Shin-Ichi Ohtake

Purification and Characterization of a 39,000-Da Serine Proteinase from the Hemolymph of a Solitary Ascidian, Halocynthia roretzi

A new endogenous serine proteinase from the cell-free hemolymph of a solitary ascidian, Halocythia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on TSKgel Toyopearl HW 65 F, ion exchange chromatography on TSKgel DEAE-Toyopearl 650 M, affinity chromatography on Arginine-Sepharose 4B, gel filtration on TSKgel Toyopearl HW 65F and hydroxyapatite chromatography on Bio-Gel HT. The serine proteinase is a single polypeptide chain whose molecular weight and isoelectric point are 39 kDa and about 7.6 pI, respectively. The most susceptible substrate was Boc-Leu-Gly-Arg-4-methyl-coumaryl-7-amide (MCA), and activity was optimal at pH 8. The enzyme was relatively stable at high temperatures; about 50% activity was retained even at 60 degrees C for 30 min in 50 mM Tris-HCl, pH 8.0, containing 0.5 M NaCl, and 0.05% Brij-35. The enzyme was characterized by the inhibitory effects of synthetic or natural inhibitors, substrate specificity toward 26 peptidyl-MCAs, proteinase activity toward natural proteins and complex formation with a serine proteinase inhibitor (58 kDa) previously found in H. roretzi hemolymph, indicating that the enzyme was a member of serine proteinases and strongly inhibited by the 58 kDa serine proteinase inhibitor as well as human antithrombin III. We also demonstrated the clotting enzyme activity of the purified serine proteinase toward bovine fibrinogen and Limulus coagulogen, a fibrinogen-like clottable protein of horseshoe crabs.

Purification and characterization of a 58, 000-Da proteinase inhibitor from the hemolymph of a solitary ascidian, Halocynthia roretzi

A new endogenous proteinase inhibitor from the cell-free hemolymph of a solitary ascidian, Halocynthia roretzi, was purified by a combination of ammonium sulfate fractionation, hydrophobic interaction chromatography on Ether-Toyopearl and affinity chromatography on Heparin-Sepharose. The purity of the inhibitor was examined by SDS-PAGE, gel-permeation chromatography, reversed-phase chromatography, isoelectric focusing, immunological analysis and amino-terminal amino acid sequence analysis. The inhibitor is a single polypeptide chain whose molecular weight, isoelectric point and the first 10 amino-terminal amino acid sequences are 58 kDa, pI 9.2 and NH2-Thr-Lys-Lys-Asp-Gly-Glu-Glu-Lys-Val-Ala, respectively. The purified protein inhibits plasma enzyme(s) of H. roretzi, and the rate of inhibition to the plasma enzyme(s) activity was accelerated by incubation with dextran sulfate, but the effect was neutralized by further incubation with polycation, such as polybrene or protamine sulfate. The inhibitory activity was not affected appreciably by pH 7-10 but ceased completely below pH 5 or by heating at 50 degrees C for 30 min.

Common Cell‐Surface Antigens Functioning in Self‐Recognition Reactions by Both Somatic Cells and Gametes in the Solitary Ascidian Halocynthia roretzi

The “contact reaction” is an extremely rapid allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the solitary ascidian Halocynthia roretzi. It has been proposed that regulation of the alloreactivity of hemocytes may be involved in preference for fertilization or self‐sterility in this species. To identify the receptors and target ligands involved both in self‐recognition by somatic cells and self‐discrimination by gametes, we produced monoclonal antibodies (mAbs) that inhibit the ACR mediated by hemocytes and tested their effects on fertilization. Six different mAbs that inhibit the ACR were prepared and categorized into three groups. Although all three mAbs seemed to have the same ability to inhibit the ACR, almost constant and statistically significant inhibition (CRB1.1) and infrequent but significant inhibition (CRB2.1, and CRB3.1) of the ACR were observed in the same pairs of animals. Pretreatment of the unfertilized eggs with CRB1.1, CRB2.1, and CRB3.1, resulted in the constant and statistically significant inhibition, infrequent but significant inhibition, and no inhibition, respectively, of fertilization. Antigens recognized by CRB1.1 (CRB1.1 antigens) were detected on the cell surface of all types of hemocytes and on the vitelline coat and follicle cells of unfertilized eggs. CRB2.1 and CRB3.1 antigens were detected on the surface of certain types of hemocytes and follicle cells, but not on the vitelline coat. CRB mAbs were directed against different epitopes in the N‐linked glycan on glycoproteins. These common carbohydrate antigens on somatic cells and gametes may function in some recognition processes in ACR and fertilization in H. roretzi.

Evidence for neurosecretory control of the optic gland in terrestrial pulmonates

The optic gland in terrestrial pulmonates secretes gonadotropic hormone. The present study investigates the fine structure of the optic gland to clarify the control of secretion. Animals used were the slug, Limax marginatus, and the snail, Euhadra peliomphala. The optic gland cell has a process and the cytoplasm is filled with large granules about 750-1300 nm in diameter with a thin cortex in L. marginatus and 800-1700 nm delimited by a thick peripheral layer in E. peliomphala. These gland cells are characterized by well-developed granular endoplasmic reticulum. Granule formation was seen in the region of the Golgi apparatus. During the breeding season, the medial neurosecretory cells of the brain are active in the production and release of secretory materials. Although no neurosecretory cell bodies occur in the tentacular ganglion, neurosecretory axons penetrate into the optic gland cells. Cobalt filling reveals that axons of the medial neurosecretory cells project to the tentacular ganglion, near the optic gland. These results suggest that the optic gland is controlled by a neurohormone originating from the medial neurosecretory cells of the brain.

