A chitin-binding lectin from stinging nettle rhizomes with antifungal properties.

Rhizomes of stinging nettle contain a small-sized lectin that exhibits binding specificity toward chitin. This lectin inhibits growth of several phytopathogenic and saprophytic chitin-containing fungi in vitro. The antifungal action of the nettle lectin differs from the action of chitinases, which are a ubiquitous class of antifungal plant proteins. Moreover, the nettle lectin acts synergistically with chitinase in inhibiting fungal growth. The nettle lectin may be a promising candidate for possible applications in the genetic engineering of disease-resistant crops.

Arabinoxylooligosaccharides from wheat bran inhibit Salmonella colonization in broiler chickens.

The objective of this in vivo experiment was to evaluate the influence of arabinoxylooligosaccharides (AXOS) on shedding and colonization of Salmonella Enteritidis in broilers. Arabinoxylooligosaccharides, which are oligosaccharides derived from arabinoxylans by partial hydrolysis, have a beneficial effect on feed conversion ratios when added to broiler diets. Additionally, AXOS have been shown to promote the growth of bifidobacteria in the cecocolonic compartment of the gastrointestinal tract. To investigate the impact of AXOS on colonization of broilers with Salmonella, 224 one-day-old chicks were divided into 4 groups and given either unsupplemented feed or feed supplemented with 0.2% AXOS-3-0.25, 0.2% AXOS-9-0.25, or 0.4% AXOS-9-0.25 throughout the experiment. The AXOS-3-0.25 and AXOS-9-0.25 both have an ara-binose-to-xylose ratio of 0.25 and have an average degree of polymerization of 3 and 9, respectively. At 14 d posthatch, each animal was orally inoculated with 2.5 x 10(9) cfu of Salmonella Enteritidis. Cloacal swabs, taken at regular times, showed a significant reduction of Salmonella presence in the group given 0.4% AXOS-9-0.25 compared with the control group. This reduction was observed in the 1- to 11-d postinfection period. Colonization of the ceca as well as the translocation of Salmonella to the spleen was significantly reduced at 3 and 7 d postinfection in the 0.4% AXOS-9-0.25 group. A similar, although more moderate, decrease in colonization was observed in the group given 0.2% AXOS-9-0.25. It was concluded that dietary addition of AXOS provides dose-dependent protection against oral infections with Salmonella Enteritidis in poultry.

The effect of arabinoxylooligosaccharides on upper gastroduodenal motility and hunger ratings in humans.

BACKGROUND AND AIMS: Prebiotics such as Arabinoxylooligosaccharides (AXOS) are non-digestible, fermentable food ingredients stimulating growth/activity of colonic bacteria with enhanced carbohydrates fermentation (CF) in humans. The migrating motor complex (MMC) of the gastrointestinal tract has been recently identified as an important hunger signal, but no data are available yet on the role of acute CF on MMC activity and related hunger ratings. Thus, we aimed to study the effect of acute AXOS CF on MMC and hunger in humans. METHODS: A total of 13 healthy volunteers were randomized in a single-blind crossover placebo-controlled study where 9.4 g of AXOS or 10 g of maltodextrin and 1 g of unlabelled lactose ureide (LU) were given 12 hours prior to the study and, in the next morning, together with a pancake containing 500 mg of 13 C-LU. In 10 hours after the meal, 13 CO2 and hydrogen excretion were determined every 15 minutes while hunger/appetite ratings every 2 minutes through a VAS questionnaire. Five hours after the meal, antroduodenal motility was measured using HRM. KEY RESULTS: AXOS significantly increased CF (158 ± 81 vs 840 ± 464 H2 ppm*minute, placebo vs AXOS, P < .05) without affecting the orocecal transit time (OCTT). AXOS had no significant effect on the occurrence, origin, and duration of phase III and on the total number, origin, and duration of phases I and II. Hunger and appetite scores prior and after phase III were not affected by AXOS. CONCLUSIONS: AXOS acutely increases colonic fermentation, but this neither affects OCTT, activity of the MMC, nor interdigestive hunger scores in man.

The complexity of disease signaling in Arabidopsis.

Not more than 10 years ago it was generally accepted that pathogen-inducible defense mechanisms in plants are triggered through a central signaling cascade that regulates a multicomponent defense response. Now we know that the plant defense system is regulated through a complex network of various signaling cascades.

The effect of arabinoxylooligosaccharides on gastric sensory-motor function and nutrient tolerance in man.

