Isolation and localization of a spectrin-like protein from echinoderm sperm.

Thyone sperm undergo an explosive acrosome reaction resulting in the extension of a 90 microns long acrosomal process. In unreacted sperm, profilamentous actin is sequestered within the profilactin cup (Tilney: Journal of Cell Biology 69:73-89 1976), which consists of four major polypeptides: actin, profilin, and a 250/235 kDa equimolar doublet (TS 250/235). Dialysis of profilactin preparations into an actin assembly buffer resulted in the formation of acrosomal-like macromolecular aggregates containing actin, TS 250/235, and several other polypeptides as detected by SDS-PAGE. TS 250/235 was purified by subjecting extracts of pH solubilized profilactin cups to DEAE and phosphocellulose ion exchange chromatography. TS 250/235 demonstrated immunocrossreactivity with affinity purified polyclonal antibodies raised against S. purpuratus egg spectrin. As determined by biotinylated-calmodulin overlays, both subunits of TS 250/235 bound calmodulin in a Ca(++)-sensitive manner. Electron microscopy of low angle, rotary shadowed replicas of TS 250/235 revealed an elongate rod-shaped molecule with an average contour length of 203 nm. By indirect immunofluorescence, TS 250/235 was found to be uniformly distributed throughout the profilactin cup of the unreacted sperm. This distribution of TS 250/235 correlated with the location of monomeric actin as determined by localization studies utilizing fluorescent-DNase-1. Upon sperm activation, the cellular distribution of TS 250/235 dramatically changed and was observed both along the length and at the base of the extended acrosomal process.

[A comparative study of the biogenic amine receptors in the muscle tissue of mollusks, echinoderms and tunicates]. / Sravnitel'noe izuchenie retseptorov biogennykh aminov v myshechnoi tkani molliuskov, iglokozhikh i obolochnikov.

Smooth muscles of Mollusca, Echinodermata and Tunicata contain one class of adrenoreceptors with the dissociation constant and maximal specific binding 2.5 and 82 in Anodonta, 2.3 and 320 in Holothuria, 4.9 pM and 232.5 fmol/mg of protein in ascidia. Catecholamines and their antagonists can be ranged in the row as follows: isoproterenol greater than adrenalin greater than propranolol-noradrenaline greater than phentolamine. Negative regulation of the beta-adrenoreceptor affinity to isoproterenol by means of guanine nucleotides (GN) was shown. The muscular tissues of Mollusca, Echinodermata and Tunicata have only one class of the serotonin receptors with the dissociation constant and maximal specific binding 120 and 13.2 in Anodonta, 88 and 192 in Holothuria, 2.6 pM and 54 fmol/mg of protein in ascidia. The GTP negative regulation of serotonin receptors affinity to the hormone was found. The GN regulation of the above receptors affinity to agonist suggests that muscle tissue of the above species has specific GTP [correction of GTR]-binding proteins capable of coupling with these receptors.

Occurrence of chondroitin sulfate E in glycosaminoglycan isolated from the body wall of sea cucumber Stichopus japonicus.

Glycosaminoglycan was isolated from the body wall of sea cucumber Stichopus japonicus by a method consisting of enzymatic digestion, gel filtration, and ion-exchange chromatography. One gram of sea cucumber glycosaminoglycan was composed of 2.50 mmol of sulfate, 0.47 mmol of N-acetylgalactosamine (GalNAc), 0.53 mmol of glucuronic acid (GlcA), 1.73 mmol of fucose, and a small amount of peptide. When mildly hydrolyzed with 0.1 N H2SO4, this glycosaminoglycan released two products, one consisting of fucose plus sulfate and the other of fucose only. Partially hydrolyzed glycosaminoglycan thus obtained was composed of sulfate, GalNAc, GlcA, and fucose at a molar ratio of 3:2:2:1. Partially hydrolyzed glycosaminoglycan was easily digested with chondroitinase AC II. In ion-exchange chromatography, the digest exhibited four sharp peaks whose retention times agreed with those of unsaturated 0-(delta Di-0S), mono-(delta Di-4S and delta Di-6S), and di-(delta Di-SE) sulfated disaccharide, respectively. The disaccharide unit of sea cucumber glycosaminoglycan was composed of 22.4% chondroitin sulfate E, 11.2% chondroitin, 10.4% chondroitin 4-sulfate, and 56.0% chondroitin 6-sulfate.

