Gretchen and Charles Lambert
home page: http://depts.washington.edu/ascidian/
Number 59 September 2006
Thanks again to all of you who have written in the last several months about AN and how valuable it has been and still is. A surprisingly frequent comment was similar to that by George Mackie: “It gave us ascidian fans a feeling of community, which I for one will miss.” Because we’ve done AN for 30 years, other comments were similar to Don Deibel’s: “AN has been a part of my professional life for my entire career. I will miss it.” We greatly appreciate your kind words and have decided to continue with AN for a few more years at least.
wrote: “One thing we'll all miss I am sure is the updates on what the both of
you have been up to. That is not available on any database that I know of!”
We’re not sure everyone is all that interested, but here goes the latest
installment! We spent a few weeks at the
Friday Harbor Labs during June and July, then 3 exciting weeks in
There are 181 new publications in this issue of the newsletter! Keep up the good work and to assure that your new publications will appear in AN please don’t forget to send us hard copy color reprints (preferred as we do not have a color printer) or PDFs; thank you.
*Ascidian News is not part of the scientific literature and should not be cited as such.
1. Second announcement of 2007 Intl. Tunicata Conference From Christian Sardet, Station Zoologique, Observatoire, CNRS, Univ PM Curie, Villefranche sur Mer 06230, France firstname.lastname@example.org The meeting will be Saturday June 23 (arrivals) to Wednesday June 27 in Residence Delcloy situated in St. Jean Cap Ferrat about a mile from the Marine Station. To see the conference site: http://www.vacancesbleues.com/destinationRechercheMono.do, click on France then Mediterranee then St. Jean Cap Ferrat and click on the 360 panoramics to see the facilities. We will establish a specific web site, and draft a preliminary program in Sept. /October. We hope you will participate and enjoy Good Science and the French Riviera.
The Villefranche team Christian Sardet, Janet Chenevert, Clare Hudson, Alex McDougall,
Hitoyoshi Yasuo, Remi Dumollard: Things are going well and ascidians are
firmly established with 3 groups (Sardet, McDougall, Yasuo)
and a dozen researchers, Post Docs, technicians and students working on Ciona
and Phallusia. http://biodev.obs-vlfr.fr/recherche/.
The appendicularian work is reduced in Villefranche but is now strong in
We are looking for a new Director of Developmental/Cell Biology Research Unit at Villefranche-sur-mer. For further details contact our search committee email@example.com
SITES AND FILMS
I have put an extensive collection of films of ascidian maturation, fertilization, development on the internet at
I also have 2 other fun sites The Bioclips site: http://www.bioclips.com/
with some multimedia stories featuring ascidian eggs and embryos see "Polarity inside the egg cortex" and "Sparks of life". The Portal http://www.biologymultimedia.com with access to a large collection of films about cells issued from the new DVD "Exploring the living Cell" 3 hours of documents about the discovery, diversity, research and debates about cells: see: http://www.bioclips.com/dvd/content.pdf
2. While you have
your 2007 calendar handy, please add
Schedule from Mary Carman, organizer, Woods Hole Oceanog. Inst. (firstname.lastname@example.org): arrival Monday Oct. 1 at Rodd-Brudenell River Hotel (30 min. from Charlottetown in the Brudenell River Provincial Park); Oct. 2, morning field trip/charter boat outing to view aquacultured mussel lines and walk to nearby floating docks, followed by afternoon taxonomic workshop given by Gretchen and Charles Lambert at Atlantic Veterinary College (ascidians collected during the field trip or brought from home will be welcome). Oct. 3-4 will include invited plenary talks, contributed research presentations, posters, and discussions at the hotel (banquet evening Oct. 3), with departure Oct. 5. A special block of rooms has been reserved for us at the hotel; view their website at www.roddhotelsandresorts.com
Registration and call
for abstracts will open
Invasive ascidians are impacting ecosystems, creating a nuisance for the aquaculture industry, and are a major component of fouling communities. The aim of this conference is to bring together marine biologists, shellfishery scientists, representatives of the shellfish industry, members of local, state, and federal agencies concerned with coastal resources, and representatives from sponsoring organizations concerned with invasive ascidians, to explore the biology, ecology, impacts, management options for control, and other relevant topics. Extended abstracts and full papers will be published.
