Assuntos
Pneumopatias/genética , Linfopenia/genética , Doenças da Imunodeficiência Primária/genética , Proteínas rac de Ligação ao GTP/genética , Adulto , Linfócitos B/imunologia , Progressão da Doença , Proteínas Ativadoras de GTPase/metabolismo , Mutação com Ganho de Função , Doença Enxerto-Hospedeiro/tratamento farmacológico , Guanosina Trifosfato/metabolismo , Transplante de Células-Tronco Hematopoéticas , Heterozigoto , Humanos , Memória Imunológica/imunologia , Imunossupressores/uso terapêutico , Lactente , Pneumopatias/imunologia , Pneumopatias/fisiopatologia , Pneumopatias/cirurgia , Transplante de Pulmão , Linfopenia/imunologia , Masculino , Simulação de Acoplamento Molecular , Neutrófilos , Doenças da Imunodeficiência Primária/imunologia , Doenças da Imunodeficiência Primária/terapia , Recidiva , Infecções Respiratórias/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T/imunologia , Proteínas rac de Ligação ao GTP/imunologia , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/ultraestrutura , Proteína RAC2 de Ligação ao GTPRESUMO
Mevalonate kinase deficiency (MKD) is an autoinflammatory disorder caused by mutations in the MVK gene resulting in decreased activity of the enzyme mevalonate kinase (MK). Although MK is required for biosynthesis of all isoprenoids, in MKD, in particular, the timely synthesis of geranylgeranyl pyrophosphate appears to be compromised. Because small guanosine triphosphatases (GTPases) depend on geranylgeranylation for their proper signaling function, we studied the effect of MK deficiency on geranylgeranylation and activation of the two small GTPases, RhoA and Rac1. We demonstrate that both geranylgeranylation and activation of the two GTPases are more easily disturbed in MKD cells than in control cells when the flux though the isoprenoid biosynthesis pathway is suppressed by low concentrations of simvastatin. The limited capacity of geranylgeranylation in MKD cells readily leads to markedly increased levels of nonisoprenylated and activated GTPases, which will affect proper signaling by these GTPases.
Assuntos
Fibroblastos/enzimologia , Deficiência de Mevalonato Quinase/enzimologia , Fosfotransferases (Aceptor do Grupo Álcool)/deficiência , Prenilação de Proteína , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Membrana Celular/enzimologia , Ativação Enzimática , Fibroblastos/efeitos dos fármacos , Humanos , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Deficiência de Mevalonato Quinase/genética , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Transporte Proteico , Transdução de Sinais , Sinvastatina/farmacologiaRESUMO
The Structural Proteomics In Europe (SPINE) consortium contained a workpackage to address the automated X-ray analysis of macromolecules. The aim of this workpackage was to increase the throughput of three-dimensional structures while maintaining the high quality of conventional analyses. SPINE was able to bring together developers of software with users from the partner laboratories. Here, the results of a workshop organized by the consortium to evaluate software developed in the member laboratories against a set of bacterial targets are described. The major emphasis was on molecular-replacement suites, where automation was most advanced. Data processing and analysis, use of experimental phases and model construction were also addressed, albeit at a lower level.
Assuntos
Cristalografia por Raios X/métodos , Proteômica/métodos , Algoritmos , Automação , Interpretação Estatística de Dados , Bases de Dados Factuais , Modelos Químicos , Modelos Moleculares , Conformação Proteica , Controle de Qualidade , SoftwareRESUMO
In this report, we describe the validation of a rapid, single-step, microtiter plate method for quantifying bacterial adherence, based on fluorescent labeling of microorganisms with cell-permeable fluorescent DNA-binding probes. We have tested the binding to saliva-coated microtiter plates of bacteria, including Helicobacter pylori and viridans streptococci (S. mitis, S. gordonii, S. sanguis), known to interact with salivary components. Furthermore, we tested the short-term and longer-term temporal stability of a saliva-mediated adherence of these bacteria in a healthy population (N=30). The assay exhibited excellent reliability statistics, yielding within-assay variability coefficients ranging from 4.9% to 11%. A range of approximately 5 x 10(4)-1 x 10(7) cells could be detected. This method may be generally applicable to study surface binding of virtually any microbial species, while obviating the need of radioactive materials or specific antibodies for quantification, thus providing a procedure that is useful to both basic and clinical research.