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PubMed2000

Intermediate filament proteins in echinoderm coelomocytes.

The presence and organization of intermediate filament (IF) proteins in petaloid coelomocytes from two species of echinoderms, the sea urchin Strongylocentrotus droehachiensis and the sea cucumber Cucumaria frondosa, were studied. Two monoclonal antibodies (IFA and Ah6) and one polyclonal antibody (W3-1) that together recognize invertebrate as well as vertebrate IF proteins were used to probe coelomocytes by immunofluorescence and immunoblotting methods. All three antibodies cross-reacted with a single Mr 68,000 sea urchin lamin, as well as two putative lamin isoforms of approximately Mr 70,000 and 68,000 in sea cucumber coelomocytes. Both IFA and Ah6 labeled granular material in the cytoplasm of sea urchin coelomocytes; by contrast, IFA labeling revealed a striking network of reticular material irregularly arrayed within the central regions of the sea cucumber coelomocyte cytoplasm. In addition, foci of Ah6-positive material were present in coelomocyte nuclei from both species. Comparison of immunoblotting patterns among whole cell and isolated nuclear preparations suggest that the cytoplasmic IF-like material is composed of Mr 46,000 and 58,000 polypeptides, while Mr 215,000 and 185,000 proteins are candidates for the immunoreactive nuclear foci. Further study of the functions of these non-filamentous arrays of IF proteins may furnish valuable insights into the evolution of IF function within vertebrate cells, particularly with respect to certain cytoplasmic and nuclear regulatory functions with which IF proteins have been speculated to be involved.

Amino Acid Sequence↗
PubMed2000

Pharmacological identification of acetylcholine receptor subtypes in echinoderm smooth muscle (Sclerodactyla briareus).

Contractions of an echinoderm (sp. Sclerodactyla briareus) smooth muscle, the longitudinal muscle of the body wall (LMBW), were evoked by acetylcholine (ACh) and agonists: epibatidine, muscarine and nicotine (in order of force generation: ACh>muscarine=epibatidine>nicotine). ACh-induced contractions were blocked by atropine by 50%, and methoctramine, by 30%. ACh responses were also blocked by 25% by methyllycaconitine (MLA) but not by D-tubocurarine (dTC). Muscarine initiated large contractions that were completely blocked by atropine. To elucidate possible muscarinic ACh receptor (mAChR) subtypes, muscarinic agonists (oxotremorine, pilocarpine) and antagonists (methoctramine, pirenzepine) were tested. Oxotremorine, pilocarpine, and pirenzepine each enhanced resting tonus and potentiated ACh-induced contractions (order of potency: pilocarpine>oxotremorine=pirenzepine). Muscarine, oxotremorine or pirenzepine generated phasic, rhythmic contractions. Nicotine-induced contractions were almost completely blocked by dTC but were not altered by atropine. Large contractions evoked by epibatidine were potentiated by dTC whereas atropine had no effect on them. MLA blocked spontaneous rhythmicity. Cholinesterase inhibitors, neostigmine or physostigmine, caused marked potentiation of ACh-induced contractions and initiated rhythmic slow wave contractions in previously quiescent muscles. The present pharmacological evidence points to the co-existence of excitatory nicotinic ACh receptor (nAChRs) and mAChRs where nAChRs possibly modulate tone, and the mAChRs initiate and enhance rhythmicity.

Acetylcholine↗
PubMed2004

Apparent selection intensity for the cytochrome oxidase subunit I gene varies with mode of reproduction in echinoderms.

When most amino acid substitutions in protein-coding genes are slightly deleterious rather than selectively neutral, life history differences can potentially modify the effective population size or the selective regime, resulting in altered ratios of non-synonymous to synonymous substitutions among taxa. We studied substitution patterns for the mitochondrial cytochrome oxidase subunit I (COI) gene in a sea star genus (Leptasterias spp.) with an obligate brood-protecting mode of reproduction and small-scale population genetic subdivision, and compared the results to available COI sequences in nine other genera of echinoderms with pelagic larvae: three sea stars, five sea urchins and one brittle star. We predicted that this life history difference would be associated with differences in the ratio of non-synonymous (dN) to synonymous (dS) substitution rates. Leptasterias had a significantly greater dN/dS ratio (both between species and within species), a significantly smaller transition/transversion rate ratio, and a significantly lower average nucleotide diversity within species, than did the non-brooding genera. Other explanations for the results, such as altered mutation rates or selective sweeps, were not supported by the data analysis. These findings highlight the potential influence of reproductive traits and other life history factors on patterns of nucleotide substitution within and between species.

