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Dihydrochalcones (DHCs) are flavonoids produced as a side branch of the phenylpropanoid pathway. DHCs are found at high concentrations in apples (Malus spp.) but not in pears (Pyrus spp.) or other members of the Rosaceae. Biosynthesis of DHCs in apple has been hypothesized to occur via reduction of p-coumaroyl CoA by a Malus × domestica hydroxycinnamoyl CoA double-bond reductase (MdHCDBR) followed by the action chalcone synthase to produce phloretin or via direct reduction of naringenin chalcone to phloretin via an unknown enzyme. In this study, we report that genetic downregulation of MdHCDBR does not reduce DHC concentrations in apple leaves. We used comparative transcriptome analysis to identify candidate naringenin chalcone reductases (NCRs), designated MdNCR1a-c, expressed in apple leaves but not fruit. These MdNCR1 genes form an expanded gene cluster found exclusively in apple. Transient expression of MdNCR1 genes in Nicotiana benthamiana leaves indicated they produced DHCs at high concentrations in planta. Recombinant MdNCR1 utilized naringenin chalcone to produce phloretin at high efficiency. Downregulation of NCR genes in transgenic apple reduced foliar DHC levels by 85-95%. Reducing DHC production redirected flux to the production of flavonol glycosides. In situ localization indicated that NCR proteins were likely found in the vacuolar membrane. Active site analysis of AlphaFold models indicated that MdNCR1a-c share identical substrate binding pockets, but the pockets differ substantially in related weakly active/inactive NCR proteins. Identifying the missing enzyme required for DHC production provides opportunities to manipulate DHC content in apple and other fruits and has other applications, e.g., in biofermentation and biopharming., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Previous clinical studies indicate that monoamine oxidase-B (MAO-B) inhibition by blackcurrants must be predominantly attributed to bioactives other than anthocyanins. In this natural products discovery study, MAO-A/B inhibitory phytochemicals were isolated from blackcurrants, and a double-blind crossover study investigated the efficacy of freeze-dried whole-fruit blackcurrant powder in inhibiting MAO-B compared with blackcurrant juice in healthy adults. Platelet MAO-B inhibition was comparable between powder (89% ± 6) and juice (91% ± 4), and it was positively correlated with MAO-modulated plasma catecholamines, subjective alertness, and reduced mental fatigue, assessed using the Bond-Lader questionnaire. Sarmentosin, a nitrile glycoside, and its hydroxycinnamoyl esters were identified as novel MAO-A/B inhibitors from blackcurrant in vitro , and sarmentosin was demonstrated to inhibit platelet MAO-B activity in vivo . These findings confirm sarmentosin as the primary bioactive for MAO-A/B inhibition in blackcurrants, as well as its bioavailability and stability during freeze-drying, and suggest that consuming blackcurrant powder and juice may positively affect mood in healthy adults.

Purpose: The benefits of volunteering among youth are well documented. However, research is limited on volunteering among youth with disabilities. This study examined prevalence and associations of volunteering among youth with disabilities., Methods: We analyzed data from the 2016-2018 National Survey of Children's Health ( n = 42 204). Prevalence estimates were calculated for sociodemographic and household factors, volunteering, and functional limitations. Bivariate and multivariable associations between participation in volunteering activities and variables of interest were assessed., Results: Youth with disabilities had lower prevalence of volunteering compared to youth without disabilities (48.4% vs. 55.6%). Youth with hearing and vision impairments had the lowest prevalence of volunteering (2.1% and 2.5%, respectively). Correlates of volunteering included health status (adjusted odds ratios (aOR)=1.81; 95% confidence interval (CI) 1.13-2.91), sex, and age., Conclusions: Our study found low prevalence of volunteering among youth with disabilities. Since youth with disabilities face unique barriers to social inclusion, it is important to identify and reduce the barriers to participation among this group. Volunteer opportunities in the community should be designed to be both environmentally and programmatically accessible to youth with disabilities. These efforts should include accommodations and other support so that participants can fully experience all of the benefits of volunteering.Implications for rehabilitationOur multiyear nationally representative study found low prevalence of volunteering among youth with disabilities, particularly those with hearing and vision impairments.Since youth with disabilities face unique barriers to social inclusion, it is important to identify and reduce the barriers to participation among this group.Our findings support the need for environmentally and programmatically accessible volunteering opportunities for youth with disabilities.These efforts should include accommodations and other support so that participants can fully experience all of the benefits of volunteering.

Objectives: To examine the prevalence of tobacco use by product type among youths with cognitive disability; the prevalence of tobacco dependence among youths with cognitive disability; and the relationship between age of tobacco use initiation and cognitive disability., Study Design: This cross-sectional study analyzed data from the 2019 National Youth Tobacco Survey (NYTS). Participants were a nationally representative sample of 19 018 students in grades 6-12. Estimates were calculated for ever use, current use, age of tobacco use initiation, and tobacco dependence. Associations between use patterns and cognitive disability status were examined using bivariate analyses and multivariable logistic regression., Results: Compared with youths without cognitive disability, youths with cognitive disability had significantly greater odds of ever using any tobacco product (aOR, 1.49; 95% CI, 1.31-1.70), currently using any tobacco product (aOR, 1.41; 95% CI, 1.26-1.58), and currently using electronic cigarettes (e-cigarettes), cigarettes, cigars, hookahs, roll-your-own cigarettes, and heated tobacco products, specifically. They had higher prevalence and odds of reporting younger age of tobacco use initiation (aOR, 1.25; 95% CI, 1.10-1.43). Higher prevalence and odds of tobacco dependence were also observed among youths with cognitive disability compared with youths without cognitive disability (P < .001)., Conclusions: These findings reinforce the importance of developing early primary prevention efforts to reduce or delay tobacco use among adolescents with cognitive disability. They also suggest the need to address co-occurring disorders during tobacco cessation programs with this high-risk group., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Gene expression and phytohormone contents were measured in response to elevating ascorbate in the absence of other confounding stimuli such as high light and abiotic stresses. Young Arabidopsis plants were treated with 25 mM solutions of l-galactose pathway intermediates l-galactose (l-gal) or l-galactono-1,4-lactone (l-galL), as well as L-ascorbic acid (AsA), with 25 mM glucose used as control. Feeding increased rosette AsA 2- to 4-fold but there was little change in AsA biosynthetic gene transcripts. Of the ascorbate recycling genes, only Dehydroascorbate reductase 1 expression was increased. Some known regulatory genes displayed increased expression and included ANAC019 , ANAC072 , ATHB12 , ZAT10 and ZAT12 . Investigation of the ANAC019 / ANAC072 / ATHB12 gene regulatory network revealed a high proportion of ABA regulated genes. Measurement of a subset of jasmonate, ABA, auxin (IAA) and salicylic acid compounds revealed consistent increases in ABA (up to 4.2-fold) and phaseic acid (PA; up to 5-fold), and less consistently certain jasmonates, IAA, but no change in salicylic acid levels. Increased ABA is likely due to increased transcripts for the ABA biosynthetic gene NCED3 . There were also smaller increases in transcripts for transcription factors ATHB7 , ERD1 , and ABF3 . These results provide insights into how increasing AsA content can mediate increased abiotic stress tolerance.

