RESUMO
INTRODUCCIÓN: El Xpert MTB/RIF Ultra (Ultra) ha mejorado dramáticamente el diagnóstico de la tuberculosis (TBC). Con él ha nacido la categoría de trazas, que es la menor carga bacilar detectable por este examen. OBJETIVO: Describir las características clínicas de los pacientes con presencia de trazas en el Ultra y evaluar la confirmación de la TBC como diagnóstico clínico. MATERIALES Y MÉTODOS: Estudio descriptivo de serie de casos. Se extrajo la información de fichas clínicas de pacientes con positividad a trazas. Se confrontaron datos clínicos, microbiológicos e histopatológicos. RESULTADOS: Se analizaron 21 pacientes. La edad promedio fue de 52 años. Todos los casos presentaron baciloscopias negativas. Cuatro cultivos en medio líquido MGIT fueron positivos, dos en pleura parietal, uno en líquido pleural y otro en expectoración. En pleura parietal, tres casos presentaron granulomas con necrosis caseosa y un granuloma esbozos de necrosis. En tejido pulmonar se observaron dos casos con granulomas con esbozos de necrosis y dos con granulomas no necrotizantes. Tres pacientes tenían el antecedente de TBC previa, se interpretó la positividad de trazas en ellos como falsos positivos. Finalmente se diagnosticaron 13 casos como TBC activa, donde cinco de ellos fueron TBC pleurales. La mayor concordancia clínica, microbiológica e histopatológica fue en muestras de líquido y tejido pleural. DISCUSIÓN: Se debe interpretar con cautela los hallazgos de esta prueba en muestras de vía aérea; el análisis multidisciplinario (clínica, imágenes, microbiología, histología) es crucial en las decisiones de nuestras conductas clínicas futuras. El hallazgo de trazas en pleura tiene, a nuestro parecer, un alto valor diagnóstico en el estudio de la tuberculosis en esta localización.
INTRODUCTION: Xpert MTB/RIF Ultra has dramatically changed the diagnosis of tuberculosis. A new category called traces appeared, which is the smallest amount of bacillar load detectable. OBJECTIVE: Describe the clinical characteristics of patients that present traces in Xpert MTB/RIF Ultra test, and to evaluate the confirmation of tuberculosis as clinical diagnosis. METHODS: We perform a descriptive case series study. Information was recovered from clinical records of patients with positive test for traces. Clinical, histopathological and microbiological results were confronted. RESULTS: Twenty one patients were analyzed. The mean age was 52 years-old. All cases had negative smear microscopy and four MGIT cultures were positive, two in pleural fluid and another in sputum. In parietal pleura, three cases presented granulomas with caseous necrosis, and one showed granuloma with very little necrosis. In pleural tissue we observed two cases of granulomas with traces of necrosis and two with non-necrotizing granulomas. Three patients had history of previous tuberculosis and positive traces, the test was interpreted as a false positive result. Finally, active tuberculosis was diagnosed in 13 cases, and five of them were pleural tuberculosis. The highest clinical, microbiological and histopathological agreement was in fluid and pleural tissue samples. DISCUSSION: The findings of Xpert MTB/RIF Ultra in airway samples must be interpreted carefully. Multi-disciplinary analysis is crucial in future clinical decisions. The finding of traces in pleura has, in our opinion, a high diagnostic value in the study of tuberculosis in this location.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso , Tuberculose Pleural/diagnóstico , Tuberculose Pleural/microbiologia , Tuberculose Pulmonar/diagnóstico , Tuberculose Pulmonar/microbiologia , Técnicas Bacteriológicas/métodos , Escarro/microbiologia , Tuberculose Pleural/patologia , Tuberculose Pulmonar/patologia , Mycobacterium tuberculosisRESUMO
The Oman-United Arab Emirates ophiolite has been used extensively to document the geological processes that form oceanic crust. The geometry of the ophiolite, its extension into the Gulf of Oman, and the nature of the crust that underlies it are, however, unknown. Here, we show the ophiolite forms a high velocity, high density, >15 km thick east-dipping body that during emplacement flexed down a previously rifted continental margin thereby contributing to subsidence of flanking sedimentary basins. The western limit of the ophiolite is defined onshore by the Semail thrust while the eastern limit extends several km offshore, where it is defined seismically by a ~40-45°, east-dipping, normal fault. The fault is interpreted as the southwestern margin of an incipient suture zone that separates the Arabian plate from in situ Gulf of Oman oceanic crust and mantle presently subducting northwards beneath the Eurasian plate along the Makran trench.
