Isolation by HPLC and characterisation of the bioactive fraction of New Zealand manuka (Leptospermum scoparium) honey

Using HPLC a fraction of New Zealand manuka honey has been isolated, which gives rise to the non-peroxide antibacterial activity. This fraction proved to be methylglyoxal, a highly reactive precursor in the formation of advanced glycation endproducts (AGEs). Methylglyoxal concentrations in 49 manuka and 34 non-manuka honey samples were determined using a direct detection method and compared with values obtained using standard o-phenylenediamine derivatisation. Concentrations obtained using both the methods were similar and varied from 38 to 828 mg/kg.     

Submerged fermentation in wheat substrates for production of Monascus pigments

Cereal grains are normally used as solid substrates for the production of Monascus metabolites. However, solid fermentation in these substrates requires complex control systems, whereas in liquid culture the control of the fermentation is simpler and consequently significant reductions in fermentation times can be achieved. In the same way, the use of submerged culture can benefit the production of many secondary metabolites and decrease production costs by reducing the labour involved in solid-state methods. A flour composed of a mixed variety of Canadian hard wheat was used as sole nutrient source to produce the pigments of Monascus purpureus Went (IMI 210765). Supplementation with NH₄Cl promoted biomass and orange dye formation, whereas the use of zinc sulphate favoured red dyes production. In submerged fermentations significant differences in final pigment yields were observed in the use of wheat-based broth at different concentrations in the presence of bran particles and/or gluten protein. It has been found that the viscosity of the broth had a significant effect on the growth morphology and production of pigments. Gluten-free wheat flour at concentrations of 3–5% was found to be the most suitable for liquid Monascus culture. The subsequent use of passive immobilization of Monascus served to enhance red pigment yields and to facilitate the downstream processing of the dyes.     

DNA adduct levels in fish from pristine areas are not detectable or low when analysed using the nuclease P1 version of the 32P-postlabelling technique

In order to understand and apply DNA adduct formation in fish liver as a biomarker for aquatic pollution, information concerning the natural background levels in non-contaminated organisms, caused by endogenous compounds, is of fundamental importance. In this study, DNA adducts were analysed in liver of 11 fish species from arctic and sub-arctic areas in the northern Atlantic using the nuclease P1 version of the ^{332P-postlabelling technique. The collected fish were assumed not to have been influenced by anthropogenic pollution apart from possible long-range transported pollutants. As polycyclic aromatic hydrocarbons (PAHs) are thought to be fundamental in forming the type of DNA adducts detected by the method used, biliary PAH metabolite levels were measured in a selection of the investigated species. In all investigated individuals, the levels of PAH metabolites were undetectable. Controlled on-site exposure experiments with benzo[a]pyrene (polar cod) and laboratory experiments with crude oil (polar cod and Atlantic cod) were conducted. DNA adducts were formed in both these species. The field-sampled fish showed undetectable levels of DNA adducts or levels just above the detection limit. The present study supports the assumption that when DNA adducts are detected by the nuclease P1 version of the 3ng method in fish liver, it can be interpreted as DNA damage caused by pollutants.}     

Anti-predatory chemical defenses of ascidians: secondary metabolites or inorganic acids?

Both secondary metabolites and inorganic acids have been hypothesized to protect adult ascidians from predation, raising the possibility of alternative defensive strategies in these sessile, soft-bodied, benthic invertebrates. The objective of this investigation was to determine if ascidian species from the Western Atlantic have these chemical defenses against fish predators, and if so, to determine their location within the body of the ascidian. The palatability of crude organic extracts of whole ascidians, as well as the dissected tunics, viscera, and gonads (when possible) were determined at natural volumetric concentrations using laboratory feeding assays with the bluehead wrasse, Thalassoma bifasciatum. Acidified food pellets were also assayed to determine the effect of lowered pH on predation. Sixteen of the 17 species tested had deterrent organic extracts from some region of the body (Aplidium constellatum, Aplidium stellatum, Ascidia interrupta, Ascidia nigra, Botrylloides sp., Clavellina picta, Didemnum candidum, Didemnum vanderhosti, Diplosoma listerianum, Ecteinascidia turbinata, Eudistoma capsulatum, Eudistoma hepaticum, Rhopalaea abdominalis, Styela plicata, Symplegma rubra, and Trididemnum solidum). The location of the deterrent secondary metabolites was isolated in the gonad in all three solitary species, raising the possibility that these defenses are passed on to eggs or larvae. Nine ascidian species sequestered acid in their tunics (A. interrupta, A. nigra, A. stellatum, D. candidum, D. vanderhosti, E. capsulatum, E. hepaticum, R. abdominalis, and T. solidum) at levels that were effective in deterring fish predation (pH≤3.0). Only one species (Botrylloides nigrum) had neither chemical defense. Results of this study indicate that there is not a clear trade-off between the presence of secondary metabolites and inorganic acid defenses in ascidians, suggesting that these defenses are redundant, or that alternative chemical defenses may have evolved for different predators or for different stages in the life history of the ascidians producing them.     