Animals↗
PubMed1974

Progesterone metabolism by the echinoderms Asterias rubens and Marthasterias glacialis.

The echinoderms Asterias rubens and Marthasterias glacialis metabolize injected [4-(14)C]progesterone to give labelled 3beta-hydroxy-5alpha-pregnan-20-one and 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one. These radioactive products are converted by the animals into conjugated forms that are soluble in aqueous methanol, and which have mobilities on t.l.c. similar to the asterosaponins.

Animals↗
PubMed1975

The conversion of cholest-5-en-3beta-ol into cholest-7-en-3beta-ol by the echinoderms Asterias rubens and Solaster papposus.

1. The echinoderms Asterias rubens and Solaster papposus (Class Asteroidea) metabolize injected [4(-14)C]cholest-5-en-3beta-ol to produce labelled 5alpha-cholestan-3beta-ol and 5alpha-cholest-7-en-3beta-ol. 2. Conversion of 5alpha-[4(-14)C]cholestan-3beta-ol into 5alpha-cholest-7-en-3beta-ol was demonstrated in A. Rubens. 3. Incubations of A. rubens with [4(-14)C]cholest-4-en-3-one resulted in the production of labelled 5alpha-cholestan-3-one, 5alpha-cholestan-3beta-ol and 5alpha-cholest-7-en-3beta-ol. 4. [4(-14)C]Sitosterol was metabolized by A. rubens to give 5alpha-stigmastan-3beta-ol and 5alpha-stigmast-7-en-3beta-ol. 5. The significance of these results in relation to the presence of alpha7 sterols in starfish is discussed.

Animals↗
PubMed1992

Purification and characterization of echinoderm casein kinase II. Regulation by protein kinase C.

Casein kinase II (CKII) is one of several protein kinases that become activated before germinal-vesicle breakdown in maturing sea-star oocytes. Echinoderm CKII was purified over 11,000-fold with a recovery of approximately 10% by sequential fractionation of the oocyte cytosol on tyrosine-agarose, heparin-agarose, casein-agarose and MonoQ. The purified enzyme contained 45, 38 and 28 kDa polypeptides, which corresponded to its alpha, alpha' and beta subunits respectively. The beta-subunit was autophosphorylated on one major tryptic peptide on serine residues, whereas the alpha'-subunit incorporated phosphate into at least two tryptic peptides primarily on threonine residues. Western-blotting analysis of sea-star oocyte extracts with two different anti-peptide antibodies that recognized conserved regions of the alpha-subunit indicated that the protein levels of the alpha- and alpha'-subunits of CKII were unchanged during oocyte maturation. The purified CKII was partly inactivated (by 25%) by preincubation with protein-serine/threonine phosphatase 2A, but protein-tyrosine phosphatases had no effect. The beta-subunit of CKII was phosphorylated on a serine residue(s) up to 0.54 mol of P/mol of beta-subunit by purified protein kinase C, and this correlated with a 1.5-fold enhancement of its phosphotransferase activity with phosvitin as a substrate. CKII was not a substrate for the maturation-activated myelin basic protein kinase p44mpk from sea-star oocytes, nor for cyclic-AMP-dependent protein kinase. These studies point to possible regulation of CKII by protein phosphorylation.

Animals↗
PubMed2002

Essential role of growth factor receptor-mediated signal transduction through the mitogen-activated protein kinase pathway in early embryogenesis of the echinoderm.

In this study it was shown that growth factor receptors (GFR) play a crucial role in early embryogenesis of the echinoderms Hemicentrotus pulcherrimus and Clypeaster japonicus by transmitting signals to the mitogen-activated protein kinase (MAPK) pathway. The phosphorylation ratio of extracellular signal-regulated kinase 1 (ERK1) changed dynamically during early embryogenesis and showed a peak at the swimming blastula (sBl) stage. Suramin, an inhibitor of GFR, when applied during the sBl stage perturbed morphogenesis, including primary mesenchyme cell (PMC) migration, cell proliferation, archenteron elongation, spiculogenesis, pigment cell differentiation and phosphorylation of myosin light chains (MLC). Genistein, a receptor-type protein tyrosine kinase inhibitor, severely inhibited PMC migration, gastrulation and the phosphorylation of MLC. Manumycin A, a Ras inhibitor, inhibited spiculogenesis and invagination. PD98059, a MAPK/ERK kinase inhibitor, perturbed early PMC migration and pigment cell differentiation, but not spiculogenesis and gastrulation (although these two events were significantly delayed). PMC ingression was not perturbed by genistein, suramin, manumycin A or PD98059. All of the inhibitors perturbed the phosphorylation of ERK1, which was completely restored by exogenous platelet-derived growth factor (PDGF)-AB. PDGF-AB also partially restored elongation of the archenteron by restoring cell proliferation that had been perturbed by suramin.

