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Biomedical subjects

M Esteban

Publications and source records attributed to M Esteban.

At least 91 records · Page 5Linked to original sources
PubMed1996

Open reading frame 5 of porcine reproductive and respiratory syndrome virus as a cause of virus-induced apoptosis.

The gene product of open reading frame 5 (p25) of porcine reproductive and respiratory syndrome (PRRS) virus has been expressed by coinfection of culture cells with vaccinia virus expressing the T7 RNA polymerase and a recombinant vaccinia virus encoding the open reading frame 5 gene under the T7 promoter and the encephalomyocarditis virus internal ribosome entry site. In spite of the reported efficiency of the expression system, very poor accumulation of p25 protein was observed and a strong cytotoxicity was produced in the doubly infected cells. This cell toxicity was shown to occur by induction of apoptosis, as indicated by nucleosome ladder formation, chromatin condensation, and rRNA degradation. Apoptosis induction was also observed after infection of cultured cells with an adapted PRRS virus strain and after infection of swine macrophage cells with a PRRS virus field strain. Contrary to the observations made for other cases of virus-induced apoptosis, we could not prevent p25 protein-induced apoptosis by using a cell line permanently expressing Bcl-2 protein.

Animals↗
PubMed1996

Autophosphorylation sites participate in the activation of the double-stranded-RNA-activated protein kinase PKR.

The interferon-induced RNA-dependent protein kinase PKR is found in cells in a latent state. In response to the binding of double-stranded RNA, the enzyme becomes activated and autophosphorylated on several serine and threonine residues. Consequently, it has been postulated that autophosphorylation is a prerequisite for activation of the kinase. We report the identification of PKR sites that are autophosphorylated in vitro concomitantly with activation and examine their roles in the activation of PKR. Mutation of one site, threonine 258, results in a kinase that is less efficient in autophosphorylation and in phosphorylating its substrate, the initiation factor eIF2, in vitro. The mutant kinase is also impaired in vivo, displaying reduced ability to inhibit protein synthesis in yeast and mammalian cells and to induce a slow-growth phenotype in Saccharomyces cerevisiae. Mutations at two neighboring sites, serine 242 and threonine 255, exacerbated the effect. Taken together with earlier results (S. B. Lee, S. R. Green, M. B. Mathews, and M. Esteban, Proc. Natl. Acad. Sci. USA 91:10551-10555, 1994), these data suggest that the central part of the PKR molecule, lying between its RNA-binding and catalytic domains, regulates kinase activity via autophosphorylation.

Amino Acid Sequence↗
PubMed1996

Characteristics of serum protein binding of felodipine.

The protein binding of felodipine in concentrations ranging from 3 to 65 nmol L-1 has been characterized in pooled serum, and in isolated human plasma proteins: albumin (HSA) and a1-acid glycoprotein (AAG). Protein binding was determined by an ultrafiltration technique using an Amicon Micropartition System. Serum protein binding of felodipine (16 nmol L-1) was measured in five groups of individuals: I: healthy subjects (n = 16). II: patients with chronic renal disease before and after dialysis (n = 10). III: patients with liver disease (n = 9). IV: diabetics Type I (n = 10) and Type II (n = 12) and V: cancer patients (n = 12). Concentrations of HSA, AAG, lipoproteins and non esterified fatty acids (NEFA) were also measured. The drug was extensively bound in pooled serum and the protein binding was essentially unchanged over the concentrations of felodipine studied (99.60 +/- 0.31% at 3 nmol L-1; 99.70 +/- 0.15% at 65 nmol L-1). In albumin solution (40 g L-1) felodipine was also highly bound. The mean of percentage bound was not significantly different from that in serum and was also independent of the felodipine concentrations (98.57 +/- 0.35 at 3 nmol L-1; 98.31 +/- 0.90 at 65 nmol L-1). The extent of binding to AAG was significantly lower than in serum (p < 0.01) and HSA (p < 0.01) and was independent of felodipine concentrations (85.64 +/- 2.25 at 3 nmol L-1; 85.68 +/- 2.3 at 65 nmol L-1). The percentage of bound felodipine in group II (before dialysis) was significantly lower than in group I (p < 0.001). The variability in the percentage of bound felodipine was greater in group II, before dialysis, than in the rest of the groups. After dialysis, protein binding was similar to that in group I. HSA did not change and AAG was increased. NEFA was significantly higher after dialysis when compared with group I (p < 0.01). In vitro carbamylation of serum did not change felodipine protein binding. HSA was decreased significantly in group III patients (p < 0.05). However, protein binding did not change. Binding of felodipine in the rest of the groups was not significantly different from that in group I. Linear regression analysis of the data for all individuals indicated that the binding of felodipine was related to serum lipoproteins and that age, HSA, AAG, and NEFA were not significant determinants of binding.

