Isolation and characteristics of trypsin from pyloric ceca of the starfish Asterina pectinifera

Trypsin was purified from pyloric ceca of the starfish Asterina Pectinifera by ammonium sulfate precipitation, gel filtration, and cation-exchange chromatography. Final enzyme preparation was nearly homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and its molecular weight was estimated as approximately 28000. Optimum pH and temperature of A. pectinifera trypsin for hydrolysis of N(alpha)-p-Tosyl-L-arginine methyl ester hydrochloride were approximately pH 8.0 and 55 degrees C, respectively. A. pectinifera trypsin was unstable at above 50 degrees C and below pH 5.0, and was not activated by adding Ca(2+). The N-terminal amino acid sequence of A. pectinifera trypsin, IVGGHEF, was found.     

Larval Development of Starfishes as Revealed by Long-term Culture of the Embryos

Gametes were obtained from adult starfishes kept in closed cycle aquaria with artificial sea water by stimulation with 1-methyl-adenine ( Asterina pectinifera, Aphelasterias japonica ) or during spontaneous spawning ( Lysastrosoma anthosticta ). The eggs were fertilized and their development until the midbipinnaria stage ( Aph. japonica, L. anthosticta ) and settlement and metamorphosis ( A. pectinifera ) was described using scanning electron microscopy. Special attention was payed to the ciliary band and ciliary cover formation.     

Piericidins C5 and C6: new 4-pyridinol compounds produced by Streptomyces sp. and Nocardioides sp

Piericidins C5 (1) and C6 (2), two new members of the piericidin family, were isolated from a Streptomyces sp. and a Nocardioides sp., together with known piericidins C1 (3), C2 (4), C3 (5), C4 (6), D1 (7), and A3 (8). The structures were determined on the basis of their spectroscopic data. Both new compounds inhibited cell division of fertilized starfish (Asterina pectinifera) eggs at the minimum inhibitory concentration of 0.09 microg/mL.     

The adaptive value of cued seed dispersal in desert plants: Seed retention and release in Mammillaria pectinifera (Cactaceae), a small globose cactus

Serotiny, or delayed seed dispersal, is common in fluctuating environments because it hedges the risks of establishment. Mammillaria pectinifera (Cactaceae) facultatively expels fruits in the year they are produced or retains them to disperse the seed over several years. We tested whether M. pectinifera increased fruit expulsion as a response to increased rainfall. While no fruit expulsion was observed in 1997, a dry year, in the wetter 1998 around 20% of all fruits formed were expelled from the maternal plant. A greenhouse experiment showed that high moisture results in the plants expelling all their fruits. Because in 1998 establishment was five times higher than in 1997, this response seems to be highly adaptive: Active fruit expulsion and consequent seed release increases the probability of establishment during pulses of high precipitation.     

Absence of Species Specificity of Germinal Vesicle Factor Required for the Cytoplamic Cycle During Meiotic Division

Germinal vesicles (GV) of starfish oocytes are known to contain some factor(s) indispensable for inducing cyclic cytoplasmic activity, which may be involved in driving meiotic cell cycle. We have examined species specificity of the factor by injecting enucleated oocytes of the starfish, Asterina pectinifera , with GV contents of either different starfish ( Asterias amurensis, Astropecten scoparius ) or sea cucumber ( Holothuria moebi ). The injected oocytes showed cyclic changes in cortical tension after treatment with 1-methyladenine. Thus the nature of the factor appears similar among echinoderms. Growing oocytes of A. pectinifera much smaller (75% in diameter) than fully-grown oocytes did contain the factor to rescue the enucleated oocytes, even though such small oocytes themselves did not respond to 1-methyladenine. This result suggests that the factor begins to accumulate at the earlier stage of oogenesis.     

Germ Cell Differentiation in Starfish: The Posterior Enterocoel as the Origin of Germ Cells in Asterina pectinifera

The origin of germ cells of Asterina pectinifera was traced back to the posterior enterocoel (PE) of 2-day bipinnaria by two steps. First, the cellular cluster, composed of presumptive germ cells in the coelomic epithelium at brachiolaria stage, was confirmed to be the origin of the aboral haemal sinus located near the hydroporic canal (HC-AHS) by continuous observation of the formation process of HC-AHS. Second, the origin of the cluster was traced back to the PE of 2-day bipinnaria by comparison of the number of the presumptive germ cells in microsurgically PE-removed bipinnariae with that of non-operated control larvae. A summary of the differentiation of germ cells in Asterina pectinifera is given/presented.     

The nucleotide sequences of 5S rRNAs from a sea-cucumber, a starfish and a sea-urchin

The nucleotide sequences of 5S rRNA from three echinoderms, a sea-cucumber Stichopus oshimae, a starfish Asterina pectinifera and a sea-urchin Hemicentrotus pulcherrimus have been determined. These 5S rRNAs are all 120 nucleotides long. The echinoderm sequences are more related to the sequences of proterostomes animals such as mollusc, annelids and some others (87% identity on average) than to those of vertebrates (82% identity on average).     

