A microarray for monitoring the production of mycotoxins in food

A microarray, which covers most of the known relevant mycotoxin biosynthesis genes, has been developed. The microarray carries oligonucleotides of the fumonisin, the aflatoxin, the ochratoxin, the trichothecene (type A and B) and the patulin biosynthesis pathways. For trichothecene producing Fusaria the biosynthesis cluster of trichothecene producing Fusarium sporotrichioides (type A) and of Gibberrella zeae (type B, teleomorph of F. graminearum) have been spotted. The aflatoxin cluster carries oligonucleotides specific for Aspergillus flavus. The ochratoxin pattern is specific for ochratoxin A producing Penicillia, the fumonisin cluster is specific for G. moniliformis (teleomorph of F. verticillioides) and the patulin genes have been obtained from Penicillium expansum. The microarray is designed in a way that newly identified pathway genes can be added easily at any time. The microarray was used to detect the activation of all gene clusters under conditions conducive for mycotoxin biosynthesis. According to the results the obtained signals were specific under the hybridization conditions used and only insignificant cross-hybridizations occurred. The microarray was used to demonstrate differences in mycotoxin pathway gene expressions after growth on various media for trichothecene and ochratoxin A biosynthesis. It was used further to study and compare the expression kinetics of the trichothecene biosynthesis genes of Fusarium on different trichothecene supporting media. An expression pattern indicative for trichothecene biosynthesis could be identified.     

Two-step enzymatic reaction for the synthesis of pure structured triacylglycerides

Structured triacylglycerides with medium-chain fatty acids (caprylic acid) in sn1- and sn3-positions and a long-chain unsaturated fatty acid (oleic or linoleic acid) in the sn2-position of glycerol (MLM) were synthesized by lipase catalysis in a two-step process. First, pure 2-monoacylglycerides (2-MG) were synthesized by alcoholysis of triacylglycerides (triolein, trilinolein, or peanut oil) in organic solvents with 1,3-regiospecific lipases (from Rhizomucor miehei, Rhizopus delemar, and Rhizopus javanicus). The 2-MG were purified by crystallization and obtained in up to 71.8% yield. These 2-MG were esterified in a second reaction with caprylic acid in n-hexane to form almost pure MLM. For 2-MG obtained from peanut oil, the final product contained more than 90% caprylic acid in the sn1- and sn3-positions, whereas the sn2-position was composed of 98.5% unsaturated long-chain fatty acids. Reaction conditions for both steps were optimized with respect to source and immobilization of lipase, water activity, and solvent.     

Eicosapentaenoic and docosahexaenoic acid affect mitochondrial and peroxisomal fatty acid oxidation in relation to substrate preference

Decreased triacylglycerol synthesis within hepatocytes due to decreased diacylglycerol acyltransferase (DGAT) activity has been suggested to be an important mechanism by which diets rich in fish oil lower plasma triacylglycerol levels. New findings suggest that eicosapentaenoic acid (EPA), and not docosahexaenoic acid (DHA), lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation and decreased availability of fatty acids for triacylglycerol synthesis. To contribute to the understanding of the triacylglycerol-lowering mechanism of fish oil, the different metabolic properties of EPA and DHA were studied in rat liver parenchymal cells and isolated rat liver organelles. EPA-CoA was a poorer substrate than DHA-CoA for DGAT in isolated rat liver microsomes, and in the presence of EPA, a markedly lower value for the triacyl[³H]glycerol/diacyl[³H]glycerol ratio was observed. The distribution of [1-¹⁴C]palmitic acid was shifted from incorporation into secreted glycerolipids toward oxidation in the presence of EPA (but not DHA) in rat liver parenchymal cells. [1-¹⁴C]EPA was oxidized to a much greater extent than [1-¹⁴C]DHA in rat liver parenchymal cells, isolated peroxisomes, and especially in purified mitochondria. As the oxidation of EPA was more effective and sensitive to the CPT-I inhibitor, etomoxir, when measured in a combination of both mitochondria and peroxisomes, we hypothesized that both are involved in EPA oxidation, whereas DHA mainly is oxidized in peroxisomes. In rats, EPA treatment lowered plasma triacylglycerol and increased hepatic mitochondrial fatty acid oxidation and carnitine palmitoyltransferase (CPT)-I activity in both the presence and absence of malonyl-CoA. Whereas only EPA treatment increased the mRNA levels of CPT-I, DHA treatment increased the mRNA levels of peroxisomal fatty acyl-CoA oxidase and fatty acid binding protein more effectively than EPA treatment. In conclusion, EPA and DHA affect cellular organelles in relation to their substrate preference. The present study strongly supports the hypothesis that EPA, and not DHA, lowers plasma triacylglycerol by increased mitochondrial fatty acid oxidation.     

