Aluminum-induced deposition of (1,3)-β-glucans (callose) inTriticum aestivum L.

Aluminum (Al)-induced damage to leaves and roots of two Al-resistant (cv. Atlas 66, experimental line PT741) and two Al-sensitive (cv. Scout 66, cv. Katepwa) lines ofTriticum aestivum L. was estimated using the deposition of (1, 3)--glucans (callose) as a marker for injury. Two-day-old seedlings were grown for forty hours in nutrient solutions with or without added Al, and callose deposition was quantified by spectrofluorometry (0–1000 µM Al) and localized by fluorescence microscopy (0 and 400 µM Al). Results suggested that Al caused little damage to leaves. No callose was observed in leaves with up to 400 µM Al treatment. In contrast, root callose concentration increased with Al treatment, especially in the Al-sensitive lines. At 400 µM Al, root callose concentration of Al-sensitive Scout 66 was nearly four-fold that of Al-resistant Atlas 66. After Al treatment, large callose deposits were observed in the root cap, epidermis and outer cortex of root tips of Scout 66, but not Atlas 66. The identity of callose was confirmed by a reduced fluorescence in Al-treated roots: firstly, after adding an inhibitor of callose synthesis (2-deoxy-D-glucose) to the nutrient solution, and secondly, after incubating root sections with the callosedegrading enzyme -D-glucoside glucohydrolase [EC 3.2.1.21]. Root callose deposition may be a good marker for Al-induced injury due to its early detection by spectrofluorometry and its close association with stress perception.Abbreviations DDG 2-deoxy-D-glucose - PAS periodic acid - Schiffs reagent - PE pachyman equivalents     

Suppression of fungal β-glucan-induced plant defence in soybean (Glycine max L.) by cyclic 1,3-1,6-β-glucans from the symbiont Bradyrhizobium japonicum

The production of antimicrobial phytoalexins is one of the best-known inducible defence responses following microbial infection of plants or treatment with elicitors. In the legume soybean (Glycine max L.), 1,3-1,6--glucans derived from the fungal pathogen Phytophthora sojae have been identified as potent elicitors of the synthesis of the phytoalexin, glyceollin. Recently it has been reported that during symbiotic interaction between soybean and the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110 the bacteria synthesize cyclic 1,3-1,6--glucans. Here we demonstrate that both the fungal and the bacterial -glucans are ligands of -glucan-binding sites which are putative receptors for the elicitor signal compounds in soybean roots. Whereas the fungal -glucans stimulate phytoalexin synthesis at low concentrations, the bacterial cyclic 1,3-1,6--glucans appear to be inactive even at relatively high concentrations. Competition studies indicate that increasing concentrations of the bacterial 1,3-1,6--glucans progressively inhibit stimulation of phytoalexin synthesis in a bioassay induced by the fungal 1,3-1,6--glucans. Another type of cyclic -glucan, a 1,2--glucan from Rhizobium meliloti, that does not nodulate on soybean, seems to be inactive as elicitor and as ligand of the -glucan-binding sites. These results may indicate a novel mechanism for a successful plant-symbiont interaction by suppressing the plant's defence response.Abbreviations HG-APEA 1-[2-(4-aminophenyl)ethyl]amino-l-[hexaglucosyl]deoxyglucitol - HG-AzPEA l-[2-(4-azidophenyl)-ethyl]amino-l-[hexaglucosyl]deoxyglucitol - IC₅₀ concentration for half-maximal displacement We thank Ines Arlt for excellent technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 369), the Bundesministerium für Bildung, Wissenschaft, Forschung und Technologie, Fonds der Chemischen Industrie (J.E.), and USDA CSRS NRI Competitive Research grant 93373059233 (A.A.B.).     

Large-scale Preparation of (R)-(–)-1,3-Butanediol from the Racemate by Candida parapsilosis

Large-scale preparation of (R)-(–)-1,3-butanediol (R-BDO), an important chiral synthon, from the racemate by Candida parapsilosis IFO 1396 was investigated. We found that ethanol accumulated during culture enhanced the secondary alcohol oxido-reduction activity of cells. Large-scale preparation of R-BDO was done using a fermentor. 3092 g of R-BDO was obtained from the racemate by the use of this strain with 94.0% enantiomeric excess.     

Biosynthesis of (1,3)(1,4)-β-glucan and (1,3)-β-glucan in barley (Hordeum vulgare L.)