Adult↗
PubMed1996

[Analysis of patients with acute pancreatitis (AP), (1984-1994) decade. Comparison with a past series].

Between 1984-1994 records of 190 patients with acute pancreatitis (AP) were reviewed. Diagnosis was confirmed by surgery in 141, by necropsy 1 (75%), and for the remaining patients, by CT and clinical evaluation. Female 122 were female (64%), male 68 (36%), mean age 41 years (range 7-82 years). The etiologic factors were gallstones in 129 (68%) with 4 deaths (3%), alcoholic in 26 (14%) with 5 (19%) deaths, idiopathic in 24 (13%) and miscellaneous 11 (5%), the last 2 without mortality. The overall mortality was 5% (9/190). Surgical treatment was indicated in 141 (74%) and endoscopic treatment in 2 (1%), 47 patients (25%) received only medical treatment. One-hundred and fifty-four patients (81%) were mild forms with a Ranson mean score 2.2 without mortality. In this group, emergency surgery was indicated in 7 (6%), early in 3 (3%), late in 1 (0.9%) and elective in 99 (90%). In the severe group were 36 patients (19%) with a mean Ranson score of 4.2. Nine patients died (25%), 31 were surgically treated, by emergency surgery in 17 (47%), 1 (3%) early surgery and 13 (36%) late surgery. The mortality rate of this group was compared with previous series of the same hospital (1975-1984 series). The 1984-1994 group showed a significant overall mortality decrease 4.7% vs 12.7% (p = 0.0047); 3.1% vs 11.2% (p = 0.0087) for the gallstones group; without mortality in the idiopathic and miscellaneous form compared with 18.7% and 20% respectively of the previous series; no mortality was observed in the mild AP compared with 5% of the previous series. The mortality in the severe form was 25% vs 40.8% (1975-1984 group). We consider that the decreased mortality could be attributed to the improvement in the diagnosis, early recognition of the severe forms, systemic supportive care in the severe forms, the use of antibiotics with wide spectrum and deeper penetration in the pancreas and specially in the change of the surgical timing. Although, in the first period the option was the early pancreatic surgery, in the second this was indicated only in particular cases.

Acute Disease↗
PubMed1995

Inhibition of vaccinia virus DNA replication by inducible expression of nitric oxide synthase.

Nitric oxide (NO) exerts multiple biologic roles in animal cells through differential regulation of three distinct forms of NO synthase encoded by separate genes. Macrophage-inducible nitric oxide synthase (iNOS) has been correlated with inhibition of viral growth, but little is known about the mechanism of this effect. To study the antiviral role of NO, we have generated a vaccinia virus (VV) recombinant expressing iNOS under the control of Escherichia coli LacI operator/repressor elements. When cultured cells of various origins are infected with this recombinant virus, there is inducible expression of iNOS in the presence of isopropylthio-beta-galactoside, as determined by Western blot and by detection of nitrite, a NO oxidation product. The levels of nitrite increase with time after infection, correlating with marked inhibition of VV DNA and late protein synthesis. Expression of VV early proteins is not affected by NO. Inhibition of VV DNA synthesis is likely to be in part a consequence of NO-mediated inhibition of viral ribonucleotide reductase, as this inhibition can be partially overcome by addition of deoxyribonucleosides. Inhibition of the essential viral functions by NO results in a reduction of virus yields by 50 to 90%, depending on the cell line. Thus, our results demonstrate a direct antiviral effect of NO, with inhibition of VV replication occurring at the level of DNA synthesis.

Animals↗
PubMed1995

Quantification of antigen specific CD8+ T cells using an ELISPOT assay.

