Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 163 records · Page 9Linked to original sources

PubMed1994

Hybrid proteins between Pseudomonas exotoxin A and poliovirus protease 2Apro.

Two hybrid proteins between Pseudomonas aeruginosa exotoxin A (PE) and poliovirus protease 2Apro have been generated. One hybrid protein contains the poliovirus 2Apro sequence replacing the region of PE corresponding to amino acids 413-607. The other hybrid contains in addition the transforming growth factor sequence. The two hybrid proteins were efficiently synthesized in E. coli cells using the inducible pET vectors. Both hybrid toxins cleaved p220 (eIF-4 gamma) when the recombinant plasmids were transfected in COS cells infected with recombinant vaccinia virus bearing the T7 RNA polymerase gene.

ADP Ribose Transferases↗

PubMed1994

Entry of animal viruses and macromolecules into cells.

The entry of animal viruses into cells is mediated by conformational changes in certain virion-particle components. These changes are triggered by the binding of virions to receptors and are influenced by low pH during receptor-mediated endocytosis. These conformational alterations promote the interaction of some viral proteins with cellular membranes thereby leading to transient pore formation and the disruption of ionic and pH gradients. The entry of toxins that do not possess receptors on the cell surface is promoted during the translocation of the virus genome or the nucleocapsid to the cytoplasm. A model is now presented which indicates that efficient virus translocation through cellular membranes requires energy, that may be generated by a protonmotive force. The entry of some animal viruses, as promoted by low pH, should thus only take place when a pH gradient and/or a membrane potential exist, but will not take place if these are dissipated, even if virion particles are present in an acidic environment.

Animals↗

PubMed1994

Concanamycin A blocks influenza virus entry into cells under acidic conditions.

The selective inhibitor of the vacuolar proton-ATPase, concanamycin A, powerfully blocks influenza virus entry into cells, if present during the initial times of virus infection. Attachment of virus particles to cells is not prevented by concanamycin A, rather the exit of influenza virus from endosomes is the step blocked by this macrolide antibiotic. Inhibition of influenza virus entry into cells by concanamycin A or by nigericin takes place under acidic conditions. Moreover, if the pH gradient is abolished by pre-incubation of cells in acidic pH, influenza virus entry does not occur even in the absence of any inhibitors. These results indicate that acidic conditions per se are not sufficient to promote virus entry into cells; rather this step of virus infection requires a pH gradient.

Animals↗

PubMed1994

Concanamycin A: a powerful inhibitor of enveloped animal-virus entry into cells.

Concanamycin A, a selective inhibitor of the vacuolar proton ATPase, blocks the infection of animal cells by vesicular stomatitis virus, Semliki Forest virus and influenza virus even when the drug is present at the low concentration of 5 nM. Nevertheless the antibiotic prevents neither the attachment, to cells, of Semliki Forest virus nor its subsequent internalization. Under certain conditions, described in this communication, virus entry is prevented even when the pH of the medium is low, thus suggesting that a pH gradient, rather than low pH per se, is required to drive the entry, into cells, of these enveloped animal viruses.

Animals↗

PubMed1994

Influenza virus M2 protein modifies membrane permeability in E. coli cells.

The M2 protein of influenza virus is an integral membrane protein with ion channel activity. This protein has been expressed in E. coli cells in an inducible manner. Expression of the M2 protein causes rapid lysis of BL21(DE3) pLysS E. coli cells upon induction with IPTB. M2 protein increases membrane permeability to a number of hydrophylic molecules, such as ONPG, uridine or impermeant translation inhibitors. The behaviour of M2 in bacteria resembles that of other viral proteins, such as poliovirus 3A and Semliki Forest virus 6K.

Cell Membrane Permeability↗

PubMed1994

Semliki Forest virus 6K protein modifies membrane permeability after inducible expression in Escherichia coli cells.

Semliki Forest virus encodes a small protein, known as 6K, that is associated with cellular membranes in the infected cells. This protein has been cloned and expressed in an inducible manner using pET vectors in Escherichia coli cells. Two different plasmids have been utilized; either the 6K gene is placed directly under the T7 promoter (pET3-6K) or the lac operator is located between the T7 promoter and the 6K gene (pET11-6K). In both systems, efficient synthesis of the 6K protein is achieved by induction with isopropyl-1-thio-beta-D-galactopyranoside plus rifampicin. The synthesis of the 6K protein is very toxic for E. coli causing increased membrane permeability and cell lysis as shown by alterations in permeability to either choline or hygromycin B. These results indicate that the togavirus 6K is a membrane-active protein that shows structural and functional similarities to poliovirus 3A protein. The function that the 6K protein could play during the virus replication cycle is discussed in the light of these findings.

