RESUMEN
The Yang-Baxter equation is an important tool in theoretical physics, with many applications in different domains that span from condensed matter to string theory. Recently, the interest on the equation has increased due to its connection to quantum information processing. It has been shown that the Yang-Baxter equation is closely related to quantum entanglement and quantum computation. Therefore, owing to the broad relevance of this equation, besides theoretical studies, it also became significant to pursue its experimental implementation. Here, we show an experimental realization of the Yang-Baxter equation and verify its validity through a Nuclear Magnetic Resonance (NMR) interferometric setup. Our experiment was performed on a liquid state Iodotrifluoroethylene sample which contains molecules with three qubits. We use Controlled-transfer gates that allow us to build a pseudo-pure state from which we are able to apply a quantum information protocol that implements the Yang-Baxter equation.
RESUMEN
We have produced and characterized spin-squeezed states at a temperature of 26 °C in a nuclear magnetic resonance quadrupolar system. The experiment was carried out on 133Cs nuclei of spin I=7/2 in a sample of lyotropic liquid crystal. The source of spin squeezing was identified as the interaction between the quadrupole moment of the nuclei and the electric field gradients present within the molecules. We use the spin angular momentum representation to describe formally the nonlinear operators that produce the spin squeezing on a Hilbert space of dimension 2I+1=8. The quantitative and qualitative characterization of this spin-squeezing phenomenon is expressed by a squeezing parameter and squeezing angle developed for the two-mode Bose-Einstein condensate system, as well as by the Wigner quasiprobability distribution function. The generality of the present experimental scheme points to potential applications in solid-state physics.
RESUMEN
We examined whether bovine monocyte-derived and bone marrow (BM) dendritic cells (DCs) regulate antibody production in activated peripheral blood B cells. DCs were generated from monocytes and BM progenitors in the presence of bovine recombinant granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4). Monocyte-derived DCs promoted B cells activated by the anti-CD3 triggered CD4(+) T cells or through immunoglobulin M (IgM) receptor to increase the level of IgG secretion. Furthermore, the addition of DCs triggered B cells activated through IgM receptors to produce IgG2 and IgA, thus inducing an isotype switch. BM-derived DCs increased the production of IgG in B cells activated by the anti-CD3 triggered CD4(+) T cells, but unlike monocyte-derived DCs did not have any effect on B cells activated through surface IgM. These data suggest that the regulation of humoral immune responses in cattle depends on the origin of DCs and the mode of B cell activation.
Asunto(s)
Linfocitos B/inmunología , Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Inmunoglobulinas/biosíntesis , Monocitos/inmunología , Animales , Células de la Médula Ósea/citología , Complejo CD3/inmunología , Linfocitos T CD4-Positivos/inmunología , Bovinos , Diferenciación Celular , Células Cultivadas , Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Inmunoglobulina A/inmunología , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Interleucina-4/inmunología , Monocitos/citologíaRESUMEN
We have explored the potential of Trypanosoma brucei as a eukaryotic expression system. Procyclic forms, which correspond to an insect-adapted stage, can easily be cultured in vitro. The cells grow to densities approximately 10-fold greater than higher eukaryotic cells and are not infectious for mammals. An expression vector which can stably integrate into the genome was used to express high levels of recombinant bovine interleukin-4 (IL-4). Trypanosome-derived IL-4 is released into the medium and is biologically active. The recombinant protein down-regulates CD14 expression in human macrophages and inhibits NO production by stimulated bovine macrophages.
