RESUMEN
OBJECTIVE: To evaluate the safety and efficacy of intravitreal bevacizumab (Avastin) as treatment for choroidal neovascularisation (CNV) associated with angioid streaks METHODS: A non-randomised, interventional case series conducted on eyes with subfoveal CNV associated with angioid streaks. Intravitreal bevacizumab (1.25 mg in 0.05 ml) was injected into nine eyes of six patients between August 2005 and December 2007. Treatment efficacy was assessed based on pre- and post-treatment visual acuity and optical coherence tomography (OCT). RESULTS: With a mean follow-up of 19 months (range 10 to 28 months), the best corrected visual acuity improved by three or more lines in four eyes (44.4%), remained within two lines of baseline in four eyes (44.4%) and decreased by three or more lines in one eye (11.1%). Central foveal thickness (CFT) measured by OCT decreased an average of 67.7 microm (range +11 to -175 microm) with an average improvement in standardised change in macular thickening of 46.6% (range -12% to +84.5%). No injection-related complications or drug-related side effects were observed. CONCLUSIONS: Intravitreal bevacizumab for the treatment of subfoveal CNV secondary to angioid streaks mildly reduced central foveal thickness with a trend toward stabilisation of visual acuity. Additional follow-up and a larger patient cohort are needed to evaluate the long-term effects of this treatment.
Asunto(s)
Inhibidores de la Angiogénesis/administración & dosificación , Estrías Angioides/complicaciones , Anticuerpos Monoclonales/administración & dosificación , Neovascularización Coroidal/tratamiento farmacológico , Anciano , Anciano de 80 o más Años , Inhibidores de la Angiogénesis/efectos adversos , Anticuerpos Monoclonales/efectos adversos , Anticuerpos Monoclonales Humanizados , Bevacizumab , Neovascularización Coroidal/complicaciones , Neovascularización Coroidal/fisiopatología , Femenino , Angiografía con Fluoresceína , Fóvea Central/fisiología , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Tomografía de Coherencia Óptica , Resultado del Tratamiento , Agudeza Visual/efectos de los fármacosRESUMEN
BACKGROUND: It is generally accepted that, during evolution, replicating RNA molecules emerged from pools of random polynucleotides. This prebiotic RNA world was followed by an era of RNA-mediated catalysis of amide-bond formation. RNA would thus have provided the machinery responsible for the assembly of peptides and the beginning of the protein world of today. Naturally occurring ribozymes, which catalyze the cleavage or ligation of oligonucleotide phosphodiester bonds, support the idea that RNA could self-replicate. But was RNA constrained to this path and were RNA-acylated carriers required before RNA could catalyze the formation of amide bonds? RESULTS: We have isolated RNA catalysts that are capable of mediating amide-bond synthesis without the need for specifically designed templates to align the substrates, and we have kinetically characterized these catalysts. The rate enhancement observed for these RNA amide synthases exceeds the noncatalyzed amidation rate by a factor of approximately 10(4). In addition, Cu2+ ions caused a change in the affinity of RNA for the substrate rather than being directly involved in amide-bond formation. CONCLUSIONS: The discovery of these new amide synthases shows how functionally modified nucleic acids can facilitate covalent-bond formation without templating. Previously unforeseen RNA-evolution pathways can, therefore, be considered; for example, to guide amide-bond formation, en route to the protein world, it appears that substrate-binding pockets were formed that are analogous to those of protein enzymes.
Asunto(s)
Amida Sintasas/biosíntesis , Amida Sintasas/metabolismo , ARN Catalítico/metabolismo , Amida Sintasas/química , Secuencia de Bases , Cationes Bivalentes , Cobre/metabolismo , Evolución Molecular Dirigida/métodos , Cinética , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Catalítico/química , ARN Catalítico/aislamiento & purificación , Homología de Secuencia de Ácido Nucleico , Especificidad por SustratoRESUMEN
Using the systematic evolution of ligands by exponential enrichment (SELEX) method, we have identified oligonucleotides that bind to human IgE with high affinities and high specificity. These ligands were isolated from three pools of oligonucleotides, each representing 10(15) molecules: two pools contained 2'-NH2 pyrimidine-modified RNA with either 40 or 60 randomized sequence positions, and the third pool contained ssDNA with 40 randomized sequence positions. Based on sequence and structure similarities, these oligonucleotide IgE ligands were grouped into three families: 2'-NH2 RNA group A ligands are represented by the 35-nucleotide truncate IGEL1.2 (Kd = 30 nM); 2'-NH2 RNA group B ligands by the 25-nucleotide truncate IGEL2.2 (Kd = 35 nM); and the ssDNA group ligands by the 37-nucleotide truncate DI 7.4 (Kd = 10nM). Secondary structure analysis suggests G quartets for the 2'-NH2 RNA ligands, whereas the ssDNA ligands appear to form stem-loop structures. Using rat basophilic leukemia cells transfected with the human high-affinity IgE receptor Fc epsilon RI, we demonstrate that ligands IGEL1.2 and D17.4 competitively inhibit the interaction of human IgE with Fc1 epsilon RI. Furthermore, this inhibition is sufficient to dose-dependently block IgE-mediated serotonin release from cells triggered with IgE-specific Ag or anti-IgE Abs. Therefore, these oligonucleotide ligands represent a novel class of IgE inhibitors that may prove useful in the fight against allergic diseases.
