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Ascidians belonging to Phlebobranchia accumulate vanadium to an extraordinary degree (≤ 350 mM). Vanadium levels are strictly regulated and vary among ascidian species; thus, they represent well-suited models for studies on vanadium accumulation. No comprehensive study on metal accumulation and reduction in marine organisms in relation to their symbiotic bacterial communities has been published. Therefore, we performed comparative 16S rRNA amplicon sequence analyses on samples from three tissues (branchial sac, intestine, and intestinal lumen) involved in vanadium absorption, isolated from two vanadium-rich (Ascidia ahodori and Ascidia sydneiensis samea) and one vanadium-poor species (Styela plicata). For each sample, the abundance of every bacteria and an abundance value normalized to their abundance in seawater were calculated and compared. Two bacterial genera, Pseudomonas and Ralstonia, were extremely abundant in the branchial sacs of vanadium-rich ascidians. Two bacterial genera, Treponema and Borrelia, were abundant and enriched in the intestinal content of vanadium-rich ascidians. The results suggest that specific selective forces maintain the bacterial population in the three ascidian tissues examined, which contribute to successful vanadium accumulation. This study furthers the understanding of the relationship between bacterial communities and metal accumulation in marine life.

For the first time, vanadium of biological origin, extracted from centrifugal fraction of vanadium-storing blood cells of the Ascidia sydneiensis samea species, was characterized as regards its isotopic composition and content of natural radioactive elements potassium (K), thorium (Th) and uranium (U). The natural abundance of vanadium isotopes has been confirmed with high accuracy, thus excluding a possible selectivity within bio-chemical reactions of vanadium concentration in blood cells from seawater. A large potassium concentration (up to 5500 × 10−6 g g−1) was found in the blood cell samples. The concentration of thorium was determined to be about 30 × 10−9 g g−1, while the uranium concentration was about 150 × 10−9 g g−1. Hence, a highly efficient two-stage purification approach with a total vanadium recovery of better than 70% was developed and applied. The final concentrations of K

Most ascidian species settle on underwater substrates during a short free-swimming tadpole larval period. During this process, "rapid adhesion" occurs on adhesive papillae located at the anterior region of the cephalenteron. Settled and transformed ascidians subsequently expand the attachment area by "slow adhesion" with ampullae. In the present study, we attempted to identify the ultrastructures related to the adhesion process and adhesive materials in the ascidian tunic and to elucidate the biological function of vanadium in adhesion. We focused on an adhesive organ named the adhesive projection, which is newly generated by the adhered tunic to enlarge the bonding area between ascidian and substrate. Based on its structure and the presence of vanadiumcontaining blood cells, the adhesive projection was considered to be a large tunic vessel. At the adhered tunic, eosinophilic regions and migrated tunic cells were observed, but metal deposition was not detected. We speculate that the eosinophilic materials were components of the adhesive glue, and these are likey produced in epithelial cells, tunic cells, or both. Furthermore, using imaging mass spectrometry, we identified eight tunic-specific molecules as glue candidates.

A novel flow-through column electrolytic cell was proposed as a detector to obtain current signals for supercritical fluid chromatography. The electrochemical cell consisted of two electrodes and its holder, and a working and a counter electrode were fabricated from 192 carbon strings, which were composed of 400 carbon fibers of 10 μm in diameter filled into a heat-shrinkable tube. These electrodes were placed in the center of a holder made from polyether ether ketone blocks and they were separated by polytetrafluoroethylene membrane filters. To evaluate the sensitivity of this cell, a standard solution of ferrocene was injected into the supercritical fluid chromatography system connected to the electrolytic cell. The ferrocene was eluted through a silica gel column using a mixture of a mobile phase of supercritical CO2 and a modifier of methanol containing ammonium acetate. The current peak area of ferrocene correlated to the ferrocene concentration in the range of 10–400 μmol/L (r = 0.999). Moreover, the limit of detection on the column estimated from a signal-to-noise ratio of 3 was 9.8 × 10−13 mol. This article is protected by copyright. All rights reserved

Ascidians are marine animals that belong to the same phylogenetic group (Phylum Chordata) as human beings do. One of the three suborders in ascidians can accumulate a high level of vanadium ions in blood cells. Ascidia gemmata has been reported to accumulate the highest levels of vanadium at 350 mM, which is 107-fold higher than the vanadium concentration in seawater. In the last two decades, many genes and proteins related to vanadium accumulation and reduction have been revealed by molecular biological and biochemical methods. Modern omics approach enhanced the comprehensive identification of factors related to this phenomenon. In this review article, first, we would like to summarize the history of studies on vanadium accumulation in ascidians briefly. Then, we would like to overview recent advances by omics studies. How ascidians selectively accumulate vanadium is discussed from biochemical properties of proteins responsible for each step, and why ascidians accumulate vanadium is discussed in relation to the underwater adhesion.Ascidians are marine animals that belong to the same phylogenetic group (Phylum Chordata) as human beings do. One of the three suborders in ascidians can accumulate a high level of vanadium ions in blood cells. Ascidia gemmata has been reported to accumulate the highest levels of vanadium at 350 mM, which is 107-fold higher than the vanadium concentration in seawater. In the last two decades, many genes and proteins related to vanadium accumulation and reduction have been revealed by molecular biological and biochemical methods. Modern omics approach enhanced the comprehensive identification of factors related to this phenomenon. In this review article, first, we would like to summarize the history of studies on vanadium accumulation in ascidians briefly. Then, we would like to overview recent advances by omics studies. How ascidians selectively accumulate vanadium is discussed from biochemical properties of proteins responsible for each step, and why ascidians accumulate vanadium is discussed in relation...

