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[Spectrophotometric determination of the stichoposide A from the holothurian Stichopus japonicus S].

The absorption spectrum of stichoposid A with sulfuric acid, vanillin and alkaline was studied for the purpose of possibly using the reactions with these substances for quantitative determination of stichoposid A in biological materials. The reaction with 1 N NaOH was shown to be the most useful since it is highly specific with respect to the glycoside aglycone and makes it possible to investigate stichoposid A interaction with the cells of the yeast microflora.

Animals↗

Arginine kinase evolved twice: evidence that echinoderm arginine kinase originated from creatine kinase.

Arginine kinase (AK) was isolated from the longitudinal muscle of the sea cucumber Stichopus japonicus. Unlike the monomeric 40 kDa AKs from molluscs and arthropods, but like the cytoplasmic isoenzymes of vertebrate creatine kinase (CK), the Stichopus enzyme was dimeric. To explore the evolutionary origin of the dimeric AK, we determined its cDNA-derived amino acid sequence of 370 residues. A comparison of the sequence with those of other enzymes belonging to the phosphagen kinase family indicated that the entire amino acid sequence of Stichopus AK is apparently much more similar to vertebrate CKs than to all other AKs. A phylogenetic tree also strongly suggests that the Stichopus AK has evolved from CK. These results support the conclusion that AK evolved at least twice during the evolution of phosphagen kinases: first at an early stage of phosphagen kinase evolution (its descendants are molluscan and arthropod AKs) and secondly from CK later in metazoan evolution. A comparison of the amino acid sequence around the guanidino specificity (GS) region (which is a possible candidate for the guanidine substrate recognition site in the phosphagen kinase family) of the Stichopus enzyme with those of other phosphagen kinases showed that the GS region of the Stichopus enzyme was of the AK type: five amino acid deletions in the flexible loop region that might help to accommodate larger guanidine substrates in the active site. The presence of the AK-type deletions in the Stichopus AK, even though it seems that the enzyme's most immediate ancestor was probably CK, strongly suggests that the GS region has a role in substrate specificity. Stichopus AK and presumably other echinoderm AKs seem to have evolved from the CK gene; the sequence of GS region might have been replaced by the AK type via exon shuffling. The presence of an intron near the GS region in the Stichopus AK gene supports this hypothesis.

Amino Acid Sequence↗

Neural control of muscle relaxation in echinoderms.

Smooth muscle relaxation in vertebrates is regulated by a variety of neuronal signalling molecules, including neuropeptides and nitric oxide (NO). The physiology of muscle relaxation in echinoderms is of particular interest because these animals are evolutionarily more closely related to the vertebrates than to the majority of invertebrate phyla. However, whilst in vertebrates there is a clear structural and functional distinction between visceral smooth muscle and skeletal striated muscle, this does not apply to echinoderms, in which the majority of muscles, whether associated with the body wall skeleton and its appendages or with visceral organs, are made up of non-striated fibres. The mechanisms by which the nervous system controls muscle relaxation in echinoderms were, until recently, unknown. Using the cardiac stomach of the starfish Asterias rubens as a model, it has been established that the NO-cGMP signalling pathway mediates relaxation. NO also causes relaxation of sea urchin tube feet, and NO may therefore function as a 'universal' muscle relaxant in echinoderms. The first neuropeptides to be identified in echinoderms were two related peptides isolated from Asterias rubens known as SALMFamide-1 (S1) and SALMFamide-2 (S2). Both S1 and S2 cause relaxation of the starfish cardiac stomach, but with S2 being approximately ten times more potent than S1. SALMFamide neuropeptides have also been isolated from sea cucumbers, in which they cause relaxation of both gut and body wall muscle. Therefore, like NO, SALMFamides may also function as 'universal' muscle relaxants in echinoderms. The mechanisms by which SALMFamides cause relaxation of echinoderm muscle are not known, but several candidate signal transduction pathways are discussed here. The SALMFamides do not, however, appear to act by promoting release of NO, and muscle relaxation in echinoderms is therefore probably regulated by at least two neuronal signalling systems acting in parallel. Recently, other neuropeptides that influence muscle tone have been isolated from the sea cucumber Stichopus japonicus using body wall muscle as a bioassay, but at present SALMFamide peptides are the only ones that have been found to have a direct relaxing action on echinoderm muscle. One of the Stichopus japonicus peptides (holothurin 1), however, causes a reduction in the magnitude of electrically evoked muscle contraction in Stichopus japonicus and also causes 'softening' of the body wall dermis, a 'mutable connective tissue'. It seems most likely that this effect of holothurin 1 on body wall dermis is mediated by constituent muscle cells, and the concept of 'mutable connective tissue' in echinoderms may therefore need to be re-evaluated to incorporate the involvement of muscle, as proposed recently for the spine ligament in sea urchins.

