Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 181 records · Page 10Linked to original sources

PubMed1993

Entry of poliovirus into cells does not require a low-pH step.

The requirement of a low-pH step during poliovirus entry was investigated by using the macrolide antibiotic bafilomycin A1, which is a powerful and selective inhibitor of the vacuolar proton-ATPases. Thus, viruses such as Semliki Forest virus and vesicular stomatitis virus that enter cells through endosomes and need their acidification, are potently inhibited by bafilomycin A1, whereas poliovirus infection is not affected by the antibiotic. The presence of lysosomotropic agents such as chloroquine, amantadine, dansylcadaverine, and monensin during poliovirus entry did not inhibit infection, further supporting the idea that poliovirus does not depend on a low-pH step to enter the cytoplasm. The effect of bafilomycin A1 on other members of the Picornaviridae family was also assayed. Encephalomyocarditis virus entry into HeLa cells was not affected by the macrolide antibiotic, whereas rhinovirus was sensitive. Coentry of toxins, such as alpha-sarcin, with viral particles was potently inhibited by bafilomycin A1, indicating that an active vacuolar proton-ATPase is necessary for the early membrane permeabilization (coentry of alpha-sarcin) induced by poliovirus to take place.

Amantadine↗

PubMed1993

HLA molecule expression in cutaneous squamous cell carcinomas: an immunopathological study and clinical-immunohistopathological correlations.

A correlation between the lack of MHC class I gene expression on murine tumor cells and their ability to grow and metastasize has recently been established. This paper studies HLA-ABC and HLA-DR antigen expression in tumor cells and mononuclear infiltrate of 26 cutaneous squamous cell carcinomas (SCC). Our results showed a heterogeneous expression of HLA class I molecules in these tumors. No significant correlation between the degree of HLA class I molecule expression and anatomical-clinical parameters was found. Class II antigen expression was correlated to the histopathological type and to the degree of cell differentiation. Most mononuclear cells infiltrating the tumor were T-lymphocytes. No correlation with anatomical-clinical parameters was found.

Antigens, CD↗

PubMed1992

Inducible expression of a toxic poliovirus membrane protein in Escherichia coli: comparative studies using different expression systems based on T7 promoters.

The poliovirus 3AB gene has been cloned and overproduced in T7 expression vectors using different approaches to allow reduction of basal levels of expression. Expression of the poliovirus 3AB gene is highly toxic for E. coli cells, due to drastic changes induced in membrane permeability of the bacteria that lead to cell lysis when the T7 lysozyme is present. The best production of 3AB was achieved with the T7/lac system in cells lacking T7 lysozyme, where this toxic protein was synthesized to high levels and during several hours in the absence of cell lysis. These results show the efficient synthesis of a highly damaging membrane protein and open the possibility to apply heterologous gene expression in E. coli to other lytic proteins.

Bacteriophage T7↗

PubMed1992

Cloning and inducible synthesis of poliovirus nonstructural proteins.

The poliovirus nonstructural protein-encoding genes have been cloned and expressed in Escherichia coli using the inducible system described by Studier and Moffat [J. Mol. Biol. 189 (1986) 113-130] and Studier [J. Mol. Biol. 219 (1991) 37-44]. The two genes encoding the poliovirus proteases, 2Apro and 3Cpro, were cloned together with their flanking regions in order to test the ability of the polyprotein precursors synthesized to cause proteolytic cleavage and generate mature forms. Both proteases were synthesized and showed activity upon induction in this system. Previously, it had not been possible to produce the three poliovirus nonstructural proteins, 2B, 2C and 3A, and some of their precursors, 2C3AB, 2C3A and 3AB, at high levels in E. coli cells. We report the cloning of their genes using PCR techniques and their efficient expression from pET vectors upon induction with IPTG (isopropyl-beta-D-thiogalactopyranoside). Moreover, some of these proteins, e.g., 3AB, 3A and 2B, are quite toxic for E. coli cells and lysed them upon production. Our results demonstrate the usefulness of this inducible system using the pET vectors to express these toxic poliovirus proteins.