RESUMO
Xpert MTB/RF is an automatic methodology that uses the polymerase chain reaction to detect in less than two hours the presence of Mycobacterium tuberculosis and also informs about the sensibility to rifampicin. This technology has good sensibility and even better specificity in respiratory samples. In non-respiratory samples the sensibility is lower. We analyzed the results of samples sent to the Laboratory of Tuberculosis of the Instituto Nacional del Tórax-Chile (National Thorax Institute) during the last two years since we started to use this technology. We analyzed 529 samples, 384 (73%) of them were respiratory in origin and 145 (27%) were non-respiratory. Only 43 samples were positive for Mycobacterium tuberculosis, 33 from respiratory samples, and 10 from other sources. 17 smear negative samples were culture positive for Mycobacterium tuberculosis; 15 of them were detected by Xpert; 351 samples were culture negative, 17 of them were positive by Xpert, the majority in patients under treatment for tuberculosis or with old tuberculosis. Ten of 10 culture positive patients from non-respiratory samples were positive with Xpert. The interpretation of Rifampicin resistance, when its prevalence in a given population is low, like is the case in Chile, requires confirmation by using standard methods.
Xpert MTB/RIF es un método automatizado basado en la reacción de la polimerasa en cadena que permite en menos de dos horas detectar la presencia de ADN de Mycobacterium tuberculosis y, además, informa la susceptibilidad a la Rifampicina. Esta técnica, que está disponible en el Instituto Nacional del Tórax (INT) desde hace dos años, tiene una buena sensibilidad y una mejor especificidad en muestras respiratorias. En muestras no respiratorias el rendimiento, aunque inferior, sigue siendo clínicamente útil. Se analizaron 529 muestras enviadas al Laboratorio de Tuberculosis del INT, comparándolas con el cultivo de Koch en medio sólido que es el "gold standard". El 73% (384) de las muestras fueron respiratorias y sólo 145 (27%) no respiratorias (líquido pleural, orina y líquido cefalorraquídeo). Del total de las muestras, 43 resultaron positivas para tuberculosis, de ellas, 33 eran de origen respiratorio y 10 no respiratorio. Todos los pacientes que tenían baciloscopias positivas fueron confirmados por el Xpert. De los 17 enfermos con baciloscopias negativas y cultivos positivos, 15 fueron detectados con el Xpert. De los 351 pacientes con cultivos negativos de las muestras respiratorias, 17 resultaron positivos por el Xpert, pero 8 de éstos estaban en tratamiento o habían tenido una tuberculosis anteriormente. En las muestras no respiratorias, de 10 pacientes con cultivos positivos el Xpert detectó los 10, demostrando también una excelente sensibilidad. En cuanto a la resistencia a Rifampicina, cuando la prevalencia de ésta en una población es baja, como ocurre en Chile, requiere confirmación por las técnicas estándar.
