Elucidation of carotenoid patterns in citrus products by means of comprehensive normal-phase x reversed-phase liquid chromatography

A novel approach for carotenoid analysis has been developed. Orange essential oil and juice carotenoids were separated by means of comprehensive dual-gradient elution HPLC, using normal phase with a microbore silica column in the first dimension (first D), reversed phase with a monolithic C18 column in the second dimension (second D), and a 10-port switching valve as an interface. An on-line photodiode array detector was used in order to obtain absorption spectra. Peak identification was obtained by combining retention data with the UV-visible spectra.     

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.     

The regulatable MAL32 promoter in Saccharomyces cerevisiae: characteristics and tools to facilitate its use

Here we describe a set of tools to facilitate the use of maltose and the MAL32 promoter for regulated gene expression in yeast, alone or in combination with the GAL1 promoter. Using fluorescent protein reporters we find that under non‐inducing conditions the MAL32 promoter exhibits a low basal level of expression, similar to the GAL1 promoter, and that both promoters can be induced independently of each other using the respective sugars, maltose and galactose. While their repression upon glucose addition is immediate and complete, we found that the MAL32 and GAL1 promoters each exhibit distinct induction kinetics. A set of plasmids is available to facilitate the application of the MAL32 promoter for chromosomal modifications using PCR targetting and for plasmid based gene expression. Copyright © 2016 John Wiley & Sons, Ltd.     

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.     

Synthesis of mono-, di-, tri- and tetracarboxy azaphthalocyanines as potential dark quenchers

Mono-, di-, tri- and tetracarboxy-substituted metal-free azaphthalocyanines (AzaPc) were synthesized from 5,6-bis(diethylamino)pyrazine-2,3-dicarbonitrile and 6-(5,6-dicyano-3-(diethylamino)pyrazin-2-ylamino)hexanoic acid using a statistical condensation approach. AzaPc bearing eight diethylamino peripheral substituents was also isolated from the mixture. Analysis of the distribution of congeners in the statistical mixture using optimized HPLC method (Phenomenex Synergy RP Fusion column, acetonitrile/tetrahydrofuran/water (pH 5.5) 50:20:30) was performed. The analysis showed optimal ratios of starting materials to be 3:1 for AAAB, 1:3 for ABBB and 1:1 for AABB/ABAB types of the congeners. The distribution of the congeners corresponded well with calculated values indicating similar reactivity of both starting materials and no sterical constraint between adjacent isoindole units in the AzaPc ring. All investigated AzaPc showed no fluorescence, extremely low singlet oxygen quantum yields (Φ_? < 0.005) in monomeric form and strong absorption in a wide range from 300 nm to almost 700 nm. Such properties are highly promising for future investigation of these compounds as dark quenchers of fluorescence in DNA hybridization probes.     

Enhanced antiproliferative and apoptotic response of HT-29 adenocarcinoma cells to combination of photoactivated hypericin and farnesyltransferase inhibitor manumycin A

Several photodynamically-active substances and farnesyltransferase inhibitors are currently being investigated as promising anticancer drugs. In this study, the combined effect of hypericin (the photodynamically-active pigment from Hypericum perforatum) and selective farnesyltransferase inhibitor manumycin (manumycin A; the selective farnesyltransferase inhibitor from Streptomyces parvulus) on HT-29 adenocarcinoma cells was examined. We found that the combination treatment of cells with photoactivated hypericin and manumycin resulted in enhanced antiproliferative and apoptotic response compared to the effect of single treatments. This was associated with increased suppression of clonogenic growth, S phase cell cycle arrest, elevated caspase-3/7 activity and time-dependent total cleavage of procaspase-3 and lamin B, cleavage of p21Bax into p18Bax and massive PARP cleavage. Moreover, we found that the apoptosis-inducing factor is implicated in signaling events triggered by photoactivated hypericin. Our results showed the relocalization of apoptosis-inducing factor (AIF) to the nuclei after hypericin treatment. In addition, we discovered that not only manumycin but also photoactivated hypericin induced the reduction of total Ras protein level. Manumycin decreased the amount of farnesylated Ras, and the combination treatment decreased the amount of both farnesylated and non-farnesylated Ras protein more dramatically. The present findings indicate that the inhibition of Ras processing may be the determining factor for enhancing the antiproliferative and apoptotic effects of combination treatment on HT-29 cells.