Polyphenols within fruits and vegetables may contribute to health benefits due to their consumption, with the anthocyanin sub-set also adding colour. The Lemonade™ apple variety has green skin and white flesh, with low anthocyanin content, while some apple varieties have high anthocyanin content in both the skin and flesh. Effects of red compared with white-fleshed apples were studied in healthy human subjects in a randomized, placebo-controlled, cross-over intervention trial. Twenty-five healthy subjects consumed dried daily portions of the red-fleshed or placebo (white-fleshed) apple for two weeks, followed by one-week washout and further two-week crossover period. During the study, volunteers provided faecal samples for microbiota composition analysis and blood samples for peripheral blood mononuclear cell (PBMC) gene expression analysis. Subtle differences were observed in the faecal microbiota of subjects that were fed the different apples, with significant ( p < 0.05) reductions in relative abundances of Streptococcus , Ruminococcus , Blautia , and Roseburia , and increased relative abundances of Sutterella , Butyricicoccus , and Lactobacillus in subjects after consuming the red apple. Changes in PBMC gene expression showed 18 mRNA transcripts were differentially expressed between the two groups, of which 16 were immunoglobulin related genes. Pathway analysis showed that these genes had roles in pathways such as immunoglobulin production, B cell-mediated immunity, complement activation, and phagocytosis. In conclusion, this study shows that anthocyanin-rich apples may influence immune function compared to control apples, with changes potentially associated with differences in the faecal microbiota.

Background: The number of students with disabilities attending postsecondary institutions is increasing. However, research on substance use among this population is limited., Objective: This study examined disparities in the prevalence of illicit drug use and drug use disorders among college students with disabilities and their counterparts without disabilities., Methods: Data from the 2017 National Survey on Drug Use and Health were analyzed. We estimated prevalence and odds of disability, illicit drug use, and illicit drug dependence or abuse in a subsample of college students (n = 6,189)., Results: A majority of college students reporting a disability had a cognitive limitation. Students with any disability had a higher prevalence of illicit drug use and significantly higher odds of ever use of illicit drugs (AOR = 1.47; 95% CI 1.20-1.79). Compared to their peers with no disabilities, they were more likely to have misused any psychotherapeutic in the past year (AOR = 1.38; 95% CI 1.08-1.76), and had nearly twice the odds of misusing prescription pain relievers in the past month (AOR = 1.97; 95% CI 1.11-3.49). Additionally, students with disabilities had three times the odds of meeting criteria for past-year dependence or abuse of any illicit drug (AOR = 3.01; 95% CI 2.06-4.40)., Conclusion: This study documented a higher prevalence of drug use and drug use disorders among college students with disabilities compared to their nondisabled peers. Understanding the risk factors for substance use in this population is critical for developing effective prevention and treatment strategies., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to report., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Whole kiwifruit ('Hayward' and 'Zesy002') were examined for their bioaminergic potential after being subjected to in vitro gastrointestinal digestion and colonic fermentation. Controls included the prebiotic inulin and water, a carbohydrate-free vehicle. The dopamine precursor l-dihydroxyphenylalanine (L-DOPA) and the serotonin precursor 5-hydroxytryptophan were increased in the kiwifruit gastrointestinal digesta ('Hayward' > 'Zesy002') in comparison to the water digesta. Fermentation of the digesta with human fecal bacteria for 18 h modulated the concentrations of bioamine metabolites. The most notable were the significant increases in L-DOPA ('Zesy002' > 'Hayward') and γ-aminobutyric acid (GABA) ('Hayward' > 'Zesy002'). Kiwifruit increased Bifidobacterium spp. and Veillonellaceae (correlating with L-DOPA increase), and Lachnospira spp. (correlating with GABA). The digesta and fermenta were incubated with Caco-2 cells for 3 h followed by gene expression analysis. Effects were seen on genes related to serotonin synthesis/re-uptake/conversion to melatonin, gut tight junction, inflammation and circadian rhythm with different digesta and fermenta from the four treatments. These indicate potential effects of the substrates and the microbially generated organic acid and bioamine metabolites on intestinal functions that have physiological relevance. Further studies are required to confirm the potential bioaminergic effects of gut microbiota-kiwifruit interactions.

The groups of plant flavonoid metabolites termed anthocyanins and proanthocyanins (PA) are responsible for pigmentation in seeds, flowers and fruits. Anthocyanins and PAs are produced by a pathway of enzymes which are transcriptionally regulated by transcription factors (TFs) that form the MYB-bHLH-WD40 (MBW) complex. In this study, transcriptomic analysis of purple-pigmented kiwifruit skin and flesh tissues identified MYBC1, from subgroup 5 of the R2R3 MYB family, and WRKY44 (highly similar to Arabidopsis TTG2) as candidate activators of the anthocyanin pathway. Transient over-expression of MYBC1 and WRKY44 induced anthocyanin accumulation in tobacco leaves. Dual luciferase promoter activation assays revealed that both MYBC1 and WRKY44 were able to strongly activate the promoters of the kiwifruit F3'H and F3'5'H genes. These enzymes are branch points of the pathway which specifies the type of anthocyanin accumulated. Stable over-expression of MYBC1 and WRKY44 in kiwifruit calli activated the expression of F3'5'H and PA-related biosynthetic genes as well as increasing levels of PAs. These results suggest that while previously characterised anthocyanin activator MYBs regulate the overall anthocyanin biosynthesis pathway, the PA-related TFs, MYBC1 and WRKY44, more specifically regulate key branch points. This adds a layer of regulatory control that potentially balances anthocyanin and PA levels.

Emerging evidence from human intervention trials indicates health benefits of consuming blackcurrant fruit, including improvements to cognitive performance, modulation of blood flow, regulation of blood glucose and inhibition of enzymes underpinning normal cognitive function. Of particular relevance is our previous demonstration of monoamine oxidase (MAO)-A and B inhibition after the consumption of a New Zealand "Blackadder" blackcurrant juice in humans. The current study uses a double-blind, placebo-controlled, randomised cross- over design to assess the pharmacodynamics of the effects on platelet MAO-B inhibition and associated substrates, plasma prolactin levels and blood glucose levels after consumption of a single serve of "Blackadder" blackcurrant juice standardised to 500 mg polyphenols. Eight healthy male (20--35 years) participants completed the trial. Measurements were obtained at baseline 15, 30, 45, 60, 100, 120, 150, 180, 240 mins and 24 h post dose. A fast, absolute and reversible inhibition of blood platelet MAO-B ( P  < 0.001) and a significant but delayed reduction in plasma prolactin ( P  < 0.001) were observed following the consumption of "Blackadder" blackcurrant juice when compared to a placebo control. No interpretable changes in substrates of MAO or associated metabolites were seen. These data provide a clear time course of the reversible inhibition of MAO-B after the single consumption of a of New Zealand "Blackadder" blackcurrant juice standardised at 500 mg of polyphenols and, therefore, provide a therapeutic window on which to base future nutritional interventions.

Background: Affective responses experienced during exercise are a significant determinant on exercise adherence. We have previously demonstrated that consumption of New Zealand (NZ) blackcurrants preserves cognition by attenuating the feeling of fatigue. This positive affective response correlated with the ability of blackcurrant polyphenols to support monoamine neurotransmission via inhibition of monoamine oxidase-B (MAO-B) activity. Here we explore how the consumption of a NZ blackcurrant juice (BJ) influenced affective responses and potential ergogenic action on the motivation to adhere to a low impact walking exercise., Methods: In a parallel randomized controlled study (Trial registration #: ACTRN12617000319370p, registered 28th February 2017, http://www.anzctr.org.au/ ), 40 healthy sedentary male and female participants drank a BJ or matched placebo (PLA) (n = 20 per group), 1 h prior to a self-motivated treadmill walk, where heart rate and affective responses (exertion [ES] or feeling / mood [FS]) scores) were recorded at 3 or 5 min intervals. Blood glucose, lactate, malondialdehyde (MDA) and platelet MAO-B activity were measured pre- and post-exercise and comparisons were conducted using with Student's t-tests. Subjective data were analysed using 2-way ANOVA with appropriate post hoc tests., Results: Consuming a BJ 1 h prior to exercise caused a 90% decline in platelet MAO-B activity. The exercise had no significant (p > 0.05) effect on blood lactate, glucose or plasma MDA levels. Assessment of affective responses over the first 60 mins (adjusting for participant drop-out) revealed a time-dependent ES increase in both groups, with ES reported by participants in the BJ group consistently lower than those in the PLA group (p < 0.05). FS declined in PLA and BJ groups over 60 mins, but an inverse relationship with ES was only observed within the PLA group (r 2  = 0.99, p = 0.001). Whilst the average time walked by participants in the BJ group was 11 mins longer than the PLA group (p = 0.3), and 30% of the BJ group achieving > 10 km compared to only 10% for the PLA group (p = 0.28), statistical significance was not achieved., Conclusion: Our findings demonstrate that drinking a polyphenolic-rich NZ blackcurrant juice 1 h prior to exercise supports positive affective responses during a self-motivated exercise.

