BioMetals (v.26, #2)

Inhibitory zinc sites in enzymes by Wolfgang Maret (197-204).
Several pathways increase the concentrations of cellular free zinc(II) ions. Such fluctuations suggest that zinc(II) ions are signalling ions used for the regulation of proteins. One function is the inhibition of enzymes. It is quite common that enzymes bind zinc(II) ions with micro- or nanomolar affinities in their active sites that contain catalytic dyads or triads with a combination of glutamate (aspartate), histidine and cysteine residues, which are all typical zinc-binding ligands. However, for such binding to be physiologically significant, the binding constants must be compatible with the cellular availability of zinc(II) ions. The affinity of inhibitory zinc(II) ions for receptor protein tyrosine phosphatase β is particularly high (K i = 21 pM, pH 7.4), indicating that some enzymes bind zinc almost as strongly as zinc metalloenzymes. The competitive pattern of zinc inhibition for this phosphatase implicates its active site cysteine and nearby residues in the coordination of zinc. Quantitative biophysical data on both affinities of proteins for zinc and cellular zinc(II) ion concentrations provide the basis for examining the physiological significance of inhibitory zinc-binding sites in proteins and the role of zinc(II) ions in cellular signalling. Regulatory functions of zinc(II) ions add a significant level of complexity to biological control of metabolism and signal transduction and embody a new paradigm for the role of transition metal ions in cell biology.
Keywords: Zinc; Enzyme inhibition; Enzyme active sites

Zinc-rich inhibitor of apoptosis proteins (IAPs) as regulatory factors in the epithelium of normal and inflamed airways by Eugene Roscioli; Rhys Hamon; Susan Lester; Chiara Murgia; Janet Grant; Peter Zalewski (205-227).
Integrity of the airway epithelium (AE) is important in the context of inhaled allergens and noxious substances, particularly during asthma-related airway inflammation where there is increased vulnerability of the AE to cell death. Apoptosis involves a number of signaling pathways which activate procaspases leading to cleavage of critical substrates. Understanding the factors which regulate AE caspases is important for development of strategies to minimize AE damage and airway inflammation, and therefore to better control asthma. One such factor is the essential dietary metal zinc. Zinc deficiency results in enhanced AE apoptosis, and worsened airway inflammation. This has implications for asthma, where abnormalities in zinc homeostasis have been observed. Zinc is thought to suppress the steps involved in caspase-3 activation. One target of zinc is the family of inhibitor of apoptosis proteins (IAPs) which are endogenous regulators of caspases. More studies are needed to identify the roles of IAPs in regulating apoptosis in normal and inflamed airways and to study their interaction with labile zinc ions. This new information will provide a framework for future clinical studies aimed at monitoring and management of airway zinc levels as well as minimising airway damage and inflammation in asthma.
Keywords: Airway epithelium; Apoptosis; Asthma; Inflammation; Zinc

Comparative study of human liver ferritin and spleen tissues from healthy human and patient with primary myelofibrosis was carried out using Mössbauer spectroscopy with a high velocity resolution at 295 and 90 K and with a low velocity resolution at 20 K. The results obtained demonstrated that the iron content in patient’s spleen in the form of iron storage proteins was about ten times larger than that in normal tissue. However, in the case of patient with primary myelofibrosis the magnetic anisotropy energy barrier differed from that in normal case and, probably, the iron core size was supposed to be slightly larger than that in both normal spleen tissue and normal human liver ferritin in contrast to well-known data for iron overload in patients with thalassemia accompanied by the iron-core size increase. Therefore, the iron overload in the case of patient with primary myelofibrosis may be related to increase in the ferritin content mainly. It was also found that Mössbauer hyperfine parameters for normal and patient’s spleen and normal human liver ferritin demonstrated some small differences related, probably, to some small structural variations in the ferritin iron cores of patient’s spleen.
Keywords: Iron storage proteins; Spleen tissues; Mössbauer spectroscopy; Primary myelofibrosis

