BioMetals (v.25, #3)

Iron, brain and neuropsychiatric problem by Viroj Wiwanitkit (487-487).

Molecular mechanistic model of plant heavy metal tolerance by Ganesh Thapa; Ayan Sadhukhan; Sanjib Kumar Panda; Lingaraj Sahoo (489-505).
Plants being sessile are susceptible to heavy metals (HMs) toxicity and respond differentially to hostile environments. The toxicity of HM is governed by the type of ion and its concentration, plant physiology and stage of plant growth. Plants counteract the HMs stress by overexpressing numerous stress related proteins, glutathione mediated tolerance pathways and signaling proteins involving networks of various stress regulations. Though the response may vary and be specific in its stress networks regulation for each HM. The intricacy of HM tolerance response involves the set of molecular regulation, which demands to be understood to yield HM tolerant plant. Topical advancements in molecular biology and genomics have facilitated studies in transcriptomics and proteomics to identify regulatory genes implied in HM tolerance in plants. The integration of resources obtained through these studies will be of extreme significance, combining the diverse fields of plant biology to dissect the actual HM stress response network. In this review, we put an endeavor to describe the specific aspects of the molecular mechanisms of a plant response to HMs which may contribute to better understanding of the mode of HMs action and overlaps in metal sensing and signaling/crosstalk to other stresses.
Keywords: Hyperaccumulator plants; HMs; Metal transporters; Thiols; cis-Regulatory elements; Phytochelatins; Phytoengineering

Zinc is an essential trace element in cells. However, its high level in cytoplasm promotes activation of stress signaling pathways and may lead to cell death. In the present study we used Drosophila melanogaster blood cells (haemocytes), obtained from the third instar larvae, to study the effects of high concentrations of Zn2+ on programmed cell death (PCD). We analyzed the activity of caspases, the level of caspase inhibitor protein DIAP1 and metallothioneins, as well as calcium concentrations and activity of mitochondria in haemocytes exposed to 0.35 and 1.7 mM concentrations of Zn. The obtained results showed that rapid increase of [Zn2+] i in the cytoplasm up-regulates metallothionein MtnB but not MtnA gene expression in cells treated with Zn2+ in both concentrations. Excess of Zn2+ also induced activation of the initiator caspase Dronc, associated with the mitochondrial pathway of PCD, and the effector caspase DrICE. In turn, the activity of receptor-regulated Dredd caspase was not changed. The level of DIAP1 decreased significantly in haemocytes in the presence of high Zn2+ concentration in comparison to untreated cells. Moreover, mitochondrial membrane potential was significantly decreased after exposure to Zn ions. These results indicate that high concentration of Zn2+ in the cytoplasm of haemocytes induces PCD via a mitochondrial pathway and that caspases play a pivotal role in this process.
Keywords: Apoptosis; Metallothioneins; Mitochondria; Haemolymph; Fruit fly

Proteomic study of the yeast Rhodotorula mucilaginosa RCL-11 under copper stress by Verónica Irazusta; Cristina Estévez; María Julia Amoroso; Lucía I. C. de Figueroa (517-527).
In order to understand the mechanism involved in Rhodotorula mucilaginosa RCL-11 resistance to copper a proteomic study was conducted. Atomic absorption spectroscopy showed that the copper concentration in the medium decreased from 0.5 to 0.19 mM 48 h after inoculation of the yeast. Analysis of one-dimensional gel electrophoresis of crude cell extracts revealed expression of differential bands between cells with and without copper. In order to study this difference, two-dimensional electrophoresis of R. mucilaginosa RCL-11 exposed to Cu for 16, 24, and 48 h was carried out. Identification of differentially expressed proteins was performed by MALDI-TOF/TOF. Ten of the 16 spots identified belonged to heat shock proteins. Superoxide dismutase, methionine synthase and beta-glucosidase were also found over-expressed at high copper concentrations. The results obtained in the present work show that when R. mucilaginosa RCL-11 is exposed to 0.5 mM copper, differential proteins, involved in cell resistance mechanisms, are expressed.
Keywords: Proteomic; Copper overload; Yeast; Bioremediation