3C Viral Proteases↗

PubMed1992

Expression of poliovirus nonstructural proteins in Escherichia coli cells. Modification of membrane permeability induced by 2B and 3A.

The poliovirus nonstructural proteins 2B, 2C, 2C3A, 2C3AB, 3A, and 3AB have been cloned and efficiently expressed in Escherichia coli cells. Each individual protein, or combinations of some of them, were cloned using polymerase chain reaction techniques and correspond to the genuine poliovirus protein plus an additional methionine. The system used to express them uses pET vectors containing the promoter of gene 10 of phage T7. Expression of protein 2C in BL21 (DE3) pLysS cells, which express the T7 lysozyme, is not toxic, and the bacteria synthesize this protein for several hours after induction. In contrast, the expression of proteins 2B, 3A, or 3AB is not tolerated by BL21 (DE3) pLysS cells which could make them only for a limited period of time. Protein 3AB was particularly toxic and induced a rapid lysis of the recombinant clone after its induction with isopropyl-1-thio-beta-D-galactopyranoside alone or with both isopropyl-1-thio-beta-D-galactopyranoside and rifampicin. Further analyses showed that 3AB induced profound modifications in membrane permeability to o-nitrophenyl-beta-D-galactopyranoside, labeled uridine, and nonpermeant translation inhibitors. Cloning and expression of proteins 2B, 3A, and 3AB in BL21 (DE3) cells that do not contain the T7 lysozyme lead to a more sustained expression of these proteins without detectable cell lysis. Changes in permeability to low molecular weight compounds such as radioactive uridine, o-nitrophenyl-beta-D-galactopyranoside, and hygromycin B readily appeared upon induction of 2B, 3A, and 3AB. Our results indicate that the poliovirus nonstructural polypeptides 2B and 3A (or 3AB) are lytic for the bacteria. In fact, both proteins 2B and 3A contain hydrophobic domains in a potential amphipathic helix; this is one characteristic shared with a number of membrane-active peptides.

Autoradiography↗

PubMed1992

Localization of African swine fever viral antigen, swine IgM, IgG and C1q in lung and liver tissues of experimentally infected pigs.

An immunohistological study was carried out on lungs and livers of pigs experimentally infected with two different African swine fever virus (ASFV) isolates. ASFV antigen, swine immunoglobulins (IgM and IgG) and (Clq) complement were demonstrated in both organs at different stages of infection. The ASFV antigen was mainly found in mononuclear phagocytic system (MPS) cells. Immunoglobulins and complement were observed in plasma, infected and non-infected phagocytic cells and cell debris. These findings suggest the presence, in acute infection, of immune complexes which may be involved in immunopathogenic mechanisms.

African Swine Fever↗

PubMed1992

Involvement of membrane traffic in the replication of poliovirus genomes: effects of brefeldin A.

Brefeldin A (BFA) is a macrolide antibiotic that has multiple targets in vesicular transport and blocks membrane traffic between the cis- and trans-Golgi compartments, leading to the disruption of the trans-Golgi apparatus (for a review see Pelham, 1991, Cell 67, 449-451). Consequently, BFA interferes with the maturation of viral glycoproteins and suppresses the formation of infectious viruses that contain a lipid envelope. We report that this antibiotic strongly inhibits poliovirus replication even though this virus lacks a lipid envelope and does not encode any glycoproteins. Addition of BFA from the beginning of poliovirus infection blocks the synthesis of late proteins but has no effect on p220 cleavage, indicating that the input viral RNA is translated to produce active 2Apro. The presence of BFA at later times has no effect on poliovirus protein synthesis, indicating that this step is not a direct target for the antibiotic. Indeed, the target of BFA is viral RNA synthesis, because addition of the antibiotic at any time after poliovirus infection drastically reduces the incorporation of labeled uridine into poliovirus RNA. Both plus- and minus-stranded RNA syntheses are diminished when BFA is present from the beginning of infection, but plus-stranded RNA synthesis is more affected when the inhibitor is added at later times. The replication of poliovirus RNA takes place in close association with membrane vesicles that fill the cytoplasm of the infected cells. Little is known about the origin and function of these vesicles that form part of the viral replication complexes. Our findings suggest that the replication of poliovirus genomes may require the maturation of membranous vesicles from a vesicular compartment that is affected by BFA. The effects of BFA on late protein synthesis by other animal viruses varies according to the virus species examined. Among picornaviruses, rhinoviruses are sensitive to the antibiotic, whereas encephalomyocarditis virus is resistant. A negative-stranded RNA virus such as vesicular stomatitis is blocked by BFA, whereas vaccinia virus, a cytoplasmic DNA virus, is resistant.