An ELISPOT assay to detect and determine the number of antigen specific CD8+ T cells was standardized using cloned murine CD8+ T cells specific for the epitope SYVPSAEQI of a rodent malaria antigen. This assay is based on the detection of IFN-gamma secretion by single cells after their stimulation with antigen. The interferon secretion is visualized as spots revealed by using enzyme labeled anti-IFN-gamma monoclonal antibodies. Using known numbers of cloned murine CD8+ T cells it was determined that the assay detects 80-95% of these CD8+ T cells. The optimal culture conditions for the stimulation of the CD8+ T cells were determined and the antigen concentration, number of antigen presenting cells and supplement of growth factors required to perform the assay were defined. This ELISPOT assay can be performed with spleen cells from immunized mice, and provide the precise number of antigen specific CD8+ T cells present in mixed lymphocyte populations. This method is more sensitive than the chromium-51 release assay, and much simpler than the conventional precursor frequency analysis, providing the number of antigen specific CD8+ T cells in 36-48 h.

Amino Acid Sequence↗
PubMed1995

Enhanced proteolytic processing of the human immunodeficiency virus type 1 envelope protein in murine Ltk(-) cells.

Proteolytic processing of the human immunodeficiency virus type 1 (HIV-1) envelope (Env) precursor glycoprotein (gp160) to produce the mature gp120 and gp41 proteins is required for virus infection and virus-induced cell fusion. It has also been suggested that cleavage of gp120 at the immunodominant V3 loop region is required for virus-to-cell and cell-to-cell fusion. In this investigation we have studied the proteolytic processing of the HIV-1 Env in cells of various origins (human, monkey, and mouse) infected with a vaccinia virus recombinant expressing the entire gp160 protein (VV-env-1). We have observed that in murine Ltk(-) cells, in addition to the proteolytic cleavage of gp160 at the gp120/gp41 site, there is also extensive intracellular proteolytic processing of gp160 at the V3 loop and at a novel site located at the C terminus of gp41. Similar proteolytic processing of the Env precursor was observed after treatment of extracts of VV-env-1-infected monkey cells with thrombin, a trypsin-like protease that has been shown to cleave the gp120 at the V3 loop. Our findings suggest that murine Ltk(-) cells could be a good model system for structural studies of Env with different HIV isolates and in searches for proteinase inhibitors that could prevent HIV-1 infection of susceptible cells by blocking proteolysis of Env.

Animals↗
PubMed1995

Assembly of vaccinia virus: incorporation of p14 and p32 into the membrane of the intracellular mature virus.

The cytoplasmic assembly of vaccinia virus begins with the transformation of a two-membraned cisterna derived from the intermediate compartment between the endoplasmic reticulum and the Golgi complex. This cisterna develops into a viral crescent which eventually forms a spherical immature virus (IV) that matures into the intracellular mature virus (IMV). Using immunoelectron microscopy, we determined the subcellular localization of p32 and p14, two membrane-associated proteins of vaccinia virus. p32 was associated with vaccinia virus membranes at all stages of virion assembly, starting with the viral crescents, as well as with the membranes which accumulated during the inhibition of assembly by rifampin. There was also low but significant labelling of membranes of some cellular compartments, especially those in the vicinity of the Golgi complex. In contrast, anti-p14 labelled neither the crescents nor the IV but gave strong labelling of an intermediate form between IV and IMV and was then associated with all later viral forms. This protein was also not significantly detected on identifiable cellular membranes. Both p32 and p14 were abundantly expressed on the surface of intact IMV. Our data are consistent with a model whereby p32 would become inserted into cellular membranes before being incorporated into the crescents whereas p14 would be posttranslationally associated with the viral outer membrane at a specific later stage of the viral life cycle.

Animals↗
PubMed1995

Vaccinia virus A17L gene product is essential for an early step in virion morphogenesis.

