A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

We describe a whole-mount RNA in situ hybridization (ISH) method optimized for detection of the cellular and subcellular distributions of specific mRNA within Drosophila testes and male genital tract. Digoxygenin (dig)-labeled antisense RNA probes are in vitro transcribed from a template synthesized by (RT)-PCR; the probe length is reduced by hydrolysis. Testes and male genital tracts are dissected from adult flies, fixed and processed for hybridization. Both probe and fixed testes can be stored before use. Extensive post-hybridization washing reduces the background. Detection is through alkaline phosphatase-conjugated anti-dig antibodies followed by a color reaction. This protocol is suitable for low-medium throughput applications with parallel processing of 2-48 samples, and takes 4-5 d to complete. We have used this protocol, which is similar to other RNA ISH protocols, but optimized for whole-mount Drosophila testes, to document the expression of about 1,000 genes in Drosophila melanogaster male genital tract.

Phytochelatins are small cysteine-rich non-ribosomal peptides that chelate soft metal and metalloid ions, such as cadmium and arsenic. They are widely produced by plants and microbes; phytochelatin synthase genes are also present in animal species from several different phyla, but there is still little known about whether these genes are functional in animals, and if so, whether they are metal-responsive. We analysed phytochelatin production by direct chemical analysis in Lumbricus rubellus earthworms exposed to arsenic for a 28 day period, and found that arsenic clearly induced phytochelatin production in a dose-dependent manner. It was necessary to measure the phytochelatin metabolite concentrations directly, as there was no upregulation of phytochelatin synthase gene expression after 28 days: phytochelatin synthesis appears not to be transcriptionally regulated in animals. A further untargetted metabolomic analysis also found changes in metabolites associated with the transsulfuration pathway, which channels sulfur flux from methionine for phytochelatin synthesis. There was no evidence of biological transformation of arsenic (e.g. into methylated species) as a result of laboratory arsenic exposure. Finally, we compared wild populations of earthworms sampled from the field, and found that both arsenic-contaminated and cadmium-contaminated mine site worms had elevated phytochelatin concentrations.

We have investigated the response dynamics of the estrogen-dependent genes vitellogenin (VTG) and the vitelline envelope proteins (VEPs) as well as circulating VTG in immature female rainbow trout (Oncorhynchus mykiss) exposed to 17beta-estradiol (E2) and 17alpha-ethinylestradiol (EE2) for periods of 7 and 14 d. Gene responses were quantified by measurement of messenger RNA (mRNA) in liver extracts using a chemiluminescent hybridization protection assay. Circulating VTG was measured by a homologous enzyme-linked immunosorbent assay. Exposure to both E2 and EE2 induced concentration-dependent increases in all biomarkers. The data presented indicate that VEP genes may be more sensitive to estrogens than the VTG gene. The biomarker lowest-observed-effect concentrations (biomarkerLOEC) in the 14-d study with E2 were 14 ng/L (VTG protein, VTG mRNA, VEPbeta, and VEPgamma) or 4.8 ng/L (VEPalpha). The EE2 was 5- to 66-fold more potent depending on the biomarker studied. In the 7-d study, all biomarkers were elevated after 48-h exposure to E2, with biomarkerLOECs of 30 ng/L (VTG protein, VTG mRNA, and VEPgamma) or 9.7 ng/L (VEPalpha and VEPbeta). Vitellogenin mRNA was induced up to 1,000-fold above baseline, and this translated into an increase of approximately 50,000-fold in circulating VTG. In conclusion, all biomarkers responded to estrogen exposure at environmentally relevant concentrations.

A cDNA library was constructed from macroalgae adapted to prolonged elevated environmental copper levels. To investigate the possible existence of a metallothionein (MT) gene, the library was screened with degenerate probes designed using plant MT cysteine-rich motifs. A gene was identified (1229 bp) with a putative open reading frame (204 bp) encoding a 67-amino-acid protein exhibiting several characteristic features of MT proteins, including 16 cysteine residues (24%) and only one aromatic residue. Although the protein sequence showed high identity with plant and invertebrate MTs, it contained a unique 'linker' region (14 amino acid residues) between the two putative metal-binding domains which contained no cysteine residues. This extended linker is larger than the tripeptide found in archetypal vertebrate MTs, but does not conform either with the 40-amino-acid linkers commonly found in plant MT sequences. An S-peptide Fucus MT fusion protein expressed in Escherichia coli exhibited a relative molecular mass of approximately 14 kDa. The recombinant fusion bound seven Cd ions, of which 50% were dissociated at pH 4.1. Under anaerobic conditions, the Cd ions were displaced by Cu(I), which associated with the protein at a ratio of 13:1. Laboratory exposure of F. vesiculosus to elevated copper resulted in induction of the MT gene. Thus this paper describes, for the first time, an MT gene identified from macroalgae which is induced by copper exposure and whose encoded protein product binds cadmium and copper.

Cytokines play important roles in the wound healing process through various signalling pathways. The JAK-STAT pathway is utilised by most cytokines for signal transduction and is regulated by a variety of molecules, including suppressor of cytokine signalling (SOCS) proteins. SOCS are associated with inflammatory diseases and have an impact on cytokines, growth factors and key cell types involved in the wound-healing process. SOCS, a negative regulator of cytokine signalling, may hold the potential to regulate cytokine-induced signalling in the chronic wound-healing process. Wound edge tissues were collected from chronic venous leg ulcer patients and classified as non-healing and healing wounds. The expression pattern of seven SOCSs members, at the transcript and protein level, were examined in these tissues using qPCR and immunohistochemistry. Significantly higher levels of SOCS3 (P=0.0284) and SOCS4 (P=0.0376) in non-healing chronic wounds compared to the healing/healed chronic wounds were observed at the transcript level. Relocalisation of SOCS3 protein in the non-healing wound environment was evident in the investigated chronic biopsies. Thus, the results show that the expression of SOCS transcript indicated that SOCS members may act as a prognostic biomarker of chronic wounds.

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Background Chronic venous leg ulcers pose a significant burden to healthcare systems, and predicting wound healing is challenging. The aim of this study was to develop a genetic test to evaluate the propensity of a chronic ulcer to heal. Methods Sequential refinement and testing of a gene expression signature was conducted using three distinct cohorts of human wound tissue. The expression of candidate genes was screened using a cohort of acute and chronic wound tissue and normal skin with quantitative transcript analysis. Genes showing significant expression differences were combined and examined, using receiver operating characteristic (ROC) curve analysis, in a controlled prospective study of patients with venous leg ulcers. A refined gene signature was evaluated using a prospective, blinded study of consecutive patients with venous ulcers. Results The initial gene signature, comprising 25 genes, could identify the outcome (healing versus non‐healing) of chronic venous leg ulcers (area under the curve (AUC) 0·84, 95 per cent c.i. 0·73 to 0·94). Subsequent refinement resulted in a final 14‐gene signature (WD14), which performed equally well (AUC 0·88, 0·80 to 0·97). When examined in a prospective blinded study, the WD14 signature could also identify wounds likely to demonstrate signs of healing (AUC 0·73, 0·62 to 0·84). Conclusion A gene signature can identify people with chronic venous leg ulcers that are unlikely to heal.