Graph-Theoretical Analysis of Cleavage Pattern: Graph Developmental System and Its Application to Cleavage Pattern of Ascidian Egg

The skeletal structure of the embryo is represented by the graph. In the graph, the cells and the connectivities between the cells are reduced to the nodes and edges, respectively. Along cleavage history, the series of graphs is obtained. In this paper I propose a new graph developmental system(GDS) which develops the series of graphs. And I represent and analyze the cleavage pattern of the ascidian egg by GDS. In order to represent it by GDS, at first, the connectivities between the cells are labeled according to the developments of the connectivities at the next time step, and next the cells are labeled. But there are two ways of labeling the cells, then two types of GDS are defined: (1) to label according to the pattern of the connectivities of their descendant cells after two time steps (G-GDS), (2) to label according to the cell fates (C-GDS). The C-GDS of the ascidian egg produces 16 cleavage patterns at 64-cell stage non-deterministically. If some labels are distinguished at 16-cell stage, the C-GDS becomes deterministic at 64-cell stage.
GDS will be useful to simulate morphogenesis by the computer graphics.     

Transverse Vein Differentiation Associated with Gas Space Formation--Fate of the Middle Cell Layer in Leaf Sheath Development of Rice

In monocotyledons, the leaf vascular network consists of a hierarchicalsequence of vertical vascular bundles and numerous transverseveins that interconnect adjacent vertical veins. In the leafsheath of these species, especially grasses, lysigenous gascavities (gas spaces) are developed into intervascular spacesand provide a gas conducting system to non-aerial parts underflooded conditions. The spatial relationship between gas spaceformation and transverse vein differentiation was investigatedusing the leaf sheath of rice (Oryza sativa L.). Histochemicalobservation showed that patterns of differentiation of the transversevein are distinct from those of vertical vascular bundles. Onthe other hand, gas spaces are formed through the processesof cell death (collapse). Both events are initiated at a specificcell position in the middle layers of the leaf sheath, fromwhich the vascular system of the leaf is derived; this indicatesthat differentiation of transverse veins is associated withgas space formation. The cell-to-cell movement of fluoresceinisothiocyanate-conjugated dextran injected into middle layercells coincided with the area where cell collapse occurred,indicating a close relationship between the middle and adaxialcell layers, but not abaxial cell layers. A uniform cell numberbetween each transverse vein in the leaf sheath suggested theinvolvement of spatial regulation in transverse vein formationregardless of clonal history at the later stage of leaf veincanalization. Copyright 2000 Annals of Botany Company Cell collapse, leaf development, middle cell layer, microinjection, Oryza sativa L., rice, programmed cell death.     

Enhanced Melanogenesis Induced by Tyrosinase Gene-Transfer Increases Boron-Uptake and Killing Effect of Boron Neutron Capture Therapy for Amelanotic Melanoma

Specific and powerful cancer killing effect for melanoma by boron neutron capture therapy (BNCT) using DOPA analogue, ¹⁰B-p-boronophenylalanine (¹⁰B-BPA), has been established, but amelanotic melanoma is insufficiently responsive to ¹⁰B-BPA BNCT in comparison with actively melanin-producing melanoma. Although the accumulation mechanism of ¹⁰B-BPA within melanoma was not established, we have recently obtained findings suggesting that melanin monomers, key intermediates for melanin polymer formation, play a critical role in ¹⁰B-BPA accumulation. In addition, there are some kinds of human amelanotic melanomas, such as MEL2A, in which expression of tyrosinase is repressed or lacking though tyrosinase-related protein (TRP)-l and TRP-2 are well expressed. Thus, by using a similarly tyrosinase-lacking mouse amelanotic melanoma cell line, A1059, we constructed TA1059 cells by transfecting human tyrosinase-cDNA into these cells. TA1059 cells acquired higher DOPA-oxidase and DOPAchrome tautomerase activity as well as eumelanin content at even higher levels than those of B16F10 cells. TA1059 cells showed about 2.5 times higher ^p-boronophenylalanine (BPA) uptake than A1059 cells in culture. In animal experiments, by using these cell lines, tumor growth of TA1059 was significantly suppressed by ¹⁰B-BPA BNCT as compared with A1059. These findings indicate that the induction of active melanin biosynthesis by melanogenic gene-transfer effectively improves the treatment of amelanotic melanoma by BNCT.     

Pathway of mannitol formation during photosynthesis in brown algae

Eisenia bicyclis, Arame, was allowed to photosynthesize in seawatercontaining H¹⁴CO₃^–, and ¹⁴C-mannitol was isolated fromits fronds. The ratio of ¹⁴C-total/¹⁴C₁ + ¹⁴C₆ in the ¹⁴C-mannitolwas found to be about 8.0 at 1 min-illumination, but graduallydecreased with time to 3.0, showing uniform radioactivity distribution.Mannitol therefore seems to be formed in brown algae throughthree carbon compounds. Enzymes which may be involved in the possible biosynthetic pathwayof mannitol, i.e. aldolase, hexose diphosphatase, mannitol-1-phosphataseand glucosephosphate isomerase were present in extracts fromseveral brown algae. Some of their properties are discussed. ¹Contribution from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 187. ²Present address: Reseach Institute, Seikagaku Kogyo Co., Ltd.,Yamato-machi, Kitatama-gun, Tokyo, Japan. (Received December 13, 1968; )     

Establishment of the Embryo-derived Stem (ES) Cell Lines from Mouse Blastocysts: Effects of the Feeder Cell Layer.

