Spinous injury caused by a sea urchin.

A bather on holiday in Kenya injured a finger on a spiny marine creature living on the sea bed. A skin biopsy specimen from the injured finger contained several black spines about 0.5 mm in diameter and up to 1.5 cm in length. Spines removed from the specimen were embedded in plastic resin to facilitate transverse sectioning. Light microscopical examination using crossed polarisers showed an ornate symmetrical structure brightly illuminated against a dark background. These features are characteristic of sea urchin (Echinoderm) spines which are composed of ornately formed calcite crystals covered by an epithelium. The spines of sea mice, on the other hand, are chitinous in nature; they are also much finer and lack the ornate symmetry of sea urchin spines.

A toxic substance from the sea urchin Toxopneustes pileolus induces histamine release from rat peritoneal mast cells.

A toxic substance (P-II fraction), fractionated from the pedicellariae of the sea urchin Toxopneustes pileolus, dose-dependently caused the histamine release from rat peritoneal mast cells. The histamine release induced by P-II fraction increased with time, while compound 48/80 caused a more rapid histamine release. The dose-response curve for P-II fraction was studied with concentration 0.03-2.0 mg/ml. This reaction was dependent on Ca2+ and temperature. When glucose (5.5 mM) was omitted during the incubation step, the histamine release induced by P-II fraction was significantly reduced as compared to that of compound 48/80. Pyruvate reversed this reduction. On the other hand, the histamine release induced by P-II fraction was effectively potentiated by the addition of glucose (11.0 mM), but not that by compound 48/80. These results suggest that P-II fraction-induced histamine release differs from that of compound 48/80 disregards to the effects of glucose, because this histamine release appears to be more sensitive to the glycolytic pathway than compound 48/80-induced histamine release.

Immunological evidence for the presence in sea urchin embryos of a cell adhesion protein similar to mouse uvomorulin (E-cadherin).

A tryptic fragment (88 kDa), obtained from external digestion of sea urchin embryos carried out in the presence of calcium, shows immunological cross-reactivity with polyclonal and monoclonal antibodies (DECMA-1) against mouse teratocarcinoma uvomorulin. Fab fragments obtained from anti-mouse terato-carcinoma uvomorulin mono- and polyclonal antibodies, and from polyclonal antibodies against the partially purified 88-kDa tryptic fragment, decompact early sea urchin embryos and block reaggregation of dissociated sea urchin blastula cells. These data indicate the presence of an uvomorulin-like protein in sea urchin embryos and suggest an important role for this protein in embryonic development.

Biochemical and immunological characterization of collagen molecules from echinothurioid sea urchin Asthenosoma ijimai.

Collagens collected from the test (the external hard covering of invertebrates) of the sea urchin, Asthenosoma ijimai, were characterized biochemically and immunologically. The amino-acid composition was typical of that of mammalian collagens. Crystals of segment-long-spacing showed that the molecules of sea urchin collagen were 300 nm long. Selective salt precipitation revealed that the collagen has the same solubility characteristics as type I collagen. The collagen was denatured at 23.1 degrees C. Anti-sea urchin collagen antisera were immunologically cross-reacted with collagens of the same species and the starfish Asterina pectinifera. However, the antisera showed no or slight responses to collagens of bovine type I, II, III, IV and V. The collagen molecules contained four alpha-chains, named alpha 1(SU), alpha 2(SU), alpha 3(SU) and alpha 4(SU), respectively. All of the four alpha-chains were eluted in the same fraction on gel filtration chromatography. Chains of alpha 1(SU) and alpha 2(SU) were extracted earlier than alpha 3(SU) and alpha 4(SU) during pepsin digestion. Other biochemical and immunological analyses clearly demonstrated that test of sea urchins contains two genetically different, but biochemically similar, species of collagens, one of which is composed of alpha 1(SU) and alpha 2(SU) chains, and the other of alpha 3(SU) and alpha 4(SU).

