Biomedical subjects

L Pasamontes

Publications and source records attributed to L Pasamontes.

23 records · Page 2Linked to original sources

PubMed1990

Structural organization of poliovirus RNA replication is mediated by viral proteins of the P2 genomic region.

Transcriptionally active replication complexes bound to smooth membrane vesicles were isolated from poliovirus-infected cells. In electron microscopic, negatively stained preparations, the replication complex appeared as an irregularly shaped, oblong structure attached to several virus-induced vesicles of a rosettelike arrangement. Electron microscopic immunocytochemistry of such preparations demonstrated that the poliovirus replication complex contains the proteins coded by the P2 genomic region (P2 proteins) in a membrane-associated form. In addition, the P2 proteins are also associated with viral RNA, and they can be cross-linked to viral RNA by UV irradiation. Guanidine hydrochloride prevented the P2 proteins from becoming membrane bound but did not change their association with viral RNA. The findings allow the conclusion that the protein 2C or 2C-containing precursor(s) is responsible for the attachment of the viral RNA to the vesicular membrane and for the spatial organization of the replication complex necessary for its proper functioning in viral transcription. A model for the structure of the viral replication complex and for the function of the 2C-containing P2 protein(s) and the vesicular membranes is proposed.

Antibodies, Monoclonal↗

PubMed1987

Association of polioviral proteins of the P2 genomic region with the viral replication complex and virus-induced membrane synthesis as visualized by electron microscopic immunocytochemistry and autoradiography.

Using high resolution electron microscopic autoradiography and immunocytochemistry with monoclonal antibodies against poliovirus proteins of the P2 genomic region, the location of these proteins in respect to the virus-induced vesicle formation and the viral RNA synthesis was followed during the viral replication cycle. It was found that P2 proteins become rER associated soon after their synthesis. At the site of protein and rER interaction, electron-dense patches appear. Simultaneously, membrane protrusions grow and form vesicles which finally budd off, carrying the patches on their outer surface. As shown by autoradiography, these patches are the site of viral RNA replication and, therefore, they represent the poliovirus replication complex. The vesicles with the replication complex, including replicating and replicated viral RNA, move away from the rER to form a continuously growing vesiculated area in the center of the infected cell, where virus maturation takes place. A likely function of the 2C protein is to attach the replication complex, or some of its components, to the vesicular membranes.

Antibodies, Monoclonal↗

PubMed1986

Production of monoclonal and monospecific antibodies against non-capsid proteins of poliovirus.

Non-capsid poliovirus proteins of the P2 region in extracts of infected cells were solubilized by SDS, separated by PAGE, electroeluted from the gels and used to immunize mice. The sera obtained were rendered monospecific by extensive absorption with proteins from uninfected cell extracts, and the spleen cells of these animals were used to establish antibody-secreting hybridomas. The monoclonal and the monospecific antibodies recognized denatured antigen (proteins 2C, 2BC and P2) in ELISA, immunoblot and immunoprecipitation tests and also combined with the native proteins of the P2 region in immunoprecipitation. In addition, the antibodies could be used successfully for immunofluorescence and electron microscopic immunocytochemistry.

Antibodies, Monoclonal↗

PubMed1986

Electron microscopic immunocytochemistry. Silver enhancement of colloidal gold marker allows double labeling with the same primary antibody.

Electron microscopic sections, immunocytochemically labeled with colloidal gold, can be prepared for double labeling by applying the "EM-silver enhancement" procedure. This method, a photographic, so-called physical, development, increases the size of the gold marker to a predeterminable value and thereby inactivates the anti-species antibody present on the gold grain, thus allowing the labeling of a second antigen with antibody raised in the same species.

Antibodies, Monoclonal↗

PubMedDate not supplied

Direct identification of recombinant vaccinia virus plaques by PCR.

A fast method for the identification of recombinant vaccinia viruses directly from individual plaques is described. Plaques are picked, resuspended in PBS-A and processed for PCR using two 'universal' primers. The amplified sequences are analyzed by agarose gel electrophoresis. This procedure allows discrimination between spontaneously arising TK-negative mutants, which do not carry the inserted gene, and the desired TK-negative recombinants resulting from insertional inactivation of the TK gene.

Base Sequence↗