Kenzo Takata

Nuclear and cytoplasmic ribonucleic acids of calf thymus

1. 1. It has been shown that there are at least two kinds of nuclear ribonucleic acid in calf thymus tissue, one of which is associated with a fraction soluble in neutral phosphate buffer and has the same nucleotide composition as that of cytoplasmic RNA. On the other hand the RNA associated with the nuclear residue has a different nucleotide composition. 2. 2. Electrophoretically homogeneous ribonucleoprotein samples were isolated from a nuclear fraction soluble in neutral phosphate buffer and from microsomal fractions. Both showed the same electrophoretic mobilities and RNA content. 3. 3. The experiment of 32P incorporation into nuclear RNA and cytoplasmic RNA indicated that not all the RNA in the cytoplasm can be derived from the nuclear RNA soluble in phosphate buffer, assuming that no exchange reaction of cytoplasmic RNA phosphorus taked place.

A technique for testing macromolecular samples in solution for morphogenetic effects on the isolated ectoderm of the amphibian gastrula.

Abstract A method is described for testing the morphogenetic effects of a macromolecular sample in solution on the isolated presumptive ectoderm of the amphibian embryo. As an example of its application, results of experiments are reported in which the inducing effects of several nondialyzable fractions of guinea pig bone marrow separated by chromatography with DEAE-cellulose were tested. A protein sample having 96% mesodermal effect at a concentration of 50 μg/ml was obtained; it did not contain a detectable amount of RNA.

Use of lectins as probes for analyzing embryonic induction

SummaryLectins were used as probes to investigate the mechanism of embryonic induction. Concanavalin (Con A) and gorse agglutinin out of 7 species of lectins tested were found to have strong neural-inducing effect on the presumptive ectoderm of newt gastrulae. Their effects were abolished by the addition of α-methyl-D-mannoside and α-L-fucose, respectively. Succinyl-Con A had a weak inducing activity in comparison to Con A. Autoradiography of3H-Con A-treated explants revealed that Con A bound to the inner surface, but not to the outer surface of ectoderm and was successively incorporated into cytoplasm.3H-Thymidine incorporation was lower in the first half and higher in the second half of the 60 h cultivation period in Con A-treated explants as compared to controls.Con A-Sepharose had a strong inductive effect. This suggests that neural induction is caused through Con A binding to the plasma membrane, but not through incorporation into the cytoplasm of the ectoderm cells.

Inductive Capacities for the Dorsal Mesoderm of the Dorsal Marginal Zone and Pharyngeal Endoderm in the Very Early Gastrula of the Newt, and Presumptive Pharyngeal Endoderm as an Initiator of the Organization Center*

The organization center of Cynops pyrrhogaster was divided into Parts 1, 2 and 3 of equal size (0.3×0.4 mm2) with presumptive fates as pharyngeal, pharyngeal+prechordal+trunk notochord, and trunk‐tail notochord, respectively. Movements and changes in size and shape of each part were followed through gastrulation. Differentiation tendencies of each part were examined under three conditions: I, isolated; II, sandwiched with presumptive ectoderm; 111, sandwiched with presumptive ectoderm after preculture in isolation for various times. In I, Parts 2 and 3 differentiated into dorsal mesoderm. In II, each part induced dorsal mesoderm and neural tissues, the frequency being highest in Part 2 and lowest in Part 3. In III, Parts 1 and 2 realized their presumptive fates, through changes in inductive capacities from trunk‐tail to head. This change progressed rapidly in Part 1, and slowly in Part 2. Part 3 required induction by neighbouring Part 2 to realize its presumptive fate. Changes of inductive capacity of Parts 1 and 2 respectively, were chronologically similar in normal development and in preculture experiments. Lastly, the primary presumptive pharyngeal zone at blastula was proposed to act as an initiator of the organization center, its programmed information being transmitted to Part 2, and then to Part 3.

Glycoproteins responsive to the neural-inducing effect of Concanavalin A in Cynops presumptive ectoderm

To examine the possible occurrence of receptors in the ectodermal cell surface which apparently mediates the neural-inducing stimulus, a further experiment by using Con A was done in combination with the enzyme treatments. The presumptive ectoderm explants of Cynops gastrula were first treated with neuraminidase to remove sialic acid. Prior to the Con A treatment, the explants were treated with almond glycopeptidase, which cleaves the asparagine linkage between protein and oligosaccharide in glycoprotein and releases the oligosaccharide moiety intact containing mannose residue from the substrate. No neural induction occurred. When the explants were not treated with almond glycopeptidase, the neural induction frequency was found to be the same as that of the explants treated with only Con A. Biochemical analyses showed that when the fixed ectoderm explants were treated with almond glycopeptidase, several oligosaccharides were released and then fractionated by means of Bio-Gel P-4 filtration. Based on the strict specificity of almond glycopeptidase, these oligosaccharides are unmistakably asparagine-linked oligosaccharides with mannose residues. We discuss the hypothesis of involvement of glycoproteins in the first step of molecular events in the neural induction mechanism.

