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Discovering macromolecules and understanding the associated mechanisms involved in underwater adhesion are essential for both studying the fundamental ecology of benthos in aquatic ecosystems and developing biomimetic adhesive materials in industries. Here, we employed quantitative proteomics to assess protein expression variations during the development of the distinct adhesive structure - stolon in the model fouling ascidian, Ciona robusta. We found 16 adhesive protein candidates with increased expression in the stolon, with ascidian adhesive protein 1 (AAP1) being particularly rich in adhesion-related signal peptides, amino acids, and functional domains. Western blot and immunolocalization analyses confirmed the prominent AAP1 signals in the mantle, tunic, stolon, and adhesive footprints, indicating the interfacial role of this protein. Surface coating and atomic force microscopy experiments verified AAP1′s adhesion to diverse materials, likely through the specific electrostatic and hydrophobic amino acid interactions with various substrates. In addition, molecular docking calculations indicated the AAP1′s potential for cross-linking via hydrogen bonds and salt bridges among Von Willebrand factor type A domains, enhancing its adhesion capability. Altogether, the newly discovered interfacial protein responsible for permanent underwater adhesion, along with the elucidated adhesion mechanisms, are expected to contribute to the development of biomimetic adhesive materials and anti-fouling strategies. Discovering macromolecules and studying their associated mechanisms involved in underwater adhesion are essential for understanding the fundamental ecology of benthos in aquatic ecosystems and developing innovative bionic adhesive materials in various industries. Using multidisciplinary analytical methods, we identified an interfacial protein - Ascidian Adhesive Protein 1 (AAP1) from the model marine fouling ascidian, Ciona robusta. The interfacial functions of AAP1 are achieved by electrostatic and hydrophobic interactions, and the Von Willebrand factor type A domain-based cross-linking likely enhances AAP1's interfacial adhesion. The identification and validation of the interfacial functions of AAP1, combined with the elucidation of adhesion mechanisms, present a promising target for the development of biomimetic adhesive materials and the formulation of effective anti-fouling strategies. [Display omitted] [ABSTRACT FROM AUTHOR]

The glycoprotein hormone (GPH) system is fundamentally significant in regulating the physiology of chordates, such as thyroid activity and gonadal function. However, the knowledge of the GPH system in the primitive chordate ascidian species is largely lacking. Here, we reported an ancestral GPH system in the ascidian (Styela clava), which consists of GPH α subunit (Sc-GPA2), GPH β subunit (Sc-GPB5), and the cognate leucine-rich repeat-containing G protein-coupled receptor (Sc-GPHR). Comparative structure analysis revealed that distinct from vertebrate GPH β subunits, Sc-GPB5 was less conserved, showing an atypical N-terminal sequence with a type II transmembrane domain instead of a typical signal peptide. By investigating the presence of recombinant Sc-GPA2 and Sc-GPB5 in cell lysates and culture media of HEK293T cells, we confirmed that these two subunits could be secreted out of the cells via distinct secretory pathways. The deglycosylation experiments demonstrated that N-linked glycosylation only occurred on the conserved cysteine residue (N78) of Sc-GPA2, whereas Sc-GPB5 was non-glycosylated. Although Sc-GPB5 exhibited distinct topology and biochemical properties in contrast to its chordate counterparts, it could still interact with Sc-GPA2 to form a heterodimer. The Sc-GPHR was then confirmed to be activated by tethered Sc-GPA2/GPB5 heterodimer on the Gs-cAMP pathway, suggesting that Sc-GPA2/GPB5 heterodimer-initiated Gs-cAMP signaling pathway is evolutionarily conserved in chordates. Furthermore, in situ hybridization and RT-PCR results revealed the co-expression patterns of Sc-GPA2 and Sc-GPB5 with Sc-GPHR transcripts, respectively in ascidian larvae and adults, highlighting the potential functions of Sc-GPA2/GPB5 heterodimer as an autocrine/paracrine neurohormone in regulating metamorphosis of larvae and physiological functions of adults. Our study systematically investigated the GPA2/GPB5-GPHR system in ascidian for the first time, which offers insights into understanding the function and evolution of the GPH system within the chordate lineage. [ABSTRACT FROM AUTHOR]

• DOM exposure results in higher lipophilic contaminant uptake trend in mucous feeder. • Despite favorable emulsification properties terrDOM does not lead to more uptake. • The pattern in Ciona matches observations made during a previous study on Mytilus. • Less contaminant accumulated in mucous feeder Ciona than in Mytilus. The widely reported increase of terrestrial dissolved organic matter (terrDOM) in northern latitude coastal areas ("coastal darkening") can impact contaminant dynamics in affected systems. One potential impact is based on differences in chemical adsorption processes of the molecularly larger terrDOM compared to marine DOM (marDOM) that leads to increased emulsification of lipophilic contaminants with terrDOM. Filter feeders filter large amounts of water and DOM daily and thus are directly exposed to associated contaminants through both respiration and feeding activity. Thus, increased exposure to terrDOM could potentially lead to an increase in bioaccumulation of lipid soluble contaminants in filter feeders. To assess the effect of DOM on bioaccumulation in filter feeders, we exposed the mucous based filter feeding ascidian Ciona intestinalis (formerly known as Ciona intestinalis Type B), to the lipophilic veterinary drug teflubenzuron (log K OW : 5.39) in combination with four DOM treatments: TerrDOM, marDOM, a mix of the two called mixDOM, and seawater without DOM addition. The exposure lasted for 15 days, after which the individuals in all DOM treatments showed a trend towards higher bioaccumulation of Teflubenzuron than those in the seawater control. However, there was considerable overlap in posterior distributions. Against our expectations, marDOM resulted in the highest bioaccumulation factor (BAF), followed by mixDOM, with terrDOM resulting in the lowest BAF except for seawater (kinetic BAF L/kg median, 2.5 %–97.5 % percentile marDOM 94, 74–118; mixDOM 82, 63–104; terrDOM 79; 61-99; seawater 61, 44–79). All BAFs were below the level of concern according to the EU REACH regulation (BAF < 2000 L / kg) and, therefore, likely not environmentally problematic in the examined context. However, the results show that DOM can act as a dietary vector; thus, different combinations of contaminants, DOM, and filter feeding organisms should be tested further. [ABSTRACT FROM AUTHOR]

Deuterostome invertebrates, including echinoderms, hemichordates, cephalochordates, and urochordates, exhibit common and species-specific morphological, developmental, physiological, and behavioral characteristics that are regulated by neuroendocrine and nervous systems. Over the past 15 years, omics, genetic, and/or physiological studies on deuterostome invertebrates have identified low-molecular-weight transmitters, neuropeptides and their cognate receptors, and have clarified their various biological functions. In particular, there has been increasing interest on the neuroendocrine and nervous systems of Ciona intestinalis Type A, which belongs to the subphylum Urochordata and occupies the critical phylogenetic position as the closest relative of vertebrates. During the developmental stage, gamma-aminobutylic acid, D-serine, and gonadotropin-releasing hormones regulate metamorphosis of Ciona. In adults, the neuropeptidergic mechanisms underlying ovarian follicle growth, oocyte maturation, and ovulation have been elucidated. This review article provides the most recent and fundamental knowledge of the neuroendocrine and nervous systems of Ciona , and their evolutionary aspects. • Deuterostomes possess a variety of endocrine, neuroendocrine, and nervous systems. • Ciona , a deuterostome invertebrate, is the closest relative of vertebrates. • Metamorphosis is regulated by neurotransmitters and neuropeptides in Ciona larva. • Ovarian follicle development in Ciona adults is regulated by neuropeptides. • Recent studies highlight common and unique neuroendocrine and nervous systems of Ciona in deuterostomes. [ABSTRACT FROM AUTHOR]