Amino acid sequence of a major globin from the sea cucumber Paracaudina chilensis.

The sea cucumber Paracaudina chilensis (Echinodermata) contains three major globins I, II and III in coelomic cells. The complete amino acid sequence of globin I has been determined. It is composed of 157 amino acid residues, is acetylated at the N-terminus, and has a characteristic N-terminal extension of 9-10 residues when compared with vertebrate globins. The sequence of Paracaudina globin I showed slightly higher homology with human alpha globin (25%) rather than with the invertebrate Anadara alpha globin (22%). Paracaudina globin I also showed strong homology (59%) with globin D from another sea cucumber, Molpadia arenicola (Mauri, F.C. (1985) Ph.D. dissertation, University of Texas). The globin sequences from the phylum Echinodermata have an important position in the molecular evolution of the globins, because they are the invertebrate group most closely related to the vertebrates.

Purification and immunocytolocalization of protein phi 0 from sperm cells of the echinoderm Holothuria tubulosa.

Histones in chromatin from germ cells of the echinoderm Holothuria tubulosa are retained throughout spermatogenesis. However, some alterations occur in the histone complement of the mature sperm, including the presence of a germ-line-specific H1 subcomponent unusually rich in arginine, and the appearance of a basic component termed phi 0. Histones from ripe sperm have been extracted in a preparative scale to allow for isolation and purification of protein phi 0. Polyclonal antibodies against phi 0 have been produced and purified by affinity chromatography. The specificity of the antibodies to phi 0 has been assessed by enzyme-linked immunosorbent assays, competition experiments, and Western immunoblotting analysis. No cross-reactivity of the antibodies with the remainder histone fractions has been observed. Immunocytolocalization of protein phi 0 by immunogold labeling has revealed that this protein is essentially confined to chromatin from ripe sperm, whereas it is wholly absent from less advanced germ cell types. From these observations, together with biochemical studies previously reported, it is inferred that protein phi 0 may well be instrumental in the known chromatin transitions occurring in this organism during germ cell development.

Relaxing action of a holothurian toxin on mammalian smooth muscle.

1. The crude epidermal mucous secretion produced by the sea cucumber Holothuria mexicana Ludwig inhibits the tonus and spontaneous mechanical activity of rabbit ileum at concentrations of 0.1 mg/ml and higher. 2. This effect, similar to that of epinephrine, is attributed to a compound referred to as mucotoxin (MuTX). 3. MuTX also relaxes strips of the rabbit aorta contracted by 10(-8) M norepinephrine and exerts a similar, less marked, effect on the same strips contracted in high potassium solutions. 4. These observations suggest that the relaxing effect of MuTX on mammalian smooth muscle is not mediated by an adrenergic mechanism.

Occurrence of a unique fucose-branched chondroitin sulfate in the body wall of a sea cucumber.

The sulfated polysaccharides in the body wall of the sea cucumber occur as three fractions that differ markedly in molecular mass and chemical composition. The fraction containing a high molecular mass component has a high proportion of fucose and small amounts of galactose and amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction (F-2), which represents the major portion of the sea cucumber-sulfated polysaccharides, contains approximately equimolar quantities of glucuronic acid, N-acetyl galactosamine, and fucose, and has a sulfate content higher than that in the other two fractions. The structure of fraction F-2 was examined in detail. This polysaccharide has an unusual structure composed of a chondroitin sulfate-like core, containing side chain disaccharide units of sulfated fucopyranosyl linked to approximately half of the glucuronic acid moieties through the O-3 position of the acid. These unusual fucose branches obstruct the access of chondroitinases to the chondroitin sulfate core of F-2. However, after partial acid hydrolysis, which removes the sulfated fucose residues from the polymer, fraction F-2 is degraded by chondroitinases into 6-sulfated and nonsulfated disaccharides.

Phylogenetic study of the oxytocin-like immunoreactive system in invertebrates.

