An enzyme-linked immunosorbent assay-based system for determining the physiological level of poly(ADP-ribose) in cultured cells.

PolyADP-ribosylation is mediated by poly(ADP-ribose) (PAR) polymerases (PARPs) and may be involved in various cellular events, including chromosomal stability, DNA repair, transcription, cell death, and differentiation. The physiological level of PAR is difficult to determine in intact cells because of the rapid synthesis of PAR by PARPs and the breakdown of PAR by PAR-degrading enzymes, including poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3. Artifactual synthesis and/or degradation of PAR likely occurs during lysis of cells in culture. We developed a sensitive enzyme-linked immunosorbent assay (ELISA) to measure the physiological levels of PAR in cultured cells. We immediately inactivated enzymes that catalyze the synthesis and degradation of PAR. We validated that trichloroacetic acid is suitable for inactivating PARPs, PARG, and other enzymes involved in metabolizing PAR in cultured cells during cell lysis. The PAR level in cells harvested with the standard radioimmunoprecipitation assay buffer was increased by 450-fold compared with trichloroacetic acid for lysis, presumably because of activation of PARPs by DNA damage that occurred during cell lysis. This ELISA can be used to analyze the biological functions of polyADP-ribosylation under various physiological conditions in cultured cells.

Characterization of O-GlcNAcylation in starfish (Asterina pectinifera) development from fertilization to bipinnaria larva.

Though O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) of nucleocytoplasmic proteins has been found in many multicellular organisms, its presence or absence in Echinodermata is unknown. Here we report the occurrence of O-GlcNAcylation in starfish (Asterina pectinifera) oocytes and the apparent O-GlcNAcylation pattern in starfish early development. O-GlcNAcylation might participate in the regulation of starfish development at the mid-blastula stage and thereafter.

The Starfish Asterina pectinifera: Collection and Maintenance of Adults and Rearing and Metamorphosis of Larvae.

Here we describe methods for (a) collecting starfish during their breeding period; (b) maintaining adults with fully grown gonads in laboratory aquaria; (c) rearing fertilized eggs to brachiolaria larvae, and (d) inducing larvae to metamorphose into juveniles under laboratory conditions. Such protocols should facilitate various analyses of starfish development throughout the entire life cycle of these model organisms.

Anti-fertilization activity of a spirocyclic sesquiterpene isocyanide isolated from the marine sponge Geodia exigua and related compounds.

(-)-10-epi-Axisonitrile-3, a spirocyclic sesquiterpene isocyanide obtained from the marine sponge Geodia exigua, immobilized sperm of sea urchin and starfish to block fertilization at the minimum effective concentration of 0.4 microg/ml. On the other hand, fertilized eggs developed normally to the gastrula stage in the presence of a 250-times higher concentration of the isocyanide. Analysis by (31)P NMR revealed an accumulation of phosphocreatine and a depletion of inorganic phosphate in the isocyanide-treated sperm, suggesting that (-)-10-epi-axisonitrile-3 inhibited the phosphocreatine shuttle participating in the high-energy phosphate metabolism, thereby immobilizing sperm to block fertilization. No analogs of (-)-10-epi-axisonitrile-3 containing different functionalities or isocyanides with different carbon skeleton exhibited such activity.

Achromosomal Division of Early Starfish Embryos Cultured in the Presence of Actinomycin D: (actinomycin D/RNA synthesis/starfish/blastulation).

Embryos of the starfish Asterina pectinifera were examined for their ability to undergo the early events of embryonic development in the presence of actinomycin D, a most widely used inhibitor of RNA synthesis. Fertilized eggs continued to divide eight or nine times in the presence of 25 µg ml-1 actinomycin D, although delay of development was observed. Chromatin disintegrated in the blastomeres of actinomycin D-treated embryos specifically at the 32-cell stage and the nucleus was undetectable at later stages. Before the 32-cell stage, RNA synthesis was not affected by the presence of actinomycin D whereas DNA synthesis was severely inhibited. The stage when achromosomal divisions cease and embryos begin to die corresponds to the period just before onset of blastulation, suggesting that the presence of the nucleus and chromosomes is a prerequisite for blastula formation and development beyond the 512-cell stage in this species.

