Intracellular transport by an anchored homogeneously contracting F-actin meshwork

Actin-based contractility orchestrates changes in cell shape underlying cellular functions ranging from division to migration and wound healing. Actin also functions in intracellular transport, with the prevailing view that filamentous actin (F-actin) cables serve as tracks for motor-driven transport of cargo. We recently discovered an alternate mode of intracellular transport in starfish oocytes involving a contractile F-actin meshwork that mediates chromosome congression. The mechanisms by which this meshwork contracts and translates its contractile activity into directional transport of chromosomes remained open questions. Here, we use live-cell imaging with quantitative analysis of chromosome trajectories and meshwork velocities to show that the 3D F-actin meshwork contracts homogeneously and isotropically throughout the nuclear space. Centrifugation experiments reveal that this homogeneous contraction is translated into asymmetric, directional transport by mechanical anchoring of the meshwork to the cell cortex. Finally, by injecting inert particles of different sizes, we show that this directional transport activity is size-selective and transduced to chromosomal cargo at least in part by steric trapping or "sieving." Taken together, these results reveal mechanistic design principles of a novel and potentially versatile mode of intracellular transport based on sieving by an anchored homogeneously contracting F-actin meshwork.     

Acrosomal ATPase in starfish and bivalve mollusk spermatozoa

ATPase activity was found in acrosomes of starfish and bivalve mollusk spermatozoa, using a cytochemical method with electron microscopy. The activity was located in central material of the starfish acrosome and in material lining the acrosomal membrane of the Mytilus acrosome, as well as in the basal part of the starfish acrosome. The ATPase activity in the former material was preferably activated by Ca²⁺, while that in the starfish basal material was preferably activated by Mg²⁺. Both types of activity persisted during and after the acrosome reaction. ATPase activity was also observed in the region of the axial filament complex of the flagella, in centrioles and in a basal matrix. ATPase in the acrosome also hydrolysed other nucleoside triphosphates. However, there was no detectable phosphatase activity, and little pyrophosphatase or 5′-nucleotidase activity. Evidence was obtained that adenylate kinase may be included in the acrosome. A possible role of the ATPase activity in the acrosome reaction is discussed.     

Polar steroids from <Emphasis Type="Italic">Solaster endeca</Emphasis> starfish and the physiological activity of polar steroids from three starfish species

Four polyhydroxylated steroids, new (20R)-5α-cholestan-3β,6α,8,15α,24,26-hexaol (I) and known (20R,25S)-5α-cholestan-3β,6α,8,15β,16β,26-hexaol, (20R,25S)-5α-cholestan-3β,6α,15β,16β,26-pentaol, and marthasterone sulfate were isolated from the Solaster endeca starfish inhabiting the Sea of Okhotsk and characterized. Steroid (I) contains a 24,26-dihydroxylated side chain, which is unique for starfish polyols. The isolated steroids and related metabolites from two starfish species of the Evasterias genus (in total, 15 compounds) were weakly cytotoxic in a human HeLa cell culture and some of them were inhibitors of non-specific esterase from mouse Ehrlich carcinoma. The effects of these compounds on the p53 protein activity were studied in a yeast two-hybrid test system and both inhibitors and stimulators of this activity were found among them.     

Antibacterial and antilarval activity of deep-sea bacteria from sediments of the West Pacific Ocean

Abstract

Deep-sea microorganisms are a new source of bioactive compounds. In this study, crude ethyl acetate extracts of 176 strains of deep-sea bacteria, isolated from sediments of the West Pacific Ocean, were screened for their antibacterial activity against four test bacterial strains isolated from marine biofilms. Of these, 28 deep-sea bacterial strains exhibited antibacterial activity against one or more of the bacteria tested. Active deep-sea bacterial strains belonged mainly to the genera of Pseudomonas, Psychrobacter and Halomonas. Additionally, antilarval activity of 56 deep-sea bacterial strains was screened using Balanus amphitrite larvae. Seven bacterial strains produced metabolites that had strong inhibitive effects on larval settlement. None of these metabolites showed significant toxicity. The crude extract of one deep-sea Streptomyces strain could completely inhibit larval settlement at a concentration of 25 μg ml^?1.     

