Starfish Asterina Pectinifera Research Articles

The Starfish Asterina pectinifera: Collection and Maintenance of Adults and Rearing and Metamorphosis of Larvae.

Here we describe methods for (a) collecting starfish during their breeding period; (b) maintaining adults with fully grown gonads in laboratory aquaria; (c) rearing fertilized eggs to brachiolaria larvae, and (d) inducing larvae to metamorphose into juveniles under laboratory conditions. Such protocols should facilitate various analyses of starfish development throughout the entire life cycle of these model organisms.

Mass Species of the Sea Star Asterina Pectinifera as a Potential Object of Mariculture

Starfish Asterina (= Patiria) pectinifera) is widely distributed along the western coast of the Pacific Ocean from Sakhalin Island to the Yellow Sea. This is the most numerous species in the Peter the Great Bay of the Sea of Japan. Asterina tissues in large quantities contain a mixture of carotenoids, the most famous of which is astaxanthin. The developed technology of complex processing of A. pectinifera starfish allows to obtain biologically active collagen peptides and carotenoid preparations enriched with astaxanthin, exhibiting immunomodulatory, anti-inflammatory and antioxidant activities. They can be used as a raw material for obtaining new medicinal, cosmetic and food products.

Characterization of a novel hatching enzyme purified from starfish Asterina pectinifera.

Hatching enzyme is a protease which can degrade the membrane of egg. In this study, a hatching enzyme was purified from starfish (Asterina pectinifera) with 6.34 fold of purification rate, 5.04 % of yield, and 73.87 U/mg of specific activity. The molecular weight of starfish hatching enzyme was 86 kDa, which was reduced to 62 kDa after removal of N-linked oligosaccharides. The optimal pH and temperature of the hatching enzyme activity were pH 7.0 and 40 °C, respectively, while those of stability were pH 8 and 20 °C. The kinetic parameters, V max, K m, Kcat and K cat /K m values were 0.197 U/ml, 0.289 mg/ml, 112.57 s−1, and 389.52 ml/mg s, respectively. Zn2+ increased the enzyme activity by 167.28 %, while EDTA, TPCK, TGCK, leupeptin, PMSF, and TLCK decreased. In addition, Ca2+, Mg2+, and Cu2+ did not affect the enzyme activity. The starfish hatching enzyme activity pretreated with EDTA was recovered by Zn2+. Therefore, the starfish hatching enzyme was classified as a serine-zinc protease.

The Total Synthesis of Starfish Ganglioside GP3 Bearing a Unique Sialyl Glycan Architecture.

The total synthesis of ganglioside GP3, which is found in the starfish Asterina pectinifera, has been accomplished through stereoselective and effective glycosylation reactions. The sialic acid embedded octasaccharide moiety of the target compound was constructed by [4+4] convergent coupling. A tetrasaccharyl donor and acceptor that contained internal sialic acid residues were synthesized with an orthogonally protected N-Troc sialic acid donor as the key common synthetic unit, and they underwent highly stereoselective glycosidation. The resulting sialosides were subsequently transformed into reactive glycosyl acceptors. [4+4] coupling furnished the octasaccharide framework in 91 % yield as a single stereoisomer. Final conjugation of the octasaccharyl donor and glucosyl ceramide acceptor produced the protected target compound in high yield, which underwent global deprotection to successfully deliver ganglioside GP3.

Relaxin-like gonad-stimulating peptide is highly conserved in starfish Asterina pectinifera

Relaxin-like gonad-stimulating peptide (RGP) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Recently, RGP was purified from the radial nerves of starfish Asterina pectinifera, which belongs to the Order Valvatida in the Class Asteroidea. A. pectinifera is an endemic Japanese species, inhabiting rocky shores from northern to southern Japanese waters. This study examined whether genetic variation or polymorphism is found in RGP. Comparing cDNA sequences of RGP in A. pectinifera from 10 local populations in Japanese waters, we found that the coding DNA sequences (CDSs) were exactly the same. This result indicated that RGP is a highly conserved peptide in A. pectinifera. Furthermore, the CDS of RGP identified in Certonardoa semiregularis, which also belongs to Order Valvatida, was completely consistent with that of A. pectinifera. Thus, this also suggested that the chemical structure of A. pectinifera RGP is conserved among starfish of the Order Valvatida beyond species.

