Andreas Seubert

Cytosolic Monothiol Glutaredoxins Function in Intracellular Iron Sensing and Trafficking via Their Bound Iron-Sulfur Cluster

Iron is an essential nutrient for cells. It is unknown how iron, after its import into the cytosol, is specifically delivered to iron-dependent processes in various cellular compartments. Here, we identify an essential function of the conserved cytosolic monothiol glutaredoxins Grx3 and Grx4 in intracellular iron trafficking and sensing. Depletion of Grx3/4 specifically impaired all iron-requiring reactions in the cytosol, mitochondria, and nucleus, including the synthesis of Fe/S clusters, heme, and di-iron centers. These defects were caused by impairment of iron insertion into proteins and iron transfer to mitochondria, indicating that intracellular iron is not bioavailable, despite highly elevated cytosolic levels. The crucial task of Grx3/4 is mediated by a bridging, glutathione-containing Fe/S center that functions both as an iron sensor and in intracellular iron delivery. Collectively, our study uncovers an important role of monothiol glutaredoxins in cellular iron metabolism, with a surprising connection to cellular redox and sulfur metabolisms.

Cadmium Is a Novel and Independent Risk Factor for Early Atherosclerosis Mechanisms and In Vivo Relevance

Objectives—Although cadmium (Cd) is an important and common environmental pollutant and has been linked to cardiovascular diseases, little is known about its effects in initial stages of atherosclerosis. Methods and Results—In the 195 young healthy women of the Atherosclerosis Risk Factors in Female Youngsters (ARFY) study, cadmium (Cd) level was independently associated with early atherosclerotic vessel wall thickening (intima-media thickness exceeding the 90th percentile of the distribution; multivariable OR 1.6[1.1.–2.3], P=0.016). In line, Cd-fed ApoE knockout mice yielded a significantly increased aortic plaque surface compared to controls (9.5 versus 26.0 mm2, P<0.004). In vitro results indicate that physiological doses of Cd increase vascular endothelial permeability up to 6-fold by (1) inhibition of endothelial cell proliferation, and (2) induction of a caspase-independent but Bcl-xL-inhibitable form of cell death more than 72 hours after Cd addition. Both phenomena are preceded by Cd-induced DNA strand breaks and a cellular DNA damage response. Zinc showed a potent protective effect against deleterious effects of Cd both in the in vitro and human studies. Conclusion—Our research suggests Cd has promoting effects on early human and murine atherosclerosis, which were partly offset by high Zn concentrations.

A Specific Role of the Yeast Mitochondrial Carriers Mrs3/4p in Mitochondrial Iron Acquisition under Iron-limiting Conditions

The yeast genes MRS3 and MRS4 encode two members of the mitochondrial carrier family with high sequence similarity. To elucidate their function we utilized genome-wide expression profiling and found that both deletion and overexpression of MRS3/4 lead to up-regulation of several genes of the “iron regulon.” We therefore analyzed the two major iron-utilizing processes, heme formation and Fe/S protein biosynthesis in vivo, in organello (intact mitochondria), and in vitro (mitochondrial extracts). Radiolabeling of yeast cells with 55Fe revealed a clear correlation between MRS3/4 expression levels and the efficiency of these biosynthetic reactions indicating a role of the carriers in utilization and/or transport of iron in vivo. Similar effects on both heme formation and Fe/S protein biosynthesis were seen in organello using mitochondria isolated from cells grown under iron-limiting conditions. The correlation between MRS3/4 expression levels and the efficiency of the two iron-utilizing processes was lost upon detergent lysis of mitochondria. As no significant changes in the mitochondrial membrane potential were observed upon overexpression or deletion of MRS3/4, our results suggest that Mrs3/4p carriers are directly involved in mitochondrial iron uptake. Mrs3/4p function in mitochondrial iron transport becomes evident under iron-limiting conditions only, indicating that the two carriers do not represent the sole system for mitochondrial iron acquisition.

