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The ecological effects of novel grass–endophyte associations used in agriculture have not been widely studied. Previous studies of asexual Epichloe-infected Lolium perenne suggest that endophyte concentration is altered in high sugar grasses (HSGs) selectively bred to produce higher concentrations of water-soluble carbohydrates relative to conventional cultivars. We investigated whether differences are due to the effects of altered carbohydrates, or genetic background, by growing multiple cultivars in both high-sugar trait expression and non-expression conditions (using light/temperature treatments). Endophyte and alkaloid concentrations were measured in three HSG and three NSG (normal-sugar grass) cultivars infected with Lp19 or AR37 endophyte strains. Low molecular weight (LMW) carbohydrates had a small effect, explaining

One of the most expanded upper Campanian–Maastrichtian successions worldwide has been cored in a series of boreholes in eastern Denmark. A high-resolution holostratigraphic analysis of this part of the Chalk Group has been undertaken on these cores, notably Stevns-1, in order to provide a record of changes in chalk facies, water depths and sea-water temperatures. Combined lithological data, a suite of petrophysical logs including gamma ray (GR) logs, nannofossil and dinoflagellate palaeontology, stable carbon isotopes, seismic reflection and refraction sections form the basis for the definition of two new formations and six members, three of which are new, and for recognition of Boreal nannofossil subzones UC15e BP to UC20d BP . The upper Campanian–lowermost Maastrichtian Mandehoved Formation is subdivided into the Flagbanke and Boesdal Members and the Maastrichtian Mons Klint Formation is subdivided into the Hvidskud, Rordal, Sigerslev, Kjolby Gaard Marl and Hojerup Members. The Boesdal and Rordal Members show high GR values and a pronounced chalk-marl cyclicity. The Rordal and the thin Kjolby Gaard Marl Members have a regional distribution and can be traced over most of the Danish Basin, whereas the Hojerup Member is restricted to the easternmost part of Sjaelland. The other members consist of rather featureless white chalk.

Tea is an infusion made from the dried leaves of Camellia sinensis L. and is the second most consumed beverage in the world. It has been shown that factors such as fermentation methods, cultivar, geographical origin and season can affect the biochemical composition of tea. In this study, the biochemical composition of green, oolong and black commercial tea samples from around the world was studied using a non-targeted method utilising reversed phase ultra high pressure liquid chromatography (UHPLC) and high resolution mass spectrometry. Principal component analysis of green, oolong and black tea extracts clearly showed that fermented tea can be resolved from non-fermented tea. When the non-targeted data were combined with the supervised multivariate technique, partial least squares discriminant analysis, the method was able to clearly distinguish ‘country of origin’ within green tea and to a lesser extent within a black tea sample set, plus provide indicative marker ions for the country of origin. Many of the significant components detected in this study are unknowns, emphasising the importance of un-biased non-targeted analytical techniques. This study highlights the potential efficacy of non-targeted UHPLC–mass spectrometry when combined with multivariate statistics to differentiate fermented from non-fermented tea and provide potential indicators of provenance of tea samples for further examination.

Alkaline phosphatases (ALP) in arbuscular mycorrhizal (AM) fungi have been suggested to be involved in transfer of phosphate from the mycorrhizal fungus to the host plant, but exact mechanisms are still unknown, partially due to the lack of molecular information. We isolated a full-length cDNA (FmALP) from the AM fungus Funneliformis mosseae (syn. Glomus mosseae) showing similarity with putative ALP genes from Rhizophagus intraradices (syn. Glomus intraradices) and Gigaspora margarita. For functional characterisation FmALP was expressed heterologously in the yeast Pichia pastoris. The recombinant FmALP protein had a pH optimum of 9.5, and catalysed the hydrolysis of glycerolphosphate and, to a lesser extent of glucose-1- and 6-phosphate, confirming it to be an alkaline phosphatase belonging to the family of alkaline phosphomonoesterases (EC 3.1.3.1). FmALP did not catalyse the hydrolysis of ATP or polyP. Relative FmALP transcript levels were analysed in intra- and extraradical hyphae isolated from F. mosseae infected ryegrass (Lolium perenne) using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). FmALP was highly expressed in intraradical hyphae at low Pi supply, and its expression was repressed by high Pi supply. Taken together this study provides evidence for mycorrhizal alkaline phosphatases playing a role in P mobilisation from organic substrates under P starvation conditions.