Asunto(s)
Interleucina-4/biosíntesis , Trypanosoma brucei brucei/genética , Animales , Bovinos , Vectores Genéticos , Humanos , Interleucina-4/genética , Interleucina-4/farmacología , Receptores de Lipopolisacáridos/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Nitritos/metabolismo , Proteínas Recombinantes/biosíntesis , Trypanosoma brucei brucei/metabolismoRESUMEN
Transmission of Trypanosoma brucei by the tsetse fly entails several rounds of differentiation as the parasite migrates through the digestive tract to the salivary glands of its vector. Differentiation of the bloodstream to the procyclic form in the fly midgut is accompanied by the synthesis of a new coat consisting of EP and GPEET procyclins. There are three closely related EP isoforms, two of which (EP1 and EP3) contain N-glycans. To identify the individual EP isoforms that are expressed early during synchronous differentiation in vitro, we exploited the selective extraction of GPI-anchored proteins and mass spectrometry. Unexpectedly, we found that GPEET and all isoforms of EP were coexpressed for a few hours at the onset of differentiation. At this time, the majority of EP1 and EP3 molecules were already glycosylated. Within 24 hours, GPEET became the major surface component, to be replaced in turn by glycosylated forms of EP, principally EP1, at a later phase of development. Transient transfection experiments using reporter genes revealed that each procyclin 3' untranslated region contributes to differential expression as the procyclic form develops. We postulate that programmed expression of other procyclin species will accompany further rounds of differentiation, enabling the parasite to progress through the fly.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/crecimiento & desarrollo , Moscas Tse-Tse/parasitología , Regiones no Traducidas 3'/genética , Secuencia de Aminoácidos , Animales , Diferenciación Celular , Genes Protozoarios/genética , Genes Reporteros/genética , Glicosilfosfatidilinositoles/metabolismo , Espectrometría de Masas , Glicoproteínas de Membrana/genética , Datos de Secuencia Molecular , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Protozoarias/genética , Análisis de Secuencia de ADN , Transfección , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/metabolismo , Moscas Tse-Tse/anatomía & histologíaRESUMEN
Procyclins are the major surface glycoproteins of insect-form Trypanosoma brucei. The procyclin expression sites are polycistronic and are transcribed by an alpha-amanitin-resistant polymerase, probably RNA polymerase I (Pol I). The expression sites are flanked by transcription units that are sensitive to alpha-amanitin, which is a hallmark of Pol II-driven transcription. We have analysed a region of 9.5 kb connecting the EP/PAG2 expression site with the downstream transcription unit. The procyclin expression site is longer than was previously realized and contains an additional gene, procyclin-associated gene 4 (PAG4), and a region of unknown function, the T region, that gives rise to trans-spliced, polyadenylated RNAs containing small open reading frames (ORFs). Two new genes, GU1 and GU2, were identified in the downstream transcription unit on the opposite strand. Unexpectedly, the 3' untranslated region of GU2 and the complementary T transcripts overlap by several hundred base pairs. Replacement of GU2 by a unique tag confirmed that sense and antisense transcription occurred from a single chromosomal locus. Overlapping transcription is stage specific and may extend > or = 10 kb in insect-form trypanosomes. The nucleotide composition of the T. brucei genome is such that antisense ORFs occur frequently. If stable mRNAs can be derived from both strands, the coding potential of the genome may be substantially larger than has previously been suspected.
Asunto(s)
ADN sin Sentido/genética , Proteínas Protozoarias/genética , Transcripción Genética , Trypanosoma brucei brucei/genética , Regiones no Traducidas 3' , Animales , Regulación de la Expresión Génica , Glicoproteínas/genética , Proteínas Asociadas a Microtúbulos/genética , Datos de Secuencia Molecular , Mutación , ARN Polimerasa I/genética , ARN Polimerasa I/metabolismo , Análisis de Secuencia de ADNRESUMEN
The activation of innate immune responses by genomic DNA from bacteria and several nonvertebrate organisms represents a novel mechanism of pathogen recognition. We recently demonstrated the CpG-dependent mitogenic activity of DNA from the protozoan parasite Babesia bovis for bovine B lymphocytes (W. C. Brown, D. M. Estes, S. E. Chantler, K. A. Kegerreis, and C. E. Suarez, Infect. Immun. 66:5423-5432, 1998). However, activation of macrophages by DNA from protozoan parasites has not been demonstrated. The present study was therefore conducted to determine whether DNA from the protozan parasites B. bovis, Trypanosoma cruzi, and T. brucei activates macrophages to secrete inflammatory mediators associated with protective immunity. DNA from Escherichia coli and all three parasites stimulated B-lymphocyte proliferation and increased macrophage production of interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-alpha), and nitric oxide (NO). Regulation of IL-12 and NO production occurred at the level of transcription. The amounts of IL-12, TNF-alpha, and NO induced by E. coli and protozoal DNA were strongly correlated (r2 > 0.9) with the frequency of CG dinucleotides in the genome, and immunostimulation by DNA occurred in the order E. coli > or = T. cruzi > T. brucei > B. bovis. Induction of inflammatory mediators by E. coli, T. brucei, and B. bovis DNA was dependent on the presence of unmethylated CpG dinucleotides. However, at high concentrations, E. coli and T. cruzi DNA-mediated macrophage activation was not inhibited following methylation. The recognition of protozoal DNA by B lymphocytes and macrophages may provide an important innate defense mechanism to control parasite replication and promote persistent infection.
