cDNA cloning and expression of Contractin A, a phospholipase A2-like protein from the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus.

Venomous sea urchins contain various biologically active proteins that are toxic to predators. Contractin A is one such protein contained within the globiferous pedicellariae of the venomous sea urchin Toxopneustes pileolus. This protein exhibits several biological activities, such as smooth muscle contraction and mitogenic activity. N-terminal amino acid residues of Contractin A have been determined up to 37 residues from the purified protein. In this study, we cloned cDNA for Contractin A by reverse transcription-PCR using degenerate primers designed on the basis of its N-terminal amino acid sequence. Analysis of the cDNA sequence indicated that Contractin A is composed of 166 amino acid residues including 31 residues of a putative signal sequence, and has homology to the sequence of phospholipase A2 from various organisms. In this study, recombinant Contractin A was expressed in Escherichia coli cells, and the protein was subjected to an assay to determine lipid-degrading activity using carboxyfluorescein-containing liposomes. As a result, Contractin A was found to exhibit Ca(2+)-dependent release of carboxyfluorescein from the liposomes, suggesting that Contractin A has phospholipase A2 activity, which may be closely associated with its biological activities.

Nutrient-related changes in the toxicity of field blooms of the cyanobacterium, Cylindrospermopsis raciborskii.

Nutrients have the capacity to change cyanobacterial toxin loads via growth-related toxin production, or shifts in the dominance of toxic and nontoxic strains. This study examined the effect of nitrogen (N) and phosphorus on cell division and strain-related changes in production of the toxins, cylindrospermopsins (CYNs) by the cyanobacterium, Cylindrospermopsis raciborskii. Two short-term experiments were conducted with mixed phytoplankton populations dominated by C. raciborskii in a subtropical reservoir where treatments had nitrate (NO3 ), urea (U) and inorganic phosphorus (P) added alone or in combination. Cell division rates of C. raciborskii were only statistically higher than the control on day 5 when U and P were co-supplied. In contrast, cell quotas of CYNs (QCYNS ) increased significantly in treatments where P was supplied, irrespective of whether N was supplied, and this increase was not necessarily related to cell division rates. Increased QCYNS did correlate with an increase in the proportion of the cyrA toxin gene to 16S genes in the C. raciborskii-dominated cyanobacterial population. Therefore, changes in strain dominance are the most likely factor driving differences in toxin production between treatments. Our study has demonstrated differential effects of nutrients on cell division and strain dominance reflecting a C. raciborskii population with a range of strategies in response to environmental conditions.

Stability of toxin gene proportion in red-pigmented populations of the cyanobacterium Planktothrix during 29 years of re-oligotrophication of Lake Zürich.

BACKGROUND: Harmful algal blooms deteriorate the services of aquatic ecosystems. They are often formed by cyanobacteria composed of genotypes able to produce a certain toxin, for example, the hepatotoxin microcystin (MC), but also of nontoxic genotypes that either carry mutations in the genes encoding toxin synthesis or that lost those genes during evolution. In general, cyanobacterial blooms are favored by eutrophication. Very little is known about the stability of the toxic/nontoxic genotype composition during trophic change. RESULTS: Archived samples of preserved phytoplankton on filters from aquatic ecosystems that underwent changes in the trophic state provide a so far unrealized possibility to analyze the response of toxic/nontoxic genotype composition to the environment. During a period of 29 years of re-oligotrophication of the deep, physically stratified Lake Zürich (1980 to 2008), the population of the stratifying cyanobacterium Planktothrix was at a minimum during the most eutrophic years (1980 to 1984), but increased and dominated the phytoplankton during the past two decades. Quantitative polymerase chain reaction revealed that during the whole observation period the proportion of the toxic genotype was strikingly stable, that is, close to 100%. Inactive MC genotypes carrying mutations within the MC synthesis genes never became abundant. Unexpectedly, a nontoxic genotype, which lost its MC genes during evolution, and which could be shown to be dominant under eutrophic conditions in shallow polymictic lakes, also co-occurred in Lake Zürich but was never abundant. As it is most likely that this nontoxic genotype contains relatively weak gas vesicles unable to withstand the high water pressure in deep lakes, it is concluded that regular deep mixing selectively reduced its abundance through the destruction of gas vesicles. CONCLUSIONS: The stability in toxic genotype dominance gives evidence for the adaptation to deep mixing of a genotype that retained the MC gene cluster during evolution. Such a long-term dominance of a toxic genotype draws attention to the need to integrate phylogenetics into ecological research as well as ecosystem management.