Aim: To evaluate blackcurrant anthocyanin-rich extract (BAE) consumption on time- and dose-dependent plasma anthocyanin bioavailability and conduct a pilot study to explore the potential effect of BAE in promoting recovery from exercise-induced oxidative stress, and maintenance of circulating neutrophil function. Methods: Time- and dose-dependent blackcurrant anthocyanin bioavailability was assessed using LC-MS in 12 participants over 6 h after the ingestion of a placebo or BAE containing 0.8, 1.6, or 3.2 mg/kg total anthocyanins. In a separate pilot intervention exercise trial, 32 participants consumed either a placebo or 0.8, 1.6, or 3.2 mg/kg BAE (8 individuals per group), and then 1 h later performed a 30 min row at 70% VO 2 max. Blood was collected during the trial for oxidative, antioxidant, inflammatory, and circulating neutrophil status. Results: Consumption of BAE caused a time- and dose-dependent increase in plasma anthocyanins, peaking at 2 h after ingestion of 3.2 mg/kg BAE (217 ± 69 nM). BAE consumed 1 h prior to a 30 min row had no effect on plasma antioxidant status but hastened the recovery from exercise-induced oxidative stress: By 2 h recovery, consumption of 1.6 mg/kg BAE prior to exercise caused a significant ( P < 0.05) 34 and 32% decrease in post-exercise plasma oxidative capacity and protein carbonyl levels, respectively, compared to placebo. BAE consumption prior to exercise dose-dependently attenuated a small, yet significant ( P < 0.01) transient 13 ± 2% decline in circulating neutrophils observed in the placebo group immediately post-exercise. Furthermore, the timed consumption of either 1.6 or 3.2 mg/kg BAE attenuated a 17 ± 2.4% ( P < 0.05) decline in neutrophil phagocytic capability of opsonised FITC- Escherichia coli observed 6 h post-exercise in the placebo group. Similarly, a dose-dependent increase in neutrophil surface expression of complement receptor-3 complex (CR3, critical for effective phagocytosis of opsonised microbes), was observed 6 h post-exercise in both 1.6 and 3.2 mg/kg BAE intervention groups. Conclusions: Consumption of BAE (>1.6 mg/kg) 1 h prior to exercise facilitated recovery from exercise-induced oxidative stress and preserved circulating neutrophil function. This study provides data to underpin a larger study designed to evaluate the efficacy of timed BAE consumption on post-exercise recovery and innate immunity.

Anthocyanins are a group of secondary metabolites that colour fruit and flowers orange, red, purple or blue depending on a number of factors, such as the basic structure, co-pigmentation, metal ion complexation and vacuolar pH. The biosynthesis of anthocyanin is regulated at the transcriptional level by a group of transcription factors, the MYB-bHLH-WD40 (MBW) complex. In this study, the purple colouration in several kiwifruit ( Actinidia) species was identified and characterised as red cyanidin-based and blue delphinidin-based anthocyanins. The differential pigmentation in the skin and flesh can be attributed to the differential ratio of cyanidin and delphinidin derivatives accumulated in the total anthocyanin profile. The expression of anthocyanin biosynthetic genes chalcone synthase ( CHS ), flavonoid 3-O-glucosyltransferase ( F3GT ), flavonoid 3'-hydroxylase ( F3 ' H ) and flavonoid 3'5'-hydroxylase ( F3 ' 5 ' H ) is crucial for anthocyanin accumulation. However, the balance of expression of the F3 ' H and F3 ' 5 ' H genes appears responsible for the ratio of cyanidin and delphinidin derivatives, while a lack of CHS , F3GT and MYB110 expression is responsible for a lack of total anthocyanins. The transcriptional regulation of the F3 ' H and F3 ' 5 ' H promoters by the R2R3 MYB transcription factor MYB110 is markedly different in tobacco transient assays. When kiwifruit MYB10 or MYB110 are over-expressed in Actinidia chinensis both cyanidin-based and delphinidin-based anthocyanins are elevated, but F3 ' H and F3 ' 5 ' H genes are not strongly correlated with MYB expression. These results suggest that the core kiwifruit anthocyanin pathway genes are dependent on characterised MYB transcription factors, while other regulatory proteins are more directly responsible for the expression of the F3 ' H and F3 ' 5 ' H genes., Competing Interests: The authors declare that they have no conflict of interest.

Background: Spinal cord stimulation (SCS) is now established as the primary treatment for failed back surgery syndrome (FBSS). Commonly, patients with chronic pain and FBSS often report symptoms of anxiety and depression resulting from this condition. These factors can modulate and amplify the pain experience, therefore, further challenging treatment success., Aims: This study examined the efficacy of SCS on alleviating the symptoms of anxiety and depression associated with chronic pain as well as pain intensity in a group of patients with FBSS., Methods: A convenience sample (n = 26) was selected for participation. Questionnaires [Hospital Anxiety and Depression Scale (HADS) and Brief Pain Inventory Short Form (BPI-SF)] were completed and examined pre and post spinal cord implant., Results: Analysis of the data 1 year following SCS indicates that there was a statistical significant improvement in the symptoms of depression and anxiety reported as well as pain intensity in all participants (p < 0.001). Both anxiety and depression scores on the HADS were significantly lower compared to baseline (p < 0.001). Pain intensity scores decreased by ≥50% from baseline in all participants. Opioid analgesia was discontinued by 90% (n = 8) of participants., Conclusion: Whilst it is already recognised that SCS reduces pain in FBSS, this study demonstrated that it also reduced the symptoms of anxiety and depression with an associated reduction in opioid consumption.

The polyphenol profile of apple (Malus × domestica) is dominated by the dihydrochalcone glycoside phloridzin, but its physiological role is yet to be elucidated. Biosynthesis of phloridzin occurs as a side branch of the main phenylpropanoid pathway, with the final step mediated by the phloretin-specific glycosyltransferase UGT88F1. Unexpectedly, given that UGTs are sometimes viewed as 'decorating enzymes', UGT88F1 knockdown lines were severely dwarfed, with greatly reduced internode lengths, narrow lanceolate leaves, and changes in leaf and fruit cellular morphology. These changes suggested that auxin transport had been altered in the knockdown lines, which was confirmed in assays showing that auxin flux from the shoot apex was increased in the transgenic lines. Metabolite analysis revealed no accumulation of the phloretin aglycone, as well as decreases in many non-target phenylpropanoid compounds. This decreased accumulation of metabolites appeared to be mediated by the repression of the phenylpropanoid pathway via a reduction in key transcript levels (e.g. phenylalanine ammonia lyase, PAL) and enzyme activities (PAL and chalcone synthase). Application of exogenous phloridzin to the UGT88F1 knockdown lines in tissue culture enhanced axial leaf growth and partially restored some aspects of 'normal' apple leaf growth. Together, our results strongly implicate dihydrochalcones as critical compounds in modulating phenylpropanoid pathway flux and establishing auxin patterning early in apple development., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.)