Mixed membrane preparations from the coleoptiles and first leaves of young barley (Hordeum vulgare L. cv. Triumph) plants catalysed the synthesis of 55% methanol-insoluble labelled material from UDP[U-¹⁴C]glucose, the main components of which were identified as (1,3)(1,4)-- and (1,3)--D-glucans. The membrane preparations also catalysed the transformation of UDP-glucose into labelled low-molecular-weight products, mainly glucose (by phosphatase action), glucose-1-phosphate (by phosphodiesterase action) and glyco(phospho)lipids (by glycosyltransferase action). The formation of (1,3)(1,4)--glucans, (1,3)--glucans, and the other reactions competing for UDP-glucose, were monitored simultaneously and quantitatively by a novel procedure based on enzymatic analysis, thin-layer chromatography and digital autoradiography. Thus it was possible (i) to optimise conditions to obtain (1,3)(1,4)--glucan synthesis or (1,3)--glucan synthesis in isolation, and (ii) to study the influence of temperature, pH, cofactors, substrate concentration etc. on the (1,3)(1,4) and (1,3)--glucan synthesis reactions even when both occurred together. The synthesis of both -glucans was optimal at 20°C. In Tris-HCl buffer, the pH optima for (1,3)(1,4)--glucan synthesis and (1,3)--glucan synthesis were pH 8.5 and pH 7.0, respectively. Both glucan-synthesis reactions required Mg²⁺: (1,3)--glucan synthesis was optimal at 2 mM, whereas (1,3)(1,4)--glucan synthesis continued to increase up to 200 mM Mg²⁺, when the ion was supplied as the sulphate. (1,3)--Glucan synthesis was Ca²⁺ dependent and this dependence could be abolished by proteinase treatment. The K _m with respect to UDP-glucose was 1.5 mM for (1,3)--glucan synthesis and approximately 1 mM for (1,3)(1,4)--glucan synthesis. The (1,3)(1,4)--glucan formed in vitro had the same ratio of trisaccharide to tetrasaccharide structural blocks irrespective of the experimental conditions used during the synthesis: its enzymatic fragmentation pattern was indistinguishable from that of barley endosperm (1,3)(1,4)--glucan. This indicates either a single synthase enzyme, which is responsible for the formation of both linkage types, or two enzymes which are very tightly coupled functionally.Abbreviations G4G4G3G Glc(1,4)Glc(1,4)Glc(1,3)Glc (-linked) - UDP-Glc uridine-5-diphosphate glucose We are grateful to the Commission of the European Communities for the award of Training Fellowships to Christine Vincent and Martin Becker.     

The effect of 1-β-d-arabinofuranosyl-cytosine on the expression of the common fragile site at 3p14

Summary The effect of the G₂-treatment of 1--d-arabino-furanosyl-cytosine (araC) on the expression of the common fragile site at 3p14 (FRA3B) was studied. A significantly increased frequency of FRA3B induced by G₂ treatment of araC was found in the lymphocytes grown in folate-deficient medium (positive rate 100%). A relatively low frequency of FRA3B was also induced in the cultures with folate in four of the seven subjects. These is a synergistic effect between araC and growth in folate-deficient medium on the induction of FRA3B. The results suggest that the DNA lesions related to the expression of FRA3B induce the long-patch repair and that the low DNA polymerase activity and inefficient repair process during G₂ phase is involved in the expression of FRA3B.     

Hydrolysis of β-1,3/1,6-glucan by glycoside hydrolase family 16 endo-1,3(4)-β-glucanase from the basidiomycete Phanerochaete chrysosporium

When Phanerochaete chrysosporium was grown with laminarin (a β-1,3/1,6-glucan) as the sole carbon source, a β-1,3-glucanase with a molecular mass of 36 kDa was produced as a major extracellular protein. The cDNA encoding this enzyme was cloned, and the deduced amino acid sequence revealed that this enzyme belongs to glycoside hydrolase family 16; it was named Lam16A. Recombinant Lam16A, expressed in the methylotrophic yeast Pichia pastoris, randomly hydrolyzes linear β-1,3-glucan, branched β-1,3/1,6-glucan, and β-1,3-1,4-glucan, suggesting that the enzyme is a typical endo-1,3(4)-β-glucanase (EC 3.2.1.6) with broad substrate specificity for β-1,3-glucans. When laminarin and lichenan were used as substrates, Lam16A produced 6-O-glucosyl-laminaritriose (β-d-Glcp-(1–>6)-β-d-Glcp-(1–>3)-β-d-Glcp-(1–>3)-d-Glc) and 4-O-glucosyl-laminaribiose (β-d-Glcp-(1–>4)-β-d-Glcp-(1–>3)-d-Glc), respectively, as one of the major products. These results suggested that the enzyme strictly recognizes β-d-Glcp-(1–>3)-d-Glcp at subsites −2 and −1, whereas it permits 6-O-glucosyl substitution at subsite +1 and a β-1,4-glucosidic linkage at the catalytic site. Consequently, Lam16A generates non-branched oligosaccharide from branched β-1,3/1,6-glucan and, thus, may contribute to the effective degradation of such molecules in combination with other extracellular β-1,3-glucanases.     

