A sensitive multi-electrode was developed to observe the spatio-temporal evolution of extracellular electric potential around plasmodia of starved Physarum polycephalum. As the starvation proceeded, oscillation of the electric potential in all places was observed, with a large reduction in the frequency and a great increase in the amplitude. This was followed by morphological change about one hour later. In spite of the great change in the oscillation, however, there was a specific relationship between the frequency and amplitude: (amplitude) × (frequency)² = constant.
Under anaerobic conditions, the oscillation was greatly suppressed and the morphological changes were completely suppressed. This strongly suggested that some activity related to respiration was very important to the proliferation of the starved plasmodia. In addition, in the presence of the proton pump in-hibitor diethylstilbestrol (DES), the spatial uniformity and the amplitude change of the electric potential oscillation were greatly reduced, although the development of the frequency of the electric potential was very similar to that in its absence. DES also inhibited the morphological change of starved plasmodium. The correlation between the oscillation phenomena and the mor-phological change is discussed.

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We observed the dynamic changes in the localization of microfilaments during the exocytic secretion of rat parotid and submandibular gland acinar cells, and obtained results which led us to propose a new concept of microfilament function in exocytosis. With the electron microscopy, NBD-Phallacidin (NBD-PL) fluorescence technique and immunohistochemistry for myosin, microfilaments consisting of F-actin and myosin were localized mainly underneath the luminal plasma membrane. Microfilaments were not detectable around the secretory granules which were stored in the cytoplasm, but were clearly observed around them whose membranes were continuous with the luminal plasma membrane. When viewed with NBD-PL and myosin fluorescence, the area of fused granule membranes revealed bright fluorescence in association with the luminal border, so that the luminal membrane undergo-ing exocytosis appeared like a 'bunch of grapes'. When excess exocytosis was stimulated by isoproterenol (IPR), the number of individual 'grapes' increased dramatically, indicating that the secretory granules are surrounded by microfilaments after the fusion with the luminal membrane. Microfilaments thus continuously undercoat the luminal membrane during exocytosis although the exocytic process involves the dilation and subsequent reduction of the luminal membrane due to the addition and removal of secretory granule mem-branes. This reduction of the dilated luminal membrane follwoing exocytosis was, however, inhibited when the microfilaments were disrupted by cytochalasin D. Following this treatment, the lumina was expanded extraor-dinarily and the secretory products remained in the enlarged lumina, showing that the release of secretory products is inhibited when the microfilament func-tion is disturbed.
These results indicate that 1) microfilaments are localized mainly underneath the luminal plasma membrane and act as an obstacle to exocytosis when cells are at the resting phase and 2) at the secretory phase microfilaments allow exocytosis by disorganizing their barrier system and then, by encircling the discharged secretory granule membranes, provide forces for the extrusion of secretory products through the action of the acto-myosin contractile system.

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We studied the effects of colchicine on the intracellular transport ofsecretory proteins in rat liver parenchymal cells using the direct im-munoenzyme technique. Livers were perfusion-fixed 0.5, 1, and 2 h after injec-tion of colchicine. Vibratome sections of the fixed liver were stained using perox-idase-conjugated Fab' of anti-albumin or anti-fibrinogen. By light microscopy, reaction deposits showing albumin and fibrinogen were observed in the cytoplasmic granules of hepatocytes. Such stained granules decreased 30 min after injection, but later increased gradually and crowded in the cytoplasm. The Golgi complex stained for the proteins decreased after 30 min but increased in the juxtanuclear region after 60 min. The analysis of serial sections showed that colchicine severely disturbed the spatial relationship between the Golgi apparatus and the bile canaliculus. We obtained similar results by electron microscopy; a positive reaction for albumin and fibrinogen was observed in a small number of the cytoplasmic granules after 30 min. After 1 h of treatment, most of the Golgi complexes were fragmented and lost their stacked cisternae. However, they reappeared accompanied with vacuolated cisternae and secretory granules, which were partially stained for albumin and fibrinogen. After 2 h, the secretory granules positive for both proteins accumulated further. Some of them lined a long the plasma membrane, and others made a cluster in the cytoplasm. The profiles showing exocytosis were very rarely seen. These results showed that in the first 30 min, colchicine primarily disturbs partially the Golgi assembly but does not affect the post Golgi secretory pathway much. Later, the drug affects both the post Golgi pathway and the Golgi assembly, and it causes a marked accumulation of secretory granules.

