High compared with standard gentamicin dosing for chorioamnionitis: a comparison of maternal and fetal serum drug levels.

OBJECTIVE: To compare umbilical cord and maternal serum peak gentamicin concentration, gentamicin elimination, and clinical outcomes between women who received once-daily compared with standard, thrice-daily dosing for clinical chorioamnionitis. METHODS: We randomly assigned 38 laboring women, at least 34 weeks gestation, with clinical chorioamnionitis, into 1 of 2 gentamicin dosing groups: 5.1 mg/kg every 24 hours (once-daily; n = 18), or 120 mg followed by 80 mg every 8 hours (standard; n = 20). We measured maternal serum peak and delivery gentamicin concentrations and cord serum levels at delivery. Polynomial curve fitting was used to summarize gentamicin elimination. We also compared maternal and neonatal outcomes. RESULTS: Demographic characteristics of the 2 groups were similar. Median maternal peak gentamicin levels were higher with once-daily (18.2 microg/mL) compared with standard dosing (7.1 microg/mL) (P < .001). Maternal serum levels decreased below 2 microg/mL by 10 hours in the once-daily group and by 5 hours in the standard dosing group. Extrapolated peak cord serum levels were 6.9 microg/mL in the once-daily and 2.9 microg/mL in the standard dosing arm. Cord levels decreased below 2 microg/mL by 10 hours in the once-daily and by 5 hours in the standard dosing group. We found no differences in maternal or neonatal outcomes. CONCLUSION: Peak maternal serum gentamicin levels ranged from 13 to 25 microg/mL after a dose of 5.1 mg/kg. Single-dose gentamicin resulted in fetal serum peak levels that were closer to optimal neonatal values. Gentamicin clearance in the term fetus was similar to published values for the newborn infant. No adverse effects of high-dose therapy were noted.

Interplay between site-specific mutations and cyclic nucleotides in modulating DNA recognition by Escherichia coli cyclic AMP receptor protein.

Mutagenesis of various amino acids in Escherichia coli cyclic AMP receptor protein (CRP) has been shown to modulate protein compressibility and dynamics [Gekko et al. (2004) Biochemistry 43, 3844-3852]. Cooperativity of cAMP binding to CRP and the apparent DNA binding affinity are perturbed [Lin and Lee (2002) Biochemistry 41, 11857-11867]. The aim of this study is to explore the effects of mutation on the surface chemistry of CRP and to define the consequences of these changes in affecting specific DNA sequence recognition by CRP. Furthermore, the role of the interplay between mutation and specific identity of the bound cyclic nucleotide in this DNA recognition was explored. In the current study, effects of eight site-specific mutations (K52N, D53H, S62F, T127L, G141Q, L148R, H159L, and K52N/H159L) on DNA recognition of four sequences (Class I (site PI of lac), Class II (site PI of gal), and synthetic sequences that are hybrids of Classes I and II sites) modulated by three different cyclic nucleotides (cAMP, cCMP, and cGMP) were investigated. All mutations altered the surface chemistry of CRP as evidenced by the change in elution properties of these proteins from different matrixes. While T127L, S62F, K52N, and H159L exhibited unexpected behavior under combinations of specific experimental conditions, such as the identity of bound cyclic nucleotide and DNA sequence, in general, results showed that the affinities of CRP for DNA were sequence-dependent, increasing in the order of lacgal26 < gal26 < lac26 < gallac26 for all the mutants in the presence of 200 microM cAMP. The apparent association constants significantly increased in the order of no cyclic nucleotide approximately cGMP < cCMP < cAMP for all the examined DNA sequences. Linear correlation between the DeltaG for CRP-DNA complex formation and the cooperativity energy for cAMP binding was observed with gallac26, gal26, and lacgal26; however, the slope of this linear correlation is DNA sequence dependent. Structural information was presented to rationalize the interplay between CRP sequence and cyclic nucleotides in defining the recognition of DNA sequences.

Ability of E. coli cyclic AMP receptor protein to differentiate cyclic nucelotides: effects of single site mutations.

Escherichia coli cyclic AMP receptor protein (CRP) is a global transcriptional regulator which controls the expression of many different genes. Although different cyclic nucleotides can bind to CRP with almost equal affinity, only in the presence of cAMP could wild-type CRP bind to specific DNA sequences. Molecular genetic studies have identified a class of mutants, CRP*, which either do not require exogenous cAMP for activation or can be activated by cGMP. Thus, these mutants might aid in identifying the structural elements that are involved in the modulation of CRP to correctly differentiate the messages embedded in cyclic nucleotides. In this in vitro study, five CRP* mutants, namely, D53H, S62F, G141Q, G141K, and L148R, were tested for their abilities to bind the lac promoter sequence and the effects of cyclic nucleotides in modulating DNA sequence recognition. For comparison, non-CRP* mutants K52N, T127L, H159L, and K52N/H159L were studied. cCMP and cGMP can replace cAMP as an allosteric effector in all of these CRP mutants except S62F and non-CRP* mutants. The D53H, G141Q, G141K, and L148R mutants exhibit significantly higher affinity for the lac promoter sequence than wild-type CRP while S62F and the non-CRP* mutants exhibit reduced affinity. To probe the pathway of communication, the energetics of subunit assembly in these mutants were monitored by sedimentation equilibrium, and the conformational states of these mutants were probed by proteolysis and accessibility of Cys178 to chemical modifications. Results from these studies imply that signals due to mutations are mostly transmitted through the subunit interface. Thus, residues in CRP outside of the cyclic nucleotide binding site modulate the ability of CRP to differentiate these three cyclic nucleotides through long-range communication. Furthermore, this study shows that CRP* mutations do not impart any unique properties to CRP except that the DNA binding constants are shifted to a regime of higher affinity.