Shinichiro Kusunoki

Inhibitory effect of α-hydrazinoornithine on egg cleavage in sea urchin eggs

Abstract In fertilized sea urchin eggs which are kept in sea water containing α-hydrazinoornithine (αHO) at a concentration above 1 mM from the time of fertilization, cleavage is delayed markedly. The third cleavage is almost completely blocked by 3 mM αHO. Hydrazine, as well as ornithine, exerts no harmful effect on egg cleavage. αHO causes competitive inhibition of ornithine decarboxylase (ODC) in the egg homogenate. Polyamine levels decrease in fertilized eggs treated with αHO. The addition of ornithine (above 3 mM) to an egg culture containing αHO prevents the αHO-induced delay of cleavage. Putrescine (0.2–0.5 mM), which is the product of the reaction catalyzed by ODC, also relieves egg cleavage from the inhibited state. The same effect occurs in the presence of spermidine (0.2–0.5 mM) or spermine (0.1–0.8 mM). Especially, spermine (0.5 mM) completely cancels the inhibitory effect of αHO on egg cleavage. Egg cleavage is delayed only slightly in the presence of each polyamine (above 2 mM).

Cyclic change in polyamine concentrations in sea urchin eggs related with cleavage cycle

Abstract Spermidine and spermine are found in unfertilized eggs of the sea urchin, Hemicentrotus pulcherrimus . Putrescine becomes detectable and concentrations of spermidine and spermine increase in the eggs upon fertilization. Then, concentrations of these polyamines decrease after respective peaks in polyamine concentrations. The peaks in the concentrations are found at 15 minutes post fertilization for putrescien, at 30 minutes for spermidine and at 30–40 minutes for spermine respectively. Levels of polyamines elevate again and reduce after the 2nd concentration peaks of respective compounds, and then the first cleavage of the eggs takes place. Cyclic change in each polyamine concentration is also observed after the first cleavage, and egg cleavage occurs at decreasing phase of polyamine concentrations.

Cell-surface arylsulfatase A and B on sinusoidal endothelial cells, hepatocytes, and Kupffer cells in mammalian livers

Arylsulfatase A (ARSA) and B (ARSB) have been regarded as lysosomal enzymes because of their hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of their enzymatic activity. Using sea urchin embryos, we previously demonstrated that the bulk of ARS is located on the cell surface of the epithelium, colocalizing with sulfated polysaccharides, and that it does not exhibit enzymatic activity. To examine whether ARSA and ARSB exist on the cell surface in mammalian tissues, we raised antibodies against ARSA and ARSB and examined immunohistochemically their localization in the liver using light and electron microscopy. Here we show that mammalian ARSA and ARSB exist on the cell surface of sinusoidal endothelial cells, hepatocytes, and sinusoidal macrophages (Kupffer cells), as well as in the lysosome. They are also colocalized with heparan sulfate proteoglycan. These results suggest that ARSA and ARSB also may function in the cell surface of mammals. This is the first report to show cell-surface localization of ARS in mammalian somatic cells. The extracellular localization of ARS will provide new insight for human ARS deficiency disorders, such as metachromatic leukodystrophy and mucopolysaccharidosis VI.

Hbox1 and Hbox7 are involved in pattern formation in sea urchin embryos

In spite of their potential importance in evolution, there is little information about Hox genes in animal groups that are related to ancestors of deuterostome. It has been reported that only two Hox genes (Hbox1 and Hbox7) are expressed significantly in sea urchin embryos. Expression of Hbox1 protein is restricted to the aboral ectoderm, and Hbox7 expression is restricted to oral ectoderm, endoderm and secondary mesenchyme cells in sea urchin embryos after the gastrula stage. With the aim of gaining insight into the role of Hbox1 and Hbox7 in sea urchin development, Hbox1 and Hbox7 overexpression experiments were performed. Overexpression of Hbox1 repressed the development of oral ectoderm, endoderm and mesenchyme cells. On the contrary, overexpression of Hbox7 repressed the development of aboral ectoderm and primary mesenchyme cells. The data suggest that Hbox1 and Hbox7 are expressed in distinct non‐overlapping territories, and overexpression of either one inhibits territory‐specific gene expression in the domain of the other. It is proposed that an important function of both Hbox1 and Hbox7 genes is to maintain specific territorial gene expression by each one, in its domain of expression, while repressing the expression of the other in this same domain.

CHANGES IN ACTIVITIES OF THYMIDYLATE SYNTHASE AND DIHYDROFOLATE REDUCTASE IN SEA URCHIN EGGS AFTER FERTILIZATION

Thymidylate synthase activity in sea urchin eggs increases just after fertilization and decreases 30 min later. Then, cyclic variation in the activity occurs in association with the cleavage cycle. Dihydrofolate reductase activity in fertilized eggs is almost the same as in unfertilized eggs and shows no marked change within 3 hr after fertilization. Aminopterin, an analogue of dihydrofolate, inhibits dihydrofolate reductase, and arrests cleavage. On incubation in sea water containing aminopterin (20‐100μM) from the time of fertilization, the development of Clypeaster and Pseudocentrotus eggs was arrested at the 32–64 cell stage, and that of Anthocidaris eggs was arrested at the morula stage. Dihydrofolate (100μM) counteracts the inhibitory effect of aminopterin on egg cleavage. Thymidine at concentrations above 10μM also prevents inhibition by aminopterin. Other deoxyribonucleosides at concentrations of 10μM to 100μM do not affect inhibition of egg cleavage by aminopterin. Deoxyadenosine at concentrations above 5 mM inhibits egg cleavage, but other deoxyribonucleosides have no effect.