Assuntos
Humanos , Tuberculose/diagnóstico , Reação em Cadeia da Polimerase/métodos , Chile , Interpretação Estatística de Dados , Estudos Retrospectivos , Microscopia/métodosRESUMO
Functional analyses of PDB (Paget's disease of bone)-associated mutants of the p62 [also known as SQSTM1 (sequestosome 1)] signalling adaptor protein represent an interesting paradigm for understanding not only the disease mechanism in this skeletal disorder, but also the critical determinants of ubiquitin recognition by an ubiquitin-binding protein. The 11 separate PDB mutations identified to date all affect the C-terminal region of p62 containing the UBA domain (ubiquitin-associated domain), a ubiquitin-binding element. All of these mutations have deleterious effects on ubiquitin binding by p62 in vitro, and there is evidence of an inverse relationship between ubiquitin-binding function and disease severity. The effects on ubiquitin-binding function of most of the mutations can be attributed to either reduced UBA domain stability, and/or the mutations affecting the presumed ubiquitin-binding interface of the UBA domain. However, a subset of the mutations are more difficult to rationalize; several of these affect sequences of p62 outside of the minimal ubiquitin-binding region, providing insights into non-UBA domain sequences within the host protein which mediate ubiquitin-binding affinity. The p62 mutations are presumed to result in activation of (osteoclast) NF-kappaB (nuclear factor kappaB) signalling. Understanding how loss of ubiquitin-binding function of p62 impacts on signal transduction events in osteoclasts will undoubtedly further our understanding of the disease mechanism in PDB at the molecular level.
Assuntos
Mutação , Osteíte Deformante/genética , Proteínas/genética , Ubiquitina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Humanos , Fenótipo , Ligação Proteica , Proteínas/metabolismo , Proteína Sequestossoma-1RESUMO
Ubiquitin-associated (UBA) domain mutations of SQSTM1 are an important cause of Paget's disease of bone (PDB), which is a human skeletal disorder characterized by abnormal bone turnover. We previously showed that, when introduced into the full-length SQSTM1 protein, the disease-causing P392L, M404V, G411S, and G425R missense mutations and the E396X truncating mutation (representative of all of the SQSTM1 truncating mutations) cause a generalized loss of monoubiquitin binding and impaired K48-linked polyubiquitin binding at physiological temperature. Here, we show that the remaining three known PDB missense mutations, P387L, S399P, and M404T, have similar deleterious effects on monoubiquitin binding and K48-linked polyubiquitin binding by SQSTM1. The P387L mutation affects an apparently unstructured region at the N terminus of the UBA domain, some five residues from the start of the first helix, which is dispensable for polyubiquitin binding by the isolated UBA domain. Our findings support the proposal that the disease mechanism in PDB with SQSTM1 mutations involves a common loss of ubiquitin binding function of SQSTM1 and implicate a sequence extrinsic to the compact globular region of the UBA domain as a critical determinant of ubiquitin recognition by the full-length SQSTM1 protein.
Assuntos
Osso e Ossos/metabolismo , Mutação de Sentido Incorreto/genética , Osteíte Deformante/genética , Osteíte Deformante/metabolismo , Proteínas/genética , Ubiquitinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação/genética , Osso e Ossos/patologia , Osso e Ossos/fisiopatologia , Predisposição Genética para Doença/genética , Humanos , Osteíte Deformante/fisiopatologia , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Proteínas/química , Proteínas/metabolismo , Proteína Sequestossoma-1RESUMO
Mutations affecting the UBA (ubiquitin-associated) domain of SQSTM1 (Sequestosome 1) (p62) are a common cause of Paget's disease of bone. The missense mutations resolve into those which retain [P392L (Pro(392)-->Leu), G411S] or abolish (M404V, G425R) the ability of the isolated UBA domain to bind Lys-48-linked polyubiquitin. These effects can be rationalized with reference to the solution structure of the UBA domain, which we have determined by NMR spectroscopy. The UBA domain forms a characteristic compact three-helix bundle, with a hydrophobic patch equivalent to that previously implicated in ubiquitin binding by other UBA domains. None of the mutations affect overall folding of the UBA domain, but both M404V and G425R involve residues in the hydrophobic patch, whereas Pro-392 and Gly-411 are more remote. A simple model assuming the isolated UBA domain is functioning as a compact monomer can explain the effects of the mutations on polyubiquitin binding. The P392L and G411S mutations do however have subtle local effects on secondary structure, which may become more relevant in full-length SQSTM1. Identification of the in vivo ubiquitylated substrates of SQSTM1 will be most informative in determining the functional significance of the SQSTM1-ubiquitin interaction, and consequences of the disease-associated mutations.