Background Acoels are primitive bilaterians with very simple soft bodies, in which many organs, including the gut, are not developed. They provide platforms for studying molecular and developmental mechanisms involved in the formation of the basic bilaterian body plan, whole-body regeneration, and symbiosis with photosynthetic microalgae. Because genomic information is essential for future research on acoel biology, we sequenced and assembled the nuclear genome of an acoel, Praesagittifera naikaiensis. Findings To avoid sequence contamination derived from symbiotic microalgae, DNA was extracted from embryos that were free of algae. More than 290x sequencing coverage was achieved using a combination of Illumina (paired-end and mate-pair libraries) and PacBio sequencing. RNA sequencing and Iso-Seq data from embryos, larvae, and adults were also obtained. First, a preliminary ∼17–kilobase pair (kb) mitochondrial genome was assembled, which was deleted from the nuclear sequence assembly. As a result, a draft nuclear genome assembly was ∼656 Mb in length, with a scaffold N50 of 117 kb and a contig N50 of 57 kb. Although ∼70% of the assembled sequences were likely composed of repetitive sequences that include DNA transposons and retrotransposons, the draft genome was estimated to contain 22,143 protein-coding genes, ∼99% of which were substantiated by corresponding transcripts. We could not find horizontally transferred microalgal genes in the acoel genome. Benchmarking Universal Single-Copy Orthologs analyses indicated that 77% of the conserved single-copy genes were complete. Pfam domain analyses provided a basic set of gene families for transcription factors and signaling molecules. Conclusions Our present sequencing and assembly of the P. naikaiensis nuclear genome are comparable to those of other metazoan genomes, providing basic information for future studies of genic and genomic attributes of this animal group. Such studies may shed light on the origins and evolution of simple bilaterians.

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: AsTrx1, A thioredoxin gene, was cloned from the vanadium-rich ascidian, Ascidia sydneiensis samea, by PCR. AsTrx1 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 AsTrx1 was also examined by RT-PCR. Results: When reduced AsTrx1 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 AsTrx1 caused a significant loss of reductase activity. Vapp and Kapp 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 Kapp value was 2.7-fold lower than that observed in the glutathione cascade. The AsTrx1 gene was expressed at a very high level in blood cells, in which Vanabins 1–4 were co-expressed. Conclusions: AsTrx1 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., This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (Nos. 20570070, 21570077, 22224011, 25120508 and 25440170).

Ascidians (tunicates or sea squirts) accumulate extremely high levels of vanadium as the reduced form V(III) in extremely acidic vacuoles in their blood cells. Several key proteins related to vanadium accumulation have been isolated from vanadium-rich ascidians and their physiological functions characterized. Of these, vanabins are small, cysteine-rich proteins that have been identified only in vanadium-rich ascidians. Our previous study revealed that Vanabin2 can act as a V(V)-reductase. The current study examines the role of cysteine and several other amino acid residues of Vanabin2 in V(V)-reduction. When all eighteen cysteine residues of Vanabin2 were substituted with serine residues, the V(V)-reductase activity was lost. Substitutions of three, structurally clustered cysteines in three different regions resulted in a moderate decrease in reductase activity, suggesting that more than a single cysteine pair is responsible for the V(V)-reductase activity of Vanabin2. Mutations in the V(IV)-binding domains caused either an increase or decrease in activity but no mutation caused the complete loss of activity. These results suggest that some pairs, but more than a single pair, of cysteine residues are necessary for the V(V)-reductase activity of Vanabin2.

Xenacoelomorpha has recently been proposed as an animal taxon that includes acoels, nemertodermatids, and xenoturbellids. Their flattened bodies are very simple and lack discrete organs. The Acoela and Nemertodermatida (which comprise Acoelomorpha) were traditionally regarded as early-diverged extant orders of the class Turbellaria of the phylum Platyhelminthes. Recent anatomical studies and molecular phylogenetic studies demonstrate that the two groups belong to the phylum Xenacoelomorpha together with Xenoturbellida. However, debate remains in regard to whether Xenacoelomorpha is monophyletic, and whether xenacoelomorphs are sisters to all other bilaterians or have close affinity to ambulacrarians. The present study addresses the first question by examining the presence or absence of diagnostic peptide sequences shared by the three taxa. Hox genes have been used to investigate the phylogenetic relationships of metazoans. It has been shown that lophotrochozoans, rotifers, and chaetognaths share diagnostic peptide sequences in the C-terminal region of the Lox5 (Hox5/6/7) homeodomain proteins, which supports the clustering of these taxa. Examination of the decoded genome of the acoel Praesagittifera naikaiensis and reported xenacoelomorph Hox genes revealed that acoels share a peptide NLK(S/T)MSQ(V/I)D, which starts immediately after the homeodomain sequence of the central Hox4/5/6. In addition, we found another diagnostic peptide, KEGKL, in the C-terminal region of the anterior Hox1, which is shared by all the three groups of xenacoelomorphs, but not other bilaterians. Furthermore, two acoels, Praesagittifera naikaiensis and Symsagittifera roscoffensis, share another peptide SG(A/P)-PGM in the posterior Hox9/11/13. These results support the designation of the phylum Xenacoelomorpha, in which Acoela is a discrete group.