Antiviral Agents↗

PubMed1992

Lack of direct correlation between p220 cleavage and the shut-off of host translation after poliovirus infection.

Poliovirus induces a drastic inhibition of host protein synthesis soon after infection of susceptible cells. The correlation between this inhibition and the cleavage of p220, a polypeptide that forms part of protein synthesis initiation factor elF-4F, has been examined in detail. Measurements of protein synthesis at half-hourly intervals after infection with poliovirus show the lack of direct correlation between p220 cleavage and the blockade of cellular translation. Moreover, the use of inhibitors of poliovirus RNA synthesis helped to dissociate those two events more clearly. Thus, in the presence of guanidine or Ro 09-0179 when little shut-off was induced by poliovirus extensive proteolytic degradation of p220 took place. When HeLa cells infected with poliovirus are placed at 28 degrees the inhibition of host protein synthesis is prevented and cellular translation continues for at least 8 hr, albeit at a reduced level compared to cells incubated at 37 degrees. At 28 degrees, cleavage of p220 is observed and about 80% of p220 is degraded after 6 hr of incubation at that temperature. Strikingly, when cells in which more than 50% of p220 is cleaved are shifted to 37 degrees, cellular translation recuperates to 100%, in spite of the fact that no detectable p220 is present. Furthermore, if poliovirus-infected cells are incubated for 2 hr at 37 degrees to permit the cleavage of p220 and then are shifted to 28 degrees in the presence of guanidine, cellular proteins are synthesized at the same level as uninfected HeLa cells incubated at 28 degrees. These results show that translation of cellular mRNAs takes place in cells containing a cleaved p220 and indicate that this cleavage is not directly responsible for the shut-off of host translation induced by poliovirus.

Antiviral Agents↗

PubMed1992

Distribution of ASFV antigens in pig tissues experimentally infected with two different Spanish virus isolates.

Lymph nodes, spleen, liver, lung and kidney obtained from pigs experimentally infected with two African Swine Fever Virus (ASFV) isolates of differing virulence were fixed by perfusion with glutaraldehyde and embedded in paraffin. An immunoperoxidase technique using a polyclonal anti-ASFV serum was performed on tissue sections in order to detect ASFV antigen. The distribution of ASFV antigen in such infected organs is shown and the differences between both infections compared and discussed. Monocytes, macrophages, hepatocytes, endothelial cells, neutrophils and epithelial cells were found to contain ASFV antigens.

African Swine Fever↗

PubMed1992

Gliotoxin: inhibitor of poliovirus RNA synthesis that blocks the viral RNA polymerase 3Dpol.

The mode of action of gliotoxin against poliovirus has been analyzed in detail. This fungal metabolite inhibits the appearance of poliovirus proteins when present from the beginning of infection but has no effect on viral translation when added at late times. In agreement with previous findings, this toxin potently inhibited the incorporation of [3H]uridine into poliovirus RNA soon after its addition to the culture medium. Analysis of the synthesis of poliovirus plus- or minus-stranded RNA in the presence of gliotoxin suggests that this compound effectively hampered both processes. This result contrasts with the mode of action of other inhibitors of poliovirus RNA synthesis, such as guanidine or flavones, that selectively block plus-stranded RNA synthesis and suggests that the target of gliotoxin differs from the target of guanidine, i.e., poliovirus protein 2C. Indeed, gliotoxin was found to be a potent inhibitor of poliovirus RNA synthesis in cell-free systems, using membranous crude replication complexes, a reaction that is not blocked by guanidine or Ro 09-0179. Moreover, in vitro activity of the purified poliovirus polymerase 3Dpol was efficiently inhibited by gliotoxin. These results indicate that this toxin acts on the poliovirus polymerase 3Dpol, providing the first description of an inhibitor of this viral enzyme.

