RESUMEN
The aim of this prospective clinical trial was to investigate the efficacy and toxicity of a short-term, maintenance-free chemotherapy protocol in feline lymphoma. Twenty-six cats with confirmed diagnosis of high-/intermediate-grade lymphoma were treated with a 12-week protocol consisting of cyclic administration of l-asparaginase, vincristine, cyclophosphamide, doxorubicin and prednisolone. Complete (CR) and partial remission (PR) rates were 46 and 27%, respectively. Median duration of first CR was 394 days compared with a median PR duration of 41 days. No factor was identified to significantly influence the likelihood to reach CR. Overall survival amounted to 78 days (range: 9-2230 days). Median survival in CR cats was 454 days and in PR cats was 82 days. Toxicosis was mainly low grade with anorexia seen most frequently. In cats achieving CR, maintenance-free chemotherapy may be sufficient to attain long-term remission and survival. Factors aiding in prognosticating the likelihood for CR, strategies enhancing response and targeting chemotherapy-induced anorexia need to be identified in future.
Asunto(s)
Antineoplásicos/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Enfermedades de los Gatos/tratamiento farmacológico , Linfoma no Hodgkin/veterinaria , Animales , Antibióticos Antineoplásicos/administración & dosificación , Antibióticos Antineoplásicos/efectos adversos , Antineoplásicos/efectos adversos , Antineoplásicos Alquilantes/administración & dosificación , Antineoplásicos Alquilantes/efectos adversos , Antineoplásicos Hormonales/administración & dosificación , Antineoplásicos Hormonales/efectos adversos , Antineoplásicos Fitogénicos/administración & dosificación , Antineoplásicos Fitogénicos/efectos adversos , Protocolos de Quimioterapia Combinada Antineoplásica/efectos adversos , Asparaginasa/administración & dosificación , Asparaginasa/efectos adversos , Gatos , Ciclofosfamida/administración & dosificación , Ciclofosfamida/efectos adversos , Doxorrubicina/administración & dosificación , Doxorrubicina/efectos adversos , Femenino , Linfoma no Hodgkin/tratamiento farmacológico , Masculino , Estadificación de Neoplasias , Prednisolona/administración & dosificación , Prednisolona/efectos adversos , Inducción de Remisión , Análisis de Supervivencia , Vincristina/administración & dosificación , Vincristina/efectos adversosRESUMEN
AIM: Deep rectovaginal fistulas are a rare entity and pose a delicate challenge for the surgeon. The present study introduces different operative interventions involved in transperineal omental flap surgery. METHOD: A retrospective analysis of all patients treated with a low or mid rectovaginal or enterovaginal fistula at the Department of Surgery of the University Hospital of Schleswig-Holstein, Campus Luebeck, was performed. Treatment results were discussed with respect to aetiology, localization, morbidity and outcome. RESULTS: Between the years 2000 and 2010, a total of nine patients with a low or mid rectovaginal fistula were treated at our clinic. After local fistulectomy, all patients were additionally treated by a laparoscopically assisted omental flap reconstruction of the rectovaginal and perineal space. Eight of the nine patients received a protective ileostomy or colostomy. Only the patient with a history of Crohn's disease had no ileostomy raised. At a median follow-up of 22 months, no patient experienced recurrence of a rectovaginal fistula. Perioperative mortality was zero and minor complications were observed in 22%. Major complications were an anastomotic insufficiency after low anterior resection that was treated without further interventions. Another complication was a persistent fistula within the sphincter that needed re-operation and bovine plug repair combined with a mucosa flap. CONCLUSIONS: Complete omental reconstruction of the rectovaginal space appears decisive in the operative therapy of deep rectovaginal or enterovaginal fistulas. Comparative studies on standard therapies are necessary although direct comparison of case series is difficult.