BACKGROUND: Intestinal microbiota regulates gastrointestinal sensory-motor function. Prebiotics such as arabinoxylan-oligosaccharide (AXOS) are non-digestible, fermentable food ingredients beneficially affecting intestinal microbiota, colon activity, and improving human health. We wanted to investigate whether acute AXOS or maltodextrin (placebo) administration may alter gastric sensitivity (GS), accommodation (GA), nutrient tolerance (NT) in man. METHODS: Thirteen HV (6 M, 32.2 ± 1.8 years; BMI 22.3 ± 0.2) underwent two 48 h treatment periods with oral 4 × 9.4 g AXOS or 4 × 10 g maltodextrin (at least 1 week wash-out) for gastric barostat assessment of GS, gastric compliance (GC), GA to a liquid test meal, on day 1, and NT drink test, on day 2. Oro-cecal transit-time (OCTT), colonic fermentation (CF) were assessed simultaneously with (13) C-lactose ureide, H2 breath tests. KEY RESULTS: Arabinoxylan-oligosaccharide significantly increased CF on day 1 and 2 (565 ± 272 vs 100 ± 24, 365 ± 66 vs 281 ± 25 H2 ppm/min, AXOS vs maltodextrin, both p < 0.05), not the OCTT. AXOS did not alter GC, sensitivity before and after the meal. Gastric accommodation was not significantly influenced by AXOS (volume increment: 171 ± 33 vs 130 ± 28 mL, AXOS vs maltodextrin, p = NS). On day 1, AXOS fermentation was associated with significantly higher postprandial bloating scores (960 ± 235 vs 396 ± 138 mm*min, AXOS vs maltodextrin, p < 0.05). On day 2, AXOS did not affect maximal NT (946 ± 102 vs 894 ± 97 mL, AXOS vs maltodextrin, p = NS), increased the bloating score (1236 ± 339 vs 675 ± 197 mm*min, AXOS vs maltodextrin, p < 0.05). CONCLUSIONS & INFERENCES: Acute AXOS administration, associated with increased CF, does not affect GA, is not associated with increased meal-induced satiety or perception scores.

Synergistic Enhancement of the Antifungal Activity of Wheat and Barley Thionins by Radish and Oilseed Rape 2S Albumins and by Barley Trypsin Inhibitors.

Although thionins and 2S albumins are generally considered as storage proteins, both classes of seed proteins are known to inhibit the growth of pathogenic fungi. We have now found that the wheat (Triticum aestivum L.) or barley (Hordeum vulgare L.) thionin concentration required for 50% inhibition of fungal growth is lowered 2- to 73-fold when combined with 2S albumins (at sub- or noninhibitory concentrations) from radish (Raphanus sativus L.) or oilseed rape (Brassica napus L.). Furthermore, the thionin antifungal activity is synergistically enhanced (2- to 33-fold) by either the small subunit or the large subunit of the radish 2S albumins. Three other 2S albumin-like proteins, the barley trypsin inhibitor and two barley Bowman-Birk-type trypsin inhibitor isoforms, also act synergistically with the thionins (2- to 55-fold). The synergistic activity of thionins combined with 2S albumins is restricted to filamentous fungi and to some Gram-positive bacteria, whereas Gram-negative bacteria, yeast, cultured human cells, and erythrocytes do not show an increased sensitivity to thionin/albumin combinations (relative to the sensitivity to the thionins alone). Scanning electron microscopy and measurement of K+ leakage from fungal hyphae revealed that 2S albumins have the same mode of action as thionins, namely the permeabilization of the hyphal plasmalemma. Moreover, 2S albumins and thionins act synergistically in their ability to permeabilize fungal membranes.

Specific binding sites for an antifungal plant defensin from Dahlia (Dahlia merckii) on fungal cells are required for antifungal activity.

Dm-AMP1, an antifungal plant defensin from seeds of dahlia (Dahlia merckii), was radioactively labeled with t-butoxycarbonyl-[35S]-L-methionine N-hydroxy-succinimi-dylester. This procedure yielded a 35S-labeled peptide with unaltered antifungal activity. [35S]Dm-AMP1 was used to assess binding on living cells of the filamentous fungus Neurospora crassa and the unicellular fungus Saccharomyces cerevisiae. Binding of [35S]Dm-AMP1 to fungal cells was saturable and could be competed for by preincubation with excess, unlabeled Dm-AMP1 as well as with Ah-AMP1 and Ct-AMP1, two plant defensins that are highly homologous to Dm-AMP1. In contrast, binding could not be competed for by more distantly related plant defensins or structurally unrelated antimicrobial peptides. Binding of [35S]Dm-AMP1 to either N. crassa or S. cerevisiae cells was apparently irreversible. In addition, whole cells and microsomal membrane fractions from two independently obtained S. cerevisiae mutants selected for resistance to Dm-AMP1 exhibited severely reduced binding affinity for [35S]Dm-AMP1, compared with wild-type yeast. This finding suggests that binding of Dm-AMP1 to S. cerevisiae plasma membranes is required for antifungal activity of this protein.

