Number 51 June 2002
After spending most of the winter working on manuscripts, in
April we enjoyed three weeks touring Italy and were shown the remarkable
rearing facility at the Stazione Zoologica in Naples by Dr. Elisabetta
Tosti and her associates. We were very much impressed by the facility
and their success in rearing Ciona for many different studies.
Not only do they maintain genetically defined stocks but also their installation
will solve the supply problem. In early June we participated in a
rapid assessment survey for nonindigenous species on both sides of the
Panama Canal, led by Dr. Andrew Cohen. The team consisted of 11 specialists
in various marine groups. We sampled numerous sites and worked at
Smithsonian Tropical Research Institute labs on both the Pacific and Caribbean
sides. There were many more ascidians on the Caribbean side especially
on marina floats and pier pilings, nearly all of which could be considered
introduced. Ascidia sydneiensis, Styela canopus, Herdmania
pallida, Microcosmus exasperatus and several other species were
very abundant. We also greatly enjoyed seeing the canal, its locks
and its complex flooding system, which periodically reverses the flow of
rivers. However, the bugs were quite vicious on the Atlantic coast and
caused a great deal of discomfort to many of the crew.
We will spend a week identifying ascidian voucher specimens at the Smithsonian Environmental Research Center in Maryland in late June. In July we’ll return to Roscoff in Brittany, France, to work for 6 weeks. It has been 5 years since our last visit. Gretchen will assist Dr. Billie Swalla on the localization of gene products in ascidian embryos and Charley will work on germinal vesicle breakdown in ascidian oocytes. We will be at the Friday Harbor Labs for most of September and early October. We hope to meet many of you and renew old acquaintances in the near future.
*Ascidian News is not part of the scientific literature and should not be cited as such.
NEWS AND VIEWS
1. It is with a very great deal of sadness that we report the death of Dr. R.H. Millar on 22 May. He was a tireless investigator of ascidians who contributed greatly to many aspects of their lives. Dr. Millar always published using only his initials but his first name was Robin. Dr. Millar received his Ph.D. from the University of Glasgow under the guidance of Prof. C.M. Yonge and worked at the Marine Biological Station in Millport until about 1970, when he moved to the Dunstaffnage Marine Research Laboratory at Oban, Scotland. From the 1940’s to the 1980’s he published numerous papers on many aspects of the lives of ascidians including structure, reproduction, development, systematics and distribution. In 1953 he published his Ph.D. thesis, an invaluable work simply titled “Ciona”, a thorough analysis of the biology of Ciona intestinalis that served many of us going back to our undergraduate days and is still a widely used classic reference. He had an encyclopedic knowledge of numerous taxa and his keen-eyed dissections made his taxonomic monographs particularly useful to all who have followed him. His review “The Biology of Ascidians” (Adv. Mar.Biol. 1971 vol. 9:1-100) summarizes most of what was known about the lives of ascidians up to that time and will certainly reward you to re-read it if only to see how far we have come. We look at this every few years and always find something we did not know or had forgotten. In addition to his work on ascidians Dr. Millar made many contributions to oyster research and was also involved in administration of the Millport and Oban laboratories. Unfortunately, we never had the opportunity to meet him but we did correspond with him occasionally and we both consult his publications frequently. The community of ascidiologists has lost a key contributor and resource and we all mourn his passing.
Dr. Patricia Mather knew Robin Millar very well and sent us this
I knew him as a wonderful colleague, a careful, thorough and objective observer of ascidians, an innate taxonomist and a dear friend. He was a large man in every sense of the word - generous and kind, with a wonderful, dry sense of humour. I met him first in 1950, when, as a graduate student at Plymouth I made a visit to Millport. He took me out dredging in the Clyde, it was a rough day and very uncomfortable and I'll never forget his kindness. We had started working on ascidians at more or less the same time. Robin published his Ciona monograph after he returned from the war, and at about the same time, I had started working on Harold Thompson's collection of Australian ascidians when, as a very young woman, I was appointed to CSIRO Fisheries Division in New South Wales. I missed him so much after he retired from working on ascidians - suddenly I was very alone. A lot of the things that were to occupy a lot of my attention (like the resolution of the clearly polyphyletic Euherdmaniinae) were things that we had discussed, and often subjects that he had raised. I never lacked a response from him during the greater part of the years I worked on the Ascidiacea and I respected his opinion enormously. His principal contribution to the understanding of the Ascidiacea is in their taxonomy, and he had a vast experience of them-from those in his own Scottish waters, to collections from South Africa, the Brazilian coast, Caribbean, Antarctic, western Pacific and the deep water ascidians in the Galathea and the Vema collections. He also leaves with me his concern for the precise use of words- he hated the use of "to" when "or" is intended (such as in "2 or 3"); he intensely disliked the use of the word endemic, when indigenous is intended (thereby leaving me with a life long battle with most of my biological colleagues). He served as deputy Director of the Dunstaffnage Laboratory at Oban for a number of years. After his retirement he excluded ascidians from his interests- Joan, his garden and his water colours occupying him entirely. I was so pleased when he sent me his water colour of a Scottish loch that now hangs on my study wall, where it will remain in memory of a gifted scientist and dear friend who established a precise and scholarly basis for modern studies on the taxonomy of the Ascidiacea.
