Altering the proclivity towards daptomycin resistance in methicillin-resistant Staphylococcus aureus using combinations with other antibiotics.

Daptomycin (DAP) is increasingly used as a part of combination therapy, particularly in complex methicillin-resistant Staphylococcus aureus (MRSA) infections. While multiple studies have reported the potential for synergy between DAP and adjunctive anti-infectives, few have examined the influence of adjunctive therapy on the emergence of DAP resistance. This study examined eight adjunctive antimicrobial combinations with DAP in vitro and the emergence of DAP resistance over time (up to 4 weeks) using clinical isolates of DAP-susceptible MRSA (MIC, 0.5 µg/ml) in which DAP resistance subsequently developed during patient therapy (MIC, 3 µg/ml). In addition to DAP susceptibility testing, selected strains were examined for phenotypic changes associated with DAP resistance, including changes to cell wall thickness (CWT) and cell membrane alterations. The addition of either oxacillin or clarithromycin in medium containing DAP significantly inhibited the development of DAP resistance through the entirety of the 4-week exposure (10- to 32-fold MIC reduction from that of DAP alone). Combinations with rifampin or fosfomycin were effective in delaying the emergence of DAP resistance through the end of week one only (week one MIC, 0.5 µg/ml; week four MIC, 24 µg/ml). Cell wall thickening was observed for all antibiotic combinations regardless of their effect on the DAP MIC (14 to 70% increase in CWT), while changes in cell membrane fluidity were variable and treatment dependent. DAP showed reduced activity against strains with DAP MICs of 1 to 12 µg/ml, but cell membrane integrity was still disrupted at concentrations achieved with doses greater than 10 mg/kg of body weight. The emergence of DAP resistance in MRSA is strongly influenced by the presence of subinhibitory concentrations of adjunctive antimicrobials. These data suggest that combining DAP with oxacillin or clarithromycin may delay the development of DAP resistance in cases requiring prolonged antibiotic therapy.

Advancing matrix-assisted laser desorption/ionization-mass spectrometric imaging for capillary electrophoresis analysis of peptides.

In this work, the utilization of matrix-assisted laser desorption/ionization-mass spectrometric imaging (MALDI-MSI) for capillary electrophoresis (CE) analysis of peptides based on a simple and robust off-line interface has been investigated. The interface involves sliding the CE capillary distal end within a machined groove on a MALDI sample plate, which is precoated with a thin layer of matrix for continuous sample deposition. MALDI-MSI by time of flight (TOF)/TOF along the CE track enables high-resolution and high-sensitivity detection of peptides, allowing the reconstruction of a CE electropherogram while providing accurate mass measurements and structural identification of molecules. Neuropeptide standards and their H/D isotopic formaldehyde-labeled derivatives were analyzed using this new platform. Normalized intensity ratios of individual ions extracted from the CE trace were compared to MALDI-MS direct analysis and the theoretical ratios. The CE-MALDI-MSI results show potential for sensitive and quantitative analysis of peptide mixtures spanning a wide dynamic range.

The virucidal EB peptide protects host cells from herpes simplex virus type 1 infection in the presence of serum albumin and aggregates proteins in a detergent-like manner.

The linear cationic amphiphilic EB peptide, derived from the FGF4 signal sequence, was previously shown to be virucidal and to block herpes simplex type I (HSV-1) entry (H. Bultmann, J. S. Busse, and C. R. Brandt, J. Virol. 75:2634-2645, 2001). Here we show that cells treated with EB (RRKKAAVALLPAVLLALLAP) for less than 5 min are also protected from infection with HSV-1. Though protection was lost over a period of 5 to 8 h, it was reinduced as rapidly as during the initial treatment. Below a 20 µM concentration of EB, cells gained protection in a serum-dependent manner, requiring bovine serum albumin (BSA) as a cofactor. Above 40 µM, EB coprecipitated with BSA under hypotonic conditions. Coprecipitates retained antiviral activity and released active peptide. NaCl (≥0.3 M) blocked coprecipitation without interfering with antiviral activity. As shown for ß-galactosidase, EB below 20 µM acted as an enzyme inhibitor, whereas above 40 to 100 µM EB, ß-galactosidase was precipitated as was BSA or other unrelated proteins. Pyrene fluorescence spectroscopy revealed that in the course of protein aggregation, EB acted like a cationic surfactant and self associated in a process resembling micelle formation. Both antiviral activity and protein aggregation did not depend on stereospecific EB interactions but depended strongly on the sequence of the peptide's hydrophobic tail. EB resembles natural antimicrobial peptides, such as melittin, but when acting in a nonspecific detergent-like manner, it primarily seems to target proteins.

