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Peptides found in marine life have various specific activities due to their special growth environment, and there is increasing interest in the isolation and concentration of these biofunctional compounds. In this study, the protein hydrolysate of the marine worm Urechis unicinctus was prepared by enzymolysis and enriched by using mesoporous materials of silica MCM-41 and SBA-15 and carbon CMK-3. The differences in pore structures and elemental composition of these materials lead to differences in surface area and hydrophobicity. The adsorption capacities of peptides were 459.5 mg g-1, 431.3 mg g-1, and 626.3 mg g-1 for MCM-41, SBA-15 and CMK-3, respectively. Adsorption kinetics studies showed that the pseudo-second-order model fit the adsorption process better, where both external mass transfer and intraparticle diffusion affected the adsorption, while the Langmuir model better fit the adsorption of peptides on MCM-41 and SBA-15 and the Freundlich model was more suitable for CMK-3. Aqueous acetonitrile (ACN, 50/50, v/v) yielded the most extracted peptides. MALDI-TOF mass spectrometry of the extracted peptides showed that the three mesoporous materials, especially the CMK-3, gave good enrichment results. This study demonstrates the great potential of mesoporous materials in the enrichment of marine biofunctional peptides.

The nutritional content of kian marine worm has never been reported. This study aimed to profile the nutritional content of worms from the Kei Islands, Maluku. Nutritional profiles were tested by proximate assays, atomic absorption spectroscopy (AAS), and High-Performance Liquid Chromatography (HPLC) of amino acids. The proximate analysis resulted in 71.16 percent of protein, 12.05 percent of water, 2.63 percent of carbohydrates, 2.21 percent of fat, 2.14 percent of crude fiber and 0.44 percent of ash. Mineral contents of Kian marine worm were 785.28 mg/kg potassium, 585.14 mg/kg magnesium, 180.98 mg/kg calcium, 0.89 mg/kg sodium and 0.44 mg/kg phosphorus. Kian marine worms contained 9 essential amino acids and 11 essential amino acids with levels of essential amino acids up to 206.94 mg/g and non-essential amino acids was 233.21 mg/g. Nutritional content in kian marine worms can be used as a potential high protein food resource.

International audience; The amanitins (namely α- and β-amanitin) contained in certain mushrooms are bicyclic octapeptides that, when ingested, are responsible for potentially lethal hepatotoxicity. M101 is an extracellular hemoglobin extracted from the marine worm Arenicola marina. It has intrinsic Cu/Zn-SOD-like activity and is currently used as an oxygen carrier in organ preservation solutions. Our present results suggest that M101 might be effective in reducing amanitin-induced hepatotoxicity and may have potential for therapeutic development.

Acrorhagin I (U-AITX-Aeq5a) is a disulfide–rich peptide identified in the aggressive organs (acrorhagi) of the sea anemone Actinia equina. Previous studies (Toxicon 2005, 46:768–74) found that the peptide is toxic in crabs, although the structural and functional properties of acrorhagin I have not been reported. In this work, an Escherichia coli (BL21 strain) expression system was established for the preparation of 13C,15N–labelled acrorhagin I, and the solution structure was determined using NMR spectroscopy. Structurally, acrorhagin I is similar to B–IV toxin from the marine worm Cerebratulus lacteus (PDB id 1VIB), with a well–defined helical hairpin structure stabilised by four intramolecular disulfide bonds. The recombinant peptide was tested in patch–clamp electrophysiology assays against voltage-gated potassium and sodium channels, and in bacterial and fungal growth inhibitory assays and haemolytic assays. Acrorhagin I was not active against any of the ion channels tested and showed no activity in functional assays, indicating that this peptide may possess a different biological function. Metal ion interaction studies using NMR spectroscopy showed that acrorhagin I bound zinc and nickel, suggesting that its function might be modulated by metal ions or that it may be involved in regulating metal ion levels and their transport. The similarity between the structure of acrorhagin I and that of B-IV toxin from a marine worm suggests that this fold may prove to be a recurring motif in disulfide-rich peptides from marine organisms.

The processes permeating the relationships between bioturbation and microorganisms remain poorly understood due to the difficulty of traditional techniques in quantifying their two- and three-dimensional aspects. We used cutting-edge technologies to address the macro- and microorganisms' interactions under metal-contamination. Bioturbation (mucus-lined gallery perimeter, mucus-lined gallery surface area, and gallery water volume) positively influence the carbohydrate consumption rate by the bacterial consortium, elevating bacterial metabolic activity, despite metal-contamination. Synchrotron-based 2D-μXRF revealed that the mucous lining by marine worm during bioturbation as the primary carbon source enhances metal immobilization by bacterial biofilm, improving the bacterial metabolic activity. Bioturbation thus can positively affect bacterial consortium that can use the mucus as a carbon source, which enhances the resistance to metals through biofilm formation in metal-contaminated sediments.

In situ hybridization (ISH) is a very informative technique to present cellular distribution patterns of specific genes (e.g., mRNA and ncRNA) in tissues. The sipunculid worm Sipunculus nudus is a crucial fishery resource as it has high nutritional and medicinal values. Currently, the research on the molecular biology of Sipunculus nudus is still in its infancy. The purpose of this article is to develop a sensitive method for localizing specific mRNA in Sipunculus nudus coelomic fluid. The protocol includes detailed steps of ISH, including digoxigenin-labeled antisense and sense riboprobe preparation, coelomic fluid collection and section preparation, specific riboprobe hybridization, antibody incubation, coloration and post-coloration treatments. The representative results obtained from a successful experiment using this method are demonstrated. The protocol should be applicable to other Sipuncula species as well.

