Protease in ascidian endostyle

Summary A proteolytic activity at pH 7.6 was determined in the endostyle of the ascidianStyela plicata. With the use of specific inhibitors, it was demonstrated that a tryptic-like alkaline protease, TLCK-dependent, is involved.The author wishes to thank Prof. A Minganti for his critical revision of the text.     

Myoneural junctions in larval ascidian tail

Zusammenfassung Elektronenmikroskopische Untersuchungen der Neuriten in der Peripherie des dorsalen Nervenstranges im Schwanz der Aszidien-LarveAmaroucium constellatum zeigen, dass die in der Nähe liegenden Schwanzmuskelzellen Verbindungen mit den Neuriten eingehen, die den neuromuskulären Kontaktstellen im Skelettmuskel der Wirbeltiere ähnlich sind. Diese neuromuskulären Verbindungen verteilen sich über die Gesamtlänge des Schwanzes und sind offenbar die Innervation der Schwanzmuskulatur.

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Supported by grants No. NS-07495 and No. NS-07197 from the NIH, U.S. Public Health Service.     

Autonomous fluorescence of ascidian blood cells with special reference to identification of vanadocytes

Summary A tunichrome that has been suggested to be involved in the accumulation of vanadium ions in ascidian blood cells produces an autonomous fluorescence upon excitation with blue-violet light. However, we have found that signet ring cells, which contain large amounts of vanadium, do not fluoresce upon such excitation. The strongest fluorescence due to the tunichrome was observed in morula cells, which do not contain vanadium.     

The presumptive territory of the mesoderm in the ascidian germ

Riassunto Col metodo delle marche con granuli di gesso colorato, sono stati modificati gli schemi diConklin riguardanti i territori presuntivi del mesenchina e della muscolatura nel germe di Ascidie.     

The mitochondrial pattern in the development of the ascidian egg

Riassunto Con il verde Janus è stata seguita la distribuzione dei mitocondri lungo lo sviluppo dell'uovo di Ascidie. I mitocondri hanno una distribuzione diffusa nell'uovo vergine: ma dopo la fecondazione sono trasportati al polo vegetativo dell'uovo e successivamente nella porzione subequatoriale dorsale. Vengono poi gradualmente segregati nelle cellule della linea muscolare. Sono queste anche le cellule più ricche in citocromoossidasi, e che presentano un'attività ossido-riduttiva più intensa.     

Involvement of prolyl endopeptidase in ascidian fertilization

Inhibitory effects on fertilization of the ascidian of three benzyloxycarbonyl(Z)-aminoacyl prolinals and Z-Gly-Pro-chloromethyl ketone added before and after insemination were examined. The results suggest that the prolyl endopeptidase is involved in the process of fertilization, especially in a process taking place between chorion elevation and cell cleavage.     

Involvement of prolyl endopeptidase in ascidian fertilization

Summary Inhibitory effects on fertilization of the ascidian of three benzyloxycarbonyl(Z)-aminoacyl prolinals and Z-Gly-Pro-chloromethyl ketone added before and after insemination were examined. The results suggest that the prolyl endopeptidase is involved in the process of fertilization, especially in a process taking place between chorion elevation and cell cleavage.Acknowledgment. We are indebted to Dr T. Numakunai of the Marine Biological Station of Tohoku University for collecting the ascidians and to Dr M. Hoshi of the Tokyo Institute of Technology for his helpful discussion. We are also grateful to Dr T. Yoshimoto and Prof. D. Tsuru of Nagasaki University for their generous gift of Z-Gly-Prochloromethyl ketone. This work was supported by a Grant-in-Aid from the Ministry of Education, Science and Culture of Japan.     

Incorporation of phenylalanine-H3 in the fragments of the fertilized ascidian egg

Riassunto E'stata studiata l'incorporazione di fenilalanina-H³ nelle metà animali e vegetative delle uova fecondate di Ascidie tagliate subito dopo l'emissione del 1° e del 2° globulo polare, allo scopo di vedere se le potenzialità di sviluppo delle metà vegetative fossero legate con un diverso metabolismo proteico. I risultati hanno mostrato che entrambe le metà incorporano fenilalanina-H³.     