Studies of two low-molecular-weight endo-1(1→4)-β-d-xylanases constitutively synthesised by the cellulolytic fungus Trichoderma koningii

Two endo-(1→4)-β-d-xylanases (xylanases 1 and 2), which were constitutively synthesised by the fungus Trichoderma koningii, were purified to homogeneity on gel-filtration media and by isoelectric focusing. They had molecular weights of 29,000 (xylanase 1) and 18,000 (xylanase 2), and isoelectric pHs of 7.24 (xylanase 1) and 7.3 (xylanase 2); neither enzyme was associated with carbohydrate. Xylanase 1 had an optimum at the remarkably high temperature of 60–65°. Each enzyme liberated a different range of oligosaccharides from oat-straw arabinoxylan, but only xylanase 1 released l-arabinose and d-xylose. Both xylanases were free from cellulase activity.     

Bi-antennary oligo-(N-acetyllactosamino)glycans of I-type are galactosylated preferentially at the GlcNAcβ1-6Gal linked arms by α1,3-galactosyltransferase of bovine thymus

1,3-Galactosylation of radiolabelled bi-antennary acceptors Gal1-4GlcNAc1-3(Gal1-4GlcNAc1-6)Gal-R (R=1-OH, 1-4GlcNAc or 1-4Glc) with bovine thymus 1,3-galactosyltransferase was studied. At all stages of the reactions the three acceptors reacted faster at the 1 6 linked arm than at the 1 3 linked branch. Hence, in addition to the doubly 1,3-galactosylated products, practically pure Gal1-4GlcNAc1-3(Gal1-3Gal1-4GlcNAc1-6)Gal-R could be obtained from the three acceptors in reactions that had proceeded to near completion. The isomeric mono-1,3-galactosylated products were identified by using exoglycosidases to remove the branches unprotected by 1,3-galactoses and by subsequently identifying the resulting linear glycans chromatographically.Abbreviations Gal d-galactose - GlcNAc N-acetyl-d-glucosamine - Lac lactose - LacNAc Gal1-4GlcNAc - MH maltoheptaose - MP maltopentaose - MT maltotriose - MTet maltotetraose - WGA wheat germ agglutinin - 3 position 3 of the galactose unit of LacNAc or Lac - 6 position 6 of the galactose unit of LacNAc or Lac     

Hydrolysis of oligosaccharides of the β-(1→4)-linked d-xylose series by an endo(1→4)-β-d-xylanase from the anaerobic rumen fungus Neocallimastix frontalis

An endo-(14)--d-xylanase from Neocallimastix frontalis was purified by anion-exchange chromatography. The enzyme had an apparent molecular mass of 30 kDa on SDS-PAGE and exhibited maximum activity at 50°C and at pH values between 6.0 and 6.6. Kinetic studies on the hydrolysis of xylo-oligosaccharides, ranging from xylobiose to xylodecaose, showed that xylohexaose and xyloheptaose were the preferred substrates for the enzyme and that xylobiose, xylotriose and xylotetraose were not hydrolysed. Xylose was not a product of the hydrolysis of any of the xylo-oligosaccharide substrates tested. The enzyme appeared to have a strong preference for the hydrolysis of the internal glycosidic bonds of the oligosaccharides, which is typical of endo-(14)--d-xylanase activity, but it differed from other fungal endo-(14)--d-xylanases in that it had uniform action on the various internal linkages in the xylo-oligosaccharides.V. Garcia-Campayo, S.I. McCrae and T.M. Wood are with The Rowett Research Institute, Greenburn Road, Bucksburn, Aberdeen AB2 9SB, UK     

Hyphal in vitro growth of the arbuscular mycorrhizal fungus Glomus mosseae is affected by chitinase but not by β-1,3-glucanase

Purified basic chitinase or #-1,3-glucanase or a combination of the two enzymes were applied to hyphae of the arbuscular mycorrhizal fungus Glomus mosseae grown in vitro. Chitinase applied to the hyphal tip produced an inhibition of hyphal extension, lysis of the apex and alterations of the growth pattern of the fungus. No effect was observed, however, when chitinase was applied to subapical parts of the hyphae or when glucanase was applied to any part of the hyphae. Application of a combination of the two enzymes to the hyphal tip produced an effect similar to that of chitinase alone.     

