Microtubules are major cytoskeletal components of the mitotic spindles in echinoderm eggs. Well preserved isolated spindles from the first cleavage of sea urchin eggs reveal that 3, 000 to 5, 000 orderly arranged microtubules are present. Considering the sensitive nature of spindle bi-refringence, it is highly probable that the spindle assembly is regulated by the labile association of tubulin molecules and the involvement of kinetic centers such as the kinetochores and MTOCs in the centrosome region.
Heavy water (D₂O) increases the spindle volume and birefringence in living and dividing sea urchin eggs. Through the in vitro experiments, we confirmed that the initial rate and the final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of D₂O. Yields were higher as the D₂O concentration increased. D₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concent-ration in the presence of D₂O. We obtained linear van't Hoff plots and calculated thermodynamic parameters which were positive and increased with the elevation of the D₂O concentration. The enhancement of the polymerization of tubulin by D₂O in vitro could, therefore, be the result of the strengthening of intra-and/or inter-molecular hydrophobic interactions of tubulin molecules. We believe that the increase in length and number of microtubules of the mitotic spindles in dividing cells of eukaryotes with D₂O is caused by the direct involvement of D₂O in the polymerization of tubulin.
When fertilized sea urchin eggs were exposed to dilute solutions of alkyl-resorcinols, i.e. T-1, an isolate from the culture medium of Pseudomonas sp., its derivatives or analogs such as Ansamycin, Curvularin or Macbecin I, spindles were more or less arrested in metaphase and became either barrel-shaped or ex-tremely enhanced. T-1 and Curvularin, which induced barrel-shaped spindles, possessed the same structural features with respect to 1, 3-benzenediol system with long lipophilic side chains. Structural modification of one of the hydroxyl groups of Curvularin provides a remarkable increase in chemical efficacy. This effect can be considered to be a freezing of the state of dynamic equilibrium or partial association/dissociation of tubulin molecules along with the microtubules by the given drugs. The barrel-shaped spindles consisted of bundles of straight microtubules of equal length, and we believe this might reflect the stabilized state of tubulin polymers caused by the local increase of molecular hydrophobicity. The anaphase sequence was usually delayed and chromosomes moved very slowly. We found that this unusual spindle shape was induced by the spread, discoidal redistribution of centrosomal MTOCs, and comparable with the irregular sea urchin spindles induced with mercaptoethanol.
The finding of the TO series compounds turned our attention to 1, 4-benzo-quinones with long hydrophilic side chains, and Macbecin I was recognized to be a unique mitotic arrester. This compound could be a lead compound to search new mitotic arresters and its analogues obtained by synthesis. The unique in vivo assay system developed by us using the mitosis in dividing sea urchin eggs can be widely applied for the experimental survey of new mitotic arresters. Fur-ther massive screening effort would provide us more opportunity to find new drugs which can be used as the molecular probes to analyze the physico-chemical basis of the assembly and/or disassembly of cytoskeleton.

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The relation of the total cellular content of sialic acid to phenotypic expression of B16 mouse melanoma cells was examined by using phenotype-modifying reagents and more than 10 cloned cell lines with spon-taneous phenotypic variations. The sialic acid content changed in a growth phase-dependent manner with a peak in the early log phase of growth. This peak completely disappeared when cells were treated with 5-bromodeoxyuridine (BrdU), suggesting its relation to quasi-normal phenotypes of the treated cells. BrdU treatment also reduced the cellular sialic acid content itself and resulted in the suppression of the activity of tyrosinase, the key enzyme for melanogenesis, and a considerable increase in cell-to-substratum adhesiveness. Treatment with theophylline, in contrast, markedly elevated the sialic acid content, which was accompanied by dramatic increments in tyrosinase activity and pigmentation as well as a slight increase in adhesiveness. The results show a correlation of sialic acid level with tyrosinase expression but not with cell adhesion. From com-parison of spontaneous phenotypic variations, the correlation of sialic acid level with tyrosinase activity was confirmed, while there was only a slight cor-relation with adhesiveness. It is thus suggested that sialylation/desialylation, be-ing reflected as variations in cellular sialic acid content, is implicated in melanoma cell differentiation in terms of tyrosinase expression.