Assuntos
Mutação , Osteíte Deformante/genética , Proteínas/química , Proteínas/genética , Proteínas Adaptadoras de Transdução de Sinal , Glicina/química , Humanos , Espectroscopia de Ressonância Magnética , Mutação de Sentido Incorreto , Poliubiquitina/química , Prolina/química , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas/fisiologia , Proteína Sequestossoma-1 , Relação Estrutura-Atividade , Ubiquitina/químicaRESUMO
Spontaneous bladder rupture is a rare condition associated with significant morbidity and mortality. We describe a case that occured following a period of alcohol intoxication and presented as acute renal failure. The factors that contribute to this condition in an intoxicated person are outlined and useful clinical markers are suggested. This case demonstrates the difficulties with diagnosis and the need for a high index of suspicion.
Assuntos
Intoxicação Alcoólica/complicações , Bexiga Urinária/lesões , Injúria Renal Aguda/diagnóstico , Injúria Renal Aguda/etiologia , Adulto , Diagnóstico Diferencial , Humanos , Masculino , Ruptura EspontâneaRESUMO
NMR studies have shown that the minor groove-binding ligand Hoechst 33258 binds to the two T4/A4 tracts within the duplex d(CTTTTCGAAAAG)2 in a highly cooperative manner, such that in titration experiments no intermediate 1:1 complex can be detected. The NMR-derived structures of the free DNA and the 2:1 complex have been obtained, but can shed little light on what the origins of this cooperativity may be. Here we present the results of a series of molecular dynamics simulations on the free DNA, the 1:1 complex, and the 2:1 complex, which have been designed to enable us to calculate thermodynamic parameters associated with the molecular recognition events. The results of the molecular dynamics studies confirm that structural factors alone cannot explain the cooperativity observed, indeed when enthalpic and hydration factors are looked at in isolation, the recognition process is predicted to be slightly anticooperative. However, when changes in configurational entropy are taken into account as well, the overall free energy differences are such that the calculated cooperativity is in good agreement with that observed experimentally. The results indicate the power of molecular dynamics methods to provide reasonable explanations for phenomena that are difficult to explain on the basis of static models alone, and provide a nice example of the concept of "allostery without conformational change".
Assuntos
Bisbenzimidazol/química , DNA/química , Modelos Químicos , Bisbenzimidazol/farmacologia , Simulação por Computador , DNA/efeitos dos fármacos , Adutos de DNA/química , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Soluções , TermodinâmicaRESUMO
H alpha chemical shifts are often used as indicators of secondary structure formation in protein structural analysis and peptide folding studies. On the basis of NMR analysis of model beta-sheet and alpha-helical peptides, together with a statistical analysis of protein structures for which NMR data are available, we show that although the gross pattern of H alpha chemical shifts reflects backbone torsion angles, longer range effects from distant amino acids are the dominant factor determining experimental chemical shifts in beta-sheets of peptides and proteins. These show context-dependent variations that aid structural assignment and highlight anomalous shifts that may be of structural significance and provide insights into beta-sheet stability.