Probiotic Properties of the 2-Substituted (1,3)-β-d-Glucan-Producing Bacterium Pediococcus parvulus 2.6

Exopolysaccharides have prebiotic potential and contribute to the rheology and texture of fermented foods. Here we have analyzed the in vitro bioavailability and immunomodulatory properties of the 2-substituted (1,3)-β-d-glucan-producing bacterium Pediococcus parvulus 2.6. It resists gastrointestinal stress, adheres to Caco-2 cells, and induces the production of inflammation-related cytokines by polarized macrophages.Lactic acid bacteria (LAB) are industrially important microorganisms for fermented food production. The recent widespread application of LAB and bifidobacteria for elaboration of functional food is attributable to the accumulating scientific evidence showing their beneficial effects on human health (3, 16). Most commercialized probiotics are limited to a few strains of bifidobacteria, lactobacilli, and streptococci, most of which produce exopolysaccharides (EPS) (27, 30). This fact, together with reports on immunomodulating ability as well as anticarcinogenic and cholesterol-lowering activities of EPS-producing LAB (25), suggests that the beneficial properties of these microorganisms for human health may be due to the biological activities of these prebiotic biopolymers (25, 26), whose producing bacteria are also frequently used to improve the texture and taste of dairy products (5, 11, 25). The future development of functional foods will be aimed at the diversification of this class of food, and therefore the identification and characterization of further bacteria with probiotic potential, isolated from habitats different from those of the currently used organisms (digestive tract and dairy products), will increase the biodiversity and utility of this class of microorganisms.LAB strains belonging to the genera Pediococcus, Lactobacillus, and Oenococcus, isolated from cider and wine, produce a 2-substituted (1,3)-β-d-glucan EPS (4, 6, 7, 12, 17). One of these strains is Pediococcus damnosus 2.6 (also known as ropy and 2.6R), originally isolated from cider (8) and later renamed Pediococcus parvulus 2.6 (32). Curing of its 35-kDa pPP2 plasmid generated the isogenic, nonropy (2.6NR), non-EPS-producing strain (8). The plasmidic gtf gene determinant for EPS production was cloned into Escherichia coli, and determination of its DNA sequence revealed that it encodes a protein, named GTF glycosyltransferase, belonging to the COG1215 membrane-bound glycosyltransferase family (32). Cloning of the gtf gene and functional expression of its encoded glycosyltransferase in Streptococcus pneumoniae (32) and Lactococcus lactis revealed that this enzyme is indeed responsible for the synthesis of the β-d-glucan (33).The GTF glycosyltransferase has identity (33%) only with the Tts glycosyltransferase of Streptococcus pneumoniae serotype 37 (19). The latter enzyme catalyzes the biosynthesis and secretion of this organism''s capsule (18), which is a β-d-glucan similar to the EPS synthesized by Pediococcus, and anti-serotype 37 antibodies also agglutinate Streptococcus pneumoniae (32) and Lactococcus lactis strains that overexpress gtf (4, 33), as well as LAB strains naturally carrying this gene (4, 32).Analysis of the rheological properties of the β-d-glucan synthesized by P. parvulus 2.6 showed that it has potential utility as a biothickener (29). In addition, human ingestion of oat-based food elaborated with P. parvulus 2.6 resulted in a decrease of serum cholesterol levels, boosting the effect previously demonstrated for (1,3)-β-d-glucans in oats (21). Therefore, this LAB is a potential probiotic strain useful for elaboration of functional food.In this work, we performed a comparative analysis of the β-d-glucan producer P. parvulus 2.6 and its isogenic nonropy strain, using in vitro models that simulate the conditions in the human gastrointestinal (GI) tract.Cultures of the strains were grown to early stationary phase in MRS medium (Pronadisa, Madrid, Spain) at 30°C under anaerobic conditions. Aliquots containing 3.4 × 10⁷ cells of each bacterium were independently subjected to agglutination tests with S. pneumoniae type 37-specific antiserum (Statens Serum Institut, Copenhagen, Denmark), as previously described (32), and production of EPS was examined under a microscope (Fig. ​(Fig.1).1). Agglutination of the cultures, detected by phase-contrast microscopy as previously described (33), showed that immunoprecipitation of strain 2.6R occurred with antibodies against pneumococcal serotype 37 (Fig. ​(Fig.1A).1A). As expected, these antibodies did not react with strain 2.6NR (Fig. ​(Fig.1B).1B). This type of analysis, coupled with plate counting, revealed that growth of P. parvulus 2.6 up to the beginning of the stationary phase was an optimal condition for EPS production without a loss of viability (results not shown). Therefore, the strains were grown to an optical density at 620 nm (OD₆₂₀) of 1.2 (10⁹ CFU ml⁻¹) as described above and subjected to conditions of the human gut by use of an in vitro model which approximates exposure to saliva, the pH gradient of the stomach, and intestinal stress (Fig. ​(Fig.2),2), as previously described (9), with the following modifications. For gastric (G) stress analysis, bacteria were treated for 20 min, after exposure to