3. From Bill Smith, UC Santa Barbara:
An application for
an NIH program entitled Tools for Genetic and Genomic Studies in Emerging Model
Organisms, submitted by Bill Smith and Mike Levine, has been awarded. This
four-year, $1 million award will provide funds for the construction of new
ascidian culturing facilities at
Van Name W.G. 1945. The North and South American ascidians. Bull.of the Am. Mus. of
Nat. Hist. vol.84:1-476 is available online at http://digitallibrary.amnh.org/dspace/handle/2246/1186.
It is a very large pdf—209 mb.
However, it is downloadable if you have access to high speed broadband. It can
then be printed out (be sure to print double sided because of the large number
of pages). Once downloaded, the advantage of having a digital copy is that it
is searchable! You can easily find all the species listed for
The chordate Gateway vector set is now available to the ascidian community. Gateway technology allows to bypass the main problems encountered in traditional restriction enzyme-mediated cloning strategies. AgnХs Roure in the Lemaire lab in Marseille (France) generated a collection of Gateway vectors dedicated to overexpress native or fusion proteins in ascidians as well as to study the activity of cis-regulatory elements. 2 kinds of vectors were developed. The pSPE3 series is designed to synthetise mRNA in vitro, which can be micro-injected into embryos. The pSP1.72 series are transgenesis vectors designed to be electroporated into embryos. In both series, an ORF of interest can be introduced into an RfA Gateway cassette flanked by attR1 and attR2 recombination sites. Various vectors harbour N or C-terminal fusion tags (fluorescent protein or epitope) allowing to track the subcellular localisation of protein products. In addition, AgnХs developped a second, attR3-attR5 flanked Gateway cassette used to receive a cis-regulatory region (in attR3-attR5). This second cassette is placed upstream the ORF-accepting attR1-attR2 cassette in the pSP1.72 series. These vectors were all tested in vivo and shown to be functional in ascidian embryos. The whole collection is freely available upon request (email@example.com). All details about the Gateway technology, vectors, sequences and procedures can be found in « V2.0 Chordate Gateway Vectors Manual » which is downloadable from our lab web site: http://crfb.univ-mrs.fr/ciona/lemaire/.
1. Society for
Two hearts beat as one: Experimental compartmentalization of the Ciona heart. B. Davidson, W. Shi, J. Beh, L. Christiaen, M. Levine. Dev. Biol. 295: 334 #29. firstname.lastname@example.org
The evolution of the complex, multi-chambered vertebrate heart may have involved either sub-division of the ancestral heart field or progressive addition of supplementary cardiac lineages. The single-chambered condition of the ancestral chordate heart has apparently been maintained within the tunicates, including Ciona intestinalis. Here, specific manipulations of progenitor cell recruitment cause compartmentalization of the Ciona heart. We present evidence that FGF signaling induces cardiac mesoderm within a subset of competent cells. Targeted inhibition of FGF signaling blocks heart formation, and a similar loss is obtained with a constitutive repressor form of the RTK transcriptional effector, Ets1/2 (Ets-WRPW). Conversely, targeted expression of a constitutively active form of Ets1/2, Ets-VP16, throughout the heart field doubles the number of heart progenitor cells. These excess heart cells produce an unexpected phenotype: the transformation of a one-chambered heart into a functional multi-compartment organ. These results suggest that progenitor cell recruitment was an important step during the emergence of the vertebrate multi-chambered heart. We propose that variability in the distribution of progenitor cells represents a general mechanism for potentiating evolution of novel internal structures.
2. 77th annual meeting of the Zool. Soc. of Japan, Shimane Univ., Matsue, Japan, 21-24 September 2006.
a. Estrogen alters
the gene expression profile in the ascidian, Ciona intestinalis. R.
Koyanagi1,2, M. Yamashita3,
The gene expression profile of the ascidian Ciona intestinalis in the presence of an estrogen, 17b-estradiol (E2) was analyzed. Mature adult animals were exposed to nanomolar concentrations of E2 under several different conditions. The gene expression profile of the experimental individuals has been explored using a series of DNA microarray analysis. Although no homologous gene for mammalian intracellular estrogen receptors was found in the C. intestinalis genome, the results of statistical study on the expression profiles revealed E2-dependent alteration of the profiles as well as the genes up- or down-regulated depending on the exposure time. On the other hand, the different (100-fold) concentration of E2 gave no significant effect on the profiles, suggesting that the concentration is not the primal parameter to drive the change in the gene expression profiles at the conditions tested in this study. These results show the presence of a signaling pathway to respond E2 stimulation which is independent of known vertebrate receptor and suggest E2 or estrogenic compounds as a potential endocrine disruptor for C. intestinalis. Further analysis on the expression profile will give a good scaffold to understand the molecular mechanism behind this phenomenon.
b. Roles of spermosin
L-chain in fertilization of the ascidian Halocynthia roretzi. M.