Further interesting reminiscences of Dr. Millar from Dr. Ivan Goodbody,
Univ. of the West Indies, Kingston, Jamaica:
I was deeply saddened to receive the news that Robin Millar had died. I first met Robin in the early 1950s when he was still at the Millport laboratory. I immediately recognised in him a kindred spirit with enthusaism for marine biology and indeed natural history in general. He quickly introduced me to oysters and induced me to eat them raw as he collected them from the sea. He took me to some coastal rapids (at Loch Fyne, I think), to see large aggregations of Ascidiella aspersa living in the fast flowing water. Around 1968 I found myself in possession of several undescribed species of ascidian from Jamaica but I lacked the experience to describe them unaided. I took them to Oban and sought help from Robin. He was only too willing to share his knowledge and expertise with me and we spent many useful and rewarding days together in his laboratory and in the process formed a lasting friendship. In the paper we wrote afterwards on New Species of Ascidian from the West Indies, all the line drawings were made by Robin. During the day as we worked on the specimens, Robin made sketches with a ball-point pen and next morning would come in with ready-to-publish line drawings. I soon learnt that outside marine biology Robin had two other great interests - art and gardening. He and Joan lived in a small house north of Oban where Robin engaged in these two pursuits surrounded by a lovely garden and with rooms decorated with his paintings. Nevertheless it came as a shock when Robin retired and declared that he was taking no further interest in ascidians but was going home to paint and grow his garden - in both of which he was supremely happy. His large and valuable collection of reprints he donated to the marine laboratory at Millport. I do not think he was ever content working at Dunstaffnage and missed the smaller and more intimate surroundings of the old Millport lab. We have lost a very good friend and a great ascidian taxonomist, but his legacy remains in the papers he published and is there for the next generation's benefit.
2. From Shigeki Fujiwara, Dept. of Biol., Fac. of Sci., Kochi
Univ., Kochi, Japan:
The Japan Ascidian Club (informal meeting of ascidian biology, held at the annual meeting of the Zool. Soc. of Japan) has changed. The new organizers since 2001 are Hiroshi Wada at Seto Marine Biol. Laboratory, Kyoto Univ., Wakayama (email@example.com) and Yutaka Satou at Kyoto Univ. Dept. of Zool., Graduate Sch. of Sci., Kyoto (firstname.lastname@example.org). The name of the meeting is "Hoya no Seibutsugaku Danwa-kai" (Hoya=ascidian(s), no=of, Seibutugaku=biology, Danwa=informal talk, kai=meeting), shortened to Hoya-no-Kai (see AN #37, 1995). The meeting was established more than 15 years ago. It does not require definite membership; everyone who attends the meeting are members that year.
3. Much ado about vanadium: Later in this newsletter there are several contributions by Hitoshi Michibata and his colleagues on vanadium in ascidians. Recently we heard a rumor that Dr. Donald P. Abbott was asked by the war dept. during WWII to work on a project to extract vanadium from tunicates to use in making an atomic bomb. We wondered if there was the slightest shred of truth in that so we asked his widow Izzie Abbott and she replied, “Such a request was made of Don, but he showed them how much vanadium was in the tunicates that took it up, and it was just too small to bother with, and as I remember that was the end of it.”
WORK IN PROGRESS
1. A molecular analysis of ascidian metamorphosis reveals activation
of an innate immune response. Brad Davidson and Billie J.
