated in the calcareous alkaline soil although uptake of Zn from the ZnO NPs occurred doubling the Zn level compared to control plants. This increase occurred with a low level of Zn in the soil solution as expected from the interactions of Zn with the soil components at the alkaline pH. Soluble Zn in the acid soil was 200-fold higher and shoot levels were tenfold higher than from the alkaline soil correlating with phytotoxicity. Mitigation of toxicity was not observed in plants grown in sand amended with a commercial preparation of humic acid: growth, shoot uptake and solubility of Zn from the NPs was not altered by the humic acid. Thus, variation in humic acid between soils may not be a major factor influencing plant responses to the NPs. These findings illustrate that formulations of ZnO NPs to be used as a soil amendment would need to be tuned to soil properties to avoid phytotoxicity yet provide increased Zn accumulations in the plant.
Keywords: Bioavailability; Humic acid; Nanoparticles; Soil; Wheat; Zn

The role of NOS-mediated ROS accumulation in an early phase Cu-induced acute cytotoxicity in MCF-7 cells by Lingying Zhong; Lumei Wang; Lurong Xu; Qunlu Liu; Linlei Jiang; Yuee Zhi; Wei Lu; Pei Zhou (113-122).
Copper (Cu) ion is essential for the biological systems, however, high level of CuCl2 exposure causes detrimental effects, which leads to cell apoptosis. Nitric oxide (NO) is an efficient cell signal messenger, which plays an important role in cell apoptosis. However, the potential mechanism of an early phase Cu-induced acute cytotoxicity through the nitric oxide synthase (NOS) signaling pathway and its interaction has not been studied. In this report, we provide data showing that high level of CuCl2 could rapidly decrease the NO production with the release of Ca2+ and Zn2+, and then modulate the transcriptional and translational expression of NOSs in MCF-7 cells. The reactive oxygen species (ROS) level in cells was increased after high level of CuCl2 exposure, which led to the alpha subunit of eukaryotic initiation factor 2 phosphorylation. By using the free radical scavenger N-acetyl-l-cysteine or the NOS substrate l-arginine, it demonstrated that NOS played a critical role on the Cu-induced ROS generation, which further led to the oxidative stress and cell apoptosis. These results suggested that Cu-induced apoptosis was associated with the oxidative stress, and through the NOS-mediated signaling pathway.
Keywords: Copper (Cu); Nitric oxide synthase (NOS); Reactive oxygen species (ROS); Oxidative stress; Alpha subunit of eukaryotic initiation factor 2 (eIF2α) phosphorylation; Apoptosis

Impact of heavy metal stresses on the growth and auxin homeostasis of Arabidopsis seedlings by Rui Wang; Jiehua Wang; Lin Zhao; Shaohui Yang; Yingjin Song (123-132).
The phytohormone auxin is an essential mediator in many aspects of plant development. Its dynamic and differential distribution within the plant is regulated by a variety of environmental cues including heavy metal stimuli. In the present study, we first evaluated the toxic effects of seven heavy metals including Pb2+, Cd2+, Hg2+, Ni2+. Zn2+, Co2+ and Cu2+ in their excess on the model plant, Arabidopsis thaliana. Various morphological defects including loss in fresh weight and leaf area, decrease of the primary root length and stimulation of the lateral root density occurred to a different extent among seven heavy metals. Next, using an indicative DR5:GUS reporter line of Arabidopsis, the auxin accumulation and distribution within plant seedlings were found to be dramatically and differentially affected by these heavy metals. We further analyzed the transcriptional changes of 27 selected auxin homeostasis-related genes by qRT-PCR technique and found that upon various heavy metals, the expressions of the candidate genes were distinctly altered in shoots and roots. Our data indicated that when confronted with excessive heavy metals, plants could dynamically and differentially regulate the transcription of auxin-related genes to adjust the location and effective accumulation of auxin within the plant for better adaptation and survival under the adverse environment.
Keywords: Arabidopsis thaliana ; Heavy metal; Auxin homeostasis; DR5:GUS ; Transcriptional regulation

Boric acid induces cytoplasmic stress granule formation, eIF2α phosphorylation, and ATF4 in prostate DU-145 cells by Kimberly A. Henderson; Sarah E. Kobylewski; Kristin E. Yamada; Curtis D. Eckhert (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.
Keywords: Boron; Boric acid; eIF2α; ATF4; DU-145 cells