Blotting, Northern↗

PubMed1992

Significant anti-HIV activity of new modified polyanionic polymers in vitro.

The anti-HIV activities of two new polyanionic polymers (AM 242 and AM 612) were investigated in cell culture-based and biochemical antiviral assays. These compounds inhibited the reverse transcriptases from HIV-1 and HIV-2, using enzyme purified from virions and either a ribosomal RNA or gapped duplex DNA as the template. With the ribosomal RNA template, AM 242 and AM 612 had ID50 values of 1.1 and 0.10 micrograms/ml against the HIV-1 reverse transcriptase. In vitro cell based assays determined that both compounds significantly inhibited both the cytopathic effects associated with HIV-1 infection and the replication of virus in infected cells. AM 242 had an IC50 of approximately 1.0 micrograms/ml, while that of AM 612 was 0.19 micrograms/ml. These two active polyanionic polymers were effective in inhibiting the growth of a panel of HIV-1 isolates and were also active against HIV-2. Although the compounds were toxic at high concentration, they had antiviral activity over a wide range of nontoxic concentrations, yielding a high selectivity index. AM 612 was 100% protective for CEM cells from 320 ng/ml to 1 microgram/ml. Both compounds caused a significant increase in cellular proliferation as determined by the concentration-dependent increase in incorporation of radioactive precursors into cellular macromolecules.

Anions↗

PubMed1992

Kupffer cells and PIMs in acute experimental African swine fever.

An ultrastructural study of Kupffer cells and pulmonary intravascular macrophages (PIMs) of healthy and African Swine Fever (ASF)-infected pigs was carried out. A vascular perfusion method was performed in order to obtain an optimal intravascular morphology and tissue fixation. The infection developed acute ASF lesions in both organs. Both Kupffer cells and PIMs were studied at different stages of infection. The differences observed in both macrophagic cells from uninfected and infected tissues are shown and discussed.

African Swine Fever↗

PubMed1992

Detection of African swine fever viral antigens in paraffin-embedded tissues by use of immunohistologic methods and polyclonal antibodies.

Tissues obtained from pigs inoculated with African swine fever virus (ASFV), fixed by vascular perfusion using glutaraldehyde, and embedded in paraffin or araldite were used for an immunohistologic electron microscopic study. To detect ASFV antigens, 4 methods were used on paraffin sections with or without pretreatment of the tissues. Use of biotinylated anti-ASFV antiserum combined with avidin-biotin complex and peroxidase proved to be the most suitable method, and antigen was detected in tissues infected with 2 ASF viruses of different virulence. Use of the glutaraldehyde fixation method should ensure optimal morphologic (structural and ultrastructural) data while allowing an immunohistologic study, and add to knowledge of the pathogenesis of ASF.

African Swine Fever↗

PubMed1991

Cerulenin, an inhibitor of lipid synthesis, blocks vesicular stomatitis virus RNA replication.

The replication of genomes of animal viruses in the cytoplasm of susceptible cells is usually coupled to specialized membrane structures. The inhibitor of lipid synthesis cerulenin blocks the formation of vesicular stomatitis virus polypeptides when added to cells soon after virus entry, but has much less effect on viral translation, or the acylation of the glycoprotein G, when cerulenin is added later during infection. By contrast, cerulenin powerfully blocks viral RNA synthesis or the incorporation of glycerol into lipids when present at any time after VSV-infection. These findings suggest that the synthesis of VSV RNA is dependent on continuous synthesis of lipids.

Cerulenin↗

PubMed1991

Mechanism of inhibition of HSV-1 replication by tumor necrosis factor and interferon gamma.

