Antiviral activity of phorbol myristate acetate and possible relationships with interferon action.

Signal-induced turnover of membrane phospholipids represents a fundamental transducing mechanism that induces a signal cascade resulting in mobilization of calcium, activation of protein kinase C by diacylglycerol, release of arachidonic acid and stimulation of cyclic GMP production. In this pathway tumor-promoting phorbol esters such as phorbol myristate acetate (PMA) may substitute for diacylglycerol. The interferon-like antiviral effect of PMA described here suggests that the inositol phospholipid-diacylglycerol-protein kinase C signal-transducing mechanism may be involved in interferon action.

Inositol phospholipid turnover and protein kinase C translocation are stimulated by poly(I).poly(C) in human amnion cells (UAC).

Polyinosinic-polycytidylic acid, a potent inducer of inducer of interferon (IFN) production and activator of some IFN-induced enzymes, inhibits [3H]uridine incorporation into the RNA of vesicular stomatitis virus even in the absence of IFN synthesis, transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C. Since IFNs also have similar activities these results suggest that IFN induction and IFN function are realised through common biochemical pathways.

Interferon (IFN)-like antiviral effect is induced by unspecific cross-linking of cell surface receptors.

Treatment of human amniotic cells (UAC) with Cytodex 1 (DEAE-dextran) results in the development of an antiviral state of the cells, as proven by studying (i) the cytopathic effect and (ii) [3H]uridine incorporation into the RNA of vesicular stomatitis virus (VSV) after VSV infection. The same treatment transiently triggers the breakdown of inositol phospholipids and activates the translocation of protein kinase C (PKC). On the basis of these data it can be suggested that cross-linking of cell surface receptors by a solid carrier bearing covalently bound positive charges may result in IFN-like effects.

Phospholipase C and phospholipase A2 are involved in the antiviral activity of human interferon-alpha.

Treatment of human amniotic cells (UAC) with human interferon-alpha (Hu-IFN alpha) or phorbol myristate acetate (PMA) resulted in translocation of protein kinase C (PK-C) activity from the cytosol fraction to that of the membranes. Analysis of 32P incorporation into phospholipid fractions and studies of alterations in fatty acid content for the major phospholipids of IFN-treated cells suggest that phospholipases C and A2 are activated by Hu-IFN alpha. Addition of neomycin (an inhibitor of phospholipase C), as well as mepacrine (an inhibitor of phospholipase A2) to IFN-treated cells inhibited the antiviral activity of Hu-IFN alpha in the vesicular stomatitis virus (VSV)-UAC system used. These observations indicate that (i) activation of PK-C and (ii) diacylglycerol formation, arachidonic acid and/or lysophosphatidylcholine release are important steps in the mechanism of action of IFN.

Ethanol-induced signal transducing mechanism associated with a transient antiviral state in human amniotic cells.

Short-term effects of ethanol on human amnion cells were investigated by studying the cellular signaling processes and the replication of vesicular stomatitis virus. Treatment of human amniotic cells with ethanol transiently triggers the breakdown of inositol phospholipids, stimulates intracellular [Ca2+]i mobilization and activates the translocation of protein kinase C. Activation of this signal transduction mechanism is associated with the development of an antiviral state, as proven by studying 3H-uridine incorporation into the RNA of vesicular stomatitis virus. Induction of the antiviral state in human amniotic cells correlates with the solubility of the alcohols in the lipid membrane of the cells.

Ultrastructural changes of human amniotic cells induced by natural human interferon-alpha.

Human interferon-alpha (Hu-IFN alpha) and phorbol myristate acetate (PMA), a direct activator of protein kinase C (PK-C), induce the translocation of protein kinase C from the cytosol to the membrane fraction. By the use of transmission (TEM) and scanning (SEM) electron microscopy we have shown that treatment of human amniotic cells (UAC) with Hu-IFN alpha resulted in profound changes in the shape, volume and ultrastructure of the cells. Most treated cells had enlarged nuclei with marginal condensation of chromatin. Nucleolar segregation, disintegration and clumping of nucleolar components were also observed. The number of interdigitating cell processes decreased and the cell surface microvilli became shortened. Similar ultrastructural alterations were induced by PMA also. All these functional and morphological data strongly support the hypothesis that protein kinase C is a key factor in IFN-mediated cell reactions.

