An autoradiographic investigation of the proliferative activity of pyloric caeca and gondas of Asterias rubens

The proliferative activity of the pyloric caeca of Asterias rubens was investigated. Autoradiographic experiments using intracoelomically injected (methyl-³H)-thymidine were performed throughout the year and incorporation into pyloric caeca and into gonads was studied. Tritiated thymidine was found to be incorporated mainly in the coelomic lining of both organs. Cell divisions in the coelomic lining may be necessary for the growth of these organs, for the production of coelomocytes or, in the case of the pyloric caeca, for growth of the digestive epithelium. Proliferative activity of the digestive epithelium of the pyloric caeca was only observed in the median duct. It is hypothesized that new cells, arising from mitosis, grow from the median duct to the side lobes and differentiate into storage cells, for example. The existence of a mitosis-inducing or mitosis-stimulating substance is discussed. In the ovaries follicle cells were found to incorporate (methyl-³H)-thymidine; in the testis, proliferation of the germinal epithelium occurred simultaneously in all spermatogenic columns. First, the spermatogonia and then later the spermatocytes became labeled. Absorption of substances from the coelomic fluid is discussed.     

Histochemical observations on the pyloric caeca of Asterias rubens (Echinodermata,asteroidea) in relation to the reproductive cycle

The pyloric caeca of the starfish Asterias rubens were investigated histochemically during the reproductive cycle. The median duct and the side lobes reacted differently. The median duct reacted positively for acid phosphatases and glucose-6-phosphate dehydrogenase, whereas the side lobes reacted positively for alkaline phosphatases, neutral lipids, and fatty acids. In the transition zone between the median duct and the side lobes, the reaction for alkaline phosphatases and neutral lipids increased toward the side lobes. The function of the enzymes and the histochemical results are discussed in relation to the function of the pyloric caeca and to the reproductive cycle.     

Isolation and characterization of a sialidase from the starfish Asterias rubens

The starfish Asterias rubens contains a soluble sialidase (1.4 mU/mg homogenate protein), which was purified over 500-fold to apparent homogeneity by ammonium sulfate precipitation, gel filtration and affinity chromatography on immobilized 2-deoxy-2,3-didehydroneuraminic acid. The native sialidase has a molecular mass of 230 kDa (gel filtration) and consists of 4 subunits of each 63 kDa, as determined by SDS-gel electrophoresis. Its isoelectric point is at pH 4.9, the activity is optimum at pH 4.2 and 37 degrees C, and it hydrolyses preferably 4-methylumbelliferyl-alpha-N-acetyl-neuraminic acid, followed by sialyllactose and glycoproteins. The hydrolysis rate is decreased or stopped by the presence of O-acetyl groups on the sialic-acid residue to be cleaved. N-Glycoloyl residues also retard enzyme action, as well as alpha(2-6) bonds when compared with alpha(2-3) linkages. This relatively stable enzyme is inhibited by mercury or copper ions, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and by the increase of ionic strength. The evolutionary significance of starfish sialidase is discussed.     

Progesterone and oestrone levels in the gonads and pyloric caeca of the male sea star Asterias rubens: A comparison with the corresponding levels in the female sea star

• 1.1. Levels of progesterone and oestrone were estimated by means of RIA in the gonads and pyloric caeca of male specimens of Asterias rubens throughout the reproductive cycle.
• 2.2. The patterns obtained appeared to be sex-specific on comparison with those of female specimens of A. rubens and it was concluded that progesterone and oestrone in the testes are involved in the regulation of gametogenesis.
• 3.3. The levels of progesterone in the pyloric caeca were about ten times higher than those in the testes; in the same sequence levels of oestrone were only slightly higher.
• 4.4. The onset of a new reproductive cycle, taking the abrupt decrease of the pyloric caeca-index as a marker, coincides with a strong decrease of the progesterone level (P < 0.001) and an increase of the oestrone level (P < 0.01) in the pyloric caeca.
• 5.5. It is supposed that progesterone and oestrone in the pyloric caeca are concerned with making available the materials required for gametogenesis.

