The action of <Emphasis Type="Italic">Laminaria japonica</Emphasis> extractives on 1,3-β-D-glucanase,a digestive enzyme of the <Emphasis Type="Italic">Strongylocentrotus intermedius</Emphasis> sea urchin

Nutritional attractiveness of the brown alga Laminaria japonica for the sea urchin Strongylocentrotus intermedius was studied. The composition of L. japonica was analyzed after one and two years of its life under natural conditions, in its seedlings, and in the alga partially degraded by natural factors. Substances extracted with various solvents were tested for the presence of inhibitors and activators of 1,3-β-D-glucanase, a digestive enzyme of the sea urchin. Ethanolic extract of freshly harvested L. japonica was found to suppress the enzyme activity. Substances present in ethanolic extracts of the alga after one or two years of its life cycle and in the alga, partly degraded by natural factors, activated the sea urchin enzyme. This fact is in agreement with earlier natural observations concerning the nutritional attractiveness of such L. japonica samples for Strongylocentrotus intermedius.     

The role of circadian rhythm and environmental factors in the regulation of sea urchin spawning

Simultaneous video recording of the sea urchin spawning behavior and recording of environmental factors enabled us to found synergism of the effects of circadian rhythm and phytoplankton on the spawning activity of the sea urchin Strongylocentrotus intermedius (Agassiz, 1864).     

Distribution of intracellular fucoidan hydrolases among marine bacteria of the family Flavobacteriaceae

A search for fucoidan-degrading enzymes and other O-glycosylhydrolases has been performed among 51 strains of marine bacteria of the family Flavobacteriaceae isolated from red, green, and brown algae, as well as from the sea urchin Strongylocentrotus intermedius and the holothurian Apostichopus japonicus. Over 40% of the studied strains synthesized fucoidanases. The marine bacteria Mesonia algae KMM 3909(T) (an isolate from green alga Acrosiphonia sonderi), as well as Maribacter sp. KMM 6211 and Gramella sp. KMM 6054 (associants of the sea urchin S. intermedius), were the best producers of fucoidanases. Xylose effectively induced the biosynthesis of fucoidanases in these strains. None of the 15 strains of marine bacteria belonging to the genus Arenibacter produced polysaccharide hydrolases.     

The interaction of DNA with sperm-specific histones of the H1 family

We have studied the interactions of DNA with sperm-specific histones of the H1 family of sea urchin Strongylocentrotus intermedius, sea starfish Aphelasterias japonica and bivalve mollusk Chlamis islandicus using circular dichroism and DNA melting analysis. It was shown that echinoderm's sperm H1 protein has additional alpha-helical domains in its C-terminus and it demonstrates stronger DNA compaction. The differential melting curves of DNA-protein complexes have two peaks. The low temperature peak characterized the melting temperature of free DNA within the complex. The higher temperature peak characterizes the melting temperature of DNA bond to protein. DNA is found to be in the most stable state in the complexes with mollusk sperm H1 protein.     

Purification and characterization of trypsin-like enzyme from sea urchin eggs: substrate specificity and physiological role

A trypsin-like enzyme has been purified to homogeneity from eggs of the sea urchin, Strongylocentrotus intermedius. The purified enzyme efficiently hydrolyzed Z-Phe-Arg-4- methylcoumaryl -7-amide (MCA) and Pro-Phe-Arg-MCA among 12 peptidyl-Arg (or Lys)- MCAs . The substrate specificity of the enzyme was closely similar to that of the enzyme activity in the egg cortical granule exudate. Among various peptidyl-argininal (Arg-H) derivatives, Z-Phe-Arg-H and Z-Phe-Leu-Arg-H showed the strongest inhibition against both the activity of the purified enzyme and the elevation of vitelline coat. Thus, the trypsin-like enzyme of sea urchin possesses a narrow substrate specificity and participates at least in the elevation of vitelline coat during fertilization.     

Study of dynein ATPase by oxygen isotope exchange H2(18)O in equilibrium with [16O]-Pi

The oxygen isotope exchange reactions catalyzed by sea urchin Strongylocentrotus intermedius spermatozoa dynein I were studied with a view of comparing molecular mechanisms of ATP hydrolysis by dynein and myosin ATPases. It was demonstrated that the isotope exchange takes place during ATP hydrolysis and during enzyme incubation with ADP and Pi and is absent when the enzyme is incubated with Pi. It was assumed that the molecular mechanisms of ATP hydrolysis by dynein I and myosin are identical.     

