laminaran and curdlan

1,3-β-Glucans are a class of natural polysaccharides with unique pharmacological properties and the ability to form single- and triple-helical structures that can be formed into resilient gels with the application of heat and humidity. The pharmacological capabilities of 1,3-β-glucans include the impartation of tumor inhibition, resistance to infectious disease, and improvements in wound healing. Curdlan is a linear 1,3-β-glucan that has been used extensively to study the nature of these helical structures and gels, and Curdlan sulfates have found ongoing application in the inhibition of HIV infection. 1,3-β-Glucan gels have been used in food science as stabilizers and encapsulating agents, in nanoscience as scaffolds to build nanofibers and nanowires, and in drug delivery to form nanoparticles and create helical micelles encapsulating polynucleotides. 1,3-β-Glucans are beginning to have enormous significance due to their dual nature as structure-forming agents and pharmacological substances, and research is especially focused on the application of these polymers in animal nutrition and drug delivery.

Topics: Animals; Anti-Infective Agents; Anticarcinogenic Agents; beta-Glucans; Carbohydrate Conformation; Drug Delivery Systems; Gels; HIV Infections; Hot Temperature; Humans; Immunologic Factors; Molecular Structure; Polysaccharides, Bacterial; Wound Healing

Heparin has been the drug of choice in clinical pre-surgical and post-surgical prophylaxis of thrombotic events. However, because of its side-effects, such as bleeding and other disadvantages (i.e. chemical inhomogeneity and variability of its physiological activities), alternatives to heparin are an important field of research. A necessary procedure in the development of new drugs is the evaluation of structure-activity relationships. Genuine neutral polysaccharides were chemically modified and examined for their anticoagulant activities. The linear beta-1,3-glucan curdlan, an easily available bacterial polysaccharide, served as the basic polymer. It could be established that the anticoagulant activity was dependent on the degree of sulfation and the molecular weight. For heparin, the sulfation pattern, i.e. the actual location of the sulfate groups along the heparin chain, was of importance in addition to the degree of sulfation. Therefore, we investigated whether there was also a relationship between the substitution pattern of the curdlan sulfates and their anticoagulant activity. For determination of the substitution pattern of the sulfated polysaccharides, a method was developed that is based on synthesis of the partially alkylated alditol acetates of the polymer and examination of these derivatives using combined gas chromatography-mass spectrometry. In addition to the analytical data, the structure-activity relationship of anticoagulative curdlan sulfates is presented.

Topics: Animals; beta-Glucans; Blood Coagulation Tests; Dimethylformamide; Drug Evaluation; Fibrinolytic Agents; Gas Chromatography-Mass Spectrometry; Glucans; Glycosaminoglycans; Heparin; Heparinoids; Humans; Methylation; Molecular Weight; Neovascularization, Pathologic; Polysaccharides; Structure-Activity Relationship; Sulfates; Thrombosis

RNA aptamersare nucleic acids that are obtained using the systematic evolution of ligands by exponential enrichment (SELEX) method. When using conventional selection methods to immobilize target proteins on matrix beads using protein tags, sequences are obtained that bind not only to the target proteins but also to the protein tags and matrix beads. In this study, we performed SELEX using β-1,3-glucan recognition protein (GRP)-tags and curdlan beads to immobilize the acute myeloid leukaemia 1 (AML1) Runt domain (RD) and analysed the enrichment of aptamers using high-throughput sequencing. Comparison of aptamer enrichment using the GRP-tag and His-tag suggested that aptamers were enriched using the GRP-tag as well as using the His-tag. Furthermore, surface plasmon resonance analysis revealed that the aptamer did not bind to the GRP-tag and that the conjugation of the GRP-tag to RD weakened the interaction between the aptamer and RD. The GRP-tag could have acted as a competitor to reduce weakly bound RNAs. Therefore, the affinity system of the GRP-tagged proteins and curdlan beads is suitable for obtaining specific aptamers using SELEX.

Topics: Aptamers, Nucleotide; beta-Glucans; Glucans; Ligands; RNA

Heterologous endo-xanthanase (EX) from the thermophilic planktomycete Thermogutta terrifontis strain was obtained using Penicillium verruculosum 537 (ΔniaD) expression system with the cellobiohydrolase 1 gene promoter. Homogeneous EX with a molecular weight of 23.7 kDa (pI 6.5) was isolated using liquid chromatography methods. This xanthan degrading enzyme also possesses the enzymatic activity towards CM-cellulose, β-glucan, curdlan, lichenan, laminarin, galactomannan, xyloglucan but not towards p-nitrophenyl derivatives of β-D-glucose, mannose and cellobiose. The temperature and pH optima of EX were 55°C and 4.0, respectively; the enzyme exhibited 90% of its maximum activity in the temperature range 50-60°C and pH 3-5.