NGIWYamide-induced contraction of tube feet and distribution of NGIWYamide-like immunoreactivity in nerves of the starfish Asterina pectinifera

NGIWYamide, a neuropeptide recently isolated from sea cucumbers, was tested on tube feet of the starfish Asterina pectinifera. NGIWYamide (10(-6)-10(-4) M) caused contraction of isolated tube feet. NGIWYamide-like immunoreactivity (NGIWYa-LI) was investigated with an antiserum against NGIWYamide. NGIWYa-LI was found in the radial nerve cord (RNC), the marginal nerve, and the tube feet. Both ectoneural and hyponeural parts of the RNC showed NGIWYa-LI. Immunoreactive cell bodies were found in both parts of RNC. Extensive labeling in the basal region of the ectoneural part suggests that a substantial proportion of axons in this part contains NGIWYamide or a similar substance. In tube feet, NGIWYa-LI was found in the sub-epithelial nerve plexus and in the basal nerve ring. Double labeling along with 1E11, a neuron-specific monoclonal antibody developed from A. pectinifera, indicated that the structures with NGIWYa-LI are neurons. These results suggest that NGIWYamide or an NGIWYamide-like peptide exists in starfish and functions as a neurotransmitter or a neuromodulator.     

Duration of the mitotic cycle at the period of synchronous cleavage division (t0) in the starfish Asterina pectinifera (Müller et Troschel)

The duration of a single division (tau 0) in a period of synchronous cleavage, time of development before the start of cleavage, and rotation, hatching and gastrulation stages in A. pectinifera at different temperatures were estimated. The range of temperatures optimal for embryonic and early larval development of the starfish was determined.     

Effect of Cysteine and Its Derivatives on 1-Methyladenine Production by Starfish Follicle Cells

Spawning of the gametes in the starfish, Asterina pectinifera , induced by a gonad-stimulating substance (GSS) was inhibited by cysteine. This inhibitory effect of cysteine was due to the reduction of GSS activity and the inhibition of 1-methyadenine (1-MeAde) production in follicle cells. Homocysteine and cysteamine also reduced GSS activity, but cystamine had no effect on the activity. This suggests that the loss of GSS activity is related to a SH group. Furthermore, the effect of cysteine on 1-MeAde production was investigated using isolated follicle cells. The half maximum decrease of the GSS-dependent 1-MeAde production in follicle cells was obtained with the concentration of 2.0 mM L-cysteine. A similar effect was observed with D-cysteine. Homo cysteine and cysteamine also reduced the GSS-dependent 1-MeAde production. But methionine, serine, glutamic acid and aspartic acid did not affect the 1-MeAde production of follicle cells. In addition, cystamine also inhibited the GSS-dependent 1-MeAde production. These suggest that both a SH group and a SS bond of these compounds are effective on the inhibition of the GSS-dependent 1-MeAde biosynthesis. Cysteine and homocysteine also inhibited concanavalin A (Con A).induced 1-MeAde production of follicle cells, but they had no effect on binding of Con A to the surface of follicle cells.     

Molecular cloning of two toxic phospholipases A2 from the crown-of-thorns starfish Acanthaster planci venom

The full-length cDNAs encoding two toxic phospholipases A2 (AP-PLA2-I and -II) from the crown-of-thorns starfish Acanthaster planci venom were individually cloned by RT-PCR, 3'RACE and 5'RACE. In common with both AP-PLA2s, the precursor protein is composed of a signal peptide, a propeptide and a mature protein (136 and 135 residues for AP-PLA2-I and -II, respectively). The four motifs (Ca2+-binding loop, Ca2+-binding site, active site and catalytic network) characteristic of groups I and II PLA2s are well conserved in both AP-PLA2s. In addition to this, the presence of the elapid and pancreatic loops and the involvement of a propeptide in the precursors suggested that AP-PLA2s are highly analogous to the group IB PLA2s. However, when compared to the amino acid sequence of bovine pancreatic PLA2, the representative group IB PLA2, AP-PLA2s require some amino acid insertions and deletions in the region 76-100, as previously observed for the starfish Asterina pectinifera PLA2s. Furthermore, the phylogenetic tree made clearly demonstrated that AP-PLA2s and A. pectinifera PLA2s are distinguishable from the group IB PLA2s as well as other PLA2s, being classified into a new group.     

Characterization of O-GlcNAcylation in starfish (Asterina pectinifera) development from fertilization to bipinnaria larva

Though O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins has been found in many multicellular organisms, its presence or absence in Echinodermata is unknown. Here we report the occurrence of O-GlcNAcylation in starfish (Asterina pectinifera) oocytes and the apparent O-GlcNAcylation pattern in starfish early development. O-GlcNAcylation might participate in the regulation of starfish development at the mid-blastula stage and thereafter.     

Bacterial expression and characterization of starfish phospholipase A(2)

Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris--HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.     