Akasaka, Y. Harada, and H. Sawada. Sugashima Mar. Biol. Lab., Graduate Sch. of
We previously reported that two sperm trypsin-like proteases, acrosin and spermosin, are involved in fertilization of the ascidian Halocynthia roretzi, and that C-terminal CUB domain of ascidian proacrosin and L-chain of spermosin are capable of binding to the vitelline coat components. But, the roles in fertilization of these regions of sperm trypsin-like proteases have not been studied well. In this context, we made the antibodies against HrProacrosin CUB-domain, HrSpermosin L2 region, and HrSpermosin L1 (BL2) region, and their effects on fertilization were examined. Although anti-CUB-domain antibody showed weak or no inhibition toward fertilization, anti-L2 antibody potently inhibited the fertilization. In contrast, anti-L1 (BL2) antibody increased the fertilization ratio in a concentration-dependent manner. These results suggest that spermosin plays a key role in ascidian fertilization and that Pro-rich L1 (BL2) region of spermosin type I may be involved in the block to fertilization. Further studies are necessary to elucidate the mechanism of stimulation in fertilization ratio by anti-spermosin L1 (BL2) antibody.
c. Vitelline-coat protein CiVC100, a candidate allorecognition protein during fertilization of the ascidian Ciona intestinalis. Y. Harada, Y. Takagaki, T. Saito, and H. Sawada. email@example.com firstname.lastname@example.org
Ascidians are hermaphroditic, releasing both sperm and eggs almost simultaneously, but many species, such as Halocynthia roretzi and Ciona intestinalis, exhibit self-sterility. It is reported that the barrier against self-sperm resides on the vitelline coat and that the barrier is impaired or removed by short treatment of eggs with weak acid. We previously reported that a highly polymorphic 70-kDa vitelline-coat protein HrVC70, consisting of 12 epidermal-growth-factor (EGF)-like repeats, is a promising candidate for the allorecognition molecule during fertilization of the ascidian H. roretzi and that this protein is easily detached from the vitelline coat by short treatment with weak acid. Here, we show that a 100-kDa vitelline-coat-component CiVC100 is detached by acid treatment from the isolated vitelline coat of another ascidian, C. intestinalis. Based on the protein sequences of the protease-digested fragments of CiVC100, we attempted to identify this protein from C. intestinalis genome database. Interestingly, CiVC100 showed no homology to HrVC70, but turned out to be an apolipoprotein ortholog.
d. Functional analysis of HrUrabin, a sperm GPI-anchored protein capable of binding to a candidate allorecognition protein on the vitelline coat, HrVC70, in Halocynthia roretzi.
Y. Nakagawa, Y. Harada, and H. Sawada. email@example.com
Ascidians are hermaphrodites, but several ascidians, including Halocynthia roretzi, show strict self-sterility because of unknown molecular mechanisms. We previously reported that a highly polymorphic vitelline-coat sperm-receptor HrVC70 is a candidate allorecognition protein and that a sperm GPI-anchored 35-kDa glycoprotein HrUrabin in lipid raft fraction is capable of binding to HrVC70 in in vitroassay conditions. Here, we investigated the role of HrUrabin in fertilization by using a specific antibody against HrUrabin. We found that anti-HrUrabin antibody potently inhibited the fertilization in a concentration-dependent manner. Concerning the binding ability of sperm to HrVC70, the number of nonself-sperm bound to an HrVC70-agarose bead was significantly higher than that of self-sperm, as reported previously. Under these conditions, anti-HrUrabin antibody almost completely blocked the binding of both self- and nonself-sperm to HrVC70-agarose beads. These results indicate that HrUrabin plays a pivotal role in fertilization, in particular in the sperm binding process to HrVC70, and that there may be a sperm-derived novel allorecognition molecule in addition to HrUrabin.
3. Marine Genomics Congress,
endonuclease mRNA is expressed in the oocytes of ascidian Ciona intestinalis.
