Anew species of lysophosphatidic acid was isolated from myxoamoebae of a true slime mold, Physarum polycephalum, and structural studies were performed. The purified substance was subjected to nuclear magnetic resonance spectroscopy (NMR), infrared spectroscopy (IR), fast atom bombardment mass spectroscopy (FAB/MS), alkaline hydrolysis and tandem mass spectroscopy (MS/MS), and the results suggested this substance to be lysophosphatidic acid composed of a cyclic phosphate and cis-ll, 12-methylene octadecanoic acid. The effects of the LPA on DNA polymerases were studied and compared with the effects of PHYLPA, which had been isolated as a specific inhibitor of eukaryotic DNA polymerase α (6). It showed a specific inhibitory activity on eukaryotic DNA polymerase α, but no activity on the repair-type, or mitochondrial DNA polymerases.
Folimycin (concanamycin A) specifically inhibited vacuolar-type ATPase as far as examined. Folimycin blocked excretion of the glycoprotein (G protein) of vesicular stomatitis virus into the medium and, instead, G protein was accumulated intracellularly. The intracellularly accumulated G protein electrophoresed a little faster than mature one. The N-glycan of the G protein was endoglycosidase H-sensitive, and terminal galactose and N-acetylglucosamine were not detected essentially on sequential digestion with exoglycosidases, indicating that processings known to occur in the Golgi apparatus do not take place in the presence of folimycin. The oligosaccharide chain of the G protein was determined to have a composition of Man8GlcNAc2 as analyzed by Bio-Gel P-4 column chromatography and high-performance liquid chromatography following digestion with α- and then with β-mannosidase. Activities of mannosidase I and glycosyltransferases prepared from baby hamster kidney cells were not inhibited as far as examined, indicating that the incompleteness of the N-glycosidic chain in folimycin-treated cells is not caused by inhibition of processing enzymes. Taken together these observations suggest that folimycin blocks the intracellular translocation of G protein before the step of trimming by mannosidase I which is confined to the cis compartment of the Golgi. The intracellular localization of G protein as revealed by fluorescence microscopy was in good accordance with this assumption.
The cytoskeletal network of cells is postulated to play a role in the signal transduction pathways of growth promoting stimuli. Weshow here that cytoskeletal active drugs modulate the mitogenic signal transduction pathway of the tumor promoter TPAin 3T3-L1 cells. Compounds which act on microtubules (vinblastine sulfate) and micro filaments (cytochalasin B) have opposite effects on DNA synthesis. Vinblastine sulfate leads to stimulation, whereas cytochalasin B causes potent inhibition of DNA synthesis in response to TPA. These drugs are cell cycle specific and apparently exert their regulatory action distal to activation of protein kinase C by TPA. The expression of four genes necessary for DNA synthesis in response to tumor promoters was examined: two nuclear proto-oncogenes (c-myc and c-fos), a transcription factor (c-jun/AP-1) and a key enzyme involved in polyamine synthesis (ornithine decarboxylase). c-jun mRNAlevels are not modulated during the action of cytoskeletal disrupting drugs on TPA-mediated mitogenesis, whereas c-myc and c-fos mRNA levels are similarly enhanced. Expression of ornithine decarboxylase mRNA and protein is increased by vinblastine sulfate but decreased by cytochalasin B in TPA treated cells. These data indicate that changes in cytoskeletal organization may play a role in regulating the levels of an enzyme critical for DNA synthesis.
To obtain biologically active substances that are useful in the study of cell structures and functions, we examined several biological activities of an extract of silkworm faeces with hot buffer. Wedetected a lectinlike substance in the extract and purified it. When the extract of silkworm faeces was added to a culture of quail myoblasts transformed with temperaturesensitive Rous sarcoma virus (QM-RSVcells), the plated cells which were cultured at 35.5°C (the permissive temperature for RSVthat suppresses myogenic differentiation) became detached from the dishes and the cells in suspension aggregated. However, when the same amount of extract was added to plated QM-RSVcells cultured at 41°C, which is a nonpermissive temperature for RSV, the cells did not become detached. Other kinds of plated cells examined also did not become detached. Unlike plated cells, nonplated cells of all kinds aggregate in suspensionuponthe addition of the extract. This active substance was purified by monitoring its induction of cell aggregation. The purified substance was found to be a lectinlike glycoprotein with an apparent molecular mass of about 60 kDa. Further studies showed that the binding sites of this glycoprotein are sugar chains on the cell surface and that mannoseis an epitope.
The interaction of βIII-depleted tubulin with taxol was investigated. A monoclonal antibody against the βIII tubulin isotype was immobilized on a sepharose 4B column and used to remove the βIII tubulin isotype from unfractionated tubulin. The assembly of βIII-depleted tubulin in the presence of taxol was enhanced compared to unfractionated tubulin. The critical concentration of unfractionated tubulin in the presence of 10 μM taxol is 0.4 mg/ml, while the critical concentration of βIII-depleted tubulin is 0.16 mg/ml. At different concentrations of taxol, the assembly of βIII-depleted tubulin is increased relative to that of un fractionated tubulin and reaches the maximumat about a 1 : 1 ratio of tubulin and taxol. The assembly of un fractionated tubulin and βIII-depleted tubulin has also been studied by electron microscopy. After 2 minutes at 37°C, unfractionated tubulin assembly in the presence of 10 μM taxol results only in ribbon-like and ring structures; there are no visible microtubules. By 5 minutes, microtubules appear and increase in length. The assembly of βIII-depleted tubulin in the presence of 10 μM taxol occurs more quickly. In contrast to the case with un fractionated tubulin, βIII-depleted tubulin assembles within 2 minutes into microtubules which increase in length with time. At 30 minutes, microtubules assembled from βIII-depleted tubulin are shorter than the microtubules assembled from unfractionated tubulin. There is no visible difference between the microtubules assembled from unfractionated tubulin and βIII-depleted tubulin. Taxol-induced βIII-depleted tubulin assembly is more resistant to the inhibiting effect of podophyllotoxin and colchicine. It is also less sensitive to the inhibiting effect of cold temperature.
We investigated the expression of laminin in two cell lines with different metastatic potentials established from murine Lewis lung carcinoma. Immunostaining of the cells with anti-laminin antibody and Northern blot analysis of laminin mRNA demonstrated that the high metastatic clone expressed less laminin than the low metastatic one. In contrast, expressions of 67 kDa-laminin receptor were at similar levels between these two lines. These findings showthe possibility that endogenous laminin may contribute to the difference in metastatic properties in the murine Lewis lung carcinoma cell lines examined.