Calcium ion (Ca2+) is involved in the protein-mediated larval adhesion of fouling ascidians, yet the effects of environmental Ca2+ on larval adhesion remain largely unexplored. Here, the larvae of fouling ascidian C. robusta were exposed to different concentrations of Ca2+. Exposures to low-concentration (0 mM and 5 mM) and high-concentration (20 mM and 40 mM) Ca2+ significantly decreased the adhesion rate of larvae, which was primarily attributed to the decreases in adhesive structure length and curvature. Changes in the expressions of genes encoding adhesion-, microvilli-, muscle contraction-, and collagen-related proteins provided a molecular-level explanation for adhesion rate reduction. Additionally, larvae likely prioritized their energy towards immunomodulation in response to Ca2+ stresses, ultimately leading to adhesion reduction. These findings advance our understanding of the influencing mechanisms of environmental Ca2+ on larval adhesion, which are expected to provide references for the development of precise antifouling strategies against ascidians and other fouling species. [Display omitted] • Exposures to high- and low-concentrations of Ca2+ reduced larval adhesion rate. • Adhesive structure changes attributed to the reduction in adhesion rate. • Expression changes of adhesion-related genes explained adhesion reduction. • Collagen, microvilli, and muscle-related genes were related to adhesion reduction. • Larvae prioritized energy towards immunomodulation, leading to adhesion reduction. [ABSTRACT FROM AUTHOR]

Abstract The toxicity of natural marine iodoarenes or their synthetic counterparts is widely unknown despite the fact that triiodothyronine and thyroxine are members of this class. In this work we aimed to expand such knowledge on iodinated marine natural products and tested an ascidian (Didemnum rubeum) metabolite, N -(3,5-diiodo-4-methoxyphenethyl)benzamide, together with closely related synthetic iodinated tyramides: N -(2,5-diiodo-4-methoxyphenethyl)benzamide, N -(3-iodo-4-methoxyphenethyl)benzamide, N -(4-methoxyphenethyl)benzamide, and N -(3-iodo-4-methoxyphenethyl)formamide, for their effect on the viability of rat macrophages, as well as acute toxicity on Artemia salina. The tested tyramides exerted a varying degree of toxicity towards brine shrimps, but in certain cases, the determined lethal concentrations were even lower than those of known toxicants (e.g. strychnine sulfate, SDS). The toxicity was highly dependent on the structure of these mutually related compounds, while the natural one was shown to be the most toxic. In the case of macrophage cultures, the tested tyramides exerted much less toxicity but were found to have an effect on the functioning of these normal immune cells. The samples of the tyramides were obtained by synthesis, and were fully structurally and spectrally characterized, which also provided corroboration of the proposed structure of the natural product originally isolated in minute amounts. Graphical abstract Image 1 Highlights • An iodinated marine natural product and four related tyramides were synthesized and fully spectrally characterized. • Their acute toxicity was determined in the brine shrimp model, and some of them turned out to be more toxic than strychnine. • Their effect on rat macrophage viability and functioning (MPO activity and adhesion ability) was tested. • Macrophage functional properties were affected (especially by the natural product) while the viability was mainly unaltered. • The toxicity was highly dependent on structural variations, but the presence of an I atom and benzamide moiety were crucial. [ABSTRACT FROM AUTHOR]

Innate immune responses face infectious microorganisms by inducing inflammatory responses. Multiple genes within distinct functional categories are coordinately and temporally regulated by transcriptional ‘on’ and ‘off’ switches that account for the specificity of gene expression in response to external stimuli. Mechanisms that control transcriptional and post-transcriptional regulation are important in coordinating the initiation and resolution of inflammation. Macrophage migration inhibitory factor (MIF) is an important cytokine that, in Ciona robusta , is related to inflammatory response. It is well known that in C. robusta, formerly known as Ciona intestinalis, the pharynx is involved in the inflammatory reaction induced by lipopolysaccharide (LPS) injection in the body wall. Using this biological system, we describe the identification of two C. robusta MIFs ( Cr MIF1 and Cr MIF2). The phylogenetic tree and modeling support a close relationship with vertebrate MIF family members. Cr MIF1 and Cr MIF2 possess two evolutionally conserved catalytic sites: a tautomerase and an oxidoreductase site with a conserved CXXC motif. Real-time PCR analysis shows a prompt expression induced by LPS inoculation in Cr MIF1 and a late upregulation of Cr MIF2 and in silico analyses of 3’UTR show a cis-acting GAIT element and a CPE element in 3’-UTR, which are not present in the 3’-UTR of Cr MIF1, suggesting that different transcriptional and post-transcriptional control mechanisms are involved in the regulation of gene expression of MIF during inflammatory response in C. robusta . [ABSTRACT FROM AUTHOR]

Tritaeta gibbosa is an epibiotic amphipod that lives in self-constructed invaginations in the tissue of various hosts as diverse as sponges, soft corals, ascidians and echinoderms. Here, some aspects of the relationship of T. gibbosa to different hosts are described. The main focus, however, is the colonisation of the holothurian Ocnus planci. The association between this holothurian species and T. gibbosa was studied by means of histology and scanning electron microscopy. The investigations revealed that T. gibbosa does not damage the host tissue. Only some stretching, thinning, thickening, and displacement of the holothurian cuticle, epidermis and dermis was observed. This suggests that the invagination is formed by pressure caused by a specific behaviour of T. gibbosa . The amphipod takes advantage of the properties of the mutable connective tissue characteristic for echinoderms. The majority of invaginations are situated at the holothurian’s anterior end. Furthermore, most invaginations are perpendicularly oriented to the long axis of the holothurian body. This seems to improve the exploitation of food particles coming from the tentacles of O. planci . The amphipod specimens are subadult to adult and there is a strong bias toward females as is generally the case in benthic T . gibbosa populations. Although some new information about the ecology of T. gibbosa is added by this study, some general questions such as the tissue reactions of the various hosts, the ontogeny and juvenile behaviour, and the type of association to the host still need investigation. [ABSTRACT FROM AUTHOR]

The aim of this work was to evaluate the Ascidian Embryo Teratogenicity assay (AET) as new alternative invertebrate model to test the developmental effects of the co-exposure to ethanol and fluconazole. Ciona intestinali s embryos were exposed to the azolic fungicide fluconazole, (FLUCO, 7.8–250 μM), to ethanol (Eth, 0.01–0.5%) and to their mixture (0.01% Eth + FLUCO 7.8–250 μM) from neurula to larval stage. At the end of the exposure period, larvae were morphologically evaluated and benchmark analysis performed by using the PROAST modelling software. Both compounds were teratogenic in a concentration-related manner, particularly affecting the pigmented organs. The co-exposure to Eth enhanced the effects of FLUCO, the additive hypothesis was not rejected by the modelling. The results demonstrated that AET could be considered a good vertebrate-free alternative model for toxicological investigation in embryos. [ABSTRACT FROM AUTHOR]