DNA repair is probably one of the most important processes to be performed in the oocytes and zygote, at the time of fertilization and immediately after, in order to allow complete embryonic development. APEX/Ref1, Apurinic/apyrimidinic endonuclease-Red-Ox factor1 is capable of initiating the repair of apurinic/apyrimidic (AP) sites, the most common decay, in damaged DNA. In vertebrate models, the enzyme is also supposed to play an important role in response to oxidative stress. The DNA-binding activity of APEX is modulated by a post translational mechanism involving reduction oxidation and so at least partly mediated by ROS. Preliminary data indicate that APEX mRNA is expressed in human spermatozoa and oocytes and in pre-implantation embryos. In order to investigate if the level of expression of APEX was evolutionarily conserved, we extended our study to oocytes ad spermatozoa of ascidian Ciona intestinalis, an organism intensively studied in developmental biology and, more recently, proposed as a model to study meiotic regulation. From an evolutionary point of view, tunicates (appendicularians, salps and sea squirts) have very recently been re-evaluated as the closest relatives of vertebrates, more than cephalochordates, like amphioxus. This important discovery has been made possible since the advent of genomics that actually provide the opportunity for phylogenetics to resolve a number of outstanding evolutionary questions. In this respect, the draft copy of the C. intestinalis genome became publicly available providing new insights into origin and evolution of chordates. Based on this evidence, we used C. intestinalis gametes as comparative model to study the conservativity of APEX function. Our data indicate that APEX transcripts were detected in oocytes and embryos, but not in spermatozoa, of C. intestinalis. Of phylogenetic significance is also the observation that ascidian APEX lacks redox transcriptional activity.
4. 5th Int'l Conf. on Vanadium Chemistry & Biochemistry, Am. Chem. Soc. meeting, San Francisco, Sept. 10-14, 2006
a. Towards the biological reduction mechanism of vanadyl
ion in the blood cells of vanadium-sequestering tunicates. P. Frank, E.J. Carlson, R.M.K. Carlson, B. Hedman and K.O.
Hodgson. Dept. of Chem.,
Nearly one hundred years after Henze reported high concentrations of vanadium and acid in some ascidians, the mechanism for the reduction of ambient V5+ to cellular V3+ remains unknown. We will report the results of x-ray absorption spectroscopic (XAS) measurements that queried the fate of vanadyl ion following uptake by living blood cells from the tunicate Ascidia ceratodes. These new results, in addition to previous results from XAS experiments and insights from the known inorganic chemistry of vanadium, will form the basis of a proposed mechanism for the biological reduction of vanadyl ion. The new field of vanadium redox-enzymology, long suspected but virtually undetected until now, has thus achieved infancy and awaits growth.
b. Genes and proteins involved in vanadium accumulation by ascidians. H. Michibata, M. Yoshinaga, M. Yoshihara, N. Kawakami, and T. Ueki. (firstname.lastname@example.org)
Several species of ascidians (tunicates) accumulate high levels of vanadium in blood cells known as vanadocytes. The intracellular vanadium concentration can be as high as 350 mM, which is 107 times the concentration in seawater. Vanadium accumulated in the ascidians is reduced to the +3 oxidation state via the +4 oxidation state. From a vanadium-rich ascidian, Ascidia sydneiensis samea, genes and proteins, such as Vanabin family, enzymes in the pentose phosphate pathway, metal-ATPase, glutathione S-transferase and SO4-2 transporter, likely to be involved in vanadium accumulation, have been isolated. Molecular physiological roles of these proteins will be discussed.
c. Metal ion
selectivity and affinity of wild type and mutant Vanabins. T. Ueki and H.
Michibata. (email@example.com) Dept. Biol. Sci. and Mar. Biol. Lab., Grad.
Ascidians are well known to accumulate high levels of vanadium ion in the vacuole of one or more type(s) of blood cells. We previously identified five low molecular weight vanadium-binding proteins, designated Vanabin1, 2, 3 ,4 and P, from a vanadium-rich ascidian Ascidia sydneiensis samea. EPR and NMR analyses suggested that lysines and arginines in Vanabin1 and Vanabin2 mainly contribute as coordination sites for vanadium(IV) ions. We performed in vitro mutagenesis of Vanabin2 to modify lysines, arginines and some amino acid residues in possible binding sites, and assessed the metal binding ability of mutants by immobilized metal ion affinity chromatography. Mutation of some of lysines and arginines affected the vanadium binding ability of Vanabin2.