Measurement of total iron in Helicobacter pylori-infected gastric epithelial cells by Sebastian E. Flores; Andrew S. Day; Jacqueline I. Keenan (143-150).
Despite the evidence suggesting a role for Helicobacter pylori in the induction of systemic iron deficiency anaemia, little is known about the possibility of infection-associated changes in cellular iron homeostasis at the gastric epithelium. In this study we compared four different techniques for measuring iron in AGS cells, a gastric epithelial cell line that is widely used to model to H. pylori infection in vitro. Inductively coupled plasma-mass spectrometry proved to be an efficient method, but only when large numbers of cells were used. Two colorimetric assays that included the use of concentrated hydrochloric acid with or without potassium ferrocyanide detected iron in the micromolar but not the nanomolar range in cell-free standards. However, the third colorimetric assay that incorporated ferrozine proved to be highly accurate at detecting iron in the nanomolar range, and was able to detect iron in AGS cells, Moreover, using this assay, we were able to show that the level of iron in H. pylori-infected AGS cells is significantly increased when compared to uninfected cells.
Keywords: Iron measurement; Gastric epithelial cells; Helicobacter pylori

Lead induced changes in phosphorylation of PSII proteins in low light grown pea plants by Wasilewska Wioleta; Drożak Anna; Bacławska Ilona; Kąkol Kamila; Romanowska Elżbieta (151-162).
Light-intensity and redox-state induced thylakoid proteins phosphorylation involved in structural changes and in regulation of protein turnover. The presence of heavy metal ions triggers a wide range of cellular responses including changes in plant growth and photosynthesis. Plants have evolved a number of mechanisms to protect photosynthetic apparatus. We have characterized the effect of lead on PSII protein phosphorylation in pea (Pisum sativum L.) plants grown in low light conditions. Pb ions affected only slightly photochemical efficiency of PSII and had no effect on organization of thylakoid complexes. Lead activated strongly phosphorylation of PSII core D1 protein and dephosphorylation of this protein did not proceed in far red light. D1 protein was also not degraded in this conditions. However, phosphorylation of LHCII proteins was not affected by lead. These results indicate that Pb2+ stimulate the phosphorylation of PSII core proteins and by disturbing the disassembly of supercomplexes play a role in PSII repair mechanism. LHCII phosphorylation could control the distribution of energy between the photosystems in low light conditions. This demonstrates that plants may respond to heavy metals by induction different pathways responsible for protein protection under stress conditions.
Keywords: Lead; Low light; Pea; Photosystem II; Protein phosphorylation; Thylakoids

Selenium (Se) is an essential dietary trace element, which acts as an antioxidant. Heat shock proteins (HSPs) are a family of intracellular proteins whose synthesis is greatly increased upon exposure of cells to environmental stressors including oxidative metabolites, heavy metals, amino acid analogues and so on. However, little is known about the role of HSPs in oxidative stress damage induced by Se deficiency in the chicken liver. The aim of this study was to investigate the effects of Se deficiency on the expression levels of HSPs (Hsps27, 40, 60, 70, and 90) and oxidative indexes in the chicken liver. A total of 300 1-day-old sea blue white laying hens were divided into two groups (n = 150/group), and each of those groups was randomly divided into groups so that the trials were conducted in triplicate. The Se-deficient group (−Se) was fed a Se-deficient corn-soy basal diet (the Se content was 0.02 mg/kg); the Se-adequate group as control (+Se) was fed the same basal diet supplemented with Se at 0.2 mg/kg (sodium selenite). The liver tissue was collected and examined for pathological observations, oxidative indexes, mRNA and protein levels of HSPs genes at 15, 25, 35, 45, 55 and 65 days old. The histopathological analysis showed that liver tissues were injured seriously in the Se-deficient group. The oxidative indexes data showed that the malondialdehyde (MDA) level increased and the activity of L-glutathione (GSH) and glutathione peroxidase (GSH-Px) in the chicken liver decreased in Se-deficient group (p < 0.05). Additionally, the mRNA levels of HSPs (27, 40, 60, 70, and 90) increased significantly (p < 0.05) in the Se-deficient group compare to the corresponding control group. Meanwhile, the protein expression of HSPs (60, 70, and 90) also increased significantly (p < 0.05) in the Se-deficient group. These results suggested that oxidative stress and the levels of HSPs expression levels in chicken liver can be influenced by dietary Se deficiency. And HSPs played an important role in the protection of the liver after oxidative stress due to Se deficiency.
Keywords: Chicken liver; Selenium deficiency; Heat shock protein; Oxidative stress