Asunto(s)
Linfocitos B/inmunología , Babesia bovis/genética , ADN Protozoario/farmacología , Interleucina-12/biosíntesis , Activación de Linfocitos/efectos de los fármacos , Macrófagos/metabolismo , Mitógenos/farmacología , Óxido Nítrico/biosíntesis , Trypanosoma brucei brucei/genética , Trypanosoma cruzi/genética , Factor de Necrosis Tumoral alfa/biosíntesis , Animales , Bovinos , Metilación de ADN , Fosfatos de Dinucleósidos/farmacología , Interleucina-12/genética , Óxido Nítrico/genética , ARN Mensajero/análisis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Trypanosoma brucei, the protozoan parasite causing sleeping sickness, is transmitted by a tsetse fly vector. When the tsetse takes a blood meal from an infected human, it ingests bloodstream form trypanosomes that quickly differentiate into procyclic forms within the fly's midgut. During this process, the parasite loses the 10(7) molecules of variant surface glycoprotein that formed its surface coat, and it develops a new coat composed of several million procyclin molecules. Procyclins, the products of a small multigene family, are glycosyl phosphatidylinositol-anchored proteins containing characteristic amino acid repeats at the C terminus [either EP (EP procyclin, a form of procyclin rich in Glu-Pro repeats) or GPEET (GPEET procyclin, a form of procyclin rich in Glu-Pro-Glu-Glu-Thr repeats)]. We have used a sensitive and accurate mass spectrometry method to analyze the appearance of different procyclins during the establishment of midgut infections in tsetse flies. We found that different procyclin gene products are expressed in an orderly manner. Early in the infection (day 3), GPEET2 is the only procyclin detected. By day 7, however, GPEET2 disappears and is replaced by several isoforms of glycosylated EP, but not the unglycosylated isoform EP2. Unexpectedly, we discovered that the N-terminal domains of all procyclins are quantitatively removed by proteolysis in the fly, but not in culture. These findings suggest that one function of the protease-resistant C-terminal domain, containing the amino acid repeats, is to protect the parasite surface from digestive enzymes in the tsetse fly gut.
Asunto(s)
Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias , Trypanosoma brucei brucei/metabolismo , Moscas Tse-Tse/parasitología , Glicoproteínas Variantes de Superficie de Trypanosoma/metabolismo , Secuencia de Aminoácidos , Animales , Técnica del Anticuerpo Fluorescente , Glicoproteínas de Membrana/biosíntesis , Datos de Secuencia Molecular , Factores de TiempoRESUMEN
Many cell surface proteins are attached to membranes via covalent glycosylphosphatidylinositol (GPI) anchors that are posttranslationally linked to the carboxy-terminus of the protein. Removal of the GPI lipid moieties by enzymes such as GPI-specific phospholipases or by chemical treatments generates a soluble form of the protein that no longer associates with lipid bilayers. We have found that the removal of lipid moieties from the anchor can also have a second, unexpected effect on the antigenicity of a variety of GPI-anchored surface molecules, suggesting that they undergo major conformational changes. Several antibodies raised against GPI-anchored proteins from protozoa and mammalian cells were no longer capable of binding the corresponding antigens once the lipid moieties had been removed. Conversely, antibodies raised against soluble (delipidated) forms reacted poorly with intact GPI-anchored proteins, but showed enhanced binding after treatment with phospholipases. In the light of these findings, we have reevaluated a number of publications on GPI-anchored proteins. Many of the results are best explained by lipid-dependent changes in antigenicity, indicating this might be a widespread phenomenon. Since many pathogen surface proteins are GPI-anchored, researchers should be aware that the presence or absence of the GPI lipid moieties may have a major impact on the host immune response to infection or vaccination.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/fisiología , Animales , Anticuerpos , Glicosilfosfatidilinositoles/análisis , Mamíferos , Proteínas de la Membrana/inmunología , Conformación Proteica , Procesamiento Proteico-Postraduccional , Trypanosoma brucei brucei/fisiología , Trypanosoma congolense/fisiología , Glicoproteínas Variantes de Superficie de Trypanosoma/química , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunologíaAsunto(s)