Polychaete fan worms and ascidians accumulate high levels of vanadium ions. Several vanadiumbinding proteins, known as vanabins, have been found in ascidians. However, no vanadium-binding factors have been isolated from the fan worm. In the present study, we sought to identify vanadiumbinding proteins in the branchial crown of the fan worm using immobilized metal ion affinity chromatography. A nucleoside diphosphate kinase (NDK) homolog was isolated and determined to be a vanadium-binding protein. Kinase activity of the NDK homologue, PoNDK, was suppressed by the addition of V(IV), but was unaffected by V(V). The effect of V(IV) on PoNDK precedes its activation by Mg(II). This is the first report to describe the relationship between NDK and V(IV). PoNDK is located in the epidermis of the branchial crown, and its distribution is very similar to that of vanadium. These results suggest that PoNDK is associated with vanadium accumulation and metabolism in P. occelata.

Ascidians are hyperaccumulators that have been studied in detail. Proteins and genes involved in the accumulation process have been identified, but regulation of gene expression related to vanadium accumulation remains unknown. To gain insights into the regulation of gene expression by vanadium in a genome-wide manner, we performed a comprehensive study on the effect of excess vanadium ions on a vanadium-rich ascidian, Ciona intestinalis, using a microarray. RT-PCR and enzyme activity assay were performed from the perspective of redox and accumulation of metal ions in each tissue. Glutathione metabolism-related proteins were significantly up-regulated by VIV treatment. Several genes involved in the transport of vanadium and protons, such as Nramp and V-ATPase, were significantly up-regulated by VIV treatment. We observed significant up-regulation of glutathione synthesis and degradation pathways in the intestine and branchial sac. In blood cells, expression of Ci-Vanabin4, glutathione reductase activity, glutathione levels, and vanadium concentration increased after VIV treatment. VIV treatment induced significant changes related to vanadium exclusion, seclusion, and redox pathways in the intestine and branchial sac. It also induced an enhancement of the vanadium reduction and accumulation cascade in blood cells. These differential responses in each tissue in the presence of excess vanadium ions suggest that vanadium accumulation and reduction may have regulatory functions. This is the first report on the gene regulation by the treatment of vanadium-rich ascidians with excess vanadium ions. It provided much information for the mechanism of regulation of gene expression related to vanadium accumulation., This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (#21570077 to H. M. and #20570070 to T. U.) and a grant from the Toray Science Foundation (03-4402 to T. U.).

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 (Km and Vmax) of AsNramp-mediated VO2+ transport at pH 8.5 were 90 nM and 9.1 pmol/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+. Conclusions: 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., This work was supported in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, Japan (#17370026, #18570070, #20570070, and #21570077).

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 V-V state in the natural environment, but in ascidians, most of vanadium is reduced to V-III via V-IV 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 V-IV transporters and enzymes catalyzing the redox reactions of V-V/V-IV are addressed in detail. The function of accumulated V-III is discussed in relation to redox reactions. (C) 2011 Elsevier B.V. All rights reserved.

The discovery of high levels of vanadium-containing compounds in ascidian blood cells goes back to 1911. Ascidians, which are also known as tunicates or sea squirts, belong to a subphylum of the Chordata, between the vertebrates and invertebrates. This discovery attracted the attention of an interdisciplinary group of chemists, physiologists, and biochemists, in part because of interest in the possible role of vanadium in oxygen transport as a prosthetic group in respiratory pigments, which was later shown not to be such a role, and in part because of the fact that high levels of vanadium were unknown in other organisms. The intracellular concentration of vanadium in some ascidian species can be as high as 350 mm, which is 107times that in seawater. Vanadium ions, which are thought to be present in the +5 oxidation state in seawater, are reduced to the +3 oxidation state via the +4 oxidation state and are stored in the vacuoles of vanadium-containing cells called vanadocytes, where high levels of protons and sulfate ions are also found. Recently, many proteins and genes that might be involved in the accumulation and reduction of vanadium have been isolated. In this review, we not only trace the history of vanadium research but also describe recent advances in our understanding of the field from several viewpoints: (i) vanadium-accumulating blood cells, (ii) the energetics of vanadium accumulation, (iii) the redox mechanism of vanadium, (iv) the possible role of sulfate, and (v) the physiological roles of vanadium.

Background Vanabins are a unique protein family of vanadium-binding proteins with nine disulfide bonds. Possible binding sites for VO 2+ 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 VO 2+ . Mutation at K24/K38/R41/R42 (site 2) decreased the maximum binding number, but only slightly increased the overall affinity for VO 2+ . 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 VO 2+ . General significance These results suggested that the site 1 is a high affinity binding site for VO 2+ , while the site 2 composes a moderate affinity site for multiple VO 2+ .