A comparative immunological analysis of CoCl2 treated cells with in vitro hypoxic exposure by Shweta; K. P. Mishra; S. Chanda; S. B. Singh; L. Ganju (175-185).
The hypoxic preconditioning of mammalian cells has been shown to have beneficial effects against hypoxic injuries. However, very little information is available on the comparative analysis of immunological responses to hypoxic and hypoxia mimetic exposure. Therefore, in the present study, mouse peritoneal macrophages and splenocytes were subjected to hypoxia exposure (0.5 % O2) and hypoxia mimetic Cobalt chloride (CoCl2) treatment to evaluate their effect on immune response and delineate the underlying signaling mechanisms. The results obtained indicated that super oxide generation increased while TLR4 expression and cell surface markers like CD25, CD40 and CD69 were suppressed in both the treatments as compared to normoxia. Cobalt chloride treatment increased NF-κB expression, nitric oxide (NO) and iNOS expression, cytokines TNF-α and IL-6 as compared to hypoxia exposure. Our study showed that CoCl2 stabilizes HIF-1α to create hypoxia like conditions but it mainly influences the inflammatory response via NF-κB signaling pathway by skewing the production of proinflammatory molecules like TNF-α, IL-6 and NO.
Keywords: Cobalt chloride; Hypoxia; Inflammatory response; NF-κB; iNOS; Nitric oxide; Proinflammatory cytokines

Endogenous concentrations of biologically relevant metals in rat brain and cochlea determined by inductively coupled plasma mass spectrometry by Stacia R. Wegst-Uhrich; Elizabeth J. Mullin; Dalian Ding; Senthilvelan Manohar; Richard Salvi; Diana S. Aga; Jerome A. Roth (187-196).
Manganese (Mn), iron (Fe), copper (Cu), and zinc (Zn) are essential nutrients which aid in the proper functioning of cells, but high concentrations of these metals can be toxic to various organs. Little is known about the endogenous concentrations of these metals in the cochlea, the auditory portion of the inner ear which is extremely small and difficult to access. To fill this gap, a trace quantitative digestion and inductively coupled plasma mass spectrometry method was developed to determine the concentrations of these metals in the stria vascularis, organ of Corti, and spiral ganglion, three critically important parts of the cochlea (≤1.5 mg); these values were compared to those in specific brain regions (≤20 mg) of rats. Rats were sacrificed and the cochlea and brain regions were carefully isolated, digested, and analyzed to determine baseline concentrations of Mn, Fe, Cu, and Zn. In the cochlea, Mn, Fe, Cu, and Zn concentrations ranged from 3.2–6, 73–300, non-detect, and 13–200 µg/g respectively. In the brain, Mn, Fe, Cu, and Zn concentrations ranged from 1.3–2.72, 21–120, 5.0–10.6, and 33–47 µg/g respectively. Significant differences (p < 0.05) were observed between the tissue types within the cochlea, and between the cochlea and brain. This validated method provides the first quantitative assessment of these metals in the three key subdivisions of the cochlea compared to the levels in the brain; Mn, Fe, and Zn levels were considerably higher in the cochlea than brain.
Keywords: ICP-MS; Cochlea; Trace analysis; Auditory pathway; Iron; Manganese; Zinc; Copper

Molecular characterization of a homolog of the ferric-uptake regulator, Fur, from the marine bacterium Marinobacter algicola DG893 by Ryan A. Barker; Jerrell Tisnado; Lisa A. Lambert; Astrid Gärdes; Mary W. Carrano; Paul N. Carrano; Christopher Gillian; Carl J. Carrano (197-206).
Full length recombinant iron regulatory protein, Fur, has been isolated and characterized from the algal-associated marine bacterium Marinobacter algicola DG893. Under nondenaturing conditions the Fur protein behaves on size exclusion chromatography as a dimer while it is monomeric under SDS PAGE conditions. ICP-MS and fluorescence quenching experiments show that Mb-Fur binds a single metal ion (Zn, Mn, or Co) per monomer. Electrophoretic mobility shift assays were used to probe the interaction of Mb-Fur with the purported Fur box in the promoter region upstream of the vibrioferrin biosynthetic operon. Interaction of Mb-Fur with a 100 bp DNA fragment containing the Fur box in the presence of 10 µM Mn, Co or Zn(II) resulted in decreased migration of DNA on a 7.5 % polyacrylamide gel. In the absence of the Fur protein or the metal, no interaction is seen. The presence of EDTA in the binding, loading or running buffers also abolished all activity demonstrating the importance of the metal in formation of the promoter-repressor complex. Based on a high degree of similarity between Mb-Fur and its homolog from Pseudomonas aeruginosa (PA) whose X-ray structure is known we developed a structural model for the former which suggested that only one of the several metal binding sites found in other Fur’s would be functional. This is consistent with the single metal binding stoichiometry we observed. Since the purported metal binding site was one that has been described as “structural” rather than “functional” in PA and yet the monometallic Mb-Fur retains DNA Fur box binding ability it reopens the question of which site is which, or if different species have adapted the sites for different purposes.
Keywords: Iron; Ferric uptake regulator; Metal binding; Homology model; Recombinant protein

