Mode of Action of the Antimicrobial Peptide Aureocin A53 from Staphylococcus aureus

We investigated the mode of action of aureocin A53 on living bacterial cells and model membranes. Aureocin A53 acted bactericidally against Staphylococcus simulans 22, with >90% of the cells killed within a few minutes. Cell death was followed by lysis, as indicated by a clearing of the cell suspension and Gram staining. Aureocin A53 rapidly dissipated the membrane potential and simultaneously stopped biosynthesis of DNA, polysaccharides, and protein. Aureocin A53 induced a rapid release of preaccumulated glutamate and Rb⁺. Experiments on model membranes demonstrated that aureocin A53 provoked significant leakage of carboxyfluorescein (CF) exclusively from acidic liposomes but only at relatively high concentrations (0.5 to 8 mol%). Thus, the bactericidal activity of aureocin A53 derives from membrane permeation via generalized membrane destruction rather than by formation of discrete pores within membranes. Tryptophan emission fluorescence spectroscopy demonstrated interaction of aureocin A53 with both acidic and neutral membranes, as indicated by similar blue shifts. Since there was no significant aureocin A53-induced CF leakage from neutral liposomes, its appears that the peptide does interact with neutral lipids without provoking membrane damage.     

Glyoxalase II in Saccharomyces cerevisiae: in situ kinetics using the 5,5'-dithiobis(2-nitrobenzoic acid) assay.

The determination of glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) activity is usually accomplished by monitoring the decrease of absorbance at 240 nm due to the hydrolysis of S-d-lactoylglutathione. However, it was not possible, using this assay, to detect any enzyme activity in situ, in Saccharomyces cerevisiae permeabilized cells. Glyoxalase II activity was then determined by following the formation of GSH at 412 nm using 5,5'-dithiobis(2-nitrobenzoic acid). Using this method we characterized the kinetics of glyoxalase II in situ using S-d-lactoylglutathione as substrate and compared the results with those obtained for cell-free extracts. The specific activity was found to be (4.08 +/- 0.12) x 10(-2) micromol min-1 mg-1 in permeabilized cells and (3.90 +/- 0.04) x 10(-2) micromol min1 mg-1 in cell-free extracts. Kinetic parameters were Km 0.36 +/- 0.09 mM and V (7.65 +/- 0.59) x 10(-4) mM min-1 for permeabilized cells and Km 0.15 +/- 0.10 mM and V (7.23 +/- 1.04) x 10(-4) mM min-1 for cell-free extracts. d-Lactate concentration was also determined and increased in a linear way with permeabilized cell concentration. gamma-Glutamyl transferase (EC 2.3.2.2), which also accepts S-d-lactoylglutathione as substrate and hence could interfere with glyoxalase II assays, was found to be absent in Saccharomyces cerevisiae permeabilized cells.     

Environmental impact trade-offs in building envelope retrofit strategies

Purpose

The use of high levels of thermal insulation is a common practice towards reducing the energy consumption of the existing building stock; however, the embodied burdens associated with the additional insulation material are usually not taken into account and questions regarding the risks of over-specifying the insulation levels have been emerging, particularly for mild climate regions. This article addresses the issue presenting an integrated approach that combines life cycle assessment and thermal dynamic simulation to assess alternative retrofit strategies for the roof and exterior walls of two dwellings (from the beginning of the twentieth century), in the historic city center of Coimbra, Portugal. A comprehensive analysis of alternative insulation thicknesses (no insulation, 40, 80, and 120 mm of expanded polystyrene) was made to identify optimal thickness levels minimizing life cycle (LC) environmental impacts for a single-family house and an apartment.

Methods

Embodied and operational impact trade-offs were calculated for six impact categories: climate change, ozone depletion, terrestrial acidification, freshwater eutrophication, marine eutrophication, and non-renewable primary energy. The operational energy was calculated using a dynamic thermal modeling software (EnergyPlus). The functional unit selected for this study was 1 m² of living area over a period of 50 years.

Results and discussion

The single-family house embodied impacts account for 26–57 % of total LC impacts. For insulation thicknesses larger than 80 mm, the embodied impacts are greater than operational impacts. For the apartment, embodied impacts account for 25–49 % of total LC impacts. The environmental benefits of additional insulation are very low (<3 %) for thicknesses of more than 80 mm for both roof and exterior walls. For thicknesses above the tipping point (where total LC impacts are minimized), the marginal impacts of additional insulation are higher than the benefits. The results for the apartment show that optimal insulation thicknesses (LC tipping point) range from 30 to 40 mm for the roof and from 60 to 80 mm for the exterior walls. The LC tipping point for the single-family house is achieved by combining 80–100 mm of roof insulation with 60–80 mm of exterior wall insulation.

Conclusions

Extra insulation levels in temperate climates can lead to higher embodied impacts, without significant reduction in operational impacts, which can result in higher total LC impacts. The results show that a tipping point can be identified, and recommendations are provided for the roof and exterior wall retrofits of buildings from the beginning of the twentieth century.

     

Antimalarial activity enhancement in hydroxymethylcarbonyl (HMC) isostere-based dipeptidomimetics targeting malarial aspartic protease plasmepsin

Plasmepsin (Plm) is a potential target for new antimalarial drugs, but most reported Plm inhibitors have relatively low antimalarial activities. We synthesized a series of dipeptide-type HIV protease inhibitors, which contain an allophenylnorstatine-dimethylthioproline scaffold to exhibit potent inhibitory activities against Plm II. Their activities against Plasmodium falciparum in the infected erythrocyte assay were largely different from those against the target enzyme. To improve the antimalarial activity of peptidomimetic Plm inhibitors, we attached substituents on a structure of the highly potent Plm inhibitor KNI-10006. Among the derivatives, we identified alkylamino compounds such as 44 (KNI-10283) and 47 (KNI-10538) with more than 15-fold enhanced antimalarial activity, to the sub-micromolar level, maintaining their potent Plm II inhibitory activity and low cytotoxicity. These results suggest that auxiliary substituents on a specific basic group contribute to deliver the inhibitors to the target Plm.