Diopatra claparedii which is colloquially known as Ruat Sarung can be found along the west coast of Peninsular Malaysia. The species has a unique ability to regenerate anterior and posterior segments upon self-amputation or injury, thus having potential as a wound healing promoter. In this study, the wound healing potential of D. claparedii aqueous extract on acute wound model in rats was revealed for the first time. Various concentrations (0.1%, 0.5%, and 1.0% w/w) of D. claparedii ointment were formulated and tested on Sprague Dawley rats through topical application on full-thickness skin wounds for 14 days. The wound healing effects were investigated via behaviour observation, wound contraction, and histopathological analysis. Quality assessment was performed via skin irritation test, microbial contamination test (MCT), and heavy metal detection. The study also included test for antibacterial activities and detection of bioactive compounds in D. claparedii. One percent of D. claparedii ointment showed rapid wound healing potential with good soothing effects and more collagen deposition in comparison to the commercial wound healing ointments such as acriflavine (0.1% w/v) and traditional ointment gamat (sea cucumber extract) (15.0% w/v). No local skin irritation, microbial contamination, and insignificant concentration of heavy metals were observed, which indicate its safe application. Moreover, the aqueous extract of D. claparedii exhibited antibacterial activities against Escherichia coli and Pseudomonas aeruginosa with minimum inhibitory concentration (MIC) value at 0.4 g/ml. 1H NMR analysis of the aqueous extract of D. claparedii revealed some metabolites that might be responsible for its wound healing properties such as amino acids, halogenated aromatics, organic acids, vitamins, and others. Altogether, these results suggested that the aqueous extract of D. claparedii could be utilised as an alternative natural wound healing promoter.

The first enantioselective total synthesis and structural reassignment of (-)-thelepamide, a cytotoxic tetraketide-amino acid from the marine worm Thelepus crispus, is reported. A convergent approach provides access to all thelepamide diastereomers in six steps from four simple building blocks. Key features of the synthesis include the application of Melchiorre's organocatalytic thia-Michael reaction and a sonication-assisted assembly of an unprecedented N,O-acetal-hemiacetal moiety. The corrected structure was confirmed by NMR-DFT analysis.

The hemoglobin from the marine worm Amphitrite ornata has been found to possess multiple enzymatic activities in addition to its O2-transport function. Named dehaloperoxidase (DHP), this globin employs four mechanisms for substrate oxidation as a defense mechanism against toxic metabolites, spanning electron- (peroxidase and oxidase) and O-atom (peroxygenase and oxygenase) transfer. As such, DHP can be defined as a multifunctional catalytic hemoglobin. Given that its peroxidase function is already well established in the literature, this review aims to provide further structural and mechanistic details into the other three activities performed by DHP, with a particular emphasis on its peroxygenase activity.

Symsagittifera roscoffensis is a small marine worm found in the intertidal zone of sandy beaches around the European shores of the Atlantic. S. roscoffensis is a member of the Acoelomorpha, a group of flatworms formerly classified with the Platyhelminthes, but now recognised as Xenacoelomorpha, a separate phylum of disputed affinity. We have used electron microscopy to examine the process of spermiogenesis (the final stage of spermatogenesis) in S. roscoffensis, by which spermatids form highly elongated spermatozoa. Their nuclei are long and thread-like, running most of the cell’s length and during the process a pair of flagella are fully incorporated into the cell body. Two previously undescribed inter-organelle contact sites form at different stages of spermiogenesis. Strikingly, there is an extensive nucleus-plasma membrane contact site. Golgi-derived granules containing electron-dense filaments line up along the spermatid plasma membrane, undergo a conformational change, and donate material that forms a peri-nuclear layer that cements this contact site. We also show in earlier stage spermatids that the same granules are associated with microtubules, presumably for traffic along the elongating cell. We identify a second spermiogenesis-specific contact site where sheaths engulfing each internalising flagellum contact the nuclear envelope. Finally, detailed studies of the spermatozoon axonemes show that the central keel has varying numbers of microtubules along the length of the cell, and is likely to be a centriole derivative.Summary sentenceDuring spermiogenesis in the acoel flatworm Symsagittifera roscoffensis, two previously unidentified contact sites contribute to the structure of the mature spermatozoon and the axonemal structures show direct continuity between doublet and dense core microtubules.

The natural phenomenon of bioluminescence is light production fueled by biochemical processes. In general, bioluminescence is observed as very short (millisecond) flashes, or a lasting glow in bact...

For the purpose of advancing knowledge of the structural variations available in the natural cephalostatins contained in the marine worm Cephalodiscus gilchristi, the isolation and structure of the 20th member (1) has been accomplished (10(-7) % yield). In turn cephalostatin 20 (1) proved to be enough for an initial SAR study comprising six important human cancer cell lines. A parallel objective was aimed at the possible discovery of a natural cephalostatin with a more accessible structure for total synthesis and/or synthetic modifications, but with powerful cancer cell growth inhibition.

Lectins including flowering plant lectins, algal lectins, cyanobacterial lectins, actinomycete lectin, worm lectins, and the nonpeptidic lectin mimics pradimicins and benanomicins, exhibit anti-HIV activity. The anti-HIV plant lectins include Artocarpus heterophyllus (jacalin) lectin, concanavalin A, Galanthus nivalis (snowdrop) agglutinin-related lectins, Musa acuminata (banana) lectin, Myrianthus holstii lectin, Narcissus pseudonarcissus lectin, and Urtica diocia agglutinin. The anti-HIV algal lectins comprise Boodlea coacta lectin, Griffithsin, Oscillatoria agardhii agglutinin. The anti-HIV cyanobacterial lectins are cyanovirin-N, scytovirin, Microcystis viridis lectin, and microvirin. Actinohivin is an anti-HIV actinomycete lectin. The anti-HIV worm lectins include Chaetopterus variopedatus polychaete marine worm lectin, Serpula vermicularis sea worm lectin, and C-type lectin Mermaid from nematode (Laxus oneistus). The anti-HIV nonpeptidic lectin mimics comprise pradimicins and benanomicins. Their anti-HIV mechanisms are discussed.

