Biomedical subjects

M Duarte

Publications and source records attributed to M Duarte.

At least 19 recordsLinked to original sources

PubMed2003

Wild-type Escherichia coli producing microcins B17, D93, J25, and L; cloning of genes for microcin L production and immunity.

For the first time, an Escherichia coli strain producing four microcins (Mcc), B17, D93, J25, and L, and showing immunity to Mcc V was isolated and characterized. Each of the gene clusters encoding the production of Mcc B17, D93, and L was cloned separately. The gene cluster for Mcc L was cloned within a 13.5-kb HindIII-SalI fragment, which includes the Mcc V immunity gene, cvi.

Bacteriocins↗

PubMed2002

Efficacy and safety of a home-made non-valved spacer for bronchodilator therapy in acute asthma.

UNLABELLED: The aim of this study was to assess the efficacy and safety of an alternative spacer to deliver salbutamol in acute asthma. A prospective randomized study was performed with 196 patients with acute asthma, aged 4-15 y. The clinical features, oxygen saturation (SaO2) and the best of three peak expiratory flow rate (PEFR) measurements were recorded in a standardized questionnaire. Each patient received up to three conventional doses of salbutamol through an alternative home-made non-valved spacer (NVS) (500 ml mineral water plastic bottle) or an oxygen-driven nebulizer (ODN). The clinical and functional respiratory parameters (PEFR and SaO2) were assessed 15 min after each administration of the bronchodilator. In the ODN (n = 99) and NVS (n = 97) groups, PEFR (p > 0.05) and SaO2 (p > 0.05) measurements improved in all stages of the treatment. During the asthma attacks, the ODN group needed a prolonged observation in the emergency room (p = 0.000000). Side effects of salbutamol, e.g. increased heart rate, were observed in 17.2% of the patients in the ODN group and 4.1% in the NVS group (p = 0.003). CONCLUSION: Both devices showed comparable efficacy in mild and moderate acute attacks, but the use of the alternative NVS resulted in a shorter observation period in the emergency room. The frequency of side effects was significantly higher in the ODN group than in the NVS group.

Acute Disease↗

PubMed2002

Cytokine gene transfer into dendritic cells for cancer treatment.

Bone marrow-derived dendritic cells have been used to treat established experimental tumors by unleashing a cellular immune response against tumor antigens. Such antigens are artificially loaded onto dendritic cells' antigen-presenting molecules by different techniques including incubation with synthetic antigenic determinants, tumor lysates or nucleic acids encoding for those relevant antigens. Ex vivo gene transfer with viral and non-viral vectors is frequently used to obtain expression of the tumor antigens and thereby to formulate the therapeutic vaccines. Efficacy of the approaches is greatly enhanced if dendritic cells are transfected with a number of genes which encode immunostimulating factors. In some cases, such as with IL-12, IL-7 and CD40L genes, injection inside experimental malignancies of thus transfected dendritic cells induces complete tumor regression in several models. In this case tumor antigens are captured by dendritic cells by still unclear mechanisms and transported to lymphoid organs where productive antigen presentation to T-cells takes place. Many clinical trials testing dendritic cell-based vaccines against cancer are in progress and partial clinical efficacy has been already proved. Transfection of genes further strengthening the immunogenicity of such strategies will join the clinical club soon.

Bone Marrow Cells↗

PubMed2001

Organization of the human tarbp2 gene reveals two promoters that are repressed in an astrocytic cell line.

TRBP1 and TRBP2 are isoforms of a double-stranded RNA-binding protein that differ in their N-terminal end and were each identified by binding to human immunodeficiency virus type 1 (HIV-1) trans-activation-responsive RNA. TRBP1 and TRBP2 also bind and modulate the function of the double-stranded RNA-activated protein kinase, protein kinase R. Both proteins increase long terminal repeat expression in human and murine cells, and their gene has been mapped to human chromosome 12. We have isolated and characterized the complete tarbp2 gene (5493 bp) coding for the two TRBP proteins. Two adjacent promoters initiate transcription of alternative first exons for TRBP1 and TRBP2 mRNAs that are spliced onto common downstream exons. TRBP2 transcription and translation start sites are localized within the first intron of TRBP1. TRBP promoters are TATA-less but have CCAAT boxes, a CpG island, and several potential binding sites for transcriptional factors. Promoter deletion analysis identified two regions from position -1397 to -330 for TRBP1 and from position -330 to +38 for TRBP2 that are important for promoter function. TRBP2 promoter activity was expressed at a higher level compared with TRBP1 promoter. In addition, a specific down-regulation of TRBP1 and TRBP2 promoter activity was identified in human astrocytic cell line U251MG compared with HeLa cells. This minimal TRBP promoter activity may account for minimal HIV-1 replication in astrocytes.