Amino Acid Sequence↗

Fibrinolysis relating substances in marine creatures.

1. Extracts with physiological saline solution were obtained from about 20 species of invertebrates and seaweed. Tosyl-L-Arg-MeOH hydrolysing and fibrin plate lytic activity were detected in the invertebrates Stichopus japonicus, Crassost gigas, Tapes japonica, and Kintai-gai as well as the seaweed Codiales codium. 2. These activities were all labile against heat (at 65 degrees C for 1 hr). Except for the extract from Stichopus japonicus, lytic activities against fibrin plates with and without plasminogen were similar. 3. The extract from S. japonicus showed plasminogen activating potency as well as the existence of urokinase (UK) activity enhancing factor. 4. On the other hand, the extract of the seaweed Hizikia fusiformis showed a strong UK inhibiting activity. 5. A fraction of fibrinolytic enzyme was obtained from the extract of S. japonicus by absorption to the celite affinity chromatography. It was orally administered to rabbits at a dosage of 40 mg/kg/day. 6. Fibrinolytic activity was determined periodically on the eugloblin fraction of plasma samples collected from these animals. 7. As compared with the pretreatment value, the activity increased about 2 times (P less than 0.01) and 3 times (P less than 0.005) after 4 and 8 weeks, respectively, of the treatment. 8. After 8 weeks of treatment, the kidney of treated rabbits was extracted with 2 M KCl. The activity of tissue plasminogen activator (free-type TPA) was revealed to be enhanced significantly (P less than 0.001) in the extracts. 9. The fibrinolytic enzyme increased in the blood was recognized by zymography to be mainly the UK type plasminogen activator with mol. wt of 53,000.

Animals↗

Thermal inactivation and unfolding of a dimeric arginine kinase.

Thermal inactivation and unfolding of the dimeric arginine kinase (AK) from sea cucumber Stichopus japonicus was investigated. The activation energy was calculated to be 388 kJ/mol. Based on the analysis of the denaturation course at 58 degrees C, a model is suggested for the thermal unfolding of this dimeric AK. In addition, the effect of free Mg(2+) and the potential biological significance on the thermal unfolding of dimeric AK is discussed.

Animals↗

Expression of the otx gene in the ciliary bands during sea cucumber embryogenesis.

The Otx gene encodes a homeodomain transcription factor that has a highly conserved role in brain formation of both flies and vertebrates. To deduce evolutionary relationship of the chordate central nervous system to the larval or adult nervous system of nonchordate deuterostomes, we characterized the expression of the Otx gene (Sj-Otx) throughout the entire embryonic and larval development of the sea cucumber Stichopus japonicus. Sj-Otx transcripts were detected in fertilized eggs and in the posterior part of the archenteron of gastrulae. However, the expression was downregulated as embryos developed into auricularia larvae. Sj-Otx was expressed again in the ciliary bands of late auricularia larvae, just before metamorphosis to doliolaria larvae. The expression domain corresponded to the domains moving to the mouth during metamorphosis and sinking into the buccal cavity, but not to the five transverse ciliary bands of the doliolaria. The expression gradually disappeared during further development and was not detected in juveniles. These results indicate that the gene responsible for chordate brain formation is expressed in the ciliary bands of auricularia larvae.

Amino Acid Sequence↗

Isolation and characterization of a fucoidan-degrading marine bacterium.