Gangliosides of sea urchin embryos. Their localization and participation in early development

The influence of antibodies to gangliosides of sea urchin Strongylocentrotus intermedius eggs on early embryos of this species was studied. gamma-Globulins were isolated from rabbit anti-ganglioside serum by micropreparative electrophoresis. These gamma-globulins produced anomalies in the development of embryos permeabilized in Triton X-100. The anomalies were not observed when anti-ganglioside gamma-globulins were added to the incubation medium together with gangliosides or when the permeabilized embryos were incubated with gamma-globulins of normal rabbit serum. Pretreatment of S. intermedius embryos with serotonin, tryptamine or some other indole derivatives led to the disappearance of ganglioside determinants from the cell surface and sharply increased immunofluorescence within the cell. Such pretreatment of embryos increased the amount of cell-associated gangliosides more than threefold as compared to untreated embryos. Serotonin was shown to bind specifically to sea urchin gangliosides immobilized on octyl-Sepharose. These observations suggest that cell-surface gangliosides, after binding drugs, are internalized and that serotonin and its antagonists inhibit the transport of newly synthesized gangliosides to the cell-surface membrane.     

Lytic activity and biochemical properties of lysozyme in the coelomic fluid of the sea urchin strongylocentrotus intermedius

Lysozyme was identified in the coelomic fluid including coelomocytes of the sea urchin Strongylocentrotus intermedius, and its lytic activity and biochemical properties were examined in this study. The urchin lysozyme was electrophoretically fractionated to a single lytic band of about 14 kDa. No distinct difference in the lytic activity of this enzyme was found between urchins held at two temperatures, 11 degrees and 25 degrees C. The lysozyme of this species was purified through several procedures: salting out with ammonium sulfate, precipitation by ethanol saturation, gel filtration with a Biogel column, and an affinity chromatography with a heparin Sepharose column. The combination method of Biogel filtration and affinity chromatography resulted in the most purified lysozyme fraction, but we could not obtain a single protein band in SDS-PAGE. In addition, anti-hen egg white lysozyme (HEWL) antibody was produced and confirmed to react specifically with the urchin lysozyme in this study. Therefore, the HEWL antibody may be available for examining the lytic activity of lysozyme at an individual level to determine the biodefense activity of sea urchins. Copyright 1999 Academic Press.     

Use of biological methods in the analysis of the purinergic activity of etimizol

To analyse etimizol purinergic activity, methods of biological analysis were applied using isolated smooth muscle strips of the guinea-pig caecum and developing embryo cultures of the sea urchin (Strongylocentrotus intermedius). Stimulating etimizol action in 10(-8)-10(-12) M concentration was observed on embryonal development in the sea urchin to be eliminated by co-action of etimizol and P1-purinoceptor antagonist theophylline. The analogous action of the preparations was established using isolated smooth muscle strips of the guinea-pig caecum.     

Thymidine kinase from sea urchin oocytes

Thymidine kinase was isolated and purified 1600-fold from sea urchin (Strongylocentrotus intermedius) oocytes. The molecular mass of the enzyme is 66 kDa, pI is 5.2. The enzyme activity needs Mg2+, ATP and the corresponding phosphate acceptor. The pH optimum of the enzyme activity is at 9.0-9.5. The isolated enzyme does not exhibit any strict substrate specificity and can phosphorylate thymidine, deoxycytidine and some other pyrimidine nucleosides and their derivatives, the phosphorylation rate being maximal for thymidine, ATP or dATP. The TMP formed via the enzymatic reaction does not influence the thymidine kinase activity.     

Effect of 1,3;1,6-β-D-glucans on Developing Sea Urchin Embryos

The effect of 1,3;1,6-beta-D-glucooligo- and polysaccharides with different structures (from 1 to 10 kDa of molecular mass; from 10-25% of beta-1,6-linked glucose residues content) on the developing embryos of sea urchin, Strongylocentrotus intermedius, was evaluated for the screening of potential positive stimulants. 1,3;1,6-beta-D-glucans with a molecular mass of between 6-10 kDa and at concentrations of 0.05-0.25 mg/ml shown the best modulator effect on the sea urchin embryos. 1,3;1,6-beta-D-glucans increased the survival of the sea urchin embryos up to 2.5-fold compared with the control animals.     