Glicoproteínas de Membrana/genética , Proteínas Protozoarias/genética , Trypanosoma congolense/clasificación , Trypanosoma congolense/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Secuencia Conservada , Cartilla de ADN/genética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Mensajero/química , ARN Mensajero/genética , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
Differentiation is a means by which unicellular parasites adapt to different environments. In some cases, the developmental program may be modulated by interactions with the host, but the mechanisms are largely unknown. Trypanosoma brucei is transmitted between mammals by tsetse flies. The development of the procyclic form in the tsetse midgut is marked by the synthesis of a new glycoprotein coat, composed of EP and GPEET procyclins, that is important for survival. Here we demonstrate that the composition of the coat changes in response to extracellular signals in vitro and during development in vivo. EP and GPEET are coinduced when differentiation is initiated. Subsequently, EP expression is maintained, whereas GPEET is repressed after 7-9 days. The timepoint at which GPEET is repressed coincides with the appearance of parasites in a new compartment of the fly midgut. In culture, down-regulation of GPEET can be prevented by exogenous glycerol or accelerated by hypoxia. Regulation is post-transcriptional, and is conferred by the GPEET 3' untranslated region. The same sequence also regulates expression of a reporter gene in the fly. The finding that GPEET is expressed during a defined window during the establishment of infection suggests that it has a specific function in host-parasite interactions rather than a generalized role in shielding underlying membrane molecules.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Anaerobiosis , Animales , Ciclo Celular , Sistema Digestivo/parasitología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Glicerol/farmacología , Glicoproteínas/genética , Proteínas Protozoarias , Transcripción Genética , Transfección , Trypanosoma brucei brucei/genética , Moscas Tse-Tse/parasitologíaRESUMEN
Bloodstream forms (BSF) and procyclic culture forms (PCF) of African trypanosomes were incubated with a variety of lectins in vitro. Cessation of cell division and profound morphological changes were seen in procyclic forms but not in BSF after incubation with concanavalin A (Con A), wheat germ agglutinin and Ricinus communis agglutinin. These lectins caused the trypanosomes to cease division, become round and increase dramatically in size, the latter being partially attributable to the formation of what appeared to be a large 'vacuole-like structure' or an expanded flagellar pocket. Con A was used in all further experiments. Spectrophotometric quantitation of extracted DNA and flow cytometry using the DNA intercalating dye propidium iodide showed that the DNA content of Con A-treated trypanosomes increased dramatically when compared to untreated parasites. Examination of these cells by fluorescence microscopy showed that many of the Con A-treated cells were multinucleate whereas the kinetoplasts were mostly present as single copies, indicating a disequilibrium between nuclear and kinetoplast replication. Immunofluorescence experiments using monoclonal antibodies (mAb) specific for paraflagellar rod proteins and for kinetoplastid membrane protein-11 (KMP-11), showed that the Con A-treated parasites had begun to duplicate the flagellum but that this had only proceeded along part of the length of the cells, suggesting that the cell division process was initiated but that cytokinesis was subsequently inhibited. Tunicamycin-treated wild-type trypanosomes and mutant trypanosomes expressing both high levels of non-glycosylated procyclins and procyclin isoforms with truncated N-linked sugars were resistant to the effects of Con A, suggesting that N-linked carbohydrates on the procyclin surface coat were the ligands for Con A binding. This was supported by data obtained using mutant parasites created by deletion of all three EP procyclin isoforms, two of which contain N-glycosylation sites, by homologous recombination. The knockout mutants showed reduced binding of fluorescein-labelled Con A as determined by flow cytometry and were resistant to the effects of Con A. Taken together the results show that Con A induces multinucleation, a disequilibrium between nuclear and kinetoplast replication and a unique form of cell death in procyclic African trypanosomes and that the ligands for Con A binding are carbohydrates on the EP forms of procyclin. The possible significance of these findings for the life cycle of the trypanosomes in the tsetse fly vector is discussed.