Screening and cDNA cloning of Kv1 potassium channel toxins in sea anemones.

When 21 species of sea anemones were screened for Kv1 potassium channel toxins by competitive inhibition of the binding of (125)I-α-dendrotoxin to rat synaptosomal membranes, 11 species (two species of Actiniidae, one species of Hormathiidae, five species of Stichodactylidae and three species of Thalassianthidae) were found to be positive. Furthermore, full-length cDNAs encoding type 1 potassium channel toxins from three species of Stichodactylidae and three species of Thalassianthidae were cloned by a combination of RT-PCR, 3'RACE and 5'RACE. The precursors of these six toxins are commonly composed of signal peptide, propart and mature peptide portions. As for the mature peptide (35 amino acid residues), the six toxins share more than 90% sequence identities with one another and with κ(1.3)-SHTX-She1a (Shk) from Stichodactyla helianthus but only 34-63% identities with the other type 1 potassium channel toxins.

Molecular cloning of two toxic phospholipases A2 from the crown-of-thorns starfish Acanthaster planci venom.

The full-length cDNAs encoding two toxic phospholipases A2 (AP-PLA2-I and -II) from the crown-of-thorns starfish Acanthaster planci venom were individually cloned by RT-PCR, 3'RACE and 5'RACE. In common with both AP-PLA2s, the precursor protein is composed of a signal peptide, a propeptide and a mature protein (136 and 135 residues for AP-PLA2-I and -II, respectively). The four motifs (Ca2+-binding loop, Ca2+-binding site, active site and catalytic network) characteristic of groups I and II PLA2s are well conserved in both AP-PLA2s. In addition to this, the presence of the elapid and pancreatic loops and the involvement of a propeptide in the precursors suggested that AP-PLA2s are highly analogous to the group IB PLA2s. However, when compared to the amino acid sequence of bovine pancreatic PLA2, the representative group IB PLA2, AP-PLA2s require some amino acid insertions and deletions in the region 76-100, as previously observed for the starfish Asterina pectinifera PLA2s. Furthermore, the phylogenetic tree made clearly demonstrated that AP-PLA2s and A. pectinifera PLA2s are distinguishable from the group IB PLA2s as well as other PLA2s, being classified into a new group.

Isolation and characterization of a novel protein toxin from the Hawaiian box jellyfish (sea wasp) Carybdea alata.

The box jellyfish (sea wasp) Carybdea alata Reynaud, 1830 (Cubozoa) is distributed widely in the tropics. The sting of C. alata causes severe pain and cutaneous inflammation in humans. We successfully isolated C. alata toxin-A (CaTX-A, 43 kDa) and -B (CaTX-B, 45 kDa) for the first time from the tentacle of C. alata collected at a site along the Hawaiian shore. The experimental results showed that CaTX-A, but not CaTX-B, is present in the nematocyst, the organ responsible for stinging. Both CaTX-A and -B showed potent hemolytic activity, with CaTX-A being lethally toxic to crayfish when administered via intraperitoneal injection. Furthermore, we sequenced the cDNA encoding CaTX-A. The deduced amino acid sequence of CaTX-A (463 amino acids) showed 43.7% homology to Carybdea rastoni toxins (CrTXs) but not with any other known proteins. Therefore, these jellyfish toxins potentially represent a novel class of bioactive proteins. Secondary structure analysis of CaTX-A and CrTXs suggested the presence of amphiphilic alpha-helices, which are also seen in several known hemolytic or cytolytic protein toxins, including peptide toxins.