RESUMEN
Three variations to the structure of the nicotinamide adenine dinucleotide (NAD)-dependent L-lactate dehydrogenase from Bacillus stearothermophilus were made to try to change the substrate specificity from lactate to malate: Asp197----Asn, Thr246----Gly, and Gln102----Arg). Each modification shifts the specificity from lactate to malate, although only the last (Gln102----Arg) provides an effective and highly specific catalyst for the new substrate. This synthetic enzyme has a ratio of catalytic rate (kcat) to Michaelis constant (Km) for oxaloacetate of 4.2 x 10(6)M-1 s-1, equal to that of native lactate dehydrogenase for its natural substrate, pyruvate, and a maximum velocity (250 s-1), which is double that reported for a natural malate dehydrogenase from B. stearothermophilus.
Asunto(s)
Geobacillus stearothermophilus/enzimología , L-Lactato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Sitios de Unión , Geobacillus stearothermophilus/genética , Cinética , L-Lactato Deshidrogenasa/metabolismo , Modelos Moleculares , Conformación Proteica , Especificidad por SustratoRESUMEN
A site-directed mutant of Bacillus stearothermophilus lactate dehydrogenase (lactate:NAD+ oxidoreductase, EC 1.1.1.27) has been engineered in which the conserved hydrophobic residue isoleucine-250 has been replaced by the more hydrophilic residue asparagine. This isoleucine forms a large part of a water-accessible, hydrophobic surface in the active site of the apo-enzyme which is covered by the B-face of the nicotinamide ring when coenzymes are bound. Reduction in the area of this hydrophobic surface results in the mutant tetramer being more thermally stable than the wild-type enzyme.
Asunto(s)
L-Lactato Deshidrogenasa , Sitios de Unión , Fenómenos Químicos , Química Física , Geobacillus stearothermophilus/enzimología , Geobacillus stearothermophilus/genética , Isoleucina , Mutación , TemperaturaRESUMEN
Cytomegaloviurs (CMV) infections are a recognized problem in the first six months after renal transplantation. Studies have suggested that primary infections produce symptomatic disease, whereas reactivation infections are usually asymptomatic. Two patients are described who developed fatal CMV infections in the second year after transplantation. Both patients had typical CMV disease with fever, pneumonitis, and hepatitis. Results of serologic studies in one patient were characteristic of primary infection, with seroconversion at the time of disease and appearance of specific IgM antibodies. The other patient had a similar antibody response at the time of his illness, but serial antibody tests showed that he had had a transient seroconversion earlier, in the second month after transplanation, that was not associated with clinical symptoms. These patients indicate that CMV infection must be considered in the differential diagnosis of serious febrile illnesses even in the late posttransplantation period and may occur either as the result of primary or reactivation infection. Serologic studies at the time of illness may not allow distinction between the types of infection.
Asunto(s)
Infecciones por Citomegalovirus/etiología , Trasplante de Riñón , Complicaciones Posoperatorias , Adulto , Anticuerpos Antivirales/análisis , Citomegalovirus/inmunología , Infecciones por Citomegalovirus/inmunología , Humanos , Inmunoglobulina M/análisis , Masculino , Persona de Mediana Edad , Trasplante HomólogoRESUMEN
In a further exploration of idiosyncratic reactions to injections of contrast media, a systematic study of immediate or delayed hypersensitivity in guinea pigs injected with contrast media analogs was performed. No evidence of immediate or delayed hypersensitivity could be ascertained to either contrast media analogs alone, or analogs bound to protein carriers. This does not represent conclusive evidence against the notion of antibody formation as a factor in the production of some adverse reactions, but makes such a pathogenesis less likely.
Asunto(s)
Ácido Acetrizoico/inmunología , Formación de Anticuerpos , Diatrizoato/inmunología , Hipersensibilidad Tardía/inmunología , Hipersensibilidad Inmediata/inmunología , Yodobenzoatos/inmunología , Ácido Metrizoico/inmunología , Animales , Especificidad de Anticuerpos , Complejo Antígeno-Anticuerpo , Antígenos , Medios de Contraste/efectos adversos , Cobayas , Masculino , Ovalbúmina , Anafilaxis Cutánea Pasiva , Albúmina Sérica Bovina , Pruebas CutáneasRESUMEN
A single case of back pain due to Blastomyces dermatitidis infection limited solely to the skeleton is reported with a discussion of the differential diagnosis. This rare disease must be differentiated from other destructive bone lesions such as tuberculosis or tumors because of the availability of effective treatment.