Neuroendocrine control of the dorsal bodies in the giant African snail, Achatina fulica

Summary In the giant African snail, Achatina fulica, the ultrastructure of the dorsal bodies was examined in an attempt to characterize the neural control of their activity. In the dorsal body many large ellipsoidal lipid droplets with a thick electron-dense cortex and secretory granules with an electron-dense core were found. In a bundle of commissural nerves that originated from the cerebral ganglion, two types of axon were detected: type I axons contained spherical granules and type II axons contained ellipsoidal granules. These axons innervated the dorsal body cells. Our results suggest that each cell of the dorsal bodies is under the control of two axons from the cerebral ganglion: one that is stimulatory and another that may be inhibitory.

Mutual and directional allogeneic cytotoxic reaction of hemocytes in the solitary ascidian Halocynthia roretzi revealed by one-step quantitative fluorimetric assay.

Abstract A novel one-step microplate cytotoxicity assay using the cytoplasmic fluorescent viability dye calcein AM was established for simple, rapid, sensitive, and quantitative measurements of the allogeneic cytotoxic reaction (ACR) mediated by hemocytes in the ascidian Halocynthia roretzi. The mutual and directional ACR was distinguishable by the assay using the hemocytes from pairs of animals with different alloreactivities. The ACR assay may allow more precise genetic analysis of the gene that controls allore-activity of hemocytes, since the mutual and directional ACR may be related to levels of expression or numbers of the gene product or products on the target cells. The directional ACR will be useful in elucidating the cellular and molecular mechanisms of self-recognition in H. roretzi, since it allowed us to equate hemocytes from one animal with “effector cells” and those from the other animal of the pair with “target cells”. In addition, the quantitative ACR assay in a large number of samples is possible and it will allow production of monoclonal antibodies that may recognize receptors or ligands functioning in self-recognition processes by the H. roretzi hemocytes.

The Viriform Cell of Halocynthia roretzi: Fine Structure, Distribution, and Appearance

The viriform cell (VC) in the tunic of Halocynthia roretzi is rod shaped (about 15x5 µm) and is characterized by intracellular eosinophilic and electron-dense granules (about 1–2 µm in diameter). The VC is confirmed to be a native tunic cell by the DNA fingerprint method. Light and electron microscopic observations and immunohistochemical findings demonstrated that VCs appeared in the tunic and hemocoel of about 3-month-old specimens but were not seen in younger juvenile specimens (2 weeks after metamorphosis). There were more VCs in the tunic matrix of 1-year-old specimens than in 3-month-old specimens. It seems that the cells migrated from the hemocoel through the epidermis. In mature specimens, the density of VCs was very high not only in the inner tunic matrix but also in the tunic of growing attachment villi. When the villi became brown, broken VCs were observed at the area just under the cuticle, where the subcuticle forms later. VCs also crowded at the repairing surface of the tunic after artificial injury. These findings suggest that VCs may play roles in subcuticle formation and wound healing.

Common Cell Surface Ligands Functioning in Allogeneic Cytotoxic Reaction and Fertilization in Halocynthia roretzi

Two distinct oligosaccharide ligands that function in both the allogeneic contact/cytotoxic reaction (ACR) by hemocytes and gamete fertilization were identified by means of the monoclonal antibodies (Mabs), CRB1 and CRB2, which inhibit both the ACR and fertilization in Halocynthia roretzi. The CRB1 epitope, determined to be the “core” α1–6 fucosylated oligosaccharide epitope (CFOE) in hybrid-type N-glycans, was detected on the cell surfaces of all types of hemocytes as well as on those of follicle cells and the vitelline coat of eggs. The CRB2 epitope, which was indicated as an outer-side sulfated and phosphorylated ojigosaccharide epitope (OSPOE) in complex-type N-glycans, was demonstrated on the cell surfaces of some types of hemocytes and egg follicle cells. Both epitopes, OSPOES and CFOE, may function as ligands in the initial attachment and binding, respectively, in the sperm-egg interaction. These epitopes may also function in the same way in the self-recognition of the ACR. We propose that the fucose residue in CFOE is among the key molecules, and perhaps the most important, involved in self-recognition in the ACR and fertilization.

Hemopoiesis in Solitary Ascidians

The gut-associated hemocyte aggregates in Halocynthia roretzi contained a large number of proliferating small basophilic cells, while circulating hemolymph contained proliferating hemocytes. The gut-associated hemocyte aggregates developed into a large accumulation of proliferating hemocytes as Halocynthia roretzi grew. Those gut-associated hemocyte aggregates may be the main hemopoietic site of this animal, while small hemopoietic sites may also be distributed in other tissues.

Study of Color Variation in the Solitary Ascidian Halocynthia roretzi, Collected in the Inland Sea of Japan

Abstract In the vicinity of Yashiro Island in the Inland Sea of Japan, the solitary ascidian (tunicate) Halocynthia roretzi with tunics of various colors were collected. Samples of these animals were sorted into three groups on the basis of visual observation of tunic color. The red group includes animals with dark-red, light-red, or orange tunics. The pink group includes animals with tunic colors ranging between red and white. The white group includes only animals with completely white tunics. Animals in the white group lacked color internally, with the exception of the hepatopancreas and the gonads in breeding season; the epidermis and gill basket were white. In contrast, animals of both the red group and the pink group were colored internally, with red-orange epidermis and yellow gill basket. Alloreactivity was tested by mixed-hemocyte incubation between different animals belonging to the same color group and between animals belonging to different color groups. Alloreactivity between animals of the white group was 56.3%, between animals of the pink group was 60.0%, and between animals of the red group was 69.3%. The relatively high frequency of compatible combinations among the white animals is discussed.