Animals↗
PubMedDate not supplied

Gene expression and larval evolution: changing roles of distal-less and orthodenticle in echinoderm larvae.

We describe the expression of the homeobox genes orthodenticle (Otx) and distal-less (Dlx) during the larval development of seven species representing three classes of echinoderms: Holothuroidea, Asteroidea, and Echinoidea. Several expression domains are conserved between species within a single class, including Dlx expression within the brachiolar arms of asteroid larvae and Otx expression within the ciliated bands of holothuroid larvae. Some expression domains are apparently conserved between classes, such as the expression of Dlx within the hydrocoel (left mesocoel) in all three classes. However, several substantial differences in expression domains among taxa were also evident for both genes. Some autapomorphic (unique derived) features of gene expression are phylogenetically associated with autapomorphic structures, such as Dlx expression within the invaginating rudiment of euechinoids. Other autapomorphic gene expression domains are associated with evolutionary shifts in life history from feeding to nonfeeding larval development, such as Otx expression within the ciliated bands of a nonfeeding holothuroid larva. Similar associations between evolutionary changes in morphology and life history mode with changes in regulatory gene expression have also been observed in arthropods, urochordates, and chordates. We predict that recruitment of regulatory genes to a new developmental role is commonly associated with evolutionary changes in morphology and may be particularly common in clades with complex life cycles and diversity of life history modes. Caution should be used when making generalizations about gene expression and function based on a single species, which may not accurately reflect developmental processes and life histories of the phyla to which it belongs.

Animals↗
PubMed1971

Bioelectric responses of the echinoderm egg to fertilization.

The fertilization reaction of echinoderm eggs (Lytechinus pictus, a sea urchin, and Dendraster excentricus, a sand dollar) was followed with intracellular electrodes. Membrane potential and K(+) activity were recorded. The unfertilized egg of Lytechinus has a membrane potential of -8 mV, inside negative. Within 5 sec after the addition of sperm, a fertilization action potential develops, going to +10 mV, inside positive. The time from the initial depolarization to a return to the original -8 mV is 120-150 sec. The repolarization continues until a potential of -10 to -14 mV is reached, at which point it pauses for 3-4 min. At 6-8 min after fertilization, a further and relatively rapid hyperpolarization begins, going to -60 to -65 mV by 15-25 min after fertilization and remaining constant at these values. The membrane potential of the unfertilized egg appears to depend on a general permeability to anions. The fertilization action potential seems to reflect a prolonged influx of sodium. The final depolarization to -60 mV is attributable to the development of potassium conductance. Simultaneous measurements with a K(+) ion-selective electrode gives constant readings of about 240 mM K(+) in the unfertilized eggs throughout the fertilization process. Similar results were obtained with Dendraster eggs. The resting potential of the unfertilized eggs was -7 mV; the action potential on activation attained +18 mV; the repolarization paused at -16 to -24 mV and the final potential attained was -70 mV. The electrical changes after fertilization with spermatozoa or activation with Pronase were identical.

Action Potentials↗
PubMed1992

Paleozoic record of morphologica diversity in blastozoan echinoderms.

There has been extensive debate about the magnitude and implications of morphological diversity in early Paleozoic animals, with some workers using apparently rapid initial diversification to infer unusual evolutionary processes. Analysis of discrete morphological characters shows that initial morphological diversification in the echinoderm subphylum Blastozoa was so pronounced that morphological diversity relative to taxonomic diversity was greatest in the Cambrian, whereas morphological diversity itself was greatest in the Middle and Upper Ordovician. Thus, a small number of Cambrian taxa sparsely occupied a large range in morphological space, whereas subsequent diversification involved expansion and filling of morphospace. A measure of clade-shape asymmetry and a method for statistical testing of clade shape are used to show that morphological diversity is significantly concentrated early in the history of the Blastozoa. The subphylum represents the highest biologic level at which temporal patterns of morphological diversity have been analyzed. Because this study is based on explicit morphological analysis, not taxonomic proxies for morphological diversity, the results are not artifacts of taxonomic practice.

Journal Article↗
PubMed1990

Preservation of a complex satellite DNA in two species of echinoderms.