Ascidians are good monitors for assessing water quality, since they filter large volumes of water; however, little is known about how xenobiotics, including metals, can affect ascidian hemocytes. Metals can be either toxic or beneficial to health, inducing many different responses. The response mechanism depends on the class of metals to which organisms are exposed: essential, nonessential, and borderline. To analyze the influence of metals from different classes on the protective mechanisms of an ascidian, we investigated the production of nitric oxide (NO) after exposure to various concentrations of Mg, Mn and Pb over different time periods. We also determined the amounts of each metal in the hemocytes. Our results indicated that especially Pb could stimulate NO production. Although Pb induced the highest NO production, cell viability was not severely altered in all Pb concentrations and time periods. Ascidians might serve as biomonitor for Pb, since their vanadocytes accumulate Pb. [ABSTRACT FROM AUTHOR]

To unveil the underlying mechanism of mitochondrial gene regulation associated with ageing and budding in the tunicate Polyandrocarpa misakiensis , mitochondrial non-coding-region (NCR)-containing reporter genes were constructed. PmNCR2.3K/GFP was expressed spatiotemporally in a pattern quite similar to mitochondrial 16S rRNA . The reporter gene expression was sensitive to high dose of rifampicin similar to mitochondrial genes, suggesting that the transcription indeed occurs in mitochondria. However, the gene expression also occurred in vivo in the cell nucleus and in vitro in the nuclear extracts. Mitochondrial transcription factor A ( PmTFAM ) enhanced reporter gene expression, depending on the NCR length. A budding-specific polypeptide TC14-3 is an epigenetic histone methylation inducer. It heavily enhanced reporter gene expression that was interfered by histone methylation inhibitors and PmTFAM RNAi. Our results indicate for the first time that the nuclear histone methylation is involved in mitochondrial gene activity via TFAM gene regulation. [ABSTRACT FROM AUTHOR]

Allorecognition is the capability of an organism to recognize its own or related tissues. The colonial ascidian Botryllus schlosseri , which comprises five genetically distinct and divergent species (Clades A-E), contains two adjacent genes that control allorecognition: fuhc sec and fuhc tm . These genes have been characterized extensively in Clade A and are highly polymorphic. Using alleles from 10 populations across the range of Clade A, we investigated the type and strength of selection maintaining this variation. Both fuhc genes exhibit higher within-population variation and lower population differentiation measures (F ST ) than neutral loci. The fuhc genes contain a substantial number of codons with >95% posterior probability of d N /d S > 1. fuhc sec and fuhc tm also have polymorphisms shared between Clade A and Clade E that were present prior to speciation (trans-species polymorphisms). These results provide robust evidence that the fuhc genes are evolving under balancing selection. [ABSTRACT FROM AUTHOR]

Lysozyme is an important defense molecule of the innate immune system. Known for its bactericidal properties, lysozyme catalyzes the hydrolysis of b-(1,4)-glycosidic bonds between the N-acetyl glucosamine and N-acetyl muramic acid in the peptidoglycan layer of bacterial cell walls. In this study, the complete coding sequence of four g-type lysozymes were identified in Ciona intestinalis . Phylogenetic analysis and modelling supported the hypothesis of a close relationship with the vertebrate g-type lysozymes suggesting that the C. intestinalis g-type lysozyme genes ( Ci Lys-g1, Ci lys-g2, Ci Lys-g3, Ci Lys-g4) share a common ancestor in the chordate lineage. Protein motif searches indicated that C. intestinalis g-type lysozymes contain a GEWL domain with a GXXQ signature, typical of goose lysozymes. Quantitative Real-Time PCR analysis results showed that transcripts are expressed in various tissues from C. intestinalis . In order to determine the involvement of C. intestinalis g-type lysozymes in immunity, their expression was analyzed in the pharynx, showing that transcripts were significantly up-regulated in response to a challenge with lipopolysaccharide (LPS). These data support the view that Ci Lys g-type are molecules with potential for immune defense system against bacterial infection. [ABSTRACT FROM AUTHOR]

[Display omitted] • First review on the extraction of tunicate-derived cellulose: alkali treatment and the kraft paper process. • Preparation methods of tunicate-derived nanocellulose are evaluated. • The unique structural composition of this cellulose and the application prospects in various fields are discussed. • Provide insight into future research prospects for this cellulose. Cellulose extracted from the outer capsule of tunicates has received much attention from researchers because of its excellent physicochemical properties. In addition, its nanocellulose has high strength, high purity, high modulus, and versatile photoelectromagnetic properties, and is gradually becoming a novel biomaterial. However, the utilization of tunicate is extremely low, and its discarded outer capsules accumulate in large quantities, resulting in a huge environmental burden and waste of resources. Therefore, it has become imperative to fully exploit and reuse tunicates. This review highlights the different extraction methods (e.g. , alkali treatment and kraft process, etc.) and the preparation techniques (e.g., acid hydrolysis, enzymatic treatment, TEMPO oxidation and spinning technique , etc.). The successful preparation and functional modification of tunicate nanocellulose provide the basis for the discovery of its new functions. The applications of tunicate and its composites in various fields (e.g. , biomedical field, energy and electronics, polymer reinforcement, food packaging field, paper industry and environmental materials, etc.) are also discussed. Finally, this review concludes with further applications and future perspectives of tunicate cellulose, to provide new ideas and references for improving the high-value integrated utilization of the outer capsule of tunicates. [ABSTRACT FROM AUTHOR]

Prochloron , a blue-green algae belonging to ancient prokaryotes, produces, like other cyanobacteria, cyclic pseudo -peptides, which are also found in its obligate symbiont ascidiae ( Lissoclinum patellum ). Although research has focused for some time on the putative metabolic function of these cyclic peptides, to date it is still not understood. Their role might be connected to the increased concentrations of divalent metal ions, especially Cu II , found in ascidiae. Dinuclear copper(II) complexes of cyclic pseudo -peptides revealed a broad hydrolytic capacity, including carboanhydrase and phosphatase activity. This study reports their β - lactamase as well as α- and β - glycosidase activity with k cat = (11.34 ± 0.91)ˑ10 − 4 s − 1 for β - lactamase, k cat = (1.55 ± 0.13)ˑ10 − 4 s − 1 for α-glycosidase and k cat = (1.22 ± 0.09)ˑ10 − 4 s − 1 for β - glycosidase activity. [ABSTRACT FROM AUTHOR]

Transforming growth factor (TGF-β) is a well-known component of a regulatory cytokines superfamily that has pleiotropic functions in a broad range of cell types and is involved, in vertebrates, in numerous physiological and pathological processes. In the current study, we report on Ciona intestinalis molecular characterisation and expression of a transforming growth factor β homologue ( Ci TGF-β). The gene organisation, phylogenetic tree and modelling supported the close relationship with the mammalian TGF suggesting that the C. intestinalis TGF-β gene shares a common ancestor in the chordate lineages. Functionally, real-time PCR analysis showed that Ci TGF-β was transcriptionally upregulated in the inflammatory process induced by LPS inoculation, suggesting that is involved in the first phase and significant in the secondary phase of the inflammatory response in which cell differentiation occurs. In situ hybridisation assays revealed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by cluster of hemocytes inside the pharynx vessels. These data supported the view that Ci TGF-β is a potential molecule in immune defence systems against bacterial infection. [ABSTRACT FROM AUTHOR]