Expression of functional Raphanus sativus antifungal protein in yeast.

Rs-AFP2 is a 51 amino acid cysteine-rich peptide isolated from radish (Raphanus sativus) seeds that exhibits potent inhibitory activity against filamentous fungi. A cDNA clone encoding the Rs-AFP2 preprotein was modified by recombinant DNA methods to allow expression in the yeast Saccharomyces cerevisiae. This peptide was expressed in yeast as a fusion protein carrying at its N-terminus the prepro-sequences derived from the precursor of the yeast pheromone mating factor alpha 1. These sequences allow secretion of the biologically active peptide in a correctly processed form. Deletion of the mating factor alpha 1 pro-peptide drastically reduced the expression level of the peptide.

Isolation and characterisation of plant defensins from seeds of Asteraceae, Fabaceae, Hippocastanaceae and Saxifragaceae.

From seeds of Aesculus hippocastanum, Clitoria ternatea, Dahlia merckii and Heuchera sanguinea five antifungal proteins were isolated and shown to be homologous to plant defensins previously characterised from radish seeds and gamma-thionins from Poaceae seeds. Based on the spectrum of their antimicrobial activity and the morphological distortions they induce on fungi the peptides can be divided into two classes. The peptides did not inhibit any of three different alpha-amylases.

A new family of basic cysteine-rich plant antifungal proteins from Brassicaceae species.

Out of seeds of 4 Brassicaceae species, 7 antifungal proteins were isolated which are nearly identical to 2 previously characterized radish seed antifungal proteins. These basic proteins, multimers of a 5 kDa polypeptide, specifically inhibit fungal growth. One of the antifungal proteins has decreased antifungal activity and an increased antibacterial activity. In addition, the previously described antifungal activity of the radish seed 2S albumins was extended to the 2S albumins of the seeds of the 4 other Brassicaceae species. A 2S albumin-like trypsin-inhibitor from barley seeds was found to have much less activity against fungi.

Arabinoxylan-oligosaccharides (AXOS) affect the protein/carbohydrate fermentation balance and microbial population dynamics of the Simulator of Human Intestinal Microbial Ecosystem.

Arabinoxylan-oligosaccharides (AXOS) are a recently newly discovered class of candidate prebiotics as - depending on their structure - they are fermented in different regions of gastrointestinal tract. This can have an impact on the protein/carbohydrate fermentation balance in the large intestine and, thus, affect the generation of potentially toxic metabolites in the colon originating from proteolytic activity. In this study, we screened different AXOS preparations for their impact on the in vitro intestinal fermentation activity and microbial community structure. Short-term fermentation experiments with AXOS with an average degree of polymerization (avDP) of 29 allowed part of the oligosaccharides to reach the distal colon, and decreased the concentration of proteolytic markers, whereas AXOS with lower avDP were primarily fermented in the proximal colon. Additionally, prolonged supplementation of AXOS with avDP 29 to the Simulator of Human Intestinal Microbial Ecosystem (SHIME) reactor decreased levels of the toxic proteolytic markers phenol and p-cresol in the two distal colon compartments and increased concentrations of beneficial short-chain fatty acids (SCFA) in all colon vessels (25-48%). Denaturant gradient gel electrophoresis (DGGE) analysis indicated that AXOS supplementation only slightly modified the total microbial community, implying that the observed effects on fermentation markers are mainly caused by changes in fermentation activity. Finally, specific quantitative PCR (qPCR) analysis showed that AXOS supplementation significantly increased the amount of health-promoting lactobacilli as well as of Bacteroides-Prevotella and Clostridium coccoides-Eubacterium rectale groups. These data allow concluding that AXOS are promising candidates to modulate the microbial metabolism in the distal colon.

Wheat germ agglutinin in wheat seedling roots: induction by elicitors and fungi.