Lithium enhances TRAIL-induced apoptosis in human lung carcinoma A549 cells by Yan Lan; Xiufeng Liu; Rong Zhang; Kai Wang; Yao Wang; Zi-Chun Hua (241-254).
Non-small cell lung cancer (NSCLC) A549 cells are resistant to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. Therefore, combination therapy using sensitizing agents to overcome TRAIL resistance may provide new strategies for treatment of NSCLC. Here, we investigated whether lithium chloride (LiCl), a drug for mental illness, could sensitize A549 cells to TRAIL-induced apoptosis. We observed that LiCl significantly enhanced A549 cells apoptosis through up-regulation of death receptors DR4 and DR5 and activation of caspase cascades. In addition, G2/M arrest induced by LiCl also contributed to TRAIL-induced apoptosis. Concomitantly, LiCl strongly inhibited the activity of c-Jun N-terminal kinases (JNKs), and the inhibition of JNKs by SP600125 also induced G2/M arrest and augmented cell death caused by TRAIL or TRAIL plus LiCl. However, glycogen synthase kinase-3β (GSK3β) inhibition was not involved in TRAIL sensitization induced by LiCl. Collectively, these findings indicated that LiCl sensitized A549 cells to TRAIL-induced apoptosis through caspases-dependent apoptotic pathway via death receptors signaling and G2/M arrest induced by inhibition of JNK activation, but independent of GSK3β.
Keywords: LiCl; TRAIL; Apoptosis; A549; G2/M arrest

Research on NO in plants has achieved huge attention in recent years mainly due to its function in plant growth and development under biotic and abiotic stresses. In the present study, we investigated Cd induced NO generation and its relationship to ROS and antioxidant regulation in Brassica juncea. Cd accumulated rapidly in roots and caused oxidative stress as indicated by increased level of lipid peroxidation and H2O2 thus, inhibiting the overall plant growth. It significantly decreased the root length, leaf water content and photosynthetic pigments. A rapid induction in intracellular NO was observed at initial exposures and low concentrations of Cd. A 2.74-fold increase in intracellular NO was recorded in roots treated with 25 μM Cd than control. NO effects on Malondialdehyde (MDA) content and on antioxidant system was investigated by using sodium nitroprusside (SNP), a NO donor and a scavenger, [2-(4-carboxy-2-phenyl)-4,4,5,5-tetramethylinidazoline-1-oxyl-3-oxide] (cPTIO). Roots pretreated with 5 mM SNP for 6 h when exposed to 25 μM Cd for 24 h reduced the level of proline, non-protein thiols, SOD, APX and CAT in comparison to only Cd treatments. However, this effect was almost blocked by 100 μM cPTIO pretreatment to roots for 1 h. This ameliorating effect of NO was specific because cPTIO completely reversed the effect in the presence of Cd. Thus, the present study report that NO strongly counteracts Cd induced ROS mediated cytotoxicity in B. juncea by controlling antioxidant metabolism as the related studies are not well reported in this species.
Keywords: Nitric oxide; Cadmium; Oxidative stress; B. juncea ; Antioxidant

The Rhizobia are a group of free-living soil bacteria known for their ability to symbiotically infect the roots of specific host plants as well as to produce siderophores in order to compete with other microorganisms for the limited availability of iron in the rhizosphere. In this study, Rhizobium leguminosarum ATCC 14479, which preferentially infects the red clover Trifolium pratense, was found to produce the trihydroxamate siderophore vicibactin (C33H55N6O15) under iron restricted conditions. In addition, two other iron-binding, siderophore-like compounds: C20H36N4O10, C31H55N6O15, were isolated and purified from the culture media. Due to the structural similarity of the latter compounds to vicibactin based on electrospray-mass spectrometry and nuclear magnetic resonance data, these heretofore unreported molecules are thought to be either modified or degraded products of vicibactin. Although vicibactin has previously been found to be commonly produced by other rhizobial strains, this is the first time it has been chemically characterized from a clover infecting strain of R. leguminosarum.
Keywords: Vicibactin; Siderophore; Iron; Rhizobium leguminosarum ; ATCC 14479; Trifolii

