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Biomedical subjects

L Carrasco

Publications and source records attributed to L Carrasco.

At least 127 records · Page 7Linked to original sources
PubMed1996

Pneumonic pasteurellosis associated with Pasteurella haemolytica in chipmunks (Tamias sibiricus).

A laboratory study was performed in order to identify the possible cause of death in chipmunks (Tamias sibiricus) imported from China with respiratory disease. Severe congestion, alveolar oedema and fibrinous pleuritis were observed. Biochemical analyses identified the causative organism as Pasteurella haemolytica. An in vitro susceptibility test using various antimicrobial agents revealed sensitivity to beta-lactams (ampicillin and amoxicillin) and streptomycin.

Amoxicillin↗
PubMed1996

The entry of reovirus into L cells is dependent on vacuolar proton-ATPase activity.

Inhibitors of vacuolar proton-ATPase activity (5 microM bafilomycin A1 or 50 nM concanamycin A) prevented infection by reovirus particles but not by infectious subviral particles (ISVPs). Neither compound affected virus attachment or internalization. However, both compounds potently blocked cleavage of the viral protein mu 1C. Finally, both reovirus particles and ISVPs efficiently translocated the toxin alpha-sarcin to the cytosol during virus entry. Bafilomycin A1 blocked translocation of alpha-sarcin by reovirus particles but not by ISVPs.

Animals↗
PubMed1996

Hybrid proteins between Pseudomonas aeruginosa exotoxin A and poliovirus 2Apro cleave p220 in HeLa cells.

Cleavage of p220, a component of the initiation factor eIF-4F, has been correlated with the inhibition of host translation during poliovirus infection. To obtain p220 cleavage in the absence of any other poliovirus gene products, hybrid proteins containing Pseudomonas aeruginosa exotoxin A and poliovirus protease 2Apro have been constructed. The addition of the hybrid molecules to cultured cells did not lead to substantial p220 cleavage. However, the simultaneous presence of the hybrid toxin with replicationally inactive chicken adenovirus particles results in efficient cleavage of p220 in the intact cells. Under these conditions, cellular translation continues unabated for several hours, arguing against a direct requirement for intact p220 in each round of the initiation of translation of cellular mRNAs.

ADP Ribose Transferases↗
PubMed1996

Immunohistochemical characterization of hemangiopericytomas and other spindle cell tumors in the dog.

The immunohistochemical expression of muscle actin has been studied in 45 canine hemangiopericytomas (CHP) using a monoclonal antibody (HHF35) and formalin-fixed, paraffin-embedded specimens. The distribution of vimentin, desmin, cytokeratins, lysozyme, factor VIII-related antigen, S-100 protein, and glial fibrillary acidic protein was studied both in CHP and in some canine soft-tissue neoplasms (seven fibrosarcomas, seven benign schwannomas, seven benign fibrous histiocytomas, and six leiomyosarcomas) used as controls for differential diagnosis. All CHP and control tumors expressed vimentin. Twenty-three CHP expressed muscle actin, whereas all control tumors analyzed were muscle actin-negative, with the exception of leiomyosarcomas. Among muscle actin- and vimentin-positive CHP, one case could be reclassified as leiomyosarcoma because it was desmin-positive, two cases expressed lysozyme, and nine cases expressed S-100 protein. Among muscle actin-negative and vimentin-positive CHP, seven expressed S-100 protein. In addition, S-100 protein was detected in five schwannomas. All CHP and control tumors analyzed were negative for cytokeratins, factor VIII-related antigen, and glial fibrillary acidic protein. Our results support the hypothesis of a pericytic origin of CHP, and suggest that muscle actin, desmin, vimentin, and lysozyme could be useful for the differential diagnosis of canine spindle cell tumors, but not all these neoplasms can be identified with these tumor tissue markers.

Actins↗
PubMed1996

In vivo replication of African swine fever virus (Malawi '83) in neutrophils.

The presence of virus replication centers in neutrophils from pigs inoculated with a highly virulent strain of African swine fever virus is described for the first time in vivo. Virus antigens were observed from 3 days post-inoculation (dpi) onwards by means of an immunohistochemical technique. At this time (3 dpi), transmission electron microscopy studies revealed the presence of large amounts of neutrophils in the vascular lumens. At 5 and 7 dpi, neutrophils with phagosomes frequently contained virus particles. In addition, within the cytoplasm of some mature and immature neutrophils, both viral particles and virus replication centers were observed at 5 and 7 dpi.

African Swine Fever↗
PubMed1996

Pulmonary intravascular macrophages in deer.

Pulmonary intravascular macrophages (PIMs) have been found in the septal capillaries of deer lungs. Lung samples from adult deer were fixed in 2.5% glutaraldehyde, and then routinely processed for electron microscopy. The main features of the PIMs were the presence of tubular invaginations in the membrane (micropinocytosis vermiformis), phagosomes, and junctions with endothelial cells. A mean of 4.4 of these junctions was recorded per cell. They comprised segments ranging from 67 to 289 nm in length, where the plasma membranes were separated by spaces from 10 to 25 nm wide. In these areas the cytoplasm underlying the membranes showed evidence of increased electron density. When PIMs were compared with alveolar macrophages, it could be seen that although the PIMs were more numerous (more than twice), they were also smaller than the alveolar macrophages (47.625 versus 101.260 microns2 respectively.

