We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region. The genes were expressed in E. coli under control of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies. Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (the conversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment. The crude orf6 gene product converted UDP-[␣-D-U-14 C]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of synthetic ␣-galactofuranose-1-phosphate. No mutase activity was detected in extracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells. The orf6 gene product was purified by anion-exchange chromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form. The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed. These observations strongly suggest that orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDPgalactopyranose mutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 ؎ 8, in accordance with that calculated for the Glf protein. N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence. UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified as flavin adenine dinucleotide.

No abstract

No abstract

GR69153 is a novel cephalosporin incorporating a catechol-substituted 7-aminothiazolyl-oxime. The antibiotic is actively transported into gram-negative cells via iron-regulated outer membrane proteins regulated by the tonB product. This transport enhances bactericidal activity most significantly at low concentrations, essentially removing the permeability barrier for antibiotic uptake.

No abstract

An in-vitro model was shown to be capable of simulating a cefuroxime serum profile equivalent to that observed in human volunteer studies, following a single dose of 250 mg cefuroxime axetil. The model was used to carry out kill kinetic studies and showed cefuroxime to lyse the four bacterial test strains, time of onset of lysis being related to the sensitivity of the respective organisms. The more sensitive Staphylococcus aureus and Haemophilus influenzae strains were subject to a higher absolute kill and showed no regrowth over the duration of the simulated serum profile. In contrast, Proteus mirabilis and Escherichia coli showed regrowth after 4 and 5 h respectively. The kill kinetic profiles of the respective organisms are discussed in relation to the pharmacokinetic analysis of the cefuroxime serum profile.