Swalla, Zool. Dept., Univ. of Washington, Seattle WA. Development (in
Ascidian metamorphosis represents a powerful model for comparative work on chordate development that has remained largely unexplored. We isolated transcripts differentially expressed during metamorphosis in the ascidian Boltenia villosa by suppressive PCR subtractions of staged larval and juvenile cDNAs. We employed a series of three subtractions to dissect gene expression during metamorphosis. We have isolated 132 different protein coding sequences, and 65 of these transcripts show significant matches to Genbank proteins. Some of these genes have putative functions relevant to key metamorphic events including the differentiation of smooth muscle, blood cells, heart tissue, and adult nervous system from larval rudiments. In addition, a significant fraction of the differentially expressed transcripts match identified genes from the innate immune system. Innate immunity confers a rapid response to pathogen-specific molecules and/or compromised self-tissues. The activation of innate immunity genes during metamorphosis may represent the programmed maturation of the adult immune system. In addition, this immune response may be necessary for phagocytosis and re-structuring of larval tissues. An innate immune-related inflammatory response may also underlie two waves of trans-epidermal blood cell migration that occur during the swimming larval period and immediately upon settlement. We characterized these trans-epidermal migrations and discovered that some migratory cells leave the animal entirely through an anterior tunnel in the tunic. We show that these cells are positioned to detect external settlement cues and hypothesize that the innate immune system may also be employed to detect and rapidly respond to environmental settlement cues.
2. Control of maturation of ascidian oocytes. Charles Lambert, Univ. of Washington Friday Harbor Labs, Friday Harbor, WA. Last summer I continued work on oocyte maturation using oocytes of Boltenia villosa. Boltenia oocytes spontaneously undergo germinal vesicle breakdown (GVBD) when dissected into natural pH 8.2 seawater but can be blocked at pH 4. In most chordates GVBD is initiated by the synthesis of cyclin which complexes with a cyclin dependent kinase to form the active maturation promoting factor (MPF). However, in some chordates and many echinoderms this is triggered by the dephosphorylation of a preexisitng MPF comprised of cyclin and a cyclin dependent kinase. This can often be inhibited by cAMP. Curiously, in ophiuroids, nemerteans and brachiopods cAMP actually induces GVBD rather than inhibiting it. Intracellular cAMP levels can be raised by the direct addition of a permeant form of cAMP, stimulation of adenylyl cyclase by forskolin or inhibition of the phosphodiesterase by methyl xanthines such as isobutyl methyl xanthine, hypoxanthine, caffeine or theophylline. In Boltenia oocytes the permeant 8-bromo cAMP activated GVBD in a dose dependent manner 4 mM gave a maximal response. Adenylyl cyclase was activated by forskolin, in a dose dependent manner with concentrations as low as .002 µm giving a maximal response. In addition, inhibition of c AMP phosphodiesterase with theophylline caused GVBD in a dose dependent manner with a maximal response at 0.5 µm, caffiene, hypoxanthine and isobutyl methylxanthine also caused GVBD at very low concentrations. Thus it appears that c-AMP elevation is important in GVBD in ascidian oocytes. Studies are now in progress to determine the role of the inner follicle cells to this process.
3. Gretchen Lambert spends most of her time on pesky squirts these days, identifying collections of mostly nonindigenous species in many parts of the world. The latest trip was a week in Panama with Charley surveying both sides of the canal with a team of 11 taxonomists. Other work includes a huge collection from a number of California sites for the Moss Landing Marine Lab and ongoing identifications for the Smithsonian Environmental Research Center in Maryland. I am also in contact with numerous aquaculturists involved in mussel and oyster culture in eastern Canada, Alaska, New Zealand, Puget Sound in Washington state, and elsewhere because of the overgrowth and smothering of the cultures by huge numbers of ascidians, primarily Ciona intestinalis and Styela clava in eastern Canada and C. intestinalis in New Zealand, and Botrylloides violaceus in the Sitka, Alaska and Puget Sound areas. The newest threat is an unidentified Didemnum sp. in NE U.S. and northern New Zealand, apparently the same species in both locations, whose populations have exploded in the past couple years. If you are plagued by pesky squirts and would like a willing ear to complain to, please send me an email at email@example.com. I am very interested in keeping track of these “outbreaks” worldwide.
1. A morphological and genetic characterization of metamorphosis
in the ascidian Boltenia villosa. Bradley Justin Davidson
Ph.D. dissertation, Dept. of Zoology, Univ. of Washington, Seattle, WA
98195. firstname.lastname@example.org Chair
of the Supervisory Committee: Dr. Billie Swalla.