1. A phylogenetic study of oxytocin (OXT)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Oncidium verrucosum, Limax marginatus and Meretrix lamarckii of the Mollusca; and Baratha brassica of the Arthropoda. 3. No immunoreactive cells were found in Bipalium sp. of the Platyhelminthes; Pomacea canaliculata, Aplysia kurodai, Bradybaena similaris and Achatina fulica of the Mollusca; and Gnorimosphaeroma rayi, Procambarus clarkii, Hemigrapsus sanguineus, Helice tridens and Gryllus bimaculatus of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 4. These results demonstrate that an OXT-immunoreactive substance is widely present not only in vertebrates but also in invertebrates. 5. OXT seems to have been introduced into these invertebrates at an early stage of their phylogenetic history.

Phylogenetic study of the arginine-vasotocin/arginine-vasopressin-like immunoreactive system in invertebrates.

1. A phylogenetic study of arg-vasotocin (AVT)/arg-vasopressin (AVP)-like immunoreactive cells was performed by the PAP method in the central nervous system of invertebrates. 2. The immunoreactivity was detected in the nerve cells of Hydra magnipapillata of the Coelenterata; Neanthes japonica and Pheretima communissima of the Annelida; Pomacea canaliculata, Aplysia kurodai, Oncidium verrucosum, Bradybaena similaris, Achatina fulica, Limax marginatus and Meretrix lamarckii of the Mollusca; Gnorimosphaeroma rayi, Hemigrapsus sanguineus, Gryllus bimaculatus and Baratha brassicae of the Arthropoda; Asterina pectinifera of the Echinodermata; and Halocynthia roretzi of the Protochordata. 3. No immunoreactivity was detected in Bipalium sp. of the Platyhelminthes, or in Procambarus clarkii and Helice tridens of the Arthropoda. 4. From these results, it appears that AVT/AVP is a phylogenetically ancient peptide which is present in a wide variety of invertebrates. 5. The actions of AVT/AVP and its presence in invertebrates are discussed.

The hemolysin-producer coelomocytes in Holothuria polii.

Using sodium metrizoate discontinuous gradients, two hemolysin-producer amebocyte populations have been separated from total circulating Holothuria polii coelomocytes. The amebocytes of population 1 are responsible for the production of the calcium-dependent and temperature-labile hemolysin, whereas those of population 2 produce the calcium-independent and temperature-stable one. The intracytoplasmic hemolysins were evidenced also by immunofluorescence. Petaloid and filipodial amebocytes were the only positive cell types.

Highly acidic glycans from sea cucumbers. Isolation and fractionation of fucose-rich sulfated polysaccharides from the body wall of Ludwigothurea grisea.

The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose-rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. In Ludwigothurea grisea, glycans can be separated into three fractions which differ in molecular mass and chemical composition. The fraction containing a high-molecular-mass component has a high proportion of fucose and small amounts of amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction, which represents the major portion of the sea cucumber polysaccharides, contains besides fucose, approximately equimolar proportions of glucuronic acid and amino sugars, and has a sulfate content higher than that in the other two fractions. Both D and L-isomers of fucose are found in these polysaccharides, and the sulfate is linked to the O-3 position of the fucose residues. The attachment position of the sulfate groups to the glucuronic acid units and amino sugars is still undetermined. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber's body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue.

[Holotoxin content in the tissues of the holothurian Stichopus japonicus at different seasons of the year and their effect on oocyte maturation]. / Soderzhanie golotoksinov v tkaniakh goloturii Stichopus japonicus v raznye sezony goda i ikh vliianie na sozrevanie ootsitov.

Seasonal variations and distribution of holotoxins in tissues of the sea cucumber Stichopus japonicus have been studied. It was shown that holotoxin content in the ovaries is higher than that in other organs during the breeding season. In the course of a year, the amount of glycosides in other holothurian organs changed a little. It has been found out that high concentrations of glycosides inhibit oocyte maturation in the holothurian Stichopus japonicus.

Membrane damage by coelomic fluid from Holothuria polii (Echinodermata).