d. Characterization of the AsGSTs, vanadium-binding glutathione transferases isolated from the vanadium-rich ascidian Ascidia sydneiensis samea. M. Yoshinaga, T. Ueki and H. Michibata
Some ascidians accumulate high levels of vanadium. We have isolated novel proteins with a homology to glutathione transferases (GSTs), designated AsGST-I and AsGST-II from the digestive system of the vanadium-accumulating ascidian Ascidia sydneiensis samea. Because AsGSTs were highly expressed in the digestive system and showed vanadium-binding activity which has never been reported for GSTs isolated from other organisms, we postulate that AsGSTs play important roles in vanadium accumulation in the ascidian digestive system which is thought to be involved in vanadium-uptake. In this study, through analysis of the recombinant AsGST-I, we examined the metal-selectivity and the GST-activity in the presence of vanadium. As a result, the vanadium-binding activity was barely inhibited in the presence of magnesium(II) or molybdate(VI) ions, which indicated the high vanadium-selectivity of AsGSTs, and the GST-activity was partly inhibited in the presence of vanadium, which suggested that vanadium may be involved in control of the GST-activity.
5. 48th Symp. of the Soc. for
Histochemistry: Histochemistry of Cell Damage and Death. Stresa,
Recognition and clearance of apoptotic cells in colonial ascidians. L. Ballarin, Dipartimento di
The colonial ascidian Botryllus schlosseri forms new zooids by blastogenesis, through the formation of palleal buds which progressively grow and mature until an adult is formed. At a temperature of 19°C, adult zooids remain active for about one week; then they contract, close their siphons and are gradually resorbed, being replaced by buds which reach functional maturity, open their siphons and begin their filtering activity as adult zooids. This recurrent generation change, known as regression or take-over, is characterised by the occurrence of diffuse programmed cell death by apoptosis. During the take-over, circulating phagocytes infiltrate in zooid tissues and engulf apoptotic cells; in addition, the frequency of haemocytes showing nuclear condensation and annexin-V labelling significantly increases. Moreover, the number of circulating phagocytes showing a globular morphology and containing ingested cells or cell debris significantly rises whereas the frequency of hyaline amoebocytes, which represent mobile, active phagocytes decreases. Phagocytes, both professional and occasional, actively recognize senescent cells and ingest them. As regards the eat-me signals, PS seems to be involved in the recognition of effete cells, as the addition of phospho-L-serine, a soluble analogue of PS, inhibits in vitro phagocytosis of apoptotic cells. CD36, a part of the receptorial complex binding thrombospondin which act as a bridging molecule between phagocyte surface and apoptotic cells, is expressed on Botryllus phagocytes: the frequency of cells recognised by anti-CD36 antibodies significantly increases during the take-over and the expression pattern changes from a patchy distribution to a uniform staining of the phagocyte surface during the take-over. Anti-CD36 antibodies significantly decreases the phagocytosis of effete cells suggesting that similarly to that described in vertebrates the thrombospondin receptor play a role in apoptotic cell removal by phagocytes.
apoptosis in the digestive tract of a protochordate. F. Cima, Dipartimento di Biologia,
Tissue degeneration which occurs during development of organisms is often of morphogenetic importance as well as proliferation and differentiation. Cyclic apoptosis of organs was progressively lost in Chordates. Botryllus schlosseri is a colonial ascidian continuously forming new zooids by blastogenesis, through the recurrent formation of palleal buds, which grow and mature until an adult is formed. Three blastogenic generations are
commonly co-present: adult, filtering zooids, their buds and budlets on buds. At a temperature of 19°C, adult zooids remain active for about one week (mid-cycle stages); then they contract, close their siphons and are gradually resorbed, being replaced by a new generation of adult zooids, represented by buds which reach functional maturity, open their siphons and begin their filtering activity (regression or take-over stage). This stage is characterized by the occurrence of diffuse programmed cell death by apoptosis in zooid tissues, as evidenced by TUNEL reaction for chromatin fragmentation and annexin V labelling for detection of exposed phosphatidylserine, whereas infiltration of circulating phagocytes, which appear engulfed with apoptotic cells, is observed. With these characteristics, colonial tunicates are suitable subjects for studies on cyclical involution and resorption of tissues. In residual zooids remaining for a long time in the centre of each colony, melanin and lipofuscins accumulate as detected with Masson-Fontana, Ziehl-Nielsen and H2O2 bleaching methods. Immunocytochemical assays to detect pro- and antiapoptotic factors reveal an opposite expression which progressively extends in tissues of adult zooids with an organ gradient starting from the branchial basket. Results support the idea that fundamental mechanisms for the induction of apoptosis are well conserved throughout Chordate evolution.