Synthesis of Novel Niobium Aluminide-Based Composites

A reactive sintering process has been used to produce almost fully dense composites with interpenetrating networks of NbAl₃ and Al₂O₃. The process involves the reaction synthesis of niobium aluminides and Al₂O₃ from compacts of intensively milled aluminum and Nb₂O₅ powder mixtures. During carefully controlled heating under an inert atmosphere, the oxide reduction by aluminum to form niobium aluminides and Al₂O₃ proceeds at temperatures below the melting point of aluminum. At temperatures of >1000°C, the reaction-formed niobium aluminides and Al₂O₃ sinter. The present paper discusses processing parameters, such as attrition milling, the heating cycle, and the metal:ceramic ratio in the starting mixture, that control microstructure development and mechanical properties.     

Structure–Activity Relationships of 2‐Sufonylpyrimidines as Quorum‐Sensing Inhibitors to Tackle Biofilm Formation and eDNA Release of Pseudomonas aeruginosa

Drug‐resistant Pseudomonas aeruginosa (PA) strains are on the rise, making treatment with current antibiotics ineffective. Hence, circumventing resistance or restoring the activity of antibiotics by novel approaches is of high demand. Targeting the Pseudomonas quinolone signal quorum sensing (PQS‐QS) system is an intriguing strategy to abolish PA pathogenicity without affecting the viability of the pathogen. Herein we report the structure–activity relationships of 2‐sulfonylpyrimidines, which were previously identified as dual‐target inhibitors of the PQS receptor PqsR and the PQS synthase PqsD. The SAR elucidation was guided by a combined approach using ligand efficiency and ligand lipophilicity efficiency to select the most promising compounds. In addition, the most effective inhibitors were rationally modified by the guidance of QSAR using Hansch analyses. Finally, these inhibitors showed the capacity to decrease biofilm mass and extracellular DNA, which are important determinants for antibiotic resistance.     

Male pheromone of swift moth,Hepialus hecta L. (Lepidoptera: Hepialidae)

(R)-6-Ethyl-2-methyl-2,3-dihydro-4H-pyran-4-one, (1R,3S,5R)-3-ethyl-1,8-dimethyl-2,9-dioxabicyclo[3.3. 1]non-7-ene, and (1R,3S,5R)-3-ethyl-1,8-dimethyl-2,9-dioxabicyclo[3.3.1]non-7-en-6-one represent the main components in the male pheromone of the swift moth,Hepialus hecta. The amounts of the three components were 40, 5, and 5 g per male, respectively. Structure elucidation of the compounds was based on spectroscopic data as compared to synthetic reference samples. The absolute configurations were determined by gas chromatography on chiral stationary phases; optically active samples served as reference compounds. Electrophysiological and behavioral experiments with natural material and synthetic samples clearly showed the three heterocyclic compounds to act as pheromones. (E, E)--Farnesene represents the main component of the scent secretion of maleHepialus humuli.     

Influence of dietary partially hydrogenated vegetable and marine oils on membrane composition and function of liver microsomes and platelets in the rat

The aim of the present study was to investigate the influence of partially hydrogenated vegetable and marine oils on membrane composition and function of liver microsomes and platelets with particular reference to the metabolism of linoleic acid and the production of arachidonic acid metabolites. Four groups of male weanling rats were fed linoleic acid supplemented diets containing 20% (w/w) of partially hydrogenated low erucic acid rapeseed oil (HLRSO), partially hydrogenated herring oil (HHO), olive oil (OO) and trierucin + triolein (TE) for 10 weeks. An additional two groups were fed partially hydrogenated low erucic acid rapeseed oil and partially hydrogenated herring oil without linoleic acid supplementation (HLRSO- and HHO-, respectively). Substantial amounts oftrans fatty acids were incorporated into liver microsomes (12.6% in group HLRSO) and platelets (7.0% in group HLRSO-). This incorporation was not dependent on the dietary linoleic acid level. Hepatic microsomal Δ⁵-desaturase activity was significantly increased after HLRSO feeding compared to OO feeding. Δ⁶-Desaturase activity did not vary in the linoleic acid supplemented groups. Both Δ⁵- and Δ⁶-desaturase activities were significantly increased in groups without linoleic acid supplementation. Docosenoic acid was incorporated into platelet phospholipids in contrast to liver microsomes. In the platelet, docosenoic acid seemed to have a special preference for phosphatidylserine. Very small amounts were incorporated into platelet phosphatidylinositol. Feeding diets HLRSO, HHO and OO did not influence rat platelet cyclooxygenase or 12-lipoxygenase activity. Platelets from rats fed TE, however, produced significantly less 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) than platelets from rats fed OO. Feeding of HLRSO- and HHO- resulted in a significantly diminished production of the arachidonic acid metabolites 12-HETE, 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT) and 6-keto-prostaglandin F_1α in stimulated platelets and aorta. Thus, high dietary levels oftrans isomers of monoenoic acids do not interfere with platelet cyclooxygenase or lipoxygenase activity provided sufficient amounts of linoleic acid are available.     

