Coelomocyte Morphology in the Holothurians Apostichopus japonicus (Aspidochirota: Stichopodidae) and Cucumaria japonica (Dendrochirota: Cucumariidae)

The cellular composition of the coelomic fluid of the Far Eastern holothurinans Apostichopus japonicus and Cucumaria japonica was studied using light and transmission electron microscopy and histochemistry. In the coelomic fluid of A. japonicus, the following types of coelomocytes were distinguished: progenitor cells; amoebocytes; vacuolated cells; small (or young) morula cells; morula cells of type I, type II, and type III; crystal cells; and vibratile cells. In the coelomic fluid of C. japonicawere found progenitor cells, amoebocytes, vacuolated cells, morula cells of type I and type II, crystal cells, and hemocytes containing a respiratory pigment. The issue of stem cell type, which gives rise to coelomocytes, is discussed.     

Mineral Content and Salt-dependent Viscosity in the Dermis of the Sea Cucumber Cucumaria frondosa

Samples from the deep dermis of the sea cucumber Cucumaria frondosa were analyzed to determine the concentrations of Na, K, Ca, and Mg in fresh tissues as well as in those that had been incubated in filtered seawater or extracted extensively in deionized water. Fresh tissues were in equilibrium with seawater with respect to the above minerals, and showed a relative concentration of K, Mg, and Ca. Water-extracted tissues were enriched in Mg and Ca and depleted in K and Na. The Ca and Mg could be removed from the tissues by extraction in a divalent cation chelator or in 0.1 M HCl. Biomechanical (creep) tests showed that dermal specimens in which the resident cells had been lysed by freezing and thawing had very low viscosities in buffered solutions of a divalent cation chelator containing either 0.03 M or 1.0 M NaCl. In contrast, their viscosities were quite high in similar solutions containing 0.3 M NaCl. The aggregation of isolated collagen fibrils in the presence of the dermal glycoprotein stiparin showed a similar dependence on the concentration of NaCl: the fibrils aggregated in 0.3 M NaCl, but not in 0.03 or 1.0 M NaCl. The above results are inconsistent with the hypothesis that collagen fibril interactions in C. frondosa dermis are regulated by cellular control of the extracellular [Ca²⁺]. The results are consistent with the hypothesis that stiparin mediates mechanical interactions between collagen fibrils in dermal tissues as well as in isolated fibrils.     

Purification, characterization and cloning of tensilin, the collagen-fibril binding and tissue-stiffening factor from Cucumaria frondosa dermis.

The inner dermis of the sea cucumber, Cucumaria frondosa, is a mutable collagenous tissue characterized by rapid and reversible changes in its mechanical properties regulated by one or more protein effectors that are released from neurosecretory cells. One such effector, tensilin, is a collagen-fibril binding protein, named for its ability to induce dermis stiffening. Tensilin was purified using an affinity column constructed from C. frondosa collagen-fibrils. The protein migrates as a single band on SDS-PAGE (Mr approximately 33 kDa) and has an isoelectric point of 5.8. Equilibrium sedimentation experiments suggest a molecular mass of approximately 28.5-29.4 kDa. Carbohydrate analysis of tensilin revealed no measurable sugar content. The molar amount of tensilin was determined to be 0.38% that of collagen and 47% that of stiparin, a constitutive matrix glycoprotein. A full-length cDNA clone for tensilin was obtained from a C. frondosa inner dermis cDNA expression library. Predicted properties derived from the deduced peptide sequence were in agreement with those of the native protein. A noted feature of tensilin's deduced peptide sequence, particularly in its N-terminal domain, is its homology to tissue inhibitor of metalloproteinases. Tensilin's C-terminal tail has no known homology to other proteins but contains a putative collagen-fibril binding site.     