As invasive species have successfully colonized a wide range of dramatically different local environments, they offer a good opportunity to study interactions between species and rapidly changing environments. Gene expression represents one of the primary and crucial mechanisms for rapid adaptation to local environments. Here, we aim to select reference genes for quantitative gene expression analysis based on quantitative Real-Time PCR (qRT-PCR) for a model invasive ascidian, Ciona savignyi . We analyzed the stability of ten candidate reference genes in three tissues (siphon, pharynx and intestine) under two key environmental stresses (temperature and salinity) in the marine realm based on three programs (geNorm, NormFinder and delta Ct method). Our results demonstrated only minor difference for stability rankings among the three methods. The use of different single reference gene might influence the data interpretation, while multiple reference genes could minimize possible errors. Therefore, reference gene combinations were recommended for different tissues - the optimal reference gene combination for siphon was RPS15 and RPL17 under temperature stress, and RPL17 , UBQ and TubA under salinity treatment; for pharynx, TubB , TubA and RPL17 were the most stable genes under temperature stress, while TubB , TubA and UBQ were the best under salinity stress; for intestine, UBQ , RPS15 and RPL17 were the most reliable reference genes under both treatments. Our results suggest that the necessity of selection and test of reference genes for different tissues under varying environmental stresses. The results obtained here are expected to reveal mechanisms of gene expression-mediated invasion success using C. savignyi as a model species. [ABSTRACT FROM AUTHOR]

The glycosaminoglycan heparin and its derivatives act strongly on blood coagulation, controlling the activity of serine protease inhibitors in plasma. Nonetheless, there is accumulating evidence highlighting different anticancer activities of these molecules in numerous types of cancer. Nano-heparins may have great biological significance since they can inhibit cell proliferation and invasion as well as inhibiting proteasome activation. Moreover, they can cause alterations in the expression of major modulators of the tumor microenvironment, regulating cancer cell behavior. In the present study, we evaluated the effects of two nano-heparin formulations: one isolated from porcine intestine and the other from the sea squirt Styela plicata , on a breast cancer cell model. We determined whether these nano-heparins are able to affect cell proliferation, apoptosis and invasion, as well as proteasome activity and the expression of extracellular matrix molecules. Specifically, we observed that nano- Styela compared to nano-Mammalian analogue has higher inhibitory role on cell proliferation, invasion and proteasome activity. Moreover, nano- Styela regulates cell apoptosis, expression of inflammatory molecules, such as IL-6 and IL-8 and reduces the expression levels of extracellular matrix macromolecules, such as the proteolytic enzymes MT1-MMP, uPA and the cell surface proteoglycans syndecan-1 and -2, but not on syndecan-4. The observations reported in the present article indicate that nano-heparins and especially ascidian heparin are effective agents for heparin-induced effects in critical cancer cell functions, providing an important possibility in pharmacological targeting. [ABSTRACT FROM AUTHOR]

Several families of ascidians accumulate extremely high levels of vanadium in their blood cells. The concentration of vanadium has been determined in each species; the highest concentration, found in Ascidia gemmata , reaches 350 mM, corresponding to 10 7 times that of sea water. How and why ascidians accumulate vanadium in a highly selective manner and at such extremely high levels have yet to be determined. To address these questions, our research group sought to identify the genes and proteins responsible for the accumulation and reduction of vanadium in vanadocytes, a type of blood cell, as well as the process of vanadium transport from sea water to blood cells through the branchial sac, intestine, and blood plasma. Here, we review the accumulation steps as a system, especially those related to the concentration and chemical species of vanadium at each step. A comprehensive analysis on each organ has already revealed several categories of protein families, such as vanadium-binding proteins and vanadium transporters. Herein, we also discuss the mechanisms by which ascidians selectively accumulate vanadium ions from a biochemical viewpoint. [ABSTRACT FROM AUTHOR]

Overfishing and nutrient enrichment are among the major local stressors to coral reefs worldwide, as they can alter the benthic reef community by promoting fast growing algae and bioeroders. The Northern coast of Pacific Costa Rica is strongly influenced by seasonal upwelling events that naturally increase nutrient concentrations between December and March. This study therefore investigated the combined effects of simulated overfishing and naturally increased nutrients on benthic community composition and succession on settlement tiles over a period of 24 weeks (October 2013 until March 2014) using exclusion cages deployed in a coral reef in the Gulf of Papagayo. Tile cover of functional groups and development of organic C and N on light-exposed and –shaded tile sites were assessed. Results revealed that the exclusion of fish significantly increased the development of organic C and N and decreased the C/N ratio on light-exposed tiles. Large filamentous algae (> 2 mm), fleshy macroalgae and the colonial ascidian Didemnum sp. (Savigny 1816) (up to 80% tile coverage) were dominant on both tile sites. A significant peak of filamentous algae growth and associated organic matter C/N ratio occurred on light-exposed tiles throughout all treatments in February when nutrient concentrations were elevated. These results suggest that both herbivore exclusion and natural eutrophication have a strong influence on the benthic reef community composition and its early succession patterns. The presence of Didemnum sp. and turf algae could represent good early warning bioindicators for local overfishing and eutrophication and may therefore be included in management and monitoring strategies. [ABSTRACT FROM AUTHOR]

In humans, IL-17 is a proinflammatory cytokine that plays a key role in the clearance of extracellular bacteria promoting cell infiltration and production of several cytokines and chemokines. Here, we report on three Ciona intestinalis IL-17 homologues ( Ci IL17-1, Ci IL17-2, Ci IL17-3). The gene organization, phylogenetic tree and modeling supported the close relationship with the mammalian IL-17A and IL-17F suggesting that the C. intestinalis IL-17 genes share a common ancestor in the chordate lineages. Real time PCR analysis showed a prompt expression induced by LPS inoculation suggesting that they are involved in the first phase of inflammatory response. In situ hybridization assays disclosed that the genes transcription was upregulated in the pharynx, the main organ of the ascidian immune system, and expressed by hemocytes (granulocytes and univacuolar refractile granulocyte) inside the pharynx vessels. [ABSTRACT FROM AUTHOR]

Background It is well-understood that ascidians accumulate high levels of vanadium, a reduced form of V(III), in an extremely acidic vacuole in their blood cells. Vanabins are small cysteine-rich proteins that have been identified only from vanadium-rich ascidians. A previous study revealed that Vanabin2 can act as a V(V)-reductase in the glutathione cascade. Methods As Trx1, a thioredoxin gene, was cloned from the vanadium-rich ascidian, Ascidia sydneiensis samea , by PCR. As Trx1 and Vanabin2 were prepared as recombinant proteins, and V(V)-reduction by Vanabin2 was assessed by ESR and ion-exchange column chromatography. Site-directed mutagenesis was performed to examine the direct involvement of cysteine residues. Tissue expression of As Trx1 was also examined by RT-PCR. Results When reduced As Trx1 and Vanabin2 were combined, Vanabin2 adopted an SS/SH intermediate structure while V(V) was reduced to V(IV). The loss of cysteine residues in either Vanabin2 or As Trx1 caused a significant loss of reductase activity. V app and K app values for Vanabin2-catalyzed V(V)-reduction in the thioredoxin cascade were 0.066 mol-V(IV)/min/mol-Vanabin2 and 0.19 mM, respectively. The K app value was 2.7-fold lower than that observed in the glutathione cascade. The As Trx1 gene was expressed at a very high level in blood cells, in which Vanabins 1–4 were co-expressed. Conclusions As Trx1 may contribute to a significant part of the redox cascade for V(V)-reduction by Vanabin2 in the cytoplasm of vanadocytes, but prevails only at low V(V) concentrations. General significance This study is the first to report the reduction of V(V) in the thioredoxin cascade. [ABSTRACT FROM AUTHOR]