Asunto(s)
Ileus/etiología , Epiplón/trasplante , Complicaciones Posoperatorias/etiología , Fístula Rectovaginal/cirugía , Recto/cirugía , Colgajos Quirúrgicos , Vagina/cirugía , Adulto , Anciano de 80 o más Años , Femenino , Humanos , Persona de Mediana Edad , Recurrencia , Estudios Retrospectivos , Colgajos Quirúrgicos/efectos adversosAsunto(s)
Ciego , Colon Ascendente/patología , Laparoscopía , Neumatosis Cistoide Intestinal/diagnóstico , Tomografía Computarizada por Rayos X , Anciano , Carcinoma de Células Escamosas/patología , Carcinoma de Células Escamosas/terapia , Ciego/patología , Diagnóstico Diferencial , Humanos , Metástasis Linfática , Masculino , Neoplasias de la Boca/patología , Neoplasias de la Boca/terapia , Recurrencia Local de Neoplasia/patología , Recurrencia Local de Neoplasia/terapia , Neumatosis Cistoide Intestinal/patología , Neumatosis Cistoide Intestinal/cirugíaRESUMEN
BACKGROUND: To date laparoscopic hepatic surgery is only common in a few centres for a specific selected patient group. The intention of this survey was to estimate the current state of affairs for laparoscopic hepatic surgery in Germany at 2008. MATERIALS AND METHODS: A questionnaire was prepared and sent out by e-mail in May 2009 to the members of the DGAV (German Society of General and Visceral Surgery). The feedback was evaluated anonymously. RESULTS: A total of 181 answers were received by 31st July 2009 (return rate of 15.9%). The return rate of basic and standard care hospitals was 9.2%, specialized hospitals 23.6%, hospitals with maximum care 50% and university hospitals had a return rate of 71.9%. The question whether laparoscopic hepatic surgery had been performed in 2008 was answered with YES by 125 (69.1%) and NO by 54 (29.8%) members. The number of laparoscopic hepatic surgery interventions (laparoscopic ultrasound, laparoscopic radiofrequency ablation and resection) in 2008 was given as more than 50 by 4 (2.2%) hospitals, between 20 and 50 by 11 (6.1%) hospitals, between 10 and 20 by 23 (12.7%) hospitals, between 5 and 10 by 45 (24.9%) hospitals and between 0 and 5 by 54 (29.8%) hospitals. In 2008 the frequency of laparoscopic ultrasound during intraoperative staging to confirm the diagnosis ranged from 2 to 250, whereby 96.4% of the hospitals had less than 50 and only 2 hospitals (2.7%) had 211 and 250 examinations, respectively. 50 hospitals carried out laparoscopic radiofrequency ablation (RFA). 69 (38.1%) of the interviewed hospitals reported hepatic laparoscopic resections (n=551). CONCLUSION: Laparoscopic liver surgery has been done in Germany in patients with benign or malignant liver lesions. Pure laparoscopy is the most common access. Atypical resections are the primarily indication followed by left lateral resections. All further types of resection have been done in a very small number. Laparoscopic liver surgery has been performed in all types of hospitals.