Asunto(s)
Muerte Celular/fisiología , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias , Receptores de Superficie Celular/metabolismo , Trypanosoma brucei brucei/fisiología , Trypanosoma congolense/fisiología , Animales , Anticuerpos Monoclonales/inmunología , Concanavalina A/metabolismo , Medios de Cultivo , Citometría de Flujo , Lectinas/metabolismo , Glicoproteínas de Membrana/inmunología , Microscopía Electrónica , Trypanosoma brucei brucei/genética , Trypanosoma congolense/genética , Tunicamicina/farmacologíaRESUMEN
The expression of procyclins is the earliest known marker of differentiation of bloodstream forms of Trypanosoma brucei to procyclic forms. We have generated transgenic bloodstream and procyclic forms in which the coding region of one procyclin gene was replaced by E. coli beta-glucuronidase (GUS). GUS activity can be monitored in a simple one-step colour reaction in microtitre plates; this assay is potentially suitable for large-scale screening for compounds that influence differentiation. GUS was stage-specifically expressed in procyclic forms and its synthesis occurred in parallel with that of procyclin when bloodstream forms were triggered to differentiate by the addition of cis-aconitate. GUS could also be induced by brief treatment with the proteases trypsin, pronase or thermolysin, but not with pepsin or thrombin. Interestingly, a combination of one of the active proteases with cis-aconitate resulted in increased GUS activity relative to either trigger alone. In contrast to cis-aconitate, protease treatment resulted in considerable cell death. Experiments with the pleomorphic strain AnTat 1.1 showed that long slender bloodstream forms were rapidly killed by proteases, whereas stumpy forms were largely resistant. Stumpy forms treated with trypsin differentiated synchronously and expressed procyclin with faster kinetics than when they were triggered by cis-aconitate. As predicted by the GUS assay, differentiation was even more rapid when both inducers were used simultaneously, with all cells expressing maximal levels of procyclin within 3 h.
Asunto(s)
Glucuronidasa/genética , Glicoproteínas de Membrana/genética , Proteínas Protozoarias , Transgenes , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/genética , Ácido Aconítico/farmacología , Animales , Animales Modificados Genéticamente , Citratos/farmacología , Endopeptidasas/farmacología , Escherichia coli/enzimología , Escherichia coli/genética , Citometría de Flujo , Regulación del Desarrollo de la Expresión Génica , Glicoproteínas de Membrana/metabolismo , Trypanosoma brucei brucei/efectos de los fármacosRESUMEN
The surface coat of procyclic forms of Trypanosoma brucei consists of related, internally repetitive glycoproteins known as EP and GPEET procyclins. Previously we showed that the extracellular domain of GPEET is phosphorylated. We now show that phosphorylation of this glycosylphosphatidylinositol-anchored surface protein can be induced in vitro using a procyclic membrane extract. Using antibodies that recognize either the phosphorylated or unphosphorylated form of GPEET, we analyzed their expression during differentiation of bloodstream forms to procyclic forms. Unphosphorylated GPEET, together with EP, was detected in cell lysates 2-4 hours after initiating differentiation whereas phosphorylated GPEET only appeared after 24 hours. Surface expression of EP and both forms of GPEET occurred after 24-48 hours and correlated with the detection of phosphorylated GPEET on immuno-blots. Electron micrographs showed that unphosphorylated GPEET was predominantly in the flagellar pocket whereas the phosphorylated form was distributed over the cell surface. In contrast, expression of a membrane-bound human placental alkaline phosphatase in procyclic forms caused the accumulation of dephosphorylated GPEET on the cell surface, while the phosphorylated form was restricted to the flagellar pocket. A GPEET-Fc fusion protein, which was retained intracellularly, was not phosphorylated. We propose that unphosphorylated GPEET procyclin is transported to a location close to or at the cell surface, most probably the flagellar pocket, where it becomes phosphorylated. To the best of our knowledge, this study represents the first localization of phosphorylated and unphosphorylated forms of a GPI-anchored protein within a cell.