Tumor necrosis factor (TNF) synergizes with interferon (IFN gamma) in the blockade of HSV-1 replication. Antibodies against IFN beta block this synergism, implying a role of IFN beta in the antiviral activity of TNF plus IFN gamma. IFN beta 1 added exogenously to Hep-2 cells shows antiviral activity against HSV-1 only at high concentrations, whereas IFN beta 2 (also known as IL-6) alone has no effect on the replication of VSV or HSV-1 even when 1,000 U/ml are present. Our results are in accordance with the idea that TNF induces IFN beta 1 and that both cytokines must be present in the culture medium to synergize with IFN gamma in order to inhibit HSV-1 replication.

Animals↗

PubMed1991

Synthesis of Semliki Forest virus RNA requires continuous lipid synthesis.

The involvement of lipid biosynthesis in the replication of Semliki Forest virus (SFV) in HeLa cells has been analyzed by the use of cerulenin, an inhibitor of lipid synthesis. The presence of this agent from the beginning of infection blocks the appearance of viral proteins. However, when the antibiotic is added at later stages of infection it has no effect on protein synthesis, the cleavage of viral proteins and their acylation by palmitic acid. Cerulenin is a powerful inhibitor of viral RNA synthesis, as analyzed by [3H]uridine incorporation, incorporation of [32P]phosphate into viral replication complexes, or Northern blot analysis of viral RNAs hybridized with minus- or plus-stranded riboprobes. Finally, analysis of phospholipids made in SFV-infected cells indicates that viral infection clearly stimulates the synthesis of phosphatidyl choline and modifies the membrane formed as analyzed by sucrose gradient centrifugation. Cerulenin blocks the synthesis of phospholipids and inhibits the formation of new membranes. These results show that, when the synthesis of lipids is blocked by cerulenin, SFV RNA replication is hampered, suggesting that the synthesis of viral RNAs needs continuous lipid synthesis and membrane formation.

Centrifugation, Density Gradient↗

PubMed1991

Effects of fatty acids on lipid synthesis and viral RNA replication in poliovirus-infected cells.

Animal viruses profoundly modify the metabolism of lipids, the synthesis of new membranes, and membrane traffic. These alterations are related to the replication of viral genomes. Addition of oleic acid from the beginning of poliovirus infection inhibits the appearance of virus polypeptides at concentrations that do not affect translation in mock-infected HeLa cells. This inhibition is due to the blockade of viral RNA synthesis. Membranes made in poliovirus-infected cells in the presence of oleic acid differ in their buoyant density from control membranes. These results suggest that the incorporation of oleic acid into membranes leads to increased membrane fluidity and decreased buoyant density, making these membranes nonfunctional for poliovirus RNA replication.

Cell Membrane↗

PubMed1991

Cell type determines the relative proportions of (-) and (+) strand RNA during poliovirus replication.

To examine the influence of the host cell type on poliovirus RNA synthesis we compared the levels of (-) and (+) strand poliovirus RNA during infection of epithelial (HeLa and HEp-2), leukocytic (U-937, HL-60 and K-562) and nerve (IMR-32) cells. The levels of (-) strand RNA were higher in IMR-32, U-937, K-562 or HL-60 cells than those in HeLa or HEp-2 cells. By contrast, (+) strand RNA content was greater in HeLa or HEp-2 cells. Although significant levels of (+) strand RNA were detected in U-937, K-562 and HL-60 cells, no viral protein synthesis was detected by polyacrylamide gel electrophoretic analysis of metabolically labelled proteins. The molar ratio of poliovirus (-) and (+) RNAs was 2-3 fold higher in IMR-32, U-937 and K-562 cells than in HeLa or HL-60 cells and 5-6 fold higher than in HEp-2 cells. Differentiation of HL-60 cells with a variety of inducers produced differential effects on poliovirus (-) and (+) RNA content and modified the molar ratio of (-)/(+) strand RNAs. These findings indicate that host cell components play a critical role in the regulation of the amount of poliovirus (-) and (+) strand RNAs synthesized during infection.

Cell Line↗