Topics: Bacterial Proteins; beta-Glucans; Cellulose; Cloning, Molecular; Galactose; Glucans; Glycoside Hydrolases; Hot Temperature; Hydrogen-Ion Concentration; Mannans; Planctomycetales; Planctomycetes; Substrate Specificity; Talaromyces; Xylans

There is evidence that major components of the fungi cell wall not only define fungal properties and survival but also are responsible for their biological activities. Some data indicate that structural components of the fungal cell wall exert stimulatory/modulatory effects on immunocompetent cells acting as pathogen-associated molecular patterns (PAMPs). Fungal components can influence the activity of certain immune cell populations by affecting cell maturation and proliferation, promoting phagocytosis, cytotoxic activity, and cell migration, as well as production of various mediators. However, there is little information available concerning the impact of fungal-derived components on peripheral blood mononuclear cell (PBMC) activation. The aim of this study was to determine whether certain fungi-associated molecules, i.e., β-(1,3)-glucans (zymosan and curdlan) and mannan activate in vitro human PBMCs to synthesize cytokines, including chemokines. We documented that PBMCs, in response to stimulation with zymosan, curdlan, and mannan, express cytokines IFN-γ and GM-CSF, and chemokine CCL3, both at protein and transcript levels, as well as cytokine IL-1β and chemokine CXCL8, at mRNA level. Our observations support the idea that fungal-derived components can activate immune cells, including PBMCs, by stimulation of cytokine/chemokine production. A thorough understanding of this interaction is of prime importance since it influence both pathophysiological and immune processes as well as anti-fungal defense mechanisms.

Topics: beta-Glucans; Cells, Cultured; Cytokines; Fungi; Glucans; Humans; Lectins, C-Type; Leukocytes, Mononuclear; Mannans; Oxazines; Protein Kinase Inhibitors; Pyridines; RNA, Messenger; Syk Kinase; Zymosan

Water-soluble β-1,3-glucan (w-glucan) prepared from curdlan is reported to possess various bioactive and medicinal properties. To develop an efficient and cost-effective microbial fermentation method for the direct production of w-glucan, a coupled fermentation system of Agrobacterium sp. and Trichoderma harzianum (CFS-AT) was established. The effects of Tween-80, glucose flow rate, and the use of a dissolved oxygen (DO) control strategy on w-glucan production were assessed. The addition of 10 g L

Topics: Agrobacterium; Antineoplastic Agents; beta-Glucans; Cell Line, Tumor; Fermentation; Glucose; Humans; Hydrolysis; Industrial Microbiology; Neoplasms; Oxygen; Polysorbates; Solubility; Trichoderma; Water

C-type lectin receptors (CLRs) comprise a large family of immunoreceptors that recognize polysaccharide ligands exposed on pathogen surfaces and are conserved among mammals. However, interspecies differences in their ligand spectrums are not fully understood. Dectin-1 is a well-characterized CLR that recognizes β-glucan. We report here that seaweed-derived fucan activates cells expressing human Dectin-1 but not mouse Dectin-1. Low-valency β-glucan components within fucan appeared to be responsible for this activation, as the ligand activity was eliminated by β-glucanase treatment. The low-valency β-glucan laminarin also acted as an agonist for human Dectin-1 but not for mouse Dectin-1, whereas the high-valency β-glucan curdlan activated both human and mouse Dectin-1. Reciprocal mutagenesis analysis revealed that the ligand-binding domain of human Dectin-1 does not determine its unique sensitivity to low-valency β-glucan. Rather, we found that its intracellular domain renders human Dectin-1 reactive to low-valency β-glucan ligand. Substitution with two amino acids, Glu

Topics: Amino Acid Substitution; Animals; beta-Glucans; Cell Line; Glucans; Humans; Lectins, C-Type; Mice; Mutagenesis; Mutation, Missense; Polysaccharides; Protein Domains; Species Specificity; Substrate Specificity

