In the yeasts, Saccharomyces cerevisiae and Schyzosaccharomycespombe, reduction of intracellular cyclic adenosine monophosphate (cAMP)is known to trigger the sporulation processes by activating various meiosis specific genes. In order to ascertain whether a similar mechanism is operative in higher plants, we carried out preliminary studies on lily microsporocytes. Measurement of cAMP levels as well as the activities of adenyl cyclase and phosphodiesterase in somatic cells and different stages of meiosis, and arrest of its in protoplasts cultured under conditions of high cAMP provided direct evidence that similar phenomena occur in plant meiocytes as earlier documented in yeasts.

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Rapid changes in morphology of PC12D cells, a subline of PC12 cells, in response to various agents were studied in relation to the subsequent outgrowth of neurites. A few minutes after addition of NGF or of dbcAMP, staining of F-actin with rhodamine phalloidin revealed the formation of ruffles around the periphery of cells. Simultaneous relocalization of F-actin to the area of ruffles occurred in response to NGF. A moderate relocalization of F-actin occurred in dbcAMP-treated cells. Other neurite-promoting agents on PC12D cells, such as bFGF, EGF and PMA, also caused ruffling and an identical redistribution of F-actin. The actin bundles then condensed into several dot-like aggregates that subsequently became the growth cones of neurites. When an inhibitor of protein kinase, K-252a, was added, only the NGF-induced morphological change was selectively decreased. By contrast, an inhibitor of protein kinase A, H-89, selectively blocked the dbcAMP induced change. These are analogous to the effects of those inhibitors on the outgrowth of neurites. These observations indicate that the formation of ruffles with the redistribution of F-actin might be one of the earliest steps in the neurite outgrowth and that the morphological changes might be triggered by the activation of specific protein kinases. Neither cytochalasin B nor colchicine prevented the series of morphological changes. However, processes formed in the presence of cytochalasin B had no filopodium and protrusions formed in the presence of colchicine were shaped like large filopodia. It appears that microtubules and micro filaments may not be absolutely required for the initiation of the rapid morphological changes, but that complete neurites might be formed with contribution by microtubules and by micro filaments.

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Normal human lung fibroblast diploid cells, WI-38, become senescent after a definite number of divisions. VA-13 is a line of immortalized cells established by transformation of WI-38 cells by SV40 virus. To determine whether SV40 large T (SV40-T) antigen is essential for this immortalization of WI-38 cells we introduced an antisense gene for T antigen into VA-13. Two morphologically different types of antisense transformant (VA-AS5-8 and VA-AS37-8) were obtained. In both antisense transformants the expression of T antigen was reduced by more than 70%as compared to that in the parent cells. The morphology of the antisense transformants indicated a partial conversion to the senescent phenotype of WI-38. The relative number of cells in the S phase of the antisense transformants was decreased as compared to that in cultures of VA-13and about 50% of cells were at G_1/0. The doubling time of the transformants was prolonged to close to the doubling time of WI-38. The level of expression of retinoblastoma protein (PRB) complexed with SV40-T antigen of the antisense transformants was significantly decreased although the level of total PRB was much higher than that in VA-13. The PRB was present exclusively in the underphosphorylated form. Thus, the decreased level of formation of the complex between SV40-T and PRB or the underphosphorylation of PRB may explain the suppression of growth of antisense transformants. Together, these results show that an antisense gene for SV40-T antigen can efficiently block the cell proliferation and the cell immortalization of VA-13 cells.

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GTP-binding proteins have been identified on the membranes of highly purified dextran-filled lysosomes (dextranosomes) and Triton-filled lysosomes (tritosomes) obtained from rat liver. Autoradiography of blots of lysosomal membrane proteins incubated with [α-₃₂P]GTP revealed the presence of several specific GTP-binding proteins with a relative molecular mass (M_r) predominantly in the range of 26-30 kDa. These GTP-binding proteins migrated slower in polyacrylamide gels than purified c-Ha-ras protein expressed in E. coli, whose apparent M_r was 23 kDain the same blot. The relative contents of GTP-binding proteins in lysosomal membranes were comparable or greater than that of plasma membranes and of microsomes. Chemical extraction showed that lysosomal GTP-binding proteins were more tightly associated with the membranes than with microsomal GTP-binding proteins. The possible involvement of lysosomal GTP-binding proteins in cellular functions including vacuolar (lysosomal) acidification and organellar dynamics are discussed.