Asunto(s)
Conducta Cooperativa , Endoscopía del Sistema Digestivo/tendencias , Gastroenterología/tendencias , Comunicación Interdisciplinaria , Laparoscopía/tendencias , Grupo de Atención al Paciente/tendencias , Especialidades Quirúrgicas/tendencias , Recolección de Datos , Difusión de Innovaciones , Predicción , Alemania , Necesidades y Demandas de Servicios de Salud/tendencias , Humanos , Especialización/tendencias , Servicio de Cirugía en Hospital/estadística & datos numéricos , Servicio de Cirugía en Hospital/tendencias , Encuestas y Cuestionarios , Ultrasonografía Intervencional/tendenciasRESUMEN
BACKGROUND: CT scanning of the lungs is the standard procedure for preoperative evaluation of central lung tumors. The extent of the tumor and infiltration of central lung structures or lung segments are decisive parameters to clarify whether surgery is possible and the extent of resection. With computer-assisted methods for the segmentation of anatomical structures based on CT data (Fraunhofer MeVis, Bremen) an enhanced, three-dimensional selective visualization is now possible. PATIENTS AND METHODS: From August 2007 through June 2009, 22 patients with central lung tumors were treated at the department of thoracic surgery, University of Schleswig-Holstein, campus Lübeck. There were 15 males and 7 females with a mean age of 60.2 years (range 41-74 years), 18 patients had a long history of smoking, while 4 patients had never smoked. Of the patients 20 had a primary lung carcinoma, 1 patient had local recurrent lung cancer after lobectomy and 1 patient had a central lung metastasis from a non-pulmonary primary carcinoma. A multi-slice detector computer tomogram (MSDCT) scan was performed in all cases. All data were three-dimensionally reconstructed and visualized using special computer-aided software (Fraunhofer MeVis, Bremen). Pulmonary lung function tests, computed postoperative lung volume, bronchoscopic findings, general condition of the patients and the three-dimensionally reconstructed CT data were used for an individual risk analysis and surgical planning. RESULTS: According to the risk analysis 14 out of the 22 patients were surgically treated, 7 patients were staged as functionally inoperable and 1 as technically inoperable. A pneumonectomy was performed in 5 cases, a lobectomy/bilobectomy in 4 cases, an extended lobectomy in 3 cases and 1 case each of a wedge resection and a sleeve resection. Of the 14 patients 2 were classified as stage Ia/b, 7 patients as stage IIa/b and 5 patients as stage IIIa. The median length of time spent in hospital was 8.5±33 days and the mortality rate was 0%. The three-dimensional visualization of the tumor and its anatomical relationship to central pulmonary vessels and the airway system was feasible in all cases. The three-dimensional reconstruction was confirmed in all cases by surgical exploration. CONCLUSION: Three-dimensional reconstruction of CT scan data is a new and promising method for preoperative presentation and risk analysis of central lung tumors. The three-dimensional visualization with anatomical reformatting and color-coded segmentation enables the surgeon to make a more precise strategic approach for central lung tumors.
Asunto(s)
Neoplasias Pulmonares/diagnóstico por imagen , Neoplasias Pulmonares/cirugía , Adulto , Anciano , Femenino , Humanos , Procesamiento de Imagen Asistido por Computador/métodos , Tiempo de Internación , Pulmón/diagnóstico por imagen , Neoplasias Pulmonares/patología , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Pruebas de Función Respiratoria , Tomografía Computarizada por Rayos X/métodosRESUMEN
Surgical treatment of giant abdominal hernias includes reduction of the hernia content and tension-free closure of the abdominal wall. Initial laparoscopy simulates the postoperative abdominal wall tension. Recognizing the need for a preoperative pneumoperitoneum in cases of chronic eventration may help to avoid "abdominal catastrophes" including bowel resection, abdominal compartment, and extended abdominal wall reconstruction. We report a 66-year-old man with an asymptomatic long-standing giant scrotal hernia who was admitted with sepsis and uremia caused by intestinal obstruction.