Alternative Splicing↗

PubMed2001

Alpha(v)beta(3) integrin-mediated adenoviral transfer of interleukin-12 at the periphery of hepatic colon cancer metastases induces VCAM-1 expression and T-cell recruitment.

We previously reported that systemic injection of recombinant adenovirus resulted in a rim of gene transduction around experimental liver tumor nodules. This zone of higher infection is dependent on the alpha(v)beta(3) integrin, acting as an adenovirus internalization receptor, which is overexpressed in tissues surrounding liver metastases. When a recombinant adenovirus encoding interleukin-12 (AdCMVIL-12) is given into a subcutaneous tumor nodule in mice also bearing concomitant liver tumors, a fraction of AdCMVIL-12 reaches the systemic circulation and infects liver tissue, especially at the malignant/healthy tissue interface. As a result of the expression at this location of the interleukin-12 transgenes, VCAM-1 is induced on vessel cells and mediates the recruitment of adoptively transferred anti-tumor cytolytic T-lymphocytes. These studies provide mechanistic explanations for the potent therapeutic synergy observed between interleukin-12 gene transfer and adoptive T-cell therapy.

Adenoviridae↗

PubMed2001

Analysis of kinematically redundant reaching movements using the equilibrium-point hypothesis.

Six subjects performed a planar reaching arm movement to a target while unpredictable perturbations were applied to the endpoint; the perturbations consisted of pulling springs having different stiffness. Two conditions were applied; in the first, subjects had to reach for the target despite the perturbation, in the second condition, the subjects were asked to not correct the motion as a perturbation was applied. We analyzed the kinematics profiles of the three arm segments and, by means of inverse dynamics, calculated the joint torques. The framework of the equilibrium-point (EP) hypothesis, the lambda model, allowed the reconstruction of the control variables, the "equilibrium trajectories", in the "do not correct" condition for the wrist and the elbow joints as well as for the end point final position, while for the other condition, the reconstruction was less reliable. The findings support and extend to a multiple-joint planar movement, the paradigm of the EP hypothesis along with the "do not correct" instruction.

Adult↗

PubMed2001

Thrombopenic purpura induced by a monoclonal antibody directed to a 35-kilodalton surface protein (p35) expressed on murine platelets and endothelial cells.

OBJECTIVE: With the aim of obtaining monoclonal antibodies (mAbs) against mouse endothelial surface antigens, immunization of rats with a mouse-derived endothelial cell line (PY4.1) and subsequent hybridoma production were performed. MATERIALS AND METHODS: One of the mAbs produced by hybridoma EOL5F5 was selected for its surface binding to endothelial cell lines, and identification of the mAb-recognized antigen was performed by immunoprecipitation. Experiments were performed to analyze the effects of EOL5F5 on systemic administration to mice. RESULTS: EOL5F5-recognized antigen was a single band of 35 kDa under reducing and nonreducing conditions, features that do not match other known differentiation antigens with comparable tissue distribution. In vivo administration of purified EOL5F5 mAb to mice (n = 20) induced intense cutaneous purpura as well as severe but transient thrombocytopenia. Expression of EOL5F5-recognized antigen was detected on platelets from which it immunoprecipitated a moiety of identical electrophoretic pattern in SDS-PAGE, as the one recognized on endothelial cells. Immunohistochemically, EOL5F5-recognized antigen (p35) also was expressed on dermal capillaries, suggesting that, in addition to thrombocytopenia, damaging effects of the antibody on endothelial cells also might cause the observed purpura. CONCLUSIONS: Our results show induction of thrombocytopenic purpura in mice with an mAb against a single antigenic determinant expressed on both platelets and endothelium. EOL5F5 mAb injection sets the stage for useful experimental models that resemble immune thrombocytopenic purpura.

