RESUMEN
Differential gene regulation in the human pathogen Neisseria meningitidis group B (MenB) and in Neisseria lactamica, a human commensal species, was studied by whole genome microarray after bacterial interaction with epithelial cells. Host-cell contact induced changes in the expression of 347 and 285 genes in MenB and N. lactamica, respectively. Of these, only 167 were common to MenB and N. lactamica, suggesting that a different subset of genes is activated by pathogens and commensals. Change in gene expression was stable over time in N. lactamica, but short-lived in MenB. A large part (greater than 30%) of the regulated genes encoded proteins with unknown function. Among the known genes, those coding for pili, capsule, protein synthesis, nucleotide synthesis, cell wall metabolism, ATP synthesis, and protein folding were down-regulated in MenB. Transporters for iron, chloride and sulfate, some known virulence factors, GAPDH and the entire pathway of selenocysteine biosynthesis were upregulated. Gene expression profiling indicates that approximately 40% of the regulated genes encode putative surface-associated proteins, suggesting that upon cell contact Neisseria undergoes substantial surface remodeling. This was confirmed by FACS analysis of adhering bacteria using mouse sera against a subset of recombinant proteins. Finally, a few surface-located, adhesion-activated antigens were capable of inducing bactericidal antibodies, indicating that microarray technology can be exploited for the identification of new vaccine candidates.
Asunto(s)
Neisseria meningitidis Serogrupo B/genética , Neisseria meningitidis Serogrupo B/patogenicidad , Neisseria/genética , Neisseria/patogenicidad , Adhesión Bacteriana/genética , Proteínas Bacterianas/genética , Vacunas Bacterianas/aislamiento & purificación , Línea Celular , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Humanos , Neisseria/inmunología , Neisseria meningitidis Serogrupo B/inmunología , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
The most popular approach for proteomics analysis is based on the combination of two-dimensional gel electrophoresis and mass spectrometry (MS). Although very effective, the method suffers from a number of limitations, the most serious one being the necessity to have expensive and sophisticated instrumentation requiring handling by skilled personnel. Here we propose an alternative approach which may offer some advantages over the current methods, at least for some specific applications. The method is based on two-dimensional gel separation of radiolabeled synthetic proteins derived from transcription/translation reactions of linear polymerase chain reaction amplified genes. The gel is autoradiographed and this is superimposed on the sample gel whose protein spots have to be identified. Matching between autoradiographs and sample gel spots allows immediate protein identification. The method has been validated identifying six proteins from a membrane protein preparation of Neisseria meningitidis MC58 strain. All proteins were correctly identified as judged by confirmation analysis with MS. The approach is particularly useful when a specific subset of proteins needs to be identified in a complex protein mixture.
Asunto(s)
Proteínas Bacterianas/química , Clonación Molecular , Electroforesis en Gel Bidimensional/métodos , Reacción en Cadena de la Polimerasa/métodos , Biosíntesis de Proteínas , Proteínas/química , Animales , Biología Computacional/métodos , Bases de Datos como Asunto , Vectores Genéticos , Concentración de Iones de Hidrógeno , Procesamiento de Imagen Asistido por Computador , Espectrometría de Masas , Modelos Biológicos , Neisseria meningitidis/metabolismo , Plásmidos/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transcripción GenéticaRESUMEN
Paracetamol/Eudragit RS, paracetamol/ethylcellulose, and paracetamol/cellulose acetate pellets of different drug/polymer ratios (w/w) were prepared by the dissolution/solvent evaporation technique. These pellets were then characterized by particle size distribution analysis, ultraviolet (UV) spectroscopy, differential thermal analysis, and scanning electron microscopy (SEM). Hard gelatin capsules were filled with each particle size fraction of these pellets, and in vitro dissolution studies were performed to verify the capability of each series of pellets to control drug release. Pellets were spherical, presented a polynucleated microcapsule structure, and under certain experimental conditions, the yield of the preparation process reached very high values. The dissolution studies pointed out the slow paracetamol release from these pellets.
Asunto(s)
Acetaminofén , Resinas Acrílicas , Analgésicos no Narcóticos , Celulosa/análogos & derivados , Química Farmacéutica/métodos , Tecnología Farmacéutica , Preparaciones de Acción Retardada , Implantes de Medicamentos , Emulsiones , Ácidos Polimetacrílicos , Soluciones EsclerosantesRESUMEN
DNA immunization is a very promising approach to the formulation of multivalent vaccines. However, little information is currently available on the immunogenicity of multi-plasmid formulations. To address this issue, we immunized mice with a combination of four plasmids encoding malarial antigens and we compared antibody responses with those obtained with single-plasmid injections. We found that when four plasmids encoding Plasmodium falciparum circumsporozoite protein, thrombospondin-related anonymous protein, major merozoite surface protein (MSP)1 and Pfs25 are co-injected into mice, Ab responses against each antigen are elicited at levels at least as high as the level obtained with single-plasmid injection. The quality of antibody production, as determined by isotype analysis, was similar when single- and multi-plasmid administrations were compared, indicating the priming of the same cytokine profile for CD4+ T helper cells. The sera from mice immunized with the four-plasmid formulation specifically recognized sporozoites, blood stage schizonts and gametes, indicating that DNA immunization induced antibody responses relevant to the native conformation. Finally and of particular interest, in the case of MSP1, the antibody response appears to be strongly potentiated by the presence of additional plasmids, indicating an adjuvant effect of DNA.