Novel pentapeptide,PALAL, derived from a bony fish elicits contraction of the muscle in starfish Patiria pectinifera

A bioactive peptide mimicking peptide‐signaling molecules has been isolated from the skin extract of fish Channa argus which caused contraction of the apical muscle of a starfish Patiria pectinifera, a deuterostomian invertebrate. The primary structure of the isolated pentapeptide comprises amino acid sequence of H‐Pro‐Ala‐Leu‐Ala‐Leu‐OH (PALAL) with a molecular mass of 483.7 Da. Pharmacological activity of PALAL, dosage ranging from 10^?9 to 10^?5 M, revealed concentration‐dependent contraction of the apical muscles of P. pectinifera and Asterias amurensis. However, PALAL was not active on the intestinal smooth muscle of the goldfish Carassius auratus and has presumably other physiological roles in fish skin. Investigation of structure‐activity relationship using truncated and substituted analogs of PALAL demonstrated that H‐Ala‐Leu‐Ala‐Leu‐OH was necessary and should be sufficient to constrict apical muscle of P. pectinifera. Furthermore, the second alanine residue was required to display the activity, and the fifth leucine residue was responsible for its potency. Comparison with PALAL's primary structure with those of other known bioactive peptides from fish and starfish revealed that PALAL does not have any significant homology. Consequently, PALAL is a bioactive peptide that elicits a muscle contraction in starfish, and the isolation of PALAL may lead to develop other bioactive peptides sharing its similar sequence and/or activity. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Generality of the action of various maturation-promoting factors

It has been known in amphibians and starfishes that a cytoplasmic factor called maturation-promoting factor (MPF), produced in maturing oocytes under the influence of the maturation-inducing hormones, can induce germinal vesicle breakdown (GVBD) and the subsequent process of meiotic maturation. The present study revealed that injection of cytoplasm of maturing starfish oocytes (starfish MPF) into immature sea cucumber oocytes brought about maturation of the recipients. Amphibian MPF obtained from mature oocytes of Xenopus laevis or Bufo bufo was found to induce maturation of starfish oocytes following injection. Cytoplasm taken from cleaving starfish blastomeres induced maturation when injected into immature starfish oocytes. The maturation-inducing activity of cytoplasm of starfish blastomeres changed along with the mitotic cell cycle during 1- to 4-cell stages so far tested and reached a peak just before cleaving. Furthermore, an extract of mammalian cultured cells, CHO or V-79, synchronized in M phase, induced GVBD in starfish oocytes following injection, whereas S phase extract had little activity. These facts suggest that MPF generally brings about nuclear membrane breakdown in both meiosis and mitosis, and that the nature of MPF is very similar among vertebrates and invertebrates.     

Cancer chemopreventive effects of starfish polysaccharide in human breast cancer cells

We investigated the effect of starfish (Asterina pectinifera) polysaccharide on the progression and metastasis of human breast cancer cells. At a concentration range of 10 ∼ 120 μg/mL the polysaccharide significantly decreased the expression of cyclooxygenase-2 (COX-2) protein induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) and of aromatase mRNA. In a wound healing assay, motility of human MDA-MB-231 breast cancer cells was prevented by the polysaccharide in a dose-dependent manner. These results indicate that starfish polysaccharide can prevent breast cancer progression and metastasis by decreasing prostaglandin E₂ and estrogen biosynthesis by COX-2 and aromatase, and by inhibiting cell motility. This report presents information regarding the effectiveness of starfish polysaccharide as a chemopreventive agent against breast cancer.     

A preliminary phylogeny of the Pterasteridae (Echinodermata,Asteroidea) and the first fossil record: Late Cretaceous of Germany and Belgium

The Pterasteridae comprises a diversified group of extant largely deep-sea starfishes. Generic diagnoses have been based classically on soft tissue characters and skeletal architecture. A preliminary phylogeny of sixteen extant species is here worked out by cladistic analysis. The resulting tree suggests monophyly of extant genera and the validity of dissociated plates for identification of genera. Fossil remains of Pterasteridae are here described for the first time. By comparison with extant species, all the skeletal remains from the lower Upper Campanian of Belgium and from the lower Maastrichtian of Germany are tentatively assigned to the genusPteraster. The fossil record of starfishes is poor, but the present Late Cretaceous pterasterids provide one more piece of evidence of the high diversity of starfishes during the Mesozoic. Known Late Cretaceous and Paleogene fossils are broadly similar, which suggests the end-Cretaceous extinction event did not cause major turnover in asteroid faunal composition. As suggested for other starfish groups, both the fossil record of deep-sea Pterasteridae in shelf settings and tree topology imply an onshore-offshore evolutionary trend.      