Assuntos
Aminoácidos/química , Ressonância Magnética Nuclear Biomolecular/métodos , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Animais , Bovinos , Ligação de Hidrogênio , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Ubiquitina/químicaRESUMO
The thermodynamics of the native<-->A state and native<-->unfolded transitions for ubiquitin have been characterized in detail using the denaturants methanol and guanidinium chloride (Gdn.HCl) both separately and in combination. Gdn.HCl destabilizes the partially folded alcohol-induced A state such that the effects of alcoholic solvents on the native<-->unfolded transition can be investigated directly via a two-state model. The combined denaturing effects of methanol and Gdn.HCl appear to conform to a simple additive model. We show that ubiquitin folds and unfolds cooperatively in all cases, forming the same "native" state; however, the thermodynamics of the N<-->U transition change dramatically between alcoholic and Gdn.HCl solutions, with folding in aqueous methanol associated with large negative enthalpy and entropy terms at 298 K with a gradual falloff in DeltaC(p) at higher methanol concentrations, as previously reported for the N<-->A transition (Woolfson, D. N., Cooper, A., Harding, M. M., Williams, D. H., and Evans, P. A. (1993) J. Mol. Biol. 229, 502-511.). Both the N<-->U and the N<-->A transitions are enthalpy driven to a similar extent. We conclude that under these conditions van der Waals interactions in the packing of the nonpolar protein core, which is common to both the N<-->U and the N<-->A transitions, appear to drive folding in the absence of entropic effects associated with release of ordered solvent (hydrophobic effect). Solvent transfer studies of hydrocarbons into alcoholic solvents, with and without Gdn.HCl, are consistent with a large enthalpic driving force for burial of a nonpolar surface, with a linear dependence of protein stability (DeltaG(N)(<-->)(U)) on cosolvent concentration reflected in a similar linear dependence of hydrocarbon solubility. The data demonstrate that the hydrophobic effect is not a prerequisite for specific stabilization of the native state or the A state and that van der Waals packing of the nonpolar core appears to be the dominant factor in stabilization of the native state.
Assuntos
Dobramento de Proteína , Ubiquitinas/química , Ubiquitinas/metabolismo , Animais , Calorimetria , Bovinos , Dicroísmo Circular , Guanidina/farmacologia , Metanol/farmacologia , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Desnaturação Proteica , Estrutura Secundária de Proteína , Solventes , Termodinâmica , Ubiquitinas/efeitos dos fármacosRESUMO
AIM: A prospective cross-sectional study was performed on 170 patients with various glomerular diseases to study the accuracy of predicting 24-hour proteinuria from the spot urine protein-creatinine ratio (Up/Uc). A cost-benefit analysis was performed for the New Zealand health economic system to obtain the best cut-off values for proteinuria. SUBJECTS, METHODS AND RESULTS: Two spot urine samples (Up/Uc1 and Up/Uc2) were collected on the same day as the collection of a 24-hour urine. A randomly chosen subsample of 50 patients provided a second set of urine samples. The correlation and precision of agreement between the two methods were examined. The predictive intervals were calculated for derived 24-hour proteinuria. The level of agreement was evaluated by the Bland-Altman method and concordance analysis. The limits of agreement were evaluated against the clinical limits of agreement. A cost-benefit analysis (CBA) was performed to obtain the optimum operating points on receiver operating characteristic (ROC) curves for the best decision threshold. Correlations of r = 0.97 and 0.99 were observed between Up/Uc1, Up/Uc2 and 24-hour proteinuria, respectively. The 95% predictive intervals were wide. A high concordance correlation coefficient was obtained. The most of the differences between the two methods fell within the clinical limits of agreement. The Up/Uc1 of 0.26 and 3.20 represent the best thresholds to detect normal and nephrotic proteinuria, respectively. CONCLUSIONS: Despite wide confidence intervals, a good correlation and precision of agreement were demonstrated between the two methods across the whole range of proteinuria, regardless of the level of renal function. The difference between the two methods was less than the biological variability in the protein excretion and its measurement, enabling the methods to be used interchangeably. The optimum thresholds for abnormal and nephrotic range proteinuria were obtained.
