Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 217 records · Page 12Linked to original sources

PubMed1988

Reovirus type 3 synthesizes proteins in interferon-treated HeLa cells without reversing the antiviral state.

Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

2',5'-Oligoadenylate Synthetase↗

PubMed1988

Inhibition of natural killer cytotoxicity by extracellular ppp(A2'p5')nA oligonucleotides.

The effect of purified 2'-5' linked polyadenylated oligomers on the human natural killer (NK) activity has been investigated. Permeabilization of NK cells to ppp(A2'p5')2A did not enhance their NK activity at any concentration tested. Furthermore, concentrations higher than 0.1 mM inhibited their NK activity. Similar results were obtained by pre-incubating non-permeabilized intact cells, indicating an extracellular effect of these compounds on cell membranes, which was further investigated. This inhibition by the oligomers was greater (50% inhibition was obtained with 50 micron), when they were added directly to the NK assay, than when the NK cells were pre-incubated. Similar inhibitory effects were observed with ppp(A2'p5')A and ppp(A2'p5')3A oligomers but not with the dephosphorylated "core" molecules or other nucleotides. The inhibition was completely or partially reversed by phorbol esters and by interferon (IFN), respectively. The inhibition was temperature and extracellular Ca2+ dependent. The oligomers did not inhibit the binding of the effector cells to the target cells. The programming for lysis was the step of the lytic mechanism affected, possibly due to the alteration of the Ca2+ influx into the NK cells observed in presence of the ppp(A2'p5')nA.

Adenine Nucleotides↗

PubMed1988

Effect of heterocyclic analogues of triphenylmethane dyes against Trypanosoma cruzi.

Several heterocyclic analogues of triphenylmethane dyes have been synthesized. Their in vitro activity has been evaluated using the epimastigote forms of Trypanosoma cruzi as an assay system. The malachite green and gentian violet dyes previously reported as trypanocidal agents were also tested for comparison. Among the compounds tested, the 2-benzothienyl-bis-(4,4'-dimethylaminophenyl) derivative proved to be the most interesting, because of its high selectivity in killing parasites. The ID50, defined as the concentration which produced 50% reduction in (3H) thymidine incorporation in DNA after 18 hours or in viable parasites after five days of incubation with the above compounds, was 0.2 ng ml-1 and less than 1 ng ml-1, respectively. The relative toxicity, measured by its effect on cultured human HeLa cells, was over 10,000-fold higher. By contrast, gentian violet only showed a ten-fold higher inhibitory effect for T. cruzi. The other derivatives synthesized were active in various degrees. According to these results, a structure-activity relationship for these compounds is proposed.

Animals↗

PubMed1988

The P2 and P3 regions of the poliovirus genome are preferentially translated at alkaline pH in infected HeLa cells.

HeLa cells infected with poliovirus exclusively synthesized proteins coded by the P2 and P3 regions of the viral genome when they were placed in an alkaline medium lacking sodium ions. The amount of viral protein synthesized was augmented by increasing the concentration of KCl. Also in the presence of KCl, the cells continued synthesizing this altered pattern of proteins for longer times. This effect occurred in the presence of guanidine, it was reversible, and the normal pattern of poliovirus proteins reappeared when control medium was added, even when guanidine was present. These findings suggest that truncated viral RNA is not made under these conditions. Pactamycin and sodium fluoride, two known inhibitors of the initiation of translation, blocked protein synthesis in cells placed in alkaline medium, indicating the possibility that initiation at internal sites of the viral mRNA may take place under these conditions. Finally, the proteins synthesized were analysed by two-dimensional gel electrophoresis. The proteins migrated as authentic viral proteins indicating that if internal initiation takes place, at least some of these initiation events occur in phase with the initiation codon present in the poliovirus genome.

Cycloheximide↗

PubMed1988

Mode of action of a new type of UDP-glucose analog against herpesvirus replication.

