Responsiveness of metallothionein and hemocyanin genes to cadmium and copper exposure in the garden snail Cornu aspersum.

Terrestrial gastropods express metal-selective metallothioneins (MTs) by which they handle metal ions such as Zn2+ , Cd2+ , and Cu+ /Cu2+ through separate metabolic pathways. At the same time, they depend on the availability of sufficient amounts of Cu as an essential constituent of their respiratory protein, hemocyanin (Hc). It was, therefore, suggested that in snails Cu-dependent MT and Hc pathways might be metabolically connected. In fact, the Cu-specific snail MT (CuMT) is exclusively expressed in rhogocytes, a particular molluscan cell type present in the hemocoel and connective tissues. Snail rhogocytes are also the sites of Hc synthesis. In the present study, possible interactions between the metal-regulatory and detoxifying activity of MTs and the Cu demand of Hc isoforms was explored in the edible snail Cornu aspersum, one of the most common European helicid land snails. This species possesses CdMT and CuMT isoforms involved in metal-selective physiological tasks. In addition, C. aspersum expresses three different Hc isoforms (CaH ɑD, CaH ɑN, CaH ß). We have examined the effect of Cd2+ and Cu2+ exposure on metal accumulation in the midgut gland and mantle of C. aspersum, testing the impact of these metals on transcriptional upregulation of CdMT, CuMT, and the three Hc genes in the two organs. We found that the CuMT and CaH ɑD genes exhibit an organ-specific transcriptional upregulation in the midgut gland of Cu-exposed snails. These results are discussed in view of possible interrelationships between the metal-selective activity of snail MT isoforms and the synthesis and metabolism of Hc isoforms.

Randomised double-blind placebo-controlled intervention study on the nutritional efficacy of a food for special medical purposes (FSMP) and a dietary supplement in reducing the symptoms of veisalgia.

OBJECTIVE: To assess whether the symptoms of veisalgia can be reduced by intense water supply and the intake of antioxidative supplements and plant extracts. METHODS: We performed the world's largest randomised double-blind placebo-controlled intervention study (214 participants) on the efficacy of a food for special medical purposes (FSMP) against veisalgia symptoms. We analysed the effectiveness of: (1) an FSMP, including distinct plant extracts, vitamins and minerals, and additional (antioxidative) compounds; (2) a dietary supplement only comprising vitamins and minerals and additional (antioxidative) compounds; and (3) a placebo containing only glucose. The study followed the CONSORT (Consolidated Standards of Reporting Trials) guidelines and trial registration was not necessary. RESULTS: Our study showed no statistically significant relationship between the variation of body water content and alcohol consumption. Contrary to common belief, the results showed that intervention with a supplement containing vitamins and minerals and additional antioxidative compounds did not lead to a statistically significant improvement in hangover symptoms. Additionally, our results confirmed a high individual variability in developing hangover symptoms depending on the amount of alcohol. Thus, standardisation of the amount of alcohol consumed in hangover studies does not necessarily contribute to the validity of the results. Finally, this study found a number of positive effects on certain hangover symptoms as a result of the FSMP, which were most likely due to the plant extracts contained within the food. CONCLUSION: This study significantly supports the finding that haemostasis of electrolytes and minerals caused by alcohol consumption might be negligible and that no significant dehydration due to alcohol consumption seems to occur. Additionally, only the FSMP provides evidence for a significant efficiency in the reduction of hangover symptoms such as headache and nausea following moderate and non-excessive alcohol consumption.

cDNA sequences of two arylphorin subunits of an insect biliprotein: phylogenetic differences and gene duplications during evolution of hexamerins-implications for hexamer formation.

Arylphorins represent a conserved class of hexameric ∼500 kDa insect hemolymph glycoproteins, rich in aromatic amino acids, which are produced in large quantities at the larval stage as reserves for metamorphosis and egg development. The recently isolated arylphorin from the moth Cerura vinula is unique in being complexed to a novel farnesylated bilin. Protein sequencing suggested the presence of two different ∼85 kDa subunits. Here, we report the complete coding sequences of two cDNAs encoding two arylphorins subunits with 67% identity and calculated physicochemical characteristics in agreement with the isolated holoprotein. Our phylogenetic analyses of the hexamerins revealed monophyletic origins not only for each of the arylphorins and methionine-rich proteins (H-type and M-type), the two major classes of hexamerins, but also for the minor groups of arylphorin-like and riboflavin-binding hexamerins. We named the latter proteins X-type (mixed type) hexamerins because they share sequence features with both major groups, and they show unique deletions and insertions at conserved sites located on the protein surface. We present a phylogenetic tree of lepidopteran hexamerins, which is in agreement with actual systematics. Overall, duplications of hexamerin genes occurred independently in several lepidopteran lineages. We also analyzed the hexamerin sequences for key parameters, which characterize each type of hexamerins. Based on the crystal structure of the homomeric arylphorin from Antheraea pernyi, we present a model for the heteromeric Cerura protein focusing on the role of N-glycan structures in stabilizing the hexamer structure.

The first complete cDNA sequence of the hemocyanin from a bivalve, the protobranch Nucula nucleus.