Asunto(s)
Enfermedades de los Genitales Masculinos/cirugía , Hernia Abdominal/cirugía , Obstrucción Intestinal/cirugía , Neumoperitoneo Artificial , Escroto/cirugía , Anciano , Humanos , Laparoscopía , Masculino , Orquiectomía , Complicaciones Posoperatorias/cirugía , Reoperación , Sepsis/cirugía , Resección Transuretral de la Próstata , Uremia/cirugíaRESUMEN
Based on current references four clinical scenarios were discussed and different management strategies were compared for secondary and primary prophylaxis of ulcer or peptic ulcer bleeding under continuous therapy with non-steroidal antiinflammatory drugs (NSAID) or low-dose-aspirin, for H.pylori-positive and H.pylori-negative patients. Used as secondary prophylaxis eradication alone is insufficient in preventing recurrent peptic ulcer or recurrent ulcer bleeding for H.pylori-positive patients who continue to take unselective NSAIDs. Maintenance therapy with PPIs or switching from nonselective NSAID to COX-2-inhibitors is required after eradication of H.pylori or primary H.pylori-negative patients. Further evaluation is needed of what kind of secondary prophylaxis - maintenance therapy with PPI or switching to COX-2-inhibitor - is more (cost-)effective. It is sufficient to use eradication of H.pylori alone as secondary prophylaxis in preventing recurrent peptic ulcer or recurrent ulcer bleeding for H.pylori-positive patients, who continue to take low-dose-aspirin. Maintenance therapy with PPI is not generally required. However it can be considered for patients with increased risk for gastrointestinal complications (previous history of peptic ulcer, age over 65 years, concomitant use of corticosteroids, anticoagulants or individual NSAID with higher risk for gastrointestinal complications, serious cardiovascular disease). Switching from low-dose-aspirin to clopidogrel is not required. Used as primary prophylaxis in preventing peptic ulcer or ulcer bleeding before starting long-term therapy with NSAIDs, COX-2-inhibitors or unselective NSAIDs concomitant with PPIs are recommended for patients with increased risk for gastrointestinal complications. Patients starting long-term therapy with unselective NSAIDs should be screened for H.pylori and eradicated. There are no valid data supporting screening for H.pylori and eradication for patients starting long-term therapy with low-dose-aspirin. Further studies are needed to evaluate a possible benefit for patients with increased risk for gastrointestinal complications.
Asunto(s)
Antiinflamatorios no Esteroideos/efectos adversos , Aspirina/efectos adversos , Inhibidores de la Ciclooxigenasa/uso terapéutico , Hemorragia Gastrointestinal/prevención & control , Infecciones por Helicobacter/tratamiento farmacológico , Helicobacter pylori , Inhibidores de la Bomba de Protones , Gastropatías/inducido químicamente , Gastropatías/prevención & control , Anciano , Antiinflamatorios no Esteroideos/administración & dosificación , Antiinflamatorios no Esteroideos/uso terapéutico , Aspirina/administración & dosificación , Aspirina/uso terapéutico , Inhibidores de la Ciclooxigenasa/administración & dosificación , Inhibidores de la Ciclooxigenasa/efectos adversos , Humanos , Úlcera Péptica/inducido químicamente , Úlcera Péptica/prevención & control , Úlcera Péptica Hemorrágica/inducido químicamente , Úlcera Péptica Hemorrágica/prevención & control , Prevención Primaria , Recurrencia , Úlcera Gástrica/inducido químicamente , Úlcera Gástrica/prevención & control , Factores de TiempoRESUMEN
Primary hemangiosarcoma of the kidney is a very rare tumor for which only 23 case reports appear in the literature worldwide. An additional case of renal angiosarcoma in an adult is reported. The patient developed local tumor recurrence with multiple metastases within 4 weeks and died although there was no evidence of metastases at the time of nephrectomy.
Asunto(s)
Hemangiosarcoma/patología , Neoplasias Renales/patología , Adulto , Resultado Fatal , Hemangiosarcoma/cirugía , Humanos , Neoplasias Renales/cirugía , Masculino , Persona de Mediana Edad , Metástasis de la Neoplasia , Recurrencia Local de Neoplasia/patología , NefrectomíaRESUMEN
We analysed the processing of small bipartite model substrates by Escherichia coli and Bacillus subtilis RNase P and corresponding hybrid enzymes. We demonstrate specific trans-cleavage of a model substrate with a 4 bp stem and a 1 nucleotide (nt) 5' flank, representing to date the smallest mimic of a natural RNase P substrate that could be processed in trans at the canonical RNase P cleavage site. Processing efficiencies decreased up to 5000-fold when the 5' flank was shortened from 3 to 1 nt. Reduction of the 5' flank to 1 nt was more deleterious than reducing the stem from 7 to 4 bp, although the 4 bp duplex formed only transiently, in contrast to the stable 7 bp duplex. These results indicate that the crucial contribution of nt -2 in the single-stranded 5' flank to productive interaction is a general feature of A- and B-type bacterial RNase P enzymes. We also showed that an Rp-phosphorothioate modification at nt -2 interferes with processing. Bacterial RNase P holoenzymes are also capable of cleaving single-stranded RNA oligonucleotides as short as 5 nt, yielding RNase P-specific 5'-phosphate and 3'-OH termini, with measured turnover rates of up to 0.7 min-1. All cleavage sites were at least 2 nt away from the 5' and 3' ends of the oligonucleotides. Some cleavage site preferences were observed dependent on the identity of the RNase P RNA subunit.