A species-specific sperm-activating peptide from the egg jelly of the sea urchin Diadema setosum.

1. A species-specific sperm-activating peptide was isolated from the egg jelly of the sea urchin Diadema setosum and the amino acid sequence was determined as follows: (formula; see text). 2. The peptide caused significant increases of respiration rates and cyclic nucleotide concentrations in D. setosum spermatozoa as low as 10(-9) M. 3. The addition of the peptide to D. setosum spermatozoa resulted in the appearance of a newly stained protein (mol. wt 128,000) on sodium dodecyl sulfate-polyacrylamide gels.

Localization of the sea urchin Spec3 protein to cilia and Golgi complexes of embryonic ectoderm cells.

Expression of the Spec3 gene of Strongylocentrotus purpuratus is associated with ectodermal ciliogenesis. An antiserum was raised against the amino terminus of the deduced Spec3 amino acid sequence and used for immunofluorescent staining. Cilia and an apical structure at the base of the stained cilium of each ectodermal cell stained intensely in gastrula and later stage embryos. Microtubule-depolymerizing agents dispersed the concentrated spot of apical staining, suggesting a localization of Spec3 antigen to the Golgi complex. Immunogold electron microscopy confirmed the localization of Spec3 antigen on cilia and in the Golgi complex. Spec3 antigen showed a diffuse punctate staining pattern in the ectodermal cytoplasm of hatching blastula when Spec3 transcripts are most prevalent, suggesting that after synthesis, Spec3 is sequestered in the Golgi complex before appearing on cilia. Whereas the predicted Mr of the Spec3 protein is 21,600, immunoblotting with S. purpuratus proteins indicated that a Spec3 antigen was concentrated in cilia and migrated as an SDS-resistant aggregate of Mr approximately 350,000. Spec3 is also concentrated in cilia of Lytechinus pictus but the protein migrated with an Mr approximately 23,000 in this species. The S. purpuratus Spec3 antigen remains associated with the ciliary axoneme after extraction of membrane proteins.

Purification and characterization of a cortical secretory vesicle membrane fraction.

A membrane fraction has been prepared by sucrose density gradient fractionation of purified cortical secretory vesicles from the eggs of the sea urchin Strongylocentrotus purpuratus. The purified cortical vesicle membrane fraction has a phospholipid to protein ratio of 1.76 and exhibits a morphology typical of biological membranes as seen by electron microscopy. The protein composition of the purified membranes was analyzed by SDS-polyacrylamide gel electrophoresis and shown to be distinct from that of eggs, cell surface complex, cortical vesicles, fertilization product, and yolk platelets. Alkaline extraction (pH 11.0) of peripheral membrane proteins increased the phospholipid to protein ratio to 2.55 and removed several polypeptides. Immunoblot analysis of the isolated cortical vesicle membrane fraction revealed low levels of contamination with two major cortical vesicle content proteins. Fractions enriched in egg plasma membranes and yolk platelet membranes also have been isolated and compared with the cortical vesicle membranes by SDS-polyacrylamide gel electrophoresis. The protein compositions of the three membrane fractions were found to contain very little overlap, indicating that the cortical vesicle membrane preparation is relatively free of contamination from these likely noncortical vesicle sources of membrane. Both the plasma membrane and cortical vesicle membrane samples were found by immunoblotting to contain actin.

Avidin-like domain in an epidermal growth factor homolog from a sea urchin.

We have found that a protein from the purple sea urchin has a carboxyl-terminal domain with striking sequence similarity to chicken avidin and bacterial streptavidin. All our evidence supports the homology of these sequences. Tetramers of avidin and streptavidin bind biotin strongly; the biotin binding site involves two to four tryptophans and probably an adjacent lysine in each chain. The presence of four tryptophans at equivalent positions in the sea urchin protein domain suggests that it may also be able to bind biotin and inhibit cell growth, as do the two other proteins. Alternatively, this domain may have acquired a new role as part of a multidomain protein.