Asunto(s)
Afinidad de Anticuerpos/efectos de los fármacos , Inmunoglobulina E/química , Oligonucleótidos/química , Oligonucleótidos/farmacología , Receptores de IgE/antagonistas & inhibidores , Receptores de IgE/química , Secuencia de Bases , Unión Competitiva/genética , Unión Competitiva/inmunología , ADN/química , Humanos , Ligandos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , Receptores de IgE/genética , Antagonistas de la Serotonina/inmunologíaRESUMEN
Transposition of Tn5 requires the binding of the transposase protein to the transposon outside end (OE) DNA sequences. Transposase mutants that increase the transposition frequency result in the formation of two distinct transposase/OE DNA complexes, observed by gel retardation analysis. The slower migrating complex I, also formed by wild-type transposase, contains protein oligomers of transposase and transposase related proteins. The faster migrating, novel complex II is caused by the binding of monomeric, proteolytic transposase fragments gamma and delta that have lost the carboxy-terminus of the protein. Transposase gamma and delta bind OE DNA with a high apparent affinity but are unable to promote transposition in vivo. We propose that the transposase protein is functionally unstable and can undergo a conformational change that reduces the activity but protects the protein from proteolysis. The transposase mutants favor the more active but proteolytically hypersensitive protein conformation.
Asunto(s)
Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN/fisiología , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleotidiltransferasas/metabolismo , Proteínas Bacterianas/genética , Elementos Transponibles de ADN/genética , ADN Bacteriano/química , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Modelos Genéticos , Mutagénesis Sitio-Dirigida , Nucleotidiltransferasas/genética , Fenotipo , TransposasasRESUMEN
Tn5 is a composite transposon consisting of two IS50 sequences in inverted orientation with respect to a unique, central region encoding several antibiotic resistances. The IS50R element encodes two proteins in the same reading frame which regulate the transposition reaction: the transposase (Tnp), which is required for transposition, and an inhibitor of transposition (Inh). The inhibitor is a naturally occurring deletion variant of Tnp which lacks the N-terminal 55 amino acids. In this report, we present the purification of both the Tnp and Inh proteins and an analysis of their DNA binding properties. Purified Tnp, but not Inh, was found to bind specifically to the outside end of Tn5. Inh, however, stimulated the binding activity of Tnp to outside-end DNA and was shown to be present with Tnp in these bound complexes. Inh was also found to exist as a dimer in solution. These results indicate that the N-terminal 55 amino acids of Tnp are required for sequence-specific binding. They also suggest that Inh inhibits transposition by forming mixed oligomers with Tnp which still bind to the ends of the transposon but are defective for later stages of the transposition reaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Nucleotidiltransferasas/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Cromatografía en Gel , Clonación Molecular , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Immunoblotting , Nucleotidiltransferasas/biosíntesis , Nucleotidiltransferasas/aislamiento & purificación , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TransposasasRESUMEN
Transposition of Tn5 in Escherichia coli is regulated by two transposon-encoded proteins: transposase (Tnp), promoting transposition preferentially in cis, and the trans-acting inhibitor (Inh). Two separate transposase mutants were isolated that replace glutamate with lysine at position 110 (EK110) and at position 345 (EK345). The EK transposase proteins increase the Tn5 transposition frequency 6- to 16-fold in cis and enhance the ability of transposase to act in trans. The purified mutant transposase proteins interact with transposon outside end DNA differently from the wild-type protein, resulting in the formation of a novel complex in gel retardation assays. During characterization of the transposase proteins in the absence of inhibitor, we found that wild-type transposase itself has a transposition-inhibiting function and that this inhibition is reduced for the mutant proteins. We present a model for the regulation of Tn5 transposition, which proposes the existence of two transposase species, one cis-activating and the other trans-inhibiting. The phenotype of the EK transposase mutants can be explained by a shift in the ratio of these two species.