Effects of wheat bran extract containing arabinoxylan oligosaccharides on gastrointestinal parameters in healthy preadolescent children.

OBJECTIVES: We assessed whether wheat bran extract (WBE) containing arabinoxylan-oligosaccharides (AXOS) elicited a prebiotic effect and modulated gastrointestinal (GI) parameters in healthy preadolescent children upon consumption in a beverage. METHODS: This double-blind randomized placebo-controlled crossover trial evaluated the effects of consuming WBE at 0 (control) or 5.0 g/day for 3 weeks in 29 healthy children (8-12 years). Fecal levels of microbiota, short-chain fatty acids, branched-chain fatty acids, ammonia, moisture, and fecal pH were assessed at the end of each treatment and at the end of a 1-week run-in (RI) period. In addition, the subjects completed questionnaires scoring distress severity of 3 surveyed GI symptoms. Finally, subjects recorded defecation frequency and stool consistency. RESULTS: Nominal fecal bifidobacteria levels tended to increase after 5 g/day WBE consumption (P = 0.069), whereas bifidobacteria expressed as percentage of total fecal microbiota was significantly higher upon 5 g/day WBE intake (P = 0.002). Additionally, 5 g/day WBE intake induced a significant decrease in fecal content of isobutyric acid and isovaleric acid (P < 0.01), markers of protein fermentation. WBE intake did not cause a change in distress severity of the 3 surveyed GI symptoms (flatulence, abdominal pain/cramps, and urge to vomit) (P > 0.1). CONCLUSIONS: WBE is well tolerated at doses up to 5 g/day in healthy preadolescent children. In addition, the intake of 5 g/day exerts beneficial effects on gut parameters, in particular an increase in fecal bifidobacteria levels relative to total fecal microbiota, and reduction of colonic protein fermentation.

Effects of a wheat bran extract containing arabinoxylan oligosaccharides on gastrointestinal health parameters in healthy adult human volunteers: a double-blind, randomised, placebo-controlled, cross-over trial.

Wheat bran extract (WBE) is a food-grade soluble fibre preparation that is highly enriched in arabinoxylan oligosaccharides. In this placebo-controlled cross-over human intervention trial, tolerance and effects on colonic protein and carbohydrate fermentation were studied. After a 1-week run-in period, sixty-three healthy adult volunteers consumed 3, 10 and 0 g WBE/d for 3 weeks in a random order, with 2 weeks' washout between each treatment period. Fasting blood samples were collected at the end of the run-in period and at the end of each treatment period for analysis of haematological and clinical chemistry parameters. Additionally, subjects collected a stool sample for analysis of microbiota, SCFA and pH. A urine sample, collected over 48 h, was used for analysis of p-cresol and phenol content. Finally, the subjects completed questionnaires scoring occurrence frequency and distress severity of eighteen gastrointestinal symptoms. Urinary p-cresol excretion was significantly decreased after WBE consumption at 10 g/d. Faecal bifidobacteria levels were significantly increased after daily intake of 10 g WBE. Additionally, WBE intake at 10 g/d increased faecal SCFA concentrations and lowered faecal pH, indicating increased colonic fermentation of WBE into desired metabolites. At 10 g/d, WBE caused a mild increase in flatulence occurrence frequency and distress severity and a tendency for a mild decrease in constipation occurrence frequency. In conclusion, WBE is well tolerated at doses up to 10 g/d in healthy adults volunteers. Intake of 10 g WBE/d exerts beneficial effects on gut health parameters.

Novel fungicidal benzylsulfanyl-phenylguanidines.