The cloning and sequencing of a tandemly arrayed repetitive DNA sequence from the sea cucumber Holothuria tubulosa has been recently described (Sainz, J., Azorín, F. and Cornudella, L. 1989. Gene 80, 57-64). We have now searched the genomes of several echinoderm species for the presence of homologous repetitive elements. A close but not identical repeated sequence has been identified in a related holothuroid, H. polii. The monomeric repeat unit is 391 bp long and has a base composition of 66.8% A and T residues, lined up in tracts of 4 nt or larger. The monomeric sequence lacks any internal subrepeat organization although it displays a substantial degree of internal redundancy in the form of inverted and direct repeats. The repeated element accounts for 0.34% of the genome which corresponds to a repetition frequency of about 0.5 x 10(5) copies per haploid complement. The intra- and interspecific homologies among monomers of the satellite DNA as derived from sequence analyses are very high, averaging 97%. The results suggest that the homogeneity of the highly reiterated DNA sequence may be attributed to evolutionary conservative trends.

Animals↗
PubMed1996

Cell cycle-dependent phosphorylation of the 77 kDa echinoderm microtubule-associated protein (EMAP) in vivo and association with the p34cdc2 kinase.

The most abundant microtubule-associated protein in sea urchin eggs and embryos is the 77 kDa echinoderm microtubule-associated protein (EMAP). EMAP localizes to the mitotic spindle as well as the interphase microtubule array and is a likely target for a cell cycle-activated kinase. To determine if EMAP is phosphorylated in vivo, sea urchin eggs and embryos were metabolically labeled with 32PO4 and a monospecific antiserum was used to immunoprecipitate EMAP from 32P-labeled eggs and embryos. In this study, we demonstrate that the 77 kDa EMAP is phosphorylated in vivo by two distinct mechanisms. In the unfertilized egg, EMAP is constitutively phosphorylated on at least five serine residues. During the first cleavage division following fertilization, EMAP is phosphorylated with a cell cycle-dependent time course. As the embryo enters mitosis, EMAP phosphorylation increases, and as the embryo exits mitosis, phosphorylation decreases. During mitosis, EMAP is phosphorylated on 10 serine residues and two-dimensional phosphopeptide mapping reveals a mitosis-specific site of phosphorylation. At all stages of the cell cycle, a 33 kDa polypeptide copurifies with the 77 kDa EMAP, regardless of phosphorylation state. Antibodies against the cdc2 kinase were used to demonstrate that the 33 kDa polypeptide is the p34cdc2 kinase. The p34cdc2 kinase copurifies with the mitotic apparatus and immunostaining indicates that the p34cdc2 kinase is concentrated at the spindle poles. Models for the interaction of the p34cdc2 kinase and the 77 kDa EMAP are presented.

Animals↗
PubMed2001

Growth factors, heat-shock proteins and regeneration in echinoderms.

The study of regeneration in armed echinoderm species, including crinoids, ophiuroids and asteroids, is attracting increasing attention. Recent interest has focused on the presence and potential role of growth factors, including members of the nerve growth factor (NGF) and transforming growth factor-beta (TGF-beta) families, in the regenerative process and their possible relationship to the normal developmental (ontogenetic) regulatory cascade. In addition, the expression patterns of the heat-shock family of stress proteins (Hsps) during regeneration are also important. Their role forms part of a normal stress response to the trauma of autotomy in combination with a putative function in tissue remodelling and associated protein turnover during regeneration. The temporal dynamics of the stress response may also be strongly indicative of environmentally adaptive pressures operating on these systems.

Amino Acid Sequence↗
PubMed2002

Is muscle involved in the mechanical adaptability of echinoderm mutable collagenous tissue?

The mutable collagenous tissue (MCT) of echinoderms has the capacity to change its mechanical properties in a time scale of less than 1 s to a few minutes under the influence of the nervous system. Although accumulating evidence indicates that the mechanical adaptability of MCT is due primarily to the modulation of interactions between components of the extracellular matrix, the presence of muscle in a few mutable collagenous structures has led some workers to suggest that contractile cells may play an important role in the phenomenon of variable tensility and to call for a re-evaluation of the whole MCT concept. This contribution summarises present information on MCT and appraises the argument implicating muscle in its unique mechanical behaviour. It is concluded that there is no evidence that the variability of the passive mechanical properties of any mutable collagenous structure is due to muscle.

Animals↗
PubMed1977

Asymmetric waveforms in echinoderm sperm flagella.

1.Dark-field, multiple-exposure photographs of live spermatozoa of a number of echinoderms were analysed. 2. Bends develop at the base in pairs, as they do in flagella with symmetrical waveforms. However, the angles of these bends do not cancel, so that microtubular sliding-of up to over 50% of that associated with bend propagation-is transferred distally. This sliding implies that (a) microtubules are not rigidly cross-linked within straight regions, and (b) bends can propagate normally in spite of appreciable extrinsic microtubular sliding. 3. Both the sperm head and the asymmetry of the waveform appear to affect the swim path of a spermatozoon.

