Usnic acid,as a biotic factor,changes the ploidy level in mosses

Lichens and mosses often share the same environmental conditions where they compete for substrate and other essential factors. Lichens use secondary metabolites as allelochemicals to repel surrounding plants and potential rivals. In mosses, endoreduplication leads to the occurrence of various ploidy levels in the same individual and has been suggested as an adaptation to abiotic stresses. Here, we show that also biotic factors such as usnic acid, an allelochemical produced by lichens, directly influenced the level of ploidy in mosses. Application of usnic acid changed the nuclei proportion and significantly enhanced the endoreduplication index in two moss species, Physcomitrella patens and Pohlia drummondii. These investigations add a new aspect on secondary metabolites of lichens which count as biotic factors and affect ploidy levels in mosses.     

Synthesis of alpha-substituted fosmidomycin analogues as highly potent Plasmodium falciparum growth inhibitors

In view of the promising antimalarial activity of fosmidomycin or its N-acetyl homologue FR900098, the objective of this work was to investigate the influence of aromatic substituents in the alpha-position of the phosphonate moiety. The envisaged analogues were prepared using a linear route involving a 3-aryl-3-phosphoryl propanal intermediate. The activities of all compounds were evaluated on Eschericia coli 1-deoxy-d-xylulose 5-phosphate reductoisomerase and against two Plasmodium falciparum strains. Compared with fosmidomycin, several analogues displayed enhanced activity towards the P. falciparum strains. Compound 1e with a 3,4-dichlorophenyl substitution in the alpha-position of fosmidomycin emerged as the most potent analogue of this series. It is approximately three times more potent in inhibiting the growth of P. falciparum than FR900098, the most potent representative of this class reported so far.     

Acyloxyalkyl ester prodrugs of FR900098 with improved in vivo anti-malarial activity

FR900098 represents an improved derivative of the new antimalarial drug fosmidomycin and acts through inhibition of the 1-deoxy-D-xylulose 5-phosphate (DOXP) reductoisomerase, an essential enzyme of the mevalonate independent pathway of isoprenoid biosynthesis. Prodrugs with increased activity after oral administration were obtained by chemical modification of the phosphonate moiety to yield acyloxyalkyl esters. The most successful compound demonstrated 2-fold increased activity in mice infected with the rodent malaria parasite Plasmodium vinckei.     

Studies on the response ofAmblyseius bibens [Acarina: Phytoseiidae] to conditions of prey scarcity

Various aspects of the ability of the predacious miteAmblyseius bibens Blommers to survive under conditions of prey scarcity were studied in the laboratory. The presence of water considerably lengthened survival in the absence of food. An ability to pierce leaves was not observed in the predator. Walking speed was little affected by hunger, but declined with inanition. Activity (=percentage time spent walking) was greatest in well fed females, and decreased with increasing hunger, and eventually with inanition. Hence, the area searched per unit time decreased with increasing hunger and inanition. Hungry predators showed greater activity on more hairy leaves. Hungry predators became more restive in the presence of a cover. A webbed area was preferred (as resting place) by the predator, whether hungry or not. Gut content, and hence initial feeding, was only representative for the state of hunger, if the availibility of water was accounted for. Duration of the complete recovery (oviposition) in the presence of ample food provided a measure of the original inanition. Many kinds of pollen might serve as alternate food.     

Use of proteomics for validation of the isolation process of clotting factor IX from human plasma

The use of proteomic techniques in the monitoring of different production steps of plasma-derived clotting factor IX (pd F IX) was demonstrated. The first step, solid-phase extraction with a weak anion-exchange resin, fractionates the bulk of human serum albumin (HSA), immunoglobulin G, and other non-binding proteins from F IX. The proteins that strongly bind to the anion-exchange resin are eluted by higher salt concentrations. In the second step, anion-exchange chromatography, residual HSA, some proteases and other contaminating proteins are separated. In the last chromatographic step, affinity chromatography with immobilized heparin, the majority of the residual impurities are removed. However, some contaminating proteins still remain in the eluate from the affinity column. The next step in the production process, virus filtration, is also an efficient step for the removal of residual impurities, mainly high molecular weight proteins, such as vitronectin and inter-alpha inhibitor proteins. In each production step, the active component, pd F IX and contaminating proteins are monitored by biochemical and immunochemical methods and by LC–MS/MS and their removal documented. Our methodology is very helpful for further process optimization, rapid identification of target proteins with relatively low abundance, and for the design of subsequent steps for their removal or purification.     

