Arsenic is a human carcinogen, causing skin, bladder, and lung cancers. Although arsenic in drinking water affects millions of people worldwide, the mechanism(s) of action by which arsenic causes cancers is not known. Arsenic probably exerts some toxic effects by binding with proteins. However, few experimental data are available on arsenic-containing proteins in biological systems. This study reports on arsenic interaction with metallothionein and established binding stoichiometries between metallothionein and the recently discovered trivalent metabolites of arsenic metabolism. Size exclusion chromatography with inductively coupled plasma mass spectrometry analysis of reaction mixtures between trivalent arsenicals and metallothionein clearly demonstrated the formation of complexes of arsenic with metallothionein. Analysis of the complexes using electrospray quadrupole time-of-flight tandem mass spectrometry revealed the detailed binding stoichiometry between arsenic and the 20 Cys residues in the metallothionein molecule. Inorganic arsenite (As(III)) and its two trivalent methylation metabolites, monomethylarsonous acid (MMA(III)) and dimethylarsinous acid (DMA(III)), readily bind with metallothionein. Each metallothionein molecule could bind with up to six As(III), 10 MMA(III), and 20 DMA(III) molecules, consistent with the coordination chemistry of these arsenicals. The findings on arsenic interaction with proteins are useful for a better understanding of arsenic health effects.

mRNA isolation for the purpose of cDNA library construction was performed in a microfluidic chip device using paramagnetic oligo-dT beads. The simple Y-intersection flow design mixes beads and sample on-chip, and uses magnetic trapping to capture, then release the beads. The capillary gel electrophoresis (CGE) detection of the total unamplified mRNA isolated on-chip, and of a reverse transcription-polymerase chain reaction (RT-PCR) amplified rare gene indicated that mRNA could be captured by oligo-dT beads on-chip, had very good integrity and was suitable for constructing a cDNA library. The limit of detection for the rare bicoid gene of Drosophila Melanogaster corresponded to the capture of approximately 2.8 ng of total mRNA from 0.85 microgram of total RNA (TRNA) within the microchip. As much as 34 ng of total mRNA was estimated to be captured from 10 micrograms of TRNA.

No abstract

Cisplatin is widely used to treat a number of cancers, and its covalent binding to DNA is believed to cause cell death; however, the roles of cisplatin-protein interactions in the mechanisms of action, toxicity, and resistance of the drug largely remain to be elucidated. Here, we investigate the interactions of cisplatin and a native rabbit metallothionein (MT), containing 1.4% zinc and 7.9% cadmium, using nanospray tandem quadrupole time-of-flight mass spectrometry (MS) and size-exclusion high-performance liquid chromatography with inductively coupled plasma MS. At near-neutral pH conditions, reactions between cisplatin and MT resulted in the formation of complexes that contained Cd 4 -Pt n -MT (n = 1-7). While zinc was displaced by cisplatin, both platinum and cadmium were bound to the same MT molecule. This is the first report to provide direct evidence for the co-binding of cadmium and platinum to MT, which suggests that the mechanism of the binding of cisplatin to the native MT may not be through the displacement of cadmium as previously proposed. A tandem MS investigation into the binding sites of the platinum and cadmium to MT showed platinum-and cadmium-related fragments, such as (PtS 2 C 2 H 7 N) + and (CdS 3 C 5 H 17 N 2 ) + , demonstrating the platinum-cysteine and cadmium-cysteine binding. In addition, detection of Cd 4 -Pt 7 -MT demonstrated more than ten metals bound to a single MT molecule. This finding was extended to the binding of MT with a five-fold excess of CdCl 2 . As many as 14 metal atoms (13 cadmium and one zinc) were detected bound to a single MT molecule, the complexes being Cd x -Zn-MT (x = 5-13). The high binding capacity of MT for cadmium and platinum is consistent with the role of MT in reduction of metal toxicity and its involvement in drug resistance.