Isolation and primary structure of a cellulase from the Japanese sea urchin Strongylocentrotus nudus

Glycoside-hydrolase-family 9 (GHF9) cellulases are known to be widely distributed in metazoa. These enzymes have been appreciably well investigated in protostome invertebrates such as arthropods, nematodes, and mollusks but have not been characterized in deuterostome invertebrates such as sea squirts and sea urchins. In the present study, we isolated the cellulase from the Japanese purple sea urchin Strongylocentrotus nudus and determined its enzymatic properties and primary structure. The sea urchin enzyme was extracted from the acetone-dried powder of digestive tract of S. nudus and purified by conventional chromatographies. The purified enzyme, which we named SnEG54, showed a molecular mass of 54kDa on SDS-PAGE and exhibited high hydrolytic activity toward carboxymethyl cellulose with an optimum temperature and pH at 35 degrees C and 6.5, respectively. SnEG54 degraded cellulose polymer and cellooligosaccharides larger than cellotriose producing cellotriose and cellobiose but not these small cellooligosaccharides. From a cDNA library of the digestive tract we cloned 1822-bp cDNA encoding the amino-acid sequence of 444 residues of SnEG54. This sequence showed 50-57% identity with the sequences of GHF9 cellulases from abalone, sea squirt, and termite. The amino-acid residues crucial for the catalytic action of GHF9 cellulases are completely conserved in the SnEG54 sequence. An 8-kbp structural gene fragment encoding SnEG54 was amplified by PCR from chromosomal DNA of S. nudus. The positions of five introns are consistent with those in other animal GHF9 cellulase genes. Thus, we confirmed that the sea urchin produces an active GHF9 cellulase closely related to other animal cellulases.     

Morpho-functional changes in the ovaries of the sea urchin Strongylocentrotus intermedius after exposure to cadmium

The ovaries were studied in the sea urchins kept in a sea water added with 1, 50 and 100 micrograms/l cadmium chloride for 5, 15, 40, 72 and 130 days. The gland reaction depended on the drug dose and exposure. A short exposure (5 and 15 days) stimulated the development of a larger, as compared with the control, number of oogonia and raised the activity of acid and alkaline phosphatases. A long exposure decreased the number of germ cells, decelerated their growth, destroyed gametes and accessory cells, inhibited the activity of alkaline phosphatase. The cadmium accumulation in the ovaries was noted only on the 130th day at concentrations of 50 and 100 micrograms/l. The monitoring of morphological and biochemical indices allowed to conclude that cadmium exerted a toxic effect on the sea urchin ovaries.     

Isolation and various properties of beta DNA polymerase from the embryos of the sea urchin Strongylocentrotus intermedius

DNA-polymerase beta was isolated from embryonic cells of the sea urchin S. intermedius and purified 1040-fold. The molecular weight of the enzyme is 40 000, sedimentation coefficient 3.2S, pI 8.5. The SH-reagent--N-ethylmaleimide--has no appreciable influence on the enzyme activity. The enzyme is thermolabile and needs four deoxyribonucleoside triphosphates, bivalent metal ions (Mg2+ or Mn2+) and primer template for its activity. The maximal activity is observed when a synthetic polymer--poly(dA).oligo(dT) is used. DNA-polymerase performs DNA synthesis via a distributive mechanism. In terms of physico-chemical properties, the enzyme can be related to DNA-polymerases beta.     

Developmental studies of sea urchin chromatin. Chromatin isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus

Chromatin was isolated from spermatozoa of the sea urchin Strongylocentrotus purpuratus. The isolated chromatin shows less absorptivity ratio of 230 nm : 260 nm and possesses less protein than does embryonic chromatin. The ratio of histone : DNA is 1.02; nonhistone : DNA 0.13; RNA : DNA 0.04. Sperm chromatin melts in two steps with T_ms 70°C and 84°C in 2.5 × 10⁻⁴, M EDTA in contrast to embryonic chromatin with a single T_m = 72°C. Disc electrophoresis of basic proteins of sperm revealed one minor component with extremely fast mobility and three major components. The one with the slowest mobility is characteristic of sperm. The embryo has in turn its characteristic histone which also migrated slowly in disc electrophoresis. Both of these unique histone fractions are selectively extracted from chromatin by 5% perchloric acid. Amino acid analyses of these chromatographically purified unique fractions show that both contain a large amount of lysine, while that from sperm, in addition, contains also a large amount of arginine. Minimal molecular weights of 33,000 for sperm and 16,200 for embryo unique histone were estimated from these analyses. Sperm chromatin supports a level of RNA synthesis in vitro with exogeneously supplied RNA polymerase about 2% that of the corresponding free DNA.     