Asunto(s)
Bacillus subtilis/enzimología , Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Oligonucleótidos/metabolismo , ARN Bacteriano/metabolismo , ARN Catalítico/metabolismo , Holoenzimas/metabolismo , Cinética , ARN Bacteriano/química , Ribonucleasa P , Especificidad por SustratoRESUMEN
It is demonstrated that acceptor stem duplexes derived from native tRNAs which contain a three-nucleotide extension at the 5'-terminus of mature tRNA are minimal substrates for ribonuclease P from both Escherichia coli and Bacillus subtilis. Variants with a cytidine at position -1 are most efficiently processed whereas the G -1 variant represents a comparatively poor substrate. An A -1 acceptor stem variant is a slightly better substrate than the G -1 variant though generally distinctly less efficient than the C -1 duplex. This is in qualitative agreement with the frequency of the occurrence of the corresponding nucleotides at position -1 in natural substrates, which is highest for pyrimidines and least for G. NMR analyses of the corresponding acceptor stems reveal that the conformation of the nucleotides at position -1 correlates with the substrate preferences of Ribonuclease P: Whereas C -1 adopts a conformation characterized by a glycosidic angle in the anti range (close to high-anti), the G -1 is clearly in syn conformation, and that of A -1 is intermediate between high-anti and syn. The riboses of nucleotides -1 are in all cases predominantly 2'-endo puckered.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Conformación de Ácido Nucleico , Procesamiento Postranscripcional del ARN , ARN Catalítico/metabolismo , Adenosina/metabolismo , Bacillus subtilis/enzimología , Composición de Base , Citidina/metabolismo , Escherichia coli/enzimología , Guanosina/metabolismo , Hidrólisis , Cinética , Resonancia Magnética Nuclear Biomolecular , Ácidos Nucleicos Heterodúplex/metabolismo , ARN de Transferencia/metabolismo , Ribonucleasa P , Especificidad por SustratoRESUMEN
The phenylalanyl-tRNA synthetase (FRS) from Thermus thermophilus has previously been shown to bind DNA. We demonstrate that the "winged" helix-turn-helix motifs in the duplicate domains B5 are the relevant structural elements for this DNA-binding property. By altering particular amino acids in the "wing", the affinity of the FRS to DNA was significantly reduced. Based on experimental data, which indicate that the FRS prefers a certain DNA structure rather than a particular consensus sequence, we propose a novel loop model for the DNA-binding mode of the FRS. In our model we assume that two segments of the same DNA molecule are bound simultaneously by both B5 domains and are aligned in parallel, while the intervening DNA forms a loop. Due to the limited flexibility of the DNA, loop formation is only possible if the respective intervening DNA stretch exceeds a certain length. Several lines of evidence support this model. (1) We demonstrate by gel retardation assays that the DNA requires a minimal number of ca 80 base-pairs to be bound by the FRS. (2) In the presence of the FRS, DNA longer than ca 80 base-pairs has a significantly increased DNase I accessibility. This agrees well with its known preferential cleavage at positions where the minor grove is on the outside of looped-out DNA molecules. (3) The initial cleavage by DNase I of >80 bp long DNA occurs in the middle of the fragment. In a looped molecule this is the position with the highest accessibility to DNase I. The function of the FRS related to DNA binding is still unknown. Since the FRS exists in the nucleus of rapidly growing mammalian cells, and protein-induced DNA bending or looping contributes to several transcription, replication, and recombination systems in both prokaryotes and eukaryotes, it is likely that the FRS, in addition to its aminoacylation function, influences common cellular processes via DNA binding.