Zinc aspartate suppresses T cell activation in vitro and relapsing experimental autoimmune encephalomyelitis in SJL/J mice by Diana Stoye; Claudia Schubert; Alexander Goihl; Karina Guttek; Annegret Reinhold; Stefan Brocke; Kurt Grüngreiff; Dirk Reinhold (529-539).
Zinc is an essential trace element with a critical role in normal growth and development and in immune homeostasis. Zinc deficiency impairs both the innate and the adaptive immune system and can be normalized by zinc supplementation. On the other end of the spectrum, high dosages of zinc diminish immune cell functions similar to zinc deficiency. Here, we investigated the influence of zinc aspartate on proliferation and cytokine production of stimulated human T cells and mouse splenocytes in vitro. Furthermore, the effect of zinc aspartate was examined in mice with experimental autoimmune encephalomyelitis (EAE), an animal model of Multiple Sclerosis (MS) with a Th1/Th17 T cell-mediated immunopathogenesis. Zinc aspartate suppressed proliferation as well as IL-2, IL-10 and IL-17 production in stimulated human T cells and mouse splenocytes. Importantly, administration of a medium range dose of 30 μg/day zinc aspartate [1.5 mg/kg body weight (BW)] in a therapeutic manner led to a significant reduction of the clinical severity of the EAE during the first relapse of the disease. A lower zinc aspartate dose (6 μg/day, 0.3 mg/kg BW) had no significant therapeutic effect on the severity of the EAE, while administration of higher zinc aspartate amounts (120 μg/day, 6 mg/kg BW) led to more severe disease. Taken together, our data suggest that zinc aspartate can modulate activation, proliferation and cytokine production of effector T cells in vitro and in vivo and that activated autoreactive T cells may be potential therapeutic targets of tightly controlled zinc supplementation in autoimmune diseases like MS.
Keywords: Zinc aspartate; IL-2; IL-10; IL-17; T cell proliferation; Experimental autoimmune encephalomyelitis (EAE)

Aluminum ammonium sulfate dodecahydrate purified from traditional Chinese medicinal herb Korean monkshood root is a potent matrix metalloproteinase inhibitor by Yehua Shen; Sen Liu; Fenghai Jin; Tianyang Mu; Cong Li; Kun Jiang; Weihua Tian; Dahai Yu; Yingqi Zhang; Xuexun Fang (541-551).
Matrix metalloproteinases (MMPs) are zinc-dependent endopeptidases and key regulators for many physiological and pathological functions. The MMP inhibitors have been shown to modulate diseases such as cancer, inflammation, and cardiovascular diseases. In this paper we tracked the MMP inhibitory activities of the traditional Chinese medicinal herb Korean Monkshood Root. The purified active ingredient was identified by the elemental analysis, infrared spectrum (IR) and X-ray diffraction as aluminum ammonium sulfate dodecahydrate. This inorganic compound showed inhibitory activities toward a number of MMP family members. In particular, it has a strong inhibitory effect toward MMP-2 and MMP-9, with IC50 values of 0.54 and 0.50 μM, respectively. Further analysis suggested that the MMP inhibitory activity is mainly due to Al3+. Cell viability assays using human fibrosarcoma HT1080 cells showed aluminum ammonium sulfate had minimal cyto-toxicity with a concentration up to 500 μM. However, within 50 μM, it exhibited significant inhibition of cell invasion. To our knowledge, there has been no previous report of inorganic form of the MMP inhibitor with strong inhibitory activity. Our results for the first time showed that aluminum ammonium sulfate is an inorganic form of MMP inhibitor with high potency, and can be used to interfere with MMP related cellular processes.
Keywords: MMP inhibitors; Aluminum ammonium sulfate dodecahydrate; Inorganic MMP inhibitor