By cDNA sequencing we have achieved the first, and complete, hemocyanin sequence of a bivalve (Nucula nucleus). This extracellular oxygen-binding protein consists of two immunologically distinguishable isoforms, here termed NnH1 and NnH2. They share a mean sequence identity of 61%, both contain a linear arrangement of eight paralogous, ca.50-kDa functional units (FUs a-h), and in both isoforms the C-terminal FU-h possesses an extension of ca. 100 amino acids. The cDNA of NnH1 comprises 11,090 bp, subdivided into a 5'utr of 75 bp, a 3'utr of 791 bp, and an open reading frame for a signal peptide of 19 amino acids plus a polypeptide of 3389 amino acids (Mr = 385 kDa). The cDNA of NnH2 comprises 10,849 bp, subdivided into a 5'utr of 47 bp, a 3'utr of 647 bp, and an open reading frame for a signal peptide of 16 amino acids plus a polypeptide of 3369 amino acids (Mr = 387 kDa). In contrast to other molluscan hemocyanins, which are highly glycosylated, the bivalve hemocyanin sequence exhibits only four potential N-glycosylation sites, and within both isoforms a peculiar indel is present, surrounding the highly conserved copper-binding site CuA. Phylogenetic analyses of NnH1 and NnH2, compared to the known hemocyanin sequences of gastropods and cephalopods, reveal a statistically sound closer relationship between gastropod and protobranch hemocyanin than to cephalopod hemocyanin. Assuming a molecular clock, the last common ancestor of protobranch and gastropods lived 494 million +/- 50 million years ago, in conformity with fossil records from the late Cambrian.

Developmental expression of two Haliotis asinina hemocyanin isoforms.

Hemocyanins are large copper-containing respiratory proteins that play a role in oxygen transport in many molluscs. In some species only one hemocyanin isoform is present while in others two are expressed. The physiological relevance of these isoforms is unclear and the developmental and tissue-specific expression of hemocyanin genes is largely unknown. Here we show that two hemocyanin genes in the gastropod Haliotis asinina, which encode H. asinina hemocyanin (HaH1) and HaH2 isoforms, are developmentally expressed. These genes initially are expressed in a small number of mesenchyme cells at trochophore and pre-torsional veliger stages, with HaH1 expression slightly preceding HaH2. These cells largely are localized to the visceral mass, although a small number of cells are present in head and foot regions. Following metamorphosis the isoforms show overlapping as well as isoform-specific expression profiles, suggesting some degree of isoform-specific function.

cDNA sequence, protein structure, and evolution of the single hemocyanin from Aplysia californica, an opisthobranch gastropod.

By protein immunobiochemistry and cDNA sequencing, we have found only a single hemocyanin polypeptide in an opisthobranch gastropod, the sea hare Aplysia californica, which contrasts with previously studied prosobranch gastropods, which express two distinct isoforms of this extracellular respiratory protein. We have cloned and sequenced the cDNA encoding the complete polypeptide of Aplysia californica hemocyanin (AcH). The cDNA comprises 11,433 bp, encompassing a 5'UTR of 77 bp, a 3'UTR of 1057 bp, and an open reading frame for a signal peptide of 20 amino acids plus a polypeptide of 3412 amino acids (Mr ca. 387 kDa). This polypeptide is the subunit of the cylindrical native hemocyanin (Mr ca. 8 MDa). It comprises eight different functional units (FUs: a, b, c, d, e, f, g, h) that have been identified immunobiochemically after limited proteolysis of AcH purified from the hemolymph. Each FU shows a highly conserved copper-A and copper-B site for reversible oxygen binding. FU AcH-h carries a specific C-terminal extension of ca. 100 amino acids that include two cysteines that may be utilized for disulfide bridge formation. Potential N-glycosylation sites are present in six FUs but lacking in AcH-b and AcH-c. On the basis of multiple sequence alignments, phylogenetic trees and a statistically firm molecular clock were calculated. The latter suggests that the last common ancestor of Haliotis and Aplysia lived 373+/-47 million years ago, in convincing agreement with fossil records from the early Devonian. However, the gene duplication yielding the two distinct hemocyanin isoforms found today in Haliotis tuberculata occurred 343+/-43 million years ago.

A new metallothionein gene from the giant keyhole limpet Megathura crenulata.

Metallothioneins (MTs) are small soluble proteins ubiquitously expressed in animals and plants. Different isoforms are present in deuterostomes and protostomes. They do not differ greatly in primary structure, but are clearly distinguishable. Here, I present the gene and the complete cDNA of a novel MT from the mollusk Megathura crenulata. This protein is closely related to the Cu-inducible MTs of the vineyard snail Helix pomatia, but has also some minor sequence features typical of Cd-inducible isoforms of H. pomatia and other molluscs. Overall, the deduced primary structure is similar to the known molluscan MTs, but in addition possesses an insertion of 5 amino acids not found in any other molluscan MTs, protostomic or deuterostomic MTs. In addition, a pentapeptide insertion, characteristic of mammalian MT-3 is present but it lacks the functional tetrapeptide CPCP within the beta-region of those MT-3 proteins that are known to suppress neuronal growth processes. The M. crenulata MT is a novel form of MT in comparison to all other known MTs. Possible functional aspects for this new MT are discussed.