Nutrient balance of shifting cultivation by burning or mulching in the Eastern Amazon – evidence for subsoil nutrient accumulation

For over a hundred years shifting cultivation with slash-and-burn land preparation has been the predominant type of land use by smallholders in the Bragantina region of the Brazilian Eastern Amazon. This study contrasts the nutrient balance of slash-and-burn agriculture with a fire-free cultivation. Therefore, one half of a 3.5-year-old (28.7 t DM ha^–1) and a 7-year-old woody fallow vegetation (46.5 t DM ha^–1) was burnt and the other half mulched, leaving the biomass as a surface residue. Subsequently, a sequence of maize, beans and cassava was cropped for 1.5 year. Burning the 3.5- and 7-year-old fallow removed 97 and 94% of the C, 98 and 96% of the N, 90 and 63% of the P-stocks, and between 45 and 70% of the cations K, Mg and Ca of the aboveground biomass by volatilization or ash-particle transfer. These losses were avoided with the slash-and-mulch land preparation. Mulching did not increase the losses of nutrients by leaching, despite the high amount of rapidly decomposing surface mulch. Also the length of preceding fallow had no significant influence on leaching losses. At a depth of 3 m, leached nutrients were quantitatively negligible in both treatments. Comparing the nutrient fluxes at soil depths of 0.9 m, 1.8 m and 3 m, the amounts of all mobile nutrients, and also of chloride and sodium were markedly reduced during percolation and must have been retained. It is likely that nutrient retention in the subsoil layer is only temporary, emphasizing the need for a rapid re-establishment of the naturally deep-rooting secondary vegetation after abandonment of sites to enable uptake of these nutrients. The overall nutrient balance was highly negative for slash-and-burn. 291 and 403 kg N ha^–1, 21 and 18 kg P ha^–1, and 70 and 132 kg K ha^–1 were removed from the burnt plots with a preceding fallow of 3.5 and 7 years, respectively. A reduced fallow period (3.5 years), which is a common trend in the region, resulted in a higher mean annual rate of nutrient loss averaged over the duration of the cycle than a fallow period of 7 years. Eliminating the burning losses by mulching brought the agricultural system back to an equilibrated or even slightly positive nutrient balance, even after a reduced fallow period. Thus, slash-and-mulch is a viable alternative to maintain agricultural productivity and ecosystem functioning.     

Molecular Cross-Talk between Gravity- and Light-Sensing Mechanisms in Euglena gracilis

Euglena gracilis is a photosynthetic flagellate. To acquire a suitable position in its surrounding aquatic environment, it exploits light and gravity primarily as environmental cues. Several physiological studies have indicated a fine-tuned relationship between gravity sensing (gravitaxis) and light sensing in E. gracilis. However, the underlying molecular mechanism is largely unknown. The photoreceptor photoactivated adenylyl cyclase (PAC) has been studied for over a decade. Nevertheless, no direct/indirect interaction partner (upstream/downstream) has been reported for PAC. It has been shown that a specific protein, kinase A (PKA), showed to be involved in phototaxis and gravitaxis. The current study reports the localization of the specific PKA and its relationship with PAC.     

2‐(1‐Ethynylpyrene)‐Adenosine as a Folding Probe for RNA—Pyrene in or out

A series of short RNA duplexes containing one or two 1‐ethynylpyrene‐modified adenine bases was synthesised. The melting behaviour of these duplexes was examined by monitoring temperature‐dependent pyrene fluorescence. In the singly modified RNA duplexes, the bases flanking the ethynylpyrene‐rA were varied to examine the sequence specificity of the fluorescence change of pyrene upon RNA hybridisation. Because an increase in pyrene fluorescence upon melting of the duplex can be correlated with intercalation of pyrene, and a decrease is usually associated with the position of pyrene outside the strand, a relationship between the flanking bases and the tendency of the dye to intercalate has been established. It was found that pyrene intercalation is less likely to take place if the modified base is flanked only by A–U base pairs. Flanking G–C base pairs, even only in the 5′‐direction of the modified base, will favour intercalation. In addition, we examined a doubly modified compound that had a pyrene located on each strand. The spectra indicated that the two pyrenes were close enough for interaction. Upon melting of the strand, a fluorescence blue shift corresponding to the dissociation of the pyrene–pyrene complex could be observed in addition to the intensity effect already known from the singly modified compounds. Two melting curves based on the different properties of the fluorophore could be extracted, leading to different melting points corresponding to the global duplex melting and to the change of local pyrene environment, respectively.