The blue glow of the mucus from Chaetopterus involves a photoprotein, iron and flavins. Identity and respective role of these components remain, however, largely unresolved today, likely because of viscosity issues and inhibition of this system by oxidizers conventionally used to track bioluminescence activity. Here, we used gentle centrifugation to obtain a mucus supernatant showing no inhibition to oxidizers, allowing for further analysis. We applied conventional chromatographic techniques to isolate major proteins associated with light emission. Luminescence ability of elutriate fractions was tested with hydrogen peroxide to track photoprotein and/or protein-bound chromophore. Fractions producing light contained few major proteins, one with similarity to ferritin. Addition to the mucus of elements with inhibitory/potentiary effect on ferritin ferroxidase activity induced corresponding changes in light production, emphasizing the possible role of ferritin in the worm bioluminescence. DNA of the protein was cloned, sequenced, and expressed, confirming its identity to a Chaetopterus Ferritin (ChF). Both ferric and ferrous iron were found in the mucus, indicating the occurrence of both oxidase and reductase activity. Biochemical analysis showed ChF has strong ferroxidase activity, which could be a source of biological iron and catalytic energy for the worm bioluminescence when coupled to a reduction process with flavins.

GGNG peptides are a group of excitatory neuropeptides identified from several annelids and a mollusk. Each member of the GGNG peptides is named EEP from the earthworm, LEP from the leech, PEP from the marine worm, and TEP from the marine snail. These peptides consisted of 15–18 amino acids, and have a ring structure made by one disulfide bond. They share the C-terminal GGNG or GGN–NH2 structure. Known bioactivities of GGNG peptides are mostly excitatory; namely, they induce or potentiate the contraction of the digestive tract and sex-associated organs. On the precursor proteins of GGNG peptides so far known, a single copy of the peptide is encoded in the N-terminal or medial region of the protein.

The lipid composition of four tissues, musculo-cutaneous, blood, internal organs, and gonads, from the marine worm Urechis unicinctus was studied. It was shown that the principal phospholipids (PL) in the studied tissues were phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Depending on the tissue type, the fraction of PC was 31.8–42.6%; PE, 16.1–25.7% (of total PL). Blood contained a high percentage of lyso-PE (20.8%). The fatty-acid (FA) composition of the tissues consisted of C16, C18, C20, and C22 families with different degrees of unsaturation. Eicosapentaenoic acid (20:5) had the highest percentage in all tissues at 15.5–26.1%. Tissues with different functions such as musculo-cutaneous and gonads contained the same amounts of 20:5 FA, 26.1% (of total FA). The high content of polyunsaturated FA and, in particular, eicosapentaenoic FA in U. unicinctus confirmed that it was valuable as not only an energy source but also a source of PL and essential FA.

A homogeneous peptide with a mass of 683 Da which inhibits HIV-1 integrase with IC50 3 × 10−5 M was separated from aqueous extracts of a marine worm Eunicidae sp. by multistage chromatography purification. The Asp-Leu-Hse-His-Ala-Gln structure was proposed for this peptide according to amino acid analysis, automated amino acid Edman sequences, and TLC with witness homoserine and MS/MS fragmentation. The proposed structure is the first example of a natural peptide containing an amino acid homoserine residue.

Total mRNA were extracted from brain of Nereis diversicolor (Annelida, Polychaeta) and were translated in vitro or in ovo. The newly synthesized polypeptides were analyzed through electrophoresis of immunoprecipitated products or the Western blotting technique using polyclonal antibodies raised against mammalian dynorphin 1-17 and mammalian alpha-neo-endorphin. Among the products translated in vitro, only one class of polypeptide of 70 kDa was recognized by anti-dynorphin 1-17 antibodies. Furthermore, some in ovo translated products as well as proteins extracted from brain of worms showed identical immunoreactivity. These polypeptides, 60-70 kDa, reacted with anti-dynorphin 1-17 and anti alpha-neo-endorphin antibodies. These results suggest the existence of epitopes common to in ovo and in vitro translated products, to polypeptides extracted from the brain and to some mammalian opioid peptides of the prodynorphin family. We postulate the presence, in the brain of N. diversicolor, of precursors of peptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Data reported in this investigation do not allow us to propose or even postulate the presence, in the brain of the worm, of one precursor molecule common to polypeptides related to mammalian dynorphin 1-17 and alpha-neo-endorphin. Furthermore, the Nereis precursor molecules exhibit a clear-cut difference in molecular mass with the mammalian prodynorphin: 70 kDa versus 30 kDa.

The changes in heme (associated with hemoglobin), hemoglobin and hematin in the coelomic fluid of marine worm,Urechis unicinctus, exposed to different concentrations of sulfide, were investigated using biochemical techniques. When exposed to different sulfide concentrations for up to 96 h, the relative amounts of the three components changed in a regular pattern suggesting that the coelomocytes play an important role in the worm's tolerance to sulfide. The possible roles of heme compounds in sulfide tolerance of this species are discussed on the basis of our experimental data.