Asunto(s)
ADN/química , ADN/metabolismo , Conformación de Ácido Nucleico , Fenilalanina-ARNt Ligasa/metabolismo , Thermus thermophilus/enzimología , Acilación , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Sitios de Unión , ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desoxirribonucleasa I/metabolismo , Secuencias Hélice-Giro-Hélice , Humanos , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fenilalanina-ARNt Ligasa/química , Fenilalanina-ARNt Ligasa/genética , Unión Proteica , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , TermodinámicaRESUMEN
The binding of Mg(2+)/Mn(2+) to acceptor stem microhelices as minimal models for precursor-tRNA(Gly) is demonstrated by NMR spectroscopy. From the evaluation of COSY and NOESY spectra, binding sites for Mg(2+)/Mn(2+) can be inferred. In particular, one binding site exists near the ribose moiety of nucleotide -1 at the position of cleavage by RNase P. From comparison with a variant possessing a deoxynucleotide at this position, it is concluded that the 2'-OH group of this nucleotide is indispensable for coordinating the divalent metal ion. Hence, this catalytically important metal ion is "pre-bound" to the precursor-tRNA before complexation with RNase P.
Asunto(s)
Endorribonucleasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/enzimología , Magnesio/metabolismo , Manganeso/metabolismo , Precursores del ARN/metabolismo , ARN Catalítico/metabolismo , ARN de Transferencia/metabolismo , Emparejamiento Base , Secuencia de Bases , Sitios de Unión , Cationes Bivalentes/metabolismo , Endorribonucleasas/química , Endorribonucleasas/genética , Escherichia coli/genética , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Precursores del ARN/química , Precursores del ARN/genética , ARN Catalítico/química , ARN Catalítico/genética , ARN de Transferencia/química , ARN de Transferencia/genética , Ribonucleasa PRESUMEN
Two polypeptides resistant against proteolytic digestion were identified in Thermus thermophilus translation initiation factor 2 (IF2): the central part of the protein (domains II/III), and the C-terminal domain (domain IV). The interaction of intact IF2 and the isolated proteolytic fragments with fMet-tRNAfMet was subsequently characterized. The isolated C-terminal domain was as effective in binding of the 3' end of fMet-tRNAf Met as intact IF2. N-Formylation of Met-tRNAfMet was required for its efficient binding to the C-terminal domain. This suggests that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is responsible for the recognition of fMet-tRNAfMet by IF2 during translation initiation. Moreover, it was demonstrated that fMet-AMP is a minimal ligand of IF2. fMet-AMP inhibits fMet-tRNAfMet binding to IF2 as well as the activity of IF2 in the stimulation of ApUpG-dependent ribosomal binding of fMet-tRNAf Met. Specific interaction of fMet-AMP with IF2 was demonstrated by 1H-NMR spectroscopy. These findings indicate that fMet-AMP and the 3' terminal fMet-adenosine of fMet-tRNAfMet use the same binding site on the C-terminal domain of IF2 and imply that the interaction between the C-terminal domain and the 3' end of fMet-tRNAfMet is primarily responsible for the fMet-tRNAfMet binding and recognition by IF2.
Asunto(s)
Adenosina Monofosfato/metabolismo , Factor 2 Eucariótico de Iniciación/metabolismo , N-Formilmetionina/metabolismo , Biosíntesis de Proteínas , ARN de Transferencia de Metionina/metabolismo , Thermus thermophilus/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , TemperaturaRESUMEN
Acceptor stem variants of tRNA(fMet) (Escherichia coli) have been characterized by nuclear magnetic resonance. The wild type contains a C1-A72 mismatch pair which is crucial for its biological function. For comparison, the mismatch was replaced by regular pairs U1-A72 and C1-G72. Further variants contain an altered discriminator base, G73, or a G1-C72/U73 combination. The stems of variants U1-A72/A73 and C1-G72/A73 have A-RNA geometry, which extends essentially to the single-strand terminus. C1-A72/G73 variant and wild type are structurally almost identical. C1 and A72 adopt peculiar conformations with C1 being largely destacked with respect to G2, while A73 stacks upon C1. The unique arrangement of the mismatch causes a distinctly different orientation of the single-strand terminus compared to variants with regular 1-72 base pairs, and to formyltransferase-complexed tRNA(fMet).