Vaccinia virus (VV) A17L gene encodes a 23-kDa protein that is proteolytically cleaved to generate a 21-kDa product that is incorporated into the viral particles. We have previously shown that the 21-kDa protein forms a stable complex with the VV 14-kDa envelope protein and suggested that the 21-kDa protein may serve to anchor the 14-kDa protein to the envelope of the virion (D. Rodríguez, J. R. Rodríguez, and M. Esteban, J. Virol. 67:3435-3440, 1993). To study the role of the 21-kDa protein in virion assembly, in this investigation we generated a VV recombinant, VVindA17L, that contains an inducible A17L gene regulated by the E. coli repressor/operator system. In the absence of the inducer, shutoff of the A17L gene was complete, and this shutoff correlated with a reduction in virus yields of about 3 log units. Although early and late viral polypeptides are normally synthesized in the absence of the A17L gene product, proteolytic processing of the major p4a and p4b core proteins was clearly impaired under these conditions. Electron microscopy examination of cells infected in the absence of isopropylthiogalactopyranoside (IPTG) revealed that virion morphogenesis was completely arrested at a very early stage, even prior to the formation of crescent-shaped membranes, which are the first distinguishable viral structures. Only electron-dense structures similar to rifampin bodies, but devoid of membranes, could be observed in the cytoplasm of cells infected with VVindA17L under nonpermissive conditions. Considering the most recent assembly model presented by Sodeik et al. (B. Sodeik, R. W. Doms, M. Ericsson, G. Hiller, C. E. Machamer, W. van't Hof, G. van Meer, B. Moss, and G. Griffiths, J. Cell Biol. 121:521-541, 1993), we propose that this protein is targeted to the intermediate compartment and is involved in the recruitment of these membranes to the viral factories, where it forms the characteristic crescent structures that subsequently result in the formation of virions.

Base Sequence↗
PubMed1994

[Mini Mental State Examination: association of the score obtained with the age and degree of literacy in an aged population].

BACKGROUND: To differentiate dementia from the normal human decline scales may be used in an attempt to identity and quantify the cognitive damage which a subject may present. The Mini Mental State Examination (MMSE) is one of those. This study was performed to identify the relation of the responses between the MMSE and the sociodemographic variables of the subjects. METHODS: The MMSE was applied as a tool to determine the presence of cognitive alterations in a prospective study of people over the age of 65 years from La Selva del Camp (Baix Camp, Catalonia, Spain). The population was differentiated according to the cut off point (24 points) with the relation of the score obtained by each subjects being studied with the sociodemographic variables of the same. RESULTS: The mean score obtained in the test by the total population was: (mean +/- SD) 23.9 +/- 4.5 points; 24.6 +/- 4.9 in the males and 22.5 +/- 5.8 in the females. In the age group 65-74 years the mean score was 24.8 +/- 3.8; 23 +/- 4.6 in those from 75-80 years and 21.4 +/- 6 in the age group over 85 years. Sex was significantly associated (p = 0.0003), as were age (p = 0.0002) and the degree of alphabetization (p = 0.0000) with women obtaining a lower MMSE score than men, the most elderly was lower than that obtained by the younger and those of less alphabetization lower than those with education. CONCLUSIONS: Although the MMSE is a good tool for the detection of cognitive alterations in a population, it must be kept in mind that the result of this test may be influenced by the age and education of the subject and should therefore not be used as an exclusive element in the diagnosis of dementia.

Age Factors↗
PubMed1994

Activation of the double-stranded RNA (dsRNA)-activated human protein kinase in vivo in the absence of its dsRNA binding domain.

The interferon-induced, dsRNA-activated human protein kinase (PKR) exerts antiviral and antiproliferative effects through inhibition of protein synthesis. Studies of structure-function relationships in PKR have shown that two dsRNA binding motifs are important for its autophosphorylation and activation by dsRNA in vitro. To correlate these findings with the activity of PKR in vivo, we examined the function of various PKR deletion mutants in cultured cells by using an inducible expression system. In a reporter gene assay, mutant forms of the kinase lacking amino acids 1-97 (delta 1-97) and 104-157 (delta 104-157), which are required for dsRNA binding in vitro, retained full activity in vivo. Deletion of amino acids 233-271 (delta 233-271), however, abolished the translational inhibitory activity of the kinase and prevented its phosphorylation. Moreover, cells infected with vaccinia virus recombinants expressing wild-type PKR, the mutant delta 104-157, delta 186-222), developed almost complete inhibition of both viral and cellular protein synthesis was upon induction of PKR. This inhibition of viral protein synthesis was not observed in cells infected with a recombinant expressing delta 233-271 mutant PKR. Our findings establish that the region encompassing amino acids 233-271 of PKR is critical for kinase activity in vivo, whereas its dsRNA binding domain is dispensable.

