RESUMEN
Frameshift mutagenesis occurs through the misalignment of primer and template strands during DNA synthesis and involves DNA intermediates that contain one or more extrahelical bases in either strand of the DNA substrate. To investigate whether these DNA structures are recognized by the proofreading apparatus of DNA polymerases, time-resolved fluorescence spectroscopy was used to examine the interaction between the Klenow fragment of DNA polymerase I and synthetic DNA primer-templates containing extrahelical bases at defined positions within the template strand. A dansyl probe attached to the DNA was used to measure the fractional occupancies of the polymerase and 3'-5' exonuclease sites of the enzyme for DNA substrates with and without the extrahelical bases. The presence of an extrahelical base at the first position from the primer 3' terminus increased the level of partitioning of the DNA substrates into the 3'-5' exonuclease site by 3-7-fold, relative to the perfectly base-paired primer-template, depending on the identity of the extrahelical base. The ability of different extrahelical bases to promote partitioning of DNA into the 3'-5' exonuclease site decreased in the following order: G > A approximately T > C. The results of partitioning measurements for DNA substrates containing a bulged adenine base at different positions within the template showed that an extrahelical base is recognized up to five bases from the primer 3' terminus. The largest effects were observed for the extrahelical base at the third or fourth positions from the primer terminus, which increased the level of partitioning of DNA into the 3'-5' exonuclease site by 8- and 18-fold, respectively, relative to that of the perfectly base-paired substrate. Steady-state fluorescence measurements of analogous primer-templates containing 2-aminopurine (AP) at the primer 3' terminus indicate that extrahelical bases increase the degree of terminus unwinding, especially when close to the terminus. In addition, steady-state kinetic measurements of removal of AP from the primer-templates indicate that the exonucleolytic cleavage activity of Klenow fragment is correlated with the increased level of partitioning of bulged DNA substrates to the 3'-5' exonuclease site relative to that of properly base-paired DNA. The results of this study indicate that misalignment of primer and template strands to generate an extrahelical base strongly promotes transfer of a DNA substrate to the 3'-5' exonuclease site, suggesting that the premutational intermediates in frameshift mutagenesis are subject to proofreading by the polymerase.
Asunto(s)
ADN Polimerasa I/química , ADN/biosíntesis , ADN/química , Mutación del Sistema de Lectura , Ácidos Nucleicos Heterodúplex/química , 2-Aminopurina/química , Emparejamiento Base , ADN/genética , ADN Polimerasa I/genética , Cartilla de ADN/química , Exodesoxirribonucleasa V , Exodesoxirribonucleasas/química , Hidrólisis , Cinética , Ácidos Nucleicos Heterodúplex/genética , Especificidad por Sustrato , Moldes GenéticosRESUMEN
Site-directed mutagenesis and time-resolved fluorescence spectroscopy were used to evaluate the contributions of individual amino acid side chains to the binding of DNA primer-templates to the 3'-5' exonuclease site of the large proteolytic fragment (Klenow fragment) of DNA polymerase I. Mutations were introduced into side chains that have been shown crystallographically to be in close proximity to a DNA 3' terminus bound at the 3'-5' exonuclease site. The wild-type residues were replaced by alanine in each case. To assess the effects of the mutations on DNA binding, time-resolved fluorescence anisotropy measurements were performed on dansyl-labeled primer-templates bound to the mutant enzymes. In contrast to techniques that simply monitor the overall binding of proteins to DNA, the time-resolved fluorescence anisotropy technique was used to determine the fractional occupancies of the polymerase and 3'-5' exonuclease active sites of Klenow fragment. Equilibrium constants describing the partitioning of DNA between the two active sites were obtained for nine different mutant enzymes bound to both matched and mismatched DNA sequences. Mutations of Leu361 and Phe473 caused the largest effects, significantly destabilizing the binding of mismatched DNA substrates to the 3'-5' exonuclease site relative to DNA bound at the polymerase site, consistent with structural data showing that the side chains of these residues are involved in intimate hydrophobic interactions with the 3' terminal and penultimate bases of the primer strand [Beese, L., and Steitz, T. A. (1991) EMBO J. 10, 25-33]. Mutations of the His660 and Glu357 side chains also resulted in significant effects on the binding of mismatched DNA to the 3'-5' exonuclease site. Surprisingly, mutation of Tyr497 increased the partitioning of mismatched DNA into the 3'-5' exonuclease site, suggesting that the tyrosine side chain in the wildtype enzyme destabilizes substrate binding, despite crystallographic data showing that Tyr497 is H-bonded to the DNA substrate. The effects of mutating the amino acid side chains that serve as ligands to two divalent metal ions bound at the 3'-5' exonuclease site, designated A and B, indicated that metal A also helps to bind DNA to the 3'-5' exonuclease site. These results demonstrate that the time-resolved fluorescence anisotropy technique can be used to quantify the energetic contributions associated with each of the crystallographically defined DNA-protein contacts at the 3'-5' exonuclease site.