Base Sequence↗

PubMed1994

Picornavirus inhibitors.

Picornaviruses are among the best understood animal viruses in molecular terms. A number of important human and animal pathogens are members of the Picornaviridae family. The genome organization, the different steps of picornavirus growth and numerous compounds that have been reported as inhibitors of picornavirus functions are reviewed. The picornavirus particles and several agents that interact with them have been solved at atomic resolution, leading to computer-assisted drug design. Picornavirus inhibitors are useful in aiding a better understanding of picornavirus biology. In addition, some of them are promising therapeutic agents. Clinical efficacy of agents that bind to picornavirus particles has already been demonstrated.

Animals↗

PubMed1994

Involvement of the vacuolar H(+)-ATPase in animal virus entry.

Semliki Forest virus (SFV) enters cells by receptor-mediated endocytosis, followed by acidification of endosomes by the action of the vacuolar H(+)-ATPase. Fusion of the viral and the endosomal membrane delivers the viral genome to the cytoplasm. Direct blockade of the vacuolar H(+)-ATPase by the selective inhibitor bafilomycin A1 (BFLA1) prevented the infection of cells by SFV, if the compound was present during the first minutes of infection. Attachment and penetration of virus particles were not the targets of the antibiotic. BFLA1 and the ionophore monensin potently blocked SFV infection even at low pH, indicating that acidic pH is not sufficient for SFV to deliver its genome to the cytoplasm, but the proper functioning of the H(+)-ATPase pump is necessary. Other enveloped RNA-containing viruses, such as vesicular stomatitis virus or influenza virus were also blocked by BFLA1, whereas no effect was observed with Sendai virus, which enters into cells by direct fusion with the plasma membrane. Enveloped DNA-containing viruses, such as herpes-viruses and vaccinia virus, infected the cells even when the vacuolar H(+)-ATPase was inhibited by BFLA1; similar behaviour was observed with poliovirus and adenovirus. Animal virus particles promote the internalization of proteins and other macromolecules during entry. BFLA1 blocked co-entry of the toxin alpha-sarcin when induced by SFV, but not when induced by Sendai virus. The inhibition of the enzyme responsible for acidification of endosomes by means of the potent inhibitor BFLA1 constitutes a selective and powerful tool to analyse the low-pH dependent mechanism(s) during virus entry and will aid in understanding the mechanisms and routes of entry of animal viruses into cells.

Amantadine↗

PubMed1994

Action of brefeldin A on translation in Semliki Forest virus-infected HeLa cells and cells doubly infected with poliovirus.

Brefeldin A (BFA) is a macrolide antibiotic that blocks membrane traffic through the vesicular system and affects the glycosylation of viral glycoproteins. Treatment of HeLa cells infected with Semliki Forest virus (SFV) with BFA enhances the synthesis of late viral proteins. Proteolytic cleavage of p107 is partially blocked and viral glycoproteins accumulate in BFA-treated cells. This enhanced synthesis of late SFV proteins is due, at least in part, to an increase in the formation of the subgenomic SFV 26S mRNA. Since BFA blocks the replication of poliovirus genomes without affecting the cleavage of the translation initiation factor p220, protein synthesis was analysed in doubly infected cells. HeLa cells infected with SFV and poliovirus at the same multiplicity predominantly synthesize poliovirus proteins. But if these cells are treated with BFA they synthesize significant amounts of SFV capsid protein C for several hours, despite the fact that p220 has been degraded.

Blotting, Northern↗

PubMed1994

Immunohistochemical distribution of vimentin, desmin, glial fibrillary acidic protein and neurofilament proteins in feline tissues.

The immunohistochemical distribution pattern of vimentin, desmin, glial fibrillary acidic protein and neurofilaments intermediate filament proteins has been analyzed in a wide range of formalin fixed, paraffin embedded tissues using polyclonal and monoclonal antibodies raised against non-feline antigens. The vimentin antibody reacted with mesenchymal cells, the desmin antibody with striated and smooth muscle fibres, the glial fibrillary acidic protein antibody with glial cells in the central and peripheral nervous system, and the neurofilament proteins antibody with neuronal cell bodies and processes. In addition, some epithelial cells were vimentin positive, perisinusoidal liver cells were desmin positive, and basal/myoepithelial cells of the mammary gland, and luteinic cells were glial fibrillary acidic protein positive. These staining patterns of feline tissues are basically similar with respect to that of corresponding tissues in other mammalian species for each of the four intermediate filament proteins studied, but some differences have been also noticed. This study confirms the broad interspecies cross-reactivity of intermediate filament proteins antisera and demonstrates their capability to differentiate particular types of feline cells and tissues.