Since Kowalevsky’s insight into the chordate nature of the ascidian larvae, numerous researchers have focused their efforts on characterizing ascidian development in order to gain insight into chordate evolution. Recent molecular analyses of solitary ascidian embryogenesis have made it clear that the genetic framework for patterning of chordate tissues is highly conserved between ascidians and vertebrates. This remarkable level of genetic homology among chordates allows for the use of ascidian development to elucidate the fundamental genetic networks that underlie more complex vertebrate developmental events. However, the almost exclusive focus on solitary ascidian embryogenesis has led researchers to neglect the majority of ascidian development. Ascidians have a complex life history in which free-swimming larvae settle and metamorphose into sessile adults. Although tail structures are fully differentiated within the larvae, these structures are resorbed upon settlement and make no contribution to the adult body plan. Instead, rudiments within the larval head differentiate to form juvenile organs such as the pharyngeal gill slits, endostyle and heart. In most solitary ascidians, juvenile differentiation occurs only after embryogenesis of the larva is complete. Thus, solitary ascidian post-embryonic development represents a crucial untapped resource for evolutionary and comparative work on chordate development. Here I present an analysis of post-embryonic development in the solitary ascidian Boltenia villosa. I begin with a general introduction to ascidian metamorphosis. Next, I present my work on a morphological and genetic characterization of post-embryonic development in Boltenia. The third chapter focuses on the acquisition of metamorphic competence in Boltenia larvae. In the fourth chapter, I present our characterization of innate immune activation during Boltenia metamorphosis. This includes the up-regulation of a number of innate immune related genes as well as a series of intriguing cell migrations that may be coordinated by immune signaling. The final chapter contains an evolutionary analysis of solitary/ colonial life history transitions, focusing primarily on the ascidians.
2. Ascidiacea (Chordata: Tunicata) from the tropical Brazilian
coast. Tito M. C. Lotufo, Ph.D. Thesis. Dept. of Zool., Instit.
of Biosci., Univ. of São Paulo, Brazil. Advisor Dr. Sergio de Almeida
Rodrigues. Current address Univ. Fed. do Ceará, Centro de
Ciências Agrárias, Dept. de Engenharia de Pesca, C. P. 12168,
60455-760 Fortaleza-CE, Brazil. email@example.com
Although ascidians are well known in many regions of the globe, information about the group on the Brazilian coast is very scanty. Most of the Brazilian coastline is in the tropical region, which is the poorest known. In order to obtain an inventory of ascidian species on the Brazilian tropical coast, surveys were conducted at different points, ranging from the intertidal to shallow subtidal depths. Another goal of the present work was to organize all available information through a revision of bibliography and visits to institutions that held representative collections. 61 visits were conducted in places along the coast of the states of Rio de Janeiro, Espírito Santo, Bahia, Alagoas, Pernambuco, Paraíba, Rio Grande do Norte e Ceará. Specimens were collected, examined and identified to the species level. An extensive taxonomic revision was made for every species, by means of literature as well as examination of types and other specimens deposited in different institutions. The present work includes synonymy lists, descriptions, pictures and remarks for each species studied. Keys for all taxa an every category were also included.
Up to the present work, 90 species of ascidians had been recorded for Brazil, of which 54 are listed for the State of São Paulo. The surveys revealed a total of 67 species, expanding the list to 98 Brazilian species. Those species are distributed in 2 orders and 3 suborders of the class, with a total of 31 genera included in 14 of the 23 families currently accepted. As an immediate result, 9 new records were registered for the Brazilian coast, along with the description of 1 new genus and 10 new species. Furthermore, 8 species have had their taxonomic status altered by synonymy or separation. The present results, together with data from literature, generated tables that were submitted to cluster analysis and a parsimony analysis of endemicity. These analyses revealed a distribution pattern similar to others observed for different benthic taxa. The region studied comprises two provinces, Brazilian Province and Paulista Province.
The full version of this thesis (in Portuguese) will be available in PDF format on the thesis/dissertations digital library (http://www.teses.usp.br).
1. 5th International Larval Biology Meeting, ,Vigo, Spain, Sept 15-20, 2002.