Cell surface changes of the presumptive ectoderm following neural-inducing treatment by concanavalin A

SummaryScanning electron microscopic studies revealed that Concanavalin A (ConA) induces characteristic changes of the cell surface and the cell architecture of the presumptive ectoderm associated with differentiation into neural tissues. In Con A-treated cells, the filopodia with which cells were connected to each other disappeared from the interior (blastocoelic) surface and the cellular adhesivity decreased significantly. Thereafter, the cells underwent from those of the control explants. After cultivation for 60 h, a certain pattern of cell arrangement, which resembled the architecture of neural tissues, was observed among randomly arranged cells in the explants treated with Con A. The morphological changes specifically observed in Con A-treated explants were different from those found in explants treated with succinyl Con A (S-Con A) orDolichos biflorus agglutinin (DBA), which is unable to induce formation of the neural tissues. The molecular organization of the plasma membrane appears to be important in the mechanism of neural induction.

Changes of chromosome length and constitutive heterochromatin in association with cell division during early development of Cynops pyrrhogaster embryo

The length of chromosomes in the presumptive ectoderms of Cynops embryos was measured at nine successive cell divisions from the 6th (cleavage stage) to the 14th (gastrula stage). Up to the 10th cell division (cleavage stage) the chromosome length remained constant. At the 11th cell division the chromosomes began to shorten and continued to shorten at every cell division up to the 14th cell division. The degree of shortening and the mode of variation in length corresponded to the respective developmental stages of cleavage, blastula and gastrula. During those periods when chromosomes became shortened, some fine C‐bands of the paracentromeric region found in earlier stages fused together. The chromatin of interphase nuclei also showed considerable changes during chromosome shortening. Besides the size reduction of interphase nuclei, the number of C‐band granules in an interphase nucleus decreased in parallel with chromosome shortening and fusion of C‐bands in mitotic chromosome.

Protein synthesis during neural and epidermal differentiation in Cynops embryo

Two‐dimensional gel electrophoresis was used to analyze protein synthesis in relation to neural and epidermal differentiation in Cynops pyrrhogaster embryo. Various regions of embryos at different developmental stages, from late morula to early neurula stages, were excised, radiolabelled with 35S‐methionine, and the pattern of protein synthesis were compared. The following four types of protein spots were observed: (1) six proteins synthesized characteristically in the epidermal region of the embryo after gastrulation, (2) two proteins synthesized in both epidermal and endodermal regions, but not in other regions, after gastrulation, (3) a protein first detected at early blastula stage, of which expression was nearly constant in presumptive epidermis region but declined in the other regions, (4) the candidate for neural plate specific protein synthesized at a very high level in ectoderm explants treated with concanavalin A, a substance which evokes neural induction.

Monitoring of 131I in milk and rain water in Japan following the reactor accident at Chernobyl and estimates of human thyroidal dose equivalents.

Iodine-131 in milk and in rain water in Nagoya, Japan, (a location 8,000 km from Chernobyl) was monitored between May and July 1986. The 131I concentration in rain water ranged from 43.1 Bq L-1 on 4 May to 15 mBq L-1 on 12 July, and that in milk ranged from 21.8 Bq L-1 on 19 May to 11 mBq L-1 on 14 July. Iodine-131 concentrations in milk were estimated to be 4 to 6 times greater than those in rain water during the first few weeks after the accident. Both concentrations decreased with approximately the same effective half-life of 5.9 +/- 0.3 d for rain water and 5.0 +/- 0.2 d for milk. The 131I concentration in milk sold in markets varied from dairy to dairy and ranged from 0.07 to 0.2 times that in fresh milk. The maximum thyroidal dose equivalents estimated for an adult man and for a baby were far lower than the population annual dose equivalent to the thyroid from natural radiation.

A Molecular Aspect of Neural Induction in Cynops Presumptive Ectoderm Treated with Lectins

Little has been reported on what happens when the competent ectoderm first receives the inductive stimulus, or the location of the cellular site on which the inductive stimulus exerts its effect. Recently, the primary involvement of cell surface in the inducing mechanism of neural tissues has been stressed (Tiedemann and Born, 1978; Grunz and Staubach, 1979; Takata et al., 1981, 1984; Yamamoto et al., 1981; Duprat et al., 1982). Experiments using Sepharose beads, on which the neural-inducing factor (vegetalizing factor: Tiedemann and Born, 1978; Con A: Takata et al., 1981) was immobilized, indicated that even if the neural-inducing factor acts on the cell surface alone, it can evoke the competent ectoderm to differentiate into neural tissues, and does not necessarily have to enter into the cytoplasm to exert its effect. This possible mode of inducing action of Con A seems to agree with the evidence that retention of a certain amount of mitogen (Con A) for a certain length of time on the cell surface is a prerequisite for stimulating lymphocytes to blasto formation, but that mitogen taken up into cytoplasm is not involved in the mechanism of stimulation of cell division (Pauli et al., 1973).