The biodiversity of marine ecosystems is diverse and a high number of species coexist side by side. However, despite the fact that most of these species share a common fertilization strategy, a high variability in terms of the size, shape, and motion of spermatozoa can be found. In this study, we have analyzed both the sperm motion parameters and the spermatozoa morphometric features of two swimmer (pufferfish and European eel) and two sessile (sea urchin and ascidian) marine species. The most important differences in the sperm motion parameters were registered in the swimming period. Sessile species sperm displayed notably higher values than swimmer species sperm. In addition, the sperm motilities and velocities of the swimmer species decreased sharply once the sperm was activated, whereas the sessile species were able to maintain their initial values for a long time. These results are linked directly to the species-specific lifestyles. Although sessile organisms, which show limited or no movement, need sperm with a capacity to swim for long distances to find the oocytes, swimmer organisms can move toward the female and release gametes near it, and therefore the spermatozoa does not need to swim for such a long time. At the same time, sperm morphology is related to sperm motion parameters, and in this study an in-depth morphometric analysis of ascidian, sea urchin, and pufferfish spermatozoa, using computer-assisted sperm analysis software, has been carried out for the first time. A huge variability in shapes, sizes, and structures of the studied species was found using electron microscopy. [ABSTRACT FROM AUTHOR]

Highlights: [•] Immunotoxic effects of ibuprofen were evaluated in Botryllus schlosseri. [•] Immunomarkers were measured after in vitro exposure of haemocytes. [•] Most of the immune parameters measured was affected by ibuprofen exposure. [Copyright &y& Elsevier]

Abstract: In the present study, the alkaline protease producing Marinobacter sp. GA CAS9 was isolated from the marine ascidian Polyclinum glabrum and identified by 16S rRNA analysis. Medium components and culture conditions for alkaline protease production were optimized using statistical optimization. Plackett–Burman design was employed to find out the optimal medium constituents and culture conditions to enhance protease production. Central composite design revealed that four independent variables, such as NaCl (60.53g/l), beef extract (14.73g/l), CuSO4 (4.73g/l) and pH (10.7) significantly influenced the protease production. Protease production obtained experimentally coincident with the predicted value and the model was proven to be adequate. The enhancement of protease from 298.34U/ml to 982.68U/ml was achieved with the optimization procedure. [Copyright &y& Elsevier]

Abstract: Toxins known to cause Paralytic Shellfish Poisoning (PSP) syndrome in humans that can have serious economic consequences for aquaculture were determined in ascidians of the genus Microcosmus. Significant concentrations of toxins were confirmed in all tested samples collected from the western coast of Istria Peninsula (Adriatic Sea, Croatia) when six people were poisoned following the consumption of fresh ascidians. Several species of bivalves that were under continuous monitoring had not accumulated PSP toxins although they were exposed to the same environmental conditions over the survey period. In the present study, HPLC-FLD with pre-column oxidation of PSP toxins has been carried out to provide evidence for the first human intoxication due to consumption of PSP toxic ascidians (Microcosmus vulgaris, Heller, 1877) harvested from the Adriatic Sea. Qualitative analysis established the presence of six PSP toxins: saxitoxin (STX), decarbamoylsaxitoxin (dcSTX), gonyautoxins 2 and 3 (GTX2,3), decarbamoylgonyautoxins 2 and 3 (dcGTX2,3), gonyautoxin 5 (GTX5) and N-sulfocarbamoylgonyautoxins 1 and 2 (C1,2), while quantitative analysis suggested STX and GTX2,3 as dominant toxin types and the ones that contribute the most to the overall toxicity of these samples with concentrations near the regulatory limit. [Copyright &y& Elsevier]

Abstract: We report the first use of K-edge X-ray absorption spectroscopy (XAS) as a direct spectroscopic probe of pH and cytosolic emf within living cells. A new accuracy metric of model-based fits to K-edge spectra is further developed. Sulfur functional groups in three collections of living blood cells and one sample of cleared blood plasma from the tunicate Ascidia ceratodes were speciated using K-edge XAS. Cysteine and cystine, the preferred thiol–disulfide model, averaged about 12% of total sulfur. Sulfate monoesters and cyclic diesters unexpectedly constituted 36% of blood cell sulfur. Soluble sulfate averaged about 25% across the three blood cell samples, while the ratio of SO4 2− to HSO4 − implied average signet ring vacuolar pH values of 0.85, 1.4, or 3.1. Intracellular (VSO4)+ was unobserved, while [V(RSO3) n ](3− n)+ was detected in the two lowest pH blood cell samples. About 5% of sulfur was distributed as mono- or dibenzothiophene or ethylene-epi-sulfide, or as a thiadiazole reminiscent of the polycarpathiamines. Blood plasma was dominated by sulfate (83%), but with 15% of an alkylsulfate ester and about 2% of low-valent sulfur. Gravimetric analysis of soluble sulfate yielded average concentrations of blood cell sulfur. Average [cysteine] and [cystine] (ranging ~10–30mM and ~20–90mM, respectively) implied blood-cell cytosolic emf values of approximately −0.20V. High cellular [cysteine] is consistent with the proposed model for enzymatic reduction of vanadate by endogenous thiol, wherein the trajectory of metal site-symmetry is controlled and directed through to a thermodynamically favored 7-coordinate V(III) product. [Copyright &y& Elsevier]

Highlights: [•] The C. intestinalis peroxinectin contains a peroxidase domain and a KGD motif. [•] The CiPxt is a new member of the peroxidase–cyclooxygenase superfamily. [•] The phylogenetic tree revealed that the CiPxt is the closest to the chordate group. [•] Sequence analysis and structure showed high similarity of CiPxt to mammalian MPO. [•] Q-PCR analyses revealed that CiPxt mRNA expression was upregulated by LPS challenge. [Copyright &y& Elsevier]

Abstract: Investigation of the secondary metabolites of the ascidian Herdmania momus led to the isolation and characterization of four new nucleoside derivatives (1–4). Structural studies showed that these derivatives represent a series of rare methylsulfinyladenosine derivatives of interconvertible transesterification isomers and/or sulfinyl epimers. The antiviral activities of these rare nucleosides were evaluated against a series of human pathogenic viruses. [Copyright &y& Elsevier]

Abstract: Muscle-specific miR-1/206 and miR-133 families have been suggested to play fundamental roles in skeletal and cardiac myogenesis in vertebrates. To gain insights into the relationships between the divergence of these miRs and muscular tissue types, we investigated the expression patterns of miR-1 and miR-133 in two ascidian Ciona species and compared their genomic structures with those of other chordates. We found that Ciona intestinalis and Ciona savignyi each possess a single copy of the miR-1/miR-133 cluster, which is only 350 nucleotide long. During embryogenesis, Ciona miR-1 and miR-133 are generated as a single continuous primary transcript accumulated in the nuclei of the tail muscle cells, starting at the gastrula stage. In adults, mature miR-133 and miR-1 are differentially expressed in the heart and body wall muscle. Expression of the reporter gene linked to the 850-bp upstream region of the predicted transcription start site confirmed that this region drives the muscle-specific expression of the primary transcript of miR-1/miR-133. In many deuterostome lineages, including that of Ciona, the miR-1/133 cluster is located in the same intron of the mind bomb (mib) gene in reverse orientation. Our results suggest that the origin of genomic organization and muscle-specific regulation of miR-1/133 can be traced back to the ancestor of chordates. Duplication of this miR cluster might have led to the remarkable elaboration in the morphology and function of skeletal muscles in the vertebrate lineage. [Copyright &y& Elsevier]

