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The first page of this article is displayed as the abstract.

Iron regulatory protein 1 (IRP-1) is a bifunctional protein involved in iron homeostasis and metabolism. In one state, it binds to specific sequences in the mRNA's of several proteins involved in iron and energy metabolism, thereby influencing their expression post-transcriptionally. In another state it contains a [4Fe–4S] iron–sulfur cofactor and displays aconitase activity in the cytosol. We have shown that this protein binds and hydrolyzes ATP, with kinetic and thermodynamic equilibrium constants that predict saturation with ATP, favouring a non-RNA-binding form at normal cellular ATP levels, and thus pointing to additional function(s) of the protein. Here we show for the first time that the RNA-binding and aconitase forms of IRP-1 can undergo interconversion dependent on the density of cells growing in culture. Thus, in high density confluent cultures, compared with low density, actively proliferating cultures, cytosolic aconitase activity is increased whereas RNA binding activity is diminished. This is accompanied by a decrease in transferrin receptor expression in confluent cells, possibly due to loss of the transcript-stabilizing activity of bound IRP-1. In high density HepG2 cultures, cytosolic glutamate and the ratio of reduced-to-oxidized glutathione were increased. We propose that increased cytosolic aconitase activity in confluent cultures may divert cytosolic citrate away from the fatty acid/membrane synthetic pathways required by dividing cells, into a glutamate-dependent maintenance of cellular macromolecular synthesis. In addition, this may confer additional protection from oxidative stress due to down-regulation of iron acquisition from transferrin and increased glutamate for glutathione synthesis.

Cytotoxicity assays of azolato-bridged dinuclear Pt(II) complexes, [{cis-Pt(NH3)2}2(μ-OH)(μ-azolato)]2+, where the azolato was pyrazolato (1), 1,2,3-triazolato-N1,N2 (2), tetrazolato-N1,N2 (3), or tetrazolato-N2,N3 (4), were performed in cisplatin-sensitive and -resistant human non-small-cell lung cancer cell lines (PC-9 and PC-14). These complexes largely circumvented the cisplatin resistance in both cell lines, with resistance factors for 1–4 in the range of 0.5–0.8 and 0.9–2.0 for PC-9 and PC-14 cells, respectively. Complex 4 exhibited approximately 10 times the cytotoxicity of 3. When 3 and 4 were reacted with 2 molar equiv. of 9-ethylguanine (9EtG), they yielded an identical product, [{cis-Pt(NH3)2(9EtG-N7)}2(μ-tetrazolato-N1,N3)]3+, that had N1,N3 platinum coordination through a Pt(II) migration process on the tetrazolate ring. The second-order rate kinetics of these isomers were almost the same as each other and faster than those of 1 and 2. The cytotoxicity of azolato-bridged complexes, except for 3, increases as their kinetic rates in the 9EtG reaction increase.

Copper transporter 1 (CTR1) is a transmembrane protein that imports copper(I) into yeast and mammalian cells. Surprisingly, the protein also mediates the uptake of platinum anticancer drugs, e.g. cisplatin and carboplatin. To study the effects of several metal-binding residues/motifs of hCTR1 on the transport of both Cu+ and cisplatin, we have constructed Hela cells that stably express a series of hCTR1 variant proteins including H22-24A, NHA, C189S, hCTR1ΔC, H139R and Y156A, and compared their abilities to regulate the accumulation and cytotoxicity of these metal compounds. Our results demonstrated that the cells expressing the hCTR1 mutants of histidine-rich motifs in the N-terminus (H22-24A, NHA) resulted in a higher basal copper level in the steady state compared to those expressing wild-type protein. However, the cellular accumulation of both copper and cisplatin in these variants was found at a similar level to that of wild type when incubated with an excess of metal compounds (100 μM). The cells expressing hCTR1 variants of H139R and Y156A exhibit lower capacities towards accumulation of copper but not cisplatin. Significantly, cells with the C189S variant partially retained the ability of the wild-type hCTR1 protein to accumulate both copper and cisplatin, while for cells expressing the C-terminus truncated variant of hCTR1 (hCTR1ΔC) this ability was absolutely abolished, suggesting that this motif is crucial for the function of the transporter.