Treatment of wheat (Triticum aestivum L.) seedlings with elicitors originating from either plant or fungal cell walls induces about a 2-fold increase of wheat germ agglutinin (WGA) in the roots. While the WGA content in roots of healthy plants normally decreases as a function of germination time, a transient accumulation of WGA could be observed in plants challenged with different fungi, including Rhizoctonia solani, Fusarium culmorum, Pythium ultimum and Neurospora crassa. Peak levels in challenged roots were 2 to 5 times as high as in control plants. Most of this induced WGA could be released from the roots by soaking them in a solution of the hapten N-acetylglucosamine. On the basis of the results obtained it is postulated that WGA may be involved in the defence of wheat against fungal attack.

Permeabilization of fungal membranes by plant defensins inhibits fungal growth.

We used an assay based on the uptake of SYTOX Green, an organic compound that fluoresces upon interaction with nucleic acids and penetrates cells with compromised plasma membranes, to investigate membrane permeabilization in fungi. Membrane permeabilization induced by plant defensins in Neurospora crassa was biphasic, depending on the plant defensin dose. At high defensin levels (10 to 40 microM), strong permeabilization was detected that could be strongly suppressed by cations in the medium. This permeabilization appears to rely on direct peptide-phospholipid interactions. At lower defensin levels (0.1 to 1 microM), a weaker, but more cation-resistant, permeabilization occurred at concentrations that correlated with the inhibition of fungal growth. Rs-AFP2(Y38G), an inactive variant of the plant defensin Rs-AFP2 from Raphanus sativus, failed to induce cation-resistant permeabilization in N. crassa. Dm-AMP1, a plant defensin from Dahlia merckii, induced cation-resistant membrane permeabilization in yeast (Saccharomyces cerevisiae) which correlated with its antifungal activity. However, Dm-AMP1 could not induce cation-resistant permeabilization in the Dm-AMP1-resistant S. cerevisiae mutant DM1, which has a drastically reduced capacity for binding Dm-AMP1. We think that cation-resistant permeabilization is binding site mediated and linked to the primary cause of fungal growth inhibition induced by plant defensins.

Datura stramonium Agglutinin : Location in the Seed and Release upon Imbibition.

The distribution of Datura stramonium agglutinin over different tissues of D. stramonium L. seeds was visualized by immunocytochemical techniques and quantified by agglutination assays. The lectin occurs predominantly in the outer seed tissues (seed coat and seed epidermis), where it is associated, at least in part, with the cell walls. Developing D. stramonium seeds secrete newly synthesized lectin polypeptides into the incubation medium, which confirms the extracellular location of the lectin. Imbibition of mature decoated seeds results in a rapid and highly specific release of lectin. Indeed, imbibition solutions contain almost exclusively the lectin together with a few other carbohydrate-binding proteins; this is indicative of the predominance of these proteins in the seed surface layer. The presence of important amounts of lectin in the outer tissues of the seed is consistent with a possible role in the mediation of cell-cell interactions.

The Urtica dioica Agglutinin Is a Complex Mixture of Isolectins.

Rhizomes of stinging nettle (Urtica dioica) contain a complex mixture of isolectins. Ion exchange chromatography with a high resolution fast protein liquid chromatography system revealed six isoforms which exhibit identical agglutination properties and carbohydrate-binding specificity and in addition have the same molecular structure and virtually identical biochemical properties. However, since the U. dioica agglutinin isolectins differ definitely with respect to their amino acid composition, it is likely that at least some of them are different polypeptides coded for by different genes.

Specific, high affinity binding sites for an antifungal plant defensin on Neurospora crassa hyphae and microsomal membranes.

Hs-AFP1, an antifungal plant defensin from seed of the plant Heuchera sanguinea, was radioactively labeled using t-butoxycarbonyl-[35S]L-methionine N-hydroxysuccinimidyl ester, resulting in a 35S-labeled peptide with unaltered antifungal activity. [35S]Hs-AFP1 was used to assess binding on living hyphae of the fungus Neurospora crassa. Binding of [35S]Hs-AFP1 was found to be competitive, reversible, and saturable with an apparent Kd of 29 nM and a Bmax of 1.4 pmol/mg protein. [35S]Hs-AFP1 also bound specifically and reversibly to microsomal membranes derived from N. crassa hyphae with a Kd of 27 nM and a Bmax of 102 pmol/mg protein. The similarity in Kd value between binding sites on hyphae and microsomes indicates that Hs-AFP1 binding sites reside on the plasma membrane. Binding of [35S]Hs-AFP1 to both hyphae and microsomal membranes could be competed to some extent by four different structurally related plant defensins but not by various structurally unrelated antimicrobial peptides. In addition, an inactive single amino acid substitution variant of the antifungal plant defensin Rs-AFP2 from Raphanus sativus seed was also unable to displace [35S]Hs-AFP1 from its binding sites, whereas Rs-AFP2 itself was able to compete with [35S]Hs-AFP1.