Identification of a major polypeptide component of the sea urchin fertilization envelope.

A monoclonal antibody (MAb No. 25-16), raised against purified cortical secretory vesicles (CVs) from the eggs of Strongylocentrotus purpuratus, has been used to identify a previously uncharacterized CV-derived polypeptide component of the sea urchin fertilization envelope (FE). MAb No. 25-16, an IgG1, bound to a group of proteins with Mr approximately 200,000 on immunoblots of CVs. This same group of proteins also was detected in fertilization product and in soft FEs prepared from early embryos, indicating that the antigen is released at fertilization by CV exocytosis and becomes incorporated into the FE. The multiple components recognized by MAb No. 25-16 apparently did not result from proteolysis during sample preparation or differential N-linked glycosylation. No simplification of the SDS-gel or immunoblot patterns was observed when samples of fertilization product or cell surface complex were prepared in the presence of a cocktail of protease inhibitors; nor was a change in mobility of any of the antigen forms detected following treatment with endoglycosidase F. Upon partial denaturation and reduction of the protein by incubation at room temperature in the presence of SDS and dithiothreitol, the antigen was shown to undergo a decrease in relative mobility on SDS-PAGE. Complete reduction and denaturation, by boiling in dithiothreitol-containing SDS sample buffer or by an on-blot reduction technique, resulted in loss of the epitope. The protein component recognized by MAb No. 25-16 underwent a striking increase in mobility on SDS-PAGE after chelation of calcium ions with EGTA. Immunogold labeling on thin sections of unfertilized eggs revealed that the antigen is located in the spiral lamellar cores of all CVs. In fertilized eggs, fixed 5 min after insemination, the antigen was detected in the FE. Based on these biochemical and immunological data, we suggest that the antigen recognized by MAb No. 25-16 is released exocytotically from the CVs into the perivitelline space at fertilization and becomes incorporated into the FE. The abundance of this antigen suggests that it may function as a structural component of the FE.

Fatty chain composition of phospholipids in sea urchin spermatozoa.

1. An analysis was made of lipids extracted from the spermatozoa of the sea urchins, Hemicentrotus pulcherrimus and Anthocidaris crassispina. 2. Nearly all the lipids from both species consisted of phospholipids (about 80%) and cholesterol (about 14%). Triglyceride and cholesterol ester were present in trace amounts. 3. The fatty acid composition of each phospholipid was determined by gas-liquid chromatography. In both species, the fatty acid consisting of phosphatidylethanolamine was of the unsaturated type for the most part, while cardiolipin was comprised to a considerable degree of saturated fatty acids. In phosphatidylcholine and phosphatidylserine from H. pulcherrimus sperm, unsaturated fatty acid content was somewhat higher than that in phospholipids from A. crassispina sperm.

Localization and possible function of 20 kDa actin-modulating protein (actolinkin) in the sea urchin egg.

We have previously described a novel actin-capping protein, a 20,000-molecular weight protein (20K protein)-actin complex (20K-A) isolated from sea urchin eggs. In the present study, the localization and possible function of this 20K protein were investigated. The 20K protein was localized in the sea urchin egg cortex. Its distribution in the cortex as revealed by immunofluorescence microscopy did not change during or after fertilization up to the first mitosis, but it was concentrated to some extent in the cleavage furrow region. Exogenously added actin polymerized on the cortex isolated from unfertilized egg; however, actin did not polymerize on the cortex extracted with 0.6 M KCl, that is, the cell membrane, which lost the 20K protein. The cell membrane preincubated with 20K-A restored the activity to grow actin filaments. When decorated with myosin subfragment 1, almost all the actin filaments showed the arrowhead configuration pointing away from the membrane, indicating that they were connected to the membrane at their barbed ends. These results strongly suggest that the 20K protein connects actin filaments to the plasma membrane of sea urchin eggs. Because of this property we call this protein "actolinkin".

A high molecular weight actin binding protein: its localization in the cortex of the sea urchin egg.