Higher animals typically rely on calcification to harden certain tissues such as bones and teeth. Some notable exceptions can be found in invertebrates: The fangs, teeth, and mandibles of diverse arthropod species have been reported to contain high levels of zinc. Considerable quantities of zinc also occur in the jaws of the marine polychaete worm Nereis sp. High copper levels in the polychaete worm Glycera dibranchiata recently were attributed to a copper-based biomineral reinforcing the jaws. In the present article, we attempt to unravel the role of zinc in Nereis limbata jaws, using a combination of position-resolved state-of-the-art techniques. It is shown that the local hardness and stiffness of the jaws correlate with the local zinc concentration, pointing toward a structural role for zinc. Zinc always is detected in tight correlation with chlorine, suggesting the presence of a zinc–chlorine compound. No crystalline inorganic phase was found, however, and results from x-ray absorption spectroscopy further exclude the presence of simple inorganic zinc–chlorine compounds in amorphous form. The correlation of local histidine levels in the protein matrix and zinc concentration leads us to hypothesize a direct coordination of zinc and chlorine to the protein. A comparison of the role of the transition metals zinc and copper in the jaws of two polychaete worm species Nereis and Glycera , respectively, is presented.

The genotoxic, cytotoxic and ontogenetic (embryo-larval) or developmental effects of tri-n-butyltin (TBT), were investigated in Platynereis dumerilii. Following the determination of maximum tolerated dose with regard to ontogenetic effects and mortality, early life stages of P. dumerilii were exposed to a range of TBT concentrations. Subsequently, the embryo-larvae were analysed for evidence of genotoxicity and cytotoxicity. Genotoxicity was assessed using cytogenetic endpoints that included the frequency of sister chromatid exchanges and chromosomal aberrations from metaphase spreads. Cytotoxicity was evaluated by determining the proliferative rate index of the growing embryo-larval cells using 5-bromodeoxyuridine labelling of the chromosomes or fluorescence plus Giemsa staining technique. TBT-exposed embryo-larvae of P. dumerilii exhibited sensitivity similar to that of other invertebrates, indicating that P. dumerilii is a suitable ecotoxicity test species. The results also suggested dose-dependent effects for genotoxic and cytotoxic end points in relation to TBT exposure. The present study highlights the need to elucidate the relative importance of direct genotoxic and indirect effects through production of genotoxic hormonal derivatives.

Most of our knowledge on plastic ingestion by zooplankton comes from experiments exposing invertebrates to plastic particles smaller than their feeding apparatus. By examining millimetre-sized marine plastics using a scanning electron microscope, we putatively identified some surface textures as feeding marks produced by invertebrates grazing upon the plastic biofilm. We observed sub-parallel linear scrapes with 5-14 μm spacing, which is similar to typical distances between teeth of the mandibular gnathobases of copepods. We also observed peculiar rounded marks close to an unidentified marine worm. Small portions of the plastic particles were apparently removed, and perhaps ingested, during these putative grazing activities. Thus, we suggest that (1) plastic biofouling induces plastic ingestion, and (2) plastic pieces must not necessarily be smaller than the organism for a feeding interaction to occur. Experiments exposing invertebrates to millimeter-sized plastics may support these suggestions.

Amphitrite ornata, a terebellid polychaete, inhabits marine environments that are contaminated by biogenically produced halometabolites. These halogenated organic compounds are toxic and quite diverse. To survive in this environment, A. ornata produces a novel dehaloperoxidase (DHP I) that detoxifies haloaromatic compounds. In this study we identified and characterized two dehaloperoxidase genes, designated dhpA and dhpB, from an A. ornata complementary DNA library. The deduced amino acid sequences (DHP A and DHP B) of the two dhp genes both contain 137 amino acid residues, but they differ at 5 amino acid positions. Allelic variation was observed for both genes as well. Polymerase chain reaction-restriction fragment length polymorphism assays of genomic DNA from 19 in individuals showed that each individual contains both the dhpA and the dhpB genes. Therefore, the two types of DHP are encoded by separate genes and are not alleles of a single gene. Furthermore, DHP A and DHP B may have different substrate specificities since they have amino acid differences in the active site.

The precise mechanisms by which sperm trigger calcium transients in eggs or oocytes during fertilization remain unknown. Based on time-lapse confocal microscopy, we show that intracellular injections of porcine sperm extracts cause the oocytes of a marine nemertean worm to undergo repetitive calcium oscillations resembling those obtained during normal fertilizations. Such findings are consistent with the view that fertilization involves a soluble sperm factor (SF) which is capable of eliciting calcium transients without binding to externally situated receptors on the oocyte plasmalemma. This study also describes for the first time the wave-like propagation patterns of SF-induced calcium transients that are generated in a heterologous combination of gametes obtained from different phyla of animals. Such cross-reactivity between distantly related taxa suggests that the intracellular signaling pathways triggered by sperm factors can be well conserved.

The marine environment receives a wide variety of chemical inputs, many of which have the potential to damage DNA or interfere with the process of cell division. Here we describe a new assay based on the early embryo and larval stages of a planktonic spawning, tube dwelling marine worm, Pomatoceros lamarckii, which for experimental purposes has the advantage of producing large numbers of ripe gametes throughout the majority of the year. One of the most promising end-points is the use of dividing cells to detect anaphase aberrations such as lagging chromosomes, tripolar anaphases, acentric fragments and chromosome bridges. Apart from the reference mutagens mitomycin C and cyclophosphamide and the well-documented spindle poison colchicine, we tested the fungicide carbendazim, a primary metabolite of the fungicide benomyl, and thiabendazole, a pesticide and antihelminthic drug; both of which are known to act as aneugens in other test systems. In addition we tested sodium hypochlorite, a widely used oxidizing agent and disinfectant, di-butylphthalate, a commercial plasticizer and suspected aneugen, and sodium chloride, a recognized non-genotoxin. Significant increases in the frequency of anaphase abnormalities occurred with most test compounds at relatively low concentrations, confirming the sensitivity of the new assay. Sodium chloride yielded a negative response except at the highest non-relevant concentrations, where some chromatid stickiness was observed. In addition, the developmental consequences of exposure to these compounds were assessed in 4-8 cell embryos and at 48 h once the embryos had metamorphosed into free swimming larvae. Mitotic inhibition and anaphase aberrations were found to be a more sensitive indicator of genotoxic exposure than larval development, although there was a suggestion of a possible mechanistic link between aneugenicity/clastogenicity and larval fitness. The new test assay provides a rapid and inexpensive method for screening chemicals and effluents destined for release into the marine environment for potential gamete effects.