Change in phosvitin kinase activity during early development of the sea urchin

Protein kinase, which phosphorylated phosvitin at the expense of ATP but did not phosphorylate casein, protamine, and histone mixture, was obtained by DEAE-cellulose column chromatography of the extract from the embryos of the sea urchin, Strongylocentrotus intermedius. This enzyme, partially purified by DEAE-cellulose column, reversibly catalyzed the reaction of phosvitin phosphorylation. This indicates that the sea urchin embryos contain phosvitin kinase. Phosvitin kinase in sea urchin embryos is somewhat different from that found in the other types of cells, which are able to phosphorylate casein as well as phosvitin. In unfertilized eggs, the activity of this enzyme was found only in the supernatant fraction obtained by centrifuging the homogenate at 10,000g for 20 min. The activity in the embryos at the swimming and the mesenchyme blastula stage was higher than in unfertilized eggs, and was localized in the sedimentable fraction obtained by centrifuging the homogenate of the embryos at 10,000g for 20 min. The highest activity of phosvitin kinase was observed in the embryos at the mesenchyme blastula stage, and the enzyme activity became quite low at the late gastrula stage. The activity and the intracellular distribution of phosvitin kinase changed during the development. The enzyme in this sedimentable fraction was not solubilized with 1% Triton X-100 but was extracted by 1 M NaCl.     

Sea urchin sperm DNP. I. Chemical composition and template properties of DNP

Electrophoretic mobility, amino acid composition and salt dissociation of histones isolated from sperm of sea urchin Strongylocentrotus intermedius and calf thymus cells were studied. The special arginine-rich histone fraction (I) has been observed in sea urchin sperm chromatin, this fraction being absent in calf thymus chromatin. Dissociation of lysine-containing histone fractions from sea urchin chromatin occured in the range of 0.7 to 1.0 M NaCl concentrations. H1 of calf thymus chromatin was totally extracted with 0.6 M NaCl. In the course of a further increase of salt concentrations (up to 1.5 M NaCl) a practically total extraction of histones from sperm chromatin was observed, while about 20% of proteins remained bound to DNA in thymus chromatin after extraction with 2.0 M NaCl. The template activity of non-extracted DNP preparations from urchin sperm was equal to 2-3% of that of totally deproteinized DNA. The template activity of DNP gradually increased at protein extraction from DNP preparations. The hybridization capacity of RNA transcribed on partially dehistonized DNP templates in vitro also increased.     

Effects of removing sea urchins (Strongylocentrotus droebachiensis): Stability of the barren state and succession of kelp forest recovery in the east Atlantic

Stability properties of the barren state of a kelp forest-sea urchin system were studied in northern Norway. The ability of the sea urchin Strongylocentrotus droebachiensis to maintain high population densities and recover from perturbations, and the succession of kelp forest revegetation, were studied experimentally by reducing the sea urchin density on a barren skerry. Additional information was obtained from community changes following a natural, but patchy, sea urchin mortality that varied between sites. On the barren grounds, high sea urchin densities (30 50 per m²) is maintained by annual recruitment. Severe reductions of sea urchin densities initiated luxuriant kelp growth, while more moderate reductions allowed establishment of opportunistic algae (during spring and early summer), but no kelps. Succession of algal growth, after the severe decline in sea urchin densities, followed a predictable pattern. At first the substrate was colonized by filamentous algae, but within few weeks they were outcompeted by the fast growing kelp Laminaria saccharina. After 3–4 years of the removal experiment, the slower-growing, long-lived kelp L. hyperborea became increasingly dominant. Increased food availability after reduction in sea urchin density led to increased individual growth of the remaining sea urchins. However, the population density did not increase, neither from recruitment nor immigration from adjacent areas with high sea urchin densities. Possibly, early establishment of a dense kelp stand, may represent a breakpoint in the ability of sea urchins to reestablish a barren state. The ability of L. saccharina quickly to invade and monopolize an area may have both positive and negative effects on the succession towards the climax L. hyperborea kelp forest. Competitive interactions may slow the process, but development of a dense stand of L. saccharina will also reduce grazing risk on scattered recruits of the more slowly growing L. hyperborea.     