Selenium effect on selenoprotein transcriptome in chondrocytes by Jidong Yan; Yuewen Zheng; Zixin Min; Qilan Ning; Shemin Lu (285-296).
Selenium is an essential micronutrient and exerts its biological functions predominantly through selenoproteins. Selenium deficiency is associated with cartilage function. This study demonstrated that all 24 selenoprotein transcripts in mouse genome were detectable in ATDC5 chondrocytes except deiodinase 1 (DIO1), DIO2, and selenoprotein V (Sel V), while all 25 selenoprotein transcripts in human genome were detectable in C28/I2 chondrocytes except glutathione peroxidase 6 (GPx6) and DIO1. In addition, gene expression of five selenoproteins (GPx1, Sel H, Sel N, Sel P, and Sel W) was up-regulated and two selenoproteins (SPS2 and Sel O) was down-regulated by sodium selenite (Se) in both ATDC5 and C28/I2 cells. Gene expression of six selenoproteins (TrxR1, Sel I, Sel M, Sel R, Sel S, Sel T) and one selenoprotein (GPx3) was up-regulated by Se in ATDC5 and C28/I2 cells, respectively. Gene expression of one selenoprotein (TrxR2) was down-regulated by Se only in ATDC5 cells. Further transcription inhibition assay showed that both transcriptional and posttranscriptional mechanisms involved in Se-regulated gene expression of GPx1, TrxR1, TrxR2, SPS2, Sel O, and Sel S. However, Se-regulated gene expression of Sel H, Sel I, Sel M, Sel N, Sel P, Sel R, Sel T, and Sel W mainly at posttranscriptional level. Moreover, new protein synthesis inhibition assay indicated that Se-mediated new protein synthesis also played roles in Se-regulated gene expression of GPx1, TrxR1, TrxR2, Sel H, Sel O, Sel P, Sel R, and Sel W. In summary, this study described the selenoprotein transcriptome, Se-regulated selenoproteins and possible mechanisms involved in chondrocytes.
Keywords: Chondrocyte; Selenium; Selenoprotein; Real-time PCR; Gene expression

Comparative study of alleviating effects of GSH, Se and Zn under combined contamination of cadmium and chromium in rice (Oryza sativa) by Fangbin Cao; Nanbo Wang; Mian Zhang; Huaxin Dai; Muhammad Dawood; Guoping Zhang; Feibo Wu (297-308).
A hydroponic experiment was conducted to study the ameliorative effects of separate or combined application of exogenous glutathione (GSH), selenium (Se) and zinc (Zn) upon 20 μM cadmium (Cd) plus 20 μM chromium (Cr) heavy metal stress (HM) in rice seedlings. The results showed that HM caused a marked reduction in seedling height, chlorophyll content (SPAD) and biomass, and activities of catalase (CAT) and ascorbate peroxidase (APX) in leaves and H+-ATPase in roots/leaves, but elevated superoxide dismutase (SOD) and guaiacol peroxidase (POD) activities in leaves with elevated malondialdehyde (MDA) accumulation both in leaves and roots over the control. The best mitigation effect was recorded in HM+GSH+Zn and HM+GSH (addition of GSH+Zn and GSH to HM solution), which greatly alleviated HM-induced growth inhibition and oxidative stress. Compared with HM alone, HM+GSH and HM+GSH+Zn markedly reduced Cr uptake and translocation but not affected Cd concentration; improved H+-ATPase activity and Fe, Zn, Mn uptake and translocation, and repressed MDA accumulation. Meanwhile exogenous GSH and GSH+Zn counteracted HM-induced response of antioxidant enzymes, via suppressing HM-induced dramatic increase of root/leaf SOD and leaf POD activities, and elevating stress-depressed leaf APX and leaf/root CAT activities.
Keywords: Adenosine triphosphatases (ATPase); Alleviating effects; Cadmium; Chromium; Combined contamination; Rice (Oryza sativa) ; Oxidative stress

Two unicellular cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 showed contrasting responses to chromate stress with EC50 of 12 ± 2 and 150 ± 15 μM potassium dichromate respectively. There was no depletion of chromate in growth medium in both the cases. Using labeled chromate, very low accumulation (<1 nmol/108 cells) was observed in Synechocystis after incubation for 24 h in light. No accumulation of chromate could be observed in Synechococcus under these conditions. Chromate oxyanion is known to enter the cells using sulfate uptake channels. Therefore, inhibition of sulfate uptake caused by chromate was monitored using 35S labeled sulfate. IC50 values of chromate for 35sulfate uptake were higher in Synechococcus as compared to Synechocystis. The results suggested that the sulfate transporters in Synechococcus have lower affinity to chromate than those from Synechocystis possibly due to differences in affinity of sulfate receptors for chromate. Bioinformatic analyses revealed presence of sulfate and chromate transporters with considerable similarity; however, minor differences in these may play a role in their differential response to chromate. In both cases the IC50 values decreased when sulfate concentration was reduced in the medium indicating competitive inhibition of sulfate uptake by chromate. Interestingly, Synechococcus showed stimulation of growth at concentrations of chromate less than 100 μM, which affected its cell size without disturbing the ultrastructure and thylakoid organization. In Synechocystis, growth with 12 μM potassium dichromate damaged the ultrastructure and thylakoid organization with slight elongation of the cells. The results suggested that Synechococcus possesses efficient strategies to prevent entry and to remove chromate from the cell as compared to Synechocystis. This is the first time a differential response of Synechococcus 7942 and Synechocystis 6803 to chromate is reported. The contrasting characteristics observed in the two cyanobacteria will be useful in understanding the basis of resistance or susceptibility to chromate.
Keywords: Chromate-resistance; Cyanobacteria; IC50 chromate; Sulfate-uptake; Synechococcus PCC 7942; Synechocystis PCC 6803

