Takahiko Shimizu

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

SummaryExternal 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.

A Novel Antimicrobial Substance from a Strain of the Bacterium, Vibrio SP

Abstract The structure of a novel antimicrobial substance (1) isolated from a strain of the bacterium, Vibrio sp., was determined to be 7,7-bis(3-indolyl)-p-cresol on the basis of spectroscopic data. The substance showed specific inhibition against Bacillus subtilis with a minimum inhibitory concentration of 70 μg/ml but did not affect the growth of other test microorganisms.

Endocytosis in Adult Eel Intestine: Immunological Detection of Phagocytic Cells in the Surface Epithelium

This study was undertaken to isolate groups of eel (Anguilla japonica) intestinal cells undergoing active endocytosis in vivo and to localize these cells within the intestinal epithelium. A monoclonal antibody (8F1) of the immunoglobulin M class containing kappa light chains was raised against such endocytic cells. Ninetythree percent of the 8F1-positive cells absorbed fluorescent isothiocyanate-conjugated dextran from the intestinal lumen in vivo during a 30-min incubation, and they retained the ability to absorb dextran in vitro. The 8F1-positive cells constitute about 15% of the whole dissociated, macromolecule-absorbing cells. This suggests that other types of endocytic cells exists in the epithelium. The 8F1-positive cell can internalize fixed Vibrio anguillarum in vivo. Immunohistochemical observations demonstrated that 8F1-positive cells are very few in number and are located exclusively in the surface epithelium of the intestine. Thus these cells may well be specified phagocytic cells, different from those playing a basic nutritional role.

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. Developmental Dynamics 232:915–927, 2005.

Selective inhibition of gastrulation in the starfish embryo by albuside B, an inosine analogue

External application of 0.2–100 μg/ml albuside B inhibits gastrulation of the starfish (Asterina pectinifera) embryo. Treated embryos retain the late blastula morphology with the vegetal plate. However, the vegetal plate is unreactive to soybean agglutinin, a probe for observing the progenitor cells of the archenteron (mesendoderm) in a normal embryo. The effective period of the treatment is limited from 4 to 6 h after fertilization, a period immediately before the onset of blastulation. RNA synthesis is unaffected during the period of sensitivity. The selectivity of the inhibition shows that albuside B may be a useful tool for studying the mechanisms of mesendoderm differentiation.

High-Sensitivity Mass Spectrometry for Analysis of Posttranslational Modifications

Pulsed fast atom bombardment ionization (pulsed-FAB) mass spectrometry has been developed to improve the sensitivity of tandem mass spectrometry (MS/MS), allowing it to be used for the analysis of very small samples. MS/MS, when used with a magnetic four-sector instrument coupled with the pulsed-FAB system, allows significant enhancement in product ion intensity of over ten-fold in magnitude over conventional FAB. MS/MS was applied to the structural analysis of a unique nuclear protein, designated p28, which was isolated from a histone fraction obtained from starfish testes. The results clearly show that protein p28 is a heterodimer composed of testicular histones H2B and H4 which are cross-linked between Gln9 of H2B and Lys5 of H4.

Effects of Okadaic Acid on Embryonic Development of the Starfish Asterina pectinifera

The effects of okadaic acid, an inhibitor of protein phosphatases types 1 and 2A, from the sponge Halichondria okadai Kadota, on the embryonic development of the starfish Asterina pectinifera, were investigated. When cultured in okadaic acid from fertilization, the embryo divided synchronously without any abnormalities such as lysis, swelling or morphological changes different from control embryos up to the 32-cell stage. However, okadaic acid prevented development before the onset of blastulation. Cytological examination showed that chromosomes condensed but did not align in a plane in the mitotic apparatus in each of the blastomeres of the treated embryo at the sixth cleavage, suggesting that this was the root cause of the arrest of further development.

A Novel Modification of the Lysine Residue at Position 12 of Histone H4 in Starfish Sperm.

Post-translational modification of core histones is essential in processes requiring chromatin remodeling. We report here a novel modification in histones of the sperm of the starfish, Asterina pectinifera, which involves an ε-(γ-glutamyl)lysine cross-link between the glutamine residue at position 9 of histone H2B and the lysine residue at position 12 of histone H4.