Timing of action of sperm proteases in ascidian fertilization

Summary The timing of action of three sperm proteases, acrosin, spermosin, and a chymotrypsin-like enzyme, in the fertilization of the ascidian,Halocynthia roretzi, was examined by adding specific protease inhibitors at various times after insemination. The results indicate that the last two enzymes both function at the early stage of the process of sperm penetration through the egg investment, while acrosin functions at the late stage.Acknowledgments. We are indebted to Dr M. Hoshi of Tokyo Institute of Technology for his helpful discussion, and to Dr T. Someno of Nippon Kayaku Kogyo Co. for his generous gifts of Z-Val-Pro-Arg-H and leupeptin. This work was supported by Grants-in-Aid from the Ministry of Education, Science and Culture of Japan, and from Naito Research Foundation.     

Presence of 3,4-dihydroxyphenylalanine-containing peptides in hemocytes of the ascidian,Halocynthia roretzi

Summary Five 3,4-dihydroxyphenylalanine (DOPA)-containing peptides have been isolated from hemocytes of the ascidian,Halocynthia roretzi. Three of them were composed of DOPA, proline, phenylalanine, histidine and arginine in different ratios, while the other two contained only DOPA and an unidentified amino acid. DOPA-containing peptides were found to exist in only one type of hemocyte.     

Lithium and phorbol ester modify the activating capacity of ascidian spermatozoa

In this paper we have shown, using the whole-cell voltage clamp technique, that two parameters of the fertilization current in ascidian eggs may be modified by exposing spermatozoa to lithium or to phorbol ester. When spermatozoa were pre-treated in 250 mM lithium sea water for up to 30 min there was a significant increase in the mean initial slope of the fertilization current, from 116±90 to 169±84 pA/s (p<0.05). The peak current increased from 1371±1079 to 1719±1052 pA (p>0.05). Pre-treatment in 200–600 nM phorbol 12-myristate 13-acetate also increased the activating capacity of ascidian sperm, as monitored by a significant increase in the initial slope current in control eggs; however, there was no increase in peak current. Furthermore, we have shown, using NH₄Cl, that an increase in intracellular pH alone is insufficient to change the activating capacity of spermatozoa. This is the first report showing that the kinetics of an egg activation event depend upon the physiological status of the spermatozoon.     

In vivo incorporation of14C-phenylalanine into ascidian tunichrome

Ascidia ceratodes exposed to¹⁴C-phenylalanine in the surrounding seawater incorporates the radiolabel into newly biosynthesized tunichrome molecules. Radioactivity can be detected in tunichrome extracted from circulating blood cells within one day following initial exposure to the radiolabel; weak activity (4 Ci/mol tunichrome=22 nmol phenylalanine/mol tunichrome) is detected in 1 to 10 days; significantly higher amounts of radiolabel (57 Ci/mol tunichrome=318 nmol phenylalanine/mol tunichrome) appear 20 days after seawater exposure. Therefore, phenylalanine can function as a precursor in the biosynthesis of tunichrome.     

In vivo incorporation of 14C-phenylalanine into ascidian tunichrome.

Ascidia ceratodes exposed to 14C-phenylalanine in the surrounding seawater incorporates the radiolabel into newly biosynthesized tunichrome molecules. Radioactivity can be detected in tunichrome extracted from circulating blood cells within one day following initial exposure to the radiolabel; weak activity (less than or equal to 4 microCi/mol tunichrome = 22 nmol phenylalanine/mol tunichrome) is detected in 1 to 10 days; significantly higher amounts of radiolabel (57 microCi/mol tunichrome = 318 nmol phenylalanine/mol tunichrome) appear 20 days after seawater exposure. Therefore, phenylalanine can function as a precursor in the biosynthesis of tunichrome.