Staphylococcus aureus sortase a-mediated incorporation of peptides: Effect of peptide modification on incorporation

The endogenous Staphylococcus aureus sortase A (SrtA) transpeptidase covalently anchors cell wall-anchored (CWA) proteins equipped with a specific recognition motif (LPXTG) into the peptidoglycan layer of the staphylococcal cell wall. Previous in situ experiments have shown that SrtA is also able to incorporate exogenous, fluorescently labelled, synthetic substrates equipped with the LPXTG motif (K(FITC)LPETG-amide) into the bacterial cell wall, albeit at high concentrations of 500 μM to 1 mM. In the present study, we have evaluated the effect of substrate modification on the incorporation efficiency. This revealed that (i) by elongation of LPETG-amide with a sequence of positively charged amino acids, derived from the C-terminal domain of physiological SrtA substrates, the incorporation efficiency was increased by 20-fold at 10 μM, 100 μM and 250 μM; (ii) Substituting aspartic acid (E) for methionine increased the incorporation of the resulting K(FITC)LPMTG-amide approximately three times at all concentrations tested; (iii) conjugation of the lipid II binding antibiotic vancomycin to K(FITC)LPMTG-amide resulted in the same incorporation levels as K(FITC)LPETG-amide, but much more efficient at an impressive 500-fold lower substrate concentration. These newly developed synthetic substrates can potentially find broad applications in for example the in situ imaging of bacteria; the incorporation of antibody recruiting moieties; the targeted delivery and covalent incorporation of antimicrobial compounds into the bacterial cell wall

Population Pharmacokinetic Modeling of Blood-Brain Barrier Transport of Synthetic Adenosine A 1 Receptor Agonists

ABSTRACT A population pharmacokinetic model is proposed for estimation of the brain distribution clearance of synthetic A 1 receptor agonists in vivo. Rats with permanent venous and arterial cannulas in combination with a microdialysis probe in the striatum received intravenous infusions of 8-methylamino-N 6 -cyclopentyladenosine (MCPA) and 2Ј-deoxyribose-N 6 -cyclopentyladenosine (2Ј-dCPA) (10 mg kg Ϫ1 ). The clearance for transport from blood to the brain was estimated by simultaneous analysis of the blood and extracellular fluid concentrations using a compartmental pharmacokinetic model. The proposed pharmacokinetic model consists of three compartments describing the time course of the concentration in blood in combination with three compartments for the brain extracellular fluid concentrations. The blood clearance was 7.4 Ϯ 0.5 for MCPA and 7.2 Ϯ 1.4 ml min Ϫ1 for 2Ј-dCPA. The in vivo microdialysis recoveries determined by the dynamic-no-net-flux method were independent of time with values of 0.21 Ϯ 0.02 and 0.22 Ϯ 0.01 for MCPA and 2Ј-dCPA, respectively. The values of the intercompartmental clearance for the distribution from blood to brain were 1.9 Ϯ 0.4 versus 1.6 Ϯ 0.3 l min Ϫ1 for MCPA and 2Ј-dCPA, respectively. It is concluded that on basis of the novel six-compartment model precise estimates of the rate of brain distribution are obtained that are independent of eventual differences in systemic exposure. The low brain distribution rates of MCPA and 2Ј-dCPA were consistent with in vitro tests. Furthermore, a slow elimination from the brain compartment was observed, indicating that the duration of central nervous system effects may be much longer than expected on the basis of the terminal half-life in blood. Blood-brain barrier (BBB) transport is a major determinant of the effect of CNS active drugs. This transport is determined by: 1) the morphology and functionality of the brain capillaries and 2) the physicochemical characteristics of the drug. Specifically, the transport of hydrophilic drugs is limited due to the presence of tight junctions between the capillary endothelial cells At present, there are several approaches to the characterization of the BBB transport that can broadly be divided into three categories: 1) in vitro assays, 2) in situ perfusion techniques, and 3) in vivo methods. Blood-brain barrier transport is often studied in vitro in cocultures of brain-capillary-endothelial cells and astrocytes To study drug transport to the brain in vivo, frequently destructive sampling techniques have been applied. Nowadays, intracerebral microdialysis is an established technique for studying the physiology, pharmacology, and pathology o

CHEMICAL COMPOSITION AND INHIBITORY ACTIVITY OF SELECTED ESSENTIAL OILS AGAINST FUNGI ISOLATED FROM MEDICINAL PLANTS

In recent years great attention was paid to biological control, the application of natural products in order to protect crops and medicinal plants against contamination with phytopathogenic and saprophytic fungi. Essential oils have been evaluated as a potentially safe replacement for chemicals used for that purpose. In this regard, antifungal potential of six essential oils was examined coupled with determination of the complete qualitative and quantitative chemical composition by GC-FID and GC-MS analysis. While essential oils of rosemary, sage, Spanish sage and black pepper were exclusively composed of monoterpenes, vetiver essential oil was entirely composed of sesquiterpenes. In essential oil of cinnamon dominates (E)-cinnamaldehyde. Applying the in vitro microdilution method, it was found that all essential oils were active in inhibiting the growth of all tested 21 pre- and post-harvest phytopathogenic and saprophytic fungi. MIC and MFC ranged from 1.2 mg ml-1 up to 22.6 mg ml-1 according to the test oil. Rosemary oil showed the best antifungal potential, followed by black pepper and cinnamon oil. Sage and Spanish sage oils also exhibited significant antifungal potential. Vetiver oil demonstrated the lowest antifungal activity. Essential oils that showed considerable antifungal potential are good candidates for further examination of their use in preventing and/or protection of medicinal plants, their seeds and dried drugs against fungal infections, both in the field and in warehouses