lysozyme, with pepsin at the following pHs: 5.0, 4.1, and 3.0. Moreover, GI stress was mimicked by exposure of the G, pH 5.0, samples to bile salts and pancreatin at pH 6.5 for 120 min. Treated bacteria (G and GI samples) were further analyzed for cell viability as previously described (9) and then compared with untreated bacteria (C samples) by using Live/Dead BacLight fluorescent stain, which permits the calculation of the percentage of live cells from the ratio of green (live) to red (dead) fluorescence. Since the presence of the EPS attached to the ropy strain could impair proper staining of the cells, prior to this analysis we established that (i) for both strains the green/red ratio correlates with the number of viable cells determined by plate counting and (ii) the dyes were taken up by P. parvulus 2.6 cells, as determined by fluorescence microscopy analysis (data not shown).Open in a separate windowFIG. 1.Detection of EPS production. The P. parvulus 2.6R (A) and 2.6NR (B) strains were subjected to agglutination tests and detection by phase-contrast microscopy. Left panels, bar = 100 μm; right panels, bar = 10 μm.Open in a separate windowFIG. 2.Analysis of cell survival after G and GI stresses. The indicated bacterial strains were left untreated (C) or subjected to various G or GI stresses as described in the text. After staining, cell viability was analyzed by measurement of green and red fluorescence. The values are the means for three independent experiments and are expressed as percentages of the green/red (G/R) fluorescence ratio for untreated control samples. The 100% control values for untreated 2.6R and 2.6NR were 10.05 and 9.98, respectively.Figure ​Figure22 depicts the results of the analysis of P. parvulus 2.6 and 2.6NR subjected to G or GI stress. Both strains showed the same pattern of resistance to stress, indicating that the presence of EPS did not confer to P. parvulus 2.6 an advantage for survival in the human digestive tract. After exposure to pH 3.0, approximately 10% cell survival was detected for both strains. In addition, the intestinal conditions caused no marked loss of viability (GI, pH 5.0, versus G, pH 5.0, samples), indicating that live bacteria could be available for interaction with intestinal epithelial cells. This interaction was investigated by using human Caco-2 cells and a ratio of 10 bacteria per epithelial cell, as previously described (9). After 1 h of exposure to bacteria, the Caco-2 cells were washed three times with phosphate-buffered saline (PBS), pH 7.1, to remove unadhered bacteria, and then the Caco-2 cells were detached by treatment with 0.5% trypsin-EDTA (Invitrogen, Barcelona, Spain) and the number of adhered bacteria was determined by plate counting. In the control experiments, after 1 h of exposure to bacteria the Caco-2 cells were detached with trypsin, as described above, but without any washing, and were plate counted to determine the total number (i.e., adhered and unadhered) of bacteria. Results from the adhesion experiments are expressed as percentages of the corresponding control value.In further experiments, two probiotic strains were used, namely, Lactobacillus acidophilus LA-5 and Bifidobacterium animalis subps. lactis BB-12 (Chr. Hansen A/S, Hørsholm, Denmark), which had previously shown high and intermediate levels of adhesion (9). All bacteria were grown to early stationary phase in MRS medium as described above, sedimented by centrifugation at 12,000 × g, and used for adhesion experiments, after resuspension in PBS, pH 7.1, at 1.25 × 10⁶ cells ml⁻¹. In addition, for analysis of the influence of the EPS on the adhesion capability of the ropy strain, two subpopulations were used, (i) prepared as indicated above (2.6R) and (ii) composed of bacterial cells washed with PBS prior to resuspension as described above (2.6R*), with the aim to remove the EPS attached to bacteria before analyzing their adhesion. Prior to that, an analysis of the bacteria by electron microscopy was performed using samples prepared as follows. Glow-discharged carbon-coated Formvar grids were placed facedown over a droplet of each culture concentrated fivefold in 0.1 M AcNH₄, pH 7. After 1 min, each grid was removed, blotted briefly with filter paper, and without being dried, negatively stained with 2% uranyl acetate for 40 s and then blotted quickly and air dried. The analysis (Fig. ​(Fig.3)3) revealed that EPS bound to P. parvulus 2.6 was indeed present and was partially removed by the washing treatment. Moreover, the analysis confirmed the absence of EPS in the 2.6NR strain. Figure ​Figure33 depicts the results of the adhesion experiments. P. parvulus 2.6 showed a high level of adherence (6.1%), similar to that of L. acidophilus LA-5 (6.6%) and considerably higher than that of the non-EPS-producing strain 2.6NR (0.25%). In addition, an intermediate adherence (1.8%) was detected for the 2.6R* subpopulation of the 2.6R strain and for B. animalis BB-12. These results strongly supported a contribution of the EPS of P. parvulus 2.6 to attachment to colon epithelial cells. Therefore, the immunomodulatory properties of the 2.6R and 2.6NR strains on macrophages were investigated. To that end, proinflammatory M1 and anti-inflammatory M2 macrophages were generated from human peripheral blood mononuclear cells, using 1,000 U ml⁻¹ granulocyte-macrophage colony-stimulating factor or macrophage colony-stimulating factor (10 ng ml⁻¹), as previously described (31), and their cytokine responses after exposure to the ropy and nonropy strains for 18 h were determined by means of enzyme-linked immunosorbent assay (34), using antibodies against tumor necrosis factor alpha (TNF-α), interleukin-8 (IL-8), and IL-10 (ELISA set; ImmunoTools, Friesoythe, Germany). With regard to proinflammatory cytokines, both bacterial strains induced high levels of TNF-α (Fig. ​(Fig.4A)4A) and IL-8 (Fig. ​(Fig.4B)4B) on M1 macrophages but had a minor (TNF-α) or absent (IL-8) effect on M2 macrophages. Both strains also induced the production of the anti-inflammatory cytokine IL-10, although the extent of cytokine release was higher in M2 macrophages (Fig. ​(Fig.4C).4C). However, the levels of TNF-α and IL-8 produced by M1 macrophages were higher in response to the 2.6NR strain (Fig. 4A and B), thus implying that elimination of the pPP2 plasmid, which encodes the P. parvulus 2.6 EPS, triggers a higher level of proinflammatory cytokines in M1 macrophages. Although a contribution by other, unknown products encoded by pPP2 cannot be ruled out, these results strongly suggest that the presence of EPS in P. parvulus 2.6 counteracts the proinflammatory activation of M1 macrophages in response to the bacterium. Consequently, EPS might act by (i) preventing recognition by M1 macrophage-expressed Toll-like receptor 2 (TLR2) of the major gram-positive pathogen-associated molecular patterns lipoteichoic acid and peptidoglycan (13) or (ii) inhibiting the intracellular signaling cascade initiated upon TLR2 engagement by both cell wall components. If the latter explanation is true, then EPS could be considered a bona fide beneficial immunomodulator.Open in a separate windowFIG. 3.Adhesion of bacterial strains to Caco-2 cells. Adhesion levels are expressed as percentages of the total number of bacteria (adhered plus unadhered) detected after exposure to Caco-2 cells for 1 h. Each adhesion assay was conducted in triplicate. The values are the means for three independent experiments in which three independent determinations were performed. (Insets) Prior to the adhesion experiments, bacteria were analyzed in a JEOL 1230 transmission electron microscope operated at 100 kV.Open in a separate windowFIG. 4.Cytokine responses of macrophages to P. parvulus strains. M1 and M2 macrophages were either left untreated (basal 18 h) or stimulated with lipopolysaccharide (LPS) from Escherichia coli O55:B5 (Sigma, Barcelona, Spain) at 10 ng ml⁻¹, P. parvulus 2.6 (2.6R), or its nonropy mutant (2.6NR), and the levels of TNF-α (A), IL-8 (B), and IL-10 (C) released were determined. Each determination was performed in triplicate, and the means and standard deviations are shown.In summary, the comparative analysis of β-glucan-producing and nonproducing strains performed in this work has provided insights into the debated issues of probiotic properties of EPS-producing LAB (3) and the role of EPS in the immunomodulation of macrophages (20).Our results indicate that P. parvulus 2.6 should be able to tolerate human GI stress and thus could be metabolically active in the colon. This supports the detected changes in short-chain fatty acid formation in the cecum, distal colon, and feces of rats fed with fermented oat-based food elaborated with this bacterium (15).The EPS produced and secreted by LAB seem to be implicated in cellular recognition and the formation of biofilms, e.g., the glucans and fructans of Streptococcus mutans play an important role in the adhesion of this bacterium to the tooth surface and in the formation of dental plaque (14), thus facilitating bacterial colonization and protection against hostile habitats. However, the involvement of these biopolymers in bacterial adhesion to the intestinal epithelium in vivo has not yet been validated (25). The results of Dols-Lafargue et al. (4) show the contribution of the 2-substituted (1,3)-β-d-glucan to biofilm formation by LAB, and our results strongly support the involvement of this EPS in adhesion to human epithelial cells.There are several reports that indicate a host immune response to LAB in which the involvement of various surface components of these bacteria is demonstrated (10, 22, 28). It has been reported that the suppressive effect on activation of macrophages exerted by Lactobacillus casei strain Shirota is associated with its EPS content (34). It is also known that the (1,3)-β-d-glucans can promote antitumor and antimicrobial activity by activating macrophages, dendritic cells, or other leukocytes (1, 24). The immune responses to eukaryote-derived glucans [either linear or with (1,6) branches] and to prokaryotic linear curdlan, used for making functional foods (tofu), have been characterized, and the activities of these molecules have been correlated with their chemical structure, molecular weight, and conformation (2, 23). However, the immunomodulating properties of β-d-glucans with (1,2) branches have not been reported until now. Therefore, this is the first report that a 2-substituted (1,3)-β-d-glucan affects the activation of human macrophages. Further experiments are in progress to characterize the influence of this β-d-glucan and of P. parvulus 2.6 on the immune response.     

