Loss of penicillinase plasmids of Staphylococcus aureus after treatment with L-ascorbic acid

When 2 clinical strains of plasmid-mediated penicillin-resistant Staphylococcus aureus were treated with 1 mM sodium ascorbate for 6 h, 12-35% colony-forming units (CFU) irreversibly lost their ability to produce beta-lactamase. Agarose gel electrophoresis showed the disappearance of the plasmid bands from the lysates of colonies in which susceptibility to penicillin was induced by ascorbate treatment. Since ascorbic acid is a compound that is completely non-toxic, the possibility of its therapeutic use in the treatment of multiresistant bacterial diseases is proposed.     

Studien zur speziellen Systematik derPirolaceae

Ohne Zusammenfassung     

Conformational Heterogeneity of Cyclosporin A in Cyclophilin 18 Binding

The immunosuppressive drug cyclosporin A (CsA) binds to its receptor protein cyclophilin 18 (Cyp18) in two distinct kinetic phases, while the mechanism remains elusive. Stopped-flow measurements coupled with titration and competition experiments were used to investigate the puzzling two-phase process of CsA and Cyp18 interaction. This study leads to the dissection of different conformational fractions of either direct fast binding or slow binding with rate-limiting conformational inter-conversion and the real-time measurement of k_on value (8.34 ± 0.22 x10⁶ M^-1s^-1) in solution. Furthermore, our study indicates that the structure of CsA during dissociation from the protein possesses a distribution of conformations different from those in solution under equilibrium condition.     

Computational analysis of pediatric ventricular assist device implantation to decrease cerebral particulate embolization

Stroke is the most devastating complication after ventricular assist device (VAD) implantation with a 19% incidence and 65% mortality in the pediatric population. Current pediatric VAD technology and anticoagulation strategies alone are suboptimal. VAD implantation assisted by computational methods (CFD) may contribute reducing the risk of cerebral embolization. Representative three-dimensional aortic arch models of an infant and a child were generated. An 8 mm VAD outflow-graft (VAD-OG) anastomosed to the aorta was rendered and CFD was applied to study blood flow patterns. Particle tracks, originating in the VAD, were computed with a Lagrangian phase model and the percentage of particles entering the cerebral vessels was calculated. Eight implantation configurations (infant = 5 and child = 3) and 5 particle sizes (0.5, 1, 2, 3, and 4 mm) were considered. For the infant model, percentage of particles entering the cerebral vessels ranged from 15% for a VAD-OG anastomosed at 90° to the aorta, to 31% for 30° VAD-OG anastomosis (overall percentages: X² = 10,852, p < 0.0001). For the child model, cerebral embolization ranged from 9% for the 30° VAD-OG anastomosis to 15% for the 60° anastomosis (overall percentages: χ² = 10,323, p < 0.0001). Using detailed CFD calculations, we demonstrate that the risk of stroke depends significantly on the VAD implantation geometry. In turn, the risk probably depends on patient-specific anatomy. CFD can be used to optimize VAD implantation geometry to minimize stroke risk.     

The Fitness Effects of Random Mutations in Single-Stranded DNA and RNA Bacteriophages

Mutational fitness effects can be measured with relatively high accuracy in viruses due to their small genome size, which facilitates full-length sequencing and genetic manipulation. Previous work has shown that animal and plant RNA viruses are very sensitive to mutation. Here, we characterize mutational fitness effects in single-stranded (ss) DNA and ssRNA bacterial viruses. First, we performed a mutation-accumulation experiment in which we subjected three ssDNA (ΦX174, G4, F1) and three ssRNA phages (Qβ, MS2, and SP) to plaque-to-plaque transfers and chemical mutagenesis. Genome sequencing and growth assays indicated that the average fitness effect of the accumulated mutations was similar in the two groups. Second, we used site-directed mutagenesis to obtain 45 clones of ΦX174 and 42 clones of Qβ carrying random single-nucleotide substitutions and assayed them for fitness. In ΦX174, 20% of such mutations were lethal, whereas viable ones reduced fitness by 13% on average. In Qβ, these figures were 29% and 10%, respectively. It seems therefore that high mutational sensitivity is a general property of viruses with small genomes, including those infecting animals, plants, and bacteria. Mutational fitness effects are important for understanding processes of fitness decline, but also of neutral evolution and adaptation. As such, these findings can contribute to explain the evolution of ssDNA and ssRNA viruses.     

Geranylgeraniol Promotes Entry of UT-2 Cells into the Cell Cycle in the Absence of Mevalonate

Although UT-2 cells, a mutant clone of Chinese hamster ovary cells, have been shown to require mevalonate for growth due to a deficiency in 3-hydroxy-3-methylglutaryl-CoA reductase, the precise mevalonate-derived product(s) essential for proliferation has not been identified. These studies show that UT-2 cells proliferate in the presence of free geranylgeraniol (GG-OH), as well as mevalonate. Cell growth was optimal when the culture medium was supplemented with 5–10 μMGG-OH. Under these growth conditions [³H]GG-OH is actively incorporated into UT-2 proteins. Prominent [³H]geranylgeranylated polypeptides in the size range (19–27 kDa) of the small GTP-binding proteins are observed by SDS–PAGE. Analysis of the butanol-soluble products released from the metabolically labeled proteins by digestion with Pronase E reveals that the proteins contain [³H]geranylgeranylated cysteine residues. Even though [³H]farnesol is also incorporated into cysteinyl residues of a different set of UT-2 proteins, farnesol added at 10 μMdid not satisfy the mevalonate requirement for cell growth. These results show that UT-2 cells divide in the presence of exogenously supplied GG-OH, providing evidence that one or more geranylgeranylated proteins are essential for entry of UT-2 cells, and probably other mammalian cells, into the cell cycle.     

Antifungal sordarins. Synthesis and structure-activity relationships of 3'-O-substituted derivatives.

A number of novel 3'-O-acyl and alkyl sordarins were synthesised for structure-activity relationship studies. Many of these derivatives exhibit high activity against Candida albicans, Candida pseudotropicalis, Candida tropicalis and Cryptococcus neoformans.     

Ezrin/Radixin/Moesin Are Required for the Purinergic P2X7 Receptor (P2X7R)-dependent Processing of the Amyloid Precursor Protein

The amyloid precursor protein (APP) can be cleaved by α-secretases in neural cells to produce the soluble APP ectodomain (sAPPα), which is neuroprotective. We have shown previously that activation of the purinergic P2X7 receptor (P2X7R) triggers sAPPα shedding from neural cells. Here, we demonstrate that the activation of ezrin, radixin, and moesin (ERM) proteins is required for the P2X7R-dependent proteolytic processing of APP leading to sAPPα release. Indeed, the down-regulation of ERM by siRNA blocked the P2X7R-dependent shedding of sAPPα. We also show that P2X7R stimulation triggered the phosphorylation of ERM. Thus, ezrin translocates to the plasma membrane to interact with P2X7R. Using specific pharmacological inhibitors, we established the order in which several enzymes trigger the P2X7R-dependent release of sAPPα. Thus, a Rho kinase and the MAPK modules ERK1/2 and JNK act upstream of ERM, whereas a PI3K activity is triggered downstream. For the first time, this work identifies ERM as major partners in the regulated non-amyloidogenic processing of APP.