Animals↗

PubMed1994

Nonradioactive northwestern analysis using biotinylated riboprobes.

A nonradioactive modification of the Northwestern assay is described and applied to the detection of RNA-binding proteins. The nonradioactive assay is based on the use of biotinylated riboprobes, which are stable and easy to handle. Chemiluminescence is generated with streptavidin-conjugated peroxidase and provides even better sensitivity than the radioactive detection method to assay RNA binding to proteins bound to nitrocellulose.

Biotin↗

PubMed1994

Morphology and changes in Clara cells in the foetal bronchioles of Swiss mice.

In this work we have studied the morphology and evolution of Clara cells in the bronchiolar mucosa of lungs from 63 Swiss mice foetuses that were classified into three groups according to age (14, 16 and 18 days). A control group composed of 21 15-day-old Swiss mice was also studied. The most salient feature of the Clara cells observed was the occurrence of two types of secretory granules and a large smooth endoplasmic reticulum. On the other hand, the Clara cells of the control group had a single secretory granule. Clara cells thus seem to take part in bronchiolar metabolism, as they were quite abundant in the early foetal groups and diminished as birth approached. This cell decrease was confirmed by the control group (15-day-old mice), the bronchioles of which contained scant cells and numerous ciliated cells.

Animals↗

PubMed1993

Brefeldin A blocks protein glycosylation and RNA replication of vesicular stomatitis virus.

Brefeldin A is a macrolide antibiotic that interferes with membrane traffic and blocks the growth of several animal viruses including vesicular stomatitis virus (VSV). The inhibition of VSV by brefeldin A takes place at least at two different steps during the growth cycle: the glycosylation of VSV G protein and the replication of viral genomes. Our results indicate that interference with membrane traffic leads not only to inhibition of viral protein glycosylation, but also to the blockade of virus genome replication in several cytoplasmic RNA-containing viruses.

Animals↗

PubMed1993

Poliovirus protein 2C has ATPase and GTPase activities.

Poliovirus protein 2C belongs to an expanding group of proteins containing a nucleotide binding motif in their sequence. We present evidence that poliovirus 2C has nucleoside triphosphatase (NTPase) activity and binds to RNA. Poliovirus 2C was expressed in Escherichia coli cells as a fusion protein with the maltose binding protein (MBP). The fusion protein MBP-2C is efficiently cut by protease Xa within the 2C region. Thus, the fusion protein as such was used to assay for the putative activities of poliovirus 2C. Deletion mutants were constructed which lacked different portions of the 2C carboxyl terminus: mutant 2C delta 1 lacked the last 169 amino acids, whereas mutant 2C delta 2 had the last 74 amino acids deleted. The fusion proteins MBP-2C, MBP-2BC, and the mutant MBP-2C delta 2 that contained the first 255 amino acids of 2C had NTPase activity. Both ATPase and GTPase activities are inhibited by antibodies directed against the MBP-2C protein. Analysis of the ability of the different proteins to bind to labeled RNA indicates that MBP-2C and MBP-2BC form a complex, whereas none of the mutants interacted with RNA, indicating that the RNA binding domain lies beyond amino acid 255. None of the fusion proteins had detectable helicase activity. We suggest that poliovirus protein 2C shows similarities to the GTPases group involved in vesicular traffic and transports the viral RNA replication complexes. These results provide the first experimental evidence that poliovirus protein 2C is an NTPase and that this protein has affinity for nucleic acids.

ATP-Binding Cassette Transporters↗

PubMed1993

Vagal system involvement in changes in small bowel motility during restraint stress: an experimental study in the dog.

Changes in small intestinal motility were studied during restraint stress in fasting animals and after food; the involvement of the vagal system in the pathophysiology of these changes was assessed. Small intestinal motility was recorded in 40 dogs using electromyography with monopolar electrodes and manometry with submucosal microballoons. Twenty animals underwent bilateral truncal vagotomy. Stress increased intestinal motility (percentage of slow waves followed by action potentials in 1 min), both in fasting conditions (P < 0.001) and post-prandially (P < 0.001). It also completely abolished the migrating motor complex-interdigestive myoelectric complex periodicity characteristic of the normal fasting pattern. In dogs subjected to vagotomy, restraint stress increased motility during fasting (P < 0.001) but decreased it after feeding (P < 0.01). Restraint stress thus increases small intestinal motility, both during fasting and after food. The vagal system is partially involved in this hypermotility response.