The mode of action of a new type of UDP-glucose analog against herpes simplex virus type 1 (HSV-1) replication was examined. The analog showed good selectivity and potent activity. At 10 micrograms/ml, P-536 inhibited the formation of infectious HSV-1 by more than 90%, whereas at 100 micrograms/ml it had no cytotoxic effects, as evidenced by phase-contrast microscopy. P-536 showed a wide spectrum of action and was active against HSV-1, adenovirus type 5, vaccinia virus, poliovirus type 1, encephalomyocarditis virus, vesicular stomatitis virus, influenza virus, and measles virus, irrespective of whether these viruses have lipidic envelopes or not. P-536 clearly inhibited protein glycosylation if added at the time when late viral proteins were being synthesized. Moreover, it also interfered with the synthesis of nucleic acids and the phosphorylation of nucleosides. If P-536 was present from the beginning of infection, HSV-1 replication was blocked at an early step and the infected cells continued to synthesize cellular proteins for long periods.

Animals↗

PubMed1987

Animal viruses promote the entry of polysaccharides with antiviral activity into cells.

Charged polysaccharides such as heparin and carrageenan show potent antiviral activity in cultured cells. Labelled [3H]heparin binds to several virion particles as evidenced by Sepharose 6B chromatography. This binding is inhibited by carrageenan. The complex [3H]heparin-HSV1 virions is able to enter cells. Thus, almost no entry of [3H]heparin is observed in control HeLa cells, whereas in the presence of HSV1 or poliovirus the amount of radioactivity internalized is enhanced. This internalization is inhibited by carrageenan, suggesting that these two sulphated polysaccharides bind to the same sites on virion particles and may share a similar antiviral mechanism of action.

Animals↗

PubMed1987

Adenovirus late protein synthesis is resistant to the inhibition of translation induced by poliovirus.

Inhibition of host protein synthesis after poliovirus infection has been suggested to be a consequence of the proteolytic degradation of a p220 polypeptide necessary to translate capped mRNAs. However, the synthesis of several adenovirus late proteins on capped mRNAs was resistant to poliovirus inhibition. Thus, the hexon protein was still made 8 h after poliovirus superinfection. The synthesis of other adenovirus proteins such as the fiber was much more sensitive to poliovirus-induced inhibition than the hexon, either in the absence or in the presence of guanidine. Detailed densitometric analyses clearly showed the differential behavior of several adenovirus late mRNAs to poliovirus shut-off of translation. This is striking in view of the fact that a common leader sequence in the 5' termini is present in the adenovirus late mRNAs. The use of 3-methyl quercetin, an inhibitor of poliovirus RNA synthesis (Castrillo, J. L., Vanden Berghe, D., and Carrasco, L. (1986) Virology 152, 219-227), showed that translation of several capped adenovirus mRNAs took place in poliovirus-infected cells after the synthesis of host proteins had ceased. The poliovirus mRNA and the adenovirus mRNA coding for the hexon protein are very efficient mRNAs and have a leader sequence of more than 740 and 250 nucleotides, respectively, with very rich secondary structures making it difficult to predict how the scanning model will operate on these two mRNAs.

Adenoviridae↗

PubMed1987

Adenovirus infection reverses the antiviral state induced by human interferon.

HeLa cells treated with human lymphoblastoid interferon do not synthesize poliovirus proteins. The antiviral state against poliovirus is reversed if cells are previously infected with adenovirus type 5. A late gene product seems to be involved in this reversion, since no effect is observed at early stages of infection or in the presence of aphidicolin.

Adenine Nucleotides↗

PubMed1987

Control of membrane permeability in animal cells by divalent cations.

The permeability of several cell lines, including HeLa, L929, 3T6 and 3T3, to various compounds is affected by the concentration of divalent cations in the culture medium. In the absence of Mg2+ ions but with 4-8 mM CaCl2 in the medium, HeLa and L929 cells become permeabilized, as measured by the entry of the aminoglycoside antibiotic hygromycin B. However, 3T3 and 3T6 cells become much more permeable when calcium and magnesium are both absent from the medium. Addition of Mg2+ above 2 mM abolishes the permeabilization induced by Ca2+. Basic pH favors permeabilization, whereas acidic pH inhibits the entry of hygromycin B. Increased entry of macromolecules, such as the toxin alpha-sarcin, horseradish peroxidase (HRP) and luciferase, is also observed under permeabilization conditions, suggesting that this method could be of general use, since it is not harmful to cells and is fully reversible. Exit of 86Rb+ ions and [3H]uridine-labelled nucleotides was also assayed. We did not observe increased release of these compounds from preloaded cells under various calcium concentrations. Finally, the effects of several inhibitors of endocytosis and other membrane functions on the permeabilization inhibitors of endocytosis and other membrane functions on the permeabilization process were also analysed. The entry of alpha-sarcin was not affected by nifedipine, dibucaine or mepacrine, but was partially inhibited by NH4Cl, amantadine and chloroquine.