The complex process of carcinogenesis begins with transformation of a single cell to favor aberrant traits such as loss of contact inhibition and unregulated proliferation – features found in every cancer. Despite cancer's widespread prevalence, the early events that initiate cancer remain elusive, and without knowledge of these events cancer prevention is difficult. Here we show that exposure to As, Cr, Ni, or vanadium (V) promotes changes in gene expression that occur in conjunction with aberrant growth. We exposed immortalized human bronchial epithelial cells to one of four metals/metalloid for four to eight weeks and selected transformed clonal populations based upon anchorage independent growth of single cells in soft agar. We detected a metal-specific footprint of cancer-related gene expression that was consistent across multiple transformed clones. These gene expression changes persisted in the absence of the progenitor metal for numerous cell divisions. Our results show that even a brief exposure to a carcinogenic metal may cause many changes in gene expression in the exposed cells, and that from these many changes, the specific change(s) that each metal causes that initiate cancer likely arise.

A ferrocene–quinoline conjugate named ferroquine (FQ or SSR97193) is active against both chloroquine-susceptible and chloroquine-resistant Plasmodium falciparum and P. vivax strains and/or isolates. FQ was shown to be efficient for the treatment of uncomplicated malaria in humans (phase IIb of clinical trials). However, the molecular basis of FQ's mechanism of action is still unknown because few approaches (such as radioactive labelling or immunofluorescence) are available for that purpose. Previous reports from our laboratory suggest that the intramolecular hydrogen bond in the lateral side chain plays a crucial role in the antimalarial activity of the drug. We used two ruthenocenic bioprobes of FQ (with and without an intramolecular hydrogen bond) to study their localization and quantification in Plasmodium falciparum-infected erythrocytes. We first used Inductively Coupled Plasma Mass Spectroscopy (ICP-MS) analysis to trace ruthenoquine (RQ, with an intramolecular hydrogen bond) and methylruthenoquine (Me-RQ, without an intramolecular hydrogen bond) in the infected red blood cells (iRBCs). We showed that RQ accumulates faster in the digestive vacuole of the iRBCs than Me-RQ. We next examined the ruthenium distribution at the ultrastructural level by transmission electron microscopy (TEM). We showed that RQ accumulates faster in the parasitic digestive vacuole (DV) close to its membranes than Me-RQ.

Zn is essential to the structure and function of numerous proteins and enzymes so requires tight homeostatic control at both the systemic and cellular level. Two families of Zn transporters – ZIP (SLC39) and ZnT (SLC30) – contribute to Zn homeostasis. There are at least 10 members of the human ZnT family, and the expression profile and regulation of each varies depending on tissue type. Little is known about the role and expression pattern of ZnT10; however in silico data predict restricted expression to foetal tissue. We show a differential expression profile for ZnT10 in adult human tissue by RT-qPCR and detect highest levels of expression in small intestine, liver and brain tissues. We present data revealing the functional activity of ZnT10 to be in the efflux direction. Using a plasmid construct to express ZnT10 with an N-terminal FLAG-epitope tag, we reveal subcellular localisation in a neuroblastoma cell line (SH-SY5Y) to be at the Golgi apparatus under standard conditions of culture, with trafficking to the plasma membrane observed at higher extracellular Zn concentrations. We demonstrate down-regulation by Zn of ZnT10 mRNA levels in cultured intestinal and neuroblastoma cell lines and demonstrate reduced transcription from the ZnT10 promoter at an elevated extracellular Zn concentration. These features of ZnT10 localisation, regulation and function, together with the discovery that ZnT10 is expressed a high levels in brain tissue, indicate that ZnT10 has a role in regulating Zn homeostasis in the brain so may have relevance to the development of neurodegenerative disease.

The first page of this article is displayed as the abstract.

The first page of this article is displayed as the abstract.

In this work, sunflowers (Helianthus annuus L.) were cultivated using soil and vermicompost as substrate, and plant irrigation was carried out using either a Zn solution or a mixed ions solution (Cd, Cu, Pb and Zn). After plant harvesting, the effects of metal-ion contamination on proteins expression (either up- or down-regulation) in sunflower leaves were evaluated using two-dimensional electrophoresis (2-DE), gel images and mass spectrometry (MALDI-QTOF MS). When Zn or mixed ions solution was added to the substrate, nine proteins showed different expressions. Another twenty-three protein spots also showed considerable variation when both treatments (Zn or mixed ions) were applied. Twelve of these proteins were successfully characterized, six of them being reported for the first time in Helianthus annuus L. Two other proteins showed new sequences that have been downloaded to the protein databank.