To monitor calcium and endoplasmic reticulum (ER) dynamics during oocyte maturation and fertilization, oocytes of the marine worm Cerebratulus lacteus were injected with the calcium-sensitive indicator calcium green dextran and/or the ER-specific probe "DiI." Based on time-lapse confocal imaging of such specimens, prophase-arrested immature oocytes failed to develop normally after insemination and typically produced non-wave-like calcium transients that were lower in amplitude and less persistent than the wave-like oscillations observed during fertilizations of mature oocytes. Accordingly, the ER of DiI-loaded immature oocytes lacked an obvious substructure, whereas ER clusters, or "microdomains," began to form in maturing specimens at about the time that these oocytes became competent to undergo normal fertilization-induced calcium dynamics and cleavage. The ER microdomains of mature oocytes typically reached widths of 1-8 micrometer and disappeared approximately 1 h after fertilization, which in turn coincided with the termination of the calcium oscillations. Collectively, these findings indicate: (i) changes in ER structure are temporally correlated with the onset and cessation of the calcium oscillations required for subsequent cleavage, and (ii) such ER reorganizations may play an important role in early development by enabling mature oocytes to generate a normal calcium response.

How sperm trigger activating calcium transients in eggs remains a central, unresolved question in fertilization biology. To determine if a soluble sperm factor can generate a fertilization-like calcium response in the absence of sperm-egg binding, aqueous extracts of sperm from the nemertean wormCerebratulus lacteuswere mixed with Ca2+-sensitive fluorescent dyes and injected into unfertilized, metaphase-I-arrested oocytes. Based on confocal imaging analyses, unfertilized oocytes that had been injected with sperm extract routinely produced oscillating Ca2+waves and resumed meiotic maturation in a manner that closely resembled normal fertilization. Calcium oscillations and maturation were typically lacking in control oocytes that had been (i) injected with buffer alone or with buffer containing added calcium, (ii) given external treatments of the sperm factor, or (iii) injected with extracts made from cells other than sperm. Boiling or protease treatment essentially abolished the potency of the sperm extract, and nonboiled extracts retained full activity in >10-kDa fractions, but not in

B-IV is a 55-residue, crustacean-selective, neurotoxin secreted by Cerebratulus lacteus, a large marine worm found along the northeastern coast of North America. The 3D structure of this molecule in aqueous solution has been determined by 1H NMR spectroscopy at 600 MHz. The molecule has a well-defined helical hairpin structure, with the branches of the hairpin linked by four disulphide bonds. The disulphide connectivities were established from the NMR data to be 1-8/2-7/3-6/4-5, which differed from those determined previously by chemical means, where 1-7 and 2-8 connectivities were found. Each branch of the hairpin is largely alpha-helical, with the helices in the N and C-terminal branches encompassing residues 11 to 23 and 34 to 49, respectively. The loop connecting the branches of the hairpin contains two inverse gamma-turns centred on residues 24 and 25, a type I beta-turn at residues 28 to 31 and a type II beta-turn at residues 30 to 33. Arg17, -25 and -34, which are important for activity, are all on the same face of the molecule, while Trp30, which is also important for activity, is on the opposite face. Structure comparisons show that the B-IV structure is quite similar to those of Rop (ColE1 repressor of primer) and the heat-stable enterotoxin B from Escherichia coli. These structural similarities are discussed in relation to possible mechanisms of action of B-IV.

Mature and hatched eggshells of Bdelloura candida, a member of a family of marine worms, have been examined to determine their structure, composition and mechanism of formation. This report concentrates on the metal content with the purpose of establishing whether or not metals that can form DOPA chelates are present, and, thereby, provide a means of cross-linking. Elevated levels of Fe, Cu and Zn were observed, all of which can form such chelates.

An in vivo genotoxicity test system has been developed using the embryo-larval stages of the marine annelid, Platynereis dumerilii (Polychaeta: Nereidae). This species is representative of an ecologically important group of marine invertebrates, it is amenable to laboratory culture and has a well defined and stable karyotype (2n=28) which is suitable for the analysis of a range of cytogenetic endpoints, including chromosomal aberrations (CAs) and sister chromatid exchanges (SCEs). An evaluation of the cell cycle kinetics using the embryo-larval stages allowed selection of exposure times for cytogenetic work. Subsequently, 12-h-old embryos were exposed to reference mutagens, dissolved in sea water, in the presence of 5-bromodeoxyuridine (BrdU) for 12 h (SCE analysis) or 8 h (CA analysis) at 15 +/- 1 degree C, by which time they had reached the first larval stage (20-24h). Dose response-relationships for cytotoxicity, SCEs and CAs were observed for both direct acting mutagens (methyl methanesulfonate, mitomycin C) and mutagens which require metabolic activation (cyclophosphamide, benzo[a]pyrene). The sensitivity of the embryo-larval stages of P. dumerilii to both direct and indirect acting mutagens, their suitability for laboratory culture, together with the presence of a good karyotype and chromosome morphology for cytogenetic analyses, makes this species a potentially valuable in vivo model for marine genotoxicity testing.