6. 12th Intl. Conf.
on Biol. Inorganic Chem., July 31-Augst 5 2005,
a. Vanadium in Biology: Accumulation Mechamism
in Ascidians. H. Michibata, T. Ueki, et al. Dept. Biol. Sci.
and Mar. Biol. Lab., Grad. Sch. Sci.,
Ascidians are well known to contain high levels of vanadium. In remarkable cases, the concentration of cellular vanadium reaches 350 mM, corresponding to about 107 times the concentration of seawater. Vanadium accumulated in ascidians is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuoles of vanadocytes. From the vanadocytes of a vanadium-rich ascidian, Ascidia sydneiensis samea, we isolated some vanadium binding proteins, designated as Vanabin. Recently, we identified five types of Vanabin: Vanabin1, Vanabin2, Vanabin3, Vanabin4 and VanabinP that are likely to be involved in vanadium accumulation processes as so-called metallochaperones. Among them, recombinant proteins of Vanabin1 and Vanabin2 bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1 в 10-5 M and 2.3 в 10-5 M, respectively. Multi-dimensional NMR experiments have revealed the first 3D structure of Vanabin2 in an aqueous solution which shows novel bow-shaped conformation, with four ?-helices connected by nine disulfide bonds. There are no structural homologues reported so far. The 15N HSQC perturbation experiments of Vanabin2 indicated that vanadyl cations, which are exclusively localized on the same face of the molecule, are coordinated by amine nitrogens derived from amino acid residues such as lysines, arginines, and histidines, as suggested by the EPR results. Recently, glutathione S-transferase (GST), known to protect organisms against oxidative stress induced by heavy metals, was extracted from digestive organs of a vanadium-rich ascidian. Recombinant protein of ascidian GST was found to bind with vanadium(IV). A significance of the vanadium-binding property is under investigation.
b. Study on vanadium-binding proteins of an ascidian Ascidia sydneiensis samea. T. Ueki, M. Yoshihara, N. Yamaguchi, K. Fukui, and H. Michibata.
Ascidians are well known to accumulate high levels of vanadium ion in the vacuole of one or more type(s) of blood cells. We previously identified five low molecular weight vanadium-binding proteins, designated Vanabins, from the vanadocytes and the ceolomic fluid of a vanadium-rich ascidian Ascidia sydneiensis samea. We examined the activities of Vanabins to bind vanadium(IV) ions by Hummel-Dreyer's method. Recombinant proteins of the two Vanabins, Vanabin1 and Vanabin2, bound to 10 and 20 vanadium(IV) ions with dissociation constants of 2.1 X 10-5 M and 2.3 X 10-5 M, respectively. EPR analysis supported these results and indicated that amine nitrogens coordinate with vanadium (IV) ions. VanabinP, which is one of most abundant proteins in ceolomic fluid, also bound to vanadium (IV) ions at a similar value (maximum 13 vanadium ions at Kd=2.8x10-5M). Although Vanabin1, Vanabin2 and VanabinP are transcribed in blood cells, their distribution patterns are different; Vanabin1 and Vanabin2 are in cytoplasm of signet ring cells (vanadocytes) while VanabinP is in coelomic fluid. These results suggested that Vanabin1 and VanabinP act as cytoplasmic vanadium carrier proteins, and VanabinP as a vanadium carrier protein in coelomic fluid, since vanadium(IV) is easily precipitated at physiological pH range. The distribution and function of Vanabins are discussed.
c. Glutathione S-transferase having vanadium-binding activity isolated from a vanadium-accumulating ascidian, Ascidia sydneiensis samea. M. Yoshinaga, K. Kamino, N. Yamaguchi, T. Ueki, and H. Michibata. (M. Yoshinaga, a PhD student of Prof. Michibata, won the best poster award, Faustus Poster Award.)