Dose- and route-dependent teratogenicity, toxicity, and pharmacokinetic profiles of the hedgehog signaling antagonist cyclopamine in the mouse.

The Hedgehog (Hh) signaling pathway is an essential regulator of embryonic development and appears to play important roles in postnatal repair and cancer progression and metastasis. The teratogenic Veratrum alkaloid cyclopamine is a potent Hh antagonist and is used experimentally both in vitro and in vivo to investigate the role of Hh signaling in diverse biological processes. Here, we set out to establish an administration regimen for cyclopamine-induced teratogenicity in the mouse. The dysmorphogenic concentration of cyclopamine was determined in vitro via mouse whole-embryo culture assays to be 2.0 microM. We administered cyclopamine to female C57BL/6J mice at varied doses by oral gavage, ip injection, or osmotic pump infusion and assessed toxicity and pharmacokinetic (PK) models. Bolus administration was limited by toxicity and rapid clearance. In vivo cyclopamine infusion at 160 mg/kg/day yielded a dam serum steady-state concentration of approximately 2 microM with a corresponding amniotic fluid concentration of approximately 1.5 microM. Gross facial defects were induced in 30% of cyclopamine-exposed litters, with affected embryos exhibiting cleft lip and palate. This is the first report describing the PKs and teratogenic potential of cyclopamine in the mouse and demonstrates that transient Hh signaling inhibition induces facial clefting anomalies in the mouse that mimic common human birth defects.

Effect of pressure on a heavy-atom isotope effect of yeast alcohol dehydrogenase.

Hydrostatic pressure causes a monophasic decrease in the (13)C primary isotope effect expressed on the oxidation of benzyl alcohol by yeast alcohol dehydrogenase. The primary isotope effect was measured by the competitive method, using whole-molecule mass spectrometry. The effect is, therefore, an expression of isotopic discrimination on the kinetic parameter V/K, which measures substrate capture. Moderate pressure increases capture by activating hydride transfer, the transition state of which must therefore have a smaller volume than the free alcohol plus the capturing form of enzyme [Cho, Y.-K.; Northrop, D. B. Biochemistry 1999, 38, 7470-7475]. The decrease in the (13)C isotope effect with increasing pressure means that the transition state for hydride transfer from the heavy atom must have an even smaller volume, measured here to be 13 mL.mol(-1). The pressure data factor the kinetic isotope effect into a semiclassical reactant-state component, with a null value of k(12)/k(13) = 1, and a transition-state component of Q(12)/Q(13) = 1.028 (borrowing Bell's nomenclature for hydrogen tunneling corrections). A similar experiment involving a deuterium isotope effect previously returned the same volume and null value, plus a pressure-sensitive isotope effect [Northrop, D. B.; Cho, Y.-K. Biochemistry 2000, 39, 2406-2412]. Consistent with precedence in the chemical literature, the latter suggested a possibility of hydrogen tunneling; however, it is unlikely that carbon can engage in significant tunneling at ambient temperature. The fact that the decrease in activation volumes for hydride transfer is equivalent when one mass unit is added to the carbon end of a scissile C-H bond and when one mass unit is added to the hydrogen end is significant and suggests a common origin.