A series of substituted benzylsulfanyl-phenylamines was synthesized, of which four substituted benzylsulfanyl-phenylguanidines (665, 666, 667 and 684) showed potent fungicidal activity (minimal fungicidal concentration, MFC ≤ 10 µM for Candida albicans and Candida glabrata). A benzylsulfanyl-phenyl scaffold with an unsubstituted guanidine resulted in less active compounds (MFC=50-100 µM), whereas substitution with an unsubstituted amine group resulted in compounds without fungicidal activity. Compounds 665, 666, 667 and 684 also showed activity against single C. albicans biofilms and biofilms consisting of C. albicans and Staphylococcus epidermidis (minimal concentration resulting in 50% eradication of the biofilm, BEC50 ≤ 121 µM for both biofilm setups). Compounds 665 and 666 combined potent fungicidal (MFC=5 µM) and bactericidal activity (minimal bactericidal concentration, MBC for S. epidermidis ≤ 4 µM). In an in vivo Caenorhabditis elegans model, compounds 665 and 667 exhibited less toxicity than 666 and 684. Moreover, addition of those compounds to Candida-infected C. elegans cultures resulted in increased survival of Candida-infected worms, demonstrating their in vivo efficacy in a mini-host model.

Membrane rafts are involved in intracellular miconazole accumulation in yeast cells.

Azoles inhibit ergosterol biosynthesis, resulting in ergosterol depletion and accumulation of toxic 14alpha-methylated sterols in membranes of susceptible yeast. We demonstrated previously that miconazole induces actin cytoskeleton stabilization in Saccharomyces cerevisiae prior to induction of reactive oxygen species, pointing to an ancillary mode of action. Using a genome-wide agar-based screening, we demonstrate in this study that S. cerevisiae mutants affected in sphingolipid and ergosterol biosynthesis, namely ipt1, sur1, skn1, and erg3 deletion mutants, are miconazole-resistant, suggesting an involvement of membrane rafts in its mode of action. This is supported by the antagonizing effect of membrane raft-disturbing compounds on miconazole antifungal activity as well as on miconazole-induced actin cytoskeleton stabilization and reactive oxygen species accumulation. These antagonizing effects point to a primary role for membrane rafts in miconazole antifungal activity. We further show that this primary role of membrane rafts in miconazole action consists of mediating intracellular accumulation of miconazole in yeast cells.

A fungicidal piperazine-1-carboxamidine induces mitochondrial fission-dependent apoptosis in yeast.

To unravel the working mechanism of the fungicidal piperazine-1-carboxamidine derivative BAR0329, we found that its intracellular accumulation in Saccharomyces cerevisiae is dependent on functional lipid rafts. Moreover, BAR0329 induced caspase-dependent apoptosis in yeast, in which the mitochondrial fission machinery consisting of Fis1 (Whi2), Dnm1 and Mdv1 is involved. Our data are consistent with a prosurvival function of Fis1 (Whi2) and a proapoptotic function of Dnm1 and Mdv1 during BAR0329-induced yeast cell death.

Arabinoxylan-oligosaccharides (AXOS) reduce preneoplastic lesions in the colon of rats treated with 1,2-dimethylhydrazine (DMH).

BACKGROUND: Prebiotics are non-digestible compounds that beneficially affect the host by stimulating the growth and/or activity of one or a limited number of resident colonic bacteria in the gut. Reported beneficial effects of prebiotics include reduced gut infections, better absorption of minerals, and notably, antitumorigenic effects. Arabinoxylan (AX)-oligosaccharides (AXOS) have been suggested to exert prebiotic effects in the gut, but their effect on colon carcinogenesis has not been studied so far. AIM OF THE STUDY: To test the effect of AXOS in a rat colon carcinogenesis model. METHODS: We determined the occurrence of two types of preneoplastic lesions [aberrant crypt foci (ACF) and mucin depleted foci (MDF)] in the colon of rats treated with the colon carcinogen 1,2-dimethylhydrazine (DMH) and fed either a control diet or a diet containing AXOS (4.8% w/w) (15 rats in each group). RESULTS: Thirteen weeks after DMH treatment, MDF counts were significantly lower in the entire colon of AXOS fed rats (MDF/colon were 7.5 +/- 0.6 and 5.5 +/- 0.6, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). Although the number of ACF in the entire colon was not significantly different between Control and AXOS fed rats, AXOS fed rats had significantly fewer ACF in the distal part of the colon than Control group rats (ACF/distal colon were 135.5 +/- 15 and 84.4 +/- 11, in Control and AXOS groups, respectively, means +/- SE, P < 0.05). CONCLUSIONS: The present study shows that dietary intake of AXOS by rats reduces the occurrence of two types of preneoplastic lesions, thus suggesting a chemopreventive effect on colon carcinogenesis that should be confirmed in a long-term carcinogenesis experiment.