A novel approach for understanding the effects of interferons--a working hypothesis.

Experimental results accumulated during the last two decades prove that there are no mechanisms specific for interferon (IFN) action. Thus, IFN effects must be realized through the relatively limited number of metabolic pathways available for cell functions. Triggering the receptor system in association with membrane phospholipid breakdown, which plays an important role in signal transduction for a variety of biologically active substances, is suitable to explain generation of the pleiotropic IFN effects, too. Prevalence of one or the other of the "positive" and "negative" pathways of this signal transducing system depending on various known and up-to-now unknown factors may be responsible for the sometimes contradictory effects of IFNs.

Signal transducing mechanisms in interferon action (a brief review).

Experimental results suggest that protein kinase C (PK-C) may play a substantial role in the action of IFNs, but the precise biochemical pathway remains unknown. Recent evidences reveal the complexity of the mechanism of IFN-action and show that the IFN-alpha, -beta and -gamma induced pathways are overlapping. We briefly discuss what is known in this field and suggest a way in which the contrasting views might be reconciled.

Effect of "osmotic stress" on protein and nucleic acid synthesis in isolated tobacco protoplasts.

The incorporation of labeled precursors into RNAs and proteins of isolated tobacco (Nicotiana tabacum L.) leaf protoplasts decreases with increasing osmotic pressure in the incubation medium. The incorporation of precursors into RNA and proteins is linear for 15-18 h after the isolation of the protoplasts, irrespective of the osmolarity of the culture media. The uptake of precursors is also affected by the osmolarity of the medium. However, the osmotic stress-induced inhibition of incorporation of precursors into RNA and proteins is also apparent if the differences in uptake are taken into consideration in the calculation. Incorporation of (32)P into TMV-RNA is also inhibited by osmotic stress. As assayed by the double labeling ratio technique, osmotic stress has less unequivocal effect on TMV protein synthesis.

Oxytocin stimulates translocation of protein kinase C and induces antiviral state in human amnion cells.

Association of protein kinase C (PKC) activity to the membrane fraction was observed in oxytocin treated human amnion cells (UAC). In addition, oxytocin was shown to induce an antiviral state and to inhibit multiplication of vesicular stomatitis virus (VSV) in UAC. These observations together with earlier findings indicate that activation of inositol phospholipid breakdown with a consecutive activation of PKC is a common signal transduction pathway in interferon action and hormonal stimulation.

Polyribosomes in protoplasts isolated from tobacco leaves.

Polyribosomes (polysomes), active in an amino acid incorporation system in vitro, were isolated from tobacco leaf protoplasts. A comparison of polysome profiles indicated that the polysome/monosome ratio is greatly decreased in isolated protoplasts as compared to the intact leaf. In isolated protoplasts, a marked accumulation of ribosomal subunits was also found. The division of protoplasts, as investigated in the 8-cell and callus stages, was associated with a(n) (at least) partial regeneration of polysome profiles characteristic for leaves. Plasmolysis of leaves attached to the plant had no great effect on the polysome profile. However, leaf excision per se resulted in a dramatic loss of polysomes, even when the leaf tissue was floated on water. It is concluded that the isolation of the cell from its normal environment, and not the osmotic stress and associated increase in RNase activity, is the most important factor responsible for the loss of polysomes in isolated protoplasts.

Viral RNA synthesis is renewed in protoplasts isolated from TMV-infected Xanthi tobacco leaves in an advanced stage of virus infection.

TMV-RNA synthesis was shown to be renewed in protoplasts isolated from TMV-infected Xanthi tobacco leaves in which virus synthesis has already slowed down or stopped. This was demonstrated by the use of isotope techniques in three different disease situations: (a) in primarily infected leaves 8-10 days after infection, (b) in secondarily infected leaves 2 mo after infection, and (c) in stable green areas ("true dark green islands" according to the terminology of Atkinson and Matthews, 1970) of leaves exhibiting mosaic symptoms.