The location of (1→3)-β-glucans in the walls of pollen tubes of Nicotiana alata using a (1→3)-β-glucan-specific monoclonal antibody

The location of the (13)--glucan, callose, in the walls of pollen tubes in the style of Nicotiana alata Link et Otto was studied using specific monoclonal antibodies. The antibodies were raised against a laminarinhaemocyanin conjugate. One antibody selected for further characterization was specific for (13)--glucans and showed no binding activity against either a cellopentaose-bovine serum albumin (BSA) conjugate or a (13, 14)--glucan-BSA conjugate. Binding was inhibited by (13)--oligoglucosides (DP, 3–6) with maximum competition being shown by laminaripentaose and laminarihexaose, indicating that the epitope included at least five (13)--linked glucopyranose residues. The monoclonal antibody was determined to have an affinity constant for laminarihexaose of 2.7. 10⁴M^–1. When used with a second-stage gold-labelled, rabbit anti-mouse antibody, the monoclonal antibody probe specifically located the (13)--glucan in the inner wall layer of thin sections of the N. alata pollen tubes.Abbreviations BSA bovine serum albumin - PBS phosphate-buffered saline - ELISA enzyme linked immunosorbent assay - DP degree of polymerization - PVC polyvinyl chloride P.J.M. is an Australian Postdoctoral Research Fellow. We wish to thank Joan Hoogenraad for her technical assistance with the tissue culture, and Althea Wright for her assistance in the preparation of this paper.     

Study of the influence of temperature on the dynamics of the catalytic cleft in 1,3-1,4-β-glucanase by molecular dynamics simulations

The dependence of some molecular motions in the enzyme 1,3-1,4-β-glucanase from Bacillus licheniformis on temperature changes and the role of the calcium ion in them were explored. For this purpose, two molecular dynamics simulated trajectories along 4 ns at low (300 K) and high (325 K) temperatures were generated by the GROMOS96 package. Several structural and thermodynamic parameters were calculated, including entropy values, solvation energies, and essential dynamics (ED). In addition, thermoinactivation experiments to study the influence of the calcium ion and some residues on the activity were conducted. The results showed the release of the calcium ion, which, in turn, significantly affected the movements of loops 1, 2, and 3, as shown by essential dynamics. These movements differ at low and high temperatures and affect dramatically the activity of the enzyme, as observed by thermoinactivation studies. The first two authors contributed equally to this work     