Safety assessment of a wheat bran extract containing arabinoxylan-oligosaccharides: mutagenicity, clastogenicity, and 90-day rat-feeding studies.

Wheat bran extract (WBE) is a food-grade preparation that is highly enriched in arabinoxylan-oligosaccharides. As part of the safety evaluation of WBE, its genotoxic potential was assessed in a bacterial reverse mutagenicity assay (Ames test) and a chromosome aberration assay on Chinese hamster lung fibroblast cells. These in vitro genotoxicity assays showed no evidence of mutagenic or clastogenic activity with WBE. The safety of WBE was furthermore evaluated in a subchronic toxicity study on rats that were fed a semisynthetic diet (AIN 93G) containing 0.3%, 1.5%, or 7.5% WBE for 13 weeks, corresponding to an average intake of 0.2, 0.9, and 4.4 g/kg body weight (bw) per day, with control groups receiving the unsupplemented AIN 93G, AIN 93G with 7.5% inulin, or AIN 93G with 7.5% wheat bran. Based on this rat-feeding study, the no-observed-adverse-effect level (NOAEL) for WBE was determined as 4.4 g/kg (bw)/d, the highest dose tested.

Synthesis and fungicidal activity of 3,5-dichloropyrazin-2(1H)-one derivatives.

We synthesized a family of 3,5-dichloropyrazin-2(1H)-one derivatives and assessed their in vitro fungicidal activity against Candida albicans. Compounds 11 and 20 were most active against C. albicans and induced accumulation of reactive oxygen species in this pathogen. Using a genome-wide approach in the yeast Saccharomyces cerevisiae, we demonstrated that genes involved in vacuolar functionality and DNA-related functions play an important role in cellular mechanisms underlying the fungicidal activity of these compounds.

In vitro activity of the antifungal plant defensin RsAFP2 against Candida isolates and its in vivo efficacy in prophylactic murine models of candidiasis.

We show that RsAFP2, a plant defensin that interacts with fungal glucosylceramides, is active against Candida albicans, inhibits to a lesser extent other Candida species, and is nontoxic to mammalian cells. Moreover, glucosylceramide levels in Candida species correlate with RsAFP2 sensitivity. We found RsAFP2 prophylactically effective against murine candidiasis.

Fungicidal activity of truncated analogues of dihydrosphingosine.

The minimal fungicidal concentration (MFC) of dihydrosphingosine (DHS), phytosphingosine (PHS), and five short-chain DHS derivatives was determined for Candida albicans and Candida glabrata. In this respect, a C15- and a C17-homologue of DHS showed a 2- to 10-fold decreased MFC as compared to native DHS (i.e. C18-DHS). DHS derivatives that were active, that is, comprising 12, 15, 17, or 18 carbon atoms, induced accumulation of reactive oxygen species (ROS) in C. albicans.

Therapeutic potential of antifungal plant and insect defensins.

To defend themselves against invading fungal pathogens, plants and insects largely depend on the production of a wide array of antifungal molecules, including antimicrobial peptides such as defensins. Interestingly, plant and insect defensins display antimicrobial activity not only against plant and insect pathogens but also against human fungal pathogens, including Candida spp. and Aspergillus spp. This review focuses on these defensins as novel leads for antifungal therapeutics. Their mode of action, involving interaction with fungus-specific sphingolipids, and heterologous expression, required for cost-effective production, are major assets for development of plant and insect defensins as antifungal leads. Studies evaluating their in vivo antifungal efficacy demonstrate their therapeutic potential.