The blood cells of ascidians accumulate extremely high levels of the transition metal vanadium. We previously isolated four vanadium-binding proteins (Vanabins 1–4) and a homologous protein (VanabinP) from the vanadium-rich ascidian Ascidia sydneiensis samea . In the present study, we identified cDNAs encoding five different Vanabin2-related proteins in A. sydneiensis samea blood cells. It was notable that the sequences of the encoded proteins vary from that of Vanabin2 at up to 14 specific positions, while both the polypeptide length and the 18 cysteine residues were completely conserved. The most divergent protein, named 14MT, differed from Vanabin2 at all 14 positions. Using immobilized metal-ion affinity chromatography, we found that Vanabin2 and 14MT have the same metal-ion selectivity, but the overall affinity of 14MT for VO 2+ is higher than that of Vanabin2. Binding number for VO 2+ ions was the same between Vanabin2 and 14MT as assessed by gel filtration. These results suggested that sequence variations were under strict evolutionary constraints and high-affinity binding sites for VO 2+ are conserved among Vanabin2 variants.

The ascidians, the so-called sea squirts, accumulate high levels of vanadium, a transition metal. Since Henze first observed this physiologically unusual phenomenon about one hundred years ago, it has attracted interdisciplinary attention from chemists, physiologists, and biochemists. The maximum concentration of vanadium in ascidians can reach 350 mM, and most of the vanadium ions are stored in the +3 oxidation state in the vacuoles of vanadium-accumulating blood cells known as vanadocytes. Many proteins involved in the accumulation and reduction of vanadium in the vanadocytes, blood plasma, and digestive tract have been identified. However, the process by which vanadium is taken in prior to its accumulation in vanadocytes has not been elucidated. In the present study, a novel vanadium-binding protein, designated VBP-129, was identified from blood plasma of the vanadium-rich ascidian Ascidia sydneiensis samea. Although VBP-129 mRNA was transcribed in all A. sydneiensis samea tissues examined, the VBP-129 protein was exclusively localized in blood plasma and muscle cells of this ascidian. It bound not only to VO(2+) but also to Fe(3+), Co(2+), Cu(2+), and Zn(2+); on the other hand, a truncated form of VBP-129, designated VBP-88, bound only to Co(2+), Cu(2+) and Zn(2+). In a pull-down assay, an interaction between VanabinP and VBP-129 occurred both in the presence and the absence of VO(2+). These results suggest that VBP-129 and VanabinP function cooperatively as metallochaperones in blood plasma.

Several species of ascidians, the so-called tunicates, accumulate extremely high levels of vanadium ions in their blood cells. We previously identified a family of vanadium-binding proteins, named Vanabins, from blood cells and blood plasma of a vanadium-rich ascidian, Ascidia sydneiensis samea. The 3-dimensional structure of Vanabin2, the predominant vanadium-binding protein in blood cells, has been revealed, and the vanadium-binding properties of Vanabin2 have been studied in detail. Here, we used Far Western blotting to identify a novel protein that interacts with Vanabin2 from a blood cell cDNA library. The protein, named Vanabin-interacting protein 1 (VIP1), was localized in the cytoplasm of signet ring cells and giant cells. Using a two-hybrid method, we revealed that VIP1 interacted with Vanabins 1, 2, 3, and 4 but not with Vanabin P. The N-terminal domain of VIP1 was shown to be important for the interaction. Further, Vanabin1 was found to interact with all of the other Vanabins. These results suggest that VIP1 and Vanabin1 act as metal chaperones or target proteins in vanadocytes.

This paper presents the digital overcurrent limitation for the peak current mode dc-dc converter to realize the highly reliable power supply system. The proposed overcurrent limitation circuit is the simple configuration because the existing function in the regulation mode is applied. The proposed method can detect the overcurrent in real time and it is possible to maintain an arbitrary current in the overcurrent limitation mode.

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.

The purpose of this paper is to present the digital overcurrent detector for the peak current mode dc-dc converter which has been already applied to the server for the data center. The proposed peak current detection circuit is composed of the RC integrator and comparator as the A-D converter for the detected current. The proposed method can detect the overcurrent by using the RC integration time in real time. Furthermore, it is possible to maintain the arbitrary current in the overcurrent limitation mode. The proposed overcurrent limitation mode is implemented by the FPGA. The effectively of proposed method are confirmed by both of simulated and experimental results.

Isolation of naturally occurring bacterial strains from metal-rich environments has gained popularity due to the growing need for bioremediation technologies. In this study, we found that the vanadium concentration in the intestine of the vanadium-rich ascidian Ascidia sydneiensis samea could reach 0.67 mM, and thus, we isolated vanadium-resistant bacteria from the intestinal contents and determined the ability of each bacterial strain to accumulate vanadium and other heavy metals. Nine strains of vanadium-resistant bacteria were successfully isolated, of which two strains, V-RA-4 and S-RA-6, accumulated vanadium at a higher rate than did the other strains. The maximum vanadium absorption by these bacteria was achieved at pH 3, and intracellular accumulation was the predominant mechanism. Each strain strongly accumulated copper and cobalt ions, but accumulation of nickel and molybdate ions was relatively low. These bacterial strains can be applied to protocols for bioremediation of vanadium and heavy metal toxicity.

Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds twenty V(IV) ions at pH 7.5, and that it has a novel bow-shaped conformation. However, the role of Vanabin2 in vanadium accumulation by the ascidian has not yet been determined. In the present study, the effects of acidic pH on selective metal binding to Vanabin2 and on the secondary structure of Vanabin2 were examined. Vanabin2 selectively bound to V(IV), Fe(III), and Cu(II) ions under acidic conditions. In contrast, Co(II), Ni(II), and Zn(II) ions were bound at pH 6.5 but not at pH 4.5. Changes in pH had no detectable effect on the secondary structure of Vanabin2 under acidic conditions, as determined by circular dichroism spectroscopy, and little variation in the dissociation constant for V(IV) ions was observed in the pH range 4.5-7.5, suggesting that the binding state of the ligands is not affected by acidification. Taken together, these results suggest that the reason for metal ion dissociation upon acidification is attributable not to a change in secondary structure but, rather, that it is caused by protonation of the amino acid ligands that complex with V(IV) ions.

Some ascidians accumulate vanadium in vanadocytes, which are vanadium-containing blood cells, at high levels and with high selectivity. However, the mechanism and physiological significance of vanadium accumulation remain unknown. In this study, we isolated novel proteins with a striking homology to glutathione transferases (GSTs), designated AsGST-I and AsGST-II, from the digestive system of the vanadium-accumulating ascidian Ascidia sydneiensis samea, in which the digestive system is thought to be involved in vanadium uptake. Analysis of recombinant AsGST-I confirmed that AsGST-I has GST activity and forms a dimer, as do other GSTs. In addition, AsGST-I was revealed to have vanadium-binding activity, which has never been reported for GSTs isolated from other organisms. AsGST-I bound about 16 vanadium atoms as either V(IV) or V(V) per dimer, and the apparent dissociation constants for V(IV) and V(V) were 1.8 x 10(-4) M and 1.2 x 10(-4) M, respectively. Western blot analysis revealed that AsGSTs were expressed in the digestive system at exceptionally high levels, although they were localized in almost all organs and tissues examined. Considering these results, we postulate that AsGSTs play important roles in vanadium accumulation in the ascidian digestive system.

Some ascidians accumulate high levels of the transition metal vanadium in their blood cells. The process of vanadium accumulation has not yet been elucidated. In this report, we describe the isolation and cDNA cloning of a novel vanadium-binding protein, designated as VanabinP, from the blood plasma of the vanadium-rich ascidian, Ascidia sydneiensis samea . The predicted amino acid sequence of VanabinP was highly conserved and similar to those of other Vanabins. The N-terminus of the mature form of VanabinP was rich in basic amino acid residues. VanabinP cDNA was originally isolated from blood cells, as were the other four Vanabins. However, Western blot analysis revealed that the VanabinP protein was localized to the blood plasma and was not detectable in blood cells. RT-PCR analysis and in situ hybridization indicated that the VanabinP gene was transcribed in some cell types localized to peripheral connective tissues of the alimentary canal, muscle, blood cells, and a portion of the branchial sac. Recombinant VanabinP bound a maximum of 13 vanadium(IV) ions per molecule with a K d of 2.8 × 10 −5 M. These results suggest that VanabinP is produced in several types of cell, including blood cells, and is immediately secreted into the blood plasma where it functions as a vanadium(IV) carrier.

The effects of hexavalent chromium on the cell growth of green paramecium, Paramecium bursaria (P. bursaria), were investigated in this study. Two strains (MB-1 and F1 generation) of P. bursaria were incubated in lettuce media supplemented with different concentrations of potassium dichromate under LL (24 hr light), LD (12 hr light : 12 hr dark) and DD (24 hr dark) conditions, and the IC50 values were obtained. The IC50 7-day value showed that the toxicity of potassium dichromate was light-sensitive in both strains of P. bursaria. The results of the toxic effect of chromium on the cell shape of P. bursaria (BWK-4) showed that the body ratio of P. bursaria increased, even if the cells were incubated for 24 hr with 0.5 µM potassium dichromate solution, indicating that the cell shape of P. bursaria is very sensitive to potassium dichromate. The average amount of chromium accumulated in green paramecium ranged from 1.72 to 15.5 pg Cr/cell in a time- and concentration-dependent manner. This finding indicates the possibility of the use of P. bursaria as a biomonitor and bioaccumulator for chromium contaminants in aquatic environments. The experiment with electrical stimuli into the culture of P. bursaria indicated that cells accumulated at the negative electrode, suggesting that P. bursaria carried a positive charge. Thus, because the positive charge does not cause any significant absorption of chromium into the cells, P. bursaria may develop a variety of mechanisms for chromium accumulation in the cell. Further research studies are required to elucidate the mechanism of chromium uptake in P. bursaria.