Tea is the second most consumed beverage in the world and its consumption has been associated with numerous potential health benefits. Factors such as fermentation methods, geographical origin and season can affect the primary and secondary metabolite composition of tea. In this study, a hydrophilic interaction liquid chromatography (HILIC) method coupled to high resolution mass spectrometry in both positive and negative ionisation modes was developed and optimised. The method when combined with principal component analysis to analyse three different types of tea, successfully distinguished samples into different categories, and provided evidence of the metabolites which differed between them. The accurate mass and high resolution attributes of the mass spectrometric data were utilised and relative quantification data were extracted post-data acquisition on 18 amino acids, showing significant differences in amino acid concentrations between tea types and countries. This study highlights the potential of HILIC chromatography combined with non-targeted mass spectrometric methods to provide a comprehensive understanding of polar metabolites in plant extracts.

Polyphenolics are an important class of biologically active compounds found in plants and are purported to have benefits for human health. To assess their significance in foods, improved methods are required for their detection and classification. In this paper, we describe a targeted MS2 approach using an ion-trap mass spectrometer for the analysis and classification of proanthocyanidins (PAs) in crude plant-derived extracts, which has been applied to compare green, oolong and black tea crude extracts and a grape seed extract. The method could separate, detect and provide qualitative information (full scan MS2 spectra) to enable chromatographic and mass spectral confirmation of PAs in the crude tea extracts. Using the developed methodology we report here for the first time the detection of PA tetramers in black tea, and PA trimers and tetramers in oolong tea.

Graphical abstract Epichloënin A (1) has been isolated from fungal cultures together with a minor variant epichloënin B, and ferriepichloënin A and a related peptide epichloëamide have been found in endophyte-infected plants. Highlights ► Epichloënin A has been characterized as a ferrichrome octapeptide by HRMSn and NMR. ► The minor heptapeptide variant epichloënin B has been characterized by HRMSn. ► Ferriepichloënin A was detected in guttation fluid of endophyte-infected plants. ► The related peptide epichloëamide was also detected in guttation fluid. ► Epichloëamide has been characterized as an octapeptide by HRMSn., A number of genes encoding non-ribosomal peptide synthetases (NRPSs) have been identified in fungi of Epichloë/Neotyphodium species, endophytes of Pooid grasses, including sidN, putatively encoding a ferrichrome siderophore-synthesizing NRPS. Targeted gene replacement and complementation of sidN in Epichloë festucae has established that extracellular siderophore epichloënin A is the major product of the SidN enzyme complex (Johnson et al., 2007a). We report here high resolution mass spectrometric fragmentation experiments and NMR analysis of an isolated fraction establishing that epichloënin A is a siderophore of the ferrichrome family, comprising a cyclic sequence of four glycines, a glutamine and three Nδ-trans-anhydromevalonyl–Nδ-hydroxyornithine (AMHO) moieties. Epichloënin A is unusual among ferrichrome siderophores in comprising an octapeptide rather than hexapeptide sequence, and in incorporating a glutamine residue. During this investigation we have established that desferrichrome siderophores with pendant trans-AMHO groups can be distinguished from those with pendant cis-AMHO groups by the characteristic neutral loss of an hydroxyornithine moiety in the MS/MS spectrum. A minor component, epichloënin B, has been characterized as the triglycine variant by mass spectrometry. A peptide characterized by mass spectrometry as the putative deoxygenation product, epichloëamide has been detected together with ferriepichloënin A in guttation fluid from ryegrass (Lolium perenne) plants infected with wild-type E. festucae, but not in plants infected with the ΔsidN mutant strain, and also detected at trace levels in wild-type E. festucae fungal culture.

The roles of the c-Jun N-terminal kinases (JNKs) in inflammatory arthritis have been investigated; however, the roles of each isotype (ie, JNK1 and JNK2) in rheumatoid arthritis and conclusions about whether inhibition of one or both is necessary for amelioration of disease are unclear. By using JNK1- or JNK2-deficient mice in the collagen-induced arthritis and the KRN T-cell receptor transgenic mouse on C57BL/6 nonobese diabetic (K/BxN) serum transfer arthritis models, we demonstrate that JNK1 deficiency results in protection from arthritis, as judged by clinical score and histological evaluation in both models of inflammatory arthritis. In contrast, abrogation of JNK2 exacerbates disease. In collagen-induced arthritis, the distinct roles of the JNK isotypes can, at least in part, be explained by altered regulation of CD86 expression in JNK1- or JNK2-deficient macrophages in response to microbial products, thereby affecting T-cell-mediated immunity. The protection from K/BxN serum-induced arthritis in Jnk1(-/-) mice can also be explained by inept macrophage function because adoptive transfer of wild-type macrophages to Jnk1(-/-) mice restored disease susceptibility. Thus, our results provide a possible explanation for the modest therapeutic effects of broad JNK inhibitors and suggest that future therapies should selectively target the JNK1 isoform.