Level of M(IP)2C sphingolipid affects plant defensin sensitivity, oxidative stress resistance and chronological life-span in yeast.

The antifungal plant defensin DmAMP1 interacts with fungal sphingolipids of mannosyldiinositolphosphorylceramide (M(IP)2C) class. We screened a Saccharomyces cerevisiae transposon (Tn) mutant library against DmAMP1 and identified one DmAMP1-resistant mutant with the Tn inserted in the M(IP)2C biosynthesis gene IPT1 (DmTn11) and one DmAMP1-hypersensitive mutant with the Tn inserted in rDNA (HsTnII). However, tetrad analysis pointed to HsTnII as a spontaneous mutant. Apparently, membranes of DmTn11 lack M(IP)2C, whereas membranes of HsTnII have increased M(IP)2C levels. In addition, DmTn11 and HsTnII are characterized by increased and reduced oxidative stress resistance/chronological life-span (CL), respectively. A putative involvement of M(IP)2C in oxidative stress and CL in yeast is discussed.

Ceramide involvement in apoptosis and apoptotic diseases.

Apoptosis is implicated in a number of diseases, including neurodegenerative diseases and AIDS. More and more, evidence is accumulating pointing to the critical role of ceramides in the induction of apoptosis. The present review summarizes (i) the molecular basis and regulation of the apoptotic machinery, (ii) the molecular role of ceramides in the induction or execution of apoptotic pathways, and (iii) evidence linking ceramide generation to various apoptotic diseases. Additionally, this review discusses putative therapeutic approaches inhibiting ceramide production in apoptotic diseases.

SKN1, a novel plant defensin-sensitivity gene in Saccharomyces cerevisiae, is implicated in sphingolipid biosynthesis.

The antifungal plant defensin DmAMP1 interacts with the fungal sphingolipid mannosyl diinositolphosphoryl ceramide (M(IP)(2)C) and induces fungal growth inhibition. We have identified SKN1, besides the M(IP)(2)C-biosynthesis gene IPT1, as a novel DmAMP1-sensitivity gene in Saccharomyces cerevisiae. SKN1 was previously shown to be a KRE6 homologue, which is involved in beta-1,6-glucan biosynthesis. We demonstrate that a Deltaskn1 mutant lacks M(IP)(2)C. Interestingly, overexpression of either IPT1 or SKN1 complemented the skn1 mutation, conferred sensitivity to DmAMP1, and resulted in M(IP)(2)C levels comparable to the wild type. These results show that SKN1, together with IPT1, is involved in sphingolipid biosynthesis in S. cerevisiae.

Currently used antimycotics: spectrum, mode of action and resistance occurrence.

The increasing incidence of fungal infections combined with the emerging problem of antifungal drug resistance have prompted investigations of the mode of action of the currently used antifungal therapeutics (antimycotics). The routinely used antimycotics can be grouped into six different classes based on their mode of action. In this review, the mode of action and antifungal spectrum of these classes are discussed, together with possible resistance development against them.

Antifungal activity of synthetic peptides derived from Impatiens balsamina antimicrobial peptides Ib-AMP1 and Ib-AMP4.

Seeds of Impatiens balsamina contain a set of related antimicrobial peptides (Ib-AMPs). We have produced a synthetic variant of Ib-AMP1, oxidized to the bicyclic native conformation, which was fully active on yeast and fungal strains; and four linear 20-mer Ib-AMP variants, including two all-D forms. We show that the all-D variants are as active on yeast and fungal strains as native peptides. In addition, fungal growth inhibition nor salt-dependency of Ib-AMP4 could be improved by more than two-fold via replacement of amino acid residues by arginine or tryptophan. Native Ib-AMPs showed no hemolytic nor toxic activity up to a concentration of 100 microM. All these data demonstrate the potential of the native Ib-AMPs to combat fungal infections.

Interactions of antifungal plant defensins with fungal membrane components.

