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PubMed2003

Expression, purification, and characterization of arginine kinase from the sea cucumber Stichopus japonicus.

The arginine kinase gene of sea cucumber Stichopus japonicus was cloned and inserted into the prokaryotic expression plasmid pET-21b. The protein was expressed in a soluble and functional form in Escherichia coli and purified by Blue Sepharose CL-6B, DEAE-32, and Sephadex G-100 chromotography with a final yield of 83 mgL(-1) of LB medium. The specific activity, electrophoretic mobility, and isoelectric focusing were all identical with those of arginine kinase that was purified from sea cucumber muscle. The fluorescence emission spectrum of arginine kinase had a maximum fluorescence at a wavelength of 330 nm upon excitation at 295 nm. These results are the first report of this purified protein.

Animals↗

PubMed2003

Urea induced inactivation and unfolding of arginine kinase from the sea cucumber Stichopus japonicus.

Urea titration was used to study the inactivation and unfolding equilibrium of arginine kinase (AK) from the sea cucumber Stichopus japonicus. Both fluorescence spectral and circular dichroism spectral data indicated that an unfolding intermediate of AK existed in the presence of 1.0 to 2.0 M urea. This was further supported by the results of size exclusion chromatography. The spectral data suggested that this unfolding intermediate shared many structural characteristics with the native form of AK including its secondary structure, tertiary structure, as well as its quaternary structure. Furthermore, according to the residual activity curve, this unfolding intermediate form still retained its catalytic function although its activity was lower than that of native AK. Taken together, the results of our study give direct evidence that an intermediate with partial activity exists in unfolding equilibrium states of AK during titration with urea.

Animals↗

PubMed2002

Enhancement of t-PA-mediated plasminogen activation by partially defucosylated glycosaminoglycans from the sea cucumber Stichopus japonicus.

Sea cucumber glycosaminoglycan (SC-GAG) was isolated from the body wall of the sea cucumber Stichopus japonicus. The SC-GAG consists of a chondroitin sulfate E-type core polymer with sulfated fucose branches attaching glycosidically to almost every disaccharide unit of the core polymer at the C-3 position of the GlcA or at C-4 and/or C-6 position(s) of GalNAc. SC-GAG was subjected to mild acid-hydrolysis, which cleaved selectively the glycosidic linkages between the core polymer and the fucose branches, resulting in two types of partially defucosylated SC-GAG derivatives. One type (type A), obtained by 3 h-hydrolysis, contained 33% of the fucose branches and the other type (type B), obtained by 6-h hydrolysis, contained 10% of the fucose branches. The molecular masses of types A and B were determined to be 8 and 4 kDa, respectively, by gel permeation HPLC. A chondroitinase ABC (Chase ABC)-digestion demonstrated that types A and B contained 46 and 66% of digestable disaccharide units, respectively, and both types contained 29% of E-type unsaturated disaccharide units bearing no fucose branches. Intact SC-GAG and types A and B were compared for t-PA-mediated plasminogen activation by an in vitro assay system. Although intact SC-GAG and type B exhibited rather weak activity at 6.25 microg/ml, type A exhibited 5 to 10-fold higher activity than intact SC-GAG and type B at the same concentration. The activity of type A was almost one-third that of purified chondroitin sulfate E (127 kDa containing 64.5% E-type disaccharide units) from squid cartilage at 6.25 microg/ml concentration. These results suggest that t-PA-mediated plasminogen activation requires the presence of E-type disaccharide units bearing no fucose branches and a molecular mass larger than 7.5 kDa in terms of the chondroitin sulfate E structure with or without fucose branching.

Animals↗

PubMed1994

Amino acid sequence of a lectin from the sea cucumber, Stichopus japonicus, and its structural relationship to the C-type animal lectin family.

The complete amino acid sequence of SJL-I, a lectin from the sea cucumber, Stichopus japonicus, was determined by sequence analysis of peptides derived on enzymatic and chemical fragmentation of the protein. SJL-I consists of 143 amino acid residues and its molecular mass was calculated to be 15,837 Da. Comparison of the sequence of SJL-I with a database revealed that SJL-I exhibits apparent homology with C-type lectins, especially with those of marine invertebrates. The highest homology (identity 28.6%) was found with echinoidin, a lectin from the sea urchin, Anthocidaris crassispina. Comparison of the sequence of SJL-I with those of other C-type lectins indicated that the conserved amino acids are relatively abundant in the C-terminal half of their carbohydrate-recognition domains (CRDs), that can be considered to be involved in binding with Ca2+ as well as carbohydrates.