Animals↗

PubMed1993

Activation of phospholipase activity during Semliki Forest virus infection.

Infection of animal cells by a number of cytolytic viruses leads to increased membrane permeability. Thus, Semliki Forest virus (SFV) infection of susceptible cells modifies the permeability of the membrane for a number of cations and metabolites (Muñoz et al. (1985), Virology 146, 203-212). The molecular basis of this modification of the cell membrane has not been investigated in detail. We report that during the infection of HeLa cells with SFV, or BHK cells with vesicular stomatitis virus, there is a significant increase in the release of choline and arachidonic acid into the culture medium, suggesting that both phospholipases (PLases) C and A2 become activated during infection. Both choline and phosphorylcholine are released into the medium as expected when PLase C is activated. Cells prelabeled with arachidonic acid release a significant amount of radioactivity from the third hour postinfection. Most of this radioactivity is present in the medium of SFV-infected cells in the form of free fatty acid, suggesting that phospholipid hydrolysis has occurred; no intact phospholipids are detected in the culture medium. Finally, the action of several inhibitors of PLases, such as zinc and cadmium ions, chloroquine, chlorpromazine, amantadine, and dansylcadaverine were assayed. Our findings indicate that the release of choline or arachidonic acid is potently blocked by some of these lipase inhibitors. Following infection by SFV HeLa cells become susceptible to the inhibition of protein synthesis by hygromycin B due to increased uptake of this antibiotic. Entry of hygromycin B was prevented by zinc ions or chloroquine, suggesting that the increase in membrane permeability in SFV-infected cells may be mediated in part by lipase activation.

Animals↗

PubMed1993

High level expression in Escherichia coli cells and purification of poliovirus protein 2Apro.

The poliovirus protease 2Apro has been produced to high levels in Escherichia coli using the inducible system that utilizes T7 RNA polymerase. The protease coding sequences that contained an additional AUG to start translation were cloned in pET vectors. Synthesis of 2Apro was induced by IPTG or IPTG plus rifampicin, the levels of the protein made being higher when IPTG alone was used. The expression of the protein is not toxic for E. coli cells and can be readily visualized by Coomassie blue staining of total bacterial protein extracts separated in polyacrylamide gels. Centrifugation of the broken bacterial cells sediments more than 95% of the 2Apro synthesized at a 95% purity level after sarkosyl treatment. Antibodies raised against 2Apro in E. coli recognize a 16K protein in poliovirus-infected cells. In addition, 2Apro shows activity in trans as measured by the cleavage of p220 in HeLa cell extracts and by cleavage of a poliovirus protein substrate that contains the junction between the P1 and P2 polypeptides.

Base Sequence↗

PubMed1993

Enhancement of phospholipase activity during poliovirus infection.

Infection of human cells with poliovirus leads to modification of phospholipase activity. Phospholipase C, which generates inositol triphosphate, is stimulated, whereas the activation of phospholipase A2 by the calcium ionophore A23187 is inhibited. Analysis of phospholipid moieties in media of HeLa cells infected with poliovirus indicates that the release of fatty acids is not enhanced during infection, suggesting that phospholipase A1 and A2 activities are not stimulated. The release of choline into the medium is significantly higher 3 h after infection, indicating that a phospholipase that has phosphatidylcholine as its substrate becomes activated. This activation requires viral gene expression because inhibitors of poliovirus gene expression added at the beginning of infection block choline release, but continuous viral protein synthesis is not required. Choline and phosphorylcholine are released into the medium, but the pools of both are gradually depleted in poliovirus-infected cells, perhaps as a consequence of their release into the medium and the increased synthesis of phospholipids that takes place in poliovirus-infected cells. Inhibitors of phospholipase activity such as mepacrine, zinc or cadmium ions significantly reduce this increased release of choline from poliovirus-infected cells. Labelling of cells with [3H]phosphatidylcholine suggests that the choline released from infected cells comes, at least in part, from the hydrolysis of this compound. These results indicate that, in addition to the activation of the phospholipase C which hydrolyses phosphatidylinositol in poliovirus-infected cells, a phospholipase C that acts on a phosphatidylcholine is also activated.

Cadmium↗