     

Biosynthesis and functions of eicosanoids generated by the coelomocytes of the starfish, Asterias rubens

Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography–mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE₂ or PGD₂, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0–25 μM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.     

Isolation and characterization of cytidine-5'-monophosphate-N-acetylneuraminate hydroxylase from the starfish Asterias rubens

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is formed by cytidine-5'-monophosphate-N-acetylneuraminic acid (CMP-Neu5Ac) hydroxylase (EC 1.14.13.45). The enzyme from mammals exhibits several unusual characteristics, raising questions about its evolution. Since echinoderms are the most primitive organisms possessing glycoconjugate-bound Neu5Gc, studies on the hydroxylase from members of this phylum may yield insights into the origin and development of the hydroxylase. Investigations on crude CMP-Neu5Ac hydroxylase in gonads from the starfish Asterias rubens revealed that it shares many properties with its mammalian counterpart. However, the echinoderm hydroxylase also exhibits fundamental differences, particularly its association with a membrane and a requirement for high ionic strength for optimal activity. Here, we describe the isolation of the CMP-Neu5Ac hydroxylase from A. rubens gonads using anion exchange chromatography and chromatography on immobilized cytochrome b(5). The enzyme was enriched 137-fold with a yield of 13%. The preparation exhibited a main polypeptide of 76 kDa, consistent with a cDNA sequence published earlier, and a minor protein of 64 kDa. A kinetic characterization showed that salt activation of this enzyme results from an increase in affinity for CMP-Neu5Ac. Evidence for the formation of a ternary complex of hydroxylase, CMP-Neu5Ac and cytochrome b(5) is also presented. The mechanistic and physiological significance of these results is discussed.     

Effects of Oestradiol-17 β on the Ovaries of the Starfish Asterias rubens

Oocyte diameters and their frequency distribution, and various other data determined for the oestradiol-17 β treated female specimens of Asterias rubens proved to be significantly different from those for the control animals. The maturation index of the treated animals is 2.4, that of the control animals 1.4. Since the treated animals show a greater heterogeneity in development than the control animals, and because the diameter of the smallest oocytes is the same for both treated and control animals, a threshold size of the oocytes may be required before oocyte growth can be stimulated by oestradiol-17β, and before substances originating in the pyloric caeca are incorporated into the oocytes. Oestradiol-17β treatment caused a tenfold increase of the oestrone level in the ovaries, whereas a non-significant increase was observed in the pyloric caeca. This may indicate that in vivo oestradiol-17β is converted into oestrone in the ovaries but not in the pyloric caeca.     

Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.     

Cloning and expression of a membrane-bound CMP-N-acetylneuraminic acid hydroxylase from the starfish Asterias rubens.

The sialic acid N-glycolylneuraminic acid (Neu5Gc) is synthesized by the action of CMP-Neu5Ac hydroxylase. The enzyme from various mammals has been purified, characterized and sequenced by cDNA cloning. Although functional sequence motifs can be postulated from comparisons with several enzymes, no global homologies to any other proteins have been found. The unusual characteristics of this hydroxylase raise questions about its evolution. As echinoderms are phylogenetically the oldest organisms possessing Neu5Gc, they represent a starting point for investigations on the origin of this enzyme. Despite many similarities with its mammalian counterpart, CMP-Neu5Ac hydroxylase from the starfish A. rubens exhibits fundamental differences, most notably its association with a membrane and a requirement for high ionic strength. In order to shed light on the structural basis for these differences, the primary structure of CMP-Neu5Ac hydroxylase from A. rubens has been determined by PCR and cDNA-cloning techniques, using initial sequence information from the mouse enzyme. The complete assembled cDNA contained an ORF coding for a protein of 653 amino acids with a molecular mass of 75 kDa. The deduced amino-acid sequence exhibited a high degree of homology with the mammalian enzyme, although the C-terminus was some 60 residues longer. This extension consists of a terminal hydrophobic region, which may mediate membrane-binding, and a preceding hydrophilic sequence which probably serves as a hinge or linker. The identity of the ORF was confirmed by expression of active CMP-Neu5Ac hydroxylase in E. coli at low temperatures.     