Animals↗

PubMed2001

On complex I and other NADH:ubiquinone reductases of Neurospora crassa mitochondria.

The mitochondrial complex I is the first component of the respiratory chain coupling electron transfer from NADH to ubiquinone to proton translocation across the inner membrane of the organelle. The enzyme from the fungus Neurospora crassa is similar to that of other organisms in terms of protein and prosthetic group composition, structure, and function. It contains a high number of polypeptide subunits of dual genetic origin. Most of its subunits were cloned, including those binding redox groups. Extensive gene disruption experiments were conducted, revealing many aspects of the structure, function, and biogenesis of complex I. Complex I is essential for the sexual phase of the life cycle of N. crassa, but not for the asexual stage. In addition to complex I, the fungal mitochondria contain at least three nonproton-pumping alternative NAD(P)H dehydrogenases feeding electrons to the respiratory chain from either matrix or cytosolic substrates.

Electron Transport↗

PubMed2001

Bouncing a ball: tuning into dynamic stability.

Rhythmically bouncing a ball with a racket was investigated and modeled with a nonlinear map. Model analyses provided a variable defining a dynamically stable solution that obviates computationally expensive corrections. Three experiments evaluated whether dynamic stability is optimized and what perceptual support is necessary for stable behavior. Two hypotheses were tested: (a) Performance is stable if racket acceleration is negative at impact, and (b) variability is lowest at an impact acceleration between -4 and -1 m/s2. In Experiment 1 participants performed the task, eyes open or closed, bouncing a ball confined to a 1-dimensional trajectory. Experiment 2 eliminated constraints on racket and ball trajectory. Experiment 3 excluded visual or haptic information. Movements were performed with negative racket accelerations in the range of highest stability. Performance with eyes closed was more variable, leaving acceleration unaffected. With haptic information, performance was more stable than with visual information alone.

Adult↗

PubMed2001

Adenovirus-mediated CD40 ligand gene therapy in a rat model of orthotopic hepatocellular carcinoma.

Unresectable hepatocellular carcinoma (HCC) lacks effective therapy and entails very poor prognosis. In this study, we have explored a gene-therapeutic approach to stimulate antitumor immunity by adenoviral-mediated transfer of CD40 ligand to treat HCC in rats. In vitro infection of a rat HCC cell line (McA-RH7777) with adenoviral vector expressing CD40 ligand (AdCMVmCD40L) induced CD40L expression in a dose-dependent manner. Expression of CD40L in McA-RH7777 cells did not alter their growth rate in vitro, but it abrogated their tumorigenicity when CD40L-expressing cells were implanted into the liver of syngenic Buffalo rats. In vivo gene therapy of established orthotopic HCC nodules (6.5 mm in diameter) in Buffalo rats by intratumor injection of AdCMVmCD40L vector led to complete tumor eradication and long-term survival in 69.5% of treated animals. Therapy with AdCMVmCD40L induced strong lymphocytic infiltration of the tumoral tissue and increased apoptosis of malignant cells. The observed antitumoral effect was mediated by CD8(+) T cells and was associated with increased interleukin (IL)-12 serum levels and enhanced natural killer (NK) activity. Animals that eliminated the tumor after in vivo gene therapy developed protective antitumor immunity being resistant to rechallenge with neoplastic cells. Toxicity of the therapy with AdCMVmCD40L was slight, with only a transient increase in the level of serum transaminases and minor lymphocyte infiltration of normal liver tissue. These data demonstrate that intratumoral administration of AdCMVmCD40L may provide an efficient and safe treatment for HCC.

Adenoviridae↗

PubMed2001

Rapid identification of Escherichia coli microcin J25 producing strains using polymerase chain reaction and colony blot hybridization.

To screen, isolate, and characterize bacterial populations producing microcin J25, we report here two rapid, reliable, and sensitive methods, using polymerase chain reaction and colony blot hybridization with a digoxigenin-labelled probe. A sample of 26 Escherichia coli strains isolated from poultry intestinal contents was evaluated to detect the sequence of mcjA, the gene encoding the MccJ25 precursor. The two molecular techniques were compared with the commonly used cross-immunity tests. They generate accurate data with no obvious cross-reactions with other microcins. The results display that the producers of MccJ25 were widely distributed in the poultry intestinal habitat. The applications of these molecular methods will be useful in future studies of microcinogenic populations, and thus contribute to understand the relationships within the complex intestinal microbial ecosystem.