Rabbit erythrocyte membranes lyzed by Holothuria polii coelomic fluid, observed under the electron microscope, present lesions consisting of irregular holes which are heterogeneous in size (ranging from 50 A to 250 A) and ultrastructurally different from the ring-like structure produced by human complement. The protein pattern associated with the lyzed membrane was also examined.

Chlorinated insecticides and PCBs in Asteroidea and Holothuroidea species from Rijeka Bay, Yugoslavia.

For an evaluation of the extent of pollution in Rijeka Bay, Yugoslavia by chlorinated hydrocarbons an investigation of levels in sediments and the most abundant benthic organisms (Asteroidea and Holothuroidea species) has been carried out. Samples were collected at several stations in October 1981. The concentrations of chlorinated insecticides and polychlorinated biphenyls (PCBs) in Asteroidea species were from 0.5 to 46.0 micrograms kg-1 for DDTtotal, and from 2.5 to 435 micrograms kg-1 (wet weight) for PCBs. For the PCBs in Holothuroidea species the values ranged from 4.4 to 37.2 micrograms kg-1 and for DDTtotal from 0.9 to 48.5 micrograms kg-1. The contamination of Asteroidea and Holothuroidea samples from Rijeka Bay with persistent chlorinated hydrocarbons was compared with other data for the same species from other parts of the world. Accumulation of chlorinated hydrocarbons by Asteroidea and Holothuroidea species from sediments of Rijeka Bay is discussed with respect to concentration factors (CF). The highest CF (70.0) (dry biota/dry sediment) was obtained for the intestines of Asteroidea species for PCBs and a value of only 2.6 was obtained for DDTtotal for the skin and muscle of Holothuroidea species. The relatively low bioaccumulation of PCBs and DDTs from sediments in the Rijeka Bay by the Asteroidea and Holothuroidea species is reassuring, because of the problem of dredging and ocean dumping of sediments polluted with persistent chlorinated hydrocarbons.

Triterpene glycosides from sea cucumber Stichopus japonicus Selenka.

Triterpenoidal oligoglycosides from the far eastern sea cucumber Stitchopus japonicus Selenka collected in the Posiet Bay, Japan Sea were investigated. The glycoside fraction was shown to consist of two main components, holotoxins A1 and B1 which have been isolated by chromatography on silica gel columns. The chemical structures of the holotoxins were elucidated on the basis of chemical and physiochemical evidence (for formula see text). The proposed structures were compared with close related but not identical structures of holotoxin A and B from other collection of the same holothurian. The probable reasons of the distinction were discussed. The previously-established difference in glycoside structures between Stichopus japonicus and other representatives of the family Stichopodidae was corroborated.

Heterogeneity of the histone-containing chromatin in sea cucumber spermatozoa. Distribution of the basic protein phi 0 and absence of non-histone proteins.

Nuclei of spermatozoa of the sea cucumber Holothuria tubulosa contain the five somatic-type histones plus a sperm-specific histone H1 and a unique basic protein phi 0, which is related to H1 in amino acid composition. No proteins of the High Mobility Group (HMG) type have been detected. The structure of this chromatin has been probed nuclease digestion. Its behaviour is anomalous, since two distinct fractions of chromatin are recovered from these spermatozoa, which differ either in the presence or absence of the sperm-specific proteins H1 and phi 0. This heterogeneous distribution is not found in conventional materials, such as calf thymus or chicken erythrocytes. Proteins H1 and phi 0 are not uniformly distributed and may be localized in special regions of chromatin. Fragments containing long stretches of nucleosomes lacking both proteins can be recovered. At the same time, the chromatin fractions which contain these two proteins are shown to be less soluble. When an extensive digestion of chromatin is carried out yielding only nucleosomes and small oligomers, the H1 and phi 0 proteins redistribute themselves on chromatin, the two proteins acting in a cooperative fashion in this process. Cross-linking experiments carried out in whole cells indicate a proximity of phi 0 and H1, whereas no crosslinks have been detected between phi 0 and any of the four nucleosomal histones. The phi 0 protein may thus play a role similar to histone H1 and be only loosely associated with nucleosomal histones, but contribute to the structuration of chromatin during spermiogenesis.