A gonad-stimulating peptide of the crown-of-thorns starfish, Acanthaster planci

The crown-of thorns starfish, Acanthaster planci, has been blamed for coral mortality in a large number of coral reef systems in the Indo-Pacific region. Because population outbreaks of A. planci are closely related to reproduction, it is important to examine the mechanism of reproductive control in this starfish. Previously, a relaxin-like gonad-stimulating peptide (RGP) in starfish Asterina pectinifera has been identified as the gonadotropin responsible for final gamete maturation. On the basis of homology research on RGP cDNAs from several species, this study was carried out to identify gonadotropin in A. planci. The cDNA sequence of RGP was determined using a RACE product of mRNA from the radial nerves of A. planci. The coding DNA sequence consisted of 351 base pairs with an open reading frame encoding a peptide of 116 amino acids (aa), including a signal peptide (29 aa), B-chain (19 aa), C-peptide (44 aa), and A-chain (24 aa). The chemical structure of A. planci RGP was exactly the same as that of A. pectinifera RGP. Furthermore, synthetic RGP could induce gamete spawning and oocyte maturation in the ovarian fragments of A. planci. This strongly suggested that the RGP is a gonadotropin in A. planci.

Mesh selectivity of the drum–shaped pot for starfish Asterina pectinifera in the western coastal waters of Korea

Mesh selectivity of the drum–shaped pot for starfish Asterina pectinifera in the western coastal waters of Korea

Nucleotide sequence and expression of relaxin-like gonad-stimulating peptide gene in starfish Asterina pectinifera

Starfish gonad-stimulating substance (GSS) is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Because GSS belongs to the relaxin-like peptide family, we propose renaming for starfish gonadotropic hormone as relaxin-like gonad-stimulating peptide (RGP). This study examined the primary structure and expression regulation of the RGP gene in starfish Asterina pectinifera. RGP consisted of 3896 base pairs (bp) divided over two exons, exon 1 of 208bp and exon 2 of 2277bp, and one intron of 1411bp. Promoter sequences, CAAT and TATA boxes, were present in the 5′-upstream region of the coding DNA sequence of RGP. The transcript was 2485 bases (b) in length. The AAUAAA polyadenylation signal was found in 3′-untranslated region over 2kb away from the stop codon. This showed that only 14% of the RGP mRNA was translated into the peptide, because a size of the open-reading frame was 351 b. Furthermore, an analysis by using real-time quantitative PCR with specific primers for RGP showed that mRNA of RGP was expressed at high levels in the radial nerves. Expression was also observed in the cardiac stomachs, although the level was low, and trace levels were detected in the gonads, pyloric caeca and tube feet. This result suggests that the RGP gene is transcribed mainly in the radial nerves of A. pectinifera.

A new relaxin-like gonad-stimulating peptide identified in the starfish Asterias amurensis

Relaxin-like gonad-stimulating peptide (RGP) of starfish Asterina pectinifera was the first invertebrate gonadotropin to have its chemical structure identified. However, it is unclear whether gonadotropic hormones in other species starfish are relaxin-like peptides. Thus, this study tried to identify the molecular structure of gonadotropic hormone in Asterias amurensis. As a result, we identified A. amurensis gonadotropic hormone as the RGP (AamRGP). The DNA sequence encoding AamRGP consisted of 330 base pairs with an open reading frame encoding a peptide of 109 amino acids (aa), including a signal peptide (26 aa), B-chain (20 aa), C-peptide (38 aa) and A-chain (25 aa). Comparing with A. pectinifera RGP (ApeRGP), the amino acid identity levels between AmaRGP and ApeRGP were 58% for the A-chain and 73% for the B-chain. Furthermore, chemical synthetic AamRGP induced gamete spawning and oocyte maturation in ovarian fragments of A. amurensis. In contrast, the ovary of A. pectinifera failed to respond to the AamRGP. This suggested that AamRGP is a new relaxin-like peptide.