Fucoidan, a mixture of sulfated fucose-containing polysaccharides, was prepared from the algal bodies of Cladosiphon okamuranus (class Phaeophyceae, order Chordariales, family Chordariaceae) with a yield of 2.0% of the wet weight of the alga. To obtain enzymes that digest the fucoidan, we screened bacteria in the guy contents of the sea cucumber Stichopus japonicus for their ability to decrease the fucoidan in their culture media, and successfully isolated one bacterial strain that could decrease it. The bacterial strain was gram-negative and possessed menaquinone 7 as the predominant respiratory quinone, and the GC content of its genomic DNA was 52%. The results of the phylogenetic analysis of its 16S ribosomal DNA sequence indicated that the bacterial strain was a member of the division "Verrucomicrobia." However, as the bacterial strain is phylogenetically and phenotypically distinct from verrucomicrobial species described previously, the strain was assumed to be a new member of the division "Verrucomicrobia." When the bacterial strain was cultivated in an algal fucoidan-containing medium, the strain decreased fucoidan from C. okamuranus (44%), Nemacystus decipiens (19%), Laminaria japonica (31%), Kjellmaniella crassifolia (23%), sporophyl of Undaria pinnatifida (22%), Fucus vesiculosus (42%), and Ascophyllum nodosum (61%).

Animals↗

Distribution of alkyl and alkenyl ether-linked phospholipids and platelet-activating factor-like lipid in various species of invertebrates.

The levels of alkenylacyl, alkylacyl and diacyl subclasses of choline glycerophospholipid (CGP) and ethanolamine glycerophospholipid (EGP) fractions in 28 species of various invertebrates were studied. We found that only small amounts of either 1-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) or 1-alkenyl-2-acyl-sn-glycero-3-phosphoethanolamine (alkenylacyl-GPE) are present in most species of insects. On the other hand, almost all species examined in various phyla other than Arthropoda were shown to contain large amounts of both alkylacyl-GPC and alkenylacyl-GPE. The highest proportion of alkylacyl subclass in CGP was noted in sponge, Halichondria japonica (81.8% of CGP) and the highest proportion of alkenylacyl subclass in EGP was found in clam worm, Marphysa sanguinea (88.7% of EGP). We next surveyed the presence of platelet-activating factor (PAF)-like lipid in 45 species of invertebrates. PAF-like lipid was widely distributed among various lower animals. The highest value was obtained for sea cucumber, Stichopus japonicus, in which PAF-like lipid was present throughout the body. We also confirmed the presence of acetyltransferase activity in several lower animals. These results suggest that alkyl and alkenyl ether-linked phospholipids including PAF are physiologically important molecules particularly for invertebrates belonging to lower phyla.

Animals↗

Cells in the intestinal system of holothurians (Echinodermata) express cholecystokinin-like immunoreactivity.

The presence of cholecystokinin (CCK), originally isolated from porcine small intestine, has been reported in a diversity of invertebrates ranging, from cnidarians to protochordates, but so far, not in echinoderms. We have used immunohistochemical techniques to demonstrate the presence of cells expressing CCK-like immunoreactivity in the intestine of three species of sea cucumber: Holothuria mexicana, H. glaberrima, and Stichopus badionotus. The immunoreactivity was observed within the cytoplasm of these cells, in what appeared to be granular or vesicle-like structures. The cell bodies were present in the outer connective tissue layer of the intestine and had a neuronal appearance, sending an axon-like structure into the circular muscle and internal connective tissue. A plexus of fibers expressing CCK-like immunoreactivity was found overlying the muscle layer. Contractility of H. mexicana intestinal strips was studied under partially isometric conditions. CCK and related peptides induced relaxation of the basal muscle tension, and of tension induced by ACh application, suggesting a role for this agent in the intestinal physiology of holothurians.

Acetylcholine↗

The nucleotide sequences of 5S rRNAs from a sea-cucumber, a starfish and a sea-urchin.