Apple (Malus × domestica) accumulates bioactive ursane-, oleanane-, and lupane-type triterpenes in its fruit cuticle, but their biosynthetic pathway is still poorly understood. We used a homology-based approach to identify and functionally characterize two new oxidosqualene cyclases (MdOSC4 and MdOSC5) and one cytochrome P450 (CYP716A175). The gene expression patterns of these enzymes and of previously described oxidosqualene cyclases were further studied in 20 apple cultivars with contrasting triterpene profiles. MdOSC4 encodes a multifunctional oxidosqualene cyclase producing an oleanane-type triterpene, putatively identified as germanicol, as well as β-amyrin and lupeol, in the proportion 82 : 14 : 4. MdOSC5 cyclizes 2,3-oxidosqualene into lupeol and β-amyrin at a ratio of 95 : 5. CYP716A175 catalyses the C-28 oxidation of α-amyrin, β-amyrin, lupeol and germanicol, producing ursolic acid, oleanolic acid, betulinic acid, and putatively morolic acid. The gene expression of MdOSC1 was linked to the concentrations of ursolic and oleanolic acid, whereas the expression of MdOSC5 was correlated with the concentrations of betulinic acid and its caffeate derivatives. Two new multifuntional triterpene synthases as well as a multifunctional triterpene C-28 oxidase were identified in Malus × domestica. This study also suggests that MdOSC1 and MdOSC5 are key genes in apple fruit triterpene biosynthesis., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

The aim of this study was to provide insight into how curcumin reduces colon inflammation in the Mdr1a(-/-) mouse model of human inflammatory bowel disease using a combined transcriptomics and proteomics approach. Mdr1a(-/-) and FVB control mice were randomly assigned to an AIN-76A (control) diet or AIN-76A+0.2% curcumin. At 21 or 24weeks of age, colonic histological injury score (HIS) was determined, colon mRNA transcript levels were assessed using microarrays and colon protein expression was measured using 2D gel electrophoresis and LCMS protein identification. Colonic HIS of Mdr1a(-/-) mice fed the AIN-76A diet was higher (P<.001) than FVB mice fed the same diet; the curcumin-supplemented diet reduced colonic HIS (P<.05) in Mdr1a(-/-) mice. Microarray and proteomics analyses combined with new data analysis tools, such as the Ingenuity Pathways Analysis regulator effects analysis, showed that curcumin's antiinflammatory activity in Mdr1a(-/-) mouse colon may be mediated by activation of α-catenin, which has not previously been reported. We also show evidence to support curcumin's action via multiple molecular pathways including reduced immune response, increased xenobiotic metabolism, resolution of inflammation through decreased neutrophil migration and increased barrier remodeling. Key transcription factors and other regulatory molecules (ERK, FN1, TNFSF12 and PI3K complex) activated in inflammation were down-regulated by dietary intervention with curcumin., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Poisonings due to consumption of honeys containing plant toxins have been reported widely. One cause is the neurotoxin tutin, an oxygenated sesquiterpene picrotoxane, traced back to honeybees (Apis mellifera) collecting honeydew produced by passionvine hoppers (Scolypopa australis) feeding on sap of the poisonous shrub tutu (Coriaria spp.). However, a pharmacokinetic study suggested that unidentified conjugates of tutin were also present in such honeys. We now report the discovery, using ion trap LC-MS, of two tutin glycosides and their purification and structure determination as 2-(β-d-glucopyranosyl)tutin (4) and 2-[6'-(α-d-glucopyranosyl)-β-d-glucopyranosyl]tutin (5). These compounds were used to develop a quantitative triple quadrupole LC-MS method for honey analysis, which showed the presence of tutin (3.6 ± 0.1 μg/g honey), hyenanchin (19.3 ± 0.5), tutin glycoside (4) (4.9 ± 0.4), and tutin diglycoside (5) (4.9 ± 0.1) in one toxic honey. The ratios of 4 and 5 to tutin varied widely in other tutin-containing honeys. The glycosidation of tutin may represent detoxification by one or both of the insects involved in the food chain from plant to honey.

A Geant4 Monte Carlo simulation of the X-ray fluorescence microprobe (XFM) end-station at the Australian Synchrotron has been developed. The simulation is required for optimization of the scan configuration and reconstruction algorithms. As part of the simulation process, a Gaussian beam model was developed. Experimental validation of this simulation has tested the efficacy for use of the low-energy physics models in Geant4 for this synchrotron-based technique. The observed spectral distributions calculated in the 384 pixel Maia detector, positioned in the standard back-scatter configuration, were compared with those obtained from experiments performed at three incident X-ray beam energies: 18.5, 11.0 and 6.8 keV. The reduced χ-squared (\chi^{2}&#95;{\rm{red}}) was calculated for the scatter and fluorescence regions of the spectra and demonstrates that the simulations successfully reproduce the scatter distributions. Discrepancies were shown to occur in the multiple-scatter tail of the Compton continuum. The model was shown to be particularly sensitive to the impurities present in the beryllium window of the Maia detector and their concentrations were optimized to improve the \chi^{2}&#95;{\rm{red}} parametrization in the low-energy fluorescence regions of the spectra.

We have identified a range of food phytochemicals that inhibit Janus Kinase 2 (JAK2) and Adenosine Monophosphate Kinase (AMPK). A mutated and dysregulated form of JAK2, a tyrosine kinase, is associated with several diseases including Crohn's disease. Using an in vitro, time-resolved fluorescence (TR-FRET) assay, we tested 49 different types of food extracts, plus 10 concentrated fractions of increasing hydrophobicity from each extract, to find foods containing JAK2 inhibitors. The food extracts tested included grains, meat, fish, shellfish, dairy products, herbs, mushrooms, hops, fruits and vegetables. Several fruits were potent inhibitors of JAK2: blackberry, boysenberry, feijoa, pomegranate, rosehip and strawberry, which all contain ellagitannins, known inhibitors of kinases. These fruits are in the Rosales and Myrtales plant orders. No other foods gave >1% of the maximal JAK2 inhibitory activities of these fruits. AMPK, a sensor and regulator of energy metabolism in cells, is a serine-threonine kinase which is reported to be activated by various flavonoid phytochemicals. Using a TR-FRET assay, we tested various fruit extracts for AMPK activation and inhibition. Ellagitannin containing foods scored highly as AMPK inhibitors. Despite several reports of AMPK activation in whole cells by phytochemicals, no extracts or pure compounds activated AMPK in our assay.

Glycosides are an important potential source of aroma and flavour compounds for release as volatiles in flowers and fruit. The production of glycosides is catalysed by UDP-glycosyltransferases (UGTs) that mediate the transfer of an activated nucleotide sugar to acceptor aglycones. A screen of UGTs expressed in kiwifruit (Actinidia deliciosa) identified the gene AdGT4 which was highly expressed in floral tissues and whose expression increased during fruit ripening. Recombinant AdGT4 enzyme glycosylated a range of terpenes and primary alcohols found as glycosides in ripe kiwifruit. Two of the enzyme's preferred alcohol aglycones, hexanol and (Z)-hex-3-enol, contribute strongly to the 'grassy-green' aroma notes of ripe kiwifruit and other fruit including tomato and olive. Transient over-expression of AdGT4 in tobacco leaves showed that enzyme was able to glycosylate geraniol and octan-3-ol in planta whilst transient expression of an RNAi construct in Actinidia eriantha fruit reduced accumulation of a range of terpene glycosides. Stable over-expression of AdGT4 in transgenic petunia resulted in increased sequestration of hexanol and other alcohols in the flowers. Transgenic tomato fruit stably over-expressing AdGT4 showed changes in both the sequestration and release of a range of alcohols including 3-methylbutanol, hexanol and geraniol. Sequestration occurred at all stages of fruit ripening. Ripe fruit sequestering high levels of glycosides were identified as having a less intense, earthier aroma in a sensory trial. These results demonstrate the importance of UGTs in sequestering key volatile compounds in planta and suggest a future approach to enhancing aromas and flavours in flowers and during fruit ripening., (© 2014 The Authors The Plant Journal © 2014 John Wiley & Sons Ltd.)