Novel Use of PIT Tags in Sea Cucumbers: Promising Results with the Commercial Species Cucumaria frondosa

The lack of a reliable and innocuous mark-recapture method has limited studies that would provide essential information for the management of commercial sea cucumbers. Tagging sea cucumbers is notoriously difficult because of their plastic nature and autolysis capacities. The markers that have so far been tested, mainly on or through the body wall, were either lost rapidly or had major drawbacks (e.g. suitable only for batch identification, requiring complex analysis, causing infections, necrosis, behavioural changes and mortality). The present study explored the efficacy of passive integrated transponder (PIT) tags for individually marking sea cucumbers by assessing retention rates and long-term side effects of tags inserted in previously unstudied tissues/organs. Individuals of the species Cucumaria frondosa were tagged in the body wall, aquapharyngeal bulb and at the base of the oral tentacles. They were monitored closely for evidence of stress, infection, change in feeding and spawning behaviour and tag retention rate. Implanting the tag in an oral tentacle to reach the hydrovascular system of the aquapharyngeal bulb achieved the best retention rates in full-size individuals: from a maximum of 92% after 30 days to 68% at the end of the experimental period (300 days). Efficacy was lower in smaller individuals (84% after 30 d and 42% after 300 d). Following a slight increase in cloacal movements for 15 h post tagging, no side effect was noted in sea cucumbers tagged in the aquapharyngeal bulb via the tentacles. Feeding and spawning behaviours were not affected and no signs of infections or abnormal cell development in the vicinity of the tags were observed. This study indicates that marking sea cucumbers with 8.2 mm long PIT tags implanted via the oral tentacle is an effective technique, yielding relatively high retention rates over long periods without any detectable physiological or behavioural effects.     

The protective effect of eicosapentaenoic acid-enriched phospholipids from sea cucumber Cucumaria frondosa on oxidative stress in PC12 cells and SAMP8 mice

Alzheimer’s disease (AD) is a common neurodegenerative disorders, in which oxidative stress plays an important role. The present study investigated the effect of eicosapentaenoic acid-enriched phospholipids (EPA-enriched PL) from the sea cucumber Cucumaria frondosa on oxidative injury in PC12 cells induced by hydrogen peroxide (H₂O₂) and tert-butylhydroperoxide (t-BHP). We also studied the effect of EPA-enriched PL on learning and memory functions in senescence-accelerated prone mouse strain 8 (SAMP8) in vivo. Pretreatment with EPA-enriched PL resulted in an enhancement of survival in a dose-dependent manner in H₂O₂ or t-BHP damaged PC12 cells. EPA-enriched PL pretreatment could also reduce the leakage of lactate dehydrogenase (LDH), and increase the intracellular total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) activity compared with the H₂O₂ or t-BHP group. The down-regulated Bcl-2 mRNA level and up-regulated Bax, Caspase-9, and Caspase-3 mRNA expression induced by H₂O₂ or t-BHP could be restored by EPA-enriched PL pretreatment. These results demonstrated that EPA-enriched PL exhibited its neuroprotective effects by virtue of its antioxidant activity, which might be achieved by inhibiting the mitochondria-dependent apoptotic pathway. The neuroprotective effect of EPA-enriched PL was also verified in vivo test: the EPA-enriched PL administration prevented the development of learning and memory impairments in SAMP8 mice. Our results indicated that EPA-enriched PL could offer an efficient and novel strategy to explore novel drugs or functional food for neuronprotection and cognitive improvement.     

Effects of antimycin and 2-deoxyglucose on adenine nucleotides in human platelets. Role of metabolic adenosine triphosphate in primary aggregation, secondary aggregation and shape change of platelets

1. Human platelet-rich plasma prelabelled with [(3)H]adenine was incubated at 37 degrees C with antimycin A and 2-deoxy-d-glucose. Variations in the amounts of ATP, ADP and P(i), and in the radioactivity of ATP, ADP, AMP, IMP, hypoxanthine+inosine and adenine were determined during incubation. Adrenaline- and ADP-induced platelet aggregation and the ADP-induced shape change of the platelets were determined concurrently. 2. 2-Deoxyglucose caused conversion of [(3)H]ATP to [(3)H]hypoxanthine+inosine. The rate of this conversion increased with increasing 2-deoxyglucose concentration and was markedly stimulated by addition of antimycin, which had no effect alone. At maximal ATP-hypoxanthine conversion rates, the IMP radioactivity remained at values tenfold higher than control, whereas [(3)H]ADP and [(3)H]AMP radioactivity gave variations typical for product/substrates in consecutive reactions. The specific radioactivityof ethanol-soluble platelet ATP decreased during incubation to less than one-tenth of its original value. The amounts and radioactivity of ethanol-insoluble ADP did not vary during incubation with the metabolic inhibitors. 3. The rate of ADP- and adrenaline-induced primary aggregation decreased as the amount of radioactive ATP declined, and complete inhibition of aggregation was obtained at a certain ATP concentration (metabolic ATP threshold). This threshold decreased with increasing concentration of inducer ADP. 4. Secondary platelet aggregation (release reaction) had a metabolic ATP threshold markedly higher than that of primary aggregation. 5. Shape change was gradually inhibited as the ATP radioactivity decreased, and had a metabolic ATP threshold distinctly lower than that of primary aggregation, and which decreased with increasing concentration of ADP. 6. A small but distinct fraction of [(3)H]ATP disappeared rapidly during the combined shape change-aggregation process induced by ADP in platelets incubated with metabolic inhibitors, whereas no ATP disappearance occurred during aggregation in their absence.     