Loss of PINK1 Impairs Stress-Induced Autophagy and Cell Survival

The mitochondrial kinase PINK1 and the ubiquitin ligase Parkin are participating in quality control after CCCP- or ROS-induced mitochondrial damage, and their dysfunction is associated with the development and progression of Parkinson''s disease. Furthermore, PINK1 expression is also induced by starvation indicating an additional role for PINK1 in stress response. Therefore, the effects of PINK1 deficiency on the autophago-lysosomal pathway during stress were investigated. Under trophic deprivation SH-SY5Y cells with stable PINK1 knockdown showed downregulation of key autophagic genes, including Beclin, LC3 and LAMP-2. In good agreement, protein levels of LC3-II and LAMP-2 but not of LAMP-1 were reduced in different cell model systems with PINK1 knockdown or knockout after addition of different stressors. This downregulation of autophagic factors caused increased apoptosis, which could be rescued by overexpression of LC3 or PINK1. Taken together, the PINK1-mediated reduction of autophagic key factors during stress resulted in increased cell death, thus defining an additional pathway that could contribute to the progression of Parkinson''s disease in patients with PINK1 mutations.     

Determination of tranexamic acid concentration by solid phase microextraction and liquid chromatography-tandem mass spectrometry: first step to in vivo analysis

A solid phase microextraction (SPME) method followed by LC-MS/MS analysis was developed to determine the concentration of tranexamic acid (TA) in plasma. The use of a new biocompatible C18 coating allowed the direct extraction from complex biological samples without prior sample preparation; no matrix effect was observed. The results revealed that SPME was suitable for the analysis of polar drugs such as TA; such an application was previously inaccessible because of the limited availability of SPME coatings that can extract polar molecules. The proposed method was validated according to the bioanalytical method validation guidelines. LOD and LLOQ were 0.5 and 1.5 μg/ml, respectively. The recovery for the method was 0.19%, and the accuracy and precision of the method were <9 and <11%, respectively, with the exception of LLOQ, where the values were <16 and <13%, respectively. The performance of the proposed method was also compared against that of the standard techniques of protein precipitation and ultrafiltration. A statistical analysis indicated a clinically significant agreement among all assays. Another advantage of SPME over conventional techniques was the easy automation and feasibility of in vivo analysis; this advantage makes it possible to use the proposed method for an on-site analysis in clinical practice.     

In vivo solid-phase microextraction for monitoring intravenous concentrations of drugs and metabolites

This protocol for in vivo solid-phase microextraction (SPME) can be used to monitor and quantify intravenous concentrations of drugs and metabolites without the need to withdraw a blood sample for analysis. The SPME probe is inserted directly into a peripheral vein of a living animal through a standard medical catheter, and extraction occurs typically over 2-5 min. After extraction, the analytes are removed from the sorbent and analyzed by, for example, liquid chromatography-tandem mass spectrometry. It has been validated in comparison with conventional blood analysis, and we describe here the in vitro experiments typically conducted during method development. The new-generation biocompatible SPME probes are designed specifically for extraction of semi-volatiles and nonvolatiles directly from aqueous samples and can be steam sterilized. Sorbents are coated on fine-gauge surgical steel wire (200-μm diameter), which is more rugged and biocompatible than conventional fibers (100-μm fused silica fiber). They incorporate a binding agent that resists fouling by the biological matrix and does not cause an immune response in the experimental animal. The sorbents used (coating thickness of ～50 μm) are selected for their affinity for the types of small molecules of interest. The procedure is illustrated by the analysis of benzodiazepines with polypyrrole-coated wires inserted into peripheral blood vessels of beagles, although it can be adapted for use in smaller animals. The in vivo sampling can require as little as 1 min, in which case the entire procedure from sampling to instrumental analysis can take as little as 30 min.