Ferrous iron chelating property of low-molecular weight succinoglycans isolated from Sinorhizobium meliloti by Eunae Cho; Jae Min Choi; Hwanhee Kim; Muhammad Nazir Tahir; Youngjin Choi; Seunho Jung (321-328).
Iron is an essential nutrient for nitrogen-fixing legume root nodules, and the chelation of ferrous iron plays an important role in the mobility and availability of iron to the legume. In the present study, we investigated the iron-binding properties of low-molecular weight succinoglycans isolated from the nitrogen-fixing bacterium, Sinorhizobium meliloti. The low-molecular weight succinoglycans comprising three monomers (M1–M3), four dimers (D1–D4), and six trimers (T1–T6) of the succinoglycan repeating unit were purified by various chromatographic techniques. Interestingly, the colorimetric ferrozine method showed that the succinoglycans T6, M3, and D3 demonstrated a ferrous iron chelating ability of 83, 63, and 38 % per mg, respectively. The individual binding constants were determined as 43703, 2313, and 760 M−1 for succinoglycans T6, M3, and D3 using ultraviolet–visible spectroscopy. The complexation of succinoglycan and ferrous iron can cause structural changes, which were analyzed by circular dichroism spectroscopy. Furthermore, the complex could provide antioxidant activity through an anti-Fenton reaction. These results demonstrate that the low-molecular weight succinoglycans can effectively modulate iron biochemistry as a novel ferrous iron-acquisition system of S. meliloti.
Keywords: Low-molecular weight succinoglycans; Sinorhizobium meliloti ; Ferrous iron; Chelating ability; Fenton reaction

Protective effect of l-ascorbic acid on nickel induced pulmonary nitrosative stress in male albino rats by Shaheenkousar H. Hattiwale; Sikha Saha; Saeed M. Yendigeri; Jameel G. Jargar; Salim A. Dhundasi; Kusal K. Das (329-336).
Nickel sulfate stimulates inducible nitric oxide synthase (i-NOS) and increases serum nitric oxide concentration by overproduction of reactive nitrogen species due to nitrosative stress. The present study was undertaken to assess possible protective role of l-ascorbic acid as an antioxidant against nickel induced pulmonary nitrosative stress in male albino rats. We studied the effect of the simultaneous treatment with l-ascorbic acid (50 mg/100 g b. wt.; orally) and nickel sulfate (2.0 mg/100 g b. wt.; i.p.) on nitric oxide synthesis by quantitative evaluation of serum i-NOS activities, serum and lung nitric oxide, l-ascorbic acid and protein concentrations of Wister strain male albino rats. We have further studied histopathological changes in lung tissue after nickel sulfate treatment along with simultaneous exposure of l-ascorbic acid. Nickel sulfate treatment significantly increased the serum i-NOS activity, serum and pulmonary nitric oxide concentration and decreased body weight, pulmonary somatic index, serum and lung l-ascorbic acid and protein concentration as compared to their respective controls. Histopathological changes induced by nickel sulfate showed loss of normal alveolar architecture, inflammation of bronchioles, infiltration of inflammatory cells and patchy congestion of alveolar blood vessels. The simultaneous administration of l-ascorbic acid and nickel sulfate significantly improved all the above biochemical parameters along with histopathology of lung tissues of rats receiving nickel sulfate alone. The study clearly showed a protective role of l-ascorbic acid against nickel induced nitrosative stress in lung tissues.
Keywords: Nickel sulfate; i-NOS activity; Nitrosative stress; Lung histopathology; l-ascorbic acid