Radioprotective effect of nucleosides and nucleotides and the mechanism of action of adenosine

Adenosine and a majority of adenine mononucleotides have a radioprotective action; adenine and 2'-deoxyadenosine have no radioprotective effect, and that of 3',5'-cAMP only approaches the detectable level. Ribo- and deoxyribonucleosides and nucleotides of guanine, uracil, thymine, and cytosine have no protective action. Dipyridamole increases and alkylxanthines block the radioprotective effect of adenosine. So it follows that the radioprotective effect is realized via A-receptors of the plasmatic membrane external surface.     

Combined effect of adenosine and papaverine on the metabolism of adenine nucleotides in rat thymocytes

It is shown that in [14C]adenine-labelled thymocytes adenosine increases the content of adenine nucleotides and simultaneously accelerates their catabolism. Papaverine induces acceleration of splitting and a decrease of the specific ATP radioactivity but increases the AMP content and its specific radioactivity. The both effectors intensify considerably the outlet of total radioactive label from cells. If the papaverine effect in the extracellular medium results in accumulation mainly of hypoxanthine in the extracellular medium then the adenosine presence causes accumulation of inosine and hypoxanthine approximately in equal amounts. The release of labelled adenosine from thymocytes in all cases is an insignificant part of extracellular radioactivity. A conclusion is drawn that under conditions of the combined action of the substances under study papaverine removes the adenosine effect caused by its under study papaverine removes the adenosine effect caused by its phosphorylation with the formation of ATP and exerts the dose-depended action on adenine nucleotide metabolism in thymocytes.     

pH dependence of the effect of adenosine triphosphate and ethylenediaminetetraacetate on sodium and magnesium binding by cellular membrane fragments

The pH dependence of previously reported effects of adenosine triphosphate (ATP) and ethylenediaminetetraacetate (EDTA) on cation binding by rat liver microsomes was studied by an equilibration and washing procedure. Equilibration of microsomes in media containing 95 mM NaCl and 4 mM MgCl₂ with pH varied from 4 to 8 resulted in an increase in bound cations from zero below pH 4 to 0.90 mmoles Mg and 0.34 mmoles Na/g N at pH 8; the ratio of bound Na/bound Mg increased from 0.15 at pH 5 to 0.38 at pH 8. Addition of 5 mM EDTA to the equilibration media produced striking changes in cation binding such that bound Na/bound Mg increased from 0.30 at pH 5 to 3.90 at pH 7 and decreased to 3.55 at pH 8. In the presence of added 10 mM ATP, bound Na/bound Mg increased from 0.10 at pH 5 to a maximum of 0.80 at pH 7. The observed changes could generally be correlated with known mass law relationships, although the system containing added ATP was complicated considerably by the hydrolysis of ATP. Results demonstrate that environmental pH is an important factor in determining the effect of ATP and EDTA on the cation binding pattern of cellular membranes. Because hydrogen ion is a product of ATP hydrolysis as well as of other metabolic reactions, the described interactions may be of particular significance in the molecular mechanisms of ATP effects on cation binding and transport in living cells.     