Animals↗
PubMed1994

Interferon-gamma severely inhibits DNA synthesis of vaccinia virus in a macrophage cell line.

Interferon-gamma inhibits the growth of several viruses; however, the exact mechanism of this inhibition has not been clearly defined. In this report, we show that in a monocyte/macrophage cell line J774.G8 IFN-gamma inhibits the growth of vaccinia virus (VV) in a dose-dependent manner. This inhibition is due to a block in VV DNA synthesis and correlates with an increased generation of reactive nitrogen intermediates.

Animals↗
PubMed1994

The interferon-induced double-stranded RNA-activated protein kinase induces apoptosis.

Interferons (IFNs) exert antitumor activities, but the molecular mechanism underlying these effects is poorly understood. IFN-induced, double-stranded (ds) RNA-activated protein kinase (p68 kinase) has long been implicated in mediating the antiproliferative effects of IFN. In addition, recent studies suggest that p68 kinase may function as a tumor suppressor gene. In this investigation we showed that expression of p68 kinase in HeLa cells resulted in a rapid cell death characteristic of apoptosis. Rapid cell death was not observed in cells which expressed a mutant form of p68 kinase (lys296-->arg) indicating that cell death observed is the result of p68 kinase expression and activation. Moreover, infection of HeLa cells with the mutant vaccinia virus lacking E3L gene, which encodes a dsRNA binding protein that acts as an inhibitor of p68 kinase, also resulted in apoptosis. Thus, we propose that human p68 kinase functions as a tumor suppressor gene by actively participating in apoptosis.

Apoptosis↗
PubMed1993

Priming with recombinant influenza virus followed by administration of recombinant vaccinia virus induces CD8+ T-cell-mediated protective immunity against malaria.

Live vectors expressing foreign antigens have been used to induce immunity against several pathogens. However, for the virulent rodent malaria parasite Plasmodium yoelii, the use of recombinant vaccinia virus, pseudorabies virus, or Salmonella, expressing the circumsporozoite protein of this parasite, failed to induce protection. We generated a recombinant influenza virus expressing an epitope from the circumsporozoite protein of P. yoelii known to be recognized by CD8+ T cells and demonstrated that this vector induced class I major histocompatibility complex-restricted cytotoxic T cells against this foreign epitope. Immunization of mice with this recombinant influenza virus, followed by a recombinant vaccinia virus expressing the entire circumsporozoite protein, induced protective immunity against sporozoite-induced malaria. The sequence of immunization appears to be crucial, since a primer injection with recombinant vaccinia virus, followed by a booster injection with recombinant influenza virus, failed to induce protection. The protection induced by immunization with these recombinant viruses is mostly mediated by CD8+ T cells, as treatment of mice with anti-CD8 monoclonal antibody abolishes the anti-malarial immunity. The use of different live vectors for primer and booster injections has a synergistic effect on the immune response and might represent an effective general strategy for eliciting protective immune responses to key antigens of microbial pathogens.

Amino Acid Sequence↗
PubMed1993

The interferon-induced double-stranded RNA-activated human p68 protein kinase potently inhibits protein synthesis in cultured cells.

The role of the interferon-induced double-stranded RNA (dsRNA)-activated human p68 protein kinase as an inhibitor of protein synthesis has been inferred from work with cell-free systems, but direct proof in animal cells is lacking. To document the action of p68 protein kinase in vivo, we have used an infection-transfection system where expression of p68 is driven by a vaccinia virus promoter regulated by the lacl repressor/operator controlling elements. In cultured cells infected with vaccinia virus and transfected with a plasmid containing the p68 gene, there is synthesis of p68 when lacl repressor is inhibited with isopropyl-beta-D-thiogalactoside. When infection-transfections are carried out with the p68 gene together with the luciferase (LUC) reporter gene, a strong inhibition of LUC expression developed with time postinfection. This inhibition was not observed with a mutant form of the kinase (Lys-->Arg at position 296) and it was reversed by antisense expression of the p68 gene. During inhibition of LUC expression the protein kinase was phosphorylated, possibly as a result of autophosphorylation activated by the dsRNA forms which are known to accumulate in vaccinia virus-infected cells. Inhibition of LUC expression was at the level of translation. Our findings demonstrate that expression and activation of the human p68 protein kinase in vivo potently inhibits protein synthesis.

Cells, Cultured↗