Amino Acid Sequence↗

PubMed1994

Purification and characterization of two Ca(2+)-dependent lectins from coelomic plasma of sea cucumber, Stichopus japonicus.

Two structurally distinct lectins were purified from the coelomic plasma of holothurian, Stichopus japonicus, by affinity chromatography on a porcine stomach mucin-conjugated agarose column, gel filtration on a Superose 6 column, and ion-exchange chromatography on a HiTrap Q-FPLC. The two lectins showed apparent molecular masses of about 400 kDa (SPL-1) and 60 kDa (SPL-2) on gel filtration, but about 17 kDa on SDS-PAGE under reducing conditions. Both lectins showed hemagglutination activity toward rabbit erythrocytes in the presence of Ca2+ ions. The N-terminal amino acid sequences were highly homologous to but distinct from those of a Ca(2+)-dependent (C-type) lectin named SJL-I purified from the same species. In addition to porcine stomach mucin, the hemagglutination activity of SPL-1 was strongly inhibited by uronic acids such as galacturonic acid, and glucuronic acid, while the activity of SPL-2 was inhibited by GalNAc and galactosides. Both lectins were adsorbed on clotted coelomocytes in the presence of Ca2+ but not in the presence of inhibitory sugars or EGTA, suggesting the presence of an endogenous carbohydrate ligand(s) for plasma C-type lectins in the clot. However, coelomocyte clotting occurred normally even in the presence of inhibitory sugars, but was strongly inhibited by synthetic GRGDSP peptide or EGTA, suggesting the participation of integrin but not the lectin-carbohydrate interaction in the clotting events.

Animals↗

PubMed1994

EFFECT OF MECHANICAL VIBRATION ON ACTIVE TENSION IN THE LONGITUDINAL RETRACTOR MUSCLE OF A SEA CUCUMBER STICHOPUS JAPONICUS

1. The effect of mechanical vibration on active tension in an echinoderm somatic smooth muscle was studied using the longitudinal retractor muscle (LRM) of a sea cucumber Stichopus japonicus. 2. The steady contracture tension in LRM fibres maximally activated with 10(-3) mol l-1 acetylcholine (ACh) was reduced by vibrations (peak-to-peak amplitude, 0.52.5 % of l0, where l0 is the slack length of the muscle; frequency, 5100 Hz). The extent of reduction of active contracture tension increased with increasing amplitude of vibration, but it did not change appreciably with increasing frequency of vibration. 3. The steady contracture tension in LRM fibres submaximally activated with 10(-5) mol l-1 ACh was more markedly reduced by vibrations than was that in maximally activated fibres. 4. The vibration-induced reduction of active contracture tension disappeared when temperature was lowered from 2023 to 0 °C. 5. The development of contracture tension in LRM fibres activated with ACh was not affected by mechanical vibration. 6. These results are discussed in connection with the vibration-induced decrease in the rate of breakage of the actinmyosin linkages responsible for isometric force generation.

Journal Article↗

PubMed1982

Physiological and ultrastructural studies on the longitudinal retractor muscle of a sea cucumber Stichopus japonicus. II. Intracellular localization and translocation of activator calcium during mechanical activity.

1. The intracellular localization and translocation of activator Ca in the longitudinal retractor muscle (LRM) of a sea cucumber Stichopus japonicus were studied by fixing the LRM in a 1% OsO4 solution containing 2% K pyroantimonate. 2. In the resting LRM fibres, electron-opaque pyroantimonate precipitate was mostly localized along the inner surface of the plasma membrane and at the subsarcolemmal vesicles in close apposition to the plasma membrane. 3. In the LRM fibres fixed during the mechanical response to ACh and high [K]0, the precipitate was diffusely distributed in the myoplasm in the form of numerous particles with corresponding decrease in the amount of the precipitate at the peripheral structures. 4. Electron probe X-ray microanalysis showed the presence of Ca in the precipitate, indicating that the precipitate provides a valid measure of Ca localization. 5. These results accord with the view that, in the LRM, the contractile mechanism is activated by the release of Ca from the intracellular structures as well as by the inward movement of extracellular Ca.