Animals↗

PubMed2000

Dynamics of a bouncing ball in human performance.

On the basis of a modified bouncing-ball model, we investigated whether human movements utilize principles of dynamic stability in their performance of a similar movement task. Stability analyses of the model provided predictions about conditions indicative of a dynamically stable period-one regime. In a series of experiments, human subjects bounced a ball rhythmically on a racket and displayed these conditions supporting that they attuned to and exploited the dynamic stability properties of the task.

Algorithms↗

PubMed2000

The external calcium-dependent NADPH dehydrogenase from Neurospora crassa mitochondria.

We have inactivated the nuclear gene coding for a putative NAD(P)H dehydrogenase from the inner membrane of Neurospora crassa mitochondria by repeat-induced point mutations. The respiratory rates of mitochondria from the resulting mutant (nde-1) were measured, using NADH or NADPH as substrates under different assay conditions. The results showed that the mutant lacks an external calcium-dependent NADPH dehydrogenase. The observation of NADH and NADPH oxidation by intact mitochondria from the nde-1 mutant suggests the existence of a second external NAD(P)H dehydrogenase. The topology of the NDE1 protein was further studied by protease accessibility, in vitro import experiments, and in silico analysis of the amino acid sequence. Taken together, it appears that most of the NDE1 protein extends into the intermembrane space in a tightly folded conformation and that it remains anchored to the inner mitochondrial membrane by an N-terminal transmembrane domain.

Adenosine Triphosphate↗

PubMed2000

On the fractal properties of natural human standing.

We analyzed the temporal evolution of the displacement of the center of pressure (COP) during prolonged unconstrained standing (30 min) in non-impaired human subjects. The COP represents the collective outcome of the postural control system and the force of gravity and is the main parameter used in studies on postural control. Our analysis showed that the COP displacement during human standing displays fractal properties that were quantified by the Hurst exponent obtained from the classical rescaled adjusted range analysis. The average fractal or Hurst exponent (H) was 0.35+/-0.06. The presence of long-range correlations from a few seconds to several minutes due to the fractal characteristics of the postural control system has several important implications for the analysis of human balance.

Adult↗

PubMed2000

Intratumoral coinjection of two adenoviruses, one encoding the chemokine IFN-gamma-inducible protein-10 and another encoding IL-12, results in marked antitumoral synergy.

We have constructed a recombinant defective adenovirus that expresses functional murine IFN-gamma-inducible protein-10 (IP-10) chemokine (AdCMVIP-10). Injection of AdCMVIP-10 into s.c. tumor nodules derived from the CT26 murine colorectal adenocarcinoma cell line displayed some antitumor activity but it was not curative in most cases. Previous studies have shown that injection of similar s. c. CT26 tumor nodules with adenovirus-encoding IL-12 (AdCMVIL-12) induces tumor regression in nearly 70% of cases in association with generation of antitumor CTL activity. AdCMVIP-10 synergizes with the antitumor effect of suboptimal doses of AdCMVIL-12, reaching 100% of tumor eradication not only against injected, but also against distant noninjected tumor nodules. Colocalization of both adenoviruses at the same tumor nodule was required for the local and distant therapeutic effects. Importantly, intratumoral gene transfer with IL-12 and IP-10 generated a powerful tumor-specific CTL response in a synergistic fashion, while both CD4 and CD8 T cells appeared in the infiltrate of regressing tumors. Moreover, the antitumor activity of IP-10 plus IL-12 combined gene therapy was greatly diminished by simultaneous in vivo depletion of CD4+ and CD8+ T cells but was largely unaffected by single depletion of each T cell subset. An important role for NK cells was also suggested by asialo GM1 depletion experiments. From a clinical point of view, the effects of IP-10 permit one to lower the required gene transfer level of IL-12, thus preventing dose-dependent IL-12-mediated toxicity while improving the therapeutic efficacy of the elicited antitumor response.