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This report describes the purification and characterization of single-chain tissue-type plasminogen activator (sct-PA) present in tissue culture medium of a cell line established from human uterine muscle. The cell line used for the experiment, KW, had estrogen receptor. The PA fraction (KW-PA) was purified from the tissue culture medium of KW employing several steps of affini-ty chromatography and gel filtration in the presence of aprotinin. The final pro-duct (KW-PA) of purification, which predominantly contained the inactive form of sct-PA as well as active sct-PA to a lesser extent, revealed a single band with a molecular weight of 70, 000 on sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis both in the absence and presence of reducing agent. Electrophoretic enzymography demonstrated a single lytic zone at M_r 70, 000. When KW-sct-PA was treated with plasmin, SDS-polyacrylamide gel electrophoresis revealed two bands of Mr 37, 000 and 33, 000 under reduced con-ditions. Such plasmin treatment of KW-sct-PA enhanced the enzymatic activity as well as the [3H]DFP incorporation significantly. The KW-sct-PA demonstrated a higher affinity for lysine than did melanoma-t-PA, but the fibrin affinity of KW-sct-PA was identical with that of melanoma-t-PA. Circular dichroism (CD) analysis showed that the CD spectra of KW-sct-PA were different from those of melanoma-t-PA. These results suggest that the single-chain inactive form of t-PA which was obtained from the tissue culture medium of the cell line from human uterine muscle is activated to a two-chain form on plasmin treatment, with an accompanying significant increase in enzymatic ac-tivity.

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In order to reveal the pathway of iron release from macrophages, a ⁵⁹Fe-labelled ferric hydroxide-potassium polyvinyl sulfate com-plex (Fe-PVS) was injected intravenously into anemic rats and the level of radioactivity in the liver, spleen, bone marrow, blood plasma and red blood cells (RBC) was estimated at various time intervals after the injection. Histochemical observation of ferric iron and ferritin in the liver was also made on anemic rats treated using unlabelled Fe-PVS. Fe-PVS injection promoted the recovery of anemia causing a rapid increase in the RBC number, with ac-tivated erythropoiesis occurring in the spleen and bone marrow. Soon after the injection, most of the radio iron was found in the liver with a small amount in the circulating erythrocytes, bone marrow and spleen. The iron level in the liver decreased gradually with a rapid increase in the iron level of the erythrocytes which reached a very high level 6 days after the ⁵⁹Fe-PVS injection. Histo-chemical observations showed a heavy deposition of ferritin in the Kupffer cells 3 days after Fe-PVS injection. This deposition was minimized after 6 days with an increase in the level of ferritin in the parenchymal cells in the central area of acini. The level of radioferritin estimated biochemically in the non-parenchymal cell fractions of the liver revealed that the level dropped by about one third approximately 3.5 days after the Fe-PVS injection, showing the stimulated ferritin release at this stage. Results indicate that Kupffer cells in the liver play an important role in ferritin synthesis from the phagocytized iron com-pounds and that the iron is supplied for erythroid cell proliferation.

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The addition of dimethyl sulfoxide (DMSO) to a chemically defined, serum free medium prolonged hepatocytes survival in primary culture. DMSO exposure had a remarkable effect on morphological change and F-actin filaments distribution of hepatocytes. When hepatocytes were cultured in a medium containing 2% DMSO, the cells showed a compact and cubical shape and intracellular F-actin filaments were mainly observed in a ring-like fashion around the intercellular space. After exposure to DMSO, fibronectin fibers in the interspace between cell and substratum were not apparent.
Exposing the hepatocytes to DMSO also caused a sharp increase in cytosolic free ionized calcium ([Ca²⁺]_i). The initial increase in [Ca²⁺]_i following the addition of DMSO was not attenuated by the chelation of extracellular Ca²⁺ with EGTA. The Ca²⁺ signal in the absence of extracellular Ca²⁺ was transient and returned to the basal levels within 1-2 min, while it was maintained at a high steady state in the presence of extracellular Ca²⁺. These results suggest that DMSO may be able to increase [Ca²⁺]_i by two mechanisms, by the release of the ion from intracellular pools and, by the stimulation of influx across the plasma membrane.
The increase in [Ca²⁺]_i induced by DMSO treatment may play a role in pro-longing hepatocyte survival in culture, since [Ca²⁺]_i is one of the most important dynamic second messengers in various cellular metabolic processes.