The effect of nutritional stress on solitary and colonial ascidian
juveniles: a comparison of temperate and tropical species. M.W. Jacobs*
and K.M. Sherrard** . *Friday Harbor Labs, Univ. of Washington,
Seattle, WA. **Univ. of Chicago, Chicago, IL. firstname.lastname@example.org
Colonial ascidians produce large, well-differentiated larvae which quickly metamorphose into functional feeding juveniles, while solitary ascidians produce small, simple tadpoles which must undergo an extended period of metamorphosis after settlement before gaining the ability to feed. In both the nutrient-rich, cold water of San Juan Island near Puget Sound, USA, and the relatively nutrient-poor, warm water of the Great Barrier Reef, Australia, colonial ascidians are often dominant competitors for space in fouling communities and on the undersides of coral rubble. We examined the effect of food levels on early growth rates of seven species of tropical and temperate colonial and solitary ascidians. Despite large initial size and and advanced developmental state at settlement, colonial ascidians at San Juan Island were slower than solitary ascidians to respond to food treatment. Preliminary results from the Great Barrier Reef in the tropics indicate the opposite trend: colonial ascidians responded quickly to high food levels with elevated growth rates, while solitary ascidians showed no difference in juvenile size between treatments for the first three weeks. At the temperate site, solitary and colonial species were about equally vulnerable to starvation/food stress, but at the tropical site solitary species were more vulnerable. This variation in the relationship between embryonic provisioning and early juvenile growth and survival is surprising, and causes us to question our assumptions about the selective advantages and disadvantages of having large or small eggs. A larger sample of colonial species is required to determine whether the observed differences in response to food regime are related to environmental differences between sites or due simply to variation between species.
2. 48th meeting of the Italian Embryological Group, Grottammare, Italy June 4-7, 2002.
Effects of antagonists of 5-HT1A receptor on ascidian
embryos development. F. De Bernardi, , S. Gropelli, R. Pennati,
U. Fascio*, C. Sotgia. Dpto. di Biol.,*Centro Interdipartimentale
di Microscopia Avanzata, Univ. di Milano, Milano, Italy. email@example.com
Ascidian embryos form a tubular CNS in a similar way to that of vertebrates, by folding of the neural plate. At least four different regions are distinguishable by morphology and cell composition from rostral to caudal: the anterior sensory vesicle, containing the two pigmented sensory organs, the otolith and the ocellus; the neck; the visceral ganglion and the spinal cord (Meinertzhagen and Okamura, 2001, Trends Neurosci. 24: 101-410). There are evidences that early embryos, at cleavage and gastrulation stage, use 5-HT, together with GABA and acetylcholine, to regulate cell division and morphogenetic cell movements (Buznikov et al., 2001, Cell Tissue Res. 305: 177-186). At this moment serotonin was detected in ascidian only from swimming larva onward, but there are suggestions about the presence of serotonin and of its multiple receptors in earlier embryonic stages (Pennati et al., 2001, Develop. Growth Differ.43:647-656)
Embryos of the ascidian Phallusia mammillata has been treated for three hours with three different 5-HT1A antagonists: pindolol, spiperone and WAY-100635 (N[2-[4-(2-Methoxyphenyl)-1-piperazinyl]ethyl]-N-pyridinyl-cycloexane- carboxamide maleate) at concentrations from 0.1 to 20 µM in Millipore filtered sea water (MFSW) starting at different developmental stages When control reached the swimming larva stage , about 18 hours after fertilization, all treated and control specimens were collected and processed for immunofluorescence and histology. Among the tested antagonist, only WAY-100635 caused malformations, in a dose dependent manner, in larvae developed from treated embryos, but the incidence and the type of malformations caused by the treatment at different stages, from gastrula to early tailbud stage, were not significantly different. The larvae showed a shorter trunk region: the adhesive papillae were fused to form a single structure, were displaced more dorsally than in controls and AChE-positive cells were lacking. All the larvae showed a dramatic reduction of the anterior sensory vesicle and of the pigment of the two sensory organs: the ocellus and the otolith. Immunofluorescence experiments with an anti-tubulin monoclonal antibody specific for the neural system showed that the most anterior part of the nervous system, anterior to the visceral ganglion, was affected in a dose-dependent way. At the higher dose, it was not possible to localize the primary sensory neurons of the adhesive papillae and the papillary nerves. Moreover the neural fibers that in the control larvae connect the sensory organs to the visceral ganglion were absent or hardly detectable and also part of the anterior sensory vesicle appeared reduced or absent. On the other hand, the tail appeared always normal, with a normal spinal cord running along.
In order to understand if the reduction of the sensory vesicle was due to a failure in differentiation or to a defect in cell proliferation, we performed nuclear staining with DAPI: the counting of cell nuclei gave an average of 992 nuclei in the trunk region of the control larvae, whereas an average of 402 in the treated larvae. This suggests that the reduction of the anterior sensory vesicle can be partially due to an alteration of cell proliferation. However, the absence of papillary nerves in the larvae developed from treated embryos would also suggest that the terminal neural differentiation may be affected by the drug. A small number of larvae metamorphosed in a rather normal way: the juveniles showed a smaller oral siphon, with altered innervation. Taken together, these results suggest that 5-HT plays a role in the development of the neural system in ascidians and receptors similar to the members of 5-HT1A receptor subtype of mammals mediate the development of the most anterior nervous system of the larvae.