Asunto(s)
Disparidad de Par Base , Escherichia coli/genética , Transferasas de Hidroximetilo y Formilo/metabolismo , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN de Transferencia de Metionina/química , Adenina , Sitios de Unión , Citosina , Variación Genética , Resonancia Magnética Nuclear Biomolecular , ARN Bacteriano/metabolismo , ARN de Transferencia de Metionina/metabolismoRESUMEN
A simple system derived from the acceptor stem of tRNA(Ala) is presented which undergoes a pH-dependent dimerization. This is brought about by formation of C+-G-C base triples of the pyrimidine motif type between protonated cytidines of the 3' single-stranded end and regularly paired G-C pairs in the double-helical stem. In addition, an unusual interaction between a protonated adenine and a regular G-C pair is suggested. The equilibrium between monomer and dimer forms can be monitored via NMR spectroscopy and UV melting curve analysis. A dimerization enthalpy of 159 kJ mol(-1) was found at pH 5.0. The system could serve as a model for inter- and intra-molecular association, respectively, of single-stranded and double-helical regions to enable optimal packing of large RNA molecules.
Asunto(s)
Conformación de Ácido Nucleico , ARN Bicatenario/química , ARN Bicatenario/metabolismo , Dimerización , Concentración de Iones de Hidrógeno , Espectroscopía de Resonancia Magnética , Modelos Químicos , Espectrofotometría Ultravioleta , TemperaturaRESUMEN
We have studied a very small ribozyme described earlier which requires the presence of soft metal ions like manganese or cadmium. It consists of only three uridines as ribozyme, cleaving the sequence 5'-GAAA-3' after the guanosine. We have set out to characterize the metal ion binding in this system by NMR spectroscopy and the impact of the ribose 2'-OH group of the cleavable nucleotide upon local structure. NMR results indicate a high degree of regularity and order in the pyrimidine-rich ribozyme strand, and high flexibility within the purine-rich substrate. The guanosine 2'-hydroxy group adjacent to the cleavage site was found to have a profound effect upon the structure, apparently destabilizing a stacked arrangement. Metal ions were found to bind in a rather unspecific way, however, in the presence of higher amounts of divalent ions a preference in the vicinity of the cleavage site could be observed. 113Cd NMR spectra suggest a specific binding of Cd2+ ions to the RNA.
Asunto(s)
Cadmio/metabolismo , Conformación de Ácido Nucleico , ARN Catalítico/química , ARN Catalítico/metabolismo , Adenosina , Composición de Base , Secuencia de Bases , Sitios de Unión , Cationes Bivalentes , Variación Genética , Guanosina , Resonancia Magnética Nuclear Biomolecular , Oligorribonucleótidos/química , Mutación Puntual , ARN Catalítico/genéticaRESUMEN
A ribooligonucleotide microhelix derived from the acceptor stem of Escherichia coli tRNA(Ala) having a C3-A70 mismatch in place of the G3-U70 wobble pair in the wild-type tRNA(Ala), and a sequence variant with a regular U3-A70 base pair have been investigated by NMR. In vivo, suppressor tRNA(Ala) variants with C3-A70 (as well as several other) mismatch pairs are substrates for alanyl-tRNA synthetase (ARS), supporting the hypothesis of an 'indirect' recognition of the identity element 3-70 mismatch pair via structural modifications caused by the mispair in comparison to canonical A-RNA helices. It is demonstrated that the C-A mismatch likewise induces helix geometry alterations, in particular with respect to base stacking in the vicinity of the mismatch. However, with reference to the 'wild-type' G3-U70 microhelix, destacking in the C3-A70 acceptor stem duplex occurs in the opposite direction from the mismatch pair. Therefore it is concluded that the locally enhanced conformational flexibility or dynamics associated with the structural changes induced by the mismatch pairs could be an essential prerequisite for optimal adaptation of the tRNA(Ala) acceptor stem to the contact region of the ARS.