Phloridzin is the predominant polyphenol in apple (Malus × domestica Borkh.) where it accumulates to high concentrations in many tissues including the leaves, bark, roots and fruit. Despite its relative abundance in apple the biosynthesis of phloridzin and other related dihydrochalcones remains only partially understood. The key unidentified enzyme in phloridzin biosynthesis is a putative carbon double bond reductase which is thought to act on p-coumaroyl-CoA to produce the dihydro-p-coumaroyl-CoA precursor. A functional screen of six apple enoyl reductase-like (ENRL) genes was carried out using transient infiltration into tobacco and gene silencing by RNA interference (RNAi) in order to determine carbon double bond reductase activity and contribution to foliar phloridzin concentrations. The ENRL-3 gene caused a significant increase in phloridzin concentration when infiltrated into tobacco leaves whilst a second protein ENRL-5, with over 98% amino acid sequence similarity to ENRL-3, showed p-coumaroyl-CoA reductase activity in enzyme assays. Finally, an RNAi study showed that reducing the transcript levels of ENRL-3 in transgenic 'Royal Gala' led to a 66% decrease in the concentration of dihydrochalcones in the leaves in the one available silenced line. Overall these results suggest that ENRL-3, and its close homolog ENRL-5, may contribute to the biosynthesis of phloridzin in apple., (Copyright © 2013 Elsevier Masson SAS. All rights reserved.)

Animal models are an important tool to understand the complex pathogenesis of inflammatory bowel diseases (IBDs). This study tested the anti-inflammatory potential of a green tea extract rich in polyphenols (GrTP) in the colon of the multidrug resistance targeted mutation (Mdr1a(-/-)) mouse model of IBD. Insights into mechanisms responsible for this reduction in inflammation were gained using transcriptome and proteome analyses. Mice were randomly assigned to an AIN-76A (control) or GrTP-enriched diet. At 21 or 24 weeks of age, a colonic histological injury score was determined for each mouse, colon mRNA transcript levels were assessed using microarrays, and colon protein expression was measured using two-dimensional gel electrophoresis and liquid chromatography-mass spectrometry protein identification. Mean colonic histological injury score of GrTP-fed Mdr1a(-/-) mice was significantly lower compared to those fed the control diet. Microarray and proteomics analyses showed reduced abundance of transcripts and proteins associated with immune and inflammatory response and fibrinogenesis pathways, and increased abundance of those associated with xenobiotic metabolism pathways in response to GrTP, suggesting that its anti-inflammatory activity is mediated by multiple molecular pathways. Peroxisome proliferator-activated receptor-α and signal transducer and activator of transcription 1 appear to be two key molecules which regulate these effects. These results support the view that dietary intake of polyphenols derived from green tea can ameliorate intestinal inflammation in the colon of a mouse model of IBD, and are in agreement with studies suggesting that consumption of green tea may reduce IBD symptoms and therefore play a part in an overall IBD treatment regimen., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Three triterpene-caffeates have been isolated from skins of a russeted apple cultivar "Merton Russet" and identified by LC-MS and NMR as betulinic acid-3-cis-caffeate, betulinic acid-3-trans-caffeate, and oleanolic acid-3-trans-caffeate. Betulinic acid-3-trans-caffeate and oleanolic acid-3-trans-caffeate were also found in russeted pear skins. These compounds have not been previously reported in apples or pears, or in any other foods. Their presence was related to suberized tissue as they were only found in russet portions of the partially russeted apple cultivar "Cox's Orange Pippin" and were not detected in the waxy apple cultivar "Royal Gala". High concentrations of betulinic acid-3-trans-caffeate were found in the bark of both "Merton Russet" and "Royal Gala" trees. The three triterpene-caffeates showed anti-inflammatory activity in vitro, inhibiting NF-κB activation with IC50's of 6-9 μM. Betulinic acid-3-trans-caffeate, the predominant compound in the apples, was immuno-modulatory at around 10 μM in the in vitro and ex vivo bioassays, boosting production of the pro-inflammatory cytokine TNFα in cells stimulated with bacterial lipopolysaccharides.

Background: Consumption of high-fat diets has negative impacts on health and well-being, some of which may be epigenetically regulated. Selenium and folate are two compounds which influence epigenetic mechanisms. We investigated the hypothesis that post-weaning supplementation with adequate levels of selenium and folate in offspring of female mice fed a high-fat, low selenium and folate diet during gestation and lactation will lead to epigenetic changes of potential importance for long-term health., Methods: Female offspring of mothers fed the experimental diet were either maintained on this diet (HF-low-low), or weaned onto a high-fat diet with sufficient levels of selenium and folate (HF-low-suf), for 8 weeks. Gene and protein expression, DNA methylation, and histone modifications were measured in colon and liver of female offspring., Results: Adequate levels of selenium and folate post-weaning affected gene expression in colon and liver of offspring, including decreasing Slc2a4 gene expression. Protein expression was only altered in the liver. There was no effect of adequate levels of selenium and folate on global histone modifications in the liver. Global liver DNA methylation was decreased in mice switched to adequate levels of selenium and folate, but there was no effect on methylation of specific CpG sites within the Slc2a4 gene in liver., Conclusions: Post-weaning supplementation with adequate levels of selenium and folate in female offspring of mice fed high-fat diets inadequate in selenium and folate during gestation and lactation can alter global DNA methylation in liver. This may be one factor through which the negative effects of a poor diet during early life can be ameliorated. Further research is required to establish what role epigenetic changes play in mediating observed changes in gene and protein expression, and the relevance of these changes to health.

In the last 30 years the incidence of kiwifruit allergy has increased with the three major allergenic proteins being identified as actinidin, kiwellin, and thaumatin-like protein (TLP). We report wide variation in the levels of actinidin and TLP in 15 kiwifruit varieties from the four most widely cultivated Actinidia species. Acidic and basic isoforms of actinidin were identified in Actinidia deliciosa 'Hayward' and Actinidia arguta 'Hortgem Tahi', while only a basic isoform of actinidin was identified in Actinidia chinensis 'Hort16A'. One isoform each of kiwellin and TLP were identified in ripe fruit. The cysteine protease activity of actinidin correlated with protein levels in all species except A. arguta. Protein modeling suggested that modifications to the S2 binding pocket influenced substrate specificity of the A. arguta enzyme. Our results indicate that care is necessary when extrapolating allergenicity results from single varieties to others within the same and between different Actinidia species.

Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.