Acetylcholine↗

PubMed2003

Constituents of Holothuroidea, 13. Structure of neuritogenic active ganglioside molecular species from the sea cucumber Stichopus chloronotus.

Three ganglioside molecular species, SCG-1, SCG-2, and SCG-3, were obtained from the lipid fraction of the chloroform-methanol extract of the sea cucumber Stichopus chloronotus. On the basis of chemical and spectroscopic evidence, the structures of these gangliosides have been determined to be 1-O-[(N-glycolyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (SCG-1), 1-O-[8-O-sulfo(major)-(N-acetyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (SCG-2), and 1-O-[alpha-L-fucopyranosyl-(1-->11)-(N-glycolyl-alpha-D-neuraminosyl)-(2-->6)-beta-D-glucopyranosyl]-ceramide (SCG-3). The ceramide moieties were composed of heterogeneous long-chain base and fatty acid units. SCG-3 is the first type of ganglioside containing a fucopyranose in the sialosyl trisaccharide moiety. Moreover, these three gangliosides exhibited neuritogenic activity toward the rat pheochromocytoma PC12 cells in the presence of nerve growth factor.

Animals↗

PubMed2005

Constituents of holothuroidea, 14. Isolation and structure of new glucocerebroside molecular species from the sea cucumber Stichopus japonicus.

Five glucocerebroside molecular species, SJC-1-SJC-5, have been isolated from the less polar lipid fraction of a chloroform/methanol extract of the sea cucumber Stichopus japonicus. The structures of these glucocerebroside molecular species were determined on the basis of chemical and spectroscopic evidence. SJC-1, SJC-2, and SJC-3 are typical sphingosine- and phytosphingosine-type glucocerebroside molecular species with nonhydroxylated and hydroxylated fatty acyl moieties. SJC-4 and SJC-5 are also sphingosine-type glucocerebroside molecular species with hydroxylated fatty acyl moieties, although they are new glucocerebroside molecular species with unique sphingosine bases.

Animals↗

PubMed1993

Purification and characterization of two lectins from the sea cucumber Stichopus japonicus.

Two Ca(2+)-dependent lectins were purified from the sea cucumber Stichopus japonicus by affinity chromatography on lactosyl-Sepharose 4B and ion-exchange chromatography on Q-Sepharose. Their molecular masses were estimated to be 13kDa (SJL-I) and 15 kDa (SJL-II) on SDS-PAGE. SJL-I agglutinated rabbit erythrocytes as well as human A, B, and O-type erythrocytes, but SJL-II agglutinated only rabbit erythrocytes. Hemagglutination by SJL-I was competitively inhibited by N-acetyl-D-galactosamine and galactose-containing carbohydrates. On the other hand, only lactose, melibiose, and raffinose gave weak inhibition of hemagglutination by SJL-II, suggesting that SJL-II may have high specificity for particular complex carbohydrate(s) on the surface of rabbit erythrocytes. SJL-II was activated at ten times lower Ca2+ concentration than SJL-I. Both lectins lost activity in acidic pH, while SJL-I appeared more stable down to pH 4.5.

Amino Acids↗

PubMed1990

Glycosaminoglycan involved in the cation-induced change of body wall structure of sea cucumber Stichopus japonicus.

The body wall of sea cucumber Stichopus japonicus was treated with various concentrations of several cations, and examined for changes in toughness, taking punch force as parameter. Toughness of the body wall tended to decrease with increasing concentration of each cation, but in different modes depending mainly upon the valency of cation: e.g., the body wall completely lost toughness in 0.3 M Na+ or 0.4 M K+, whereas it retained more than half the initial toughness even in 0.4 M Ca2+ or Mg2+. Glycosaminoglycan (GAG) from the body wall was dissolved in water, and examined for viscosity changes as caused by those cations. Specific viscosity (eta sp) decreased from 0.71 (without cation) to 0.47-0.57 in the presence of 0.1 M monovalent and divalent cations. At 0.4 M, monovalent cations reduced eta sp to 0.38-0.46, but divalent cations increased eta sp to 0.56-0.63. Electron microscopy demonstrated that GAG matrix was clearly observed in the absence of cation, but disappeared in 0.4 M NaCl, forming wide free spaces in the body wall. These results all suggested that GAG is closely involved in the change of toughness of sea cucumber body wall.