Assuntos
Glomerulonefrite/urina , Proteinúria/economia , Adulto , Análise Custo-Benefício , Creatinina/urina , Estudos Transversais , Feminino , Glomerulonefrite/diagnóstico , Glomerulonefrite/economia , Humanos , Masculino , Valor Preditivo dos Testes , Estudos Prospectivos , Curva ROCRESUMO
NMR analysis and molecular dynamics simulations of d(GGTAATTACC)(2) and its complex with a tetrahydropyrimidinium analogue of Hoechst 33258 suggest that DNA minor groove recognition in solution involves a combination of conformational selection and induced fit, rather than binding to a preorganised site. Analysis of structural fluctuations in the bound and unbound states suggests that the degree of induced fit observed is primarily a consequence of optimising van der Waals contacts with the walls of the minor groove resulting in groove narrowing through: (i) changes in base step parameters, including increased helical twist and propeller twist; (ii) changes to the sugar-phosphate backbone conformation to engulf the bound ligand; (iii) suppression of bending modes at the TpA steps. In contrast, the geometrical arrangement of hydrogen bond acceptors on the groove floor appears to be relatively insensitive to DNA conformation (helical twist and propeller twist). We suggest that effective recognition of DNA sequences (in this case an A tract structure) appears to depend to a significant extent on the sequence being flexible enough to be able to adopt the geometrically optimal conformation compatible with the various binding interactions, rather than involving 'lock and key' recognition.
Assuntos
Bisbenzimidazol/análogos & derivados , Bisbenzimidazol/química , DNA/química , Conformação de Ácido Nucleico , Sequência de Bases , Gráficos por Computador , Simulação por Computador , Ligação de Hidrogênio , Modelos Moleculares , Conformação Molecular , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular/métodos , Oligodesoxirribonucleotídeos/química , Pirimidinas/químicaAssuntos
Micoses/tratamento farmacológico , Micoses/etiologia , Diálise Peritoneal Ambulatorial Contínua/efeitos adversos , Peritonite/etiologia , Peritonite/microbiologia , Rhodotorula , Idoso , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Antifúngicos/uso terapêutico , Vias de Administração de Medicamentos , Gentamicinas/administração & dosagem , Gentamicinas/uso terapêutico , Humanos , Cetoconazol/uso terapêutico , Masculino , Insuficiência Renal/terapiaRESUMO
Haematoma formation and bleeding at the femoral puncture site are the most common complications after cardiac catheterisation. Reducing the length of bedrest for patients after this procedure in one unit aimed to reduce discomfort and increase the number of patients treated. No significant clinical changes took place in the occurrence of haematoma formation or early bleeds as a result of shorter bedrest.
Assuntos
Repouso em Cama , Cateterismo Cardíaco/enfermagem , Doença das Coronárias/enfermagem , Auditoria de Enfermagem , Cuidados Pós-Operatórios/normas , Doença das Coronárias/diagnóstico , Humanos , Cuidados Pós-Operatórios/métodosRESUMO
Peptide fragments corresponding to the N- and C-terminal portions of bovine ubiquitin, U(1-35) and U(36-76), are shown by NMR to associate in solution to form a complex of modest stability (Kassn approximately 1.4 x 10(5) M(-1) at pH 7.0), with NMR features characteristic of a nativelike structure. The complex undergoes cold denaturation, with temperature-dependent estimates of stability from NMR indicating a DeltaC(p) degrees for fragment complexation in good agreement with that determined for native ubiquitin, suggesting that fragment association results in the burial of a similar hydrophobic surface area. The stability of the complex shows appreciable pH dependence, suggesting that ionic interactions on the surface of the protein contribute significantly. However, denaturation studies of native ubiquitin in the presence of guanidine hydrochloride (Gdn.HCl) show little pH dependence, suggesting that ionic interactions may be "screened" by the denaturant, as recently suggested. Examination of the conformation of the isolated peptide fragments has shown evidence for a low population of nativelike structure in the N-terminal beta-hairpin (residues 1-17) and weak nascent helical propensity in the helical fragment (residues 21-35). In contrast, the C-terminal peptide (36-76) shows evidence in aqueous solution, from some Halpha chemical shifts, for nonnative phi and psi angles; nonnative alpha-helical structure is readily induced in the presence of organic cosolvents, indicating that tertiary interactions in both native ubiquitin and the folded fragment complex strongly dictate its structural preference. The data suggest that the N-terminal fragment (1-35), where interaction between the helix and hairpin requires the minimum loss of conformational entropy, may provide the nucleation site for fragment complexation.