From the Triton-treated cortex fraction of sea urchin eggs, a high molecular weight actin binding protein (260K protein) was solubilized by a high salt solution and purified. A cosedimentation assay revealed that the 260K protein binds to actin filaments in a concentration-dependent manner. The low-shear viscosity of actin solutions largely increased in a concentration-dependent manner after addition of 260K protein. Electron microscopy showed that this protein induces the formation of large curled bundles of actin filaments. Different from fascin-induced actin bundles, no clear striations were observed within the actin bundles formed by the 260K protein. Antibodies against the 260K protein were raised in a rabbit and affinity purified. Immunoblotting analysis of Triton-solubilized cortex and various subcellular fractions showed that first only a single band reacted with the antibody and second that the 260K protein exclusively localized in the cortex fraction. Indirect immunofluorescence microscopy localized the protein in the cortex and the region of the cleavage furrow. After double staining, the fluorescence images for actin filaments and the 260K protein well correlate with each other.

Sea urchin prosome: characterization and changes during development.

A cytoplasmic particle displaying properties in common with a structure present in duck erythroblasts, termed the prosome, has been isolated from eggs and embryos of two species of sea urchin. This particle was partially purified by sedimentation in sucrose gradients containing 0.5 M KCl, and some of its physical properties and its behavior during early development were determined. The prosome sediments between 16 and 19 S and has a buoyant density of 1.30 g/cm3 in Cs2SO4 gradients. Biochemically, the particle is characterized as 20-25 polypeptides of molecular size 24-35 kDa with about 10 small RNAs. A monoclonal antibody directed against the 27-kDa protein of duck erythroblast prosome recognizes a 27-kDa protein of the sea urchin prosome. We have used this protein, as representative of the prosome, to immunologically determine the level and the subcellular localization of the particle during development. Immunoblotting and cellular fractionation studies show that the 27-kDa prosome polypeptide is present almost entirely in the postribosomal supernatant of unfertilized egg lysates. After fertilization and during early development, the total amount of 27-kDa protein per embryo remains constant, but the amount in the postribosomal supernatant decreases; there is a concomitant increase in the level of the 27-kDa protein in a rapidly sedimenting, particulate fraction containing nuclei. Immunofluorescence studies further show that the 27-kDa protein is located mainly in the cytoplasm of eggs and two-cell embryos. The subcellular location of the prosome, therefore, appears to change during development. In vivo labeling experiments have failed to detect the synthesis of either the prosome proteins or RNAs in eggs and embryos up to 48 hr of development, suggesting that this cytoplasmic particle is not synthesized de novo in early embryogenesis and thus is metabolically stable. The prosome is thus a normal cellular constituent of the sea urchin and is most likely synthesized during oogenesis and stored in the unfertilized egg.

Tryptic digestion of dynein 1 in low salt medium. Origin and properties of fragment A.

Dynein 1 was extracted from sperm flagella of the sea urchin Tripneustes gratilla with 0.6 M NaCl and dialyzed against 0.5 mM EDTA, 14 mM 2-mercaptoethanol, 5 mM imidazole/HCl buffer, pH 7.0, for 24-48 h. In some cases, fractions containing the alpha heavy chain and the beta/intermediate chain 1 complex (beta/IC1) were separated by density gradient centrifugation in the same solution. Treatment of the samples at a trypsin:protein ratio of 1:10 w/w for 32 min at room temperature yields a crude digest from which Fragment A is purified by density gradient centrifugation. The purified Fragment A consists of two principal peptides (Mr = 195,000 and 130,000) that cosediment with the peak of ATPase activity at 12.5 S, which is slightly faster than the 11 S of the original beta/IC1 complex. When digests of the separated alpha chain and of the beta/IC1 complex are followed as a function of time, the early cleavages of the two heavy chains (Mr = 428,000) resemble each other in that both lead to similarly sized peptides of Mr 316,000 and 296,000, but only in the beta/IC1 fraction does the digestion proceed to form Fragment A. The remainder of the beta chain, termed Fragment B, occurs as an Mr 110,000 peptide sedimenting at 5.7 S with no associated ATPase activity. Fragment A has a specific ATPase activity of 4.3 mumol Pi X min-1 X mg-1, with a Km of 29 microM in 0.1 M NaCl medium, and an apparent Ki for inhibition by vanadate of 1.2 microM in the absence of salt, and 22 microM in 0.6 M NaCl. Photoaffinity labeling with [alpha-32P]8-azidoadenosine 5'-triphosphate indicates that the ATP binding site on the beta chain of dynein 1 is located on the Mr 195,000 peptide of Fragment A. The possibility that Fragments A and B of the beta/IC1 complex may correspond to the head and tail regions of the tadpole-shaped particle seen by electron microscopy is discussed.

