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Characterization of Cd-binding proteins has great analytical interest due to the high toxicity of Cd to living organisms. Metallothioneins (MTs), as Cd(II)-binding proteins are of increasing interest, since they form very stable Cd chelates and are involved in many detoxification processes. In this work, inductively coupled plasma octopole reaction cell mass spectrometry and nanospray ionization time-of-flight mass spectrometry were used in parallel and combined with two-dimensional chromatography: size exclusion followed by reversed-phase high performance liquid chromatography, to study metal complexes of MT isoforms produced in hepatic cytosols of Mus musculus during exposure experiments to Cd. Exposure experiments were carried out by subcutaneous injection of a growing dose of the toxic element ranging from 0.1 to 1.0 mg of Cd per kg of body weight per day during 10 days. A control group and three exposure groups at days 2, 6 and 10 of exposure were studied, and different cadmium, copper and zinc complexes with MTs isoforms were isolated and characterized from the two most exposed groups. The results allow gaining insight into the mechanisms involved in metal detoxification by MTs, showing the changes in the stoichiometry of metal complexes-MTs along cadmium exposure.

A metallomic approach based on the use of size-exclusion chromatography (Superdex-75) with inductively coupled plasma mass spectrometry (ICP-MS) detection is combined with anion or cation exchange chromatography to characterize the biological response of the free-living mouse Mus spretus. The approach has been applied to contaminated and non-contaminated areas from Doñana National Park (southwest Spain) and the surroundings. Several areas affected by differential contamination from mining, industrial, and agricultural activities have been considered. The high presence of Mn, Cu, and Zn in liver and As and Cd in kidney is remarkable, especially in contaminated areas. The size exclusion chromatograms traced by Mn in liver cytosolic extracts are more intense than in kidney; a Mn-peak matching with the standard of 32 kDa (superoxide dismutase) is present in these organs, and its intensity is correlated with the concentration of Mn in the extracts. High-intensity peaks traced by Cu, Zn, and Cd at 7 kDa (matching with metallothionein I standard) in liver extract are triggered by the presence of contaminants. Other peaks related with molecules of 32 and 67 kDa traced by Cu and Zn can also be observed, although their intensity is higher in sites with low contamination. In kidney extracts, the presence of a Cd-peak with Mr of 7 kDa (tentatively Cd-metallothionein) with high intensity under the action of contaminants was observed, but high biological responses are also proven in the protected area of the Park, which denotes a progressive increase of diffuse contamination.

Mus musculus mice have been investigated for the total elements content in different organs (lung, liver, spleen, kidney, brain, testicle, heart and muscle) and molecular mass distribution patterns of Mn, Ni, Cu, Zn, As, Pb, Cr, Fe, Co, Se and Cd. Some differences have been found in the organs studied, with especially relevant being the Cu-containing fraction present only in the brain and the As-containing one in the liver. Other differences related to the abundance of the metallospecies have also been found. The present paper is the first step in the study of the "metallome" of this inbred laboratory species from which the genome is completely known. This organism could be used as a model in future studies focused on wild mice and the analytical approach developed could be applied to wild mice to find markers of environmental pollution. [figure: see text] The present paper is the first step in the study of the "metallome" of the inbred laboratory specie Mus musculus from which the genome is completely known. Some interesting differences have been found in the extracts from the organs that are discussed along the text.

A speciation approach based on orthogonal chromatographic systems coupled to inductively coupled plasma mass spectrometry (ICP-MS) was used to characterise the biological response of free-living mice Mus spretus to environmental pollution caused by arsenic in different areas of the Doñana National Park (south-west Spain). The relative presence of inorganic and organic forms of arsenic was studied in cytosolic extracts from high metabolic activity organs of Mus spretus mice: kidneys, liver, and brain. An instrumental coupling of size-exclusion chromatography with UV and collision/reaction cell-ICP-MS detectors (SEC-UV-ICP-ORC-MS) both in analytical and preparative scale was used for this purpose. The results showed the presence of low molecular mass (LMM) molecules linked to arsenic in these tissues especially in the kidneys, where the presence of these arsenic metabolites was higher. On the other hand, the presence of these arsenicals varied from one area to the other, which can be related to a different occurrence of contaminants. These low molecular mass fractions were collected by preparative SEC chromatography for later study with ion exchange chromatography and detection by ICP-ORC-MS, using both anionic and cationic columns. The results showed the higher presence of MMA and DMA in kidneys of mice caught in contaminated areas and the existence of small amounts of unidentified arsenicals when cation-exchange chromatography was used, which could be related to the presence of dimethylarsinoylethanol (DMAE), thioarsenic species, or arsenocholine (AsC).