Since the beginning of the last century, it has been known that ascidians accumulate high levels of a transition metal, vanadium, in their blood cells, although the mechanism for this curious biological function remains unknown. Recently, we identified three vanadium-binding proteins (vanabins), previously denoted as vanadium-associated proteins (VAPs) [Zool. Sci. 14 (1997) 37], from the cytoplasm fraction of vanadium-containing blood cells (vanadocytes) of the vanadium-rich ascidian Ascidia sydneiensis samea. Here, we describe the cloning, expression, and analysis of the metal-binding ability of vanabins. Recombinant proteins of two independent but related vanabins, vanabin1 and vanabin2, bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1x10(-5) and 2.3x10(-5) M, respectively. The binding of vanadium(IV) to these vanabins was inhibited by the addition of copper(II) ions, but not by magnesium(II) or molybdate(VI) ions. Vanabins are the first proteins reported to show specific binding to vanadium ions; this should provide a clue to resolving the problem regarding the selective accumulation of vanadium in ascidians.

X-ray microscopy enables high-resolution analysis of thick specimens such as several microns in aqueous and atmospheric pressure environments in fields of biological and material sciences. Most commonly, zone plates (ZPs) are used as optical elements in combination with the use of synchrotron radiation (SR) as an x-ray source. It is realized at the x-ray microscopy station at BL-12 of Ritsumeikan SR center. The highest spatial resolution is 45 nm. This X ray microscope has been applied in biology, medicine, and material science. Various specimens can be clearly observed. We report some applied researches of our X-ray microscope.

Blood cells of ascidians (tunicates) are known to accumulate high levels of vanadium selectively from sea water [1]. It has long been extensively studied how much vanadium is accumulated, which organ participate in the accumulation of vanadium, and what is the route of vanadium accumulation. However, they are not yet revealed completely, Micro-XANES is a powerful tool to investigate micro-distribution of chemical species of a given element. Fluorescence scanning X-ray microscope at ESRF (ID21), covering an energy range from 2 to 8 keV, has the capability of investigating chemical states of vanadium, sulfur, magnesium and calcium, In the present study we have applied this X-ray scanning microscope in fluorescence mode to elucidate the distribution of vanadium in living ascidian blood cells. Using a cryogenic sample holder, local XANES spectra was also taken successfully. The valence of the vanadium in the cellis +3 and +4,judging from XANES spectra.

Vanadium is an element with symbol V and atomic number 23. The vast majority of vanadium demand is from the steel industry, and the rest for titanium alloy and catalyst in chemical factory. Air pollution and water pollution by vanadium were recognized from early twentieth century. Increasing information on the toxicity and medicinal use enhanced the development of bioremediation of vanadium. In this chapter, the author would like to overview the history of pollution of vanadium, vanadium toxicity, bioaccumulation and bioremediation of vanadium.

Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 10(7) times that found in seawater. The vanadium ions are stored in vacuoles located within vanadium-containing blood cells, vanadocytes. To investigate the phenomenon, an expressed sequence tag analysis (EST) of a cDNA library of Ascidia sydneiensis samea blood cells was carried out. Three hundred clones were obtained and sequenced by EST analysis. A similarity search revealed that 158 of the clones (52.7%) were known genes, and 142 of the clones (47.3%) did not have any similarity to genes registered in the SwissProt database. According to the functions of their genes the identified EST clones were categorized into eight types of clones; these consisted of genes; metal-related proteins (29 clones), signal transduction (22 clones), protein synthesis (17 clones), nuclear proteins (17 clones), cytoskeleton and motility (14 clones), energy conversion (3 clones), hypothetical proteins (11 clones), and others (45 clones). The ferritin homologue has a high degree of similarity to that of mammals; the iron-binding sites of ferritin are well conserved including His-118 which is important for capturing Fe(2+), also works as a ligand for VO(2+).

Some ascidians (sea squirts) accumulate the transitional metal vanadium in their blood cells at concentrations of up to 350 mM, about 10(7) times its concentration found in seawater. There are approximately 10 different types of blood cell in ascidians. The identity of the true vanadium-containing blood cell (vanadocyte) is controversial and little is known about the subcellular distribution of vanadium. A scanning x-ray microscope installed at the ID21 beamline of the European Synchroton Radiation Facility to visualize vanadium in ascidian blood cells. Without fixation, freezing or staining realized the visualization of vanadium localized in living signet ring cells and vacuolated amoebocytes of two vanadium-rich ascidian species, Phallusia mammillata and Ascidia sydneiensis samea. A combination of transmission and fluorescence images of signet ring cells suggested that in both species the vacuoles contain vanadium.

Ascidians, especially those belonging to the Ascidiidae, are known to accumulate extremely high levels of vanadium in vanadocytes, one type of blood (coelomic) cell. Vanadium, which exists in the +5 oxidation state in seawater, is accumulated in the vanadocytes and reduced to the +3 oxidation state. We have been trying to characterize all of the polypeptides specific to vanadocytes and to specify the proteins that participate in the accumulation and reduction of vanadium. To date, we have localized three enzymes in vanadocytes: 6-phosphogluconate dehydrogenase (6-PGDH: EC 1.1.1.44), glucose-6-phosphate dehydrogenase (G6PDH: EC 1.1.1.49), and glycogen phosphorylase (GP: EC 2.4.1.1), all of which are involved in the pentose phosphate pathway. In the current study, we cloned a cDNA for transketolase, an essential and rate-limiting enzyme in the non-oxidative part of the pentose phosphate pathway, from vanadocytes. The cDNA encoded a protein of 624 amino acids, which showed 61.8% identity to the human adult-type transketolase gene product. By immunocytochemistry and immunoblot analyses, the transketolase was revealed to be a protein that was expressed only in vanadocytes and not in any of the more than ten other types of blood cell. This finding, taken together with the localized expression of the other three enzymes, strongly supports the hypothesis that the pentose phosphate pathway functions exclusively in vanadocytes.