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It has been shown that in PC12 and its subclone PC12h treatment of the cells with nerve growth factor (NGF) induces a selective decrease in the incorporation of radioactive phosphate into a 100, 000-dalton protein, designated in an earlier study as Nsp 100, in the subsequent phosphorylation of soluble extracts from cells with (γ-³²P)ATP. In the present study, we show that plant lectins, wheat germ agglutinin (WGA), concanavalin A (Con A), and lens culinaris agglutinin (LCA), inhibit the action of NGF on Nsp 100 phosphorylation in PC12h cells. Treatment of the cells with WGA, which binds to N-acetylglucosamine and sialic acid residues on glycoproteins, strongly blocked the inhibitory action of NGF on the protein phosphorylation. Con A and LCA, both of which recognize the same specific sugars (mannose, glucose), displayed only a moderate blocking effect. Unlike the native lectin, succinylated WGA, which has the ability to bind to N-acetylglucosamine but not to sialic acid residues, and other lectins examined in this study did not inhibit the action of NGF on Nsp 100. WGA-mediated inhibition of NGF action was reversed by the addition of N-acetylglucosamine and by the addition of a much lower concentration of a sialoglycoprotein, mucin, into the culture. Since the binding of succinylated WGA to N-acetylglucosamine residues of cell-surface glycoconjugates is not sufficient to prevent the action of NGF, WGA might act on sialic acid residues of the NGF receptor molecule to effect the inhibition of biological ac-tions of NGF.

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When developing sea urchin eggs were treated with sea water containing 40% D₂0 (D₂O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16-to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D₂O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occured in the larvae of Hemicentrotus pulcherrimus.
The cell cycle was longer in the presence of D₂0. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D₂O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D₂O-SW from the early development. These results are inconsistant with previous reports made by other authors (5, 9, 13).

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Melanophores in the skin of the freshwater teleost Zacco tem-mincki are light sensitive: Melanin granules, melanosomes, in the melanophores aggregate in darkness and disperse in light. Cultured melanophores of Zacco temmincki exhibited light sensitivity in the same manner as the melanophores in isolated scales. The dark-induced aggregation response became conspicuous after 2 days in culture. The appearance of the light response was later than that of the response to norepinephrine or melatonin, which induced rapid melanosome aggregation at one day in culture. The light sensitivity of the melanophores in isolated scales differed between individuals. A high correlation was observed between the degree of dark-induced aggregation in scale melanophores and that in cultured ones.

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Human epithelial cells that had grown out from a maxillary carcinoma were examined for their responsiveness to putative growth-controll-ing factors in a serum-free medium. Among the factors examined, bovine brain acidic fibroblast growth factor (FGF) at 1 to 10 ng/ml significantly promoted DNA synthesis of the cells in the presence of 5 U/ml heparin, whereas type β transforming growth factor inhibited it in a dose-dependent manner. Fetal bovine serum at 0.6% inhibited DNA synthesis of the cells by approximately 15%, but no significant influence was observed at higher concentrations up to 10%. Epidermal growth factor, bovine pituitary gland FGF and basic FGF ex-hibited no significant effect on DNA synthesis of the cells. The present result suggests that acidic FGF, a known mitogen for endothelial cells, is also mitogenic for human epithelial cells derived from maxillary carcinoma.

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Promyelocytic leukemia HL-60 cells can be induced to differen-tiate to granulocytes, under the conditions of cultures in the presence of dimethyl sulfoxide (DMSO). Examination of the binding of ¹²⁵I-labeled hemopexin to DMSO-induced HL-60 cells showed that the density of hemopex-in receptors on the induced-cells was 1.35 times that on the uninduced cells. We proposed that a specific receptor for hemopexin was present on the plasma mem-branes of polymorphonuclear leukocytes (PMNs). The binding of human [¹²⁵I]hemopexin to human PMNs at 4°C was saturable with time and with in-creasing concentrations of [¹²⁵I]hemopexin. Scatchard analysis of the binding revealed the presence of approximately 5.7 x 10⁴ binding sites per cell with an ap-parent dissociation constant (Kd) of 2.3 x 10^-9 M. [¹²⁵I]flemopexin was rapidly bound then dissociated from the cells after the release of heme, when the cells were incubated with radioactive hemopexin at 37°C. Incubation of the cells with the [⁵⁹Fe]heme-hemopexin complex resulted in an accumulation of [⁵⁹Fe]heme in the cells, with a temperature of 37°C but not that of 4°C. Oua-bain or NaF inhibited not only the binding of [¹²⁵I]hemopexin to PMNs but also the uptake of [⁵⁹Fe]heme from [⁵⁹Fe]heme hemopexin by the cells. Neither NH4 Cl nor chloroquine inhibited the uptake. Detergent extracts of ¹²⁵I-labeled PMNs were incubated with a hemopexin-coupled Sepharose CL-6B. A polypep-tide reacting with hemopexin-Sepharose was estimated to have a molecular weight of 80, 000, as determined by polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate. We propose that PMNs take up heme from hemopexin, as mediated by the 80, 000 dalton receptor for hemopexin.

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