Animals↗

PubMed1987

Proteins are cointernalized with virion particles during early infection.

Mammalian cells infected with enveloped or naked animal viruses become permeabilized to several proteins. The entry of alpha-sarcin, horseradish peroxidase, and luciferase is greatly increased during the early stages of viral infection. This process is promoted by uv-inactivated SFV, but not by heat-inactivated virions, suggesting that the process does not require viral gene expression. The entry of alpha-sarcin has been monitored both by its effects on protein synthesis and by indirect immunofluorescence. Increased entry of alpha-sarcin and luciferase is clearly observed in animal virus-infected cells by fluorescence microscopy. Chloroquine blocks the coentry of alpha-sarcin with enveloped, but not with naked, viruses. These results have implications to elucidate the mechanisms involved in virus entry.

Adenoviruses, Human↗

PubMed1987

Proteins synthesized in African swine fever virus-infected cells analyzed by two-dimensional gel electrophoresis.

At least 74 acidic and 37 basic proteins are synthesized in African swine fever virus (ASFV)-infected monkey cells not detected in uninfected cells analyzed by two-dimensional gel electrophoresis. Essentially all the proteins synthesized early during infection are also observed at late times. The use of inhibitors such as cycloheximide and phosphonoacetate has led to the identification of 34 immediate early and 13 delayed early polypeptides. Therefore 64 proteins were classified as late polypeptides. Several ASFV-induced proteins are phosphorylated as proteins a1, a4, a20, a41, a48, a49, a51, a52, a55, a58, a67, b2, b12, b28, and b32.

African Swine Fever Virus↗

PubMed1987

Baciphelacin: a new eukaryotic translation inhibitor.

Baciphelacin an antibiotic produced by Bacillus thiaminolyticus was a potent inhibitor of protein synthesis in HeLa cells and other mammalian cell lines. It had no effect on DNA or RNA synthesis. Concentrations of baciphelacin around 10(-7) M inhibited protein synthesis by 50% in intact cells. The antibiotic had no effect on protein synthesis in Saccharomyces cerevisiae or Escherichia coli, but inhibited the protozoan Trypanosoma brucei. In vitro protein synthesis in a rabbit reticulocyte cell-free system was blocked by baciphelacin. However, translation of globin mRNA in a wheat cell-free system was not affected by this antibiotic. Baciphelacin had no activity against a number of cell-free systems used to measure different steps of translation, including binding of substrates to the ribosome, peptide bond formation and polyphenylalanine synthesis. Therefore, it is assumed that it affects the initiation of translation or the charging of tRNA. Finally, the inhibition of protein synthesis by compounds structurally related to baciphelacin was tested and their effects compared to baciphelacin.

Anti-Bacterial Agents↗

PubMed1987

Effects of extracellular cations on translation in poliovirus-infected cells.

The effect of pH and different concentrations of added monovalent and divalent cations on translation in poliovirus-infected HeLa cells has been examined. A strong effect on protein synthesis was observed when the concentration of sodium ions was modified. If cells were placed in a hypotonic medium after virus adsorption, no shut-off of cellular translation took place, nor were viral proteins synthesized. An increase in the multiplicity of infection partially overcame this effect. Reversal of the shut-off of cellular translation and inhibition of viral protein synthesis was achieved when cells were placed in hypotonic medium 2 h after infection. Modification of divalent cations (calcium or magnesium) had little or no effect on the pattern of translation. On the other hand, acidic pH (below 6) inhibited both cellular and viral protein synthesis, whereas basic pH had no influence. During infection the synthesis of poliovirus proteins reached a maximum at about 4 to 6 h and then declined. This inhibition of viral translation was partially prevented if cells were placed in a medium containing a high concentration of potassium although the cytopathic effect was prevented. These results indicate that viral protein synthesis and the cytopathic effect were, to a large extent, influenced by the external monovalent ion concentration.