Vanadium(V) species are reduced to vanadium(IV) directly by NADPH in the presence of EDTA. At neutral pH, vanadium(V)–EDTA complex, [VO 2 (EDTA)] 3− (20 mM) was almost completely reduced to [VO(EDTA)] 2− directly by NADPH (200 mM) after 15 h under aerobic and anaerobic conditions. The reduction is markedly accelerated at low pH. At pH 3, the vanadium(V) complex (20 mM) was reduced by NADPH (20 mM) within 1 h. Oxygen had little effect, but inhibited the reduction to some extent at low pH. At low pH, simple vanadium(V) species were also partly reduced without the assistance of EDTA, resulting in the formation of mixed-valence (V(IV)–V(V)) polynuclear species. The accumulation and reduction of vanadium in the vanadocytes of ascidians are discussed based on the present results.

Vacuolar-type H+-ATPases (V-ATPases), which are composed of at least ten different subunits, can generate a proton-motive force by hydrolyzing ATP and acidify the contents of various intracellular organelles. Subunits A and B of V-ATPase have been detected immunologically in ascidian blood cells, predominantly in signet ring cells (vanadocytes), which accumulate vanadium in their vacuoles. The action of V-ATPase in ascidian blood cells has been demonstrated by the fact that bafilomycin A1, a specific inhibitor of V-ATPases, inhibits the acidification of the vacuoles of vanadocytes. As the next step in studying the function of V-ATPase in vanadocytes, we isolated cDNAs encoding subunits A and B of V-ATPase from the blood cells of an ascidian, Ascidia sydneiensis samea. The nucleotide sequences of the cDNAs for subunits A and B encoded proteins of 619 and 509 amino acids, respectively, both of which were highly conserved among organisms.

Ascidians have the unusual physiological ability to accumulate high levels of vanadium and reduce it to the +3 oxidation state (VIII) in vanadocytes, the vanadium-containing blood cells. We are characterizing several polypeptides specific to vanadocytes that may participate in this. This study revealed that a 100-kDa antigen, recognized by a newly prepared monoclonal antibody, S8E4, is exclusively localized in vanadocytes, and identified the antigen as glycogen phosphorylase (EC 2.4.1.1) by sequencing the encoded cDNA. Since two enzymes, glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and 6-phosphogluconate dehydrogenase (EC 1.1.1.44), both in the pentose phosphate pathway, have already been identified in vanadocytes, at least three enzymes involved in carbohydrate metabolism are localized in vanadocytes in huge amounts.

Mesoderm formation plays a crucial role in the establishment of the chordate body plan. In this regard, lancelet embryos develop structures such as the anteriorly extended notochord and the lateral divertecula in their anterior body. To elucidate the developmental basis of these structures, we examined the expression pattern of a lancelet twist-related gene, Bbtwist, from the late gastrula to larval stages. In late-gastrula embryos, the transcripts of Bbtwist were detected in the presumptive first pair of somites and the middorsal wall of the primitive gut. The expression of Bbtwist was then upregulated in the lateral wall of somites and the notochord. At the late-neurula stage, it was also expressed in the anterior wall of the primitive gut, as well as in the evaginating lateral diverticula. No signal was detected in the left lateral diverticulum when it was separated from the gut, while in the right one, the gene was expressed later during the formation of the head coelom in knife-shaped larvae, and in the anterior part of the notochord in the same larvae. In 36-h larvae, only faint expression was detected in the differentiating notochordal and paraxial mesoderm in the caudal region. These expression patterns suggest that Bbtwist is involved in early differentiation of mesodermal subsets as seen in Drosophila and vertebrates. The expression in the anterior notochord may be related to its anterior expansion. The expression in the anterior wall of the primitive gut and its derivative, the lateral diverticula, suggests that lancelets share the capability to produce a mesodermal population from the tip of the primitive gut with nonchordate deuterostome embryos. (C) 1998 Academic Press.