Animals↗

PubMed1991

Antithrombotic and anticoagulant activity of depolymerized fragment of the glycosaminoglycan extracted from Stichopus japonicus Selenka.

The antithrombotic and anticoagulant activities of depolymerized fragment (DHG-1) of glycosaminoglycan extracted from Stichopus japonicus Selenka (FGAG) were compared with those of unfractionated heparin (UFH) or low molecular weight heparin (LMWH). DHG-1 at more than 0.3 mg/kg i.v. significantly prevented death of mice treated with thrombin (800 U/kg i.v.). Under the same conditions, FGAG, UFH and LMWH significantly prevented death of mice at more than 0.3, 0.3 and 0.6 mg/kg i.v., respectively. In normal plasma, the concentration required to double the activated partial thromboplastin time (doubling APTT) of DHG-1, FGAG, LMWH and UFH were 12.0, 2.4, 5.8, and 1.2 micrograms/ml, respectively. In antithrombin III (AT III)-depleted plasma, doubling APTT of DHG-1, FGAG, and UFH were 11.3, 2.1, and 18.5 micrograms/ml, respectively. Prothrombin activation in contact-activated plasma was inhibited completely for 60 s at doubling APTT by all glycosaminoglycans used in this study. DHG-1, however, showed much less antithrombin activity than UFH as tested by thrombin clotting time in plasma and chromogenic assay in the presence of AT III. Moreover, DHG-1 showed much less inhibitory activity on factor Xa, factor IXa, and glass surface-induced factor IXa generation than UFH. These results suggested that DHG-1 is one of the promising antithrombotic agents with quite different anticoagulant property from UFH or LMWH.

Animals↗

PubMed1990

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.

Animals↗

PubMed1990

[Binding of acid mucopolysaccharides of Stichopus japanicus to washed human platelets].

The binding of acid mucopolysaccharides of Stichopus japonicus Selenka (Sjamp) to washed human platelets was studied using [3H]Sjamp. The binding was rapid, reversible and with a saturable tendency. The binding reached to equilibrium at about 10 min, and 50% of [3H]Sjamp bound to the platelets at equilibrium within the first minute. When excess unlabeled Sjamp was added to the [3H]Sjamp binding suspension, [3H]Sjamp dissociated from the platelets rapidly and rather completely. Scatchard analysis revealed a class of binding sites with Kd = 2.3 +/- 0.7 micrograms/ml and Bmax = 4.5 +/- 1.2 micrograms/10(9) platelets. Separate experiments showed that the monoclonal antibody (McAb) SZ-21, a McAb to platelet membrane glycoprotein (GP) IIIa, weakened the binding, while McAb SZ-2 and SZ-22 (to GP Ib and GP IIb respectively) were ineffective. We also observed that heparin inhibited [3H]Sjamp binding to the platelets. These results indicated that the binding sites on platelets might be the Sjamp receptors. We conclude that receptor-Sjamp binding is an important process in platelet aggregation induced by Sjamp.

Animals↗

PubMed1988

Mechanism of rabbit platelet agglutination induced by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka.