Delineation of the molecular mechanism for disulfide stress-induced aluminium toxicity by Ming J. Wu; Patricia A. Murphy; Patrick J. O’Doherty; Stephen Mieruszynski; Mark Jones; Cindy Kersaitis; Peter J. Rogers; Trevor D. Bailey; Vincent J. Higgins (553-561).
Following our previous finding that the sulfhydryl-oxidising chemical diamide induced a marked elevation of cellular Al3+ (Wu et al., Int J Mol Sci, 12:8119–8132, 2011), a further investigation into the underlying molecular mechanism was carried out, using the eukaryotic model organism Saccharomyces cerevisiae. The effects of non-toxic dose of diamide (0.8 mM) and a mild dose of aluminium sulphate (Al3+) (0.4 mM) were determined prior to the screening of gene deletion mutants. A total of 81 deletion mutants were selected for this study according to the available screening data against Al3+ only (Kakimoto et al., BioMetals, 18: 467–474, 2005) and diamide only (Thorpe et al., Proc Natl Acad Sci USA, 101: 6564–6569, 2004). On the basis of our screening data and the cluster analysis, a cluster containing the gene deletions (rpe1∆, sec72∆, pdr5∆ and ric1∆) was found to be specifically sensitive to the mixture of diamide and Al3+. However gnp1∆, mch5∆ and ccc1∆ mutants were resistant. Dithiothreitol (DTT) and ascorbate markedly reversed the diamide-induced Al3+ toxicity. Inductively-coupled plasma optical emission spectrometry demonstrated that DTT reduced the intracellular Al3+ content in diamide/Al3+-treated yeast cells six-fold compared to the non-DTT controls. These data together revealed that the pleiotropic drug resistance transporter (Pdr5p) and vacuolar/vesicular transport-related proteins (Ric1p and Sec72p) are the targets of diamide. A dysfunctional membrane-bound Pdr5p terminates the detoxification pathway for Al3+ at the final step, leading to intracellular Al3+ accumulation and hence toxicity. As Al3+ toxicity has been a problem in agriculture and human health, this study has provided a significant step forward in understanding Al3+ toxicity.
Keywords: Disulfide stress; Diamide; Aluminium; Toxicity; Yeast

Calcium channel blocker use and serum ferritin in adults with hypertension by Arch G. Mainous III; Eugene D. Weinberg; Vanessa A. Diaz; Sharleen P. Johnson; Mary M. Hulihan; Althea M. Grant (563-568).
Iron overload cardiomyopathy is becoming more prevalent, and early recognition and intervention may alter outcomes. Calcium channels are key transporters of iron under iron-overloaded conditions, and potentially represent a new therapeutic target for iron overload. The purpose of this study was to examine the relationship between Calcium channel blocker (CCB) use and serum ferritin among adults with diagnosed hypertension. We analyzed the nationally representative NHANES (National Health and Nutrition Examination Survey) 1999–2002 for adults ≥40 years with diagnosed hypertension. The association between CCBs and serum ferritin was assessed using a t-test and adjusted multiple regressions.The study population included 2143 individuals (representing 37.4 million individuals, 42.0 % males). 12.6 % of the population reported taking CCBs in the last month. Individuals taking CCBs had lower mean serum ferritin (129.3 ng/mL versus 154.5 ng/mL, p = 0.02). After adjusting for age, sex, menopause and hysterectomy status for women, race/ethnicity, and C-reactive protein, mean serum ferritin for individuals taking CCBs was 26.3 ng/mL lower than for those not taking CCBs (p = 0.01). In an adjusted regression, individuals who took CCBs and had a daily vitamin C intake of ≥500 mg had a mean serum ferritin that was 60.1 ng/mL lower than people not taking CCBs and with daily vitamin C < 500 mg (p < 0.001). In conclusion, this study found an association between use of CCBs and lower serum ferritin levels in individuals with hypertension. Further studies are needed to assess the possible use of CCBs as non-traditional chelating agents for treatment of iron overload cardiomyopathy.
Keywords: Calcium channel blockers; Serum ferritin; NHANES