Cations↗

PubMed1987

Cation content in poliovirus-infected HeLa cells.

Poliovirus-infected HeLa cells increased their permeability to monovalent ions from the third hour post-infection. At that time infected cells lost their content of potassium ions as measured by efflux of the potassium analogue 86Rb+. The rate of release of 86Rb+ from cells increased as infection proceeded, and was not sensitive to ouabain or quinidine, inhibitors of the Na+/K+ ATPase and Ca2+-induced K+ release system, respectively. The leakage of 86Rb+ was only slightly sensitive to furosemide, an inhibitor of the Na+/K+/Cl- contransport system, suggesting that the mechanism of release involved an increased passive permeability of the cell membrane. The calcium content or its efflux did not vary significantly in the infected cells. Neither were there alterations in the intracellular pH throughout infection.

Biological Transport↗

PubMed1987

Polysaccharides as antiviral agents: antiviral activity of carrageenan.

A number of polysaccharides showed good antiviral activity against several animal viruses. At 5 micrograms/ml, carrageenan prevented the cell monolayer from destruction by herpes simplex virus type 1 (HSV-1) growth. At 10 micrograms/ml, carrageenan reduced the formation of new infectious HSV-1 by almost five logs. No cytotoxic effects were detected with concentrations of carrageenan up to 200 micrograms/ml. When 10 micrograms of carrageenan per ml was added at the beginning of HSV-1 infection of HeLa cells, there was potent inhibition of viral protein synthesis, and the cells continued synthesizing cellular proteins. This did not occur if carrageenan was added 1 h after HSV-1 infection. The use of [35S]methionine-labeled virions to analyze the entry of HSV-1 or Semliki Forest virions into cells indicated that carrageenan had no effect on virus attachment or virus entry. Moreover, carrageenan did not block the early permeabilization of cells to the toxic protein alpha-sarcin. These results suggest that this sulfated polysaccharide inhibits a step in virus replication subsequent to viral internalization but prior to the onset of late viral protein synthesis.

Animals↗

PubMed1987

Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro.

Ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) eliminates foot-and-mouth disease virus from persistently infected cell cultures. The latter are 10-fold more sensitive to ribavirin than lytically infected cells. In treated cells no viral RNA or proteins could be detected by dot-blot hybridization to cDNA probes, virus and RNA infectivity assays, or immunofluorescence. A potential application of the drug for the treatment of animals carrying the virus is suggested.

Animals↗

PubMed1987

Action of 3-methylquercetin on poliovirus RNA replication.

3-Methylquercetin is a natural flavone that powerfully blocks poliovirus replication. This compound inhibits selectively poliovirus RNA synthesis both in infected cells and in cell-free systems. Poliovirus double-stranded RNA (replicative forms) is still made in the presence of this inhibitor, whereas the synthesis of single-stranded RNA and the formation of replicative intermediates are drastically blocked.

Antiviral Agents↗

PubMed1986

3-Methylquercetin is a potent and selective inhibitor of poliovirus RNA synthesis.

3-Methylquercetin (3MQ) is a natural compound isolated from Euphorbia grantii that selectively inhibits poliovirus replication, but has no effect on encephalomyocarditis virus. When the compound is present from the beginning of infection, the bulk of viral protein synthesis is prevented, but the shut-off of host protein synthesis still occurs. Addition of 3MQ 3 hr after infection has a slight effect on viral protein synthesis, suggesting that this compound blocks a step of viral replication different from translation. Indeed, poliovirus RNA synthesis is potently blocked by 3MQ, i.e., 50% inhibition at 2 micrograms/ml (6.3 X 10(-6) M). No effect on encephalomyocarditis, nor on cellular RNA synthesis is observed even at 20 micrograms/ml. The inhibitory effect of 3MQ is reversible, since cells treated with this compound from the beginning of infection start to synthesize viral RNA and proteins when the compound is removed. Strikingly, other natural compounds structurally related to 3-methylquercetin such as quercetin, naringenin, naringin, morin, catechin, kaempferol, myricetin, phloretin, phlorizdin, and rutin do not block poliovirus replication.

Animals↗