Asunto(s)
Blastomicosis/diagnóstico por imagen , Enfermedades de la Columna Vertebral/diagnóstico por imagen , Anfotericina B/uso terapéutico , Dolor de Espalda/diagnóstico por imagen , Blastomicosis/tratamiento farmacológico , Diagnóstico Diferencial , Femenino , Humanos , Pulmón/patología , Persona de Mediana Edad , Osteomielitis/diagnóstico por imagen , Columna Vertebral/diagnóstico por imagen , Tomografía Computarizada por Rayos XAsunto(s)
Intubación Intratraqueal/efectos adversos , Tráquea/lesiones , Animales , Perros , Hipotensión/complicaciones , Intubación Intratraqueal/instrumentación , Membrana Mucosa/lesiones , Respiración con Presión Positiva , Presión , Radiografía , Goma , Siliconas , Tantalio , Tráquea/irrigación sanguínea , Tráquea/diagnóstico por imagen , Traqueotomía/instrumentación , Ventiladores MecánicosAsunto(s)
Angiografía , Diatrizoato , Riñón/enzimología , Arteria Renal/diagnóstico por imagen , Urografía , Adhesividad , Animales , Aspartato Aminotransferasas/sangre , Aspartato Aminotransferasas/orina , Catalasa/sangre , Catalasa/orina , Creatina Quinasa/sangre , Creatina Quinasa/orina , Perros , Gelatina , Ácido Yotalámico , L-Lactato Deshidrogenasa/sangre , L-Lactato Deshidrogenasa/orinaAsunto(s)
Familia , Atención Domiciliaria de Salud , Salud Mental , Anciano , Humanos , Cuidados Intermitentes , Cambio Social , Estrés Psicológico , Estados UnidosRESUMEN
The R/B Enteric Differential System for identifying enteric bacteria has been evaluated with 451 "unknown" cultures from the stock culture collection of the Center for Disease Control. An average of 89.6% of these cultures were correctly identified by the R/B system, when used as recommended by the manufacturer but without the assistance of serology. This percentage ranged, however, from 47% for Klebsiella to 100% for Serratia and Providencia. Of 11 groups or genera of Enterobacteriaceae tested, only three (Enterobacter, Serratia, and Providencia) were identified with 95% or better accuracy. Four groups (Arizona, Citrobacter, Escherichia, and Salmonella) attained 90 to 95% accuracy of identification, and three groups (Edwardsiella, Proteus, and Shigella) scored between 85 and 90% accuracy. We recommend the R/B system as a screening device which is reasonably successful in grouping bacteria but not as a substitute for more exacting conventional procedures.
Asunto(s)
Técnicas Bacteriológicas , Enterobacteriaceae/clasificación , Carboxiliasas/metabolismo , Movimiento Celular , Medios de Cultivo , Enterobacteriaceae/enzimología , Enterobacteriaceae/aislamiento & purificación , Enterobacteriaceae/metabolismo , Estudios de Evaluación como Asunto , Gases/biosíntesis , Glucosa/metabolismo , Hidrógeno/biosíntesis , Indoles/biosíntesis , Liasas/metabolismo , Lisina , Ornitina , Fenilalanina , Sulfuros/biosíntesisRESUMEN
Analysis of the mechanism and structure of lactate dehydrogenases is summarized in a map of the catalytic pathway. Chemical probes, single tryptophan residues inserted at specific sites and a crystal structure reveal slow movements of the protein framework that discriminate between closely related small substrates. Only small and correctly charged substrates allow the protein to engulf the substrate in an internal vacuole that is isolated from solvent protons, in which water is frozen and hydride transfer is rapid. The closed vacuole is very sensitive to the size and charge of the substrate and provides discrimination between small substrates that otherwise have too few functional groups to be distinguished at a solvated protein surface. This model was tested against its ability to successfully predict the design and synthesis of new enzymes such as L-hydroxyisocaproate dehydrogenase and fully active malate dehydrogenase. Solvent friction limits the rate of forming the vacuole and thus the maximum rate of catalysis.