Animals↗
PubMed1996

[Etiologic and pathologic study of respiratory disease in lambs from intensive breeding facilities in southern Spain].

Between 1991 and 1993, it was observed epidemiologically that respiratory disturbances in lambs are associated with high temperatures during the summer. The etiological agent isolated is principally Mycoplasma ovipneumoniae; moreover, Actinobacillus pleuropneumoniae biovar A has been isolated in a high number of samples. Histopathologically, an interstitial bronchopneumonia was the main lesional finding; this lesion is associated with previous mycoplasma infection.

Animal Husbandry↗
PubMed1996

Virus association with lymphocytes in acute African swine fever.

This paper reports the presence of mature viral particles within the lymphocytes of samples taken from pigs inoculated with a highly virulent African swine fever (ASF) virus isolate (Malawi 83), and the adhesion of the lymphocytes to macrophages containing the virus replication sites. Virus replication in lymph-node medullar tissue macrophages was observed from 3 days post inoculation (pi). At 3 days pi, transmission electron microscopy revealed hemadsorption in some infected macrophages. At 5 and 7 days pi, a number of macrophages with virus replication were surrounded by a ring of lymphocytes. In such cases, mature viral particles were observed in membrane evaginations of the infected cell that corresponded to invaginations of the lymphocyte membrane. Also at 5 and 7 days pi, mature virions were observed within the cytoplasm of some lymphocytes. However, incomplete virions and African swine fever virus replication sites were not observed within the lymphocytes.

African Swine Fever↗
PubMed1995

Effects of poliovirus 2A(pro) on vaccinia virus gene expression.

The effects of transient expression of poliovirus 2A(pro) on p220 cleavage in COS cells have been analyzed. When 2A(pro) was cloned in plasmid pTM1 and transiently expressed in COS cells, efficient cleavage of p220 occurred after infection of these cells with a recombinant vaccinia virus bearing phage T7 RNA polymerase. High numbers of COS cells were transfected with pTM1-2A, as judged by p220 cleavage, thereby allowing an analysis of the effects of poliovirus 2A(pro) on vaccinia virus gene expression. A 40-50% cleavage of p220 by transfected poliovirus 2A(pro) was observed ten hours post infection and cleavage was almost complete (80-90%) 20-25 hours post infection with vaccinia virus. Profound inhibition of vaccinia virus protein synthesis was detectable ten hours post infection and was maximal 20-25 hours post infection. This inhibition resulted from neither a blockade of transcription of vaccinia virus nor a lack of translatability of the mRNAs present in cells that synthesize poliovirus 2A(pro). Addition of ara-C inhibited the replication of vaccinia virus and allowed the continued synthesis of cellular proteins. Under these conditions, 2A(pro) is expressed and blocks cellular translation. Finally, p220 cleavage by 2A(pro) did not inhibit the translation of a mRNA encoding poliovirus protein 2C, as directed by the 5' leader sequences of encephalomiocarditis virus. Therefore, these findings show a correlation between p220 cleavage and inhibition of translation from newly made mRNAs. Our results are discussed in the light of present knowledge of p220 function, and new approaches are considered that might provide further insights into the function(s) of initiation factor eIF-4F.

Amino Acid Sequence↗
PubMed1995

Expression of poliovirus 2Apro in mammalian cells: effects on translation.

Poliovirus protease 2Apro has been efficiently expressed in HeLa and COS cells upon transfection with vector pTM1-2A and infection with the recombinant vaccinia virus bearing the T7 RNA polymerase. The expressed poliovirus protease localizes to the cytoplasm of the transfected cells, both in the endoplasmic reticulum and in vesicles scattered in the cytoplasm. Cleavage of p220, a component of initiation factor eIF-4F, selectively occurs from 5 h post-infection in transfected cells infected with the recombinant virus. This cleavage correlates in time with the profound inhibition observed in the synthesis of vaccinia virus proteins. A similar blockade of vesicular stomatitis virus translation takes place upon 2Apro expression. Finally, the synthesis of poliovirus protein 2C from a recombinant vaccinia virus that expresses this protein under the EMC untranslated leader region is not affected by the synthesis of 2Apro. These findings lend support to the idea that translation of capped mRNAs requires the integrity of p220, while this requirement is not observed when translation of a mRNA bearing a picornavirus leader region is assayed.

Animals↗
PubMed1995

Efficient cleavage of p220 by poliovirus 2Apro expression in mammalian cells: effects on vaccinia virus.

Poliovirus protease 2A cleaves p220, a component of initiation factor eIF-4F. Polyclonal antibodies that recognize p220 and the cleaved products from different species have been raised. Transfection of several cell lines with poliovirus 2Apro cloned in different plasmids leads to efficient cleavage of p220 upon infection with VT7, a recombinant vaccinia virus that expresses the T7 RNA polymerase. Under these conditions vaccinia virus protein synthesis is severely inhibited, while expression of poliovirus protein 2C from a similar plasmid has no effect. These results show by the first time the effects of p220 cleavage on vaccinia virus translation in the infected cells.