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To elucidate the mechanism of mutual interaction between intracellular chemical rhythms in the Physarum plasmodium, external Ca²⁺ oscillation was applied to the ionophore-treated plasmodial strand and its response was measured as tension oscillation, (i) Tension oscillation is entrained and phase locked to the externally applied Ca²⁺ oscillation, (ii) Twokinds of stable phase relationship, in-phasic and anti-phasic ones, are observed between them, (iii) Transition between the two stable phase relationships is also observed. These results suggest that intracellular rhythms which control tension generation are mutually entrained by means of cytosolic Ca²⁺ oscillation in the organism and that their interactions have two kinds of stable phase relationships.

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Mature rat hepatocytes were cultured on collagen coated dishes in serum-free a-modified Eagle's minimum essential medium containing 0.1 μM insulin, 0.1 μM dexamethasone, 10 mM pyruvate and Ca²⁺ at concentrations of 0-2 mM. Survival of nondivided cells was best in medium containing 2 mM Ca²⁺. Proliferation during 5-day culture was greatest with 0.4 mM Ca²⁺, but DNA synthesis was scarcely affected by the concentration of Ca²⁺. Both the activities of alkaline phosphatase, 5 -nucleotidase, γ-glutamyltransferase and lactate dehydrogenase and the number of cell nuclei of cultures in 0.1 mM and 2 mM Ca²⁺ media were assayed over a 5-day period, and their activities were calculated as enzyme activities per unit number of cell nuclei. Alkaline phosphatase activity increased rapidly during the first day of culture in both media, and its activity in 0.1 mM medium was higher than that in 2 mM medium after culture for 3 days. The activity of 5'-nucleotidase became higher in 0.1 mM medium than in 2 mM medium from day 2 and was maximal on day 3 in both media. ϒ-Glutamyltransferase activity increased and lactate dehydrogenase activity decreased with time in culture, both activities showing no appreciable difference in the two media.

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Receptor-mediated superoxide (O₂)-generation and tyrosyl phosphorylation of neutrophil proteins, such as 58, 65, 84, 108 and 115 kDa, were enhanced by priming cells with granulocyte colony stimulating factor (G-CSF) [Akimura, K. et al. Arch. Biochem. Biophys. 298: 703-709, 1992]. To elucidate the possible involvement of tyrosyl phosphorylation of neutrophil proteins in the enhancing mechanism of O₂ generation, the effect of cepharanthine, a biscoclaurine alkaloid that inhibits phorbol 12-myristate 13-acetate (PMA)- and receptor-mediated O₂ generation, on the priming of human peripheral neutrophils (HPPMN)was studied. Both enhancement of formyl-methyonyl-leucyl-phenylalanine (FMLP)-mediated O₂ generation and tyrosyl phosphorylation of some neutrophil proteins, i.e., 115, 108 and 84 kDa proteins, by HHPMN after treatment with G-CSF were strongly inhibited by cepharanthine in a concentration- and treatment-time-dependent manner. In contrast, inhibition of PMA-mediated O₂ generation by cepharanthine was weak and independent of treatment time. These results suggest that cepharanthine might inhibit the priming step of neutrophil activation concomitantly with its inhibition of the tyrosyl phosphorylation of some neutrophil proteins that might underlie the mechanism for priming of neutrophils with G-CSF.

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The extent of actin-related gelation of extracts of thermosensitive Chinese hamster lung (CHL) cells capable of anchorage-independent growth was studied quantitatively by monitoring the total protein in the gel obtained by low-speed centrifugation. The gelation depended on the presence of ATP, KC1, MgCl₂, and a reducing agent. Micromolar concentrations of Ca²⁺ and low doses of cytochalasin B inhibited the actin-related gelation of these extracts. The gelation was more sensitive to inhibition by Ca²⁺ and cytochalasin B when the extracts were prepared from cells cultured at the permissive rather than the nonpermissive temperature. When various ts mutants were examined, the half-maximal inhibitory dose (HMID) of Ca²⁺ and cytochalasin B for gelation of extracts of cells cultured at the nonpermissive temperature was between 1.25 and 2.19 times higher than that for extracts of the same cells cultured at the permissive temperature. The values of the HMID for Ca²⁺ and cytochalasin B changed shortly after the shift in temperature of cell cultures from the nonpermissive to permissive temperature. When cell extracts were incubated at the permissive temperature in vitro for only 15 minutes, these changes in values of HMID were also observed. Analysis of polypeptides of cell extracts and gel pellets on polyacrylamide gel electrophoresis suggested that the decrease in amount of a high-molecular-weight actin-binding protein (250 kDa) may play an essential role in the increased sensitivity to inhibition by Ca²⁺ and cytochalasin B of actin-related gelation in extracts of these ts mutants.