A lectin from elder (Sambucus nigra L.) bark.

A lectin was isolated from elder (Sambucus nigra) bark by affinity chromatography on fetuin-agarose. It is a tetrameric molecule (Mr 140000) composed of two different subunits of Mr 34500 and 37500 respectively, held together by intramolecular disulphide bridges. The lectin is a glycoprotein and is especially rich in asparagine/aspartic acid, glutamine/glutamic acid, valine and leucine. It is also the first lectin isolated from a species belonging to the plant family Caprifoliaceae.

Isolation and partial characterization of a lectin from ground elder (Aegopodium podagraria) rhizomes.

A lectin has been isolated from rhizomes of ground elder (Aegopodium podagraria) using a combination of affinity chromatography on erythrocyte membrane proteins immobilized on cross-linked agarose and hydroxyapatite, and ion-exchange chromatography. The molecular structure of the lectin was determined by gelfiltration, sucrose density-gradient centrifugation and gel electrophoresis under denaturing conditions. It has an unusually high Mr (about 480000) and is most probably an octamer composed of two distinct types of subunits with slightly different Mr (about 60000). Hapten inhibition assays indicated that the Aegopodium lectin is preferentially inhibited by N-acetylgalactosamine. Nevertheless, it does not agglutinate preferentially blood-group-A erythrocytes. The ground-elder lectin is a typical non-seed lectin, which occurs virtually exclusively in the underground rhizomes. In this organ it is an abundant protein as it represents up to 5% of the total protein content. The lectin content of the rhizome tissue varies strongly according to its particular location along the organ. In addition, the lectin content changes dramatically as a function of the seasons. The ground-elder lectin differs from all other plant lectins by its unusually high molecular weight. In addition, it is the first lectin to be isolated from a species of the family Apiaceae.

Isolation and Partial Characterization of a New Lectin from Seeds of the Greater Celandine (Chelidonium majus).

Seeds of the greater celandine (Chelidonium majus L.) contain a lectin which could be isolated using a combination of affinity chromatography on chitin and ion exchange chromatography on sulphopropyl-Sephadex. The purified lectin was partially characterized with respect to its biochemical and physicochemical properties. It is a small dimeric protein composed of two different subunits of M(r) 9,500 and 11,500, respectively. Its amino acid composition is typified by high contents of glycine and cysteine. No covalently bound carbohydrate could be detected. Hapten inhibition experiments indicated that the lectin exhibits specificity towards oligomers of N-acetylglucosamine, the potency of inhibition increasing with chain length up to four residues. The greater celandine lectin is the first lectin to be isolated from a species belonging to the plant family Papaveraceae (poppy family). Although it represents a new type of plant lectin, resemblances to phytohemaglutinins from diverse taxonomic origin are obvious.

Agrobacterium-mediated transformation of apple (Malus x domestica Borkh.): an assessment of factors affecting regeneration of transgenic plants.

We have previously developed a protocol for efficient gene transfer and regeneration of transgenic calli following cocultivation of apple (cv. Jonagold) explants with Agrobacterium tumefaciens (De Bondt et al. 1994, Plant Cell Reports 13: 587-593). Now we report on the optimization of postcultivation conditions for efficient and reproducible regeneration of transgenic shoots from the apple cultivar Jonagold. Factors which were found to be essential for efficient shoot regeneration were the use of gelrite as a gelling agent and the use of the cytokinin-mimicing thidiazuron in the selective postcultivation medium. Improved transformation efficiencies were obtained by combining the hormones thidiazuron and zeatin and by using leaf explants from in vitro grown shoots not older than 4 weeks after multiplication. Attempts to use phosphinothricin acetyl transferase as a selectable marker were not successful. Using selection on kanamycin under optimal postcultivation conditions, about 2% of the leaf explants developed transgenic shoots or shoot clusters. The presence and expression of the transferred genes was verified by ß-glucuronidase assays and Southern analysis. The transformation procedure has also been succesfully applied to several other apple cultivars.