Animals↗
PubMed1994

Flexural rigidity of echinoderm sperm flagella.

The stiffness (flexural rigidity) of live sperm flagella, Triton-demembranated flagella (axonemes), trypsin-digested axonemes, and doublet microtubules of the axonemes in echinoderms was determined from the relationship between their deformation when a stream of medium was applied and the viscous resistance of the medium acting on the flagellum. The stiffness of the flagellum beating in seawater was 5.8 x 10(-21) Nm2 for bending in the direction perpendicular to the beating plane and 4.2 x 10(-22) Nm2 for bending within the beating plane. A similar difference in stiffness from the difference in bending directions was found in reactivated flagella with 1 mM ATP. The stiffness of live flagella immobilized in CO2-saturated seawater and axonemes in ATP-free medium was similar to that of beating flagella for bending in the direction perpendicular to the beating plane. The stiffness of motionless flagella significantly decreased with erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) and vanadate. The trypsin-digestion of motionless axonemes did not change their stiffness. The stiffness of doublet microtubules was 1.4 x 10(-23) Nm2 in 0.1 mM ATP medium and 6.1 x 10(-23) Nm2 in ATP-free medium. These results suggest that doublet pairs lying parallel to the beating plane of the flagellum retain fewer cross-bridges than doublet pairs lying perpendicular to the beating plane.

Adenine↗
PubMed2025

Lytic coelomocyte death is tuned by cleavage but not phosphorylation of MLKL in echinoderms.

Lytic cell death including necroptosis and pyroptosis is induced by mixed lineage kinase domain-like protein (MLKL) phosphorylation and inflammatory caspase specific cleavage Gasdermins in higher mammals, respectively. In this study, we identified a novel MLKL homolog containing a tetrapeptide recognition motif (14-LVAD-17) of inflammatory caspase from Apostichopus japonicus,which was absent of Gasdermins member by genome screening. Functional analysis revealed that AjMLKL was involved in the regulation of Vibrio splendidus AJ01 infection induced lytic coelomocyte death in a cleavage-dependent manner, but not through RIPK3-dependent phosphorylation as mammals. Mechanistically, the activated form of cysteine-aspartic specific proteases-1 (AjCASP-1) bound to the tetrapeptide site of AjMLKL and cleaved it at Asp17. Cleaved AjMLKL18-491 displayed higher binding affinities towards phosphatidylinositol phosphate and cardiolipin compared to those of un-cleaved form. In addition, cleaved AjMLKL18-491 exerted stronger ability in disrupting the membrane integrity of liposome. More importantly, AjMLKL18-491 caused a large non-selective ionic coelomocyte pore and could directly kill the invasive AJ01. Moreover, activation of inflammatory AjCASP-1 was further found to be dependent on forming an inflammasome-like complex via CASc domain of AjCASP-1 and the N-terminal Ig domains of internalized AjNLRC4. All our results proved first evidence that lytic cell death was activated through MLKL cleavage, not MLKL phosphorylation in echinoderm, which offered insights into the functional, evolutionary mechanisms of lytic cell death in invertebrates.

Animals↗
PubMed2005

Use of laboratory toxicity tests with bivalve and echinoderm embryos to evaluate the bioavailability of copper in San Diego Bay, California, USA.

Copper concentrations in parts of San Diego Bay (CA, USA) exceed ambient water quality criteria (WQC; currently 3.1 microg/L dissolved, U.S. Environmental Protection Agency [U.S. EPA]). In order to better understand the bioavailability of copper to water-column organisms in the bay, toxicity tests were performed with copper added to surface water collected from various sites in the estuary over a three-year period. The species and endpoints used, bivalve and echinoderm embryo-larval development, are among the most sensitive in the U.S. EPA's national toxicity dataset, which is used to derive WQC. No toxicity was observed in ambient bay water samples, as indicated by high proportions of normally developed larvae in control treatments, averaging 93+/-5% across all sites and all sampling events. Median effects concentrations (EC50), obtained by copper spiking of ambient water samples, ranged from 1.7 to 3.4 times lower at sites located near the mouth compared to sites near the back of the bay. These data indicate a gradient in complexation capacity increasing from the mouth to the back of the bay, which is consistent with similar trends in dissolved organic carbon and total suspended solids. For the bay as a whole, estimates for total recoverable and dissolved water-effect ratios (WER) ranged from 2.07 to 2.27 and 1.54 to 1.67, respectively. Water-effect ratios of this magnitude suggest that adoption of a somewhat higher site-specific WQC for San Diego Bay still would achieve the level of protection that is intended by the WQC guidelines.

Animals↗