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We have transfected an immortalized optic nerve-derived cell line with the cDNA's encoding the two isoforms of MAG. Our aim was to assess whether expression of L-MAG(72 KDa) and S-MAG(67 KDa) in these cells confer adhesion properties when a suspension of single cells is allowed to aggregate. The selected cell lines expressed MAGmRNAs and proteins of the appropriate molecular size, and the proteins were targeted correctly to the plasma membrane. Both L-MAG and S-MAG-expressing transfectants exhibited enhanced self-adhesive properties, aggregating with faster kinetics and forming larger aggregates than MAG-negative control cells. The interaction appears to be mostly heterophilic since MAG⁺ and MAG^-cells which were labeled with a fluorescent probe bound equally well to pre-aggregated MAG⁺ transfectants and their interaction was blocked by monoclonal anti-MAG antibodies. A further finding which supports the role of MAG in adhesion was the observation that MAG was preferentially localized at the junctions between cells, in con fluent cultures.

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Positioning of the microtubule-organizing center (MTOC)in Dictyostelium discoideum was found to be genetically regulated. We examined the wild-type strain NC-4 cells independently maintained in different laboratories, freshly recovered cells from spores stocked for over 20 years, the temperature-sensitive growth mutant HU49 isolated from NC-4, as well as strain V-12 which is the opposite mating-type to NC-4. During aggregation on nonnutrient agar plates, all these strains showed similar cell polarity, as denned by the alignment of the nucleus ahead of the MTOC. By contrast, in Ax2 and Ax3, axenic strains carrying axenic mutations on linkage groups II and III, the MTOC was usually positioned ahead of the nucleus. Cells containing axenic linkage group II but not III positioned the MTOC ahead of the nucleus. Conversely cell polarity of strains including axenic linkage group III but not II was similar to that of wild-type cells. Thus axenic linkage group II, probably axeC or other linked gene(s) not yet identified, is responsible for the location of the MTOC anterior to the nucleus during aggregation. The anterior positioning of the MTOCs was prevented by growth on bacteria in cells carrying both axenic linkage groups, but not in those carrying only axenic linkage group II.

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A novel mutation of the p53 gene has been found in a rat hepatoma cell line, FAA-HTC1.This cell line carried two kinds of abnormal p53 transcripts; one lacked the exon 8 sequence, and the other had a single base substitution G to T which resulted in a new stop codon in exon 8. In the genomic DNA, this base substitution in exon 8 was present, indicating that both transcripts were transcribed from the mutated gene. No mutation was detected in its two flanking introns. In this cell line, the exon-deleted transcript seems to be generated by exon skipping due to an unknownmechanismother than splice site mutations.

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Plasmid-borne DNAs, corresponding to 68-base oligodeoxynucleotides, synthesized in the anti-sense or sense configuration and based on the nucleotide sequences of various regions of the mouse α-globin mRNA, were introduced with the gene for xanthine-guanine phosphoribosyl transferase from E.coli(Ecogpt)into mouse erythroleukemia(MEL)cells by protoplast fusion.Specific inhibition of the synthesis of α-globin was observed only in the cells transformed with the plasmids with antisense 68-mers that corresponded to the cap site as well as the site of initiation of translation of α-globin mRNA(Oligo-A);Other plasmids with anti-sense 68-mers that corresponded to the regions of the exon/intron junctions, the individual exons, or the 3'un-translated region were ineffective.This antisense RNA efficiently reduced the production of α-globin to 9-18%of the endogenous level after induction with hexylmethylene-bis-acetoamide(HMBA).Moreover, most of the antisense transformants did not show any decrease in the expression of the c-myc gene at the early phases of differentiation of MEL cells.Thus, we propose a hypothesis that the early declineinlevels of c-myc mRNA may be independent of and uncoupled from the program of globin synthesis during the differentiation of MEL cells.

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