Partial purification and properties of phospholipase A2 from the starfish, Asterina pectinifera.

1. Phospholipase A2 (phosphatide 2-acyl-hydrolase, EC 3.1.1.4) activity was shown to occur in the supernatant fraction of a freshly prepared homogenate from the pyloric caecum of starfish (Asterina pectinifera). 2. The phospholipase A2 has been isolated and purified 130-fold by ultracentrifugation, ammonium sulfate precipitation and column chromatographic procedures. 3. The purified enzyme was stable to heat at low pH values and the optimal pH was observed at approximately 9.0. 4. The enzyme activity was activated by Ca2+ and sodium deoxycholate, and was inhibited by EDTA.     

A novel peptide from alpha5 helix of Asterina pectinifera cyclin B conjugated to HIV-Tat(49-57) with cytotoxic and apoptotic effects against human cancer cells

A series of novel peptides from various motifs of Asterina pectinifera cyclin B and their derivatives conjugated to HIV-Tat(49-57) were designed and synthesized. Their bioactivities on two human cancer cell lines were determined. Among them, Tat-a5 (KAQIRAMECNILGRKKRRQRRR) exhibited significant cytotoxic effects on cancer cell lines EC-9706 and HCT-116. Tat-a5 could arrest cancer cells at G(2)/M phase and make them apoptotic. Our results suggested that Tat-a5 could be a novel leading peptide with anticancer activity.     

PRODUCTION OF 1-METHYLADENINE INDUCED BY CONCANAVALIN A IN STARFISH FOLLICLE CELLS

Concanavalin A (Con A) was found to induce maturation of oocytes with follicular envelopes in the starfish, Asterina pectinifera . Treating a Con A sample with 85% ethanol and heat revealed that the maturation-inducing activity of the sample was not due to possible contamination with 1-methyladenine, but to Con A itself. However, Con A had little maturation inducing effect on isolated oocytes from which the follicular envelope had been removed, suggesting that its effect is indirect and probably mediated by the follicle cells. When follicle cells were incubated in seawater containing Con A, a maturation-inducing substance was found to have been produced in the incubation medium. This was purified and identified as 1-methyladenine. Therefore it is concluded that Con A has the same capacity as GSS, a gonad-stimulating peptide hormone of neural origin, to induce production of the maturation-inducing substance. Other plant lectins such as phytohemagglutinin P and wheat germ agglutinin had little effect in inducing production of 1-methyladenine in follicle cells.     

Geometrical analysis of cells becoming organized into a tensile sheet, the blastular wall, in the starfish

Abstract. A change in the intercellular relationship of developing starfish embryos ( Asterina pectinifera ) was detected by analyzing the polygonal cellular patterns. Between the seventh cleavage and the rotating blastula stage, independent spherical cells become closely packed and organize themselves further into a cell sheet whose polygonal cell boundaries will shorten. This indicates that a relaxed cell sheet which was more like a collection of independent cells turns into a tightly bound tensile sheet of an epithelial nature. The result is in good agreement with morphologic and behavioral observations of these cells in normal and experimental conditions.     

FORMATION OF JOINED LARVAE IN THE STARFISH, ASTERINA PECTINIFERA

A method for joining larvae in the starfish, Asterina pectinifera , is presented. Any number of embryos are stably united by simple contact of the cell clusters (descendents of individual denuded eggs) before reaching the early blastula stage (ca. 6 1/2 hr after insemination at 21°C). Embryos do not combine with each other after closing into hollow blastulae. This is considered to indicate the transformation of the cell cluster from a mere collection of cells to an individual, multicellular system. Biological meanings of the fertilization membrane are discussed.     

Cancer preventive agents. Part 1: chemopreventive potential of cimigenol, cimigenol-3,15-dione, and related compounds

In continuation of our previous report, cimigenol (1) and 15 related compounds were screened as potential antitumor promoters by using the in vitro short-term 12-O-tetradecanoylphorbol-13-acetate (TPA)--induced Epstein-Barr virus early antigen (EBV-EA) activation assay. Cimigenol-3,15-dione (2) displayed the greatest potency (100% inhibition at 1000 mol ratio/TPA) and consequently was further examined for antitumor-promoting activity in a two-stage carcinogenesis assay of mouse skin tumors (DMBA/TPA). In this assay, compound 2 showed significant activity, reducing the number of papillomas per mouse to 48% of the control group at 20 weeks. In addition, compounds 1 and 2 were examined for antitumor-initiating activity in a two-stage carcinogenesis assay of mouse skin tumors induced by peroxynitrite as an initiator and TPA as a promoter. Results showed that these two triterpenoids were almost equipotent with epigallocatechin gallate (EGCG) and slightly more potent than tocinol (group V), the positive controls. Thus, compounds 1 and 2 exhibited not only strong antitumor-promoting activity but also significant antitumor-initiating effect on mouse skin. These data suggest that both compounds might be valuable chemopreventors.