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Short-term and long-term (>7 months) cultured astrocytes from 14-day-old rat brain were analyzed for ganglioside content. Analysis of the extracted gangliosides by HPTLC revealed that ganglioside GM₁ was absent in 35 days and 235 days cultured astrocytes, and that the predominant ganglioside was GM3, showing a double band in both cases. A small amount of the disialogangliosides (GD₃, GD_1a) was also detected. More than 70% of radioactivities into ganglioside fractions by cultured astrocytes, in the presence of N-[³H]-acetylmannosamine, appeared in ganglioside GM3. The upper band component of GM3 increased 60% in long-term astrocyte cultures compared to 35-day-old cultures. Also, an increased GD₃ content in long-term astrocyte cultures was detected. These results suggest that the increase of GD3 and upper band GM₃ in long-term cultured astrocytes might be related to the appearance of small processes showing strong reactivity against GFAP and vimentin during astrocyte-subculture.

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We studied the molecular mechanism of the shape change of erythrocytes with a local anesthetic, lidocaine. The shape of human erythrocytes changed from discocytes to stomatocytes in the presence of lido-caine when ATP was present. But, the shape of resealed ccells which were prepared with 10 mM Tris-HC1 buffer (pH 7.4) containing 2 mM ATP-MgC1₂ and various substances was not changed from discocytes to stomatocytes with lidocaine. When intact cells and resealed cells which were prepared with various concentrations of Tris-HC1 buffer (pH 7.4) were incubated with various concen-trations of lidocaine and their membrane proteins were analyzed by SDS-PAGE, the densities of bands 62K, 28K and 22K depended on lidocaine concentration: in particular, that of band 28K changed remarkably. These mem-branous 62K-, 28K- and 22K-proteins agreed with cytoplasmic 62K-, 28K- and 22K-proteins in molecular weight. We propose that not only ATP but also the 62K-, 28K- and 22K-proteins in the cytoplasm are concerned with the shape change of human erythrocytes induced with lidocaine.

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A plasmid containing the human preprourokinase gene cDNA under the control of the simian virus 40 early region promoter was introduced into CHO-K₁ cells and recombinant cell lines secreting a relatively high level of urokinase were obtained. In the course of studying the effects of various agents on the recombinant cell lines, we found that exposure of recombinant cells to 5 mM butyrate for 24 hours resulted in a 2-3 fold increase in urokinase produc-tion. The induction by butyrate was dose-dependent. The half maximal dose was approximately 2 mM; maximal stimulation occurred at 5-10 mM. Cell growth, on the otherhand, was inhibited by butyrate concentrations greater than 2.5 mM. The responseof cells to butyrate was rapid: a significant increase in urokinase production was observed 6 hours after exposure to 5 mM butyrate. Butyrate treatment increased not only the extracellular level but also the in-tracellular level of urokinase.

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A 32-KDa calcium dependent phospholipid-binding protein was purified to homogeneity from human placenta by affinity adsorption to polyacrylamide-immobilized phosphatidylserine followed by elution with 5 mM EGTA and ion exchange chromatography. Immunochemical studies using the polyclonal antibody against the 32-KDa protein revealed that this protein was present around the nucleus in the cytoplasm but not clearly associated with cell organelles and cytoskeletons. In KB cells treated with insulin, 32-KDa protein was localized in the ruffling membranes in addition to the cytoplasm. Purified 32-KDa protein was shown to coprecipitate with skeletal muscle actin under polymerizing conditions. These findings suggest that the 32-KDa protein interacts with networks of actin filaments in cells.