Copper Stress Affects Iron Homeostasis by Destabilizing Iron-Sulfur Cluster Formation in Bacillus subtilis

Copper and iron are essential elements for cellular growth. Although bacteria have to overcome limitations of these metals by affine and selective uptake, excessive amounts of both metals are toxic for the cells. Here we investigated the influences of copper stress on iron homeostasis in Bacillus subtilis, and we present evidence that copper excess leads to imbalances of intracellular iron metabolism by disturbing assembly of iron-sulfur cofactors. Connections between copper and iron homeostasis were initially observed in microarray studies showing upregulation of Fur-dependent genes under conditions of copper excess. This effect was found to be relieved in a csoR mutant showing constitutive copper efflux. In contrast, stronger Fur-dependent gene induction was found in a copper efflux-deficient copA mutant. A significant induction of the PerR regulon was not observed under copper stress, indicating that oxidative stress did not play a major role under these conditions. Intracellular iron and copper quantification revealed that the total iron content was stable during different states of copper excess or efflux and hence that global iron limitation did not account for copper-dependent Fur derepression. Strikingly, the microarray data for copper stress revealed a broad effect on the expression of genes coding for iron-sulfur cluster biogenesis (suf genes) and associated pathways such as cysteine biosynthesis and genes coding for iron-sulfur cluster proteins. Since these effects suggested an interaction of copper and iron-sulfur cluster maturation, a mutant with a conditional mutation of sufU, encoding the essential iron-sulfur scaffold protein in B. subtilis, was assayed for copper sensitivity, and its growth was found to be highly susceptible to copper stress. Further, different intracellular levels of SufU were found to influence the strength of Fur-dependent gene expression. By investigating the influence of copper on cluster-loaded SufU in vitro, Cu(I) was found to destabilize the scaffolded cluster at submicromolar concentrations. Thus, by interfering with iron-sulfur cluster formation, copper stress leads to enhanced expression of cluster scaffold and target proteins as well as iron and sulfur acquisition pathways, suggesting a possible feedback strategy to reestablish cluster biogenesis.

Multifunctional Nanoparticles for Dual Imaging

For imaging with different modalities, labels, which provide contrast for all modalities, are required. Colloidal nanoparticles composed out of an inorganic core and a polymer shell offer progress in this direction. Both, the core and the polymer shell, can be synthesized to be fluorescent, magnetic, or radioactive. When different cores are combined with different polymer shells, different types of particles for dual imaging can be obtained, as for example, fluorescent cores with radioactive polymer shells. Properties and perspectives of such nanoparticles for multimodal imaging are discussed.

Increased serum cadmium and strontium levels in young smokers : Effects on arterial endothelial cell gene transcription

Objective—Metal constituents of tobacco have long been suspected to contribute to cardiovascular diseases. In this study, we determined the serum concentrations of aluminum, cadmium (Cd), cobalt, copper, iron, manganese, nickel, lead, strontium (Sr), and zinc of young nonsmokers, passive smokers, and smokers. Methods and Results—Cd and Sr were found to be significantly increased in smokers compared with nonsmokers. The effects of these metals on primary arterial endothelial cells were then assessed using microarray technology and real-time polymerase chain reaction (RT-PCR). The data showed that Sr does not interfere with endothelial cell transcription. In contrast, the effects of Cd in amounts delivered to the human body by smoking were dramatic. Conclusions—Arterial endothelial cells responded to Cd exposure by massively upregulating metal and oxidant defense genes (metallothioneins) and by downregulating a number of transcription factors. In addition, the mRNA of the intermediate filament protein vimentin, crucial for the maintenance of cellular shape, was reduced. Surprisingly, a number of pro-inflammatory genes were downregulated in response to Cd. The present data suggest that by delivering Cd to the human body, smoking deregulates transcription, exerts stress, and damages the structure of the vascular endothelium; furthermore, in contrast to the effects of cigarette smoke as a whole, Cd seems to possess anti-inflammatory properties.