The exposure to arsenic, a potential genotoxic carcinogen in humans, via drinking water is a serious worldwide health hazard. The arsenic content of 10 μg L−1 in drinking water, however, has been established as its guideline standard (maximum contaminant limit) that has been estimated to pose minimum risk to cancer. Since micronucleus induction in the erythrocytes of fish is a sensitive indicator of genotoxic agents in water, the piscine micronucleus assay was used in the present experiment to assess the genotoxic potential of arsenic at its various exposure levels including the guideline value for drinking water. The experiments were conducted in two different species of fishes, the pond murrel (Channa punctatus) and the goldfish (Carassius auratus). Significant increases in the frequency of micronucleated erythrocytes were documented in a dose-dependent manner in both Channa and Carassius. The fishes, however, exhibited variations in inter-specific sensitivity to micronucleus induction following arsenic exposure. The exposure level of arsenic at its guideline value for drinking water, therefore, exhibited marked genotoxicity in fishes.
Keywords: Arsenic; Genotoxicity; Fish; Guideline value; Micronucleus

Chromium levels in insulin-sensitive tissues and the thigh bone are modulated by prednisolone and high-fat diets in mice by Po-Wen Chen; Chang Lin; Chung-De Chen; Wen-Ying Chen; Frank Chiahung Mao (347-354).
Glucocorticoids (GCs) are often prescribed in clinics but many adverse effects are also attributed to GCs. It is important to determine the role of GCs in the development of those adverse effects. Here, we investigated the impact of GCs on trivalent chromium (Cr) distribution in animals. Cr has been proposed to be important for proper insulin sensitivity, and deficits may lead to disruption of metabolism. For comparison, the effect of a high-fat diet on Cr modulation was also evaluated. C57BL/6JNarl mice were fed regular or high-fat diets for 12 weeks and further grouped for treatment with prednisolone or saline. Cr levels in tissues were determined 12 h after the treatments. Interestingly, prednisolone treatment led to significantly reduced Cr levels in fat tissue in mice fed regular diets; compared to the high-fat diet alone, prednisolone plus the high-fat diet led to a further reduction in Cr levels in the liver, muscle, and fat. Notably, a single dose of prednisolone was linked with elevated Cr levels in the thigh bones of mice fed by either regular or high-fat diets. In conclusion, this report has provided evidence that prednisolone in combination with a high-fat diet effects modulation of Cr levels in selected tissues.
Keywords: Chromium; Glucocorticoids; Prednisolone; Insulin; Obese mice; Trace elements

Effect of oxygen free radicals and nitric oxide on apoptosis of immune organ induced by selenium deficiency in chickens by Zi-wei Zhang; Jiu-li Zhang; Yu-hong Zhang; Qiao-hong Wang; Shu Li; Xiao-long Wang; Shi-wen Xu (355-365).
Selenium is an essential element with antioxidant roles in immune regulation, but there is little understanding of how Se acts in apoptosis in the immune organs of birds. The aim of study was to evaluate the influence of Se deficiency on oxygen free radicals, NO and apoptosis in immune organ of chickens. 160 1-day-old chickens were randomly assigned to two groups of 80 each and were fed on a low-Se diet (0.032 mg/kg Se) or a control diet (0.282 mg/kg Se), respectively. OFR production in blood was determined on days 30, 45, 60 and 75, respectively. The iNOS–NO system activity in immune organ (thymus, spleen, bursa of fabricius) was identified by NO content and NOS activity assay on days 30, 45, 60 and 75, respectively. Apoptosis was measured by DNA ladder analysis, ultrastructural observations, TdT-mediated dUTP nick end labeling TUNEL assay and flow cytometric analysis of apoptotic DNA. The transcription of factor-associated suicide, caspase-3 mRNA was tested by fluorescence quantitative PCR. The results showed that OFR production, NO and inducible NO synthases (iNOS) activity in the low-Se group were significantly increased (p < 0.05) than in the control group. In addition, apoptosis was observed in chicken immune organ in the low-Se group. The degree and the number of apoptotic cells rose in a time-dependent manner. The expression of Fas and caspase-3 mRNA increased (p < 0.05) than in the control group. It indicated that the oxidative stress and NO played a causative role in the apoptosis of immune tissues induced by selenium deficiency.
Keywords: Selenium deficiency; NO; Apoptosis; Immune organ; Chicken

Erratum to: Effect of oxygen free radicals and nitric oxide on apoptosis of immune organ induced by selenium deficiency in chickens by Zi-wei Zhang; Jiu-li Zhang; Yu-hong Gao; Qiao-hong Wang; Shu Li; Xiao-long Wang; Shi-wen Xu (367-367).