Spondyloarthritides (SpA) occur in 1% of the population and include ankylosing spondylitis (AS) and arthropathy of inflammatory bowel disease (IBD), with characteristic spondylitis, arthritis, enthesitis, and IBD. Genetic studies implicate interleukin-23 (IL-23) receptor signaling in the development of SpA and IBD, and IL-23 overexpression in mice is sufficient for enthesitis, driven by entheseal-resident T cells. However, in genetically prone individuals, it is not clear where IL-23 is produced and how it drives the SpA syndrome, including IBD or subclinical gut inflammation of AS. Moreover, it is unclear why specific tissue involvement varies between patients with SpA. We undertook this study to determine the location of IL-23 production and its role in SpA pathogenesis in BALB/c ZAP-70(W163C)-mutant (SKG) mice injected intraperitoneally with β-1,3-glucan (curdlan).. Eight weeks after curdlan injection in wild-type or IL-17A(-/-) SKG or BALB/c mice, pathology was scored in tissue sections. Mice were treated with anti-IL-23 or anti-IL-22. Cytokine production and endoplasmic reticulum (ER) stress were determined in affected organs.. In curdlan-treated SKG mice, arthritis, enthesitis, and ileitis were IL-23 dependent. Enthesitis was specifically dependent on IL-17A and IL-22. IL-23 was induced in the ileum, where it amplified ER stress, goblet cell dysfunction, and proinflammatory cytokine production. IL-17A was pathogenic, while IL-22 was protective against ileitis. IL-22+CD3- innate-like cells were increased in lamina propria mononuclear cells of ileitis-resistant BALB/c mice, which developed ileitis after curdlan injection and anti-IL-22.. In response to systemic β-1,3-glucan, intestinal IL-23 provokes local mucosal dysregulation and cytokines driving the SpA syndrome, including IL-17/IL-22-dependent enthesitis. Innate IL-22 production promotes ileal tolerance.

Topics: Animals; Antibodies; beta-Glucans; Disease Models, Animal; Endoplasmic Reticulum Stress; Female; Ileitis; Immune Tolerance; Interleukin-17; Interleukin-22; Interleukin-23 Subunit p19; Interleukins; Intestinal Mucosa; Intestines; Male; Mice; Mice, Inbred BALB C; Mice, Knockout; Receptors, Interleukin; Spondylarthritis

Schizophyllan is a homoglucan produced by the fungus Schizophyllum commune, with a β-1,3-linked backbone and β-1,6-linked side chains of single glucose units at every other residue. Schizophyllan is commercially produced for pharmaceutical and cosmetics uses. However, surprisingly little information is available on the biodegradation of schizophyllan. Enzymes that attack schizophyllan could be useful for controlled modifications of the polymer for novel applications. Enrichment cultures were used to isolate 20 novel fungal strains from soil samples, capable of growing on schizophyllan as a sole carbon source. Three additional strains were isolated as contaminants of stored schizophyllan solutions. Strains showing the highest levels of β-glucanase activity were identified as Penicillium simplicissimum, Penicillium crustosum, and Hypocrea nigricans. β-glucanases also showed activity against the similar β-glucans, laminarin and curdlan. By comparison, commercial β-glucanase from Trichoderma longibrachiatum and laminarinase from Trichoderma sp. showed lower specific activities toward schizophyllan than most of the novel isolates. β-glucanases from P. simplicissimum and H. nigricans exhibited temperature optima of 60°C and 50°C against schizophyllan, respectively, with broad pH optima around pH 5.0. Partial purifications of β-glucanase from P. simplicissimum and P. crustosum demonstrated the presence of multiple active endoglucanase species, including a 20-25 kD enzyme from P. simplicissimum.

Topics: Aspergillus; beta-Glucans; Fungal Proteins; Glucan 1,3-beta-Glucosidase; Glucans; Hydrogen-Ion Concentration; Hydrolysis; Hypocrea; Penicillium; Polysaccharides; Schizophyllum; Sizofiran; Soil Microbiology; Substrate Specificity; Temperature; Trichoderma

Streptomyces sp Mo endo-β-1,3-glucanase was found to have hydrolyzing activity toward curdlan and released laminarioligosaccharides selectively. The molecular weight was estimated to be 36000 Da and its N-terminal amino acid sequence was VTPPDISVTN. The optimal pH was 6 and the enzyme was found to be stable from pH 5 to 8. The optimal temperature was 60 °C and the activity was stable below 50 °C. The enzyme hydrolyzed selectively curdlan containing only β-1,3 linkages. The enzyme had 89% relative activity toward Laminaria digitata laminarin, which contains a small amount of β-1,6 linkages compared with curdlan, while Eisenia bicyclis laminarin with a higher amount of β-1,6-linkages, was not hydrolyzed. Mo enzyme adsorbed completely on curdlan powder. The enzymatic hydrolysis of curdlan powder resulted in the accumulation of laminaribiose (yield 81.7%). Trisaccharide was inevitably released from the hydrolysis of laminarioligosaccharides with 5 to 7 degrees of polymerization (DP). Although the enzyme cleaved off disaccharide (DP 2) from tetrasaccharide (DP 4), the reaction rate was lower than those of DP 5 to 7. The results indicated that the active site of Mo endo-β-1,3-glucanase can efficiently recognize glucosyl residue chain of greater than DP 5 and hydrolyzes the β-1,3 linkage between the 3rd and 4th glucosyl residue.