A case of multiple sclerosis improvement following removal of heavy metal intoxication by Alessandro Fulgenzi; Sante Guido Zanella; Mario Mauro Mariani; Daniele Vietti; Maria Elena Ferrero (569-576).
Multiple sclerosis (MS) is a chronic progressive disease of the central nervous system (CNS) provoking disability and neurological symptoms. The exact causes of SM are unknown, even if it is characterized by focal inflammatory lesions in CNS accompanied by autoimmune reaction against myelin. Indeed, many drugs able to modulate the immune response of patients have been used to treat MS. More recently, toxic metals have been proposed as possible causes of neurodegenerative diseases. The objective of this study is to investigate in vivo the impact of heavy metal intoxication in MS progression. We studied the case of a patient affected by MS, who has been unsuccessfully treated for some years with current therapies. We examined his levels of toxic heavy metals in the urine, following intravenous “challenge” with the chelating agent calcium disodium ethylene diamine tetraacetic acid (EDTA).The patient displayed elevated levels of aluminium, lead and mercury in the urine. Indeed, he was subjected to treatment with EDTA twice a month. Under treatment, the patient revealed in time improved symptoms suggestive of MS remission. The clinical data correlated with the reduction of heavy metal levels in the urine to normal range values. Our case report suggests that levels of toxic metals can be tested in patients affected by neurodegenerative diseases as MS.
Keywords: Chelation therapy; Multiple sclerosis; Toxic heavy metals; EDTA

RNAβ affects the transcription process of the iron transport-biosynthesis operon encoded in the pJM1 plasmid of Vibrio anguillarum at a stem-loop structure located in the intergenic region between the fatA and angR genes. The net result is a higher level of the fatD, fatC, fatB, and fatA moiety as compared with the longer transcript encoding those genes as well as the angR and angT genes. In this work we report the secondary structure of RNAβ determined by treatment with single and double strand specific ribonucleases as well as lead acetate followed by sequencing. The generated in vitro structural data indicated that three of the four previously described loops are in agreement with the original model, however, the alteration of loop IV as well as several other structural differences in the overall shape of the molecule led to the necessity of creating a new in silico model. Using the sites of mutations in the various loops we modeled the change in the RNAβ secondary structure induced by those mutations. Mutations of loops III and IV to their complementary bases alter the overall structure of the RNAβ significantly and increase its function while mutations in loops I and II have the opposite effect, the structure is unchanged but the activity of RNAβ decreases. This indicates that loops I and II are necessary for interaction with the target mRNA. It is possible that the structural rearrangement introduced by mutations in loops III and IV promote activity and binding in loops I and II through reducing steric hindrance or increased binding to the target. This result also indicates that the exact relative positions of the critical loops are unimportant for activity.
Keywords: Antisense RNA; Iron transport; Plasmid; Vibrio anguillarum

Spectroscopic and electrochemical characterization of gold(I) and gold(III) complexes with glyoxaldehyde bis(thiosemicarbazones): cytotoxicity against human tumor cell lines and inhibition of thioredoxin reductase activity by Josane A. Lessa; Karina S. O. Ferraz; Juliana C. Guerra; Luana F. de Miranda; Carla F. D. Romeiro; Elaine M. Souza-Fagundes; Paulo Jorge S. Barbeira; Heloisa Beraldo (587-598).
Complexes [Au2(H2Gy3DH)2]Cl2 (1), [Au(H2Gy3Me)]Cl3 (2) and [Au(H2Gy3Et)]Cl3 (3) were obtained with glyoxaldehyde bis(thiosemicarbazone) (H2Gy3DH) and its N(3)-methyl (H2Gy3Me) and N(3)-ethyl (H2Gy3Et) derivatives. The bis(thiosemicarbazones) and their gold(I) and gold(III) complexes exhibited anti-proliferative activity against HL-60, Jurkat (leukemia) and MCF-7 (breast cancer) cells at 10 μmol L−1. Complex (2) was able to in vitro inhibit thioredoxin reductase (TrxR) activity, which suggests that inhibition of TrxR could be part of its mechanism of action.
Keywords: Bis(thiosemicarbazones); Gold complexes; Thioredoxin reductase; Cytotoxicity