Significance of an Increase of Intracellular Adenosine Concentration for Dormancy in Starfish Blastulae: (starfish embryos/blastula/adenosine/dormancy).

Adenosine at concentrations greater than 6 µg/ml halted embryonic development of the starfish Asterina pectinifera specifically at the 256-cell stage which corresponds to the onset of blastulation. When a fertilized egg was cultured continuously in sea water containing adenosine from fertilization, a gradual increase in intracellular concentrations of free adenosine was observed before a cessation of development took place. On the other hand, intracellular concentrations of ATP, ADP and AMP in the embryo cultured in sea water containing adenosine were nearly the same as those of an embryo cultured in sea water without adenosine. By returning the development-arrested embryo to normal sea water the embyro developed normally to the bipinnaria stage accompanied by a gradual decrease in the intracellular cencentration of adenosine. Treatment of fertilized eggs with 9-ß-d-arabinofuranosyl-9H-purine-6-amine (25 µg/ml) or 2'-deoxyadenosine (10 µg/ml) halted development specifically before the onset of blastulation in an irreversible manner. Embryos treated with 3'-deoxyadenosine (50 µg/ml) shortly after fertilization developed to healthy blastulae but hatching never occurred. These results exclude the possibilities that the action of adenosine is mediated by the inhibition of DNA synthesis or RNA polyadenylylation.

Regulation of Sperm Motility in Starfish. I. Initiation of Movement: (starfish sperm/seminal plasma/sperm propulsion/sperm best frequency).

Starfish seminal plasma has such characteristics as higher concentration of potassium ions, higher osmolality, and lower pH compared with those of sea water. These factors independently inhibited the movement of spermatozoa. Sperm movement was recorded by taking photographs of the swimming paths under a dark field microscope. Spermatozoa either did not move or swam with decreased beat frequency in isolated seminal plasma or in the presence of certain fractions of seminal plasma obtained by gel-filtration or by partitions. In normal sea water (containing calcium ions), a low pH of less than 6 resulted in a decrease in the number (%) of swimming spermatozoa, though the speed of propulsion and the beat frequency was not affected. On the other hand, in a calcium-free sea water, a large proportion of spermatozoa moved in both low and high pH conditions, but in low pH the speed of propulsion was reduced and many spermatozoa (31 %) swam in smaller circles.

Dihydrofolate Reductase in Starfish Oocytes and Embryos: Developmental Consequences of Its Inhibition by Methotrexate1 : (starfish/dihydrofolate reductase/methotrexate/DNA synthesis/early development).

Dihydrofolate reductase in immature oocytes of the starfish, Asterina pectinifera, is estimated to be 12 pg per oocyte. After completion of meiosis, the quantity of the enzyme is approximately 20 pg per egg. The content of the enzyme in the egg is kept nearly constant at this value from fertilization to the beginning of blastulation. Methotrexate, an analogue of dihydrofolate, at 20 µM did not affect meiotic maturational process and fertilization, but inhibited embryonic development at the 512-cell stage which corresponds to the beginning of blastulation. Incorporation of externally supplied deoxy[3 H]uridine into DNA of the embryos cultured in the continuous presence of 20 µM of methotrexate stopped at the 256-cell stage, suggesting that the cessassion of development of the embryo at the 512-cell stage was caused by inhibition of DNA synthesis at the preceding stage. Uptake of [3 H]methotrexate was low at early cleavage stages but increased just before blastulation. Externally supplied 1 mM of thymidine counteracted the inhibitory effect of methotrexate at 20 µM, suggesting that the starvation of the methotrexate-treated embryo for thymidine nucleotides halted DNA synthesis at the beginning of blastulation.

Development of Fertilized Starfish Eggs in Which Cytokinesis is Prevented by Iturin A-2: (starfish embryos/iturin A-2/cytokinesis/blastulation/1-methyladenine).