Abstract: Ascidians are well known to accumulate extremely high levels of vanadium in their blood cells. Several key proteins related to vanadium accumulation and physiological function have been isolated from vanadium-rich ascidians. Of these, vanadium(IV)-binding protein-129 (VBP-129) is a unique protein that has been identified from the blood plasma of an ascidian Ascidia sydneiensis samea, but its metal binding domains are not known. In this study, several deletion and point mutants of VBP-129 were generated, and their metal binding abilities were assessed by immobilized metal ion affinity chromatography (IMAC) and electron spin resonance spectroscopy (ESR). The internal partial protein, VBP-Int41, did not bind to VIV, but the two constructs, VBP-N52 and VBP-Int55, added with additional 11 or 14 neighboring amino acids bound to VIV. Mutations for cysteine-47 and lysine-50 in VBP-Int55 diminished VIV-binding in VBP-Int55, suggesting that these amino acid residues play important roles in binding VIV. ESR titration analysis revealed that VBP-129, VBP-N52 and VBP-Int55 could bind to 6, 3 and 2 VIV ions, respectively. ESR spectrum analysis indicated a N2O2 coordination geometry, which is similar to vanabins. The cysteines may contribute to the maintenance of the three-dimensional structure that is necessary for binding VIV ions. VBP-129 did not have a VV-reductase activity, as expected from its tissue localization in blood plasma. This study provided the evidences that VBP-129 possesses VIV-binding domains that make a similar coordination to VIV as those by vanabins but VBP-129 acts solely as a VIV-chaperon in blood plasma. [Copyright &y& Elsevier]

Abstract: The genetic structure and phylogeography of the solitary ascidian Halocynthia roretzi (sea pineapple) in Korea and Japan were studied based on mitochondrial DNA cytochrome oxidase subunit I (COI) gene nucleotide variations. The COI haplotypes found were divided into two clades in a statistical parsimony network. From the extensive haplotype diversity, limited nucleotide diversity, neutrality test, and mismatch distribution analysis, the species appears to have undergone rapid population expansion from an ancestral population with a small effective population size due to glacial bottlenecking in the late Pleistocene. Pairwise population F ST estimates inferred distinct genetic differentiation between the population from the northernmost east coast of Korea and the remaining pooled coastal water populations, while other populations generally exhibited low levels of differentiation. Analysis of molecular variance showed a weak but significant differentiation among three geographic population groups. Overall, the moderate population structure of H. roretzi may have been influenced by high levels of gene flow caused by hydrographic conditions such as sea currents. [Copyright &y& Elsevier]

Abstract: The chemical analysis of the ascidian Aplidium fuegiense resulted in the isolation of three novel meroterpenoids 2–4, structurally related to the main co-occurring known rossinone B (1). The structures of the new compounds were determined by interpretation of spectroscopic data. Compounds 1–4 were found to be selectively localized in the viscera of the ascidian. [Copyright &y& Elsevier]

Abstract: The farnesoid X receptors (FXRs) are the major transcriptional regulators of bile salt synthesis in vertebrates. However, the structural conservation of invertebrate FXRs has only been studied for the major model organisms and studies on additional invertebrate FXRs are clearly required to obtain better resolution of FXR phylogeny and comparative developmental insights in chordates. In the present study, the cDNA encoding the farnesoid X receptor, HrFXR, was cloned from a marine invertebrate Halocynthia roretzi. The open reading frame of HrFXR encoded 688 amino acids including a longer N-terminal region and showed overall sequence identities of 28–41% to vertebrate and Ciona intestinalis FXRs. The N-terminal activation function 1 (AF-1) and hinge domains of HrFXR displayed relatively low identities (<20%), whereas the DNA-binding and ligand-binding domains showed relatively high (>73%) and intermediate (21–50%) identities, respectively. Based on a phylogenetic analysis, HrFXR belonged to a urochordate group, which was placed differently from vertebrate FXRα and FXRβ subgroups. Real-time quantitative PCR analysis revealed that the HrFXR mRNA originated maternally and was highly expressed in adult gonads. Additionally, HrFXR mRNA levels in the gills and hepatopancreas showed significantly higher values in animals with soft tunic syndrome compared to those of normal individuals. Furthermore, direct injection of cholic acid significantly increased HrFXR transcript levels in vivo, although an expression vector containing HrFXR cDNA did not show a significant transactivation function in response to a well-known ligand for vertebrate FXR, GW4064, in HepG2 cells. These results suggest that the tunicate FXR has different structural and expressional characteristics compared to those of vertebrate FXRs. [Copyright &y& Elsevier]

Abstract: Predation during the early life history stages, when organisms are susceptible to both biotic and abiotic factors, is likely to have pervasive effects on community development. However, few studies have examined the relative importance of predation during early life-history stages on long-term community composition. Using a sessile marine community, we conducted a manipulative experiment to measure the effect of timing and extent of predator exposure. Every month over a 5month period we assessed taxa composition, dominance and taxa richness in each of the following five treatments: 1) never predated, 2) always predated, 3) early predated (during the first month of community development), 4) late predated and 5) uncaged panels. Predation was very important in determining taxa identity and dominance, reducing spatial monopolization by ascidians and increasing the area occupied by bryozoans, but overall predation did not result in a more diverse community. The localized extinction of ascidians in predation treatments was compensated by an increase in the diversity of bryozoans and barnacles. Interestingly, predation during the early stages of community development had only short-term effects on the taxa richness and composition. After 5months, predator exclusion during the early life-history stages had no effect on community composition and only current levels of predation determined differences in composition. Our results show that predation can cause changes in community composition, directly and indirectly, without necessary changing the total number of co-occurring taxa. More important, we demonstrate to our study system that although predation during the early life-history stages can be strong and have important short-term effects, predation during the adult stages determines long-term community composition. [Copyright &y& Elsevier]

Abstract: A new fascaplysin analogue, 3-bromohomofascaplysin A (1), along with two known analogues, homofascaplysin A (2) and fascaplysin (3), were isolated from a Fijian Didemnum sp. ascidian. The absolute configurations of 3-bromohomofascaplysin A (1) and homofascaplysin A (2) were determined via experimental and theoretically calculated ECD spectra. The differential activities of 1–3 against different blood-borne life stages of the malaria pathogen Plasmodium falciparum were assessed. Homofascaplysin A (2) displayed an IC50 of 0.55±0.11nM against ring stage parasites and 105±38nM against all live parasites. Given the stronger resistance of ring stage parasites against most current antimalarials relative to the other blood stages, homofascaplysin A (2) represents a promising agent for treatment of drug resistant malaria. [Copyright &y& Elsevier]