Asunto(s)
Escherichia coli/química , Conformación de Ácido Nucleico , ARN Bacteriano/química , ARN de Transferencia de Alanina/química , Espectroscopía de Resonancia Magnética , Modelos Químicos , Modelos MolecularesRESUMEN
RNA hairpins can alternatively form a dimer with a bulged loop flanked by regularly base paired regions. [1H]NMR spectroscopy and native gel electrophoresis were used to study how the sequence of nucleotides in the loop of the hairpin affect the hairpin-duplex interconversion. As a model system, a hairpin containing 7 nucleotides in the loop and 5 base pairs in the stem was used. The loop size was gradually reduced from 7 to 4 nucleotides, yielding finally the stable UNCG tetraloop. Single nucleotide mutations were performed to investigate the influence of the self-complementarity of the loop sequence on the dimerization. The results demonstrate that (1) the initial fraction of hairpin is determined by concentration of the oligonucleotide, the annealing procedure, and the relative stability of the loop, (2) the degree of self-complementarity of the loop sequence of the hairpin governs the dimerization kinetics, and (3) oligonucleotides complementary to the loop sequence decrease the dimerization rate. We propose a secondary structure-based model for the dimerization reaction of RNA hairpins in which the formation of intermolecular base pairs between self-complementary nucleotides of the loops represents the nucleation step.
Asunto(s)
Conformación de Ácido Nucleico , ARN de Transferencia de Alanina/química , Secuencia de Bases , Dimerización , Cinética , Espectroscopía de Resonancia Magnética , MutaciónRESUMEN
RNA molecules with high affinity for immobilized Ni2+ were isolated from an RNA pool with 50 randomized positions by in vitro selection-amplification. The selected RNAs preferentially bind Ni2+ and Co2+ over other cations from first series transition metals. Conserved structure motifs, comprising about 15 nt, were identified that are likely to represent the Ni2+ binding sites. Two conserved motifs contain an asymmetric purine-rich internal loop and probably a mismatch G-A base pair. The structure of one of these motifs was studied with proton NMR spectroscopy and formation of the G-A pair at the junction of helix and internal loop was demonstrated. Using Ni2+ as a paramagnetic probe, a divalent metal ion binding site near this G-A base pair was identified. Ni2+ ions bound to this motif exert a specific stabilization effect. We propose that small asymmetric purine-rich loops that contain a G-A interaction may represent a divalent metal ion binding site in RNA.
Asunto(s)
Adenina , Guanina , Níquel/metabolismo , Purinas/química , ARN/metabolismo , Composición de Base , Secuencia de Bases , Sitios de Unión , Espectroscopía de Resonancia Magnética , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Protones , ARN/química , Rayos UltravioletaRESUMEN
The interaction of N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF-Tu) was studied by affinity labelling and NMR spectroscopy. TosPheCH2Cl binds to GDP and GTP conformers of EF-Tu. The interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF-Tu x GTP does not lead to covalent labelling. [A82]EF-Tu, in which the Cys82 is replaced by Ala, has similar properties to wild-type EF-Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF-Ts) and interaction with ribosomes. This structural change did not lead to changes, compared with wild-type EF-Tu in the functionality of [A82]EF-Tu, either in the GTP or in the GDP conformation. TosPheCH2Cl binds to EF-Tu x GTP with a dissociation constant of 10 microM. The interaction of TosPheCH2Cl with EF-Tu promotes the hydration of the carbonyl group of TosPheCH2Cl. TosPheCH2Cl competes with aminoacyl-tRNA for its binding site on EF-Tu x GTP. Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl-tRNA. TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl-tRNA and binds to its binding site on EF-Tu x GTP. This rather specific interaction with EF-Tu x GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of this residue.