Adenoviridae↗

PubMed2000

Adenoviral gene transfer of interleukin 12 into tumors synergizes with adoptive T cell therapy both at the induction and effector level.

Tumors infected with a recombinant defective adenovirus expressing interleukin 12 (IL-12) undergo regression, associated with a cytotoxic T lymphocyte (CTL)-mediated antitumor immune response. In the present study we generated anti-CT26 CTLs by short-term coculture of CT26 cells and lymph node cells obtained from mice harboring subcutaneous CT26 tumors injected with an adenoviral vector expressing IL-12 (AdCMVIL-12), control adenovirus (AdCMVlacZ), or saline. Regression of small intrahepatic CT26 tumors in unrelated syngeneic animals was achieved with CTLs derived from mice whose subcutaneous tumors had been injected with AdCMVIL-12 but not with CTLs from the other two control groups. The necessary and sufficient effector cell population for adoptive transfer consisted of CD8+ T cells that showed anti-CT26 specificity partly directed against the AH1 epitope presented by H-2Ld. Interestingly, treatment of a subcutaneous tumor nodule with AdCMVIL-12, combined with intravenous adoptive T cell therapy with short-term CTL cultures, had a marked synergistic effect against large, concomitant live tumors. Expression of IL-12 in the liver in the vicinity of the hepatic tumor nodules, owing to spillover of the vector into the systemic circulation, appeared to be involved in the increased in vivo antitumor activity of injected CTLs. In addition, adoptive T cell therapy improved the outcome of tumor nodules transduced with suboptimal doses of AdCMVIL-12. Our data provide evidence of a strong synergy between gene transfer of IL-12 and adoptive T cell therapy. This synergy operates both at the induction and effector phases of the CTL response, thus providing a rationale for combined therapeutic strategies for human malignancies.

Adenoviridae↗

PubMed2000

Analysis of a binding difference between the two dsRNA-binding domains in TRBP reveals the modular function of a KR-helix motif.

Double-stranded RNA-binding proteins constitute a large family with conserved domains called dsRBDs. One of these, TRBP, a protein that binds HIV-1 TAR RNA, has two dsRBDs (dsRBD1 and dsRBD2), as indicated by computer sequence homology. However, a 24-amino-acid deletion in dsRBD2 completely abolishes RNA binding, suggesting that only one domain is functional. To analyse further the similarities and differences between these domains, we expressed them independently and measured their RNA-binding affinities. We found that dsRBD2 has a dissociation constant of 5.9 x 10-8 M, whereas dsRBD1 binds RNA minimally. Binding analysis of 25-amino-acid peptides in TRBP and other related proteins showed that only one peptide in TRBP and one in Drosophila Staufen bind TAR and a GC-rich TAR-mimic RNA. Whereas a 25-mer peptide derived from dsRBD2 (TR5) bound TAR RNA, the equivalent peptide in dsRBD1 (TR6) did not. Molecular modelling indicates that this difference can mainly be ascribed to the replacement of Arg by His residues. Mutational analyses in homologous peptides also show the importance of residues K2 and L3. Analysis of 15-amino-acid peptides revealed that, in addition to TR13 (from TRBP dsRBD2), one peptide in S6 kinase has RNA-binding properties. On the basis of previous and the present results, we can define, in a broader context than that of TRBP, the main outlines of a modular KR-helix motif required for binding TAR. This structural motif exists independently from the dsRBD context and therefore has a modular function.

Amino Acid Sequence↗

PubMed2000

Stabilographic analysis of unconstrained standing.

Natural standing is characterized by postural changes and several hypotheses have been proposed to explain these changes. In this paper, four hypotheses were investigated by quantifying the number of postural changes in the centre of pressure data from unconstrained standing in different experimental conditions, studying the effects of mechanical loading, visual conditions, and type of support surface and sole of the shoes. The subjects stood for 30 min with no specific instructions other than not to step off a force plate. There were no significant effects on the number of centre of pressure patterns associated with the postural changes due to load, vision, surface and shoes during standing; on average, approximately two centre of pressure patterns per minute were observed in all conditions. The analysis of the centre of pressure data by the commonly used statistical parameters (standard deviation, velocity, and mean frequency of the centre of pressure displacement and area of the stabilogram) also did not reveal any effect of the different conditions.

Adult↗