Ca2+ influx-linked protein kinase C activity regulates the β-catenin localization, micromere induction signalling and the oral–aboral axis formation in early sea urchin embryos

Summary Sea urchin embryos initiate cell specifications at the 16-cell stage by forming the mesomeres, macromeres and micromeres according to the relative position of the cells in the animal–vegetal axis. The most vegetal cells, micromeres, autonomously differentiate into skeletons and induce the neighbouring macromere cells to become mesoendoderm in the β-catenin-dependent Wnt8 signalling pathway. Although the underlying molecular mechanism for this progression is largely unknown, we have previously reported that the initial events might be triggered by the Ca2+ influxes through the egg-originated L-type Ca2+ channels distributed asymmetrically along the animal–vegetal axis and through the stretch-dependent Ca2+channels expressed specifically in the micromere at the 4th cleavage. In this communication, we have examined whether one of the earliest Ca2+ targets, protein kinase C (PKC), plays a role in cell specification upstream of β-catenin. To this end, we surveyed the expression pattern of β-catenin in early embryos in the presence or absence of the specific peptide inhibitor of Hemicentrotus pulcherrimus PKC (HpPKC-I). Unlike previous knowledge, we have found that the initial nuclear entrance of β-catenin does not take place in the micromeres, but in the macromeres at the 16-cell stage. Using the HpPKC-I, we have demonstrated further that PKC not only determines cell-specific nucleation of β-catenin, but also regulates a variety of cell specification events in the early sea urchin embryos by modulating the cell adhesion structures, actin dynamics, intracellular Ca2+ signalling, and the expression of key transcription factors.

Unichrom, a novel nuclear matrix protein, binds to the Ars insulator and canonical MARs.

Abstract Eukaryotic genomic DNA is organized into loop structures by attachments to the nuclear matrix. These attachments to the nuclear matrix have been supposed to form the boundaries of chromosomal DNA. Insulators or boundary elements are defined by two characteristics: they interrupt promoter-enhancer communications when inserted between them, and they suppress the silencing of transgenes stably integrated into inactive chromosomal domains. We recently identified an insulator element in the upstream region of the sea urchin arylsulfatase (HpArs) gene that shows both enhancer blocking and suppression of position effects. Here, we report that Unichrom, originally identified by its G-stretch DNA binding capability, is a nuclear matrix protein that binds to the Ars insulator and canonical nuclear matrix attachment regions (MARs). We also show that Unichrom recognizes the minor groove of the AT-rich region within the Ars insulator, which may have a base-unpairing property, as well as the G-stretch DNA. Furthermore, Unichrom selectively interacts with poly(dG)•poly(dC), poly(dA)•poly(dT) and poly(dAT)•poly(dAT), but not with poly(dGC)•poly(dGC). Unichrom also shows high affinity for single-stranded G- and C-stretches. We discuss the DNA binding motif of Unichrom and the function of Unichrom in the nuclear matrix.

Stimulation of Unfertilized Eggs of the Echiuroid, Urechis unicinctus by Polyamines

Unfertilized eggs of the echiuroid, Urechis unicinctus, were activated by polyamines, such as putrescine, spermidine and spermine at concentrations above 10 μM. Fertilization membrane elevated and germinal vesicle disappeared in unfertilized eggs kept for several min in sea water containing these polyamines. Following the addition of these polyamines, a decrease of pH value in the egg suspension, occurred in a similar manner as observed following fertilization. Several sec after the addition of polyamines to the egg suspension, the respiratoy rate increased very slightly and the sensitivity of the respiration to 2, 4‐dinitrophenol, which was lower in unfertilized eggs than in fertilized eggs, became as high as in fertilized ones. Irregular cleavage occurred in the eggs stimulated by polyamines. The incorporation of [3H]‐deoxyadenosine into DNA was initiated by adding polyamines in the unfertilized eggs preloaded with the isotope. The rate of [3H]‐leucine incorporation into protein in the preloaded unfertilized eggs was also enhanced by polyamines, in almost the same manner as observed following fertilization.

INHIBITION OF DIHYDROFOLATE REDUCTASE BY PALMITOYL-CoA AND THE REVERSAL OF THE INHIBITION BY SPERMINE AND SPERMIDINE IN THE EGGS OF THE SEA URCHIN, HEMICENTROTUS PULCHERRIMUS*

Dihydrofolate reductase activity in fertilized eggs of the sea urchin, Hemicentrotus pulcherrimus, was almost the same as in unfertilized eggs. Aminopterin inhibited the enzyme competitively with dihydrofolate (FH2). The apparent Km value for FH2 in the dihydrofolate reductase reaction was about 0.1 μM in the crude homogenate of both unfertilized and fertilized eggs. Dihydrofolate reductase in the eggs was also inhibited by palmitoyl‐CoA. The inhibition was canceled by polyamines, especially by spermine, but putrescine failed to prevent the enzyme from the inhibition. The change in long‐chain acyl‐CoA and polyamine concentrations during fertilization are discussed as possible regulatory factors of the enzyme.