3. Annual meeting of the Amer. Soc. for Cell Biology, Washington, DC, 8-12 Dec. 2001.
Do D-3 phosphoinositides signal actin polymerization during ascidian
sperm activation? Lamar Blackwell, Dave Bolger, Mohammad Hanizavareh,
Emily Zebadua, and Robert A. Koch. Calif. State Univ. Fullerton,
Dept. of Biol. Sci., Fullerton, CA. firstname.lastname@example.org
Sperm activation in the sea squirt Ascidia ceratodes is characterized by mitochondrial translocation (MTL), an actin:myosin-dependent movement known to require elevation of both intracellular pH (pHi) and free calcium ion concentration ([Ca]i). Previously, we have shown that myosin activation requires a G protein-mediated pathway involving inositol 1,4,5-trisphosphate-mediated internal Ca release and a PKC-dependent internal alkalization that precedes external Ca entry. Here, we explore signaling elements that are involved in triggering actin polymerization. In MTL assays, the actin polymerization inhibitor latrunculin (10µM) completely blocked high pH artificial sea water (ASW)-induced sperm activation (positive control), and the actin polymerization inducer jasplakinolide (7.4µM) stimulated sperm activation equal to positive controls, an action blocked by latrunculin. Dual labeling with fluorescently tagged phalloidin and DNaseI revealed that filamentous actin was distributed most heavily on the mitochondrion whereas monomeric actin was also found along the length of the tail. Sperm activation appears to increase filamentous actin on the mitochondrion. In MTL assays, the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 (50µM) blocked sperm activation induced by pH 9.4 ASW but not that induced by the G protein activator mas7 (3.5µM) or the PKC activator OAG (50µM), agents shown to be part of the myosin activation pathway. Liposomes that incorporated phosphatidylinositol 3,4,5-trisphosphate (PIP3) stimulated levels of sperm activation similar to positive controls. Indirect immunofluorescence using anti-profilin antibodies showed profilin to be present on the mitochondrion, providing a possible connection between PI3K-induced PIP3 production and actin polymerization. (Funded by CSUF University Student Research Initiative grant to MH; NIH R25-GM56820 to RAK for LB, DB & EZ; NIH R15HD36500 to RAK.) Molec. Biol. Cell 12:117a.
4. Programme IV "Anton Dohrn" Workshop New Perspectives in Tunicate
Biology, Stazione Zoologica, Punta San Pietro, Ischia Sept. 29-Oct. 2,
Aiming to draw the entire picture of the accumulation of vanadium in ascidians. H. Michibata, Marine Biol. Lab., Grad. Sch. Sci., Hiroshima Univ., Hiroshima 722-0073, Japan. email@example.com (no abstracts)
5. The 2002 Gordon Research Conference on Marine Natural Products
Chemistry, Ventura, California, Feb. 24 - March 1, 2002.
The unusual mechanism of accumulation and reduction of vanadium by ascidians. H. Michibata, Marine Biol. Lab., Grad. Sch. Sci., Hiroshima Univ., Hiroshima, Japan (no abstracts)
6. The 3rd Intl. Symp. on Chemistry & Biol. Chemistry of Vanadium. Osaka Univ., Osaka, Japan. Nov 26-29, 2002.
a) Molecular biological approaches to the accumulation and reduction
of vanadium by ascidians. H. Michibata, Marine Biol. Lab., Grad. Sch.
Sci., Hiroshima Univ., Hiroshima, Japan
About 90 years ago, M. Henze discovered high levels of vanadium in the blood (coelomic) cells of an ascidian collected from the Bay of Naples (Henze, 1911). His discovery attracted the interdisciplinary attention of chemists, physiologists, and biochemists. Two decades ago, we quantified the vanadium levels in several ascidian tissues definitively using neutron-activation analysis and revealed that some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, corresponding to about 107 times that found in seawater. Vanadium accumulated is reduced to the +3 oxidation state via the +4 oxidation state and stored in vacuoles of vanadocytes (vanadium-containing blood cells) where high levels of protons and sulfate are also contained. To investigate this unusual phenomenon, we isolated several proteins and genes that are expressed in vanadocytes. To date, three types of vanadium-binding protein, designated as Vanabins, have been isolated, with molecular masses of 12.5, 15, and 16 kDa, along with the cDNAs encoding these proteins. In addition, four types of enzyme related to the pentose phosphate pathway that produces NADPH were revealed to be located in vanadocytes. The pentose phosphate pathway participates in the reduction of vanadium(V) to vanadium(IV). The cDNA for each of the vacuolar-type H+-ATPase (V-ATPase) A, B, C, and D subunits, which are located on the vacuolar membranes of vanadocytes, has been isolated and analyzed. V-ATPase generates a proton-motive force, and is thought to provide the energy for vanadium accumulation.