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We have previously demonstrated that 5'-nucleotidase, known as a plasma membrane enzyme, is also distributed both in rat liver tritosomal membranes and contents (J. Biochem. 101, 1077-1085, 1987). When the lysosomal membranes isolated from rat livers were incubated with phosphatidylinositol-specific phospholipase C purified from B. thuringiensis, about 70% of 5'-nucleotidase activity was released from the membranes. Judging from the result by phase separation with Triton X-114, the enzyme solubilized by the phospholipase C digestion showed a hydrophilic nature such as that of the tritosomal contents. Immunoblot analysis showed that the molecular weight of 5'-nucleotidase released from the lysosomal membranes by the phospholipase C digestion was almost identical with that of the enzymes from the Tritosomal contents. The above results showed that the phosphatidylinositol-specific phospholipase C-like enzyme in the lysosomes may be responsible for the conversion of the lysosomal membrane-bound 5"-nucleotidase to the soluble form present in the lysosomal matrix.

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Cytochemical electron microscopy of cultured rat hepatoma cells (AH-130) demonstrated that thiamine pyrophosphatase (TPPase) activity was localized in the Golgi complex. When the cells were treated with brefeldin A (BFA, 2.5 μg/m1) for 10 min, the characteristic structure of the Golgi stack was no longer observed, and TPPase was cytochemically stained in the vesicular and tubular structures scattered in the cytoplasm. A longer exposue of the cells to the drug (20 min to 1 h) resulted in the distribution of the TPPase activity in the endoplasmic reticulum (ER) and nuclear envelope. Such an unusual distribu-tion of the enzyme activity, however, was reversible even in the presence of BFA. At 2 h after the exposure, the TPPase activity disappeared from the ER and was concentrated again in the vesicular and tubular structures. The enzyme activity was finally localized in the Golgi complex which was reassembled by 4 h after the exposure. The reversible effect of BFA may be due to a possible metabolism of the drug into an inert form during the incubation. Taken together, these results indicate that BFA causes a rapid disassembly of the Golgi complex and redistribution of the marker enzyme TPPase into the ER including the nuclear envelope. The spontaneous reversibility of the drug effect also favors a dynamic recycling of the Golgi marker between the ER and the Golgi complex under the conditions used.

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Some plant extracts and products are known to affect mammalian cells, tissues and organisms as they contain a toxic substance or a metabolic stimulant. Our biochemical investigations revealed that some plant saponins can increase the cellular DNA repair activity and the general recom-binase activity measured by in vitro assay (1). In the experiments described here, HeLa cells were cultured for several days with plant saponins or flavonoids and analyzed to measure i) recombination activity of the cell extract by induction of Tc^r colonies from two mutant DNAs (mutants 1 and 2, which are both tetracycline sensitive) after transformation into E. coli recA^-, and ii) repair synthesis of nuclear DNA followed by incorporation of ³H-thymidine. Saikosaponins a, bl, d, ginsenosides Rbl, Re, Rh and flavonoid baicalin caused a significant stimulation of intermolecular recombination. It is worth noting that none of the plant saponins and flavonoids had any inhibitory or toxic effect at concentrations less than 25 μg/ml in the culture media.

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We isolated mutants defective in aggregation (aggregation-less) by mutagenizing the "double-bypass" mutant HG592 of Dictyostelium discoideum as the parental strain. One of the mutants expressed the contact site A glycoprotein with an apparent molecular weight of 80 × 10³ on the cell surface in the normal developmental stage and retained EDTA-stable cell contact as well as EDTA-sensitive cell contact. However, the mutant failed to aggregate on agar plates with bacteria. This mutant was designated HG700. We could not identify any differences between this mutant and the parental strain in levels of adenylate cyclase or extracellular phosphodiesterase activity, or in its chemotaxis toward cAMP. The mutant had greatly decreased the incorporation of [³⁵S] sulfate into the particulate fractions of the cells starved for 6 h. This sug-gests that the modification by sulfation may crucially affect the mechanism of cell aggregation.

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