Synthesis and application of an inert type of 8-hydroxyquinoline-based chelating ion exchanger for sea-water analysis using on-line inductively coupled plasma mass spectrometry detection

An inert type of chelating ion exchanger is prepared by Friedel–Crafts alkylation of 8-hydroxyquinoline (8-HQ) with a previously chloromethylated poly(styrene)–divinylbenzene (PS/DVB) co-polymer. This type of Friedel–Crafts alkylation using a polymer alkylhalogenid results in an exchange capacity of 2.8 µmol Cu2+ per ml of bed volume. Because of the high formation constants of most transition and heavy metal ion complexes with 8-HQ, the low exchange capacity is still sufficient to preconcentrate a wide variety of ions from a high ionic strength sample such as sea-water. The introduction of a high-performance cation-exchange column into the elution flowpath enhanced the selectivity of the method significantly. From an initial set of 32 elements it is possible to preconcentrate 25 elements with a recovery of at least 50%. The majority of trace elements show a recovery of 60–90%. The introduction of a cation-exchange column into the elution flow path allowed the elimination of several remaining interferences. The basic performance, such as recovery and elution behaviour, as well as first results of applied analysis of sea-water using the Nearshore Seawater Reference Material for Trace Metals, CASS-2 (National Research Council of Canada) are given. The on-line detection using inductively coupled plasma mass spectrometry allows trace analysis at the low ng l–1 range in sea-water, which is the normal concentration level for most of the trace elements.

Comparison of on-line coupling of ion-chromatography with atmospheric pressure ionization mass spectrometry and with inductively coupled plasma mass spectrometry as tools for the ultra-trace analysis of bromate in surface water samples

Ion chromatography in combination with atmospheric pressure ionization mass spectrometry (API-MS) as well as with inductively coupled plasma mass spectrometry (ICP-MS) had been compared for trace analysis of bromate. The results indicate that both techniques yield comparable results, which are in excellent agreement with standard methods for bromate determination. Furthermore, both techniques showed almost equal absolute detection limits (approximately 50 pg bromate injected). Contrary to IC-API-MS, IC-ICP-MS can tolerate a higher salt concentration in the mobile phase. This allows the use of high-capacity columns combined with large sample volumes. This lowered the concentration based detection limits by one order of magnitude for IC-ICP-MS compared to IC-API-MS (0.06 microg/l vs. 0.5 microg/l). On the other hand, IC-API-MS is able to allow a positive identification of bromate even in cases when IC does not fully separate bromate from other bromine-containing species. The performance data of both IC-MS techniques have been established by participation in an international round robin test.

Germanium dioxide as internal standard for simplified trace determination of bromate, bromide, iodate and iodide by on-line coupling ion chromatography-inductively coupled plasma mass spectrometry.

The use of elemental mass spectrometry as detection for ion chromatography allows sensitive determination of several bromine and iodine species at a reasonable time scale. Lowest concentrations observable are 66 ng L(-1) for bromate, 45 ng L(-1) for iodate, 74 ng L(-1) for bromide and 151 ng L(-1) for iodide. A major drawback of previous IC-ICP-MS applications is the high consumption of time and thus the running costs. The use of GeO2 as internal standard not only allows improved external calibration, but also semiquantitative determination of bromate, bromide, iodate and iodide without any calibration procedure. Furthermore, GeO2 can be used for all known types of anion exchange columns regardless of their construction principles. It is shown, that the analyte-to-GeO2 ratio of four bromine and iodine species was nearly constant over 4 months and almost independent from the ICP-MS instrumental settings. The quantification by means of the analyte-to-GeO2 ratio for samples taken from a bromate round robin test shows that the values obtained are in excellent agreement with calibration curve and isotope dilution results.

Simultaneous determination of inorganic disinfection by-products and the seven standard anions by ion chromatography

For the first time, an ion chromatographic method for the simultaneous determination of the disinfection by-products bromate, chlorite, chlorate, and the so-called seven standard anions, fluoride, chloride, nitrite, sulfate, bromide, nitrate and orthophosphate is presented. The separation of the ten anions was carried out using a laboratory-made high-capacity anion-exchanger. The high capacity anion-exchanger allowed the direct injection of large sample volumes without any sample pretreatment, even in the case of hard water samples. For quantification of fluoride, chloride, nitrite, sulfate, bromide, nitrate, orthophosphate and chlorate, a conductivity detection method was applied after chemical suppression. The post-column reaction, based on chlorpromazine, was optimized for the determination of chlorite and bromate. The method detection limit for bromate measured in deionized water is 100 ng/l and for chlorite, it is 700 ng/l. In hard drinking water, the methods detection limits are 700 ng/l (bromate) and 3.5 microg/l (chlorite). The methods detection limits for the other eight anions, determined by conductivity detection, are between 100 microg/l (nitrite) and 1.6 mg/l (chlorate).