Strigolactones are a recently discovered class of plant hormone involved in branching, leaf senescence, root development, and plant-microbe interactions. They are carotenoid-derived lactones, synthesized in the roots and transported acropetally to modulate axillary bud outgrowth (i.e., branching). However, a receptor for strigolactones has not been identified. We have identified the DAD2 gene from petunia, an ortholog of the rice and Arabidopsis D14 genes, and present evidence for its roles in strigolactone perception and signaling. DAD2 acts in the strigolactone pathway, and the dad2 mutant is insensitive to the strigolactone analog GR24. The crystal structure of DAD2 reveals an α/β hydrolase fold containing a canonical catalytic triad with a large internal cavity capable of accommodating strigolactones. In the presence of GR24 DAD2 interacts with PhMAX2A, a central component of strigolactone signaling, in a GR24 concentration-dependent manner. DAD2 can hydrolyze GR24, with mutants of the catalytic triad abolishing both this activity and the ability of DAD2 to interact with PhMAX2A. The hydrolysis products can neither stimulate the protein-protein interaction nor modulate branching. These observations suggest that DAD2 acts to bind the mobile strigolactone signal and then interacts with PhMAX2A during catalysis to initiate an SCF-mediated signal transduction pathway., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Inflammatory bowel disease (IBD) is a collective term for conditions characterised by chronic inflammation of the gastrointestinal tract involving an inappropriate immune response to commensal micro-organisms in a genetically susceptible host. Previously, aqueous and ethyl acetate extracts of gold kiwifruit (Actinidia chinensis) or green kiwifruit (A. deliciosa) have demonstrated anti-inflammatory activity using in vitro models of IBD. The present study examined whether these kiwifruit extracts (KFE) had immune-modulating effects in vivo against inflammatory processes that are known to be increased in patients with IBD. KFE were used as a dietary intervention in IL-10-gene-deficient (Il10(-/-)) mice (an in vivo model of IBD) and the C57BL/6J background strain in a 3 × 2 factorial design. While all Il10(-/-) mice developed significant colonic inflammation compared with C57BL/6J mice, this was not affected by the inclusion of KFE in the diet. These findings are in direct contrast to our previous study where KFE reduced inflammatory signalling in primary cells isolated from Il10(-/-) and C57BL/6J mice. Whole-genome gene and protein expression level profiling indicated that KFE influenced immune signalling pathways and metabolic processes within the colonic tissue; however, the effects were subtle. In particular, expression levels across gene sets related to adaptive immune pathways were significantly reduced using three of the four KFE in C57BL/6J mice. The present study highlights the importance of investigating food components identified by cell-based assays with appropriate in vivo models before making dietary recommendations, as a food that looks promising in vitro may not be effective in vivo.

Inflammatory bowel disease (IBD) is characterized by intestinal inflammation and is believed to involve complex interactions between genetic, immunological, and environmental factors. We measured changes in the proteome associated with bacterially induced intestinal inflammation in the interleukin 10 gene-deficient (Il10(-/-)) mouse model of IBD, established effects of the dietary polyunsaturated fatty acids (PUFAs) n-3 eicosapentaenoic acid (EPA) and n-6 arachidonic acid (AA) on protein expression (using oleic acid as a control fatty acid), and compared these changes with previously observed transcriptome changes in the same model. Ingenuity pathways analysis of proteomics data showed bacterially induced inflammation was associated with reduced expression of proteins from pathways of metabolism and digestion/absorption/excretion of nutrients/ions, and increased expression of cellular stress and immune response proteins. Both PUFA treatments showed anti-inflammatory activity; EPA appeared to act via the PPARα pathway, whereas AA appeared to increase energy metabolism and cytoskeletal organization and reduce cellular stress responses, possibly enabling a more robust response to inflammation. While there was agreement between proteomic and transcriptomic data with respect to pathways, there was limited concordance between individual gene and protein data, reflecting the importance of having both gene and protein data to better understand complex diseases such as IBD.

Cysteine proteases (CPs) accumulate to high concentration in many fruit, where they are believed to play a role in fungal and insect defense. The fruit of Actinidia species (kiwifruit) exhibit a range of CP activities (e.g. the Actinidia chinensis variety YellowA shows less than 2% of the activity of Actinidia deliciosa variety Hayward). A major quantitative trait locus for CP activity was mapped to linkage group 16 in a segregating population of A. chinensis. This quantitative trait locus colocated with the gene encoding actinidin, the major acidic CP in ripe Hayward fruit encoded by the ACT1A-1 allele. Sequence analysis indicated that the ACT1A locus in the segregating A. chinensis population contained one functional allele (A-2) and three nonfunctional alleles (a-3, a-4, and a-5) each containing a unique frameshift mutation. YellowA kiwifruit contained two further alleles: a-6, which was nonfunctional because of a large insertion, and a-7, which produced an inactive enzyme. Site-directed mutagenesis of the act1a-7 protein revealed a residue that restored CP activity. Expression of the functional ACT1A-1 cDNA in transgenic plants complemented the natural YellowA mutations and partially restored CP activity in fruit. Two consequences of the increase in CP activity were enhanced degradation of gelatin-based jellies in vitro and an increase in the processing of a class IV chitinase in planta. These results provide new insight into key residues required for CP activity and the in vivo protein targets of actinidin.

Background: Some apple (Malus × domestica Borkh.) varieties have attractive striping patterns, a quality attribute that is important for determining apple fruit market acceptance. Most apple cultivars (e.g. 'Royal Gala') produce fruit with a defined fruit pigment pattern, but in the case of 'Honeycrisp' apple, trees can produce fruits of two different kinds: striped and blushed. The causes of this phenomenon are unknown., Results: Here we show that striped areas of 'Honeycrisp' and 'Royal Gala' are due to sectorial increases in anthocyanin concentration. Transcript levels of the major biosynthetic genes and MYB10, a transcription factor that upregulates apple anthocyanin production, correlated with increased anthocyanin concentration in stripes. However, nucleotide changes in the promoter and coding sequence of MYB10 do not correlate with skin pattern in 'Honeycrisp' and other cultivars differing in peel pigmentation patterns. A survey of methylation levels throughout the coding region of MYB10 and a 2.5 Kb region 5' of the ATG translation start site indicated that an area 900 bp long, starting 1400 bp upstream of the translation start site, is highly methylated. Cytosine methylation was present in all three contexts, with higher methylation levels observed for CHH and CHG (where H is A, C or T) than for CG. Comparisons of methylation levels of the MYB10 promoter in 'Honeycrisp' red and green stripes indicated that they correlate with peel phenotypes, with an enrichment of methylation observed in green stripes., Conclusions: Differences in anthocyanin levels between red and green stripes can be explained by differential transcript accumulation of MYB10. Different levels of MYB10 transcript in red versus green stripes are inversely associated with methylation levels in the promoter region. Although observed methylation differences are modest, trends are consistent across years and differences are statistically significant. Methylation may be associated with the presence of a TRIM retrotransposon within the promoter region, but the presence of the TRIM element alone cannot explain the phenotypic variability observed in 'Honeycrisp'. We suggest that methylation in the MYB10 promoter is more variable in 'Honeycrisp' than in 'Royal Gala', leading to more variable color patterns in the peel of this cultivar.

Epidemiological studies reveal that fruit consumption reduces the prevalence of airway inflammation and childhood asthma. In particular, blackcurrant polyphenolic extracts have been shown to alleviate lung inflammation. Since IL-4-stimulated eotaxin-3 (CCL26) secretion is a major factor in the continuous eosinophil recruitment observed in atopic asthma, our focus was to evaluate the effectiveness of blackcurrant polyphenolic compounds on CCL26 secretion in human alveolar epithelial cells. Our results indicate that a proanthocyanin-enriched blackcurrant extract (BC-P), but not anthocyanin-enriched blackcurrant extract suppressed both IL-4- and IL-13-stimulated CCL26 secretion in a dose-dependent manner. Furthermore pre-incubation of cells with BC-P caused a time-dependent suppression of IL-4-stimulated CCL26 secretion. Moreover, epigallocatechin (EGC), and to a lesser extent epicatechin, metabolites identified in the proanthocyanidin extract, suppressed IL-4-stimulated CCL26 secretion. EGC was also effective at reducing the cellular phosphorylated STAT-6/STAT-6 ratio. Furthermore, both BC-P and purified EGC potentiated the ability of IFN-gamma to suppress IL-4-stimulated CCL26 secretion. The progression of an allergic immune response is complex, identifying plant compounds that target specific cellular events and complement the body's own immune actions is important for the development of functional foods. Our findings support the potential for blackcurrant polyphenolic compounds to reduce eosinophil recruitment and alleviate eosinophilic-driven airway inflammation.

