ASCIDIAN NEWS*
Gretchen and Charles Lambert
206-365-3734
glambert@fullerton.edu or clambert@fullerton.edu
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.
Mike Thorndyke
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 christian.sardet@obs-vlfr.fr 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 cv@biodev.obs-vlfr.fr
SITES AND FILMS
I have put an extensive collection of films of ascidian maturation, fertilization, development on the internet at
http://biodev.obs-vlfr.fr/recherche/biomarcell/films%2Bimages/filmarchive.htm
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. (mcarman@whoi.edu): 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
4.
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
From
Patrick Lamaire,
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 (roure@ibdm.univ-mrs.fr). 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
Developmental Biology,
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. bandl@uclink.berkeley.edu
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
Sci.,
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. yharada@bio.nagoya-u.ac.jp hsawada@bio.nagoya-u.ac.jp
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. hsawada@bio.nagoya-u.ac.jp
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,
HAP/APEX/Ref-1, apurinic/apyrimidic
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. (hmichi@hiroshima-u.ac.jp)
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. (ueki@hiroshima-u.ac.jp) Dept. Biol. Sci. and Mar. Biol. Lab., Grad.
Sch. Sci.,
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,
a.
Recognition and clearance of apoptotic cells in colonial ascidians. L. Ballarin, Dipartimento di
Biologia,
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.
b. Cyclic
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
Selective metal
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
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ligands is not affected by acidification. Taken together, these results suggest
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protonation of the amino acid ligands that complex with V(IV) ions.
THESIS ABSTRACTS
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 (rkoch@fullerton.edu )
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..
In Ascidia ceratodes sperm
activation, there is evidence that activation of a Na+/H+
exchanger causes an increase in pHi and a subsequent increase in [Ca2+]i
from both intracellular and extracellular sources. However, the link between pHi, Em,
and [Ca2+]i has not been investigated. The goal of this research was to characterize
the membrane potential changes that take place during ascidian sperm
activation. A
voltage-sensitive dye was used to measure changes in sperm membrane potential
in batches of sperm. When sperm were
activated with pH 9.4 ASW and the G-protein activator mas7, a hyperpolarization
and subsequent depolarization was observed.
Ion-substitution experiments supported that the hyperpolarization is K+-dependent
and not Cl- -dependent and established an internal K+
concentration of 336mM. Valinomycin
experiments established a resting membrane potential of -68.9 mV. The osmoregulator blocker furosemide,
voltage-gated channel blocker 4-aminopyridine, and the Ca2+
low-voltage channels blockers pimozide and penfluridol inhibited
hyperpolarization. However, high, low,
intermediate KCa blockers, and the voltage-gated K+
channel blocker TEA, had no effect on the hyperpolarization. Identifying the cause of hyperpolarization is
important because disabling it inhibits sperm activation.
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