The nucleotide sequences of 5S rRNA from three echinoderms, a sea-cucumber Stichopus oshimae, a starfish Asterina pectinifera and a sea-urchin Hemicentrotus pulcherrimus have been determined. These 5S rRNAs are all 120 nucleotides long. The echinoderm sequences are more related to the sequences of proterostomes animals such as mollusc, annelids and some others (87% identity on average) than to those of vertebrates (82% identity on average).

Animals↗

Invertebrate compounds acting on the hemostatic mechanism.

Physiological secretions from some invertebrates have toxic effects on mammalian blood coagulation and fibrinolytic systems. Some of these effects occur because the substances contained in the secretions resemble the components of the hemostatic system. Some of the substances have been characterized, and have been found to have similar molecular weights or sequences, which may indicate a common ancestry. The components can be divided into five groups: antithrombic agents (group I); inhibitors and activators of the prothrombinase complex (group II); substances that affect platelet function (group III); substances that affect the fibrinolytic mechanism (group IV); and a group of miscellaneous agents whose activities are difficult to group together (group V). In group I special mention of the antithrombin agents in Hirudo medicinalis should be made. In group II, the agents affecting the prothrombinase complex are antistasin from Haementeria officinalis, ghilanten from Haementeria Ghiliani and the tick anticoagulant protein from Ornithodoros moubata, a factor V activator/inhibitor from Lonomia achelous and factor II and factor X activators from L. achelous and Lonomia obliqua. Examples of factors which affect platelet function (group III) are glossina from the black fly Glossina morsitans, calin from H. medicinalis, decorsin (a desintegrin) from Macrobdella decorsa, and FAGA from Stichopus japonicus selenka. The first three of these are inhibitors of platelet aggregation, and the last is an inducer. The plasminogen activators (group IV) from the L. achelous caterpillar and Eutriatoma maculata trigger the fibrinolytic system, whereas hementin from H. officinalis and hementerin from Haementeria depressa are directly fibrinolytic. The last group of substances (group V) include those with factor-XIIa-like activity from D. farinae, kallikrein-like activity and a factor XIII degrading enzyme from L. achelous, destabilase from H. medicinalis and prolixin S (nitroforin 2, or anti-factor-IXa) from Rhodnius prolixus. Some of these components have been well characterized, cloned and prepared in recombinant form, and seem to be very promising from the therapeutic point of view.

Animals↗

Antifungal steroid glycoside from sea cucumber.

An antifungal steroid glycoside, holotoxin, has been isolated from the sea cucumber Stichopus japonicus (Selenka). In vitro, it exhibits high activity against various fungi, including vegetable pathogens, but has scarcely any activity against Gram-positive and Gram-negative bacteria and mycobacteria in vitro.

Animals↗

Amino acid sequence of a C-type lectin CEL-IV from the marine invertebrate Cucumaria echinata.

The complete amino acid sequence of a Ca(2+)-dependent lectin, CEL-IV, from the marine invertebrate Cucumaria echinata was analyzed. The established sequence showed that CEL-IV comprises 157 amino acid residues with a molecular mass of 17,098 Da (without disulfide bonds). From comparison with other proteins, CEL-IV was apparently homologous with the C-type lectin family. The identity was relatively high with a sea cucumber (Stichopus japonicus) lectin SJL-I (40.0%) and a sea urchin (Anthocidaris crassispina) lectin echinoidin (32.6%). In CEL-IV, one interchain and two intrachain disulfide bonds were identified. Interestingly, one of the two intrachain disulfide bonds that were highly conserved among the other C-type lectins was missing, suggesting that this might be a characteristic feature of C-type lectins in the Holothuroidea.

Amino Acid Sequence↗

Efficacy of 'Gamadent' toothpaste on the healing of gingival tissues: a preliminary report.