Asunto(s)
Glicosilfosfatidilinositoles/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Fosfatasa Alcalina/genética , Animales , Transporte Biológico , Membrana Celular , Expresión Génica , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Fragmentos Fc de Inmunoglobulinas/metabolismo , Glicoproteínas de Membrana/genética , Microscopía Electrónica , Fosforilación , Proteínas Protozoarias/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Trypanosoma brucei brucei/ultraestructuraRESUMEN
Genetic immunisation is a simple method for producing polyclonal antibodies in mice. By this method, we produced antibodies against bovine interleukin-4 (BoIL-4). After a final injection with a recombinant BoIL-4 protein, nine stable hybridoma cell lines were established which secreted monoclonal antibodies (MAbs) against this cytokine. Specific binding of each of the MAbs to recombinant BoIL-4 produced by Escherichia coli, baculovirus, and Trypanosoma brucei was demonstrated in an indirect ELISA and/or in Western blotting. These MAbs recognise the same antigenic region localised in the first 47 amino acids of the mature protein. None of them was able to neutralise the biological activity of the BoIL-4 under the conditions tested but one allowed the detection of BoIL-4 by flow cytometry.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Bovinos/inmunología , Interleucina-4/inmunología , Animales , Anticuerpos Monoclonales/química , Linfocitos B/inmunología , Baculoviridae/inmunología , Unión Competitiva/inmunología , Western Blotting/veterinaria , ADN Complementario/inmunología , Ensayo de Inmunoadsorción Enzimática/veterinaria , Citometría de Flujo/veterinaria , Herpesvirus Bovino 1/inmunología , Hibridomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Plásmidos/química , Trypanosoma brucei brucei/inmunologíaAsunto(s)
Regulación de la Expresión Génica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/genética , Animales , Genes Protozoarios , Estadios del Ciclo de Vida , Glicoproteínas de Membrana/química , Proteínas Protozoarias/química , Procesamiento Postranscripcional del ARN , Transcripción Genética , Trypanosoma brucei brucei/crecimiento & desarrollo , Trypanosoma brucei brucei/metabolismo , Trypanosoma congolense/genética , Trypanosoma congolense/metabolismo , Moscas Tse-Tse/parasitologíaRESUMEN
Cytokine-mediated modulation of nitric oxide (NO) production by bacteria-stimulated bovine macrophages was studied. When Salmonella dublin, as a prototypic gram-negative organism, was used, NO generation was barely enhanced by recombinant bovine and ovine IFN-gamma, but was suppressed by IL-4. Salmonella dublin-induced NO generation was not influenced by a panel of nine other cytokines. The panel included IL-1, tumour necrosis factor (TNF) and IFN-alpha, which are active in a similar mouse macrophage model. The tested cytokines were either homologous or known to interact with bovine cytokine receptors. Recombinant bovine and ovine IFN-gamma were the only cytokines which strongly enhanced NO synthesis by macrophages exposed to the gram-positive organism, Listeria monocytogenes. Listeria-induced NO generation was strongly suppressed by recombinant human and bovine IL-4, but not by IL-10 and transforming-growth-factor-beta. Thus, two cytokines characterizing a Th1 and a Th2 response up- and down-regulate, respectively, bacteria-induced NO generation in bovine macrophages, whereas nine other cytokines had little activity in this regard. This modulation was reflected in changes in the steady state levels of mRNA coding for inducible nitric oxide synthase. Combinations of IFN-gamma and IL-4 suggested that the relative proportion of these cytokines determined whether bacteria-induced NO generation was up- or down-regulated. At saturating IL-4 concentrations, stimulation of bacteria-induced NO generation in macrophages by T cell supernatants was solely dependent on IFN-gamma. This was shown by antibody neutralization experiments and by a close correlation between the capacity of supernatants to stimulate NO generation and the IFN-gamma content, as determined by immunoassay.
Asunto(s)
Interferón gamma/inmunología , Interleucina-4/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Óxido Nítrico Sintasa/metabolismo , Animales , Bovinos , Células Cultivadas , Medios de Cultivo Condicionados , Regulación hacia Abajo , Bacterias Gramnegativas/inmunología , Interferón-alfa/inmunología , Interferón gamma/genética , Interleucinas/inmunología , Listeria/inmunología , Macrófagos/microbiología , Pruebas de Neutralización , ARN Mensajero/metabolismo , Proteínas Recombinantes/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Células Th2/inmunología , Células Th2/metabolismo , Factor de Necrosis Tumoral alfa/inmunología , Regulación hacia ArribaRESUMEN
The surface of Trypanosoma brucei brucei insect forms is covered by an invariant protein coat consisting of procyclins. There are six or seven procyclin genes that encode unusual proteins with extensive tandem repeat units of glutamic acid (E) and proline (P) (referred to as EP repeats), and two genes that encode proteins with internal pentapeptide (GPEET) repeats. Although the EP forms of procyclins have been isolated and characterized by several laboratories, evidence for GPEET procyclin has largely been confined to the expression of its mRNA. To characterize GPEET procyclin further, we isolated the protein from T. b. brucei strain 427. We found that label from [3H]myristic acid and [3H]ethanolamine was incorporated into GPEET procyclin and we demonstrated the protein's covalent modification with a glycosylphosphatidylinositol anchor. The major form of GPEET procyclin showed an apparent molecular mass of 22-32 kDa, was susceptible to proteolytic treatment and was found to be phosphorylated. Surprisingly, our results show that GPEET procyclin represents the major form of procyclin in T. b. brucei 427 culture forms and that the ratio of EP to GPEET procyclin can vary considerably between different cell lines.