Amino Acid Sequence↗
PubMed1995

Poliovirus 2Apro expression inhibits growth of yeast cells.

Poliovirus encodes two proteases, 2Apro and 3Cpro that participate in the processing of the viral polyprotein and cleave a number of host proteins. Both proteases have been cloned and expressed in an inducible manner in Saccharomyces cerevisiae cells. The expression of 2Apro, but not 3Cpro, was highly toxic for yeast cells such that growth was arrested after 5 h of induction and cell survival sharply declined. Cellular morphology was profoundly modified by expression of poliovirus 2Apro, in such a way that electron dense granules and autophagosomic bodies arise in the cytoplasm. Experiments aimed at defining the yeast function affected by 2Apro suggested that translation was not the target of protease toxicity, but showed that RNA synthesis was profoundly blocked.

Cysteine Endopeptidases↗
PubMed1995

A human virus protein, poliovirus protein 2BC, induces membrane proliferation and blocks the exocytic pathway in the yeast Saccharomyces cerevisiae.

Inducible synthesis of poliovirus protein 2BC in Saccharomyces cerevisiae arrests cell growth in the G2 phase of the cell cycle, while no effects are observed upon expression of poliovirus genes 2B or 2C, either individually or in combination. Expression of 2BC induces a number of morphological modifications in yeast cells, one of the most striking being the proliferation of small membranous vesicles that fill most of the cytoplasm. These vesicles are morphologically similar to the cytopathic vacuoles that proliferate during the infection of human cells by poliovirus. The transport and processing of several yeast proteins, including vacuolar carboxypeptidase Y, aminopeptidase I or yeast alpha-mating factor, is hampered upon expression of poliovirus 2BC, suggesting that transport of proteins through the Golgi apparatus is impaired by this viral protein. Finally, a number of 2BC variants were generated and the effects of their expression on yeast growth, cellular morphology and protein processing were analyzed. 2BC variants defective in the NTPase activity were still able to interfere with yeast growth and the exocytic system, while deletion of 30 amino acids at the N-terminus of 2BC impairs its function. These findings lend support to the idea that 2BC, but not 2B or 2C, is the protein responsible for vesicle proliferation in poliovirus-infected cells. In addition, the activity of a human virus protein in yeast cells opens new avenues to investigate the exact location at which poliovirus 2BC interferes with the vesicular system and to test the action of other animal virus proteins potentially involved in modifying the vesicular system in mammalian cells.

Base Sequence↗
PubMed1995

Mutations in the hydrophobic domain of poliovirus protein 3AB abrogate its permeabilizing activity.

Poliovirus protein 3AB contains a predicted amphipathic helix that could lead to pore formation in membranes. We have introduced various mutations in the hydrophobic domain of the protein and the membrane-modifying properties of the resulting mutants have been analyzed. Expression of wild type 3AB protein in E. coli increases the influx and efflux of different molecules such as nucleosides, lactose analogues and antibiotics. Thus, 3AB expression makes E. coli cells two orders of magnitude more sensitive to hygromycin B, a non-permeant inhibitor of translation, and causes a 15-20-fold enhancement in the efflux of uridine. Changes in membrane permeability take place under conditions where no cellular lysis is detected and when other molecules such as beta-galactosidase or polyribonucleotides are kept inside the cell. These membrane modifications can be blocked to different extents by amino acid substitutions in the membrane-spanning region of the protein. These results suggest that poliovirus protein 3AB could possess an intrinsic ability to form pores in natural membranes, thus allowing the flux of small hydrophylic molecules through them.

Amino Acid Sequence↗
PubMed1995

Poliovirus protein 2C contains two regions involved in RNA binding activity.

Poliovirus protein 2C is involved in poliovirus RNA replication, although the exact function of 2C is still unknown. Recently, it was shown that 2C can be purified to high levels when expressed as a fusion protein with maltose-binding protein (MBP). Evidence was presented that 2C has ATPase and GTPase activities; preliminary results also indicated that 2C interacts with RNA (Rodríguez, P.L., and Carrasco, L. (1993) J. Biol. Chem. 268, 8105-8110). In the present study, 20 variants of 2C have been generated, and their NTPase and RNA binding activities were analyzed. Moreover, an easy procedure to obtain genuine 2C after factor Xa cleavage of an MBP2-2C fusion protein is described. This work has determined that 2C has two regions involved in RNA binding: a NH2-terminal region located between amino acids 21 and 45 and a COOH-terminal region involving an Arg-rich region located between amino acids 312 and 319. Deletion of either the NH2- or COOH-terminal RNA-binding region abolishes RNA binding. Deletion of an internal region of protein 2C that includes the nucleotide-binding motif does not affect RNA binding, whereas this deletion destroys ATPase and GTPase activities. Therefore, the NTPase activity and the RNA binding capacity of protein 2C are located in different regions of the molecule.

Amino Acid Sequence↗