METHIONINE-ACTIVATING ENZYME IN STARFISH FOLLICLE CELLS *

In starfish follicle cells 1-methyladenine is produced under the influence of a gonad-stimulating hormonal peptide (GSS). Since such production of the substance is enhanced by the addition of L-methionine or S-adenosylmethionine in vitro, the presence of methionine-activating enzyme in the follicle cells of the starfish, Asterina pectinifera, was investigated. To detect enzyme activity, the enzyme was partially purified from the supernatant of the follicle-cell homogenate by precipitation with ammonium sulfate followed by gel-filtration on a Sephadex G-150 column. Using such a preparation of the enzyme, the production of S-adenosylmethionine from L-methionine and adenosine triphosphate was clearly demonstrated by thin-layer chromatography. GSS was found to exert no effect on the activity of the methionine-activating enzyme. The hormonal peptide, GSS, is therefore considered to take part in some reaction other than this step in the formation of 1-methyladenine.     

Comparative study on dihydrofolate reductases from <Emphasis Type="Italic">Shewanella</Emphasis> species living in deep-sea and ambient atmospheric-pressure environments

To examine whether dihydrofolate reductase (DHFR) from deep-sea bacteria has undergone molecular evolution to adapt to high-pressure environments, we cloned eight DHFRs from Shewanella species living in deep-sea and ambient atmospheric-pressure environments, and subsequently purified six proteins to compare their structures, stabilities, and functions. The DHFRs showed 74–90% identity in primary structure to DHFR from S. violacea, but only 55% identity to DHFR from Escherichia coli (ecDHFR). Far-ultraviolet circular dichroism and fluorescence spectra suggested that the secondary and tertiary structures of these DHFRs were similar. In addition, no significant differences were found in structural stability as monitored by urea-induced unfolding and the kinetic parameters, K _m and k _cat; although the DHFRs from Shewanella species were less stable and more active (2- to 4-fold increases in k _cat/K _m) than ecDHFR. Interestingly, the pressure effects on enzyme activity revealed that DHFRs from ambient-atmospheric species are not necessarily incompatible with high pressure, and DHFRs from deep-sea species are not necessarily tolerant of high pressure. These results suggest that the DHFR molecule itself has not evolved to adapt to high-pressure environments, but rather, those Shewanella species with enzymes capable of retaining functional activity under high pressure migrated into the deep-sea.     

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A₂ (PLA₂), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1–414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA₂ protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA₂ protein, suggesting the presence of multiple forms in the starfish PLA₂. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in β-wing region when aligned with the sequence of mammalian pancreatic PLA₂. In phylogenetic tree, the starfish PLA₂ should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and β-wing regions may account for the specific properties of the starfish PLA₂, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA₂ from porcine pancreas.     

FtsEX is required for CwlO peptidoglycan hydrolase activity during cell wall elongation in Bacillus subtilis

The peptidoglycan (PG) sacculus, a meshwork of polysaccharide strands cross‐linked by short peptides, protects bacterial cells against osmotic lysis. To enlarge this covalently closed macromolecule, PG hydrolases must break peptide cross‐links in the meshwork to allow insertion of new glycan strands between the existing ones. In the rod‐shaped bacterium Bacillus subtilis, cell wall elongation requires two redundant endopeptidases, CwlO and LytE. However, it is not known how these potentially autolytic enzymes are regulated to prevent lethal breaches in the cell wall. Here, we show that the ATP‐binding cassette transporter‐like FtsEX complex is required for CwlO activity. In Escherichia coli, FtsEX is thought to harness ATP hydrolysis to activate unrelated PG hydrolases during cell division. Consistent with this regulatory scheme, B. subtilis FtsE mutants that are unable to bind or hydrolyse ATP cannot activate CwlO. Finally, we show that in cells depleted of both CwlO and LytE, the PG synthetic machinery continues moving circumferentially until cell lysis, suggesting that cross‐link cleavage is not required for glycan strand polymerization. Overall, our data support a model in which the FtsEX complex is a remarkably flexible regulatory module capable of controlling a diverse set of PG hydrolases during growth and division in different organisms.     

