RESUMEN
BACKGROUND: The AACC assembled a committee to identify and validate a standard creatine kinase MB isoenzyme (CK-MB) material to improve the comparability of CK-MB mass assays. METHODS: Three protocols were used. In protocol I, various CK-MB materials prepared in different matrices were screened as candidate standards. In protocol II, participating manufacturers calibrated their systems with concentrates of human heart CK-MB and then tested 20 patient samples to evaluate calibration bias. In protocol III, participating manufacturers calibrated their immunoassay systems using recombinant CK-MB2 (rCK-MB2) diluted into their respective sample diluents and measured 50 samples. RESULTS: Candidate materials showed high recovery in stripped human serum, but bias improved only from 59% to 38%. These data led to the use of human heart CK-MB diluted in each manufacturer's sample diluent. This strategy reduced bias from 31% to 15%. Because human heart CK-MB is difficult to provide, a lyophilized source of CK-MB2 was identified. rCK-MB2 was shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reversed-phase HPLC, intrinsic protein fluorescence, circular dichroism, agarose gel electrophoresis, immunoreactivity studies, high and low temperature stability, and reconstituted stability to be equivalent to human heart CK-MB. Calibration of immunoassay systems with rCK-MB2 added into each respective manufacturer's sample diluent showed a 13% between-manufacturer bias. CONCLUSION: Lyophilized rCK-MB2 was determined suitable for use as a reference material for CK-MB mass assays.
Asunto(s)
Creatina Quinasa/normas , Calibración , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Creatina Quinasa/análisis , Creatina Quinasa/aislamiento & purificación , Electroforesis en Gel de Agar , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Humanos , Isoenzimas , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/normas , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
The histories of 56 patients with the diagnosis "osteochondrosis dissecans tali" were retrospectively analyzed with regard to etiology, localization, therapy, course, and prognosis. Some of the patients were reexamined. In 33 patients it is highly probable that the osteochondrosis dissecans was not associated with the sequelae of osteochondral fractures. The typical X-ray image is that of a medial, deep osteolysis on the trochlea of the talus, close to the margin, with one or more roundish dissections. There are usually no degenerative or posttraumatic changes. The history and frequent bilateralism tend to rule out trauma as the cause. In the authors' opinion, therefore, the term "osteochondrosis dissecans" should not be used for osteochondral fractures and their sequelae. A number of borderline cases are critically discussed.
Asunto(s)
Osteocondritis Disecante/diagnóstico por imagen , Osteocondritis/diagnóstico por imagen , Astrágalo/diagnóstico por imagen , Adolescente , Adulto , Anciano , Niño , Femenino , Estudios de Seguimiento , Fracturas Óseas/diagnóstico por imagen , Humanos , Luxaciones Articulares/diagnóstico por imagen , Masculino , Persona de Mediana Edad , Osteocondritis Disecante/cirugía , Complicaciones Posoperatorias/diagnóstico por imagen , Radiografía , Astrágalo/lesiones , Astrágalo/cirugíaRESUMEN
Human gamma-thrombin is a three (noncovalently linked)-domain enzyme which contains the known serine protease catalytic triad, Asp-His-Ser, one on each of the three noncovalent domains (Asp 99 on the A-B3 chain). While protein-folding dogma does not necessarily predict that the denatured form of this enzyme could refold to the correct conformation, a monitor of the esterase activity (Tos-Arg-OMe) shows complete recovery of native catalytically active conformation. When compared with the covalently intact alpha form which refolds from urea in less than 2 min with complete return of both clotting and esterase activity, gamma-thrombin requires up to 90 min to regain full esterase activity. The gamma-thrombin reactivation data best fit a single first order rate constant, k = 0.03 +/- 0.005 min-1. It was suggested that the gamma-thrombin renaturation process might represent first the rapid refolding, then subsequent reassociation and reisomerization of the three noncovalent domains to yield a lower energy, fully active, conformation. This study represents the only example known of the refolding (reconstitution) of a three (noncovalent)-domain protein.
Asunto(s)
Trombina , Coagulación Sanguínea , Esterasas/metabolismo , Humanos , Cinética , Conformación Proteica , Espectrometría de Fluorescencia , Trombina/metabolismo , UreaRESUMEN
Human alpha-thrombin, the two (covalently linked)-chain, highly coagulant blood-clotting enzyme was compared with its noncoagulant, yet estero/amidolytically active derivative, gamma-thrombin, a three (noncovalently associated)-domain enzyme which results from two proteolytic cleavages of the coagulant a form. Studies of their denaturation behavior by Tos-Arg-OMe esterase activity, by intrinsic fluorescence, by fluorescence of active serine-directed dansyl labels, and by monitoring the ESR of a fluorosulfonylphenyl spin-labeled inhibitor clearly demonstrated the reduced stability of the noncovalently associated gamma-thrombin form. At pH 6.5, 0.75 M NaCl, gamma-thrombin unfolds in approximately 2. 1 M urea while the more stable a form denatures at approximately 4 M urea. By monitoring active serine probes (spin label or fluorescent labels), these transitions were slightly lower, 1.0 +/- 0.1 and 2.8 +/- 0.2 M urea for spin-labeled gamma- and alpha-thrombins, respectively. Similar behavior was found for the same spin-labeled derivatives in guanidine HCl with unfolding transitions of 0.4 M and 1.0 M for spin-labeled gamma- and alpha-thrombin, respectively. These differences in structural stabilization serve as a good physical diagnostic for the two thrombin species.
Asunto(s)
Trombina , Coagulación Sanguínea , Estabilidad de Medicamentos , Espectroscopía de Resonancia por Spin del Electrón , Esterasas/metabolismo , Guanidinas , Humanos , Sustancias Macromoleculares , Conformación Proteica , Desnaturalización Proteica , Espectrometría de Fluorescencia , Trombina/metabolismo , UreaRESUMEN
This study partially replicated Martens' (1969a) social-facilitation study of motor behavior. His very robust performance findings provided impressive confirmation for Zajonc's hypothesis, and his arousal findings have since been used as evidence for a nonlearned-drive basis for social facilitation. The present study also extended Martens' investigation by examining the separate and combined effects of an audience and videotape camera. The effects due to the presence of the audience and camera were not additive; instead, the audience detrimentally affected subjects' performance consistency and the camera resulted in more trials with errors greater than 30 msec after the performance criteria had been attained. Martens' most robust findings for constant error were not replicated, nor were some of his physiological arousal findings. His pattern of constant error results over all trials is atypical of known learning strategies that subjects use to reduce error over successive trials. Overall, audience effects accounted for only a very small portion of the variance.