Assuntos
Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Dobramento de Proteína , Termodinâmica , Ubiquitinas/química , Ubiquitinas/metabolismo , Animais , Bovinos , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Cinética , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular/métodos , Fragmentos de Peptídeos/isolamento & purificação , Conformação Proteica , Estrutura Secundária de Proteína , SolventesRESUMO
The role of the non-native beta-turn sequence (NPDG) in nucleating the folding of a beta-hairpin peptide derived from the N-terminus of ubiquitin, has been examined by NMR and CD spectroscopy. The NPDG sequence, while representing a common two-residue type I turn sequence in proteins, folds to give a G1-bulged type I turn in the context of a beta-hairpin peptide, to the exclusion of other possible conformations. The turn conformation results in misalignment of the two beta strands and a beta hairpin with non-native side chain interactions. A truncated 12-residue analogue of the hairpin, in which the majority of residues in the N-terminal beta strand have been deleted, shows some weak propensity to fold into a G-bulged type I turn conformation in the absence of interstrand stabilizing interactions. The NPDG turn sequence pays some of the entropic cost in initiating folding allowing interstrand interactions, which in this case arise from the non-native pairing of residue side chains, to stabilize a significant population of the folded state. Examination of the relative abundance of the Pro-Asp type I turn, with G in the +B1 position, vs. the type I G-bulged turn PXG, in a database of high resolution structures, reveals 48 instances of PXG bulged turns for which X = Asp is by far the most common residue with 20 occurrences. Strikingly, there are no examples of a type I PD turn with G at the +B1 position, in good agreement with our experimental observations that the PDG G-bulged turn is populated preferentially in solution.
Assuntos
Fragmentos de Peptídeos/química , Ubiquitinas/química , Sequência de Aminoácidos , Bases de Dados Factuais , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Conformação Proteica , Dobramento de Proteína , SoluçõesRESUMO
NMR spectroscopy is used to show that a 20-residue beta-hairpin peptide sequence derived from ferredoxin I, with a Pro-Asp two-residue type I turn which is uncommon in beta-hairpins, is unstructured in aqueous solution but shows NOE evidence for partial folding in the presence of sodium dodecylsulphate micelles. The peptide has a number of lysine residues in the N-terminal beta-strand capable of interacting with the micelle surface and templating the partial folding of the hairpin by reducing the entropic cost of ordering the peptide backbone.
Assuntos
Micelas , Peptídeos/química , Dobramento de Proteína , Motivos de Aminoácidos , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Propriedades de SuperfícieRESUMO
The solution structure of the dodecamer duplex d(CTTTTGCAAAAG)(2)and its 2:1 complex with the bis -benzimidazole Hoechst 33258 has been investigated by NMR and NOE-restrained molecular dynamics (rMD) simulations. Drug molecules are bound in each of the two A-tracts with the bulky N-methylpiperazine ring of each drug located close to the central TG (CA) step, binding essentially to the narrow minor groove of each A-tract. MD simulations over 1 ns, using an explicit solvation model, reveal time-averaged sequence-dependent narrowing of the minor groove from the 3'-end towards the 5'-end of each TTTT sequence. Distinct junctions at the TpG (CpA) steps, characterised by large positive roll, low helical and propeller twists and rapid AT base pair opening rates, add to the widening of the groove at these sites and appear to account for the bound orientation of the two drug molecules with the N-methylpiperazine ring binding in the wider part of the groove close to the junctions. Comparisons between the free DNA structure and the 2:1 complex (heavy atom RMSD 1.55 A) reveal that these sequence-dependent features persist in both structures. NMR studies of the sequence d(GAAAAGCTTTTC)(2), in which the A-tracts have been inverted with the elimination of the TpG junctions, results in loss of orientational specificity of Hoechst 33258 and formation of multiple bound species in solution, consistent with the drug binding in a number of different orientations.