The aim of this randomised, parallel, double-blind study, in which 28 adult patients diagnosed with chronic gingivitis or early stages of chronic periodontitis were recruited, was to evaluate the efficacy of 'Gamadent' toothpaste compared to a placebo toothpaste. 'Gamadent' toothpaste has all the basic constituents of a toothpaste with the addition of a sea cucumber extract (SCE) of the species Stichopus sp. 1 to improve the healing potential of tissues. The placebo has the same basic constituents minus the extract. Out of the 28 patients, 14 were placed in the test group who used the 'Gamadent' toothpaste, and 14 patients were placed in the control group (2 control subjects defaulted and were excluded), who brushed using the placebo toothpaste. The longitudinal study was carried out over a period of 3 months with assessments made at baseline, 1 month, 2 months and 3 months after conventional therapy at the baseline visit. The clinical parameters used during the trial were Plaque Index (PI), Gingival Index (GI), Papilla Bleeding Index (PBI) and Probing Pocket Depth (PPD). A predetermined number of sites on a molar, premolar, canine and an incisor were examined and evaluated in each quadrant. After the baseline assessment, the patients had full mouth scaling and debridement as well as oral hygiene instructions. Patients were instructed to brush their teeth twice a day with the toothbrush provided (Oral-B plus, size 35) and toothpaste (test or control), using the Bass technique. At the 1-month assessment, there were significant mean reductions to baseline mean values in PI (P < 0.005) and GI (P < 0.001) in the test group as compared to the control group. At the end of the 2-month interval, significant reductions were observed in PI, PBI and PPD (P < 0.001). By the end of 3 months, there were significant differences in the mean reduction of all the parameters i.e. PI, PBI, GI and PPD (P < 0.001), between the test and control sites. In conclusion, 'Gamadent' toothpaste provided noteworthy benefits, producing statistically significant improvement in all clinical parameters compared to the placebo during the healing phase after conventional initial therapy.

Adult↗

[Characteristics of the morphological changes in the nuclear structures in oogenesis in holothurians].

Chromosomes and nucleoli were studied at the meiosis diplotene stage in oocytes of Stichopus japonicus and Cucumaria fraudatrix. The S. japonicus nuclear structures were found to change in the same way as in other holothurioidea (chromosomes turn into lampbrushes, there is a single rapidly increasing nucleolus). The C. fraudatrix oocytes are notable for their lampbrushes located in immediate vicinity of the nucleus membrane. The same modes of behavior of nuclear structures are known for three more types of Cucumaria (no more species have been studied). The specific features of behavior of the nuclear structures are discussed with regard to phylogeny of holothurioidea oogenesis.

Animals↗

Isolation and properties of a mannan-binding lectin from the coelomic fluid of the holothurian Cucumaria japonica.

A new 44-kD, C-type mannan-binding lectin (MBL-C) consisting of two identical subunits was isolated from the coelomic fluid of the holothurian Cucumaria japonica. In the direct hemagglutination assay, the lectin was effectively inhibited by highly branched mannans similar in structure to yeast mannans and composed of alpha-(1-->2)- and alpha-(1-->6)-bound D-mannopyranose residues. Hemagglutination was not inhibited by mannosaccharides, common constituents of the hydrocarbon chains of "normal" glycoproteins. The lectin reaction depends on Ca2+ concentration: maximum activity of MBL-C is observed at 10 mM Ca2+. The activity of MBL-C increases in the pH range from 5 to 7 and reaches maximum at pH 7.0. The lectin is sensitive to temperature. Heating of the lectin solution at temperatures above 40 degrees C decreases activity, while incubation at 90 degrees C for 1 h leads to complete irreversible inactivation. Carbohydrate specificity, Ca2+-dependence, and amino acid composition indicate that MBL-C belongs to the C-type mannan-binding lectins. Polyclonal antibodies against MBL-C revealed its immunochemical similarity to a mannan-binding lectin from another holothurian species, Stichopus japonicus; this provides evidence for structural homology between these proteins.

Animals↗

[Identification of sea cucumber sold in market].

OBJECTIVE: To identify the variety of the sea cucumber sold in market. METHOD: Figure and bone pieces observed for the microstructures. RESULT: 8 varieties were identified as Lessonothurea Deichmann, Thymiosycia Pearson, Microthele Brandt and Metriatyla Rowe of Holothuria, Bohadschia, Stichopus and Thelenota. CONCLUSION: The bone pieces in ora, anus, back, abdomen and tentacle of sea cucumber are different. They are the important bases to identify sea cucumber.

Animals↗