Comparison of antioxidative capacities and inhibitory effects on cholesterol biosynthesis of quercetin and potential metabolites

The flavonol quercetin is known to be rapidly metabolized after ingestion by enterocytes and bacteria in the intestinal tract which may influence the biological, e.g. antioxidative potency of this compound. Therefore, quercetin and several of its possible metabolites were compared with regard to their antioxidant activity and their capacity to inhibit hepatocellular cholesterol biosynthesis. Using the 2,2,-diphenylpicrylhydrazyl radical scavenger assay, all compounds with an ortho diphenolic structure acted as strong antioxidants. In contrast, in a cellular assay focusing on lipid peroxidation in cultured rat hepatocytes challenged with tert.-butylhydroperoxide only the lipophilic compounds quercetin and 3,4-dihydroxytoluene were active. Concerning the inhibition of cholesterol biosynthesis, 3,4-dihydroxytoluene surprisingly mimicked the effect of quercetin in primary rat hepatocytes, but much less so in HepG2 cells. All other metabolites were almost ineffective in both cell types. These results suggest that some of the biological functions of flavonoids detectable by in vitro assays may persist in vivo as long as comparably potent metabolites are systemically present.     

Bioactive beads-mediated transformation of rice with large DNA fragments containing Aegilops tauschii genes

Transformation with large DNA molecules enables multiple genes to be introduced into plants simultaneously to produce transgenic plants with complex phenotypes. In this study, a large DNA fragment (ca. 100 kb) containing a set of Aegilops tauschii hardness genes was introduced into rice plants using a novel transformation method, called bioactive beads-mediated transformation. Nine transgenic rice plants were obtained and the presence of transgenes in the rice genome was confirmed by PCR and FISH analyses. The results suggested that multiple transgenes were successfully integrated in all transgenic plants. The expression of one of the transgenes, puroindoline b, was confirmed at the mRNA and protein levels in the T₂ generation. Our study clearly demonstrates that the bioactive bead method is capable of producing transgenic rice plants carrying large DNA fragments. This method will facilitate the production of useful transgenic plants by introducing multiple genes simultaneously.     

Cunninghamella--a microbial model for drug metabolism studies--a review

Drug metabolism studies constitute an important and necessary step in the evaluation of drug efficacy and safety. In vivo drug metabolism studies suffer from many disadvantages. Hence there is a rise in validation of in vitro microbial models. This review describes the transformation studies of drugs by the fungus, Cunninghamella and correlating them with the metabolism/biotransformation in animal systems and providing technical methods to develop microbial models. Emphasis is laid on the potential of Cunninghamella fungus to mimic mammalian drug biotransformations and to use as in vitro model for drug metabolism studies and for further toxicological and pharmacological studies of metabolites.     

Developing Aspergillus as a host for heterologous expression

Filamentous fungi have long been used for production of a range of valuable products; with the advent of molecular biology, it became apparent that these fungi possess considerable potential as expression hosts for the production of heterologous proteins and small molecules. Aspergillus is an important genus, including well known species of economically significant molds, and widely used for basic genetic research. The development of a genetic engineering "toolkit" for Aspergillus, such as those existing for the simpler yeasts and bacteria, was delayed due to the added complexity of the filamentous fungi, and also to the lesser resources devoted to their study. History of the development of Aspergillus as an expression host, current state of the art and future directions are reviewed, touching on related research in other fungi when discussing the areas of greatest potential for future biotechnological applications, focusing on the large and diverse families of fungal secondary metabolites.     

The ‘mustard oil bomb’: not so easy to assemble?! Localization, expression and distribution of the components of the myrosinase enzyme system

Glucosinolates are plant secondary metabolites that are hydrolysed by the action of myrosinases into various products (isothiocyanates, thiocyanates, epithionitriles, nitriles, oxazolidines). Massive hydrolysis of glucosinolates occurs only upon tissue damage but there is also evidence indicating metabolism of glucosinolates in intact plant tissues. It was originally believed that the glucosinolate–myrosinase system in intact plants was stable due to a spatial separation of the components. This has been coined as the ‘mustard oil bomb’ theory. Proteins that form complexes with myrosinases have been described: myrosinase-binding proteins (MBPs) and myrosinase-associated proteins (MyAPs/ESM). The roles of these proteins and their biological relevance are not yet completely known. Other proteins of the myrosinase enzyme system are the epithiospecifier protein (ESP) and the thiocyanate-forming protein (TFP) that divert the glucosinolate hydrolysis from isothiocyanate production to nitrile/epithionitrile or thiocyanate production. Some glucosinolate hydrolysis products act as plant defence compounds against insects and pathogens or have beneficial health effects on humans. In this review, we survey and critically assess the available information concerning the localization, both at the tissular/cellular and subcellular level, of the different components of the myrosinase enzyme system. Data from the model plant Arabidopsis thaliana is compared to that from other glucosinolate-producing Brassicaceae in order to show common as well as divergent features of the ‘mustard oil bomb’ among these species.