Iturin A-2, a cyclic peptide obtained from cultures of Bacillus subtilis inhibited cell division but not nuclear divisions of fertilized eggs of the starfish Asterina pectinifera, resulting in the formation of non-cleaving eggs containing all the embryonic nuclei in a common cytoplasm. Fertilized eggs in which cleavage was prevented by iturin A-2 (50µg/ml) synthesized DNA, RNA and protein at comparable rates to those of normal embryos up to the onset of blastulation. In single-cell embryos, however, elevation of the level of transcription, which is a characteristic marker of blastulation, did not take place and the chromatin masses eventually dispersed in the cytoplasm. Treatment of oocytes with iturin A-2 (50µg/ml) resulted in loss of their response to the maturation-inducing substance 1-methyladenine, whose receptors are located in the cell membrane or other components of the oocyte cortex. Furthermore, iturin A-2 bound to oocyte cortices quantitatively, suggesting that its site of action is the cortices of oocytes and eggs, and that its arrest of cleavage of fertilized eggs is due to loss of the ability of the cell membrane to form a cleavage furrow.

SELECTIVE INHIBITION BY APHIDICOLIN OF THE ACTIVITY OF DNA POLYMERASE ALPHA LEADS TO BLOCKADE OF DNA SYNTHESIS AND CELL DIVISION IN SEA URCHIN EMBRYOS.

Aphidicolin at 2 µg/ml caused 90% inhibition of mitotic cell division of sea urchin embryos at the I-cell stage. However, at 40 µg/ml it did not affect meiotic maturational divisions of starfish oocytes, which do not involve DNA replication. At 2 µg/ml it caused 90% inhibition of incorporation of tritiated thymidine into DNA of sea urchin embryos but did not affect protein or RNA synthesis even at a higher concentration. At 2 µg/ml it also caused 90% inhibition of the activity of DNA polymerase α, obtained from the nuclear fraction of sea urchin embryos, but did not affect the activity of DNA polymerase ß or γ. These findings suggest that DNA polymerase α is responsible for replication of DNA in sea urchin embryos.

Petroacetylene, a new polyacetylene from the marine sponge Petrosiasolida that inhibits blastulation of starfish embryos.

A new C30 linear polyacetylene compound designated petroacetylene (1) has been isolated from the marine sponge Petrosia solida Hoshino 1981, collected off the coast of Amami-Oshima, Kagoshima Prefecture, Japan. The structure was elucidated on the basis of spectroscopic data and chemical means. Petroacetylene (1) inhibited blastulation of starfish embryos at a concentration of 3.1 µg mL(- 1) or greater.

Bromotheoynic acid, a brominated acetylenic acid from the marine sponge Theonella swinhoei.

A new brominated C(17) acetylenic acid (1) designated as bromotheoynic acid has been isolated from the marine sponge Theonella swinhoei, collected off the coast of Tanegashima, Kagoshima Prefecture, Japan. The structure was determined on the basis of the analysis of its extensive 2D NMR spectroscopic data as well as HRMS. Bromotheoynic acid (1) inhibited maturation of starfish oocytes and cell division of fertilised starfish eggs. Bromotheoynic acid (1) also inhibited proliferation of human leukaemia U937 and HL60 cells, human lung cancer A549 and H1299 cells, and human embryonic kidney 293 (HEK293) cells.

Fibrous component of the blastocoelic extracellular matrix shapes epithelia in concert with mesenchyme cells in starfish embryos.

By using a monoclonal antibody (4H11 Mab), we have investigated morphogenetic functions of a fibrous component of the blastocoelic extracellular matrix in relation to cellular activities during early development of the starfish Asterina pectinifera. The 4H11 fibers fill the blastocoele from the late-cleavage to late-gastrula stage and contain the 370-kDa proteinaceous molecule secreted only by the epithelial cells. When 4H11 Mab is introduced into the blastocoele of blastulae, the embryos reveal three distinct morphological abnormalities after the mid-gastrula stage: (1) Distribution of mesenchyme cells confined near the tip of the archenteron, (2) swelling of the posterior ectoderm, and (3) suppressed growth of the mouth, esophagus, and coelomic pouches. These abnormalities occur together with alterations in the distribution of the 4H11 fibers. In embryos recovering from the effect of 4H11 Mab, the mesenchyme cells rearrange the 4H11 fibers. We propose that 4H11 fibers play direct roles in the morphogenesis of starfish embryos by providing a dynamic scaffold not only for the mesenchyme cells but also for the epithelial cells. Moreover, 4H11 fibers have a resist force from within, in concert with the mesenchyme cells, to counter the bulging force intrinsic to the epithelia and hold the epithelia in specific positions, once the positions have been decided.