The synthesis of 3'-phosphoadenosine-5'-phosphosulfate (PAPS) from inorganic sulfate and ATP requires two enzymes, ATP sulfurylase (SUL) and adenosine-5'-phosphosulfate kinase (KIN). In bacteria, fungi, yeast and plants, the two enzymes are present on separate polypeptide chains. We have identified the first animal gene coding for these enzymes. In the marine worm, Urechis caupo (Uc), both SUL and KIN are present on a single polypeptide chain. This protein, which we call PAPS synthetase (PAPSS), is able to complement yeast mutants lacking either enzyme. The Uc PAPSS mRNA is present in oocytes, but is not translated until after fertilization. At least three adult tissues, gut, ceolomocytes and body wall, also contain the mRNA, but at lower concentrations than are found in embryos. Partial sequences of a similar gene from Caenorhabditis elegans (Ce) were detected in a search of the GenBank expressed sequence tag database. Comparison of these Uc and Ce PAPSS sequences with the sequences of cloned genes from non-animal organisms strongly suggests that the animal genes evolved through the fusion of the SUL- and KIN-encoding genes from lower organisms.

Much is known about how adult organisms protect themselves from exposure to foreign compounds (xenobiotics), but for a population to successfully colonize a habitat, all life stages of the organism must be able to grow and develop normally in that habitat. In this study, a multixenobiotic transporter is described which is similar to the multidrug transporter in mammalian cells, used by some embryos as protection from natural xenobiotics they may encounter in their environment. It is shown that some moderately hydrophobic pollutants — but not very hydrophobic pollutants — are competitive substrates of the multixenobiotic transporter in the embryos of a marine worm. Multixenobiotic resistance may protect Urechis caupo embryos from certain toxic substances by preventing their entry into cells. In this way the xenobiotics cannot interfere with cellular processes that are crucial for normal development, such as DNA synthesis, cell division, and gene transcription.

The tube-inhabiting marine worm, Cephalodiscus gilchristi has been found to produce a series of cytotoxic and antineoplastic disteroidal alkaloids. A substantial study concerned with discovery of new cell growth inhibitory constituents of this marine animal has led to isolation of cephalostatins 14 (5a) and 15 (5b). The structures were determined by interpretation of spectral data, principally high-field (400 and 500 MHz) NMR and HRFABMS combined with NMR spectral relationships to cephalostatin 1 (4) where the structure was previously assigned by X-ray crystal structure analysis. The stereochemical assignments were established employing the ROESY NMR technique. Both new cephalostatins significantly inhibited growth of the P388 lymphocytic leukemia and a selection of human cancer cell lines. The two new cephalostatins further enhance knowledge of structure–activity parameters for these potentially important steroidal alkaloids.

The project addresses the need to develop biological approaches to detoxifiy the environment. We have studied the first known hemoglobin that has a natural peroxidase function. The hemoglobin was first isolated from the marine worm (terebellid polychaete) Amphitrite ornata, but has been cloned and expressed in E. coli bacteria. During the grant period we discovered a principle of action of such peroxidases, namely internal inhibition by para-halophenols. This information permits further progress to be made in cloning and expressing bioremediation enzymes capable of degrading environmental pollutants such as 2,4-dichlorophenol, which is a byproduct of the insecticide 2,4-D and a precursor to highly toxic dioxins. We have published numerous X-ray crystal structures and nuclear magnetic resonance data to support the conclusions. We have carried out extensive enzymatic kinetic assays to demonstrate the utility of dehaloperoxidase for bioremediation applications.

Urechis caupo is a marine worm that lives and reproduces in sediments containing a variety of potentially toxic environmental chemicals (xenobiotics). Its embryos have a multixenobiotic transporter, which is similar to the multidrug transporter in mammals, as indicated by their ability to transport a variety of moderately hydrophobic compounds such as dyes, drugs, and pesticides out of the cells. The cell membranes of the embryos contain a protein of approximately 145 kD that is immunologically related to the mammalian multidrug transport protein and that can be cross-linked by a photoactivatable substrate of the mammalian multidrug transport protein. The sediments in which the worm lives contain potential substrates for the transporter, indicating that this multixenobiotic transport activity may protect Urechis embryos from naturally occurring toxic compounds. Embryos of a sea urchin from a pristine environment do not have this transport activity and are sensitive to hydrophobic toxins. These data strongly support a role for multixenobiotic transport as a mechanism of protection from environmental toxins and indicate an unsuspected mode of protection in invertebrate embryos.

Sequence-specific resonance assignments are reported for the 500-MHz 1H-NMR spectrum of the 55-residue neurotoxin B-IV, isolated from the heteronemertine worm Cerebratulus lacteus. A range of two-dimensional homonuclear correlated and NOE spectra was used in making these assignments, which include NH, C alpha H and C beta H resonances, as well as most resonances from longer-chain spin systems, with the exception of the ten Lys residues, where spectral overlap prevented complete, unambiguous assignments. The secondary structure of B-IV was identified from the pattern of sequential (i, i + 1) and medium range (i, i + 2/3/4) NOE connectivities and the location of slowly exchanging backbone amide protons. Two helices are present, incorporating residues 13-26 and 33-49, and the C-terminal five residues form a helix-like structure. A type-I reverse turn, involving residues 28-31 is present in a small loop linking the two major helices, and the N-terminus appears to be unordered at 27 degrees C, although it may adopt a more ordered conformation at lower temperatures. These elements of secondary structure, together with the four disulfide bonds in the protein, provide sufficient information to define the global fold of the molecule in solution. The pH and temperature dependence of the toxin have been investigated by 1H-NMR and the pKa values of several ionisable sidechains determined.