In this paper we suggest that the field of fungal ecology may benefit from the use of optimality models in the context of an ‘optimality research program’ (ORP). An ORP is a research program in the sense of modern philosopher of science Lakatos' [1978. The Methodology of Scientific Research Programmes: philosophical papers , vol. 1. Cambridge University Press, Cambridge] seminal work. An optimality research program has a lengthy history and record of success in the field of behavioural ecology, but has been seldom employed in fungal ecology. We discuss the ORP and provide some examples of how optimality models may be useful in fungal ecology. We suggest that such an approach may benefit experimental fungal ecologists by: providing a framework for organizing knowledge; generating hypotheses; helping in the planning of experiments; aiding in the interpretation of results; and directing the next steps of an experimental research program. We illustrate these benefits by sketching out how an ORP might be used to answer some fundamental questions about the interactions between host plants and arbuscular mycorrhizal fungi.

Based on direct infusion mass spectrometry we identified a novel alkaloid as a major component of perennial ryegrass (Lolium perenne). Initial mass spectral data suggested it to be a pyrrolizidine conjugate. As this class of alkaloids has not been described before from grasses, we isolated it to elucidate its structure. The isolated alkaloid proved to be a mixture of two stereoisomers. The structures of the two compounds as determined by 1D and 2D NMR spectroscopy, were E-thesinine-O-4′-α-rhamnoside (1) and Z-thesinine-O-4′-α-rhamnoside (2). These identifications were supported by the characterisation by GC-MS and optical rotation of (+)-isoretronecanol as the necine base released on alkaline hydrolysis of these alkaloids. 1 and 2 together with the aglycone and a hexoside were also detected in tall fescue (Festuca arundinacea). This is the first report of pyrrolizidine alkaloids produced by grasses (Poaceae).

Objectives The goal was to clarify if insulin resistance (IR) would predict cardiovascular disease (CVD) independent of the metabolic syndrome (MetSyn). Background Although the cause of MetSyn is not well defined, IR has been proposed to be an important cause. Only a small number of population-based studies have sought to clarify if IR predicts CVD independent of MetSyn. Methods This was a prospective Danish population-based study of 2,493 men and women, age 41 to 72 years, without major CVD at baseline. We defined MetSyn according to both the International Diabetes Foundation (IDF) and the National Cholesterol Education Program (NCEP) criteria, and we quantified IR by the homeostasis model assessment (HOMA-IR). Prevalence of MetSyn was 21% according to IDF criteria and 16% according to NCEP criteria. Accordingly, we defined IDF-HOMA-IR as belonging to the highest 21% of the HOMA-IR distribution, and NCEP-HOMA-IR as belonging to the highest 16% of the HOMA-IR distribution. Results Over a median follow-up of 9.4 years, the incidence of CV end points (CV death, nonfatal ischemic heart disease, and nonfatal stroke) amounted to 233 cases. In proportional hazard models, adjusting for age, gender, smoking, and low-density lipoprotein cholesterol, and with IDF-HOMA-IR and IDF-MetSyn included in the same model, the relative risk of an end point was 1.67 (95% confidence interval [CI] 1.22 to 2.29) for IDF-HOMA-IR and 1.16 (95% CI 0.84 to 1.60) for IDF-MetSyn. The corresponding figures for NCEP-HOMA-IR and NCEP-MetSyn included in the same model were 1.49 (95% CI 1.07 to 2.07) and 1.56 (95% CI 1.12 to 2.17). Conclusions In this Danish study, both HOMA-IR and NCEP-MetSyn were independent predictors of incident CVD.

The peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) is a novel transcriptional co-activator that holds an important role in lipid and glucose metabolism. PGC-1alpha is a candidate gene for the metabolic syndrome (MS) as well as type 2 diabetes. Recent studies suggested linkage between the chromosomal region of PGC-1alpha and fasting serum insulin levels, and associates a Gly482Ser polymorphism of the gene with type 2 diabetes and hypertension. In this study, we investigated whether the Gly482Ser variant is associated with the MS per se or other phenotypic traits related to this syndrome. The variant was examined, using PCR-RFLP, in the DanMONICA cohort comprising a population-based sample of 2349 subjects. MS was defined using the National Cholesterol Education Program -- Adult Treatment Panel III (NCEP-ATPIII) criteria. The allelic frequency of the Ser482 allele was 35.8% in the MS group and 35.6% in the non-MS group (P = 0.74). There were no significant differences across the three groups of genotypes with respect to any of the examined variables, including BMI, waist, fasting serum lipids, plasma glucose, serum insulin, HOMA estimates of insulin resistance and insulin secretion, 24-ambulatory blood pressure or left ventricular mass index. In conclusion, the Gly482Ser polymorphism of the PGC-1alpha gene is not associated with the metabolic syndrome, related quantitative traits or cardiac hypertrophy among Danish Caucasian subjects.

