Biomedical subjects

Luis Carrasco

Publications and source records attributed to Luis Carrasco.

23 records · Page 2Linked to original sources

PubMed2002

Interfacial domains in Sindbis virus 6K protein. Detection and functional characterization.

Alphavirus 6K is a short, constitutive membrane protein involved in virus glycoprotein processing, membrane permeabilization, and the budding of virus particles. The amino-terminal region that immediately precedes the transmembrane anchor contains a conserved sequence motif consisting of two interfacial domains separated by Asn and Gln residues. The presence of this motif confers on the 6K pretransmembrane region the tendency to partition into the membrane interface. To study the functional importance of the interfacial sequences, three different Sindbis virus 6K variants were obtained with the following modifications: 9YLW11xAAA, 18FWV20xAAA, and 9YLW11xAAA/18FWV20xAAA. Reconstituted mutant viruses were infectious and showed no defects in glycoprotein processing, although virus budding was hampered. Single 6K expression in Escherichia coli cells showed interfacial mutants to have a diminished capacity to modify membrane permeability and to have lower toxicity. In particular, the 9YLW11xAAA/18FWV20xAAA variant was expressed at high levels and did not enhance membrane permeability significantly, although it retained its integral membrane protein condition. Parallel analyses of membrane permeabilization in baby hamster kidney cells were carried out using a Sindbis virus replicon that synthesized both capsid protein and 6K. Transfection of the construct with wild-type 6K strongly increased permeability to the antibiotic hygromycin B. Replicons encoding 6K interfacial mutants induced lower membrane permeabilization. Again, the greatest impairment was observed for the 9YLW11xAAA/18FWV20xAAA variant, permeabilization activity of which was approximately 10% that of wild-type 6K. These findings show the importance of the interfacial 6K sequence for virus budding and modification of membrane permeability.

Amino Acid Sequence↗

PubMed2002

Cell killing by HIV-1 protease.

The human immunodeficiency virus protease (HIV-1 PR) was expressed both in the yeast Saccharomyces cerevisiae and in mammalian cells. Inducible expression of HIV-1 PR arrested yeast growth, which was followed by cell lysis. The lytic phenotype included loss of plasma membrane integrity and cell wall breakage leading to the release of cell content to the medium. Given that neither poliovirus 2A protease nor 2BC protein, both being highly toxic for S. cerevisiae, were able to produce similar effects, it seems that this lytic phenotype is specific of HIV-1 PR. Drastic alterations in membrane permeability preceded the lysis in yeast expressing HIV-1 PR. Cell killing and lysis provoked by HIV-1 PR were also observed in mammalian cells. Thus, COS7 cells expressing the protease showed increased plasma membrane permeability and underwent lysis by necrosis with no signs of apoptosis. Strikingly, the morphological alterations induced by HIV-1 PR in yeast and mammalian cells were similar in many aspects. To our knowledge, this is the first report of a viral protein with such an activity. These findings contribute to the present knowledge on HIV-1-induced cytopathogenesis.

Animals↗

PubMed2002

Viroporin-mediated membrane permeabilization. Pore formation by nonstructural poliovirus 2B protein.

Enterovirus nonstructural 2B protein is involved in cell membrane permeabilization during late viral infection. Here we analyze the pore forming activity of poliovirus 2B and several of its variants. Solubilization of 2B protein was achieved by generating a fusion protein comprised of poliovirus 2B attached to a maltose-binding protein (MBP) as an N-terminal solubilization partner. MBP-2B was assayed using large unilamellar vesicles as target membranes. This fusion protein was able to assemble into discrete structures that disrupted the permeability barrier of vesicles composed of anionic phospholipids. The transbilayer aqueous connections generated by MBP-2B were stable over time, allowing the passage of solutes of molecular mass under 1,000 Da. Oligomerization was investigated using fluorescence resonance energy transfer. Our data indicate that MBP-2B aggregation occurs at the membrane surface. Moreover, MBP-2B binding to membranes promoted the formation of SDS-resistant tetramers. We conclude that MBP-2B forms oligomers capable of generating a tetrameric aqueous pore in lipid bilayers. These findings are the first evidence of viroporin activity shown by a protein from a naked animal virus.

Animals↗

PubMed2002

Open vs laparoscopic repair of spigelian hernia: a prospective randomized trial.

HYPOTHESIS: The elective treatment of a spigelian hernia is still under discussion, fundamentally owing to its rarity. The purpose of the study is to analyze the elective surgical treatment of spigelian hernia. DESIGN: A prospective, randomized controlled trial. SETTING: University teaching hospital. PATIENTS AND INTERVENTIONS: Two surgeons performed 11 conventional and 11 laparoscopic repairs for a spigelian hernia, alternating roles as primary surgeon and assistant. Each time the type of technique was randomly chosen using a computerized program. MAIN OUTCOME MEASURES: Epidemiological, clinical, and surgical factors are analyzed according to treatment, ie, the open or laparoscopic approach. RESULTS: The statistical study shows no significant differences for epidemiological or diagnostic factors, but it does show significant advantages for laparoscopy in terms of morbidity (P<.05) and hospital stay (P<.001). CONCLUSION: The approach using extraperitoneal laparoscopy is the technique that offers best results in the elective treatment of spigelian hernia.

Adolescent↗

PubMed2002

Antiviral activity of seven iridoids, three saikosaponins and one phenylpropanoid glycoside extracted from Bupleurum rigidum and Scrophularia scorodonia.

As part of our screening of antiviral agents from medicinal plants, 11 compounds from plant origin (Bupleurum rigidum and Scrophularia scorodonia), three saikosaponins, seven iridoids and one phenylpropanoid glycoside were tested in vitro against herpes simplex type I (HSV-1), vesicular stomatitis virus (VSV) and poliovirus type 1. Five of these compounds showed antiviral activity against VSV. The percentages of cellular viability at the non-toxic limit concentrations of the active compounds were: verbascoside 53.6 % at 500 microg/ml, 8-acetylharpagide 32.1 % at 500 microg/ml, harpagoside 43.3 % at 450 microg/ml, scorodioside 47.8 % at 500 microg/ml and buddlejasaponin IV 56.9 % at 25 microg/ml. Although none of the saikosaponins were active against HSV-1, the iridoid scorodioside showed moderate in vitro anti-HSV-1 activity (30.6 % at 500 microg/ml). However, none of the compounds tested in this survey had any effect against poliovirus.

Animals↗