Plant-growth regulators from common starfish (<Emphasis Type="Italic">Asterias amurensis</Emphasis> Lütken) waste

Starfish waste has been shown to be an effective compost material not only in the promotion of plant growth but also in terms of having insecticidal activity. In the present study, plant growth regulation by chemicals from starfish was examined. The aqueous fraction from a hot water extract of the starfish Asterias amurensis Lütken showed plant-growth activity, while the aqueous fraction from a methanol extract inhibited growth of Brassica campestris. The lipophilic fraction from the methanol extract also exhibited a plant growth-promoting effect. The active components from each extract were identified. Asterubine from the hot water extract promoted plant growth. A ceramide from the lipophilic fraction showed root growth promoting effect, and three glucocerebrosides had promotive effects on the entire plant. Asterosaponins were identified as the main growth inhibitors in the aqueous fraction of the methanol extract. These active compounds from starfish waste could be analyzed as potential plant growth regulators in agricultural applications in the future.     

中国南海部分深海沉积物真菌多样性及其抗菌活性

【目的】从南海4个站位的深海沉积物中分离真菌,揭示其多样性并测定抗菌活性。【方法】使用4种培养方法和8种培养基,从12个深海沉积物样本中分离培养真菌,通过菌落形态观察和ITS序列系统发育分析进行鉴定。采用滤纸片扩散法和生长速率法分别测试真菌小量发酵液粗浸膏的抗细菌和抗真菌活性。【结果】共分离到125株纯培养真菌,基于形态和ITS序列分析,排重后得到18个种类型,这些真菌可以划分到12个属,大多数属于子囊菌门(Ascomycota),只有2株属于担子菌门(Basidiomycota)。4个站位可培养真菌多样性具有差异性。抑菌活性筛选显示,大多数真菌具有较好的抑菌活性;链格孢属(Alternaria)、青霉属(Penicillium)、匐柄霉属(Stemphylium)这几个属的真菌表现出对多种指示细菌有抑制作用,尤其是Alternaria tenuissima DN09、Alternaria alternata DN14和Penicillium chrysogenum DN16对G~+和G~–细菌均表现出抑制作用。【结论】本研究揭示了南海深海沉积物可培养真菌多样性和抑菌活性,为进一步利用深海沉积物来源真菌奠定了基础。     

Identification and Isolation of GTP-Binding Regulatory Protein from Starfish (Asterias amurensis) Oocyte Plasma Membranes

A method for isolating a GTP-binding regulatory protein from starfish oocytes is described. The protein consists of three subunits with molecular weights of 40, 37, and about 8 kDa. It is shown that the 40-kDa subunit has a high GTPase activity and is susceptible to ADP-ribosylation by pertussis toxin. The latter property of this subunit proved to decrease upon its incubation with nonhydrolyzable GTP analogues. These data provide evidence that the plasma membrane of starfish oocytes contains a 40-kDa GTP-binding protein with properties characteristic of the alpha subunit of the inhibitory G _i protein. The role of this protein in the transmembrane signal transmission from the 1-methyladenine receptor to intracellular effectors is discussed.     