Abstract: The palatability of two solitary and three colonial species of ascidians commonly found in sub-tropical seagrass meadows was evaluated using the abundant, sympatric, omnivorous pinfish Lagodon rhomboides as a model predator. Bite-sized pieces of fresh tissues of both solitary and one of the three colonial ascidian species were unpalatable to fish. Lipophilic and hydrophilic extracts of the three unpalatable species did not cause feeding deterrence indicating that secondary metabolites are not responsible for the lack of palatability. Distaplia bermudensis, the one colonial ascidian that was unpalatable to fish, had a highly acidified outer tunic (pH=1.5). We tested the ability of acidified agar food pellets (pH=1.5) to deter pinfish and found that the fish readily ingested acidified pellets. The toughness of the tunic of all five ascidian species was evaluated by measuring the Force (N) required to penetrate the tunic using a penetrometer. Tunic toughness is likely to explain the lack of palatability of the solitary ascidians Styela plicata and Molgula occidentalis as their tunics required a force of >34N to penetrate. Tunic toughness may be a particularly effective adaptation for ascidian defense in seagrass habitats where fish with strong crushing jaws, such as those that commonly occur in coral reef systems, are rare. [Copyright &y& Elsevier]

Abstract: Metal ions are required for physiologically essential functions such as metalloenzymatic reactions, redox reactions, electron transfer, regulation of transcription factor activity, and respiration in living cells. Generally, metal ions are homeostatically maintained at very low concentrations in the sub-micromolar to micromolar range in living cells. However, some organisms, called hyperaccumulators, collect extremely high levels of metal ions, thereby providing experimental systems in which to study the mechanisms underlying the selective accumulation of metal ions. Typical of such organisms are the ascidians, more commonly known as sea squirts or tunicates. They are sessile marine animals belonging to the chordates. Several species of ascidians are known to accumulate extremely high levels of vanadium ions in their blood cells. Vanadium is usually in the VV state in the natural environment, but in ascidians, most of vanadium is reduced to VIII via VIV during the assimilation process. In this review, we first summarize the history of the studies on vanadium accumulation in ascidians and then focus on the recent progress of molecular studies, especially on the transport and reduction of vanadium, using primarily two ascidian species, Ascidia sydneiensis samea and Ciona intestinalis. Several candidate genes for VIV transporters and enzymes catalyzing the redox reactions of VV/VIV are addressed in detail. The function of accumulated VIII is discussed in relation to redox reactions. [Copyright &y& Elsevier]

Abstract: The effects of nonylphenol (NP) on functional responses of haemocytes of the colonial ascidian Botryllus schlosseri were evaluated. Haemocytes were exposed to 0.1, 1 and 10μM NP and the effects on haemocyte viability, adhesion, morphology, lysosomal membrane stability, phagocytic activity and early apoptosis were evaluated. Haemocyte viability and adhesion were not affected by NP. Phagocytic activity and the amoebocytic index decreased significantly at all the concentrations tested, while exposure of haemocytes to 1 and 10μM NP caused a significant increase in the diffusion of Neutral Red into the cytosol. The percentage of cells positive to Annexin-V (indicative of early apoptosis) increased significantly at 1 and 10μM NP. Results obtained suggested a relationship between NP and alterations in functional responses of haemocytes in B. schlosseri. Biomarkers measured resulted sensitive, rapid and reproducible, even if their responsiveness will be evaluated after in vivo exposure of animals to NP. [Copyright &y& Elsevier]

Abstract: Two new pyridoacridine alkaloids, 13-didemethylaminocycloshermilamine D (1) and demethyldeoxyamphimedine (2), were isolated from the purple chromotype of the Western Mediterranean ascidian Cystodytes dellechiajei. This morph also contained the known shermilamine B (3), kuanoniamine D (4), N-deacetylshermilamine B (5), N-deacetylkuanoniamine D (6), styelsamines C (7), and D (8). The structures of new compounds were elucidated on the basis of spectroscopic data. A hypothetic biosynthetic pathway from the tetracyclic styelsamine D (8) was proposed for both compounds 1 and 2 and their antimicrobial potential was evaluated. [Copyright &y& Elsevier]

Abstract: Background: Vanadium is an essential transition metal in biological systems. Several key proteins related to vanadium accumulation and its physiological function have been isolated, but no vanadium ion transporter has yet been identified. Methods: We identified and cloned a member of the Nramp/DCT family of membrane metal transporters (AsNramp) from the ascidian Ascidia sydneiensis samea, which can accumulate extremely high levels of vanadium in the vacuoles of a type of blood cell called signet ring cells (also called vanadocytes). We performed immunological and biochemical experiments to examine its expression and transport function. Results: Western blotting analysis showed that AsNramp was localized at the vacuolar membrane of vanadocytes. Using the Xenopus oocyte expression system, we showed that AsNramp transported VO2+ into the oocyte as pH-dependent manner above pH 6, while no significant activity was observed below pH 6. Kinetic parameters (K m and V max) of AsNramp-mediated VO2+ transport at pH 8.5 were 90nM and 9.1pmol/oocyte/h, respectively. A rat homolog, DCT1, did not transport VO2+ under the same conditions. Excess Fe2+, Cu2+, Mn2+, or Zn2+ inhibited the transport of VO2+. AsNramp was revealed to be a novel VO2+/H+ antiporter, and we propose that AsNramp mediates vanadium accumulation coupled with the electrochemical gradient generated by vacuolar H+-ATPase in vanadocytes. General Significance: This is the first report of identification and functional analysis on a membrane transporter for vanadium ions. [Copyright &y& Elsevier]

Abstract: VSP is a transmembrane protein whose cytoplasmic region shows significant similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN). Notably, VSP exhibits a unique ability to transduce electrical signals into phosphoinositide turnover by coupling a transmembrane voltage sensor domain to the PTEN-like phosphoinositide phosphatase domain. Moreover, VSP gene is known to be widely conserved among deuterostome genomes, though the function of VSP in vivo remains largely unknown. In the present study, the expression pattern of ascidian VSP(Ci-VSP) was examined in embryos and juveniles of a marine invertebrate chordate, Ciona intestinalis. RT-PCR showed that Ci-VSP is expressed at the larval stage and that expression persists in juveniles. Whole mount in situ hybridization showed that Ci-VSP is expressed in cells of the stomach, intestine and blood cells of 2- to 3-week-old juveniles. Moreover, double staining blood cells from 2-month-old adults with Ci-VSP and Ci-PTEN probes showed that Ci-VSP-positive cells are a distinct population, separate from cells expressing Ci-PTEN. These findings suggest that in addition to its previously suggested roles in testis or sperm, Ci-VSP plays a key role in voltage-induced signal transduction in cells of the digestive system and blood. [Copyright &y& Elsevier]

Abstract: A stereospecific synthesis of both enantiomers of the marine alkaloid eudistomin X using the amino acid chiral pool is achieved. Comparison of 1H and 13C NMR chemical shifts of the synthetic product as either the free base, mono-salt or disalt with those reported for the natural product established that the ascidian metabolite was originally characterised as a mono-salt and that the absolute configuration was (10R). [ABSTRACT FROM AUTHOR]