Plant defensins are small, basic, cysteine-rich peptides that are generally active against a broad spectrum of fungal and yeast species at micromolar concentrations. Some of these defensins interact with fungal-specific lipid components in the plasmamembrane. Structural differences of these membrane components between fungal and plant cells probably account for the selective activity of plant defensins against fungal pathogens and their nonphytotoxic properties. This review will focus on different classes of complex lipids in fungal membranes and on the selective interaction of plant defensins with these complex lipids.

DmAMP1, an antifungal plant defensin from dahlia (Dahlia merckii), interacts with sphingolipids from Saccharomyces cerevisiae.

DmAMP1, an antifungal plant defensin from Dahlia merckii, was shown previously to require the presence of sphingolipids for fungicidal action against Saccharomyces cerevisiae. Sphingolipids may stabilize glycosylphosphatidylinositol (GPI)-anchored proteins, which interact with DmAMP1, or they may directly serve as DmAMP1 binding sites. In the present study, we demonstrate that S. cerevisiae disruptants in GPI-anchored proteins showed small or no increased resistance towards DmAMP1 indicating no involvement of these proteins in DmAMP1 action. Further, studies using an enzyme-linked immunosorbent assay (ELISA)-based binding assay revealed that DmAMP1 interacts directly with sphingolipids isolated from S. cerevisiae and that this interaction is enhanced in the presence of equimolar concentrations of ergosterol. Therefore, DmAMP1 antifungal action involving membrane interaction with sphingolipids and ergosterol is proposed.

Tolerance and the effect of high doses of wheat bran extract, containing arabinoxylan-oligosaccharides, and oligofructose on faecal output: a double-blind, randomised, placebo-controlled, cross-over trial.

Wheat bran extract (WBE) is a food-grade soluble fibre preparation that is highly enriched in arabinoxylan-oligosaccharides. In this placebo-controlled cross-over human intervention trial, tolerance to WBE as well as the effects of WBE on faecal parameters, including faecal output and bowel habits, were studied. After a 2-week run-in period, twenty healthy volunteers consumed WBE (15 g/d in the first week, 30 g/d in the second week), oligofructose (15 g/d in the first week, 30 g/d in the second week) and placebo (for 2 weeks) in a random order, with 2-week washout periods between each treatment period. Subjects collected a 72 h stool sample for analysis of faecal output, stool pH and stool moisture concentration. Additionally, the volunteers completed questionnaires scoring occurrence frequency and distress severity of eighteen gastrointestinal (GI) symptoms. An overall GI symptom measure was calculated to analyse the overall effect of WBE and oligofructose on GI symptoms. Intake of both 30 g/d WBE and 30 g/d oligofructose lowered stool pH, indicative of increased colonic fermentation, and increased stool moisture concentration as compared with placebo intake. Intake of 30 g/d oligofructose increased the overall GI symptom measure by 1·9-fold as compared with placebo intake. Intake of WBE at doses up to 30 g/d did not affect the overall GI symptom measure. WBE exerts beneficial effects on stool characteristics and is well tolerated at up to 30 g/d. Oligofructose exerts comparable beneficial effects on stool characteristics. However, intake of 30 g/d oligofructose appears to cause GI discomfort to some extent.

Skn1 and Ipt1 negatively regulate autophagy in Saccharomyces cerevisiae.

We demonstrated that a yeast deletion mutant in IPT1 and SKN1, encoding proteins involved in the biosynthesis of mannosyldiinositolphosphoryl ceramides, is characterized by increased autophagy and DNA fragmentation upon nitrogen (N) starvation as compared with the single deletion mutants or wild type (WT). Apoptotic features were not significantly different between single and double deletion mutants upon N starvation, pointing to increased autophagy in the double Deltaipt1 Deltaskn1 deletion mutant independent of apoptosis. We observed increased basal levels of phytosphingosine in membranes of the double Deltaipt1 Deltaskn1 deletion mutant as compared with the single deletion mutants or WT. These data point to a negative regulation of autophagy by both Ipt1 and Skn1 in yeast, with a putative involvement of phytosphingosine in this process.