Dynamics of an outbreak population of Acanthaster planci at Lizard Island, northern Great Barrier Reef (1995–1999)

Despite their significant influence on coral reef ecosystems, causes of population outbreaks of crown-of-thorns starfish (Acanthaster planci L.) are still poorly understood. Essentially, outbreaks of A. planci could arise from either (1) a single mass recruitment event or (2) the progressive accumulation of starfish from multiple cohorts. This study explored fine-scale variation in the size, distribution, and abundance of A. planci, during an outbreak at Lizard Island in the northern Great Barrier Reef, to assess the mechanism by which the outbreak occurred. Densities of A. planci around Lizard Island increased very gradually from October 1994 until December 1996, then remained at around 1.0 starfish per 200 m² until June 1998. The population of A. planci comprised individuals ranging in size from 11-cm to 62-cm diameter, representing individuals from multiple (at least four) different cohorts. These data suggest that the outbreak of A. planci at Lizard Island resulted from a prolonged build-up in starfish numbers through multiple successive recruitment events. This study shows that outbreaks of A. planci may arise independently of any sudden or substantial increase in rates of recruitment, such that any factor(s) responsible for the initial onset of outbreaks are likely to be very subtle and difficult to detect.     

Aspects of crinoid palaeontology of the North Esk Inlier,Scotland (Silurian,Llandovery, Telychian)

Abstract: The described fauna of well‐preserved Llandovery (Telychian) echinoderms from the North Esk Inlier, including six crinoids, one echinoid and seven starfish species, is mainly allochthonous. Most of these taxa are known only from starfish beds, channel fill deposits probably representing submarine mass flows and preserving a biota probably derived from elsewhere, presumably shallower water. Only one crinoid species, Pisocrinus cf. campana Miller, is recognized as a common fossil away from the starfish beds and is a biostratigraphic marker for the base of the Wether Law Linn Formation, forming part of the Skenidioides‐Cyrtia Association. Crinoid columnals preserved perpendicular to bedding (that is, in putative life position) in Lamont’s bivalve bed, Deerhope Formation, are tentatively interpreted as being in situ by comparison with a similar occurrence in the Silurian of Arisaig, Nova Scotia. Two new species of crinoid are described, the cladid Dendrocrinus? sp. and the columnal morphospecies Pentagonocyclicus (col.) lamonti sp. nov.     

The small cells of coelomic fluid and coelomic epithelium isolated from starfish <Emphasis Type="Italic">Asterias rubens</Emphasis> and <Emphasis Type="Italic">Asterias amurensis</Emphasis> (Echinodermata: Asteroidea): Comparative analysis of cell morphology and proliferative activity in vivo and in vitro

Earlier we revealed the probable candidates for the role of Asteroidea stem cells in the starfish Asterias rubens L., small coelomic epithelial cells (SECs-1) with a high nuclear–cytoplasmic ratio that were able to proliferate in vivo and in vitro. To check the existence of a similar cell type in other members of Asteroidea, the small cells in suspensions of coelomic fluid (CF) and coelomic epithelium (CE) of A. amurensis were analyzed with respect to their morphology and proportion in the total cell pool. The morphology of proliferating cells and the proliferative activity of CF and CE cells in vivo and in vitro were studied. The small cells with parameters identical to those of A. rubens SECs-1, were found both in CF and CE of related species. The subpopulation of weakly attached CE cells, highly enriched with SECs-1, was detected. These cells were able to migrate from CE and to proliferate in vivo and in vitro. Additionally, large proliferating cells were described in both starfish. The dynamics of proliferative activity in primary cell cultures of these starfish had some distinctions. Moreover, for the first time, the formation of “crystals”, the potential centres of spiculogenesis, was observed in primary culture of A. amurensis CE cells. The data prove that SECs may fulfil the common functions in two members of Asteroidea.     

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.     

Free intracellular cations in echinoderm oocytes and eggs

The concentrations of Ca²⁺, Na⁺ and H⁺ in echinoderm oocytes and eggs were measured during maturation and activation using ion-selective microelectrodes. In both oocytes and eggs, from three species of starfish and two species of sea urchin, the resting level of cytosolic Ca²⁺ was about 10^-7 M. We did not detect any change in Ca²⁺ concentration either during hormone-induced oocyte maturation (starfish) or during egg activation (starfish and sea urchin) induced by spermatozoa or chemical agents. During 1-methyl-adenine induced maturation of starfish oocytes the intracellular level of Na⁺ increased from 12–35 mM to 40–90 mM, while the pH changed from 6.6–6.8 to 7.0–7.2 Aged oocytes, with intact germinal vesicles, also had elevated levels of Na⁺ and pH.