Abstract: Sulfated glycosaminoglycans are important components of connective tissues. The pattern of sulfation is important for their biological functions. Ascidians, the closest relatives of vertebrates, have a simple chordate body plan. In the present study, we identified an almost complete set of genes encoding proteins homologous to chondroitin/dermatan sulfotransferases in the genome of the ascidian Ciona intestinalis. We found eight genes encoding 4-O-sulfotransferases, eight genes encoding 6-O-sulfotransferases, and three genes encoding uronyl 2-O-sulfotransferases. The number of sulfotransferase genes was unexpectedly large, considering that ascidians do not have a well-developed endoskeleton. In addition, most of the genes within each sub-family seemed to have arisen by gene duplication events that occurred in the ascidian lineage after divergence from the main chordate lineage. This suggests that a unique pattern of sulfation independently developed during ascidian evolution. Some of the genes identified in the present study showed tissue-specific expression in the epidermis, notochord, muscle, and central nervous system. Region-specific expression in the epidermis was also observed. The present study provides useful information for further comparative and functional analyses of sulfotransferases and proteoglycans in chordate embryos. [Copyright &y& Elsevier]

Abstract: Ascidians accumulate high levels of iron and/or vanadium in their tissues, the function of which is unknown. There is little directed study of the ecophysiological variation in the trait. We examined the variation in vanadium and iron levels in the hemocytes, body wall and tunic of the ascidian Ciona intestinalis over several seasons, including the spring bloom period. There were significant peaks in the vanadium and iron levels in the hemocytes and tunic, (up to four to five-fold higher) in March and August. The activity of G6PDH, an enzyme of a proposed reductive mechanism for vanadium, was higher in March as well. A feeding experiment showed few differences between high and low food groups in vanadium or iron levels. The season to season changes in metal levels and the differences between laboratory held animals and animals sampled directly from Woods Hole suggest that low loss rates may maintain high vanadium and iron levels, even when dissolved levels in sea water are low. This work is the first to show that vanadium and iron levels in ascidian tissues varies from season to season. [Copyright &y& Elsevier]

Abstract: Lhx3 genes are members of one of the six subfamilies of the LIM class homeobox genes. In ascidians, Lhx3 is known to play a critical role in endoderm differentiation, while in vertebrates Lhx3 is involved in the development of pituitary and subsets of motor neurons. It has been shown recently, using RT-PCR analysis, that two transcriptional isoforms a and b are differentially expressed during the larval development of Ciona intestinalis (). The present study provides an in-depth description of Lhx3 gene expression during the development of the two remote ascidian species, C. intestinalis and Halocynthia roretzi; for this, 5′RACE and whole-mount in situ hybridization (WISH) were employed. In both species, maternal expression of Lhx3a, but not Lhx3b, is evident. In H. roretzi, the maternal Lhx3a transcripts have been detected by WISH in the animal half of early cleavage stage embryos. In both species, transcriptional isoform a is also zygotically expressed in the sensory vesicle and the visceral ganglion lineages from the neurula stage onward. By contrast, Lhx3b transcripts are expressed only zygotically and localized in the endoderm, notochord and mesenchyme lineages during cleavage stage. Lhx3a, but not Lhx3b, transcripts are subjected to trans-splicing. Additionally, in C. intestinalis, other variations in the 5′ region have been identified among Lhx3a transcripts. Although some differences are present, over-all developmental expression of Lhx3 is rather well conserved between the two ascidian species, which is quite different from that of vertebrate counterparts. [Copyright &y& Elsevier]

Abstract: The gonadotropin-releasing hormone (GnRH) family peptides are most widely distributed neuropeptides and/or neurophysial hormones. GnRH is involved in diverse neuroendocrine, paracrine, autocrine, and neurotransmitter/neuromodulatory functions in the central and peripheral nervous system as well as peripheral tissues. In the present study, we show the identification of a novel GnRH-related peptide, Ci-GnRH-X, in the ascidian, Ciona intestinalis. Intriguingly, Ci-GnRH-X possesses a unique primary sequence consisting of 16 amino acids, although typical GnRH family peptides are composed of 10 amino acids. On the other hand, Ci-GnRH-X shares the GnRH consensus motifs, including the N-terminal pQHWS (‘pQ’ indicates a pyro-glutamic acid) and C-terminal Gly-amide. Reverse transcription (RT)-PCR analysis shows that the Ci-GnRH-X gene is expressed exclusively in the central nervous system. Moreover, in situ hybridization demonstrated that the Ciona GnRH-1 gene encoding Ciona GnRHs (t-GnRH-3, -5 and -6) was co-expressed with the Ci-GnRH-X gene in neurons of the cerebral ganglion. Of particular interest is that Ci-GnRH-X exhibited moderate (10–50%) inhibitory activity against t-GnRHs at their cognate receptors. Ci-GnRH-X repressed the elevation of the intracellular calcium and cAMP production by t-GnRH-6 at Ci-GnRHR-1, and cAMP production by t-GnRH-3, and t-GnRH-5 via Ci-GnRHR-3 was also inhibited by Ci-GnRH-X. In contrast, no inhibitory effect of Ci-GnRH-X at Ci-GnRHR-2 was observed. The localization and biochemical assays revealed that Ci-GnRH-X acts as an endogenous antagonist for the Ciona GnRHergic system. This is the first molecular and functional characterization of an endogenous inhibitor of GnRHs in an animal species. [Copyright &y& Elsevier]

Abstract: Background: Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO2+ in Vanabin2 from a vanadium-rich ascidian Ascidia sydneiensis samea have been detected by nuclear magnetic resonance study, but the metal selectivity and metal-binding ability of each site was not examined. Methods: In order to reveal functional contribution of each binding site, we prepared several mutants of Vanabin2 by in vitro site-directed mutagenesis and analyzed their metal selectivity and affinity by immobilized metal-ion affinity chromatography and Hummel Dreyer method. Results: Mutation at K10/R60 (site 1) markedly reduced the affinity for VO2+. Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO2+. Secondary structure of both mutants was the same as that of the wild type as assessed by circular dichroism spectroscopy. Mutation in disulfide bonds near the site 1 did not affect its high affinity binding capacity, while those near the site 2 decreased the overall affinity for VO2+. General significance: These results suggested that the site 1 is a high affinity binding site for VO2+, while the site 2 composes a moderate affinity site for multiple VO2+. [Copyright &y& Elsevier]

Abstract: Background: Several species of ascidians accumulate extremely high levels of vanadium ions in the vacuoles of their blood cells (vanadocytes). The vacuoles of vanadocytes also contain many protons and sulfate ions. To maintain the concentration of sulfate ions, an active transporter must exist in the blood cells, but no such transporter has been reported in vanadium-accumulating ascidians. Methods: We determined the concentration of vanadium and sulfate ions in the blood cells (except for the giant cells) of Ascidia sydneiensis samea. We cloned cDNA for an Slc13-type sulfate transporter, AsSUL1, expressed in the vanadocytes of A. sydneiensis samea. The synthetic mRNA of AsSUL1 was introduced into Xenopus oocytes, and its ability to transport sulfate ions was analyzed. Results: The concentrations of vanadium and sulfate ions in the blood cells (except for the giant cells) were 38 mM and 86 mM, respectively. The concentration of sulfate ions in the blood plasma was 25 mM. The transport activity of AsSUL1 was dependent on sodium ions, and its maximum velocity and apparent affinity were 2500 pmol/oocyte/h and 1.75 mM, respectively. General significance: This could account for active uptake of sulfate ions from blood plasma where sulfate concentration is 25 mM, as determined in this study. [Copyright &y& Elsevier]