The purpose of this study was to elucidate the functional differences between the CK isoforms by cloning the cDNAs of 12 CK isoforms: the M and B cytoplasmic forms and uMiCK from mouse, the M1, M2 and B cytoplasmic forms from Danio rerio , M1 and M2 cytoplasmic forms from the lower vertebrate Lampetra japonica , a cytoplasmic CK and a MiCK from the marine worm Neanthes diversicolor , and a cytoplasmic CK and a MiCK from the soft coral Dendronephthya gigantea . These were expressed in Escherichia coli as a fusion protein with maltose-binding protein, and kinetic constants ( K m , K d and k cat ) of all the recombinant enzymes, except for the unstable Dendronephthya cytoplasmic CK, were determined for the forward reaction. The kinetic constants of the M- and B-forms of the mouse and Danio cytoplasmic CKs differed significantly, with the K m for creatine ( K m Cr ) of M-CK being three- to nine-fold higher than that of B-CK, possibly reflecting differences in the concentration of creatine in muscle and brain cells. The mouse uMiCK had the lowest K m Cr value among the CK isoforms. In addition, it also exhibited a strong synergism for substrate binding ( K d / K m = 11.8). These results indicate that uMiCK has unique characteristics compared with other CK isoforms. Two subisoforms of M-CK were found in the lower vertebrate L. japonica , and the kinetic constants of recombinant M1- and M2-CKs differed significantly. The M1- and M2-CKs were expressed in skeletal muscle with a ratio of 7:3, while M1-CK was the predominant subisoform in the testis. The kinetic constants of cytoplasmic CK from the marine worm Neanthes were significantly different from those of Neanthes MiCK, possibly indicating that functional differences among CK isoforms occurred at least before the divergence of annelids from other protostome invertebrates.

Hydrogen sulfide (H2S) at concentrations of about 0.05 to 1 mmol.l(-1) appears to function as a gasotransmitter in vertebrates, analogous to nitric oxide (NO) and carbon monoxide, but the actions of H2S in invertebrate tissue have not been well studied. In this study, we investigated the role of H2S in modulating body wall muscle tone in the marine echiuran worm Urechis caupo (Echiuridae). We first determined that U. caupo body wall homogenates produce H2S upon addition of L-cysteine and pyridoxal-5'-phosphate (PLP), and that the rate is increased by addition of 2-mercaptoethanol, suggesting the presence of an activated L-serine sulfhydrase pathway. We then measured the contractile response of U. caupo body wall circular muscle strips to sodium hydrosulfide (NaHS)--which produces H2S in solution--and the NO donor sodium nitroprusside (SNP), both with and without subsequent application of acetylcholine (ACh). We found that NaHS alone stimulated contraction in muscle strips equivalent to about one-third the force of ACh alone, whereas SNP alone had no effect on muscle tone. However, simultaneous addition of NaHS with SNP elicited a much stronger contraction, reaching more than twice that of ACh alone, which could be increased further by subsequent application of ACh.

The sterol fraction from the marine worm Linneus torquatus Coe (phylum Nemertini, class Anopla, family Lineidae) has been isolated, separated by HPLC and preparative TLC on AgNO3-impregnated silica gel, and sterols identified using GC, GC-MS and NMR spectroscopy. It was shown that the fraction contains at least 12 sterols belonging mainly to Δ5,22, Δ5,24(28) and Δ5 series. The major sterol components were 24-methylcholesta-5,24(28)-dien-3β-ol, cholesta-5,22E-dien-3β-ol, 24-nor-cholesta-5,22-dien-3β-ol and cholesterol.

Three novel 2-alkylpyrrole sulfamates (1-3) were isolated from the marine worm Cirriformia tentaculata. The structures were elucidated by the interpretation of spectral data obtained on inseparable mixtures of the unstable compounds. This suite of metabolites deterred feeding by the generalist predatory fish Thalassoma bifasciatum.

International audience; In the laboratory, marine worms were fed with a mixture of algae and several aliphatic hydrocarbons for 15 days. After ingestion by the worms, 34.9% of hydrocarbons are found in the faeces and only 3.1% accumulated in the gut. The comparison between the initial mixture and the faeces shows that the worm’s digestive process lead to changes in the distribution of the n-alkane mixture. These changes are different from those only due to physical processes in the experimental conditions. In our experiment, no variation in the distribution of hydrocarbons in faeces with time and no microbial hydrocarbon biodegradation were evidenced. Our results suggest that marine worm feeding can substantially affect the fate of hydrocarbons in the sedimentary marine ecosystem by predominantly stimulating dissolution processes.

The world market for insecticides is still dominated, albeit declining, by compounds that irreversibly inhibit acetylcholinesterase (AChE), one of the most essential enzymes in the central nervous system of insects, responsible for the cleavage of the all-important neurotransmitter acetylcholine (Pittman 1971). However, as shown in Table 1, the market share of these AChE inhibitors, i.e. organophosphates and carbamates, decreased from 71% in 1987 to some 56% in 1997. Combining the AChE inhibitors and those insecticides that act on the voltage-gated sodium channel, in particular the pyrethroids, accounts for more than 75% of the world market only by these two modes of action (Table 1). One of the insecticide molecular target sites of growing importance is the nicotinic acetylcholine receptor (nAChR; Table 1); increasing considerably in value over the last decade. Only ten years ago insecticides that acted on the nAChR were of minor economic importance (

International audience; In the laboratory, marine worms were fed with a mixture of algae and several aliphatic hydrocarbons for 15 days. By comparing hydrocarbons in food and in faeces, it appeared that the worm's digestive process led to changes in the distribution of the n-alkanes mixture. These changes were different from those only due to physical processes in the experimental conditions, indicating that marine worm feeding could substantially affect the fate of hydrocarbons in the sedimentary marine ecosystem.

