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Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 253 records · Page 14Linked to original sources
PubMed1983

Effect of interferon treatment on blockade of protein synthesis induced by poliovirus infection.

Treatment of HeLa cells with lymphoblastoid interferon leads to a drastic inhibition of infective poliovirus. Even relatively high concentrations of human lymphoblastoid interferon HuIFN-alpha (Ly) (400 IU/ml) do not prevent destruction of the cell monolayer after most of the cells have been infected with poliovirus. Analysis of macromolecular synthesis in a single step growth cycle of poliovirus in interferon-treated cells detected no viral protein synthesis. In spite of this inhibition of viral translation, the shut-off of host protein synthesis in interferon-treated cells is apparent when they are infected both at low and high multiplicities. Although viral RNA synthesis is inhibited considerably in cells treated with interferon, a certain amount is detected, suggesting that some viral replication takes place. Analysis of membrane permeability after poliovirus infection shows a leakage to 86Rb+ ions and modification of membrane permeability to the translation inhibitor hygromycin B at the moment when the bulk of virus protein synthesis occurs. These changes are delayed and even prevented if cells are pretreated with interferon. A situation is described in which host protein synthesis is shut-down with no major changes in membrane permeability, as studied by the two tests mentioned above. Prevention of viral gene expression by inactivation with ultraviolet light of the input virus or by treatment with cycloheximide blocks the shut-off of protein synthesis. This does not occur in the presence of 3 mM guanidine. These observations are in agreement with the idea that some poliovirus protein synthesis takes place in interferon-treated cells and this early gene expression is necessary to block cellular protein synthesis.

Cell Membrane Permeability↗
PubMed1983

Cellular RNA is not degraded in interferon-treated HeLa cells after poliovirus infection.

A drastic inhibition of protein synthesis occurs in HeLa cells treated with human lymphoblastoid interferon and infected with poliovirus. At the time when this inhibition has been established no degradation of 32P-labelled ribosomal RNA can be detected. Isolation of the mRNAs from poliovirus-infected cells plus or minus interferon treatment, followed by translation in a reticulocyte lysate indicates that cellular mRNAs remain active. These results suggest that gross degradation of cellular RNA does not occur in interferon-treated poliovirus-infected HeLa cells and that a non-specific nuclease induced by 2'-5' A is not responsible for the inhibition of protein synthesis observed.

Cell Transformation, Viral↗
PubMed1983

Modification of membrane permeability in poliovirus-infected HeLa cells: effect of guanidine.

A drastic modification in permeability to monovalent ions occurred in HeLa cells infected with poliovirus, starting in the period from the third to the fourth h post-infection. The bulk of poliovirus protein synthesis took place from the third to the sixth h in cells in which the concentration of monovalent ions in the cytoplasm had changed considerably compared to uninfected cells. Under our conditions of infection (HeLa cells grown in monolayer), poliovirus translation continued beyond the eighth h. Modification of permeability to 86Rb+ ions induced by poliovirus infection was prevented by the addition of 2 mM-guanidine at the time of infection. However, when poliovirus replication was allowed to take place for 1 h before addition of guanidine, the membrane did become modified to some extent. The degree of leakiness to 86Rb+ ions increased when guanidine was added later. The blockage of membrane leakiness by early addition of guanidine was overcome by the addition of choline. The inhibition of cellular protein synthesis, which occurred early in infection or in the presence of guanidine, did not coincide with the modification of permeability to 86Rb+ ions. Viral protein synthesis was necessary in order to modify the membrane late in infection, since addition of 10(-5) M-cycloheximide during the first 2 h of infection prevented the leakiness to 86Rb+ ions observed from the fourth h after infection. Membrane potential, as measured by the lipophilic cation TPP+ (tetraphenylphosphonium), dropped from the fourth h post-infection. This change in the membrane was also prevented when viral gene expression was inhibited by the presence of guanidine.

Cell Membrane Permeability↗
PubMed1983

Enhancement of susceptibility of HSV-1-infected cells to natural killer lysis by interferon.