A new alpha,beta,gamma,delta-unsaturated carboxylic acid and three new cyclic peroxides from the marine sponge, Monotria japonica, which selectively lyse starfish oocytes without affecting nuclear morphology.

The marine sponge Monotria japonica contains cytolytic constituents, which have been fractionated to a new alpha,beta,gamma,delta-unsaturated carboxylic acid designated monotriajaponide A (1) and three new cyclic peroxides designated monotriajaponides B (2), C (3), and D (4), in addition to a known peroxide (5) and a known alpha,beta-unsaturated ester (6). The structures were determined on the basis of spectroscopic data. Compounds 1-5 lysed immature starfish (Asterina pectinifera) oocytes without affecting nuclear morphology at the minimum effective concentrations of 50, 6.3, 6.3, 6.3, and 13 microg/mL, respectively. On the other hand, compound 6, at the minimum effective concentration of 25 microg/mL, lysed both oocyte's plasma membrane and the nuclear envelope.

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs.

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.

Exiguamide, a new spirocyclic sesquiterpene from the marine sponge Geodia exigua that inhibits cell fate specification during sea urchin embryogenesis.

A new nitrogen-containing bicyclic spirosesquiterpene designated exiguamide which inhibited cell fate specification during sea urchin embryogenesis has been isolated from the marine sponge Geodia exigua. Its structure was determined by interpretation of spectral data and X-ray crystallographic analysis.

Inhibition of chromosome separation in fertilized starfish eggs by kalihinol F, a topoisomerase I inhibitor obtained from a marine sponge.

Kalihinol F, a naturally occurring diterpene from a marine sponge, Acanthella sp., inhibited chromosome separation in fertilized starfish (Asterina pectinifera) eggs but allows the first cleavage to occur, thereby forming unseparated metaphase chromosomes which were elongated between the two daughter cells. The chromosomes were eventually torn off in the embryonic cells. Most of the cells gradually lost the chromosomes during the cell cycle progression. The embryonic development halted at the morula stage just before the onset of blastulation. The mitotic failure occurred when kalihinol F was applied to a fertilized egg during the second meiotic process, but not after the completion of the second meiotic division. Kalihinol F inhibited topoisomerase I activity in vitro, but had no effects on activities of DNA polymerases alpha, beta, and gamma, and of topoisomerase II. These results suggest that the topoisomerase I plays an essential role in meiosis II in this species.

Accumulation of multiacetylated forms of histones by trichostatin A and its developmental consequences in early starfish embryos.

External application of 10 rig/ml (R)-trichostatin A (TSA), a potent and specific inhibitor of mammalian histone deacetylase, to the embryo of the starfish Asterina pectinifera inhibited development during the early gastrula stage before formation of mesenchyme cells. The TSA-sensitive period was limited to the mid-blastula stage before hatching. The pulse-chase experiment clearly demonstrated that TSA induced an accumulation of acetylated histone species in blastulae through inhibition of historic deacetylation. Similar blockage of development at the early gastrula stage was observed with n-butyrate, which has been known as a weak inhibitor of historic deacetylase. These results suggest an intimate role for historic acetylation-deacetylation equilibria in starfish development.

Two new analogues of the cytotoxic substance BE-52211 from Streptomyces sp.

Two new beta-hydroxy acetamides, BE-52211 B and BE-52211 C, which were structural analogues of BE-52211, were obtained as an inseparable mixture from an actinomycete, Streptomyces sp. Their structures were elucidated on the basis of spectroscopic data. They inhibited cell division of starfish embryos at a concentration of 2.5 microg/mL or greater.

Piericidins C5 and C6: new 4-pyridinol compounds produced by Streptomyces sp. and Nocardioides sp.

Piericidins C5 (1) and C6 (2), two new members of the piericidin family, were isolated from a Streptomyces sp. and a Nocardioides sp., together with known piericidins C1 (3), C2 (4), C3 (5), C4 (6), D1 (7), and A3 (8). The structures were determined on the basis of their spectroscopic data. Both new compounds inhibited cell division of fertilized starfish (Asterina pectinifera) eggs at the minimum inhibitory concentration of 0.09 microg/mL.