별불가사리 Asterina pectinifera의 유문맹낭 추출물로부터 새로운 2종류의 항균활성 펩타이드의 정제

PAP-1, a novel antimicrobial peptide isolated from pyloric caeca extract of the starfish Asterina pectinifera was purified and characterized. First, the acidified pyloric caeca extract was put through Sep-Pak C18 solid phase extraction cartridge using a stepwise gradient. Among the eluents, RM 60 (retained materials at 60% methanol) showed good antimicrobial activity against Bacillus subtilis and Escherichia coli D31 and was purified in C18 reversed-phase and ion-exchange high-performance liquid chromatography columns. The purification steps yielded two novel peptides showing strong antimicrobial activities. These peptides were named pyloric caeca A. pectinifera peptide 1 and 2 (PAP-1 and PAP-2). For the characterization of the purified peptides, the molecular weights and amino acid sequences were determined by MALDI-TOF MS and Edman degradation. The molecular weights of PAP-1 and PAP-2 were about 2951.54 Da and 2980.15 Da respectively. The amino acid sequences of PAP-1 and PAP-2 were partially determined: AIQNAGES and AIQNAAES, respectively. PAP-2 is an isoform of PAP-1, differing merely by a single residue at position 6 (glycine or alanine). The comparison of the N-terminal amino acid sequences and molecular weights of the peptides with those of other known antimicrobial peptides revealed that PAP-1 and PAP-2 have no homology with any known peptides. These findings suggest that PAP-1 and PAP-2 play a significant role in the innate defense system of starfish pyloric caeca.

Involvement of Gαs-proteins in the action of relaxin-like gonad-stimulating substance on starfish ovarian follicle cells

Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. In breeding season (stage V), GSS stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) by ovarian follicle cells. The hormonal action of GSS is mediated through the activation of its receptor, G-proteins and adenylyl cyclase. It has been reported that GSS fails to induce 1-MeAde and cyclic AMP (cAMP) production in follicle cells of ovaries during oogenesis (stage IV). This study examined the regulatory mechanism how ovarian follicle cells acquire the potential to respond to GSS by producing 1-MeAde and cAMP. Because the failure of GSS action was due to G-proteins of follicle cells, the molecular structures of Gαs, Gαi, Gαq and Gβ were identified in follicle cells of starfish Asterina pectinifera. The cDNA sequences of Gαs, Gαi, Gαq and Gβ consisted of ORFs encoding 379, 354, 353 and 353 amino acids. The expression levels of Gαs were extremely low in follicle cells at stage IV, whereas the mRNA levels increased markedly in stage V. On contrary, the mRNA levels of Gαi were almost constant regardless of stage IV and V. These findings strongly suggest that de novo synthesis of Gαs-proteins is contributed to the action of GSS on follicle cells to produce 1-MeAde and cAMP.

별 불가사리(Asterina pectinifera) 조직별 초산추출물의 생리활성 탐색

The present study was performed to examine the contraction and relaxation responses of the smooth muscles, and search for antimicrobial and antioxidant activities in the tissues, of the starfish Asterina pectinifera. Frozen samples were extracted with distilled water containing 1% acetic acid. Extracts from all tissues showed potent antimicrobial activity against Escherichia coli D31. Relatively high levels of antimicrobial activity were also detected in the body extracts. Liver, tube feet, and body extracts caused contraction responses in the dorsal retractor muscles (DRM) of the starfish. In contrast, all tissues examined exhibited contractile activity in the esophagus of squid Todarodes pacificus. In addition, liver and gonad extracts caused contraction responses upon application to the intestine of the puffer fish Takifugu pardalis. Relaxation effects on the DRM of starfish were identified in most of the extracts, while no relaxant activity was detected in body extracts. Extracts from all tissues examined also exhibited antioxidant activities. The results of this study suggest that starfish are a potential source of novel bioactive compounds.

아무르 불가사리(Asterias amurensis)의 관족으로부터 신경성 펩타이드의 분리 및 정제

Two neuropeptides were purified from the acidified tube feet extract of the starfish Asterias amurensis by C18 reversed phase and size-exclusion high-performance liquid chromatography (HPLC). The tube feet extract and the purified peptides (AST-I and AST-II) showed potent contractile activity on dorsal retractor muscle (DRM) of the starfish Asterina pectinifera and intestine (smooth muscle) of the panther puffer Takifugu pardalis. Treatment of the purified peptides with dithiothreitol (DTT) for 60 min at <TEX>$37^{\circ}C$</TEX> significantly altered their retention times, suggesting that these compounds contained disulfide bonds. The molecular weights of AST-I and AST-II were determined to be 2064.2 Da and 6137.2 Da, respectively, by MALDI-TOF mass spectrometry.

