RESUMO
Two fluorescent lysine amide analogs, in which the carboxyl end of lysine was covalently attached to dansyl or NBD groups through an ethylene glycol-based linker, were rationally designed and synthesized. Both probes showed high binding affinity to the lysine riboswitch in vitro and their fluorescence intensities decreased by riboswitch binding.
Assuntos
Corantes Fluorescentes/química , Lisina/química , Riboswitch , Amidas/química , Corantes Fluorescentes/síntese química , Estrutura MolecularRESUMO
A fluorogenic derivatization method was developed to distinguish the protein N-terminal acetylation status. The unacetylated protein selectively reacted with 4-chloro-7-nitrobenzofurazan (NBD-Cl) at neutral pH to provide high fluorescence. In contrast, the protein with N-terminal acetylation was essentially nonfluorescent under the same conditions despite the presence of many internal lysine residues. Fluorescence of the NBD-labeled protein was very stable, and only micromolar concentrations of proteins were required for reliable detection. This method also provides a general and practical way to quantify proteins when their N-terminal amino group is available.
Assuntos
4-Cloro-7-nitrobenzofurazano/metabolismo , Corantes Fluorescentes/metabolismo , Proteínas/química , Proteínas/metabolismo , Coloração e Rotulagem/métodos , 4-Cloro-7-nitrobenzofurazano/química , Acetilação , Corantes Fluorescentes/química , Estrutura Terciária de ProteínaRESUMO
N-terminal acetylation of the recombinant Z-domain protein depends on E. coli strains, expression vectors and amino acid residues near the N-terminus, and is enhanced by a high cellular level of RimJ.
Assuntos
Acetiltransferases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes/metabolismo , Proteínas Ribossômicas/metabolismo , Acetilação , Acetiltransferases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Genes Bacterianos , Estrutura Terciária de Proteína , Proteínas Ribossômicas/genéticaRESUMO
INTRODUCTION: TobraDex ophthalmic suspension (tobramycin 0.3%, dexamethasone 0.1%; Alcon Laboratories Inc, Fort Worth, Tex) is frequently used for inflammatory ocular conditions where a risk of bacterial ocular infection exists. A new formulation, TobraDex ST ophthalmic suspension (tobramycin 0.3%, dexamethasone 0.05%, Alcon), utilises a novel suspension technology to reduce viscosity and help prevent settling in the container. METHODS: A rabbit model that closely mimics the human eye and a clinical study with cataract patients was used to compare the pharmacokinetics and tissue permeability of TobraDex ST and TobraDex. An in-vitro model was used to assess the bactericidal activity using the rabbit tear concentrations of tobramycin 10 minutes after a single topical dose. RESULTS: Concentrations of both tobramycin and dexamethasone were greater in the tear film and ocular tissues of rabbits treated with TobraDex ST. There was an 8.3-fold increase in tobramycin concentration in the rabbit tear film 10 minutes after dosing with TobraDex ST compared with TobraDex. Concentrations of tobramycin and dexamethasone in ocular tissues from rabbits exposed to TobraDex ST were up to 12.5-fold greater relative to TobraDex. The in-vitro bactericidal activity (>99.9% kill, 3-log reduction) of TobraDex ST toward tobramycin-resistant and methicillin-resistant Staphylococcus aureus occurred in 90 minutes. TobraDex ST killed Streptococcus pneumoniae 3-log in 5 minutes. TobraDex had no activity toward tobramycin-resistant, methicillin-resistant S. aureus and required approximately 120 minutes for 3-log reduction of S. pneumoniae. In humans, the mean ratio of dexamethasone levels in the aqueous humour at 1 hour was 1.17 in favour of TobraDex ST. CONCLUSION: TobraDex ST demonstrated improved suspension formulation characteristics, enhanced pharmacokinetic distribution and improved bactericidal characteristics, and may provide a useful alternative as compared to TobraDex.