Does Phospholipase A2 Participate in the Acrosome Reaction of Sea Urchin Sperm?: A Pharmacological Study

We examined whether phospholipase A₂ (PLA₂) is involved in the initiation of the acrosome reaction of sperm of the sea urchin, Strongylocentrotus intermedius , using inhibitors and an activator of this enzyme. Quinacrine and p-bromophenacyl bromide (PBPB) inhibited the egg jelly-induced acrosome reaction at 100 μM, but not the ionomycin-induced one. Depression of egg jelly-induced increase of intracellular free Ca²⁺concentration ([Ca²⁺]_i) by these reagents was expected and examined using fura 2. Quinacrine interfered with the flourescence of fura 2, but PBPB was found to depress at concentrations which could inhibit the acrosome reaction. Furthermore, melittin, which is known to stimulate PLA², caused a [Ca²⁺]_i increase and a formation of acrosomal process-like structure on sperm head. These results suggest that PLA₂ participates in the early step(s) of the acrosome reaction of sea urchin sperm.     

Lack of age-associated telomere shortening in long- and short-lived species of sea urchins

The red sea urchin, Strongylocentrotus franciscanus, can live in excess of 100 years while the sea urchin Lytechinus variegatus has an estimated lifespan of only 3-4 years. In an effort to understand the molecular mechanism underlying the difference in their longevity we characterized telomere biology in these species of sea urchins. Telomerase activity was found throughout early stages of development in L. variegatus and is maintained in adult tissues of L. variegatus and S. franciscanus. Terminal restriction fragment analysis indicated a lack of age-associated telomere shortening. These data suggest that long- and short-lived sea urchins do not utilize telomerase repression as a mechanism to suppress neoplastic transformation.     

MOLECULAR PROPERTIES AND DIFFERENTIATION OF ACETYLCHOLINESTERASE IN SEA URCHIN EMBRYOS*

The two molecular forms of acethylcholinesterase (EC 3.1.1.7) in sea urchin embryos were characterized by several physical methods. The sedimentation coefficients determined by sucrose gradient centrifugation are 7.6S and 10.6S. The Stokes radii determined by gel filtration are 65 Å and 91 Å. From these parameters, molecular weights were estimated as 190,000 and 380,000; the one is twice as large as the other. Both forms have similar electric property and buoyant density in a CsCl gradient. When the enzyme solution was concentrated, the 10.6S form became predominant. These results suggest that the two forms are monomer and dimer. The sea urchin enzymes resemble globular forms of acetylcholinesterase of the electric organ of fishes. The activity of the enzyme abruptly increases in post-gastrulation embryos. Inhibition of concomitant protein synthesis by a specific inhibitor, emetine, does not affect the increase in enzyme activity. The result suggests that post-translational processes may be involved in the differentiation of this enzyme in sea urchin development. The following sea urchins were used in the study: Strongylocentrotus purpuratus, Strongylocentrotus franciscanus, and Dendraster excentricus.     

Micromere Differentiation in the Sea Urchin Embryo: Immunochemical Characterization of Primary Mesenchyme Cell-Specific Antigen and Its Biological Roles

Primary mesenchyme cell (PMC)-specific antigens in developing sea urchin embryos of five different species have been studied by using two different monoclonal antibodies, P4 and B2C2. Like B2C2 in Strongylocentrotus purpuratus (Anstrom et al. , 1987) P4 reacted with the N-linked carbohydrate in Strongylocentrotus intermedius embryo. Although both antibodies recognize the same group of glycoproteins in S. intermedius , P4 epitopes appeared earlier than B2C2 epitopes in Clypeaster japonicus embryo. PMCs of Anthocidaris crassispina blastulae raised in sulfate-deficient sea water were immuno-reactive with P4 but not with B2C2, although the embryos raised in normal sea water reacted with both antibodies at similar intensity. These results suggest that the epitopes of P4 and B2C2 are formed by glycosylation and sulfation, respectively. PMCs may display differential modification in their surface glycoprotein synthesis during differentiation. Furthermore, P4 inhibited cultured micromere descendant cells of Hemicentrotus pulcherrimus from attaching to the plastic dishes and forming spicules in vitro without detectable cytotoxic effect. P4-reactive glycoproteins may play important roles in cell-substrate interaction and spicule formation.     

Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus

Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases.     

Isolation and properties of the initiation of DNA replication in the sea urchin embryo

Using the instability of replication loops as a method for the isolation of double-stranded nascent DNA from embryos of the sea urchin Strongylocentrotus intermedius, extruded DNA enriched for replication origins was obtained. The average length of the fragments of the DNA of this fraction was estimated to be about 800 base pairs. The origin-enriched nascent DNA strands were assayed for the presence of inverted repeats. The results show that the origin-enriched DNA is also enriched in inverted repeats. The bulk of palindromes in the total nuclear DNA was estimated to be 200 base pairs in length and from the origin-enriched DNA-150 base pairs.