RESUMEN
A retrospective study was performed on 15 patients receiving 16 S-ROM mobile-bearing hinge total knee prostheses that were evaluated with at least a 2-year follow-up (range, 27-71 months). Indications for its use included severe instability and bone loss. The average patient age was 63 years (range, 33-83 years). There were 15 revision arthroplasties and 1 primary arthroplasty. Knee Society scores showed notable improvement in pain, motion, and stability (33.6 preoperatively vs 76.5 postoperatively; P <.0001) and approached significant improvement in function (29.2 preoperatively vs 43.5 postoperatively; P =.11). After excluding a patient with a traumatically ruptured patellar tendon, the probability of the latter comparison improved (P <.01). There was no evidence of loosening, and complete bone apposition was seen in nearly all cases. A high percentage of satisfactory results can be achieved when using this mobile-bearing hinge knee prosthesis for these indications.
Asunto(s)
Artroplastia de Reemplazo de Rodilla/instrumentación , Prótesis de la Rodilla , Adulto , Anciano , Anciano de 80 o más Años , Artroplastia de Reemplazo de Rodilla/métodos , Fenómenos Biomecánicos , Femenino , Estudios de Seguimiento , Humanos , Rodilla/diagnóstico por imagen , Rodilla/fisiología , Masculino , Persona de Mediana Edad , Complicaciones Posoperatorias , Radiografía , Reoperación , Estudios Retrospectivos , Resultado del TratamientoRESUMEN
Human herpesviruses can cause significant morbidity and mortality in pediatric solid organ transplant recipients. It was hypothesized that viral burden quantification by polymerase chain reaction using an internal calibration standard could aid in distinguishing between viral disease and latency. Here we report the results of a 2-year prospective study of 27 pediatric solid organ (liver, kidney, or heart) transplant recipients in which multiple samples were analyzed for levels of all eight human herpesviruses by internal calibration standard-polymerase chain reaction. Herpes simplex viruses 1 and 2, varicella-zoster virus, and Kaposi's sarcoma-associated herpesvirus were not detected in any of these samples. Human herpesvirus types 6 and 7 were detected in half of the patients, but were present at low levels, similar to those found in reference populations. Epstein-Barr virus (EBV) and cytomegalovirus (CMV) were detected in 89% and 56% of the patients, respectively. Viral burden analysis suggested distinct patient populations for CMV, with a natural cutoff of 10,000 viral targets/ml blood strongly associated with disease. In some cases, a dramatic increase in CMV levels preceded clinical evidence of disease by several weeks. EBV viral burden was relatively high in the only patient presenting with an EBV syndrome. However, two other patients without evidence of EBV disease had single samples with high EBV burden. Rapid reduction in both EBV and CMV burden occurred with antiviral treatment. These data suggest that viral burden analysis using internal calibration standard-polymerase chain reaction for CMV, and possibly other herpesviruses, is an effective method for monitoring pediatric transplant patients for significant herpesvirus infection and response to therapy.
Asunto(s)
Infecciones por Herpesviridae/virología , Herpesviridae/fisiología , Trasplante de Órganos , Reacción en Cadena de la Polimerasa/métodos , Carga Viral , Adolescente , Adulto , Niño , Preescolar , Estudios de Cohortes , Infecciones por Herpesviridae/diagnóstico , Humanos , Inmunosupresores/farmacología , Lactante , Valor Predictivo de las Pruebas , Estudios Prospectivos , Sensibilidad y Especificidad , Viremia/virologíaRESUMEN
We have determined by X-ray crystallography the structures of several variants of staphylococcal nuclease with long flexible straight chain and equivalent length cyclic unnatural amino acid side chains embedded in the protein core. The terminal atoms in the straight side chains are not well defined by the observed electron density even though they remain buried within the protein interior. We have previously observed this behavior and have suggested that it may arise from the addition of side-chain vibrational and oscillational motions with each bond as a side chain grows away from the relatively rigid protein main chain and/or the population of multiple rotamers (Wynn R, Harkins P, Richards FM. Fox RO. 1996. Mobile unnatural amino acid side chains in the core of staphylococcal nuclease. Protein Sci 5:1026-1031). Reduction of the number of degrees of freedom by cyclization of a side chain would be expected to constrain these motions. These side chains are in fact well defined in the structures described here. Over-packing of the protein core results in a 1.0 A shift of helix 1 away from the site of mutation. Additionally, we have determined the structure of a side chain containing a single hydrogen to fluorine atom replacement on a methyl group. A fluorine atom is intermediate in size between methyl group and a hydrogen atom. The fluorine atom is observed in a single position indicating it does not rotate like methyl hydrogen atoms. This change also causes subtle differences in the packing interactions.