To clarify the entire mechanism involved in the accumulation and reduction, many more genes and proteins expressed in the blood cells needed to be systematically identified. Thus, we performed an expressed sequence tag (EST) analysis of blood cells and obtained 300 cDNA clones from a blood cell library. We have, furthermore, isolated and cloned cDNA of ascidian Nramp (natural resistance-associated macrophage protein), known to transport several heavy metals, from the blood cells. Now, we are examining whether the cultured CHO-K1 cell line overexpressed a fusion protein of ascidian Nramp accumulates vanadium.
b) Analysis of metal-binding activity of vanadium-binding proteins
(Vanabins) of an ascidian Ascidia sydneiensis samea. T.
Ueki*, T. Uyama and H. Michibata, Marine Biol. Lab., Grad. Sch. Sci., Hiroshima
Univ., Hiroshima 722-0073, Japan *firstname.lastname@example.org
Ascidians have been known to accumulate high levels of vanadium ion in the vacuole of one or more type(s) of blood cells. From the signet ring cells, which are vanadium-accumulating cells, of Ascidia sydneiensis samea we previously identified several low molecular weight vanadium-binding proteins, Vanabins, and cloned cDNAs for 12.5 kDa and 15 kDa Vanabins. In the present experiment, we constructed plasmids expressing recombinant Vanabins in E. coli. We have examined the activities of the recombinant Vanabins to bind metal ions including vanadium (IV) and vanadium (V) by Hummel-Dreyer's method. As a result, 15 kDa Vanabin bound to approximately 20 vanadium (IV) or 20 vanadium (V) ions in 10mM HEPES buffer at pH 7.2 containing 20 mM NaCl. In the same buffer, 15 kDa Vanabin bound to 5 copper (II) ions, but not to iron (III) ions. In 20 mM sodium phosphate buffer at pH 7.2, 15 kDa Vanabin bound one vanadium (V) ion while it bound to 7 vanadium (IV) ions. This may be due to the similarity of the structure of phosphate and vanadate ions.
c) Hunting for vanadium-binding proteins from vanadium-accumulating
ascidian by metal-chelating column. M. Yoshinaga*, M. Aoshima,
T. Watanabe, N. Yamaguchi, T. Ueki, T. Uyama and H. Michibata, Marine Biol.
Lab., Grad. Sch. Sci., Hiroshima Univ., Hiroshima, Mukaishima 2445, Hiroshima
722-0073, Japan. * email@example.com
Ascidians accumulate high levels of vanadium in their vacuoles of vanadocytes from sea water. We have extracted vanadium-binding proteins, designated as Vanabin, from the cytoplasm of vanadocytes, cloned cDNAs encoding the proteins and prepared recombinant proteins. The analysis of their metal binding activity is in progress. In order to solve the ultimate mechanism of this unusual phenomenon, many more proteins, that are associated with the phenomenon, need to be systematically identified. The present experiment was newly planned to isolate exclusively vanadium-binding proteins not only from the vanadocytes but also from the other organs of the ascidian, Ascidia sydneiensis samea, using a metal-chelating column coupled with imidiaceticacid to which vanadium(IV) was immobilized. Soluble proteins of blood cells, plasma membrane proteins and coelomic serum proteins were submitted to the present experiments. As a result, several Vanabin-like proteins were identified in the soluble fraction. Several proteins were also hunted by the metal-chelating column when plasma membrane proteins solubilized by urea-containing buffer were applied to the column. Among them, 4.5 kDa protein sequence almost matched with that obtained by EST analysis of a cDNA library derived from vanadocytes. The predicted protein was a novel protein composed of 43 amino acids, including 4 histidine residues. Further, several vanadium-associated proteins have been extracted from the coelomic serum, A main component of the proteins is 14 kDa protein having basic amino acid-rich in N-terminal amino acids. We are now trying to determine inner amino acid sequence of this protein to examine their metal binding activity by constructing recombinant proteins.
