Analysis of noncovalent chitinase-chito-oligosaccharide complexes by infrared-matrix assisted laser desorption ionization and nanoelectrospray ionization mass spectrometry

Transferring noncovalently bound complexes from the condensed phase into the gas phase represents a challenging task due to weak intermolecular bonds that have to be maintained during the phase transition. Currently, electrospray ionization (ESI) is the standard mass spectrometric (MS) technique to analyze noncovalent complexes. Although infrared matrix-assisted laser desorption ionization (IR-MALDI)-MS also provides particular soft desorption/ionization conditions, this method has so far hardly been applied for the analysis of noncovalent complexes. In this study, we employed IR-MALDI orthogonal time-of-flight (o-TOF)-MS in combination with the liquid matrix glycerol to characterize the specific complex formation of chito-oligosaccharide (CHOS) ligands with two variants of Chitinase A (ChiA) from Serratia marcescens, the inactive E315Q mutant and the active W167A mutant, respectively. The IR-MALDI-o-TOF-MS results were compared to those obtained using nano-ESI-quadrupole (q)-TOF-MS and ultraviolet (UV)-MALDI-o-TOF-MS. Using IR-MALDI-o-TOF-MS, specific noncovalent complexes between ChiA and CHOS were detected with distributions between enzymes with bound oligosaccharides vs free enzymes that were essentially identical to those obtained by nano-ESI-q-TOF-MS. Chitinase-CHOS complexes were not detected when UV-MALDI was employed for desorption/ionization. The results show that IR-MALDI-MS can be a valuable tool for fast and simple screening of noncovalent enzyme-ligand interactions.     

Verfahren zur Verl?ngerung der Haltbarkeit von Konsummilch und ihre stofflichen Ver?nderungen: ESL-Milch

Die ESL-Technologie stellt nach der H-Milch eine weitere Entwicklung mit hohem Zusatznutzen in Bezug auf Haltbarkeit und Vorratshaltung dar, wovon sowohl Handel als auch Verbraucher gleicherma?en profitieren k?nnen. Im Vergleich zum herk?mmlich pasteurisierten Produkt bringt das ESL-Konzept entscheidende Vorteile hinsichtlich der Haltbarkeit der Milch mit sich, wirkt sich dabei aber kaum oder nur geringfügig auf die für den Verbraucher entscheidenden bzw. wertrelevanten Produkteigenschaften aus. ESL-Produkte sind in diesem Sinne also nicht nur „l?nger haltbar”, sondern unserer Einsch?tzung nach auch „l?nger frisch”. Dass gerade die Produktcharakteristika der ESL-Milch den Wünschen der Verbraucher entgegenkommen, zeigt sich an der stetig steigenden Nachfrage nach diesen Produkten und dem damit einhergehenden Absatzrückgang von herk?mmlich pasteurisierter Frischmilch. Die Verbraucher haben also durch ihr Kaufverhalten in den letzten Jahren bereits deutlich gemacht, dass sie der ESL-Milch durchaus positiv gegenüberstehen und sich mit diesem Produktkonzept keineswegs „get?uscht” fühlen, was die hier zusammengefassten Fakten eindeutig untermauern.     

Induction of Detoxification Enzymes by Feeding Unblanched Brussels Sprouts Containing Active Myrosinase to Mice for 2 Wk

Abstract: In cruciferous vegetables, myrosinase metabolizes the relatively inactive glucosinolates into isothiocyanates and other products that have the ability to increase detoxification enzyme expression. Thus, maintaining myrosinase activity during food preparation may be critical to receiving the maximum benefit of consumption of Brussels sprouts or other cruciferous vegetables. To test the importance of maintaining myrosinase activity for maximizing bioactivity, experimental diets containing 20% unblanched (active myrosinase) or 20% blanched (inactivated myrosinase) freeze-dried Brussels sprouts and a nutrient-matched control diet were evaluated in vitro and in vivo for their ability to induce detoxification enzymes. Treatment of immortalized HepG2 human hepatoma cells with the unblanched Brussels sprout diet caused a greater increase quinone activity compared to the blanched Brussels sprout diet. C3H/HeJ mice fed the unblanched Brussels sprout diets for 2 wk had significantly higher plasma sulforaphane concentrations. Liver expression of CYP1A1 and epoxide hydrolase, measured using real-time PCR, was correlated with the plasma concentration of sulforaphane. In the lung, expression of epoxide hydrolase, thioredoxin reductase, UDP glucuronosyltransferase, quinone reductase, heme oxygenase, CYP1A1, CYP1A2, and CYP1B1 were also correlated with the plasma concentration of sulforaphane. Together these data demonstrate that, as predicted by the in vitro experiment, in vivo exposure to Brussels sprouts with active myrosinase resulted in greater induction of both phase I and phase II detoxification enzymes in the liver and the lungs that correlated with plasma sulforaphane concentrations.     

A novel method to measure both the reductive and the radical scavenging activity in a linoleic acid model system

The aim of this study was to develop a combined method for measuring the total antioxidant activity, the reductive and the radical scavenging activity. Linoleic acid was used as the substrate for an iron-initiated lipid peroxidation to measure the total antioxidant activity. In addition, methyl esters of linoleic acid hydroperoxides were used as substrates to measure the reductive antioxidant activity. The radical scavenging antioxidant activity was calculated by subtracting the reductive antioxidative activity from the total antioxidative activity. As representative examples, the antioxidants alpha-tocopherol, ascorbic acid, trans-resveratrol and L-glutathione as well as commonly used food additives such as 2(3)-tert-butyl-4-hydroxyanisole (BHA) and 2,6-bis(1,1-dimethylethyl)-4-methylphenol (BHT) were analyzed. The results for the novel antioxidation test showed that alpha-tocopherol, BHA and BHT are primarily acting as radical scavengers, whereas ascorbic acid and L-glutathione show a strong reductive capacity. As linoleic acid as well as its hydroperoxides both are present in foods and in the organism, the test presented here can be considered representative of radical reactions occurring in food matrixes and in vivo. Further experiments are required to document the comprehensive applicability in foods and in vivo.     

Physical accessibility and its role in landscape development—three historical analyses from South Slovakia

Physical accessibility is recognised as an important driver or factor affecting landscape development in an increasing number of studies. This work uses cost-distance methods for analysis of landscape accessibility, and presents the analysis in three historical time periods focused on the effect of accessibility on landscape development in South Slovakia. First, from periods when the cultural landscape was forming, the effect of landscape accessibility on the location of settlements is given. The variables explaining the location of historical settlements are: slope steepness, accessibility to water expressed by distance to fluvial sediments and distance within 500 m from suitable soils for agriculture. The second example is from the second half of the twentieth century, when the small-scale agricultural landscape was transformed into large-scale fields suitable for industrial agriculture. Agricultural fields located on steeper slopes and closer to settlements were more likely to survive the collectivisation of agriculture. The third example shows accessibility as a factor affecting the abandonment of the remaining traditional agricultural landscapes (TAL) as a consequence of the economic and cultural changes triggered by the transition to an open market economy. The TAL located on less accessible areas are more likely affected by the abandonment.