Exploration of biguanido–oxovanadium complexes as potent and selective inhibitors of protein tyrosine phosphatases by Liping Lu; Xiaoli Gao; Miaoli Zhu; Sulian Wang; Qiong Wu; Shu Xing; Xueqi Fu; Zhiwei Liu; Maolin Guo (599-610).
The inhibitory effects of three biguanido–oxovanadium complexes ([VO(L1−3)2]·nH2O: HL1 = metformin, HL2 = phenformin, HL3 = moroxydine) against four protein tyrosine phosphatases (PTPs) and an alkaline phosphatase (ALP) were investigated. The complexes display strong inhibition against PTP1B and TCPTP (IC50, 80–160 nM), a bit weaker inhibition against HePTP (IC50, 190–410 nM) and SHP-1(IC50, 0.8–3.3 μM) and much weaker inhibition against ALP (IC50, 17–35 μM). Complex 3 is about twofold less potent against PTP1B, TCPTP and HePTP than complexes 1 and 2, while complex 2 inhibits SHP-1 more strongly (about three to fourfold) than the other two complexes. These results suggest that the structures of the ligands slightly influence the potency and selectivity against PTPs. The complexes inhibit PTP1B and ALP with a typical competitive type.
Keywords: Biguanido–oxovanadium complexes; Protein tyrosine phosphatases; Competitive inhibition; Selective inhibition

Exposure of Staphylococcus aureus to silver(I) induces a short term protective response by Alanna Smith; Raymond Rowan; Malachy McCann; Kevin Kavanagh (611-616).
The Ag(I) ion has well established anti-bacterial and antifungal properties. Exposure of Staphylococcus aureus to MIC80 AgNO3 (3 μg/ml) lead to an increase in the activity of superoxide dismutase, glutathione reductase and catalase at 30 min but activity declined by 60 min. In addition, exposure of cells to this metal ion for 1 h lead to increased expression of a number of proteins such as elongation factors Ts, Tu and G, fructose-bisphosphate aldolase and triosephosphate isomerase but their expression declined following 4 h exposure. ATP binding cassette transporter protein and oligoendopeptidase F showed increased expression at 4 h. While Ag(I) is a potent antimicrobial agent this work demonstrates that S. aureus can mount a short-term protective response to exposure to the metal ion but that this is eventually overcome.
Keywords: Antimicrobial; Silver(I); Staphylococcus ; Proteomics; Oxidative stress

Despite hydrogen sulfide (H2S) and nitric oxide (NO) are important endogenous signals or bioregulators involved in many vital aspects of plant growth and responses against abiotic stresses, little information was known about their interaction. In the present study, we evaluated the effects of H2S and NO on alfalfa (Medicago sativa L.) plants exposed to cadmium (Cd) stress. Pretreatment with an H2S donor sodium hydrosulfide (NaHS) and well-known NO donor sodium nitroprusside (SNP) decreased the Cd toxicity. This conclusion was supported by the decreases of lipid peroxidation as well as the amelioration of seedling growth inhibition and Cd accumulation, in comparison with the Cd-stressed alone plants. Total activities and corresponding transcripts of antioxidant enzymes, including superoxide dismutase, peroxidase and ascorbate peroxidase were modulated differentially, thus leading to the alleviation of oxidative damage. Effects of H2S above were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the specific scavenger of NO. By using laser confocal scanning microscope combined with Greiss reagent method, further results showed that NO production increased significantly after the NaHS pretreatment regardless of whether Cd was applied or not, all of which were obviously inhibited by cPTIO. These decreases of NO production were consistent with the exaggerated syndromes associated with Cd toxicity. Together, above results suggested that NO was involved in the NaHS-induced alleviation of Cd toxicity in alfalfa seedlings, and also indicated that there exists a cross-talk between H2S and NO responsible for the increased abiotic stress tolerance.
Keywords: Alfalfa seedling roots; Cd-induced oxidative stress; Hydrogen sulfide; Nitric oxide; Sodium hydrosulfide; Sodium nitroprusside