Asunto(s)
Aminoácidos/química , Nucleasa Microcócica/química , Cristalografía por Rayos X , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Conformación ProteicaRESUMEN
The structure of mitogen-activated protein (MAP) kinase p38 has been solved at 2.1-A to an R factor of 21.0%, making p38 the second low activity MAP kinase solved to date. Although p38 is topologically similar to the MAP kinase ERK2, the phosphorylation Lip (a regulatory loop near the active site) adopts a different fold in p38. The peptide substrate binding site and the ATP binding site are also different from those of ERK2. The results explain why MAP kinases are specific for different activating enzymes, substrates, and inhibitors. A model presented for substrate and activator interactions has implications for the evolution of protein kinase cascades.
Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Proteínas Quinasas Activadas por Mitógenos , Conformación Proteica , Secuencia de Aminoácidos , Animales , Proteínas Quinasas Dependientes de Calcio-Calmodulina/biosíntesis , Proteínas Quinasas Dependientes de Calcio-Calmodulina/aislamiento & purificación , Simulación por Computador , Cristalografía por Rayos X , Humanos , Ratones , Proteína Quinasa 1 Activada por Mitógenos , Modelos Moleculares , Modelos Estructurales , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Homología de Secuencia de Aminoácido , Programas Informáticos , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
The structures of several variants of staphylococcal nuclease with long flexible unnatural amino acid side chains in the hydrophobic core have been determined by X-ray crystallography. The unnatural amino acids are disulfide moieties between the lone cysteine residue in V23C nuclease and methane, ethane, 1-n-propane, 1-n-butane, 1-n-pentane, and 2-hydroxyethyl thiols. We have examined changes in the core packing of these mutants. Side chains as large as the 1-n-propyl cysteine disulfide can be incorporated without perturbation of the structure. This is due, in part, to cavities present in the wild-type protein. The longest side chains are not well defined, even though they remain buried within the protein interior. These results suggest that the enthalpy-entropy balance that governs the rigidity of protein interiors favors tight packing only weakly. Additionally, the tight packing observed normally in protein interiors may reflect, in part, the limited numbers of rotamers available to the natural amino acids.
Asunto(s)
Proteínas Bacterianas/química , Cisteína/química , Nucleasa Microcócica/química , Compuestos de Sulfhidrilo/química , Aminoácidos/química , Proteínas Bacterianas/genética , Cristalografía por Rayos X/métodos , Nucleasa Microcócica/genética , Mutación , Conformación ProteicaRESUMEN
Among the protein kinases, an absolutely conserved lysine in subdomain II is required for high catalytic activity. This lysine is known to interact with the substrate ATP, but otherwise its role is not well understood. We have used biochemical and structural methods to investigate the function of this lysine (K52) in phosphoryl transfer reactions catalyzed by the MAP kinase ERK2. The kinetic properties of activated wild-type ERK2 and K52 mutants were examined using the oncoprotein TAL2, myelin basic protein, and a designed synthetic peptide as substrates. The catalytic activities of K52R and K52A ERK2 were lower than that of wild-type ERK2, primarily as a consequence of reductions in kcat. Further, there was little difference in Km for ATP, but the Km,app for peptide substrate was higher for the K52 mutants. The three-dimensional structure of unphosphorylated K52R ERK2 in the absence and presence of bound ATP was determined and compared with the structure of unphosphorylated wild-type ERK2. ATP adopted a well-defined but distinct binding mode in K52R ERK2 compared to the binding mode in the wild-type enzyme. The structural and kinetic data show that mutation of K52 created a nonproductive binding mode for ATP and suggest that K52 is essential for orienting ATP for catalysis.