Characterization of a fasciclin I-like protein with cell attachment activity from sea urchin (Strongylocentrotus intermedius) ovaries

Fasciclin I, a neuronal cell adhesion molecule, was first identified in the grasshopper. To date, various fasciclin I-like proteins have been identified but their biological functions have not been well characterized. Here, we have purified a fasciclin I-like protein with a molecular weight of 33kDa from sea urchin (Strongylocentrotus intermedius) ovaries using hydrophobic chromatography and gel filtration. The protein was not N-glycosylated. Partial amino acid sequences of cyanogen bromide (CNBr)-cleaved fragments were highly conserved to other sea urchin fasciclin I-like proteins identified previously. The circular dichroism (CD) spectrum analysis demonstrated that the 33kDa protein contained high content of alpha-helical structure. These results suggest that the 33kDa protein is a fasciclin I-like family. Additionally, the fasciclin I-like protein promoted HT1080 human fibrosarcoma cell attachment. Further, a synthetic peptide (P1: GLREAANIAEQVDLRQVLRDVDL) of the protein corresponding to a highly conserved region of the fasciclin I-like family promoted heparin-dependent HT1080 cell attachment. Moreover, the peptide inhibited HT1080 cell attachment to the fasciclin I-like protein. These results suggest that the 33kDa protein from sea urchin ovaries isolated here is a member of the fasciclin I family and that the N-terminal region of the protein is important for cell attachment activity. The protein has a potential to be involved in biological functions in sea urchin as a cell adhesive molecule.     

Effect of noradrenaline,dopamine and adrenolytics on growth and maturation of the sea urchin,Strongylocentrotus nudus Agassiz

Both noradrenaline and dopamine inhibit growth and maturation of oocytes in sea urchins. The in vitro action of these drugs on gonads was accompanied by a decrease in intensity of incorporation of labelled RNA and protein precursors into oocytes in their cytoplasmic growth. The monoaminergic system appears to participate in the regulation of oogenesis in sea urchins besides the peptidergic neurosecretory system.     

Refertilization in eggs of the sea urchin Strongylocentrotus purpuratus

To determine the role of the sea urchin egg plasma membrane in the species-specificity of fertilization, the ability of denuded activated eggs to be heterospecifically refertilized was determined. Our initial studies included evaluating the effectiveness of three commonly used methods of vitelline envelope (VE) removal using indirect immunofluorescence microscopy with antibodies directed against the VE. Unfertilized Strongylocentrotus purpuratus eggs were extracted with 0.01 M dithiothreitol (DTT) for 3 min or digested with 1.0 mg/ml pronase for 1 hr. Eggs were also fertilized, then diluted into a divalent-free medium to produce thin, elevated envelopes (VE*s) that were mechanically removed by sieving the eggs through nylon mesh. We found that both DTT extraction and pronase digestion were not completely effective in VE removal, and mechanical removal methods gave rise to a mixed population of eggs, those that had their VEs removed and those with a collapsed envelope that was not detectable at the light microscope level. Therefore, a new method of VE removal was developed. Eggs with VE*s were prepared followed by treatment with 0.01 M DTT to solubilize the envelopes. Nearly 100% of the denuded activated eggs incorporated one or more homologous and heterologous sperm, suggesting that the egg plasma membrane does not function in determining the species-specificity of fertilization.     

The larval stages of the sea urchin, Strongylocentrotus purpuratus

The adult body plan of Strongylocentrotus purpuratus is established within the imaginal rudiment during the larval stages. To facilitate the study of these stages, we have defined a larval staging scheme, which consists of seven stages: Stage I, four-arm stage; Stage II, eight-arm stage; Stage III, vestibular invagination stage; Stage IV, rudiment initiation stage; Stage V, pentagonal disc stage; Stage VI, advanced rudiment stage; and Stage VI, tube-foot protrusion stage. Each stage is characterized by significant morphological features observed for the first time at that stage. This scheme is intended as a guide for determining the degree of larval development, and for identifying larval and adult structures. Larval anatomy was visualized using light and confocal microscopy as required on living material, whole mount fixed specimens, and serial sections. Antibody staining to localize specific gene products was also used. Detailed analysis of these data has furthered our understanding of the morphogenesis of the rudiment, and has suggested provocative questions regarding the molecular basis for these events. We intend this work to be of use to investigators studying gene expression and morphogenesis in postembryonic larvae.     

