RESUMO
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.
Assuntos
Geobacillus stearothermophilus/enzimologia , L-Lactato Desidrogenase/genética , Malato Desidrogenase/metabolismo , Sítios de Ligação , Geobacillus stearothermophilus/genética , Cinética , L-Lactato Desidrogenase/metabolismo , Modelos Moleculares , Conformação Proteica , Especificidade por SubstratoRESUMO
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oxidoreductase, EC 1.1.1.27) has been engineered in which the conserved hydrophobic residue isoleucine-250 has been replaced by the more hydrophilic residue asparagine.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.
Assuntos
L-Lactato Desidrogenase , Sítios de Ligação , Fenômenos Químicos , Físico-Química , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Isoleucina , Mutação , TemperaturaRESUMO
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.
Assuntos
Infecções por Citomegalovirus/etiologia , Transplante de Rim , Complicações Pós-Operatórias , Adulto , Anticorpos Antivirais/análise , Citomegalovirus/imunologia , Infecções por Citomegalovirus/imunologia , Humanos , Imunoglobulina M/análise , Masculino , Pessoa de Meia-Idade , Transplante HomólogoRESUMO
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.
Assuntos
Ácido Acetrizoico/imunologia , Formação de Anticorpos , Diatrizoato/imunologia , Hipersensibilidade Tardia/imunologia , Hipersensibilidade Imediata/imunologia , Iodobenzoatos/imunologia , Ácido Metrizoico/imunologia , Animais , Especificidade de Anticorpos , Complexo Antígeno-Anticorpo , Antígenos , Meios de Contraste/efeitos adversos , Cobaias , Masculino , Ovalbumina , Anafilaxia Cutânea Passiva , Soroalbumina Bovina , Testes CutâneosRESUMO
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.
Assuntos
Blastomicose/diagnóstico por imagem , Doenças da Coluna Vertebral/diagnóstico por imagem , Anfotericina B/uso terapêutico , Dor nas Costas/diagnóstico por imagem , Blastomicose/tratamento farmacológico , Diagnóstico Diferencial , Feminino , Humanos , Pulmão/patologia , Pessoa de Meia-Idade , Osteomielite/diagnóstico por imagem , Coluna Vertebral/diagnóstico por imagem , Tomografia Computadorizada por Raios XAssuntos
Intubação Intratraqueal/efeitos adversos , Traqueia/lesões , Animais , Cães , Hipotensão/complicações , Intubação Intratraqueal/instrumentação , Mucosa/lesões , Respiração com Pressão Positiva , Pressão , Radiografia , Borracha , Silicones , Tantálio , Traqueia/irrigação sanguínea , Traqueia/diagnóstico por imagem , Traqueotomia/instrumentação , Ventiladores MecânicosAssuntos
Angiografia , Diatrizoato , Rim/enzimologia , Artéria Renal/diagnóstico por imagem , Urografia , Adesividade , Animais , Aspartato Aminotransferases/sangue , Aspartato Aminotransferases/urina , Catalase/sangue , Catalase/urina , Creatina Quinase/sangue , Creatina Quinase/urina , Cães , Gelatina , Ácido Iotalâmico , L-Lactato Desidrogenase/sangue , L-Lactato Desidrogenase/urinaAssuntos
Família , Assistência Domiciliar , Saúde Mental , Idoso , Humanos , Cuidados Intermitentes , Mudança Social , Estresse Psicológico , Estados UnidosRESUMO
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.
Assuntos
Técnicas Bacteriológicas , Enterobacteriaceae/classificação , Carboxiliases/metabolismo , Movimento Celular , Meios de Cultura , Enterobacteriaceae/enzimologia , Enterobacteriaceae/isolamento & purificação , Enterobacteriaceae/metabolismo , Estudos de Avaliação como Assunto , Gases/biossíntese , Glucose/metabolismo , Hidrogênio/biossíntese , Indóis/biossíntese , Liases/metabolismo , Lisina , Ornitina , Fenilalanina , Sulfetos/biossínteseRESUMO
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.