The effect of interferon (IFN) on the natural killer (NK) activity of human PBL against HSV-1-infected HeLa cells was studied. Human PBL from several individuals did not consistently show a preferential lysis of HSV-1-, vaccinia-, or adenovirus type 5-infected cells with respect to uninfected HeLa cells. Treatment with IFN of effector PBL increased their lytic activity but did not alter the degree of preference on the lysis of the target cells shown by untreated PBL. Pretreatment with IFN of HSV-1-infected HeLa cells increased their susceptibility to lysis 5- to 10-fold. In contrast, identical pretreatment of the uninfected, adenovirus type 5- or vaccinia virus-infected HeLa cells before the assay decreased their susceptibility to NK lysis. This effect was not likely to be due to a block of the viral replication because other inhibitors like mitomycin C did not have the same effect. All target cells induced IFN synthesis in effector PBL cells. A similar level of IFN was induced by HSV-1-infected or uninfected HeLa cells. Pretreatment with IFN of HSV-1-infected, but not uninfected, HeLa cells induced 5 to 10 times more IFN by PBL, in good correlation with the increase in lytic activity. PBL treated with IFN, however, in conditions to give maximal stimulation of NK activity, presented the same preferential lysis of HSV-1-infected HeLa cells and synthesized similar levels of IFN as untreated PBL. In addition, HSV-1-infected HeLa cells were killed through different target structures than uninfected cells. Taken together, our results indicate an effect of IFN at the level of the NK target structures in HSV-1-infected HeLa cells by increasing either their number or, more likely, their affinity for NK cells independent of the effect of IFN in the effector cells or as an antiviral agent.

Cytotoxicity, Immunologic↗
PubMed1982

Translation of capped viral mRNAs in poliovirus-infected HeLa cells.

HeLa cells doubly infected with Semliki Forest virus (SFV) and poliovirus synthesize either more poliovirus proteins or more SFV late proteins depending on the time of super-infection with poliovirus. Under some conditions, the infected cells translate uncapped poliovirus mRNA and capped 26S mRNA from SFV simultaneously, even though host protein synthesis has been shut down. Vesicular stomatitis virus (VSV) protein synthesis is depressed drastically when VSV-infected cells are super-infected with poliovirus. In cells doubly infected with VSV and encephalomyocarditis (EMC) virus or with VSV and SFV, dominance of one of the viruses depends on the time of addition of the challenge virus. The influence of external conditions on the relative translation of capped or uncapped viral mRNA in doubly infected cells has also been analysed.

Encephalomyocarditis virus↗
PubMed1982

Translation of capped virus mRNA in encephalomyocarditis virus-infected cells.

The pattern of protein synthesis in HeLa cells simultaneously infected with encephalomyocarditis virus (EMCV) and Semliki Forest virus (SFV) has been analysed throughout the time course of the infection. The ratio of the picornavirus protein gamma versus the togavirus late protein C increased when the m.o.i. of EMCV was raised, and the ratio of C/gamma increased with higher multiplicities of SFV. Under some conditions, the co-infected cells simultaneously synthesized the picornavirus and togavirus proteins, and the cells exclusively translated the capped 26S mRNA from SFV at the end of the co-infection experiment. The influence of the time of addition of the second virus on the relative translation of the capped and uncapped mRNAs was also studied. When HeLa cells were co-infected with 5 p.f.u./cell of EMCV and 200 p.f.u./cell of SFV, only the synthesis of SFV proteins was apparent, whereas if SFV was added 1 to 3 h later during the course of EMCV infection the cells synthesized picornavirus and togavirus proteins. If the cells were superinfected with SFV 1 h after EMCV addition, host protein synthesis was drastically inhibited after 3 h of EMCV infection. By 5 h post-infection both kinds of virus proteins were synthesized and at 7 h post-infection the cells preferentially translated the capped 26S mRNA from SFV. If cells were first infected with SFV (10 p.f.u./cell) and co-infected or superinfected at 1 h with EMCV (50 p.f.u./cell), shut-off of host protein synthesis occurred 3 h after infection and the cells synthesized both kinds of virus proteins. However, 9 h after infection the cells synthesized SFV proteins only. When double-infected HeLa cells were placed in a hypotonic medium, they mainly synthesized the togavirus late proteins, whereas under hypertonic conditions, they translated the picornavirus RNA exclusively. These results suggest that the two kinds of mRNAs (SFV 26S mRNA and EMCV 35S mRNA) are present in the infected cells and that the relative translation of each of them depends on the external ionic conditions.

Encephalomyocarditis virus↗
PubMed1982

Molecular basis of the permeabilization of mammalian cells by ionophores.

Mammalian cells become permeable to low-molecular-weight compounds and proteins when they are treated with nigericin. Concentrations of this ionophore that have no effect on protein synthesis induce the entry of the translation inhibitors hygromycin B (Mr 550) and kappa-sarcin (Mr 16800). Treatment of HeLa cells with low concentrations of nigericin stimulate the (Na+/K+)ATPase and, as a consequence, increase the plasma membrane potential. A correlation is observed between the increase in membrane potential and the entry of antibiotics and protein toxins into cells. These results suggest a relationship between the two phenomena and help an understanding of the molecular mechanism for the passage of charged molecules and proteins into mammalian cells. This permeabilization is specific for certain ionophores, such as nigericin and monensin, and does not occur on treatment with amphotericin B, valinomycin or A23187. These compounds depolarize the plasma membrane and drastically block protein synthesis in HeLa cells under these conditions.