Characterization of the vitelline envelope of the sea urchin Strongylocentrotus purpuratus

The vitelline envelope (VE) is an extremely thin, acellular, proteinaceous coat that surrounds the extracellular surface of sea urchin eggs. Despite previous studies on VE composition, structure and function, our understanding of the envelope is still incomplete at the molecular level. We have isolated VE components from intact, unfertilized Strongylocentrotus purpuratus eggs by reduction with alkaline dithiothreitol-sea water solutions and have characterized the macromolecules by SDS-PAGE. There were eight major glycoprotein bands, including two high molecular weight components at 265 and 300 kDa, and several minor components. We have revealed, by lectin blot analysis, that most components contain mannose, while a subset of glycoproteins contain fucose and N -acetylglucosamine; galactose and sialic acid were also detected. The components in the VE preparations were compared with cell surface complex preparations by immunoblot analysis, using antisera against a VE preparation, a 305 kDa electrophoretically purified VE glycoprotein and an extracellular portion of the sea urchin egg recombinant 350 kDa sperm receptor. Serum against the recombinant sperm receptor reacted with a component of ∼350 kDa on blots, but did not react with the 300 kDa component found in VE preparations. Therefore, we suggest these two glycoproteins are not the same.     

Apoptosis in early development of the sea urchin, Strongylocentrotus purpuratus

Apoptosis provides metazoans remarkable developmental flexibility by (1) eliminating damaged undifferentiated cells early in development and then (2) sculpting, patterning, and restructuring tissues during successive stages thereafter. We show here that apoptotic programmed cell death is infrequent and not obligatory during early embryogenesis of the purple sea urchin, Strongylocentrotus purpuratus. During the first 30 h of urchin development, fewer than 20% of embryos exhibit any cell death. Cell death during the cleavage stages consists of necrotic or pathological cell death, while cell death during the blastula and gastrula stages is random and predominantly caspase-mediated apoptosis. Apoptosis remains infrequent during the late blastula stage followed by a gradual increase in frequency during gastrulation. Even after prolonged exposure during the cleavage period to chemical stress, apoptosis occurs in less than 50% of embryos and always around the pre-hatching stage. Embryonic suppression of apoptosis through caspase inhibition leads to functionally normal larvae that can survive to metamorphosis, but in the presence of inducers of apoptosis, caspase inhibition leads to deformed larvae and reduced survival. Remarkably, however, pharmacological induction of apoptosis, while reducing overall survival, also significantly accelerates development of the survivors such that metamorphosis occurs up to a week before controls.     

Cholinoreceptors of the dentis retractor muscle of the sea urchin Strongylocentrotus intermedius

Neostigmine increases and cholinergic blocking agents block contractions of Strongylocentrotus intermedius dentis retractor muscle caused by either acetylcholine or indirect stimulation. Both innervated and non-innervated parts of the muscle are sensitive to low acetylcholine concentration suggesting that there are both synaptic and extra-synaptic cholinoreceptors. Nicotinomimetics are more potent than muscarinomimetics. Both d-tubocurarine and atropine are weak blocking agents as compared to pentadecamethylene-bis-trimethylammonium or hexadecamethylene-bis-trimethylammonium, Kd being 1 X 10(-4) to 5 X 10(-5) and 5 X 10(-7) to 4 X 10(-7) M respectively. Disulphide bonds and carboxylic (or phosphate) groups were revealed in dentis retractor muscle cholinoreceptors using drugs modifying receptor structure. Cholinoreceptors of both nicotinic and muscarinic type were revealed in oesophagus longitudinal muscles of Strongylocentrotus intermedius.     

Isolation of the plasma membrane from sea urchin embryos.

A method for isolation of sea urchin embryos plasma membranes is described. Purification of the obtained fraction was assayed by several enzymatic markers and electron microscopy. The isolated plasma membranes appear to be pure from contamination of other cell membranes (endoplasmic reticulum and mitochondria), and they can therefore be used for analytical studies on the composition and structure of plasma membrane.