Asunto(s)
ADN Polimerasa I/genética , ADN Polimerasa I/metabolismo , ADN/metabolismo , Exodesoxirribonucleasas/genética , Mutagénesis Sitio-Dirigida , Sustitución de Aminoácidos/genética , Aminoácidos/genética , Aminoácidos/metabolismo , Sitios de Unión/genética , Ácidos Carboxílicos/metabolismo , Cationes Bivalentes , Compuestos de Dansilo , Polarización de Fluorescencia , Ligandos , Metales/metabolismo , Especificidad por Sustrato/genéticaRESUMEN
Congenital cricoid stenosis is a rare malformation generally diagnosed at autopsy. Out of a total of fourteen cases reported by others, only five were treated successfully. Over a 10-year period, seven patients presented with congenital cricoid stenosis at the Sophia Children's Hospital in Rotterdam. On the basis of these numbers we surmise that this anomaly is less rare than is commonly thought. All seven patients were treated successfully. A new method of treatment is described, consisting of a combination of microsurgical laryngofissure and prolonged intubation with a new, low-friction, atoxic, soft, silicone rubber nasotracheal tube. The specially designed intubation material is also recommended for the treatment of iatrogenically damaged airways. The results indicate that this method constitutes a major advance not only in the treatment of cricoid stenosis but also in the treatment of damaged airways.
Asunto(s)
Cartílago Cricoides/anomalías , Cartílagos Laríngeos/anomalías , Laringoestenosis/congénito , Preescolar , Terapia Combinada , Femenino , Humanos , Lactante , Recién Nacido , Intubación Intratraqueal/métodos , Laringoestenosis/terapia , Laringe/cirugía , MasculinoRESUMEN
Laryngotracheo-oesophageal cleft has been considered a rare congenital anomaly, diagnosed when there is respiratory distress on feeding, associated with multiple congenital disorders. A tracheo-oesophageal fistula should be excluded. Diagnosis, even in skillful endoscopic hands, is difficult. Different approaches to surgical repair have been advocated in literature. Most authors prefer the lateral approach. Only 7 patients have reportedly been treated via the anterior approach. The anterior approach offers the advantage of excellent and also easy exposure of the anomaly. The theoretical disadvantage of postoperative laryngeal instability is not substantiated by experimental work (Calcaterra, Ann Otol 83:810-813, 1974), nor by our own clinical experience in six juvenile patients who underwent laryngofissure for other reasons. A further advantage of the anterior approach is that the laryngeal nerves are not jeopardized as in the lateral approach. We have treated 4 patients. Two patients have been treated successfully via a lateral pharyngotomy approach; each of them has a left recurrent nerve palsy. The other two patients were treated via a laryngofissure, one of them successfully; in the other patient the cleft has been closed with success, but the larynx is still stenosed by excessive mucous membrane folds. Cicatricial subglottic stenosis, which may occur postoperatively, and the frequently coinciding tracheomalacia, have been successfully treated with prolonged nasotracheal intubation with siliconized silicone rubber tubes. Four patients were treated successfully. Three other patients have come to our attention. As is often the case, this disorder seems to occur more frequently than was previously thought.
Asunto(s)
Esófago/anomalías , Laringe/anomalías , Tráquea/anomalías , Esófago/cirugía , Femenino , Humanos , Lactante , Laringoscopía , Laringe/cirugía , Masculino , Faringe/cirugía , Neumonía por Aspiración/etiología , Tráquea/cirugíaRESUMEN
Diathermic coagulation and cutting in the endoscopic treatment of hypopharyngeal diverticula was replaced by CO2 laser evaporation. Endoscopic laser surgery was performed in 37 patients, with complete or almost complete relief in all cases. The rate of complications is low.
Asunto(s)
Dióxido de Carbono/uso terapéutico , Divertículo Esofágico/terapia , Endoscopía/métodos , Terapia por Láser , Enfermedades Faríngeas/terapia , Endoscopía/efectos adversos , Humanos , Rayos Láser/efectos adversos , Mediastinitis/etiología , Enfisema Subcutáneo/etiologíaRESUMEN
Fifty-nine cases of adenoid cystic carcinoma (ACC) in the head and neck were reviewed and the treatment and follow-up results from 1950 to 1983 are reported. Nearly all patients were treated with combined therapy, i.e. surgery and radiotherapy, and the actuarial survival rate was 68% after 5 years, 45% after 10 years, and 30% after 15 years. Modern radiotherapy seems a very valuable means of treatment, not only as a palliative measure.
Asunto(s)
Carcinoma Adenoide Quístico/terapia , Neoplasias de Cabeza y Cuello/terapia , Adulto , Anciano , Carcinoma Adenoide Quístico/mortalidad , Terapia Combinada , Femenino , Estudios de Seguimiento , Neoplasias de Cabeza y Cuello/mortalidad , Humanos , Masculino , Persona de Mediana Edad , Países Bajos , Estudios Retrospectivos , Factores de TiempoRESUMEN
Endoscopic laser surgery for hypopharyngeal diverticula was performed in 28 patients. Complete relief of symptoms was achieved in 22, whilst the remaining patients improved considerably. The rate of complications was low. This new application of laser surgery proved to be a safe and reliable method of treatment for dysphagia caused by a hypopharyngeal diverticulum.
Asunto(s)
Divertículo/cirugía , Hipofaringe , Terapia por Láser , Enfermedades Faríngeas/cirugía , Anciano , Fiebre/etiología , Humanos , Mediastinitis/etiología , Persona de Mediana Edad , Complicaciones Posoperatorias , Reoperación , Enfisema Subcutáneo/etiologíaRESUMEN
For endolaryngeal laser surgery an anaesthetic technique is required which provides the surgeon good access to the larynx. High frequency ventilation of the lungs using a FG 14 insufflation catheter covered by aluminium foil has proved to give a nearly optimal condition for the surgeon, while the fully paralyzed patient is very well ventilated. This has been used in more than 1000 patients, 200 of them have been treated by the laser beam in the airway. A method of total intravenous anaesthesia is described using short acting drugs. This way early awakening after surgery in the airways prevents aspiration of blood or debris into the lungs.