Skeletal muscle damage can result from disease and unaccustomed or excessive exercise. Muscle dysfunction occurs via an increased level of reactive oxygen species and hence there is potential in antioxidants as amelioration strategies. We explored the putative benefit of fruit polyphenolic extracts in reducing the susceptibility of skeletal muscle cells to oxidative stress. Muscle myotubes were simultaneously challenged with fruit extracts (1-50 microg/mL) and calcium ionophore (A23187), hydrogen peroxide, or 2,4-dinitrophenol and damage monitored by release of cytosolic enzymes. A blueberry fruit extract displayed a potent and significant dose-dependent protective capacity. Evaluation of the protective capacity of anthocyanin sub-extracts of blueberry fruit and pure individual glycosides, with identification of extract polyphenolic components using MS, suggested that malvidin galactoside and/or glucoside were the active compounds. These in vitro data support the concept that blueberry fruits or derived foods rich in malvidin glycosides may be beneficial in alleviating muscle damage caused by oxidative stress. More research on the benefits of blueberry fruit consumption in human intervention studies is warranted.

Apple extract powders from three different manufacturers were investigated for their anti-inflammatory activity, their total phenolic content, and their chemical composition. The samples represented two production batches for two products and a single batch of a third. The samples showed similar, but clearly different, anti-inflammatory activities, and had substantially different total phenolic contents, and different chemical compositions. Differences in chemical composition for batches of the same product were significant, although not as great as differences between products. The samples were fractionated into chemical classes. The most active fractions were those that contained epicatechin, catechin with phloridzin and quercetin glycosides, or those that contained procyanidin polymers. It was not possible to link activity to the presence of individual components or combinations of these. If fruit extracts are to be reliably linked to validated health benefits, then the source materials, the extraction processes, and the final composition of such products need to be more clearly defined than at present.

* High-temperature, low-light (HTLL) treatment of 35S:PAP1 Arabidopsis thaliana over-expressing the PAP1 (Production of Anthocyanin Pigment 1) gene results in reversible reduction of red colouration, suggesting the action of additional anthocyanin regulators. High-performance liquid chromatography (HPLC), liquid chromatography mass spectrometry (LCMS) and Affimetrix-based microarrays were used to measure changes in anthocyanin, flavonoids, and gene expression in response to HTLL. * HTLL treatment of control and 35S:PAP1 A. thaliana resulted in a reversible reduction in the concentrations of major anthocyanins despite ongoing over-expression of the PAP1 MYB transcription factor. Twenty-one anthocyanins including eight cis-coumaryl esters were identified by LCMS. The concentrations of nine anthocyanins were reduced and those of three were increased, consistent with a sequential process of anthocyanin degradation. Analysis of gene expression showed down-regulation of flavonol and anthocyanin biosynthesis and of transport-related genes within 24 h of HTLL treatment. No catabolic genes up-regulated by HTLL were found. * Reductions in the concentrations of anthocyanins and down-regulation of the genes of anthocyanin biosynthesis were achieved by environmental manipulation, despite ongoing over-expression of PAP1. Quantitative PCR showed reduced expression of three genes (TT8, TTG1 and EGL3) of the PAP1 transcriptional complex, and increased expression of the potential transcriptional repressors AtMYB3, AtMYB6 and AtMYBL2 coincided with HTLL-induced down-regulation of anthocyanin biosynthesis. * HTLL treatment offers a model system with which to explore anthocyanin catabolism and to discover novel genes involved in the environmental control of anthocyanins.

Three steroidal saponins, including one new and two known compounds, were isolated from the rhizomes of Paris polyphylla Smith. One- and two-dimensional NMR, LC-MS, and interpretation of hydrolytic cleavage experiments led to the identification of the structure of the new saponin as ( 25R)-spirost-5-ene-3 beta,17 alpha-diol (pennogenin) 3- O-{ O- alpha- L-rhamnopyranosyl-(1-->2)- O-[ O- beta-xylopyranosyl-(1-->5)- alpha- L-arabinofuranosyl-(1-->4)]- beta- D-glucopyranoside}. The isolated saponins were evaluated for their antifungal activity against Cladosporium cladosporioides and Candida species and showed comparable activity to chemicals used in some commercial products.

The dihydrochalcone phlorizin (phloretin 2'-glucoside) contributes to the flavor, color and health benefits of apple fruit and processed products. A genomics approach was used to identify the gene MdPGT1 in apple (Malus x domestica) with homology to the UDP-glycosyltransferase 88 family of uridine diphosphate glycosyltransferases that show specificity towards flavonoid substrates. Expressed sequence tags for MdPGT1 were found in all tissues known to produce phlorizin including leaf, flower and fruit. However, the highest expression was measured by quantitative PCR in apple root tissue. The recombinant MdPGT1 enzyme expressed in Escherichia coli glycosylated phloretin in the presence of [(3)H]-UDP-glucose, but not other apple antioxidants, including quercetin, naringenin and cyanidin. The product of phloretin and UDP-glucose co-migrated with an authentic phlorizin standard. LC/MS indicated that MdPGT1 could glycosylate phloretin in the presence of three sugar donors: UDP-glucose, UDP-xylose and UDP-galactose. This is the first report of functional characterization of a UDP-glycosyltransferase that utilizes a dihydrochalcone as its primary substrate.

The resorcylic acid lactones zearalenone ( 1), alpha-zearalenol ( 2), beta-zearalenol ( 3), alpha-zearalanol (zeranol) ( 4), beta-zearalanol (taleranol) ( 5), and zearalanone ( 6) were converted to their glucuronides on a preparative scale in good yields. Reactions were conducted with bovine uridine 5'-diphosphoglucuronyl transferase (UDPGT) as catalyst and uridine 5'-diphosphoglucuronic acid (UDPGA) as cofactor. The glucuronides were isolated by column chromatography and characterized by NMR spectroscopy and mass spectrometry. Although the principal products were 4- O-glucuronides (i.e., linkage through a phenolic hydroxyl), significant quantities of the 6'- O-glucuronides (i.e., linkage through the aliphatic hydroxyl) of alcohols 2, 4, and 5 were also isolated. In the case of 3, the 2- O-glucuronide was isolated as the minor product. Overall isolated yields of glucuronides, performed on a 20-50 mg scale, were typically ca. 80% based on the resorcylic acid lactone starting material. LC-UV-MS (2) analysis of purified specimens revealed MS (2) fragmentations useful for defining the point of attachment of the glucuronide moiety to the zearalenone nucleus.

Brevetoxins are neurotoxins associated with blooms of marine algae such as Karenia brevis and can accumulate in the marine food chain, causing intoxication of marine animals and people consuming seafood. Brevetoxin-B2 ( 5) is a toxic metabolite produced in shellfish exposed to algae that contain brevetoxin-B ( 1). S-Desoxybrevetoxin-B2 ( 4) has been proposed as a cometabolite produced during this transformation, and while LC-MS analyses suggest its presence in shellfish, it has not yet been isolated and characterized. Studies on these materials are severely constrained by the difficulty of obtaining and purifying them from natural sources. We have developed a convenient one-pot conversion of commercially available brevetoxin-B ( 1) into S-desoxybrevetoxin-B2 ( 4), and a simple method for converting 4 into brevetoxin-B2 ( 5). Full NMR and mass-spectral characterization of 4 and 5 confirmed their structures and showed that the ratio of diastereoisomers in the synthetic 4 and 5 was similar to that observed in naturally contaminated shellfish. The LD 50 values for 4, 5, and dihydrobrevetoxin-B ( 6) by ip injection in mice were 211, 400, and 250 microg/kg, respectively. The methodology for synthesis of brevetoxin metabolites should greatly facilitate toxicological, biochemical and immunochemical studies of these substances, as well as the production of analytical standards.