We report a carbohydrate-dependent supramolecular architecture in the extracellular giant hemoglobin (Hb) from the marine worm Perinereis aibuhitensis; we call this architectural mechanism carbohydrate gluing. This study is an extension of our accidental discovery of deterioration in the form of the Hb caused by a high concentration of glucose. The giant Hbs of annelids are natural supramolecules consisting of about 200 polypeptide chains that associate to form a double-layered hexagonal structure. This Hb has 0.5% (wt) carbohydrates, including mannose, xylose, fucose, galactose, glucose, N-acetylglucosamine (GlcNAc), and N-acetylgalactosamine (GalNAc). Using carbohydrate-staining assays, in conjunction with two-dimensional polyacrylamide gel electrophoresis, we found that two types of linker chains (L1 and L2; the nomenclature of the Hb subunits followed that for another marine worm, Tylorrhynchus heterochaetus) contained carbohydrates with both GlcNAc and GalNAc. Furthermore, two types of globins (a and A) have only GlcNAc-containing carbohydrates, whereas the other types of globins (b and B) had no carbohydrates. Monosaccharides including mannose, fucose, glucose, galactose, GlcNAc, and GalNAc reversibly dissociated the intact form of the Hb, but the removal of carbohydrate with N-glycanase resulted in irreversible dissociation. These results show that carbohydrate acts noncovalently to glue together the components to yield the complete quaternary supramolecular structure of the giant Hb. We suggest that this carbohydrate gluing may be mediated through lectin-like carbohydrate-binding by the associated structural chains ("linkers").

In developing a research on the cholinesterase (ChE) evolution in Invertebrata, this enzyme was studied in the unsegmented marine worm Sipunculus nudus. ChE activity was solubilized through three successive steps of extraction. These fractions are noted as low-salt (LSS), detergent (DS) and high-salt soluble (HSS) and represent 27%, 68% and 5% of total activity, respectively. LSS and DS ChE were purified to homogeneity by affinity chromatography on edrophonium-Sepharose gel. Purification factors of 1700 (LSS) and 1090 (DS) were obtained. The small amount of HSS ChE prevented a similar purification and an extensive characterization. Based on SDS/PAGE and density-gradient centrifugation, both LSS and DS enzymes show a M(r) value of about 130,000 and are likely G2 globular dimers of a 67,000 subunit. Moreover, LSS ChE seems to be an amphiphilic form including a hydrophobic domain, while DS ChE is probably linked to the cell membrane by a phosphatidylinositol anchor. Both LSS and DS enzymes hydrolyze at the highest rate propionylthiocholine. However, they also show a fairly high catalytic efficiency with other thiocholine esters as substrates, thus suggesting a wide and little-specialized conformation of the active site. Based on immunological cross-reactivity trials, LSS and DS ChE from S. nudus show a reduced structural affinity with a molluscan (Murex brandaris) enzyme. HSS ChE, an acetylcholinesterase, is also solubilized by heparin, like typical vertebrate HSS asymmetric enzymes. However, it lacks fast-sedimenting forms and an enzyme-anchoring collagenous structure.

The pyridyl ring is frequently found in natural products and drugs. While bipyridyls have served as useful scaffolds for development of industrial and pesticidal chemicals, their biological properties are still not well understood. Only 2,3'-bipyridyl, of the six isomeric bipyridyls, has been reported as a natural product in tobacco plants and in a hoplonemertine marine worm, Amphiporus angulatus (Aa), which uses its pyridyl alkaloid-rich venom to paralyze its crustacean prey and chemically defend itself against predators. Here we report for the first time the synthesis and spectroscopic properties of all eight possible methyl 2,3'-bipyridyl isomers and use this data to identify a trace alkaloidal constituent of Aa as 3-methyl-2,3'-bipyridyl. This is only the second reported instance of a methyl-bipyridyl being found as a natural product, the first being the tobacco alkaloid 5-methyl-2,3'-bipyridyl.

Some marine worms, such as Thelepus crispus and Notomastus lobatus, secrete brominated aromatic molecules and other halogenated metabolites as repellants1. Other species, such as Amphitrite ornata, do not produce repellants but are adapted to the chemical warfare of N. lobatus and cohabit with them in estuarine mudflats. The halocompounds are tolerated by A. ornata as they are degraded by dehaloperoxidase (DHP)2. We have determined the amino-acid sequence and crystal structure of DHP and find that its fold is typical of the globin family, indicating that the enzyme evolved from an oxygen carrier protein. Residues at the dimer interface do not correspond to those in tetrameric and dimeric haemoglobins and the spatial arrangement of the dimers is different. The complete amino-acid sequence is most similar to that of myoglobin from the sea hare, with 20.6% identity among 126 overlapping amino acids.

The quantitative influence of anaerobic metabolism on acid-base status and on acid-base regulation is investigated in Sipunculus nudus L. Proton generation by metabolism is calculated from theoretical predictions. The quantitative comparison of metabolic protons with non-respiratory protons found in the acid-base status is performed assuming a simplified model of the total animal. Taking the protonequivalent ion exchange between animals and ambient water into account, changes in the anaerobic acid-base status can be explained exclusively by proton generation in metabolism. It is concluded that the classical concept of acid-base physiology is adequate and that the consideration of strong ions is not required for a quantitative treatment of the acid-base status. The hypothesis that a quantitative correlation exists between metabolic and acid-base events is tested by comparing changes in acid-base status and in metabolism in animals exhibiting different metabolic rates. For this purpose, a method is developed for the calculation of intracellular pH from metabolite concentrations and extracellular acid–base parameters. Proton exchange between intra-and extracellular compartments, which is found to depend upon the total amount of accumulated non-respiratory protons, demonstrates that pHi is regulated even during anaerobiosis. The defended pH1 value, however, is lower during anaerobiosis than during subsequent recovery.

A highly crystalline form of α-chitin found in the grasping spines of the marine worm Sagitta has been examined using electron microscopy and electron diffraction. Sections cut parallel to the (100) planes show Bragg diffraction peaks out to spacings of less than 0.1 nm. The results are compared with previously reported results from α-chitin obtained from deproteinized lobster tendom.