Martina Höckner

Biosynthesis of luminescent quantum dots in an earthworm

The synthesis of designer solid-state materials by living organisms is an emerging field in bio-nanotechnology. Key examples include the use of engineered viruses as templates for cobalt oxide (Co(3)O(4)) particles, superparamagnetic cobalt-platinum alloy nanowires and gold-cobalt oxide nanowires for photovoltaic and battery-related applications. Here, we show that the earthworms metal detoxification pathway can be exploited to produce luminescent, water-soluble semiconductor cadmium telluride (CdTe) quantum dots that emit in the green region of the visible spectrum when excited in the ultraviolet region. Standard wild-type Lumbricus rubellus earthworms were exposed to soil spiked with CdCl(2) and Na(2)TeO(3) salts for 11 days. Luminescent quantum dots were isolated from chloragogenous tissues surrounding the gut of the worm, and were successfully used in live-cell imaging. The addition of polyethylene glycol on the surface of the quantum dots allowed for non-targeted, fluid-phase uptake by macrophage cells.

Shaping mechanisms of metal specificity in a family of metazoan metallothioneins: evolutionary differentiation of mollusc metallothioneins

BackgroundThe degree of metal binding specificity in metalloproteins such as metallothioneins (MTs) can be crucial for their functional accuracy. Unlike most other animal species, pulmonate molluscs possess homometallic MT isoforms loaded with Cu+ or Cd2+. They have, so far, been obtained as native metal-MT complexes from snail tissues, where they are involved in the metabolism of the metal ion species bound to the respective isoform. However, it has not as yet been discerned if their specific metal occupation is the result of a rigid control of metal availability, or isoform expression programming in the hosting tissues or of structural differences of the respective peptides determining the coordinative options for the different metal ions. In this study, the Roman snail (Helix pomatia) Cu-loaded and Cd-loaded isoforms (HpCuMT and HpCdMT) were used as model molecules in order to elucidate the biochemical and evolutionary mechanisms permitting pulmonate MTs to achieve specificity for their cognate metal ion.ResultsHpCuMT and HpCdMT were recombinantly synthesized in the presence of Cd2+, Zn2+ or Cu2+ and corresponding metal complexes analysed by electrospray mass spectrometry and circular dichroism (CD) and ultra violet-visible (UV-Vis) spectrophotometry. Both MT isoforms were only able to form unique, homometallic and stable complexes (Cd6-HpCdMT and Cu12-HpCuMT) with their cognate metal ions. Yeast complementation assays demonstrated that the two isoforms assumed metal-specific functions, in agreement with their binding preferences, in heterologous eukaryotic environments. In the snail organism, the functional metal specificity of HpCdMT and HpCuMT was contributed by metal-specific transcription programming and cell-specific expression. Sequence elucidation and phylogenetic analysis of MT isoforms from a number of snail species revealed that they possess an unspecific and two metal-specific MT isoforms, whose metal specificity was achieved exclusively by evolutionary modulation of non-cysteine amino acid positions.ConclusionThe Roman snail HpCdMT and HpCuMT isoforms can thus be regarded as prototypes of isoform families that evolved genuine metal-specificity within pulmonate molluscs. Diversification into these isoforms may have been initiated by gene duplication, followed by speciation and selection towards opposite needs for protecting copper-dominated metabolic pathways from nonessential cadmium. The mechanisms enabling these proteins to be metal-specific could also be relevant for other metalloproteins.

Physiological relevance and contribution to metal balance of specific and non-specific Metallothionein isoforms in the garden snail, Cantareus aspersus

Variable environmental availability of metal ions represents a constant challenge for most organisms, so that during evolution, they have optimised physiological and molecular mechanisms to cope with this particular requirement. Metallothioneins (MTs) are proteins that play a major role in metal homeostasis and as a reservoir. The MT gene/protein systems of terrestrial helicid snails are an invaluable model for the study of metal-binding features and MT isoform-specific functionality of these proteins. In the present study, we characterised three paralogous MT isogenes and their expressed products in the escargot (Cantareus aspersus). The metal-dependent transcriptional activation of the three isogenes was assessed using quantitative Real Time PCR. The metal-binding capacities of the three isoforms were studied by characterising the purified native complexes. All the data were analysed in relation to the trace element status of the animals after metal feeding. Two of the three C.aspersus MT (CaMT) isoforms appeared to be metal-specific, (CaCdMT and CaCuMT, for cadmium and copper respectively). A third isoform (CaCd/CuMT) was non-specific, since it was natively recovered as a mixed Cd/Cu complex. A specific role in Cd detoxification for CaCdMT was revealed, with a 80–90% contribution to the Cd balance in snails exposed to this metal. Conclusive data were also obtained for the CaCuMT isoform, which is involved in Cu homeostasis, sharing about 30–50% of the Cu balance of C. aspersus. No apparent metal-related physiological function was found for the third isoform (CaCd/CuMT), so its contribution to the metal balance of the escargot may be, if at all, of only marginal significance, but may enclose a major interest in evolutionary studies.