Early embryonic development of the nervous system of a lamprey, Lampetra japonica, was studied by using immunohistochemical techniques and by scanning electron microscopy. The earliest appearance of axons was detected at Tahara’s stage 21 - , when dorsolateral and ventral longitudinal fasciculi were present in the hindbrain and spinal cord regions. The branchiomeric nerve roots began to appear at stage 22; the fibers were joined to the dorsolateral fasciculus proximally and also extended distally into each pharyngeal arch. The anterior neural tube was divided into several neuromeres: the mid-hindbrain sulcus became apparent first, then the portion rostral to this sulcus was subdivided into two portions by the syn-parencephalic boundary. In the hindbrain around stage 23, rhombomeres developed transiently, of which, rhombomere 4 was the most distinctive. Putative crest cells forming the octavofacial nerve root anlage were selectively adhering to rhombomere 4, whereas no crest cells were found on rhombomere 3. The assignment of the crest-derived nerve anlage to rhombomeres is conserved between gnathostomes and L. japonica. The neuromerical scheme of the neural tube of L. japonica is also mostly in accordance with that in gnathostomes, sharing the basic developmental patterning of axon bundles at early developmental stages. The most distinct difference between these two groups is the topographical relationships between the hindbrain neuraxis and pharyngeal arches, as well as the otic

The effect of all-trans retinoic acid on embryogenesis was studied in a cyclo- stome, Lampetra japonica. Treatment with 0.05- 0.5 mM retinoic acid on early gastrula and early neurula resulted in loss of the pharynx and in the rostral truncation of the neural tube. The mouth, pharynx, esophagus, heart, endostyle, and rostral brain were missing with graded severity. In the severest case, the embryo consisted only of trunk segments, especially myotomes that extended to the rostral end of the axis. The effect appeared to be dose- and stage-dependent: Rostral pharyn- geal arches were more vulnerable to a lower amount of retinoic acid, and earlier treatment resulted in severer defects. The initial protrusion of the anterior axis started equally in control and retinoic acid-treated embryos, implying that the head morphogenesis is omitted in treated em- bryos. By identifying the number of myotomes based on the differentiation of hypobranchial muscles, there seemed to be no myotomes lost by retinoic acid-induced truncation. The rostral truncation, therefore, was not simply a limitation of the anterior axis but was restricted to the ventral portion; only the branchial arches disap- peared with normally developing myotomes dor- sally. The absent region can be defined as the vertebrate head in a morphological sense, includ- ing the branchiomeric and preotic paraxial re- gions as well as the heart. The results suggest the presence of distinct programs between somito- meric and branchiomeric portions of the body, providing a developmental basis for the dual- metamerical body plan of vertebrates. Dev. Dyn.

The development of peripheral nerves was studied in a Japanese marine lamprey, Lampetra japonica, in whole-mount and sectioned embryos from hatching until the earliest ammocoete. Nerve fibers were immunohistochemically stained with a monoclonal antibody against acetylated tubulin. Branchiomeric nerves first developed in a simple metamerical pattern, each associated with a single pharyngeal arch. Of those, the ophthalmicus profundus, maxillomandibular, and facial nerves later developed a highly modified branching pattern, whereas postotic nerves were less specialized and showed the stereotypical branching pattern of post-trematic nerves. The early distribution of melanocytes in myotome-free space largely overlapped with the morphology of the cranial nerve and ganglion anlage, and resembled the cephalic crest cell distribution pattern in the early chick embryo. It was suggested that the cephalic crest cell distribution, which is also inhibited by myotomes in the lamprey, would be the common basis for branchiomeric nerve patterning. In later development of the lamprey embryo, myotomes 1 through 3, which had originated in the postotic region, grew rostrally into the preotic region, laterally covering all of the branchiomeric nerves. This results in a deep position of the cranial nerves, which is not observed in gnathostomes.

In a previous study, Vanabin2, a member of a family of V(IV)-binding proteins, or Vanabins, was shown to act as a V(V)-reductase. The current study assesses the ability of Vanabin2 to reduce various transition metal ions in vitro. An NADPH-coupled oxidation assay yielded no evidence of reduction activity with the hexavalent transition metal anions, Mo(VI)O4(2-) and W(VI)O4(2-), or with three divalent cations, Mn(II), Ni(II), and Co(II). Although Cu(II) is readily reduced by glutathione and is gradually oxidized in air, this process was not affected by the presence of Vanabin2. In the experiments conducted thus far, Vanabin2 acts only as a V(V)-reductase. This high selectivity may account for the metal ion selectivity of vanadium accumulation in ascidians.

During embryogenesis of the ascidian Halocynthia roretzi, exactly eight-hundred epidermal cells are formed in the larva, and the lineage of the cells has been almost completely described. In the present study, we examined the spatio-temporal expression patterns of eight epidermis-specific genes which we already isolated. In situ hybridization of whole-mount specimens unambiguously demonstrated that the expression patterns of the eight genes were not identical, and that they were categorizable into several types. Transcripts of seven genes were restricted to presumptive epidermal cells, although transcripts of one gene were evident in the presumptive neural cells in addition to the presumptive epidermal cells. Therefore, most of the epidermis-specific genes in ascidian embryos are expressed in lineage-associated manner. We discuss these results in relation to the question of whether (a) epidermis-specific genes are expressed exclusively in presumptive epidermal cells prior to neural induction, or (b) the genes are first expressed in both epidermal precursors and precursors of the central nervous system, then the gene expression is downregulated in the latter after the completion of neural induction. Interestingly, cells of the anterior-most region as well as the dorsal midline of the tailbud embryo did not express most of the epidermis-specific genes, suggesting regional differences in the embryonic epidermis. Some other genes might be expressed in a complementary pattern in these regions.