DNA binding ability of histone-like protein HPhA is negatively affected by interaction with Pb2+ by Yanyan Wang; Ye Wang; Yang Zhan; Jinrui Zhang; Weiguo Liang; Xuexun Fang; Dahai Yu; Yan Feng (207-217).
The histone-like protein (HPhA) is highly homologous to the eukaryotic histones and has the ability to bind to the DNA molecules. In this study, we tested divalent metal ions Mg2+, Ca2+, Zn2+, Pb2+ for their effect on the recombinant HPhA (rHPhA)-DNA binding. We found that only Pb2+ was able to reduce the formation of rHPhA-DNA complex using gel mobility shift assays. Equilibrium dialysis showed that Pb2+ bound to rHPhA by a 2:1 ratio. The interaction of Pb2+ and rHPhA was further studied by spectroscopic method including fluorescence, ultraviolet visible (UV–Vis) absorption, and circular dichroism (CD) spectroscopies. Fluorescent spectroscopy results suggested that Pb2+ and rHPhA formed a complex that caused internal quenching of the fluorescence of the protein at the ground state, and the quenching is predominately static and mixed with dynamic quenching. UV–Vis absorption spectrum results showed Pb2+ caused a slightly blue shift of the maximum absorption wavelength of rHPhA which indicated the reduction of the protein’s hydrophobicity. The CD spectrum further indicated that a reduction of the α-helix content of rHPhA occurred upon binding to Pb2+. Synchronous fluorescence spectrometry analysis revealed that Pb2+ was able to affect the polarity of the amino acids that near the Trp and Tyr residues. These results together showed that Pb2+ interact with the recombinant rHPhA and this interaction negatively affect the ability of rHPhA to form complex with DNA molecules.
Keywords: Histone-like protein; Protein-DNA complex; Pb2+ ; Metal-binding proteins; Metal-protein interaction

The induction of cell death by phosphine silver(I) thiocyanate complexes in SNO-esophageal cancer cells by Zelinda Human; Appollinaire Munyaneza; Bernard Omondi; Natasha M. Sanabria; Reinout Meijboom; Marianne J. Cronjé (219-228).
Esophageal cancer is one of the least studied cancers and is found to be prominent in black South African males. It is mainly diagnosed in the late stages, and patients tend to have a low 5-year survival rate of only 10 %. Silver is generally used as an antimicrobial agent, with limited reports on anticancer studies. In this study, dimeric silver(I) thiocyanate complexes were used containing a variation of 4-substitued triphenylphosphines, including [AgSCN(PPh3)2]2 (1), [AgSCN{P(4-MeC6H4)3}2]2 (2), [AgSCN{P(4-FC6H4)3}2]2 (3) and [AgSCN{P(4-ClC6H4)3}2]2 (4). All four complexes, with their respective phosphine ligands, PPh3 (L1), P(4-MeC6H4)3 (L2), P(4-FC6H4)3 (L3) and P(4-ClC6H4)3 (L4), were subjected to in vitro toxicity studies in SNO-esophageal cancer cells, using an alamarBlue® assay. Morphological changes, including blebbing and apoptotic body formation, were observed. Phosphatidylserine externalization, a marker of apoptosis, was quantified by flow cytometry. The phosphine ligands L1L4, on their own, had minimal effect on the malignant while complexes 14 resulted in significant cell death. A 10× decreased concentration of these complexes had similar effects than cisplatin, used as the positive control. These complexes show promise as anticancer agents.
Keywords: Silver(I) thiocyanate complexes; Phosphines; Anticancer; Flow cytometry; Apoptosis; SNO-esophageal cancer

Erratum to: The lipidome of the photosynthetic bacterium Rhodobacter sphaeroides R26 is affected by cobalt and chromate ions stress by Cosima Damiana Calvano; Francesca Italiano; Lucia Catucci; Angela Agostiano; Tommaso R. I. Cataldi; Francesco Palmisano; Massimo Trotta (229-229).