d) Observation of vanadocytes of ascidians by an x-ray microscope
using synchrotron radiation. K. Takemoto1*,T. Ueki2, B. Fayard3,
M. Salome3, J. Susini3, A. Yamamoto1, H. Kihara1, S. Scippa4 and H. Michibata2.
1Dept. Physics, Kansai Medical Univ., Uyamahigashi 18-89, Hirakata, Osaka
573-1136, Japan. 2Marine Biol. Lab., Grad. Sch. Sci., Hiroshima Univ.,
Hiroshima 722-0073, Japan. 3ESRF, BP220, 38043 Grenoble cedux, France.
4Dept. Genet. Gener. Mol. Biol., Fac. Sci., Univ. Naples, 80134 Naples,
Italy. * firstname.lastname@example.org
Several species of ascidians accumulate vanadium in one or more type(s) of blood cells (coelomic cells), such as signet ring cells and some other vacuolated cells. We applied a scanning X-ray microscope at ID21 of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) to the observation of living blood cells of a Mediterranean ascidian species Phallusia mammillata and a Japanese species Ascidia sydneiensis samea. The vanadium-accumulating cells (vanadocytes) of these species contain 60mM and 13 mM vanadium in their vacuoles, respectively. The high levels of vanadium are therefore expected to give a high contrast by X-ray microscopy. Using a scanning transmission X-ray microscopy equipped with ESRF, we succeeded not only in observing living blood cells in a sealed spatial sample holder at 1 micron resolution but also in visualizing of vanadium in signet ring cells of both species and vacuolated amoebocytes of Phallusia mammillata using a x-ray fluorescence probe. Morula cells which had been thought to be vanadium-accumulating cells of both species and type-I compartment cells of Phallusia mammillata did not contain vanadium. These results are regarded as the first epoch-making one as a direct observation of vanadium in living cells.
e) Expressed sequence tag analysis of blood cells in the vanadium-rich
ascidian, Ascidia sydneiensis samea. N. Yamaguchi*, T.
Ueki, T. Uyama and H. Michibata, Mar. Biol. Lab., Graduate Sch. of Sci.,
Hiroshima Univ., Mukaishima-cho 2445, Hiroshima 722-0073, Japan *
Some species in the family Ascidiidae accumulate vanadium at concentrations in excess of 350 mM, which corresponds to about 107 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 127 of the clones (42.3%) were known genes, and 173 of the clones (57.7%) 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 seven types of clones; these consisted of genes related to nuclear proteins (21 clones), signal transduction (18 clones), the cytoskeleton (17 clones), metal ion transport or the redox of metals (16 clones), energy conversion (8 clones), ribosomal proteins (6 clones), and other proteins (41 clones). The H-subunit of ferritin 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 Fe2+, also works as a ligand for VO2+.
f) Detection of metal transport activity in cultured CHO-K1 cell-line,
which expressed AsNramp-GFP fusion protein. T. Uyama*, T. Ueki,
and H. Michibata, Marine Biol. Lab., Grad. Sch. Sci., Hiroshima Univ.,
Hiroshima., Mukaishima 2445, Hiroshima 722-0073, Japan. *email@example.com
Ascidians belonging to Ascidiacea store vanadium ion in vacuoles of vanadocytes, vanadium-containing blood cells. The concentration of vanadium attains 350mM, which is 107 times higher than that in sea water. This is thought to be the highest levels of metal concentration in any living organism. Nramp (natural resistance-associated macrophage protein) family is known to transport several heavy metals including including Fe2+, Zn2+, Mn2+, Co2+, Cd2+, Cu2+, Ni2+, and Pb2+ and is highly conserved among mammals, nematodes, yeast, and bacteria. As a first step to search for vanadium transporter in ascidians, we examined whether Nramp homolog is expressed in the vanadocytes of the vanadium-rich ascidian, Ascidia sydneiensis samea. As a result, we isolated and cloned cDNA of Nramp from the blood cells. In situ hybridization showed that ascidian Nramp homolog, named AsNramp, was expressed in the vanadocytes exclusively. To examine whether AsNramp acts as a proton-coupled vanadium transporter in ascidians, we constructed a plasmid expressing a fusion protein of AsNramp and a green fluorescent protein (GFP) under the control of cytomegalovirus(CMV) promoter. The fusion protein is overexpressed in cultured CHO-K1 cell line.
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