Anti-Bacterial Agents↗
PubMed1981

Action of nucleotide derivatives on translation in encephalomyocarditis virus-infected mouse cells.

The infection of animal cells by encephalomyocarditis (EMC) virus lead to a drastic change in membrane permeability towards low mol. wt. compounds. Addition of the nucleotide analogue GppCH2p to the culture medium resulted in a specific inhibition of protein synthesis in EMC virus-infected 3T6 cells. This inhibition was not observed when GTP or ATP were present nor in control mock-infected 3T6 cells. The induction of membrane leakiness after viral infection was not specific for 3T6 cells, as it was also detected in mouse L cells, hamster BHK-21 cells and monkey CV1 cells. The inhibitory action produced by GppCH2p in virus-infected cells was fully reversed upon addition of fresh medium. Moreover, analysis of the proteins synthesized after medium replacement showed a preferential synthesis of cellular proteins. The presence of zinc ions resulted in an inhibition of the cleavage of large viral polypeptide precursors to mature viral proteins. Under these conditions, membrane leakiness as measured by GppCH2p, was not observed. However, this seems to be an effect of zinc ions themselves on the membrane, because inhibition of mature protein formation by other means, such as the presence of amino acid analogues, did not prevent inhibition of translation by GppCH2p in virus-infected cells. Addition of the cap analogues 7mGppp and 2'-O'-mGppp, resulted in specific stimulation of viral protein synthesis in EMC virus-infected 3T6 cells. On the other hand, the presence of 7mGp had no effect on translation. We propose that a specific capping of viral mRNA takes place in the presence of these compounds, and leads to increased stability and greater efficiency in the translation of viral mRNA.

Diphosphonates↗
PubMed1981

Protein synthesis and membrane integrity in interferon-treated HeLa cells infected with encephalomyocarditis virus.

The survival of interferon (IFN)-treated cells after encephalomyocarditis (EMC) virus infection depends on both the concentration of interferon and the multiplicity of infection (m.o.i.) used. The cell survived EMC infection if a high IFN/m.o.i. ratio was used in the experiment, whereas cell death took place at low IFN/m.o.i. ratios, even if IFN is also present during infection. Analysis by polyacrylamide gel electrophoresis of the proteins synthesized in IFN-treated cells subsequently infected with EMC indicated that no virus proteins were detected at either low or high multiplicities of infection. However, at low m.o.i. the cell survived and continued synthesizing cellular proteins exclusively, whereas at high m.o.i. a drastic shut-off of host protein synthesis took place. Virus which had been inactivated by u.v. irradiation was unable to cause the shut-off of host protein synthesis, either in control or in IFN-treated cells. This result suggests that some virus gene expression occurs in cells treated with IFN, although no virus protein synthesis was detected. The synthesis of virus RNA was also strongly inhibited after treated of cells with IFN. The integrity of the cell membrane in control and in IFN-treated cells was studied by analysing the 86Rb+ ion leakage, the thymidine pool, the chronic uptake and the entry of the translation inhibitor hygromycin B, to which cells are impermeable, at different times after EMC infection. The results obtained indicate that the early membrane leakiness observed after virus infection is not prevented by IFN treatment. However, the development of late leakiness to 86Rb+ ions, thymidine and hygromycin B was not observed in IFN-treated cells.

Cell Membrane↗
PubMed1981

Thionins: plant peptides that modify membrane permeability in cultured mammalian cells.

Thionins, which are high-sulphur polypeptides present in the endosperm of wheat and related species, have ben found to prevent growth and to inhibit macromolecular synthesis in cultured mammalian cells. Baby hamster kidney (BHK) cells were markedly more sensitive to thionins than the other cell lines tested (monkey CV1, mouse L, human HeLa). A thionin concentration of 5 microgram/ml (1 microM) completely blocked translation in BHK cells. It was later found that omission of both calcium and magnesium ions from the medium strongly enhanced the inhibitory effects of thionins (BHK cells, 80% inhibition, 0.5 microgram/ml). Several lines of evidence indicate that thionins might act at the membrane level. Indeed, both the 86Rb+ content and the nucleotide pool of BHK cells were drastically decreased at thionin concentrations that inhibited translation. In addition, thionin concentrations that did not affect macromolecular synthesis in these cells, allowed inhibition of translation by antibiotics, such as hygromycin B, that are not able to cross the cell plasma membrane by themselves. Our results suggest that the inhibition of protein, RNA and DNA synthesis in BHK cells might be a consequence of membrane leakiness induced by thionin treatment. In this respect, particularly striking was the parallelism found between 86Rb+ leakage and inhibition of protein synthesis by treatment with different genetic variants of thionins (alpha 1 purothionin, alpha 2 purothionin, beta purothionin from wheat; hordothionin from barley), as well as with the viscotoxins, which are homologous polypeptides from the European mistletoe.

Animals↗