Biomedical subjects

Luis Carrasco

Publications and source records attributed to Luis Carrasco.

11 recordsLinked to original sources

PubMed2003

Viroporins.

Viroporins are a group of proteins that participate in several viral functions, including the promotion of release of viral particles from cells. These proteins also affect cellular functions, including the cell vesicle system, glycoprotein trafficking and membrane permeability. Viroporins are not essential for the replication of viruses, but their presence enhances virus growth. Comprising some 60-120 amino acids, viroporins have a hydrophobic transmembrane domain that interacts with and expands the lipid bilayer. Some viroporins also contain other motifs, such as basic amino acid residues or a domain rich in aromatic amino acids that confers on the protein the ability to interact with the interfacial lipid bilayer. Viroporin oligomerization gives rise to hydrophilic pores at the membranes of virus-infected cells. As the list of known viroporins steadily grows, recent research efforts focus on deciphering the actions of the viroporins poliovirus 2B, alphavirus 6K, HIV-1 Vpu and influenza virus M2. All these proteins can enhance the passage of ions and small molecules through membranes depending on their concentration gradient. Future work will lengthen the list of viroporins and will provide a deeper understanding of their mechanisms of action.

Amino Acid Sequence↗

PubMed2003

Mechanisms of membrane permeabilization by picornavirus 2B viroporin.

Cell infection by picornaviruses leads to membrane permeabilization. Recent evidence suggests the involvement of the non-structural protein 2B in this process. We have recently reported the detection of 2B porin-like activity in isolated membrane-protein systems that lack other cell components. According to data derived from these model membranes, four self-aggregated 2B monomers (i.e. tetramers) would be sufficient to permeabilize a single lipid vesicle, allowing the free diffusion of solutes under ca. 1000 Da. Our findings also support a role for lipids in protein oligomerization and subsequent pore opening. The lipid dependence of these processes points to negatively charged cytofacial surfaces as 2B cell membrane targets.

Amino Acid Sequence↗

PubMed2003

Individual expression of sindbis virus glycoproteins. E1 alone promotes cell fusion.

The envelope of alphavirus particles contains two major glycoproteins, E1 and E2, that participate in virus entry and assembly of new virus particles. Interactions between these glycoproteins determine their correct functioning. The expression of each glycoprotein in the absence of the other counterpart was achieved by means of electroporation of modified Sindbis virus (SV) genomes. In addition, in trans coexpression of both glycoproteins was also tested in BHK cells. Synthesis of the E1 glycoprotein alone gave rise to cell fusion after incubation in low-pH medium. In addition, expression of E1 in the absence of the E2 precursor, PE2 (E3+E2), induced the formation of cytoplasmic vacuoles in the transfected cells. The normal phenotype was recovered when PE2 was coexpressed in trans with E1. Moreover, this coexpression modified the processing of the PE2 glycoprotein. PE2 synthesized in the absence of E1 gave rise to a product, E2', whose migration was slower in SDS-polyacrylamide gel than that of genuine E2 from SV-infected cells. This alteration was corrected upon in trans coexpression of E1 and PE2. These results suggest that the two glycoproteins, E1 and PE2, interact after their expression from two separate SV genomes. Notably, BHK cells cotransfected with the two modified genomes produced SV particles. Our findings suggest that SV E1 and E2 synthesized in trans can interact with each other and participate together with capsid protein in the assembly of new virus particles.

Animals↗

PubMed2003

Cleavage of eIF4G by HIV-1 protease: effects on translation.

We have recently reported that HIV-1 protease (PR) cleaves the initiation factor of translation eIF4GI [Ventoso et al., Proc. Natl. Acad. Sci. USA 98 (2001) 12966-12971]. Here, we analyze the proteolytic activity of HIV-1 PR on eIF4GI and eIF4GII and its implications for the translation of mRNAs. HIV-1 PR efficiently cleaves eIF4GI, but not eIF4GII, in cell-free systems as well as in transfected mammalian cells. This specific proteolytic activity of the retroviral protease on eIF4GI was more selective than that observed with poliovirus 2A(pro). Despite the presence of an intact endogenous eIF4GII, cleavage of eIF4GI by HIV-1 PR was sufficient to impair drastically the translation of capped and uncapped mRNAs. In contrast, poliovirus IRES-driven translation was unaffected or even enhanced by HIV-1 PR after cleavage of eIF4GI. Further support for these in vitro results has been provided by the expression of HIV-1 PR in COS cells from a Gag-PR precursor. Our present findings suggest that eIF4GI intactness is necessary to maintain cap-dependent translation, not only in cell-free systems but also in mammalian cells.

Amino Acid Sequence↗

PubMed2003

The large millimeter telescope/el Gran Telescopio Milimétrico: a new instrument for astrobiology.

The Instituto Nacional de Astrofísica, Optica y Electrónica in Mexico and the University of Massachusetts in the U.S.A. are collaborating to build the world's largest radio telescope that operates at short millimeter wavelengths. This facility, known as the Large Millimeter Telescope (LMT) or el Gran Telescopio Milimétrico (GTM), is being sited at an altitude of 4600 m on Volcan Sierra Negra in the Mexican state of Puebla. The telescope will be a fully steerable dish with a diameter of 50 m and a surface consisting of 180 panels that are actively adjusted under computer control to correct for deformations due to gravity and temperature gradients. Instruments will include focal plane arrays to image both continuum and spectral line emission from celestial sources. The LMT/GTM will be an extremely powerful facility for studies encompassing almost all areas of astronomy, including astrobiology. In particular, the high sensitivity, angular resolution, and mapping speed will enable detailed investigations of the organic chemistry of interstellar molecular clouds, protoplanetary disks, and comets.

Chemistry, Organic↗

PubMed2003

The eukaryotic translation initiation factor 4GI is cleaved by different retroviral proteases.

The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family Retroviridae, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.

Animals↗

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↗