Matrix proteins of the teeth of the sea urchin Lytechinus variegatus.

The teeth of the sea urchin Lytechinus variegatus grow continuously. The mineral phase, a high magnesium calcite, grows into single crystals within numerous compartments bounded by an organic matrix deposited by the odontoblasts. Electron microscopic examination of glutaraldehyde-fixed Ethylene Diamine Tetra acetic acid (EDTA) demineralized teeth shows the compartment walls to be organized from multiple layers of cell membrane which might contain cytoplasmic protein inclusions. Proteins extracted during demineralization of unfixed teeth were examined by gel electrophoresis, high performance liquid chromatography, and amino acid analysis. The tooth proteins were acidic, they contained phosphoserine, and they were rich in aspartic acid. By contrast, the proteins of similarly extracted mineralized Aristotle's lantern skeletal elements were nonphosphorylated and were rich in glutamic acid. Vertebrate tooth and bone matrix proteins show similar differences. Surprisingly, an antibody to the principle rat incisor phosphoprotein showed a significant cross-reactivity with the urchin tooth protein, by dot-blot and enzyme-linked immunosorbent assay procedures. Thus, the urchin tooth proteins contain epitope regions similar to those which are phenotypic markers of vertebrate odontoblasts. Whether this is an expression of convergent or divergent evolutionary processes, it is likely that the matrix proteins play a similar role in matrix mineralization. The sea urchin tooth may thus be an excellent model for the study of odontoblast-mediated mineral-matrix relationships.

Isolation and characterization of spicule proteins from Strongylocentrotus purpuratus.

A simple method is described for the isolation of spicules from pluteus embryos of the sea urchin, Strongylocentrotus purpuratus. Radio-iodination of the demineralized matrix reveals six bands on SDS protein gels. Treatment with N-glycanase leads us to believe that some of these proteins are N-linked glycoproteins.

Characterization of yolk platelets isolated from developing embryos of Arbacia punctulata.

Yolk platelets, a major organelle of sea urchin eggs and embryos, were isolated from Arbacia punctulata and biochemically characterized over the course of development to the pluteus stage. Fractionation by sucrose gradient centrifugation revealed yolk platelets in two major density classes. The low-density yolk platelet fraction could be obtained as a very homogeneous preparation and was highly enriched in acid phosphatase activity, while depleted of mitochondrial (cytochrome c oxidase) and plasma membrane (phosphodiesterase) marker enzymes. The chemical composition of low-density yolk platelets prepared from eggs and embryos at various stages of development remained unchanged in terms of phospholipid, triglyceride, hexose, sialic acid, RNA, and protein. However, analysis of the major yolk platelet glycoproteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a number of stage-specific changes. These glycoproteins were found to be major glycoproteins of crude embryo lysates and were predominantly of the polymannose, N-linked type. The predominance of polymannose-type glycoproteins in yolk platelets was further demonstrated by their staining with concanavalin A-colloidal gold in Lowicryl-embedded sections of embryos. These studies represent the first systematic biochemical characterization of intact yolk platelets and the changes in them during early embryonic development.