Interference of benzo[a]pyrene with cytochrome P-450-mediated steroid metabolism in pyloric caeca microsomes of the sea star Asterias rubens L.

• 1.1. The effects of benzo[a]pyrene (BaP) on the metabolism of progesterone and pregnenolone, and the effects of steroids on BaP metabolism were examined in pyloric caeca microsomes of female Asterias rubens.
• 2.2. The patterns of metabolism of progesterone and pregnenolone in microsomes were similar to those found in previous studies for homogenates and tissue incubations of pyloric caeca.
• 3.3. BaP reduced the rate of hydroxylation of progesterone and pregnenolone, but had no effect on metabolite formation by non-cytochrome P-450-catalysed reactions.
• 4.4. Microsomal BaP hydroxylase activity was reduced by the presence of progesterone, but pregnenolone and testosterone had no such effect.
• 5.5. The reductions in steroid or BaP metabolism were progressive with increasing ratios of the concentration of the interfering compound to that of the assay substrate and were maximally 50% or less at ratios of × 100.
• 6.6. It is concluded that isoenzymic forms of cytochrome P-450 are present, with preferences towards either steroid or BaP metabolism. The implications of the results for the in vivo situation are discussed.

     

Analysis of monosaccharides, fatty constituents and rare O-acetylated sialic acids from gonads of the starfish Asterias rubens

A previous study (Bergwerff et al., Biochimie 74 (1992) 25-37) reported that sialic acids present in Asterias rubens gonads were essentially composed of 8-methyl-N-glycolylneuraminic acid (Neu5Gc8Me), a large part of it being acetylated in position 9. Using GC/MS of heptafluorobutyrate derivatives (Zanetta et al., Glycobiology 11 (2001) 663-676) on the chloroform/methanol soluble and insoluble fractions, we showed that most sialic acids were found in the latter and demonstrated that all sialic acids were derived from N-glycolylneuraminic acid, most of them being 8-methylated, but that the majority were also acetylated in position 4 or 7 (or both positions). GC/MS analyses of the constituents liberated using acid-catalysed methanolysis verified that major glycoprotein-bound glycans were N-linked and of the gluco-oligomannosidic type. Major fatty acids were poly-unsaturated (especially C20:4) and long-chain bases were C22:1 phytosphingosine and C22:2 6-hydroxysphingenine. Major monosaccharides found in the chloroform/methanol extract (quinovose and fucose) were derived from steroidal saponins.     

Differentiation of immune cells challenged by bacteria in the common European starfish,Asterias rubens (Echinodermata)

Amoebocytes are the main effector cells of the echinoderm immune system. In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type. The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria. Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus). Amoebocytes were collected at regular time intervals for 24 h. The cytometric characteristics and the phagocytic activity were studied by flow cytometry. Three amoebocyte groups of different size were identified. The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected. All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed. G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity. Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria. It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.     

A chemical and histochemical study of the lipides of the pyloric cecum of the starfish,Asterias forbesi

The lipides of the diverticula of Asterias forbesi have been studied by histochemical and biochemical means. Correlations between results obtained by histochemical examination of sections, and chemical analysis of isolated lipide have been made, particularly with respect to phosphatides, steroids, and aldehyde lipides. The results of the histochemical study were in good agreement with the chemical data as to the nature of the phosphatide fraction, the presence of acetone-soluble aldehyde lipides, and the composition of the free droplet fat. Homogenized diverticula were differentially centrifuged in order to establish the distribution of types of lipides in the various cellular components. In addition, data have been presented which demonstrate a direct correlation between the titer of alpha-glycerol ethers and that of acetone-soluble lipide acetals in the unsaponifiable fraction.