A stereospecific glucosyltransferase, catalysing the transfer of glucose from UDP-glucose to the 4′-hydroxy-group of cis - p -coumaric acid, has been isolated and partially purified from Sphagnum fallax Klinggr. (Klinggr. clone 1) by ion exchange chromatography, gel filtration, and hydrophobic interaction chromatography. The enzyme was purified 600-fold with a yield of 0.4%. The M r was estimated by gel filtration on a Superdex 200 column to be about 56 000. Optimal enzyme activity was observed in glycine-KOH at pH 9.3. The transferase activity was not influenced by divalent cations (1 mM) or EDTA (5 mM), but it was inhibited by NaCl (0.4 M) to 40% and by 2-mercaptoethanol (14 mM) to 30%. The enzyme showed a pronounced substrate specificity towards cis - p -coumaric acid with a K m value of 44 μM, trans - p -coumaric acid was not transformed. The apparent K m value for the cosubstrate UDP-glucose was 110 μM. The only reaction product was 4′-O-β- d -glucosyl -cis-p- coumaric acid. The substrate cis - p -coumaric acid inhibited the reaction at a concentration higher than 1 mM. The only other phenolic substrate transformed by the purified glucosyltransferase was cis -caffeic acid, with an enzyme activity of 10% compared with that using cis - p -coumaric acid as substrate.

Exogenously applied p -coumaric acid (0.1 mM) was rapidly taken up by Sphagnum fallax batch cultures, precultivated axenically in bioreactors, and metabolized to trans -sphagnum acid and a p -coumaric acid conjugate. The structure of the isolated conjugate was elucidated by NMR and IR spectroscopy and was shown to be 4′-O-β- d -glucosyl -cis-p- coumaric acid. Small amounts (80–160 nmol g −1 dry wt) of this component are also always detectable in the homogenate of untreated S. fallax plantlets. Acid hydrolysis (N HCl) of the isolated glucoside or treatment with a β-glucosidase from almonds yielded mainly cis - p -coumaric acid. Irradiation of the cis -glucoside with UV light (λ = 254 nm) resulted in the formation of 4′-O-β- d -glucosyl -trans-p- coumaric acid. The biosynthesis and accumulation of 4′-O-β- d -glucosyl -cis-p- coumaric acid is dependent on the availability of cis - p -coumaric acid in the external medium; exogenously in the dark applied trans - p -coumaric acid is not glucosylated, whereas trans -sphagnum acid is synthesized from trans - p -coumaric acid and accumulated in the light as in the dark. A cytosolic uridine diphosphate glucose (UDPG): p -coumarate glucosyltransferase, which exhibits an unusual substrate specificity for cis - p -coumaric acid, was isolated from Sphagnum fallax . The biosynthesis of phenylpropanoids in Sphagnum known up to now is discussed based on the present results.

The aim of this study was to assess differences in short- and long-term mortality between male and female patients with acute myocardial infarction (AMI). The study population consisted of 6,676 consecutive patients admitted alive with an enzyme-confirmed AMI to 27 Danish hospitals from 1990 to 1992. Five patients were excluded because of missing information. Female patients (n = 2,170) were on average 5 years older than male patients (n = 4,501, p

[ E ]-4-ethoxycarbonyl-3-(4′-hydroxyphenyl)-but-2-en-1-carboxylic acid ( trans -sphagnum acid ethyl ester) and p -hydroxyacetophenone were isolated from ethanolic extracts of Sphagnum magellanicum cell walls and [ Z ]-3-(4′-hydroxyphenyl)-pent-2-en-1,5-dicarboxylic acid ( cis -sphagnum acid) from irradiated trans -sphagnum acid solutions. The structures of these compounds were elucidated by means of NMR spectroscopy. Compartmentalization studies revealed that the main part of detectable sphagnum acid is buffer-soluble, as well as in Sphagnum collected from natural sites and in Sphagnum cultivated in bioreactors. Cultivating Sphagnum species in bioreactors under axenic conditions leads to an enhancement of the buffer-soluble part of all analysed phenolics with a concomitant decrease of their cell wall-bound amount. Sphagnum acid, sphagnum acid ethyl ester, hydroxybutenolide, p -hydroxybenzoic acid, p -coumaric acid and t -cinnamic acid are also excreted to a remarkable extent into the effluent culture media of S. fallax and S. cuspidatum . Specific compartments for the storage of endogenous free water-soluble phenolics are postulated.