Release of Relaxin-Like Gonad-Stimulating Substance from Starfish Radial Nerves by lonomycin

In starfish, the peptide hormone gonad-stimulating substance (GSS) secreted from nervous tissue stimulates oocyte maturation to induce 1-methyladenine (1-MeAde) production by ovarian follicle cells. Recently, GSS was purified from radial nerves of the starfish Asterina pectinifera and identified as a relaxin-like peptide. This study examines the mechanism of GSS secretion from radial nerves. When radial nerves isolated from A. pectinifera were incubated in artificial seawater containing ionomycin as a calcium ionophore, GSS release increased in a dose-dependent manner; 50% activity of GSS release was obtained with approximately 10 µM ionomycin. Another calcium ionophore, A23187, also stimulated GSS release from radial nerves. In contrast, membrane permeable cyclic AMP and cyclic GMP analogs failed to induce GSS release. These results suggest that GSS secretion is induced by intracellular Ca(2+) as a second messenger.

Three new steroid glycosides from the starfish Asterina pectinifera

Three new steroid glycosides, pectiniosides H–J (1–3), were isolated along with three known compounds (4–6) including a steroid glycoside and two polyhydroxysteroids, from the alcoholic extract of the starfish Asterina pectinifera. The structures of 1–3 were determined by extensive NMR and HR-ESI-MS experiments. Compounds 1–4 did not show cytostatic activity on HL-60 cells below 100 μM, while compounds 5–6 showed moderate cytostatic activity, with IG50 values of 80.3 and 40.5 μM, respectively.

The Mos-MAPK pathway regulates Diaphanous-related formin activity to drive cleavage furrow closure during polar body emission in starfish oocytes

Maintenance of spindle attachment to the cortex and formation of the cleavage furrow around the protruded spindle are essential for polar body extrusion (PBE) during meiotic maturation of oocytes. Although spindle movement to the cortex has been well-studied, how the spindle is maintained at the cortex during PBE is unknown. Here, we show that activation of Diaphanous-related formin mediated by mitogen-activated protein kinase (MAPK) is required for tight spindle attachment to the cortex and cleavage furrow closure during PBE in starfish (Asterina pectinifera) oocytes. A. pectinifera Diaphanous-related formin (ApDia) had a distinct localization in immature oocytes and was localized to the cleavage furrow during PBE. Inhibition of the Mos-MAPK pathway or the actin nucleating activity of formin homology 2 domain prevented cleavage furrow closure and resulted in PBE failure. In MEK/MAPK-inhibited oocytes, activation of ApDia by relief of its intramolecular inhibition restored PBE. In summary, this study elucidates a link between the Mos-MAPK pathway and Diaphanous-related formins, that is responsible for maintaining tight spindle attachment to the cortex and cleavage furrow closure during PBE.

Trophic structure in natural habitats of the abalone Haliotis discus hannai with distinct algal vegetation of kelp and crustose coralline algae: implication of ontogenetic niche shifts

The abundance, species composition, and stable isotope ratios of benthic organisms were investigated to determine the trophic structures in abalone (Haliotis discus hannai) habitats, which are characterized by contrasting vegetation of crustose coralline algae (CCA) and kelp beds. A size–frequency analysis revealed that juvenile abalones with shell lengths (SLs) smaller than ~30 mm primarily inhabited CCA beds, whereas adults were abundant in kelp beds. Stable isotope analyses indicated that CCA beds were composed of a single food chain, whereas kelp beds supported multiple food chains. The abalone were divided into three size groups to estimate potential species interactions during their ontogeny. A small gastropod, Homalopoma sangarense, was the most abundant species, but is suspected to be less competitive with abalone, especially in CCA beds. An abundant starfish Asterina pectinifera appeared to function as a potential predator of juvenile abalones in both CCA and kelp beds. We concluded that CCA beds are essential for immediate post-settlement processes of abalones, whereas kelp beds are more important for providing refuge and food sources for adult abalones. The present study highlights that ontogenetic niche shifting can be a successful life-history strategy to sustain the abalone population in a subtidal rocky shore ecosystem.

