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Del Giudice I.,CNR Institute of Neuroscience | Coppolecchia R.,CNR Institute of Neuroscience | Merone L.,CNR Institute of Neuroscience | Porzio E.,CNR Institute of Neuroscience | And 4 more authors.
Biotechnology and Bioengineering | Year: 2016

In vitro evolution of enzymes represents a powerful device to evolve new or to improve weak enzymatic functions. In the present work a semi-rational engineering approach has been used to design an efficient and thermostable organophosphate hydrolase, starting from a lactonase scaffold (SsoPox from Sulfolobus solfataricus). In particular, by in vitro evolution of the SsoPox ancillary promiscuous activity, the triple mutant C258L/I261F/W263A has been obtained which, retaining its inherent stability, showed an enhancement of its hydrolytic activity on paraoxon up to 300-fold, achieving absolute values of catalytic efficiency up to 105 M-1s-1. The kinetics and structural determinants of this enhanced activity were thoroughly investigated and, in order to evaluate its potential biotechnological applications, the mutant was tested in formulations of different solvents (methanol or ethanol) or detergents (SDS or a commercial soap) for the cleaning of pesticide-contaminated surfaces. © 2016 Wiley Periodicals, Inc.

Steinritz D.,Bundeswehr Institute of Pharmacology and Toxicology | Steinritz D.,Ludwig Maximilians University of Munich | Schmidt A.,Bundeswehr Institute of Pharmacology and Toxicology | Schmidt A.,University of Cologne | And 5 more authors.
Journal of Visualized Experiments | Year: 2015

Exposure to chemical substances (including alkylating chemical warfare agents like sulfur and nitrogen mustards) cause a plethora of clinical symptoms including wound healing disorder. The physiological process of wound healing is highly complex. The formation of granulation tissue is a key step in this process resulting in a preliminary wound closure and providing a network of new capillary blood vessels – either through vasculogenesis (novel formation) or angiogenesis (sprouting of existing vessels). Both vasculo- and angiogenesis require functional, directed migration of endothelial cells. Thus, investigation of early endothelial cell (EEC) migration is important to understand the pathophysiology of chemical induced wound healing disorders and to potentially identify novel strategies for therapeutic intervention. We assessed impaired wound healing after alkylating agent exposure and tested potential candidate compounds for treatment. We used a set of techniques outlined in this protocol. A modified Boyden chamber to quantitatively investigate chemokinesis of EEC is described. Moreover, the use of the wound healing assay in combination with track analysis to qualitatively assess migration is illustrated. Finally, we demonstrate the use of the fluorescent dye TMRM for the investigation of mitochondrial membrane potential to identify underlying mechanisms of disturbed cell migration. The following protocol describes basic techniques that have been adapted for the investigation of EEC. © 2015 Journal of Visualized Experiments.

von der Wellen J.,Bundeswehr Institute of Pharmacology and Toxicology | Bierwisch A.,Bundeswehr Institute of Pharmacology and Toxicology | Worek F.,Bundeswehr Institute of Pharmacology and Toxicology | Thiermann H.,Bundeswehr Institute of Pharmacology and Toxicology | Wille T.,Bundeswehr Institute of Pharmacology and Toxicology
Toxicology Letters | Year: 2016

There is an ongoing debate about the benefit of fresh frozen plasma (FFP) infusion in organophosphorus (OP) pesticide-poisoned patients. This prompted us to investigate the kinetics of OP pesticide degradation by FFP with an enzymatic assay in vitro.Degradation was rapid with shortest half-lives of 19.5 s for chlorpyrifos-oxon, 6.3 min for paraoxon-ethyl and 17.9 min for dichlorvos. Heptenophos (78.0 min), mevinphos (101.8 min), profenofos (162.3 min) and malaoxon (179.7 min) showed half-lives of up to 3 h. Substantial longer degradation half-lives of 69.7-80.8 h were determined with chlorfenvinphos and bromfenvinphos. Methamidophos and omethoate showed no degradation by FFP indicated by half-lives similar to spontaneous hydrolysis. In conclusion, degradation by FFP depends on the particular OP pesticide and the used FFP batch. © 2015 Elsevier Ireland Ltd.

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