Cadmium Protection Strategies--A Hidden Trade-Off?

Cadmium (Cd) is a non-essential transition metal which is introduced into the biosphere by various anthropogenic activities. Environmental pollution with Cd poses a major health risk and Cd toxicity has been extensively researched over the past decades. This review aims at changing the perspective by discussing protection mechanisms available to counteract a Cd insult. Antioxidants, induction of antioxidant enzymes, and complexation of Cd to glutathione (GSH) and metallothionein (MT) are the most potent protective measures to cope with Cd-induced oxidative stress. Furthermore, protection mechanisms include prevention of endoplasmic reticulum (ER) stress, mitophagy and metabolic stress, as well as expression of chaperones. Pre-exposure to Cd itself, or co-exposure to other metals or trace elements can improve viability under Cd exposure and cells have means to reduce Cd uptake and improve Cd removal. Finally, environmental factors have negative or positive effects on Cd toxicity. Most protection mechanisms aim at preventing cellular damage. However, this might not be possible without trade-offs like an increased risk of carcinogenesis.

Structural and bioinformatic analysis of the Roman snail Cd-Metallothionein gene uncovers molecular adaptation towards plasticity in coping with multifarious environmental stress

Metallothioneins (MTs) are a family of multifunctional proteins involved, among others, in stress response. The Cadmium (Cd)‐MT gene of the Roman snail (Helix pomatia), for example, encodes for a protein induced upon cadmium exposure. While our previous studies have demonstrated that the expressed Cd‐MT isoform of Roman snails assists detoxification of cadmium, the present work focuses on the potential plasticity of this gene in response to a variety of environmental stressors playing a crucial role in the specific ecological niche of H. pomatia. Our hypothesis is based on a bioinformatic approach involving gene sequencing, structural and in silico analysis of transcription factor binding sites (TFBs), and a comparison of these features with other MT genes. Our results show that the Roman snails Cd‐MT gene not only is the largest known MT gene, but also contains — apart from the regulatory promoter region — several intronic repeat cassettes of putative TFBs suggested to be involved in environmental stress response, immune competence, and regulation of gene expression. Moreover, intronic scaffold/matrix attachment regions (S/MARs) and stress‐induced duplex destabilization sites confer a high potential for epigenetic gene regulation. This suggested regulatory plasticity is also supported by physiological data showing that Cd‐MT in Roman snails can be induced differentially not only after cadmium exposure, but also in response to nonmetallic environmental stressors. It is concluded that structural analysis combined with bioinformatic screening may constitute valuable tools for predicting the potential for plasticity and niche‐specific adaptation of stress‐responsive genes in populations living under rapidly changing environmental conditions.

Nematode and snail metallothioneins

Metallobiologists have, at large, neglected soil dwelling invertebrates; exceptions are the nematode (Caenorhabditis elegans) and snails (Helix pomatia and Cantareus aspersus). This review aims to compare and contrast the molecular, protein and cellular mechanisms of the multifunctional nematode and snail metallothioneins (MTs). The C. elegans genome contains two MT genes, mtl-1, which is constitutively expressed in the pharynx and likely to act as an essential and/or toxic metal sensor, and mtl-2, which plays a negligible role under normal physiological conditions but is strongly induced (as mtl-1) in intestinal cells upon metal exposure. It has been possible to follow the intricate phenotypic responses upon the knockdown/knockout of single and multiple MT isoforms and we have started to decipher the multifunctional role of C. elegans MTs. The snails have contributed to our understanding regarding MT evolution and diversity, structure and metal-specific functionality. The H. pomatia and C. aspersus genomes contain at least three MT isoform genes. CdMT is responsible for cadmium detoxification, CuMT is involved in copper homeostasis and Cd/CuMT is a putative ancestral MT possibly only of minor importance in metal metabolism. Further investigations of nematode, snail and other invertebrate MTs will allow the development of alternative biomarker approaches and lead to an improved understanding of metallobiology, protein evolution and toxicogenomics.