Molecular Cloning, Expression, and Characterization of Starfish DNA (Cytosine-5)-methyltransferases

To determine whether and if so how a DNA methylation-dependent epigenetic mechanism for transcriptional gene silencing functions in Echinoderms, we cloned and sequenced dnmt1 and dnmt3 cDNAs of the starfish Asterina pectinifera. Since the Strongylocentrotus purpuratus genome has only two loci of DNA (cytosine-5)-methyltransferase genes encoding Dnmt1 and Dnmt3, they might constitute a sufficient set of dnmt genes in Echinoderms. The starfish Dnmt3 whose cDNA we cloned showed highest homology to a mammalian Dnmt3a2 splicing variant. Essentially all the characteristic motifs and sequences of the mammalian counterparts were found in the starfish Dnmts as well, except that a typical PCNA binding domain motif was lacking in the starfish Dnmt1. RT-PCR analysis indicated that the dnmt1 mRNA exists in both ovary and oocytes, but its levels in other tissues were very low or almost negligible. In contrast, the dnmt3 mRNA was detected only in the ovary, and not at all in the oocytes. The size of a dnmt1 transcript was about 6.5 kb on Northern blot analysis. On heterologous expression, the starfish Dnmt1 protein was expressed in insect cells in catalytically active form.

Participation of Gs-proteins in the action of relaxin-like gonad-stimulating substance (GSS) for 1-methyladenine production in starfish ovarian follicle cells

Gonad-stimulating substance (GSS) in starfish is the only known invertebrate peptide hormone responsible for final gamete maturation, rendering it functionally analogous to gonadotropins in vertebrates. Recently, we purified GSS from radial nerves in the starfish Asterina pectinifera and identified the chemical structure as a heterodimer composed of two different peptides (A- and B-chain) with disulfide cross-linkages. This study examined the hormonal action of GSS on ovarian follicle cells obtained from ovaries in growing (stage IV) and fully grown (stage V) stages, and particularly the mode of signal transduction. The action of GSS on 1-MeAde production by follicle cells in stage V was mediated through the production of cAMP. In contrast, GSS failed to induce 1-MeAde and cAMP production by follicle cells in stage IV. According to competitive experiments using radioiodinated and radioinert GSS, highly specific binding was observed in follicle cells, though their affinities and numbers in stage IV were inferior to those in stage V. Interestingly, Gsα was not detected immunologically in follicle cell membranes of stage IV. Gβ was also faint in stage IV. Although adenylyl cyclase activity in stage V was dose-dependently activated by GSS in the presence of GTP, neither GSS in the presence of GTP nor nonhydrolyzable GTP analogs were effective on the activity in stage IV. These findings strongly suggest that the failure of GSS to produce 1-MeAde is because of a lack of Gs-proteins in follicle cells at stage IV.

Interaction of Relaxin-Like Gonad-Stimulating Substance with Ovarian Follicle Cells of the StarfishAsterina pectinifera

Previously, gonad-stimulating substance (GSS), which acts as a gonadotropin, was purified from radial nerves of the starfish Asterina pectinifera and its structure was elucidated. Here, the interaction of GSS with receptors was examined in ovarian follicle cells, a target of GSS. In competitive experiments using radioiodinated and radioinert GSS, highly specific binding was observed in the microsomal/plasma membrane fraction of follicle cells. GSS scarcely bound in the cytosolic fraction. Scatchard plots showed the numbers of binding sites (NBS) in whole homogenate and the crude membrane to be 1.65 and 3.42 pmoles/mg protein, respectively. Dissociation constant (K (d)) values in these two preparations were almost the same at about 0.6-0.7 nM. Furthermore, it was shown that GSS stimulated adenylyl cyclase activity in follicle cell membranes in a dose-dependent manner that required GTP. Immunoblotting with specific antibodies for G-protein subunits after SDS-PAGE of the membrane preparation showed both stimulatory (Gs) and inhibitory (Gi) regulatory α-subunits for adenylyl cyclase and a β-subunit. The results strongly suggest that GSS interacts with G-protein-coupled receptors (GPCR) located in the follicle cell membrane to stimulate Gs-protein and adenylyl cyclase activity.