Assuntos
Bisbenzimidazol/química , Bisbenzimidazol/metabolismo , DNA/genética , DNA/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Regulação Alostérica , Sequência de Bases , Sítios de Ligação , Simulação por Computador , Citosina/metabolismo , DNA/química , Guanina/metabolismo , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Conformação Molecular , Mutação/genética , Piperazinas/química , Piperazinas/metabolismo , Poli A/química , Poli A/genética , Poli A/metabolismo , Poli T/química , Poli T/genética , Poli T/metabolismo , Prótons , Especificidade por Substrato , TermodinâmicaRESUMO
UNLABELLED: Predicting renal survival in primary focal glomerulosclerosis from the time of presentation. BACKGROUND: To predict the risk of developing chronic renal failure in patients with primary focal glomerulosclerosis (FGS) using predictors available at the time of presentation, a retrospective analysis was performed on 111 patients who were diagnosed at Christchurch Hospital from 1965 to 1998. METHODS: The predictors of outcome included age, gender, systolic and diastolic blood pressure, serum albumin, plasma creatinine, presence of hematuria, and amount of proteinuria (all at the time of presentation). An injury score (combination of percentage of sclerosed glomeruli and proportion of tubulointerstitial fibrosis) was derived from a review of the initial kidney biopsy. Log-logistic accelerated failure time parametric models were used. RESULTS: The median renal survival was 16.4 years (Kaplan-Meier estimate). The best single variable model was that using the proportion of tubulointerstitial fibrosis (global chi-square 55.99, P < 0.0001). However, inclusion of plasma creatinine significantly improved the fit of the model (global chi-square 65.04, P < 0.0001). This joint model was superior to the single-variable model. Both of the models were validated using jackknifing. CONCLUSION: For a patient with primary FGS, these models can be used to predict the risk of developing chronic renal failure at any time and the median renal survival, given the proportion of tubulointerstitial fibrosis and plasma creatinine at the time of presentation.
Assuntos
Glomerulosclerose Segmentar e Focal/mortalidade , Falência Renal Crônica/mortalidade , Adolescente , Adulto , Idoso , Biópsia , Creatinina/sangue , Feminino , Fibrose , Glomerulosclerose Segmentar e Focal/sangue , Glomerulosclerose Segmentar e Focal/patologia , Humanos , Falência Renal Crônica/sangue , Falência Renal Crônica/patologia , Glomérulos Renais/patologia , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos , Análise de SobrevidaRESUMO
NMR studies of the folding and conformational properties of a beta-hairpin peptide, several peptide fragments of the hairpin, and sequence-modified analogues, have enabled the various contributions to beta-hairpin stability in water to be dissected. Temperature and pH-induced unfolding studies indicate that the folding-unfolding equilibrium approximates to a two-state model. The hairpin is highly resistant to denaturation and is still significantly folded in 7 M urea at 298 K. Thermodynamic analysis shows the hairpin to fold in water with a significant change in heat capacity, however, DeltaCp degrees in 7 M urea is reduced. V/Y-->A mutations on one strand of the hairpin reduce folding to <10 %, consistent with a hydrophobic stabilisation model. We show that in a truncated peptide (residues 6-16) lacking the hydrophobic residues on one beta-strand, the type I' Asn-Gly turn in the sequence SINGKK is significantly populated in water in the absence of interstrand hydrophobic contacts. Unrestrained molecular dynamics simulations of unfolding, using an explicit solvation model, show that the conformation of the NG turn persists for longer than the AG analogue, which has a much lower propensity for type I' turn formation from a data base analysis of preferred turns. The origin of the high stability of the Asn-Gly turn is not entirely clear; data base analysis of 66 NG turns, together with molecular dynamics simulations, reveals no participation of the Asn side-chain in turn-stabilising interactions with the peptide backbone. However, hydration analysis of the molecular dynamics simulations reveals a pocket of "high density" water bridging between the Asn side-chain and peptide main-chain that suggests solvent-mediated interactions may play an important role in modulating phi,psi propensities in the NG turn region.