The ES ceii lines are embryo-derived stem cell lines directly isolated from the inner cell mass of mouse blastocysts using feeder cell layer. We have established a number of ES cell lines from 129 or C57BL/6 strain mice by using the feeder layer of the STO cells (from ATCC) or the primary embryonic fibroblasts, which was obtained by trypsinizing the 16-day-old BALB/c mouse fetus. The ES cell lines established on the STO feeder layer showed differentiation into various tissues in solid tumors when injected into syngenic mice. Karyotype was, however, nearly tetraploid. The ES cell lines established on the primary fibroblasts exhibited differentiation into larger variety of tissues in solid tumors. Karyotype was almost diploid and majority of the cells kept normal set of chromosomes in G-banding. We conclude that the primary fibroblasts are better feeder layer than the STO cells for establishment and maintenance of the ES cell lines.     

Metabolism of Round Spermatids: A Possible Regulation of Hexose Transport

Round spermatids (steps 1–8) were isolated from rat testes and glucose transport into the cells was examined. The exposure of spermatids to glucose resulted in an extremely low level of ATP. In contrast, the level of ATP remained constant in the presence of pyruvate. Transport of a glucose analogue, 2-deoxy-D-[³H]glucose ([³H]dGlc) into spermatids was correlated with intracellular levels of ATP and was much greater in cells with higher rather than lower levels of ATP. [³H]dGlc transport into spermatids with low levels of ATP was partially reversed when the cells were incubated with pyruvate. Inhibition of [³H]dGlc transport was exerted on Vmax and not on Km. Moreover, glucose acted as a competitive inhibitor of [³H]dGlc uptake (Km increased; Vmax unaltered). These results suggest that glucose transport into spermatids is active in vitro and probably regulated by the intracellular level of ATP.     

The Effect of Hydrocortisone on the Epidermal Proliferation in an Organ Culture of Chick Embryonic Skin

Hydrocortisone is regarded as an initiator of keratinization in embryonic skin. The present investigation dealt with the effect of hydrocortisone on the proliferation of epidermal cells during early development: Cell kinetic analyses using ³H-thymidine autoradiography were applied to a skin organ culture prepared from a 13-day chick embryo.
Hydrocortisone at a concentration between 0.01 and 1.0 μg/ml was effective in initiating a morphological change leading to the epidermal keratinization in vitro and caused a marked decrease in the mitotic and labeling indices of epidermal basal cells, the decrease being maximum at 2 days of culture previous to the morphological change.
During continuous labeling with ³H-thymidine, the number of labeled basal cells reached 100% within 2 days in the control and 4 days in the culture treated with hydrocortisone. This confirmed that the growth fraction of epidermal basal cells was 1.0 even after the administration of hydrocortisone.
The duration of each cell cycle phase at 2 days of culture was determined by percent labeled mitoses and double-labeling analyses. It was concluded that hydrocortisone extended the generation time of epidermal basal cells at this time point about three fold over the control. This extension was mainly due to the elongation of the G ₁ phase.     

K-Casein Suppresses Melanogenesis in Cultured Pigment Cells

The effects of bovine milk proteins on melanogenesis in B16 cells were examined. Both whey protein isolate and casein exhibited depigmenting properties. Among the major protein components of milk—including β-lactoglobulin, α-lactalbumin, α-, β-, and k-casein—only K-casein exhibited the depigmenting effect. However, the carboxyl terminal peptide of K-casein, glycomacropeptide, did not show this activity. Also, K-casein promoted the proliferation of the cells and inhibited the activity of tyrosinase in the cells. These results indicate that K-casein acts as a melanogenesis-suppressing modulator.     

EFFECTS OF THYROXINE AND PROLACTIN ON THE RATES OF PROTEIN SYNTHESIS IN THE THIGH BONES OF THE TADPOLE OF RANA CATESBEIANA

In thigh bones isolated from a Rana catesbeiana tadpole which has been kept in a 5 × 10⁻⁸ M thyroxine solution for several days, the rate of ¹⁴C-leucine incorporation into protein becomes higher than that in the thigh bones of control animals. Intraperitoneal injection of prolactin also results in an increase in the rate of ¹⁴C-leucine incorporation into protein in the thigh bones at a rate very similar to that in thyroxine-treated animals. In the thigh bones of the thyroxine-treated tadpoles, the rate of ¹⁴C-proline incorporation into protein is markedly higher than that of control animals. Prolactin treatment of the tadpoles also causes an increase in the rate of ¹⁴C-proline incorporation, but the rate is lower than that found in thyroxine-treated animals. The injection of prolactin into thyroxine-treated tadpoles fails to cause further increase in the rates of incorporation of these amino acids into protein. In the thigh bones of tadpoles at the climax of metamorphosis, prolactin injection does not cause any increase in the rates of ¹⁴C-labeled proline and leucine incorporation, whereas both rates become slightly higher in the thigh bones of thyroxine-treated tadpoles at this stage. The thigh bones probably become insensitive to prolactin when they are exposed to thyroxine.