Several species of ascidians accumulate vanadium in their vanadocytes, vanadium containing blood cells, at high concentration and with high selectivity. Through the accumulation process, almost vanadium ions in the +5 oxidation state are mostly reduced to the +3 oxidation state via the +4 oxidation state and stored in the vacuole of vanadocytes. For hunting new factors involved in this unique phenomenon, we have tried to isolate novel vanadium-binding proteins from tissue extracts of a vanadium-rich ascidian, Ascidia sydneiensis samea, by using a vanadium-chelating column, and consequently several vanadium-associated proteins have been isolated. We aimed at one of the proteins expressed highly in the digestive organ, and its N-terminal amino acid sequence was determined. To screen the cDNA corresponding to the protein, a degenerate primer corresponding to the amino acid sequence and cDNA library of vanadocytes were used for polymerase chain reaction. As a result, a single DNA fragment was amplified. Analysis of the DNA sequence revealed that the estimated amino acid sequence of the protein shows a striking homology with glutathione S-transferase (GST), named as AsGST. One of the most important functions of GSTs in organisms is protection against oxidative stress induced by heavy metals. Additionally glutathione (GSH), the cofactor of GSTs, is known to modulate mobilization and toxicity of metals such as cadmium and copper, and behaves as a reducing agent for metals including vanadium. Therefore, we supposed that AsGST and GSH might play important roles during the process of vanadium accumulation in ascidians. Availability of one-step isolation of the recombinant protein of AsGST cloned and expressed in E. coli. We confirmed by a vanadium-chelating column confirmed that the recombinant protein of AsGST certainly has vanadium-binding activity using a vanadium-chelating column., In addition AsGST wais disclosed to be dimeric as same as other GSTs and have has GST activity with 1-chloro-2,4-dinitrobenzene (CDNB), one of general substances of GSTs. The correlation between vanadium-binding property and GST activity is under investigation.
7. 1st European
Chemistry Congress, 27-31 Aug. 2006
binding by Vanabin2 from the vanadium-rich ascidian, Ascidia sydneiensis samea. N. Kawakami, T. Ueki, K. Matsuo, K. Gekko
& H. Michibata. Grad. Sch. Sci.,
Vanadium-binding proteins, or Vanabins, have recently been isolated from the vanadium-rich ascidian, Ascidia sydneiensis samea. Recent reports indicate that Vanabin2 binds 20 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 this 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 measured 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.
1. Confocal scanning microscopy of fluorescent lectin- and antibody-labeled Ascidia ceratodes eggs. Mia Meeyaong-Won Botkin. M.S. thesis advisor: Robert A. Koch, Laboratory for Sperm Cell Biology and Gamete Ultrastructure, Dept. of Biol. Sci., California State Univ., Fullerton, USA (firstname.lastname@example.org )
The distributions of carbohydrates and proteins on the membrane or in the extracellular matrix determine the characteristics of binding to and penetration of the egg complex by sperm. The Ascidia ceratodes egg complex consists of several layers; moving from the outside to the central oocyte, the non-cellular vitelline coat consists of the outer fibrous, central dense, inner fibrous, and perivitelline fibrous layers (OFL, CDL, IFL, and PVFL, respectively), and cellular VC (CVC) consists of the follicle (FC) and test cells (TC). This study sought to determine by confocal microscopy what glycans and extracellular matrix proteins were located in these layers of the VC. The lectins succinyl-Concanavalia ensiformis agglutinin (suc-ConA), soybean agglutinin (SBA), Limulus polyphemus agglutinin (LPA), and Peanut agglutinin labeled CVC. Ulex europaeus agglutinin-1 (UEA-1) and Maclura pomifera agglutinin (MPA) labeled FCs. Succinyl-wheat germ agglutinin (suc-WGA) labeled the FCs and OFL. Thus, mannose, glucose, N-acetyl-D-galactosamine (GalNAc), galactose, and sialic acid were found in the CVC; high amounts of fucose and low amounts of GalNAc and galactose were present in the FCs; and, N-acetyl-D-glucosamine was on the FCs and OFL. Antibody labeling showed that fibronectin and chondroitin sulfate were present in and on the FCs and TCs; fibronectin was in the IFL; keratan sulfate was on the surface of the VC and CDL; and hyaluronan was distributed on the surfaces of the CVC.
2. Characterizing membrane potential changes
during ascidian sperm activation. Reginald McNulty. M.S. thesis advisor:
Robert A. Koch, Laboratory for Sperm Cell Biology and Gamete Ultrastructure,
Dept. of Biol. Sci., California State Univ..
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