Antibacterial activity in Strongylocentrotus droebachiensis (Echinoidea), Cucumaria frondosa (Holothuroidea), and Asterias rubens (Asteroidea)

A search for antibacterial activity in different body parts of the green sea urchin Strongylocentrotus droebachiensis, the common starfish Asterias rubens, and the sea cucumber Cucumaria frondosa was conducted. Antibacterial activity was detected in extracts from several tissues in all species tested, but mainly in the coelomocyte and body wall extracts. Relatively high antibacterial activity could also be detected in gastrointestinal organs and eggs from A. rubens and in eggs from C. frondosa. Differences between active extracts regarding hydrophobicity and sensitivity to heat and proteinase K treatment indicated that several different compounds were responsible for the antibacterial activities detected. Lysozyme-like activity could be detected in several tissues from A. rubens. Haemolytic activity could be detected in all species tested, especially in the body wall extracts. Results from the current study suggest that marine echinoderms are a potential source for the discovery of novel antibiotics.     

Role of calmodulin in the effect of guanyl nucleotides on rat hippocampal adenylate cyclase: involvement of adenosine and opiates

Abstract: The adenylate cyclase activity of rat hippocampal plasma membranes can be stimulated by vaso-active intestinal polypeptide (VIP). Low concentrations (10⁻⁹ to 10⁻⁷M) of 5'-guanylyl-imido diphosphate (GppNHp) evoke a transient inhibition of the enzyme, which is followed by stimulation with increasing GppNHp concentrations (10⁻⁶ to 10⁻⁴M). Inclusion of ethyleneglycol - bis - (β - aminoethylether) - N,N' - tetraacetic acid (EGTA) during incubation abolishes the GppNHp inhibition while preserving GppNHp activation. The stimulation induced by GppNHp is amplified by VIP, but the inhibition is unaffected. Adenosine analogs and opiates are inhibitory ligands in the presence of GTP, and their effects can be reversed by the appropiate receptor antagonists, 3-isobutyl-1-methylxanthine and naloxone. Treatment of membranes with trypsin abolishes the GppNHp-induced inhibition without affecting the GppNHp stimulation. The inhibition induced by GppNHp is also abolished by EGTA treatment followed by washing, which coincides wtih a reduction in the adenosine- and opiate-mediated, GTP-dependent inhibition. The GppNHp inhibition can be restored in EGTA-treated but not in trypsin-treated membranes by addition of calcium-calmodulin but not by Ca²⁺ or Mg²⁺. Calcium-calmodulindepleted membranes lack calcium stimulation as well as GppNHp-induced inhibition, whereas untreated membranes and calcium-calmodulin-depleted membranes plus exogenous calcium-calmodulin showed calcium stimulation and GppNHp inhibition. These results suggest that calmodulin is involved in both Ca²⁺ stimulation and guanine nucleotide-mediated inhibition of rat hippocampal adenylate cyclase.     

Emerging role of extracellular nucleotides and adenosine in multiple sclerosis

Extracellular nucleotides and adenosine play important roles in inflammation. These signaling molecules interact with the cell-surface-located P2 and P1 receptors, respectively, that are widely distributed in the central nervous system and generally exert opposite effects on immune responses. Indeed, extracellular ATP, ADP, UTP, and UDP serve as alarmins or damage-associated molecular patterns that activate mainly proinflammatory mechanisms, whereas adenosine has potent anti-inflammatory and immunosuppressive effects. This review discusses the actual and potential role of extracellular nucleotides and adenosine in multiple sclerosis (MS).     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.     

Studies of nucleosides and nucleotides.: LVIII. Deamination of adenosine analogs with calf intestine adenosine deaminase

Several synthetic adeonosine analogs: 8-fluoro-, 8-azido-, 8-iodo-, 8-methylthioadenosine; 8-bromo-2′-deoxyadenosine, 8-bromoxylofuranosyladenine, 5′-benzoly-8-bromoadenosine; 8,2′-S-, 8,2′-O-, 8,2′-NH-, 8,2′-N-CH₃-, 8,3′,-S-, 8,3′-O-, 8,5′-S- and 8,5′O-cycloadenosine; 1-deaza- and 3-deazaadenosine, as well as tubercidine (7-deazaadenosine), were tested as substrates of calf intestine adenosine deaminase.It was found that the adenine base of adenosine should be in the range φ_rmCN = 0–120° (anti to syn-anti) and 8-fluoroadenosine was hydroylzed very slowly. The purine base should have N¹, N³ or N⁷ atoms for the hydrolysis and only 1-deazaadenosine revealed an inhibitory effect toward the hydrolysis of adenosine.5′-OH group should be in the position of S-configuration and must not be substituted.