Biotransformation of ent-kaur-16-ene and ent-trachylobane 7β-acetoxy derivatives by the fungus Gibberella fujikuroi (Fusarium fujikuroi)

Candol A (7β-hydroxy-ent-kaur-16-ene) (6) is efficiently transformed by Gibberella fujikuroi into the gibberellin plant hormones. In this work, the biotransformation of its acetate by this fungus has led to the formation of 7β-acetoxy-ent-kaur-16-en-19-oic acid (3), whose corresponding alcohol is a short-lived intermediate in the biosynthesis of gibberellins and seco-ring ent-kaurenoids in this fungus. Further biotransformation of this compound led to the hydroxylation of the 3β-positions to give 7β-acetoxy-3β-hydroxy-ent-kaur-16-en-19-oic acid (14), followed by a 2β- or 18-hydroxylation of this metabolite. The incubation of epicandicandiol 7β-monoacetate (7β-acetoxy-18-hydroxy-ent-kaur-16-ene) (10) produces also the 19-hydroxylation to form the 18,19 diol (20), which is oxidized to give the corresponding C-18 or C-19 acids. These results indicated that the presence of a 7β-acetoxy group does not inhibit the fungal oxidation of C-19 in 7β-acetoxy-ent-kaur-16-ene, but avoids the ring B contraction that leads to the gibberellins and the 6β-hydroxylation necessary for the formation of seco-ring B ent-kaurenoids. The biotransformation of 7β-acetoxy-ent-trachylobane (trachinol acetate) (27) only led to the formation of 7β-acetoxy-18-hydroxy-ent-trachylobane (33).     

Adenovirus-mediated expression of pig α(1,3) galactosyltransferase reconstructs Gal α(1,3) Gal epitope on the surface of human tumor cells

INTRODUCTIONGal a(1, 3) Gal (gal epitope) is a carbohydrate epitope, which is produced in large amounton the cells of pigs, mice and New World monkey(monkey of South America) by the glycosylationenzyme G alal 1 ) 4G IcNAc3- a- D- galactosyltransferase[or(1, 3)GT; EC2.4.1.511111. This enzyme is active in the Golgi appaxatus of cells and transfers galactose from the sugandonor uridine diphoSphate galactose (UDP-galactose) to the acceptor Nacetyllactosamine residue (Galaal-4GlcNAc-R…     

Biotransformation of the diterpene 15β-hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene by the fungus Fusarium fujikuroi

15β-Hydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (4) was biotransformed by the fungus Fusarium fujikuroi into 3α,11β,15β-trihydroxy-18(4→3)-abeo-ent-kaur-4(19),16-diene (5). The hydroxylation at C-3(α) in this diterpene reminds a similar reaction that occurs at C-13 in the biosynthesis of gibberellic acid in this fungus. The presence of the 15β-alcohol in the substrate directs the second hydroxylation at C-11(β), which had been observed in the incubation of ent-kaur-16-ene derivatives with this fungus when the C-19 hydroxylation was inhibited by the existence in the molecule of a 3α-OH or 3-oxo group. We also show that the angelate of the substrate is an undescribed natural product now identified as a component of the plant Distichoselinum tenuifolium.     

Identification of the site of glycation of γ-II-crystallin by (14C)-fructose

Cataract formation in diabetes may be via non-enzymic glycosylation (glycation) of lens proteins due to increased concentrations of sugars present in the lenses of diabetic patients. The objective of this project was to identify the site(s) of glycation of bovine γ-II-crystallin by [¹⁴C]fructose. γ-II-crystallin was isolated from soluble lens nucleus proteins by gel chromatography, followed by ion-exchange chromatography and was then glycated by incubation with [¹⁴C]fructose. Radioactively labelled γ-II-crystallin was cleaved with trypsin. Affinity chromatography of the tryptic peptides gave a single main peak containing the majority of the radioactivity. This indicated that fructose had reacted at a single site on the protein. Amino acid analysis of this peptide showed it to contain only lysine and a trace amount of glycine. By relating the results of the amino acid analysis to the amino acid sequence of γ-II-crystallin, it was concluded that the labelled peptide corresponded to the N-terminal dipeptide. The site of glycation of bovine γ-II-crystallin by fructose was thereby identified as the α-NH₂ group of the N-terminal glycine.