We have previously reported the isolation of an Escherichia coli K12 mutant that is extremely sensitive to mutagenesis by low doses of ethylating agents. We now show by Southern analysis that the mutation involves a gross deletion covering at least the ogt and fnr genes and that no O6-alkylguanine-DNA-alkyltransferase activity is present in cell-free extracts of an ada::Tn10 derivative of these bacteria. Confirmation that sensitisation to ethylation-induced mutagenesis was attributable to ogt and not to any other loci covered by the deletion was obtained by constructing derivatives. Thus an ogt::kanr disruption mutation was introduced into the parental ogt + bacteria, and the ogt::kanr mutation was then eliminated by cotransduction of ogt + with the closely linked Tet r marker (zcj::Tn10). The Δ(ogt-fnr) deletion or ogt::kanr disruption mutants were highly sensitive to ethyl methanesulphonate-induced mutagenesis, as measured by the induction of forward mutations to l-arabinose resistance (Ara1). Furthermore, the number of Arar mutants increased linearly with dose, unlike the case in ogt + bacteria, which had a threshold dose below which no mutants accumulated. Differences in mutability were even greater with propyl methanesulphonate. Overproduction of the ogt alkyltransferase from a multicopy plasmid reduced ethylmethanesulphonate-induced mutagenesis in the ogt mutant strains and also methylmethanesulphonate mutagenesis in ada − bacteria. A sample of AB 1157 obtained from the E. coli K12 genetic stock centre also had a deletion covering the ogt and fnr genes. Since such deletions greatly influence the mutagenic responses to alkylating agents, a survey of the presence of the ogt gene in the E. coli K12 strain being used is advisable.

Background Alternative splicing is a widespread mechanism of gene expression regulation. Previous analyses based on conventional RT-PCR reported the presence of an unspliced c-fos transcript in several mammalian systems. Compared to the well-defined knowledge on the alternative splicing of fosB, the physiological relevance of the unspliced c-fos transcript in regulating c-fos expression remains largely unknown. This work aimed to investigate the functional significance of the alternative splicing c-fos pre-mRNA. Results A set of primers was designed to demonstrate that, whereas introns 1 and 2 are regularly spliced from primary c-fos transcript, intron 3 remains unspliced in part of total transcript molecules. Here, the two species are referred to as c-fos-2 (+ intron 3) and spliced c-fos (- intron 3) transcripts. Then, we used a quantitatively rigorous approach based on real-time PCR to provide, for the first time, the actual steady-state copy numbers of the two c-fos transcripts. We tested how the mouse-organ context and mouse-gestational age, the synthesis and turnover rates of the investigated transcripts, and the serum stimulation of quiescent cells modulate their absolute-expression profiles. Intron 3 generates an in-frame premature termination codon that predicts the synthesis of a truncated c-Fos protein. This prediction was evaluated by immunoaffinity chromatography purification of c-Fos proteins. Conclusion We demonstrate that: (i) The c-fos-2 transcript is ubiquitously synthesized either in vivo or in vitro, in amounts that are higher or similar to those of mRNAs coding for other Fos family members, like FosB, ΔFosB, Fra-1 or Fra-2. (ii) Intron 3 confers to c-fos-2 an outstanding destabilizing effect of about 6-fold. (iii) Major determinant of c-fos-2 steady-state levels in cultured cells is its remarkably high rate of synthesis. (iv) Rapid changes in the synthesis and/or degradation rates of both c-fos transcripts in serum-stimulated cells give rise to rapid and transient changes in their relative proportions. Taken as a whole, these findings suggest a co-ordinated fine-tune of the two c-fos transcript species, supporting the notion that the alternative processing of the precursor mRNA might be physiologically relevant. Moreover, we detected a c-Fos immunoreactive species corresponding in mobility to the predicted truncated variant.

Mutants sensitive to moderate H2O2 concentrations were selected in a HfrphoA (S33)Escherichia coli strain after mutagenesis withN-methyl,N′-nitro,N-nitrosoguanidine (NG). Of the sensitive strains, 31% were catalase-deficient and retained glutathione reductase levels similar to those of the parental strain, whereas 69% still had normal catalase and glutathione reductase activities. Mutants supersensitive to low H2O2 concentrations were selected in a catalase-deficient strain (CGR201) after mutagenesis with NG. Of these, 20% were glutathione reductase-deficient, and the remaining 80% were unaffected in this enzymatic activity. Compared with the parental strain S33, H2O2 was 5 to 12 times more toxic for the sensitive mutants, and 19 to 21 times for the supersensitive ones.

The intracellular concentrations of total glutathione, GSSG and protein X S-SG, the total excreted glutathione concentration, and the susceptibility towards GSH-reacting compounds were assayed in strains of Escherichia coli deficient in biosynthesis and/or reduction of glutathione. A deficiency in glutathione reductase displaced the glutathione status towards the oxidized forms. This displacement was more clearly appreciated in strains additionally deficient in glutathione biosynthesis. A deficiency in catalase activity also produced an increase in the oxidation of glutathione. The most severe changes were observed in the concentrations of protein-glutathione mixed disulfides and in the amount of glutathione excreted to the medium. Increased sensitivities towards compounds known to interact with cellular GSH were observed in glutathione reductase deficient strains, although these effects were enhanced in strains additionally deficient in GSH biosynthesis.