This study modeled, in vitro, the potential effect of conjugative (phase II) metabolism on the cytoprotective capacity of fruit flavonoids against oxidative stress. Flavonoid aglycones were compared with their corresponding isomeric mixtures of glucuronides for their ability to enhance the survival of cultured human Jurkat T and neuroblastoma cells stressed with hydrogen peroxide. Various polyphenolic compounds were tested as substrates in vitro for an ovine liver glucuronyl transferase preparation. Flavonoids and their glycoside derivatives were found to be good substrates, whereas phenolic acids were either poor or nonsubstrates. Five common flavonoids were glucuronidated to prepare mixtures for bioassay testing. Glucuronidation generally weakened the cytoprotective capacities of flavonoids (in the presence of H(2)O(2)), but some compounds were weakened much more than others. The concentration that halved cell death was well below 0.5 microM for most flavonoids tested, but glucuronidation increased median effective concentration values to a range of 1-16 microM. This compares with the generally accepted physiological range (0.1-10 microM) for circulating dietary polyphenolics detected in the body. Therefore, some flavonoids may retain a reduced cytoprotective capacity in vitro, after glucuronidation, whereas others may be effectively inactivated.

Yessotoxins from a large-scale culture (226 L) of Protoceratium reticulatum strain CAWD129 were harvested by filtration followed by solid-phase extraction. The extract was purified by column chromatography over basic alumina and reverse-phase flash chromatography to afford pure yessotoxin (193 mg). Isolation of yessotoxin was greatly facilitated by selection of a strain which did not produce analogues that interfered with yessotoxin isolation. In addition to yessotoxin, numerous minor yessotoxins were detected by LC-MS in other fractions. From one of these, an early eluting minor analogue with the same molecular weight as yessotoxin and a similar mass spectrometric fragmentation pattern was isolated. This analogue was identified by NMR and mass spectrometry as a novel yessotoxin analogue containing a furan ring in the side chain. This finding reveals biosynthetic flexibility of the yessotoxin pathway in P. reticulatum and confirms earlier findings of production of many minor yessotoxin analogues by this alga. Production of these analogues appeared to be a constitutive trait of P. reticulatum CAWD129.

Actinidin is a cysteine protease found in Actinidia Lindl. (kiwifruit) species that affects the nutraceutical properties, processing characteristics and allergenicity of the fruit. Given the increased consumption of kiwifruit worldwide and the release of new varieties from different Actinidia species, the expression of actinidin mRNA and protein in a range of kiwifruit tissues was examined. Ten different actinidin mRNAs were identified encoding mature proteins of similar molecular weight (~24 kDa), but with predicted pIs ranging from acidic (pI 3.9) to basic (pI 9.3). In A. deliciosa 'Hayward' (green-fleshed kiwifruit) and A. chinensis 'Hort16A' and EM4 (gold-fleshed kiwifruit), actinidin mRNAs for acidic and basic proteins were expressed at comparable levels throughout ripening. Actinidin mRNA expression was highest in fruit at harvest, expression decreased as fruit ripened and was much lower in the core compared with outer pericarp tissue. Two-dimensional gel electrophoresis, combined with western analysis and liquid chromatography mass spectrometry (LC-MS) identified low levels of a novel basic actinidin protein in ripe A. deliciosa and A. chinensis fruit. Extremely high levels of an acidic actinidin protein were detected in A. deliciosa fruit and EM4, but this acidic protein appeared to be absent in 'Hort16A', the most important commercial cultivar of A. chinensis. Analyses on native gels indicated that both the basic and acidic actinidin isoforms in A. deliciosa were active cysteine proteases. Immunolocalisation showed that actinidin was present in small cells, but not large cells in the outer pericarp of mature A. deliciosa fruit at harvest. Within the small cells, actinidin was localised diffusely in the vacuole, associated with the plasma membrane, and in a layer in the plastids near starch granules. The presence of multiple forms of actinidin and varying protein levels in fruit will impact on the ability to breed new kiwifruit varieties with altered actinidin levels.

To simulate the effects of digestion and metabolism on the survival of different polyphenolic compounds, extracts of blueberry and apple were deglycosylated by acid hydrolysis, followed by enzymic glucuronidation under neutral conditions, yielding approximately 5% overall recovery of polyphenolics. The major polyphenolics before and after the treatment were compared, to estimate which species are likely to be present in the intestinal lumen, undegraded and available for absorption, after consumption of the fruit. Whereas blueberry extract consisted predominantly of anthocyanins, epicatechin and caffeoyl quinate esters, the major components of the treated extract were quercetin glucuronides and (unglucuronidated) caffeoyl quinates, with only traces of anthocyanidin derivatives. In apple extract, compositional changes were less marked, but caffeoyl quinates, procyanidins and quercetin were enriched at the expense of caffeic acid, epicatechin and catechin. Hydrophobic compounds like phloretin and quercetin were extensively glucuronidated, whereas caffeic acid and caffeoyl quinate were not. These results suggest that the major polyphenolic components of a fruit are not necessarily the most important contributors to any health benefits because the polyphenolic composition in the intestinal lumen and consequently, in the circulation, may be considerably different.

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

Desacetyl alpha-MSH predominates over alpha-MSH during development, but whether it is biologically active and has a physiological role is unclear. We compared the effects of 0.3 microg.g(-1).day(-1) desacetyl alpha-MSH with that of 0.3 microg.g(-1).day(-1) alpha-MSH on postnatal body growth by administering the peptides subcutaneously daily for postnatal days 0-14 and also used a two-dimensional gel electrophoresis gel-based proteomic approach to analyze protein changes in hypothalami, the relay center for body weight and growth regulation, after 14 days of treatment. We found that the growth rate between days 1 and 10 was significantly decreased by desacetyl alpha-MSH but not by alpha-MSH, but by day 14, a time reported for development of a mature pattern of hypothalamic innervation, both peptides had significantly increased neonatal growth compared with PBS-treated control rats. Desacetyl alpha-MSH significantly increased spleen weight, but alpha-MSH had no effect. alpha-MSH significantly decreased kidney weight, but desacetyl alpha-MSH had no effect. Both desacetyl alpha-MSH and alpha-MSH significantly decreased brain weight. By 14 days, both peptides significantly changed expression of a number of hypothalamic proteins, specifically metabolic enzymes, cytoskeleton, signaling, and stress response proteins. We show that peripherally administered desacetyl alpha-MSH is biologically active and induces responses that can differ from those for alpha-MSH. In conclusion, desacetyl alpha-MSH appears to be an important regulator of neonatal rat growth.

Several polyphenolic compounds, including flavonoids and phenolic acids, were compared with their per-methylated forms in both chemical and cell-based assays for antioxidant capacity. Methylation largely eliminated "chemical" antioxidant capacity, according to ferric reducing antioxidant power and oxygen radical absorbance capacity assays. Methylation, however, only moderately reduced protection of human Jurkat cells in culture, from hydrogen peroxide-mediated cytotoxicity, at physiologically relevant concentrations. Neither methylated nor un-methylated compounds were detectably metabolized by the cells. It appears that the protective mechanism of polyphenolic antioxidants against high concentrations of hydrogen peroxide in human cells may be largely unrelated to chemical antioxidant capacity.

Two novel pectenotoxins (PTXs), PTX-13 and -14, were isolated from extracts of Dinophysis acuta collected from the west coast of South Island, New Zealand. The compounds were identified as oxidized analogues of PTX-2 by NMR spectroscopic and LC-MS studies. PTX-13 (32R-hydroxyPTX-2) corresponds to the unidentified analogue PTX-11x reported by [Suzuki et al., 2003. Liquid chromatography-mass spectrometry of spiroketal stereoisomers of pectenotoxins and the analysis of novel pectenotoxin isomers in the toxic dinoflagellate Dinophysis acuta from New Zealand. J. Chromatogr. A 992, 141-150]. PTX-13 underwent slow deuteration at the 13beta-position during NMR analysis. PTX-14 corresponds to the 32,36-dehydration product of PTX-13, and may be an artifact.