Coping with cadmium exposure in various ways: the two Helicid snails Helix pomatia and Cantareus aspersus share the metal transcription factor‐2, but differ in promoter organization and transcription of their Cd‐metallothionein genes

Gastropods are able to withstand fluctuating availabilities of nonessential trace elements such as cadmium by induction of Cd-specific metallothionein isoform (Cd-MT) expression. As in other species, the induction mechanism involves the binding of metal-regulatory transcription factors (MTF-1 or MTF-2) to metal responsive elements (MREs) in the MT promoter regions. Cd-dependent transcription of Cd-MT genes was assessed by quantitative real time PCR in two helicid gastropods, Helix pomatia and Cantareus aspersus, over a period of eight days. The promoter regions of the Cd-MT genes of the two species were sequenced and compared regarding the position of MREs and other relevant potential transcription factor binding sites (TFBs). Cd-MT gene transcription is induced after Cd exposure in Helix pomatia and Cantareus aspersus, showing a transient peak in Helix pomatia, contrasting with a persistent induction rate in Cantareus aspersus. Since the existence of MTF-2 was verified in both species, differing transcription patterns of Cd-MT genes must be due to functional differences in their metal-responsive promoter regions. Both promoters contain a proximal cluster of three MREs overlapping with TFBs for the transcriptional regulator Sp1. In contrast to Cantareus aspersus, however, the Cd-MT gene of Helix pomatia hosts an additional distal MRE overlapping with a Sp1 binding site and a CACCC box. Inhibitory effects of MRE overlapping Sp1 binding sites were observed in other MT genes. We therefore suggest that transient Cd-MT transcription upon Cd(2+) exposure in Helix pomatia may be the result of an inhibitory action of the distal MRE cluster.

Evolutionary concepts in ecotoxicology: tracing the genetic background of differential cadmium sensitivities in invertebrate lineages.

In many toxicological and ecotoxicological studies and experimental setups, the investigator is mainly interested in traditional parameters such as toxicity data and effects of toxicants on molecular, cellular or physiological functions of individuals, species or statistical populations. It is clear, however, that such approaches focus on the phenotype level of animal species, whilst the genetic and evolutionary background of reactions to environmental toxicants may remain untold. In ecotoxicological risk assessment, moreover, species sensitivities towards pollutants are often regarded as random variables in a statistical approach. Beyond statistics, however, toxicant sensitivity of every species assumes a biological significance, especially if we consider that sensitivity traits have developed in lineages of species with common evolutionary roots. In this article, the genetic and evolutionary background of differential Cd sensitivities among invertebrate populations and species and their potential of adaptation to environmental Cd exposure will be highlighted. Important evolutionary and population genetic concepts such as genome structure and their importance for evolutionary adaptation, population structure of affected individuals, as well as micro and macroevolutionary mechanisms of Cd resistance in invertebrate lineages will be stressed by discussing examples of work from our own laboratory along with a review of relevant literature data and a brief discussion of open questions along with some perspectives for further research. Both, differences and similarities in Cd sensitivity traits of related invertebrate species can only be understood if we consider the underlying evolutionary processes and genetic (or epigenetic) mechanisms. Keeping in mind this perception can help us to better understand and interpret more precisely why the sensitivity of some species or species groups towards a certain toxicant (or metal) may be ranked in the lower or higher range of species sensitivity distributions. Hence, such a perspective will transcend a purely statistical view of the sensitivity distributions concept, and will enhance ecotoxicology in many respects.

Whole Genome Amplification from Microdissected Chromosomes

Over the last years various whole genome amplification (WGA) methods have been established for genetic investigations from a limited number of cells or small quantities of DNA but not for molecular analysis of isolated chromosomes, which is important for the direct investigation of haplotypes or molecular rearrangements of derivative chromosomes in clinical cytogenetics and oncology. Here, the results of a pilot study in which the GenomePlex Single Cell Kit® linker adapter PCR approach (Sigma-Aldrich, Vienna, Austria) was modified for WGA of glass needle based microdissected chromosomes are presented. Compared with two other WGA strategies (Improved-Primer Extension Preamplification PCR and Multiple Displacement Amplification) the GenomePlex Single Cell Kit® shows a higher rate of successfully amplified markers, a lower WGA drop out rate and faster feasibility.

Molecular characterization of a de novo ring chromosome 6 in a growth retarded but otherwise healthy woman

The phenotype of patients with a ring chromosome 6 can be highly variable ranging from almost normal to severe malformations and mental retardation. Size and structure of the ring chromosome as well as the level of mosaicism are important factors for the clinical phenotype. Here, we report on a 25‐year‐old woman with short stature, minor scoliosis, normal fertility, appropriate psychomotor development, minor dysmorphisms, and a de novo ring chromosome 6. Conventional karyotyping as well as molecular cytogenetic and molecular investigations of DUSP22 on 6p and RP1‐191N21.4 on 6q by a new technical approach indicated breakpoints less than 240 kb and less than 190 kb proximal to the telomeres of 6p and 6q, respectively. In addition, formation of the ring chromosome from the paternal chromosome was demonstrated. Thus this case clearly shows that in patients with ring chromosomes without loss of euchromatic material mitotic instability of the ring chromosome is the most important reason for growth retardation and minor congenital anomalies.