Hiraku Shimada

Carbonic anhydrase activity in developing sea urchin embryos with special reference to calcification of spicules

Eggs and embryos of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus did not exhibit significant changes in carbonic anhydrase activity during early development. Acetazolamide inhibited enzyme activity in homogenates of embryos and inhibited the formation of calcified spicules in a culture of micromeres at concentrations between 40 and 100 microM. Acetazolamide allowed intact embryos to develop to quasi-normal plutei but inhibited calcium deposition in the spicules. It is suggested that carbonic anhydrase contributes to CaCO3 deposition in the spicule.

DNA synthesis in isolated nuclei from the brains of rats at different post-partal stages and the infant rat brain cytosol factor stimulating the DNA synthesis in infant rat brain nuclei.

Abstract [3H]TTP incorporation into DNA of isolated nuclei from the brains of rats at different post-partal stages, as well as the effects of the adult and the infant rat brain cytosols upon the DNA synthesis in the isolated nuclear system, have been investigated. The nuclear system requires the four deoxyribonucleoside triphosphates, ATP and Mg2+ for a maximum activity and DNA synthesis proceeds linearly within 30 min of incubation. A large portion of [3H]TTP incorporated into DNA is sensitive to actinomycin D inhibition and seems to be DNA polymerase dependent, while the remaining incorporation (approx. 20 % in the infant nuclear system and approx. 40 % in the adult nuclear system) is less sensitive to actinomycin D inhibition, proceeds without dATP, dCTP and dGTP and seems to be due to the terminal DNA nucleotidyltransferase activity. The nuclear activity of [3H]TTP incorporation into DNA increases after birth, reaches a maximum on the fifth day and then declines rapidly, reaching the adult level (i. e. one half the maximum level) on the 22nd day after birth. [3H]TTP incorporation into DNA of isolated infant rat brain nuclei is stimulated specifically by the infant rat brain cytosol. The active principle in the infant rat brain cytosol seems to be thermostable and low molecular, and differs from the stimulatory factor present in the serum.

Change in the cellular level of AP4A is correlated with the initiation of DNA replication in sea urchin embryos

Abstract The cellular level of AP4A (14.4 pmole/104 unfertilized eggs) in the embryos of sea urchin, Strongylocentrotus nudus, dropped to near-zero level within 40 min after fertilization, and, thereafter, a marked and rapid elevation of AP4A level accompanied by an abrupt decrease was observed before every S phase. When the initiation of the second S phase was inhibited, the AP4A level, which rose as in normal embryos, did not drop at all. The cyclic fluctuation of the AP4A level was found to be independent of nuclear division. The present results suggest that AP4A is somehow related to the initiation of DNA replication.

Melittin, a Component of Bee Venom, Activates Unfertilized Sea Urchin Eggs

Melittin, which is known to stimulate phospholipase A, in many cells, caused as much elevation of fertilization membranes and increase in respiration of unfertilized eggs of the sea urchins Anthocidaris crassispina and Hemicentrotus pulcherrimus as normal fertilization.

Purification and characterization of arylsulfatase from sea urchin (Hemicentrotus pulcherrimus) embryos

Abstract 1. 1. Arylsulfatase was extracted from sea urchin (Hemicentrotus pulcherrimus) plutei and purified to electrophoretical homogeneity by means of DEAE-cellulose, acetone fractionation and Sepharose CL-6B, successively. 2. 2. The molecular weight of this enzyme was approx, 670,000. The molecular weight of a single subunit was approx. 63,000. The Km value for p-nitrophenyl sulfate was 0.59 mM. 3. 3. This enzyme was competitively inhibited by the sulfate ion and was classified as the type II arylsulfatase. The pH optimum was between 5.0 and 6.0.

Serum factors stimulating DNA synthesis in the isolated nuclear system from rat liver.

Abstract 3H-TTP incorporation into DNA by the isolated rat liver nuclei was stimulated by the rat serum in proportion to its concentration. Dialysis and gel-filtration of the serum indicated the presence of two factors: one is low-molecular and another is high-molecular. The high-molecular factor is thermolabile while the low-molecular one is thermostable. The latter is resistant to pronase-treatment and can not be adsorbed on charcoal. The sera from normal and partially hepatectomized rats showed similar stimulatory effect.

Discontinuous DNA replication in developing sea urchin embryos

Abstract Embryos of the sea urchin, Hemicentrotus pulcherrimus, growing synchronously and entering the third “S” phase at 120 min after fertilization, were pulse-labeled with [3H]thymidine for various periods ranging from 15 sec to 20 min, and the size of nascent DNA was analyzed by centrifugation in an alkaline sucrose gradient. It was found that pulse-labeling for 15 sec gave rise to a sedimentation profile with a major radioactivity peak at the position corresponding to a molecular weight of 4 × 104 daltons. One-minute of labeling, however, gave a major radioactive band around the position corresponding to 1.4 × 106 daltons. Upon increasing the labeling time, the radioactivity peaks or bands shifted toward the increasing molecular weights. Finally, most of the radioactive DNA was found to sediment at the bottom when the embryos were exposed to [3H]thymidine for 15 min or longer. The time span of the S phase in the cleavage embryos was about 15 min. The results of pulse and chase experiments also supported the discontinuous mechanism of DNA replication in the cleavage embryos.

Developmental Timing of Synthesis and Translation of Arylsulfatase mRNA in Sea Urchin Embryo

In the sea urchin (Hemicentrotus pulcherrimus) embryo, the arylsulfatase activity is known to increase dramatically after the mesenchyme blastula stage. In the present experiment, we have studied the mechanism underlying this increase in the arylsulfatase activity by using the inhibitors of transcription and translation. Inhibition of transcription by actionmycin D before hatching prevented the increase in the arylsulfatase activity, while inhibition of transcription after hatching failed to prevent the increase in the enzyme activity. Inhibition of translation by emetine always reduced the arylsulfatase activity. These results have been interpreted as that the arylsulfatase activity in the blastula embryo is supported by the pre‐synthesized mRNA, and that the marked increase in the enzyme activity which is observed from the mesenchyme‐blastula stage to the prism stage is due to the transcription of the arylsulfatase gene which occurs during the development from hatching to the mesenchyme‐blastula stage.

Purification of Acetylcholinesterase from Sea Urchin (Hemicentrotus pulcherrimus) Embryos by Affinity Chromatography

Only one form of acetylcholinesterase (AchE) was detected in Hemicentrotus pulcherrimus embryos. In H. pulcherrimus embryos as well as in the other sea urchin embryos, AchE activity begins to increase rapidly after gastrula stage.

Effects of emetine on initiation of DNA synthesis in embryonic cells of sea urchin

The effects of the inhibition of protein synthesis by emetine on the initiation of DNA synthesis and mitosis in the fertilized eggs of the sea urchin, Anthocidaris crassispina, were studied. The initiation of DNA synthesis was completely prevented, and no delayed initiation was observed in eggs in which protein synthesis was completely inhibited by emetine, while delayed initiation of DNA synthesis occurred after prolonged culture in eggs in which protein synthesis was incompletely inhibited by emetine. In both cases, neither mitosis nor cleavage was observed. Synthesis of some protein factor seems to be necessary for the initiation of DNA synthesis.