The effects of acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus Selenka) (SJAMP) on rabbit platelets were studied. Using citrated platelet-rich plasma (PRP), washed platelets, and formaldehyde fixed platelets from 10 New Zealand white rabbits, we investigated the effects of platelet inhibitors and various plasma and its fractions on SJAMP-induced agglutination. It was found that the tracing of platelet agglutination induced by SJAMP showed a single phase without a lag period. The lowest concentration of SJAMP required for the agglutination of rabbit platelets was approximately 2 micrograms/ml, and the magnitude of agglutination induced by SJAMP was concentration dependent. In 8 out of 10 rabbits, the platelets in PRP were agglutinated by 10 micrograms/ml of SJAMP. Platelet inhibitors, such as aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose and EDTA did not inhibit the agglutination induced by SJAMP. Washed rabbit platelets were not agglutinated by SJAMP even though the concentration of SJAMP was raised up to 50 micrograms/ml. When rabbit plasma, serum, or 50-60% ammonium sulfate saturated plasma fraction was added to the reaction mixture, agglutination of washed platelets by SJAMP was recovered completely. But human plasma or fibrinogen did not have any effect on the reactivity of washed rabbit platelets to SJAMP. From these data we conclude that the SJAMP-induced rabbit platelet agglutination is independent of energy metabolism but requires plasma cofactor(s) other than fibrinogen. The plasma cofactor is present in 50-60% ammonium sulfate saturated plasma fraction.

Animals↗

PubMed1988

Aggregation of human platelets by acidic mucopolysaccharide extracted from Stichopus japonicus Selenka.

The acidic mucopolysaccharide extracted from sea cucumber (Stichopus japonicus Selenka) (SJAMP) has been shown to cause platelets to aggregate. Using citrated platelet-rich plasma (PRP), washed platelets and formaldehyde-fixed platelets from humans, we investigated the effects of platelet inhibitors and various plasmas and their fractions on SJAMP-induced platelet aggregation. It was found that the lowest concentration of SJAMP required for the aggregation of human platelets was about 0.4 micrograms/ml and the magnitude of aggregation induced by SJAMP was concentration dependent. The platelets were aggregated by SJAMP at 10 micrograms/ml in 25 out of 28 (89%) normal subjects tested. Platelet inhibitors such as PGE1, aspirin, indomethacin, apyrase, antimycin, 2-deoxy-D-glucose and EDTA inhibited by 70 to 100% the aggregation induced by SJAMP. Washed platelets alone were not aggregated by SJAMP. In the presence of fibrinogen, washed platelets were aggregated by SJAMP but formaldehyde-fixed platelets were not. These data indicate that the SJAMP-induced human platelet aggregation requires extracellular calcium, fibrinogen, and energy metabolism. The second phase of aggregation is dependent upon the release of ADP, and cyclooxygenase pathway.

Animals↗

PubMed1987

Wahlia pulchella n. sp., a turbellarian flatworm (Neorhabdocoela: Umagillidae) from the intestine of the sea cucumber Stichopus californicus.

Wahlia pulchella n. sp. is described from the intestine of the aspidochirote holothuroid Stichopus californicus. The new species differs from other members of the genus in having the following combination of features: the pharynx lies at the end of the first quarter or third of the body, the intestine extends anterior to the brain, the testes are only slightly lobed, and the penis stylet is straight and less than half the length of the adult worm. Wahlia pulchella has a "secondary uterus" in which there may be up to 12 egg capsules. The validity of the genera of holothuroid-inhabiting umagillids characterized by 2 ovaries and a secondary uterus is discussed. It is suggested that Ozametra Marcus, 1949, be placed in synonymy under Wahlia Westblad, 1930. New diagnoses of Wahlia and Seritia Cannon, 1982, are presented.

Animals↗

PubMed1982

[Unusual way of regenerating intestinal epithelium during seasonal intestinal transformation in Stichopus japonicus holothurians (possibility of an "external cambium")].

In Stichopus japonicus during summer months a profound morphofunctional rearrangement of the digestive tract resulting in its renovation is noted. It includes certain destructive-atrophic changes in the wall of the intestinal tube and some reparative processes accompanying them. In some species a spontaneous throwing out of the intestine with its subsequent restoration takes place. The mechanisms of the intestinal tube regeneration after its partial atrophy and after its spontaneous throwing out have much in common between them and with those previously described after an experimental throwing out. It is possible that under natural conditions (as well as under experimental ones) certain forms of wandering cells, migrating from the sublayers and/or from the surrounding structures (mesenterium and others), participate in the regenerative histogenesis of the intestinal epithelium. A suggestion is made that these cells are of coelomesodermal origin and play the role of the external cambium at sharp seasonal rearrangements of the holothurian intestinal tract.

Aging↗