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Quinasas Dependientes de Calcio-Calmodulina/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Mutación Puntual , Secuencia de Aminoácidos , Sitios de Unión/genética , Proteínas Quinasas Dependientes de Calcio-Calmodulina/química , Secuencia Conservada , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Cinética , Lisina/química , Lisina/genética , Proteína Quinasa 1 Activada por Mitógenos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Proteína Básica de Mielina/metabolismo , Proteínas de Neoplasias/metabolismo , Péptidos/genética , Péptidos/metabolismo , Conformación Proteica , Especificidad por SustratoRESUMEN
The non-heme iron-dependent metalloenzyme, rat hepatic phenylalanine hydroxylase (EC 1.14.16.1; phenylalanine 4-monooxygenase (PAH) was overexpressed in Escherichia coli and purified to homogeneity, allowing a detailed comparison of the kinetic, hydrodynamic, and spectroscopic properties of its allosteric states. The homotetrameric recombinant enzyme, which is highly active and contains 0.7-0.8 iron atoms per subunit, is identical to the native enzyme in several properties: Km, 6-methyltetrahydropterin = 61 microM and L-Phe = 170 microM; Vmax = 9 s-1 (compared to 45 microM, 180 microM, and 13 s-1 for the rat hepatic enzyme). L-Phe and lysolecithin treatment induce the rPAHT-->rPAHR (where r is recombinant) allosteric transformation necessary for rPAH activity. Characteristic changes in the fluorescence spectra, increased hydrophobicity, a large activation energy barrier, and a 10% volume increase of the tetrameric structure are consistent with a significant reorganization of the protein following allosteric activation. However, optical and EPR spectroscopic data suggest that only minor changes occur in the primary coordination sphere (carboxylate/histidine/water) of the catalytic iron center. Detailed steady state kinetic investigations, using 6-methyltetrahydropterin as cofactor and lysolecithin as activator, indicate rPAH follows a sequential mechanism. A catalytic Arrhenius Eact of 14.6 +/- 0.3 kcal/mol subunit was determined from temperature-dependent stopped-flow kinetics data. rPAH inactivates during L-Phe hydroxylation with a half-life of 4.3 min at 25 degrees C, corresponding to an Arrhenius Eact of 10 +/- 1 kcal/mol subunit for the inactivation process. Catechol binding (2.4 x 10(6) M-1) is shown to occur only at catalytically competent iron sites. Ferrous rPAH binds NO, giving rise to an ST = 3/2 spin system.
Asunto(s)
Hígado/enzimología , Fenilalanina Hidroxilasa/química , Fenilalanina Hidroxilasa/metabolismo , Conformación Proteica , Regulación Alostérica , Animales , Western Blotting , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Activación Enzimática , Escherichia coli , Expresión Génica , Cinética , Lisofosfatidilcolinas/farmacología , Sustancias Macromoleculares , Peso Molecular , Fenilalanina Hidroxilasa/aislamiento & purificación , Fosforilación , Plásmidos , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Espectrofotometría , TermodinámicaRESUMEN
Bipyramidal crystals of the recombinant calmodulin from Trypanosoma brucei rhodesiense were obtained by vapor diffusion against 55% (v/v) 2-methyl-2,4-pentanediol in 0.05 M cacodylate buffer, pH 5.6. When few nucleation events occurred, crystals grew to 0.25 x 0.25 x 1.20 mm. The space group of the crystal is I4(1)22, with unit cell dimensions a = b = 56.88 A, c = 230.11 A, alpha = beta = gamma = 90 degrees, z = 16. The molecular mass and volume of the unit cell suggest that there is one molecule in the asymmetric unit. The I/sigma (I) ratio for data at 3.0 A resolution was 3.67, indicating that the final structure can be refined at higher resolution. Molecular replacement methods and the PC-refinement technique have not yet yielded the structure under a variety of search conditions. We are currently investigating the multiple isomorphous replacement approach to determine this crystal structure.