RESUMO
The purpose of this quality improvement project was to define best practices for identifying appropriate patients for genomic testing and improve timeliness for ordering tests and reporting results. An interdisciplinary team of surgeons, radiologists, medical oncologists, and nurses agreed that the RN navigator would be the key person to facilitate timely access to genomic profiling. AT A GLANCE: Genomic profiling has become the standard of care for patients with early-stage breast cancer to assist in developing individualized treatment plans. Nurse navigators can play a key role in improving timeliness of care. The APN-RN model led to improvements in turnaround time and complicance with the National Comprehensive Cancer Network's recommendations for genomic testing.
Assuntos
Neoplasias da Mama/enfermagem , Tomada de Decisão Clínica/métodos , Testes Genéticos , Papel do Profissional de Enfermagem , Navegação de Pacientes/organização & administração , Feminino , Humanos , Relações Enfermeiro-PacienteRESUMO
DNA ligases are the enzymes essential for DNA replication, repair and recombination in all organisms. The bacterial DNA ligases involved in DNA replication require NAD(+) for activity, but eukaryotic and viral DNA ligases require ATP. Because of their essential nature, unique structures and widespread existence in nature, bacterial DNA ligases represent a class of valuable targets for identifying novel and selective antibacterial agents. In this study, we cloned and expressed the ligA gene from Streptococcus pneumoniae, and characterized this ligA-encoded NAD(+)-dependent DNA ligase. We then screened small molecule chemical libraries using a biochemical assay and identified a new small molecule with a structure of 2,4-diamino-7-dimethylamino-pyrimido[4,5-d]pyrimidine. We show that this small molecule is a specific inhibitor of bacterial NAD(+)-dependent DNA ligases. Biochemical studies show that this molecule inhibits NAD(+)-dependent DNA ligases, but not ATP-dependent enzymes. The molecule inhibits NAD(+)-dependent DNA ligases competitively with respect to NAD(+) and specifically inhibits enzyme adenylation, but not DNA adenylation or ligation. Labeling studies establish that this molecule inhibits the incorporation of thymidine into DNA and that overexpression of DNA ligase in the cell abolishes this inhibition. Finally, microbiological studies show that this molecule exhibits a broad spectrum of antibacterial activity. Together, this study shows that this small molecule inhibitor identified is specific to bacterial NAD(+)-dependent DNA ligases, exhibits a broad spectrum of antibacterial activities, and has the potential to be developed into an antibacterial agent.
Assuntos
Antibacterianos/química , DNA Ligases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Pirimidinas/farmacologia , Streptococcus pneumoniae/enzimologia , Proteínas de Bactérias/antagonistas & inibidores , Sequência de Bases , DNA Ligases/genética , DNA Ligases/isolamento & purificação , Inibidores Enzimáticos/química , Humanos , Cinética , Pirimidinas/química , Bibliotecas de Moléculas Pequenas , Streptococcus pneumoniae/genéticaRESUMO
Acyl carrier protein (ACP) synthase (AcpS) catalyzes the transfer of the 4'-phosphopantetheine moiety from coenzyme A (CoA) onto a serine residue of apo-ACP, resulting in the conversion of apo-ACP to the functional holo-ACP. The holo form of bacterial ACP plays an essential role in mediating the transfer of acyl fatty acid intermediates during the biosynthesis of fatty acids and phospholipids. AcpS is therefore an attractive target for therapeutic intervention. In this study, we have purified and characterized the AcpS enzymes from Escherichia coli, Streptococcus pneumoniae, and Mycoplasma pneumoniae, which exemplify gram-negative, gram-positive, and atypical bacteria, respectively. Our gel filtration column chromatography and cross-linking studies demonstrate that the AcpS enzyme from M. pneumoniae, like E. coli enzyme, exhibits a homodimeric structure, but the enzyme from S. pneumoniae exhibits a trimeric structure. Our biochemical studies show that the AcpS enzymes from M. pneumoniae and S. pneumoniae can utilize both short- and long-chain acyl CoA derivatives but prefer long-chain CoA derivatives as substrates. On the other hand, the AcpS enzyme from E. coli can utilize short-chain CoA derivatives but not the long-chain CoA derivatives tested. Finally, our biochemical studies show that M. pneumoniae AcpS is kinetically a very sluggish enzyme compared with those from E. coli and S. pneumoniae. Together, the results of these studies show that the AcpS enzymes from different bacterial species exhibit different native structures and substrate specificities with regard to the utilization of CoA and its derivatives. These findings suggest that AcpS from different microorganisms plays a different role in cellular physiology.
Assuntos
Acil Coenzima A/metabolismo , Proteínas de Bactérias/metabolismo , Escherichia coli/enzimologia , Transferases (Outros Grupos de Fosfato Substituídos)/fisiologia , Acil Coenzima A/química , Proteínas de Bactérias/química , Mycoplasma pneumoniae , Especificidade da Espécie , Streptococcus pneumoniae , Especificidade por Substrato , Transferases (Outros Grupos de Fosfato Substituídos)/química , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismoRESUMO
Era, an essential GTPase, appears to play an important role in the regulation of the cell cycle and protein synthesis of bacteria and mycoplasmas. In this study, native Era, His-tagged Era (His-Era) and glutathione S-transferase (GST)-fusion Era (GST-Era) proteins from Escherichia coli were expressed and purified. It was shown that the GST-Era and His-Era proteins purified by 1-step affinity column chromatographic methods were associated with RNA and exhibited a higher GTPase activity. However, the native Era protein purified by a 3-step column chromatographic method had a much lower GTPase activity and was not associated with RNA which had been removed during purification. Purified GST-Era protein was shown to be present as a high- and a low-molecular-mass forms. The high-molecular-mass form of GST-Era was associated with RNA and exhibited a much higher GTPase activity. Removal of the RNA associated with GST-Era resulted in a significant reduction in the GTPase activity. The RNA associated with GST-Era was shown to be primarily 16S rRNA. A purified native Era protein preparation, when mixed with total cellular RNA, was found to bind to some of the RNA. The native Era protein isolated directly from the cells of a wild-type E. coli strain was also present as a high-molecular-mass form complexed with RNA and RNase treatment converted the high-molecular-mass form into a 32 kDa low-molecular-mass form, a monomer of Era. Furthermore, a C-terminally truncated Era protein, when expressed in E. coli, did not bind RNA. Finally, the GTPase activity of the Era protein free of RNA, but not the Era protein associated with the RNA, was stimulated by acetate and 3-phosphoglycerate. These carbohydrates, however, failed to activate the GTPase activity of the C-terminally truncated Era protein. Thus, the results of this study establish that the C-terminus of Era is essential for the RNA-binding activity and that the RNA and carbohydrates modulate the GTPase activity of Era possibly through a similar mechanism.