Asunto(s)
Especificidad de Anticuerpos , Antígenos de Protozoos/inmunología , ADN/inmunología , Plásmidos/inmunología , Plasmodium falciparum/inmunología , Vacunación , Animales , Antígenos de Protozoos/genética , Linfocitos T CD4-Positivos/inmunología , ADN/administración & dosificación , Femenino , Inmunidad , Proteína 1 de Superficie de Merozoito , Ratones , Ratones Endogámicos BALB C , Precursores de Proteínas/genética , Precursores de Proteínas/inmunología , Proteínas Protozoarias/genética , Proteínas Protozoarias/inmunologíaRESUMEN
A series of mutants of human IL-1 receptor antagonist (IL-1ra) has been designed by comparison of IL-1ra and IL-1beta structures in order to increase receptor antagonist capacity. Upon in vitro and in vivo assay of IL-1 antagonism, the IL-1ra mutants DoB 0039 (N91-->R), DoB 0040 (T109-->A) and DoB 0041 (N91/T109-->R/A) could inhibit IL-1beta effects more efficiently than wild-type IL-1ra, with DoB 0041 being the most active. Analysis of the receptor-binding capacity of the IL-1ra mutants showed that all three mutants could inhibit binding of IL-1alpha or IL-1beta to IL-1RI-bearing cells more efficiently than wild-type IL-1ra. Conversely, binding of IL-1beta to IL-1RII-bearing cells could be inhibited by DoB 0041 much less efficiently than by wild-type IL-1ra. It is known that the two types of IL-1 receptors (IL-1RI and IL-1RII) play different roles in the regulation of IL-1 activity, with IL-1RI being solely responsible for cell triggering upon IL-1 binding, whereas IL-1RII acts as a scavenger of IL-1 and can thus be considered as a natural IL-1 inhibitor. Thus, the enhanced inhibitory capacity of DoB 0041 as compared with wild-type IL-1ra is explained in terms of better binding to the activating receptor IL-1RI and poorer interaction with the inhibitory receptor IL-1RII.
Asunto(s)
Receptores de Interleucina-1/antagonistas & inhibidores , Sialoglicoproteínas/metabolismo , Animales , Línea Celular , Femenino , Humanos , Proteína Antagonista del Receptor de Interleucina 1 , Ratones , Ratones Endogámicos C3H , Mutación , Unión Proteica , Receptores de Interleucina-1/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Sialoglicoproteínas/genéticaRESUMEN
The N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, the enzyme used for the industrial production Of D-amino acids, was cloned, sequenced, and expressed in Escherichia coli. The protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidizing conditions and highly sensitive to hydrogen peroxide. To investigate the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site-directed mutagenesis in which the five residues were substituted by either Ala or Ser. Only the mutant carrying the Cys172 substitution was catalytically inactive, and the other mutants maintained the same specific activity as the wild type enzyme. The crucial role of Cys172 in enzymatic activity was also confirmed by chemical derivatization of the protein with iodoacetate. Furthermore, chemical derivatizations using both acrylamide and Ellman's reagent revealed that (i) none of the five cysteines is engaged in disulfide bridges, (ii) Cys172 is easily accessible to the solvent, (iii) Cys193 and Cys250 appear to be buried in the protein core, and (iv) Cys243 and Cys279 seem to be located within or in proximity of external loops and are derivatized under mild denaturing conditions. These data are discussed in light of the possible mechanisms of enzyme inactivation and catalytic reaction.
Asunto(s)
Amidohidrolasas/metabolismo , Cisteína , Rhizobium/enzimología , Alanina , Amidohidrolasas/biosíntesis , Amidohidrolasas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Cromatografía Líquida de Alta Presión , Cartilla de ADN , Genes Bacterianos , Yodoacetatos/farmacología , Ácido Yodoacético , Cinética , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Plásmidos , Mutación Puntual , Proteínas Recombinantes/análisis , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , SerinaRESUMEN
Upon structure comparison between IL-1 beta and its antagonist IL-1ra, single or multiple residues along the IL-1 beta sequence were replaced with the corresponding amino acids present in the IL-1ra protein, in the attempt to identify sites important for receptor binding and for biologic activity on the two molecules. Ten of fifteen mutant proteins had activity comparable to that of wild-type IL-1 beta in three different biologic assays and in receptor binding, indicating that the introduced changes did not influence the functional structure of the protein. Conversely, three mutants (SMIL-9: 127/263 R/T-->W/Y; SMIL-10: 125/127/263/265 T/R/T/Q-->R/W/Y/E; SMIL-15:222/227 I/E-->S/S) showed an increased binding capacity for IL-1RI, not paralleled by increased agonist activity, indicating that the introduced IL-1ra residues could be involved in the nonagonist IL-1RI binding site. On the other hand, two mutants showed diminished binding capacity with concomitant decrease in biologic activity. Both mutants (SMIL-1, five substitutions in the loop 202-214; and SMIL-3, total replacement of the loop 164-173 with the IL-1ra stretch 52-55) included substitutions of residues allegedly important for agonist binding to IL-1RI. Mutant SMIL-3 showed the most profound reduction in binding capacity for IL-1RI (CDw121a) and a more than 1,000-fold reduced biologic activity both in vitro and in vivo, but it retained full capacity of binding to IL-1RII (CDw121b) and acted as a selective antagonist of IL-1RII. From these results the following conclusions can be drawn. IL-1 beta binds to IL-1RI and to IL-1RII through different sites, and the loop 164-173 appears as one of the areas involved in the selective interaction with IL-1RI. Agonist (IL-1 beta) and nonagonist (IL-1ra) binding to IL-1RI occur through distinct sites, with loops 164-173 and 202-214 of IL-1 beta identified as two of the sites selectively involved in agonist binding to the activating receptor.