Topics: beta-Glucans; Disaccharides; DNA, Bacterial; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Hydrogen-Ion Concentration; Hydrolysis; Molecular Weight; Polysaccharides; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Streptomyces; Substrate Specificity; Temperature

Interleukin-23 (IL-23), a cytokine produced primarily by dendritic cells, is involved in host defense against gut pathogens and promotes innate immunity and inflammatory responses through the IL-23/interleukin-17 axis. We previously reported that extracts from edible mushrooms enhanced antimicrobial α-defensin production n HL60 cells. Because IL-23 is involved in defensin production, we hypothesized that edible mushrooms may modulate its secretion and gut inflammation. Eight-week-old C57BL/6 mice were fed the AIN76 diet or the same diet supplemented with 5% white button (WBM), portabella, or shiitake mushrooms. To assess in vivo and in vitro cytokine secretion, 7 to 8 mice per group received 3% dextran sodium sulfate (DSS) in drinking water during the last 5 days of the 6-week feeding period. To delineate the mechanisms by which mushrooms alter IL-23 secretion, J.744.1 cells were incubated with (100 μg/mL) WBM, portabella, and shiitake extracts without and with 100 μg/mL curdlan (a dectin-1 agonist) or 1 mg/mL laminarin (a dectin-1 antagonist). The dectin-1 receptor is a pattern-recognition receptor found in phagocytes, and its activation promotes antimicrobial innate immunity and inflammatory responses. In DSS-untreated mice, mushrooms significantly increased IL-23 plasma levels but decreased those of interleukin-6 (IL-6) (P < .05). In DSS-treated mice, mushroom-supplemented diets increased IL-6 and IL-23 levels (P < .05). Mushroom extracts potentiated curdlan-induced IL-23 secretion, and mushroom-induced IL-23 secretion was not blocked by laminarin in vitro, suggesting the involvement of both dectin-1-dependent and dectin-1-independent pathways. Although all mushrooms tended to increase IL-6 in the colon, only WBM and shiitake tended to increase IL-23 levels. These data suggest that edible mushrooms may enhance gut immunity through IL-23.

Topics: Animals; Anti-Infective Agents; beta-Glucans; Cell Line; Colitis; Dextran Sulfate; Dietary Supplements; Female; Glucans; Immunity, Innate; Inflammation; Interleukin-17; Interleukin-23; Interleukin-6; Lectins, C-Type; Macrophages; Mice; Mice, Inbred C57BL; Organ Size; Polysaccharides; Regression Analysis; Shiitake Mushrooms; Thymus Gland; Up-Regulation

Over the years several β-glucan transferases from yeast and fungi have been reported, but enzymes with such an activity from bacteria have not been characterized so far. In this work, we describe the cloning and expression of genes encoding β-glucosyltransferase domains of glycosyl hydrolase family GH17 from three species of proteobacteria: Pseudomonas aeruginosa PAO1, P. putida KT2440 and Azotobacter vinelandii ATCC BAA-1303. The encoded enzymes of these GH17 domains turned out to have a non-Leloir trans-β-glucosylation activity, as they do not use activated nucleotide sugar as donor, but transfer a glycosyl group from a β-glucan donor to a β-glucan acceptor. More particularly, the activity of the three recombinant enzymes on linear (β1 → 3)-linked gluco-oligosaccharides (Lam-Glc(4-9)) and their corresponding alditols (Lam-Glc(4-9)-ol) was studied. Detailed structural analysis, based on thin-layer chromatography, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, electrospray ionization mass spectrometry, and 1D/2D (1)H and (13)C nuclear magnetic resonance data, revealed diverse product spectra. Depending on the enzyme used, besides (β1 → 3)-elongation activity, (β1 → 4)- or (β1 → 6)-elongation, or (β1 → 6)-branching activities were also detected.

Topics: Azotobacter vinelandii; beta-Glucans; Enzyme Assays; Glucans; Glucosyltransferases; Models, Molecular; Molecular Structure; Polysaccharides; Protein Conformation; Pseudomonas aeruginosa; Pseudomonas putida

β-Glucans are well known for their immunomodulatory capacities in humans and mice. For this reason, together with the European ban on growth-promoting antibiotics, β-glucans are intensively used in pig feed. However, as shown in the present study, there is much variation in the stimulatory capacities of β-glucans from different sources. Since dendritic cells (DCs) are the first cells that are encountered after an antigen is taken up by the intestinal epithelial cell barrier, we decided to investigate the effect of two concentrations (5 and 10 μg/ml) of five commercial β-glucan preparations, differing in structure and source, on porcine monocyte-derived dendritic cells (MoDCs). Although all β-glucans gave rise to a significant reduction of the phagocytic activity of DCs, only Macrogard induced a significant phenotypic maturation. In addition to Macrogard, zymosan, another β-glucan derived from Saccharomyces cerevisiae, and curdlan also significantly improved the T-cell-stimulatory capacity of MoDCs. Most interesting, however, is the cytokine secretion profile of curdlan-stimulated MoDCs, since only curdlan induced significant higher expression levels of interleukin-1β (IL-1β), IL-6, IL-10, and IL-12/IL-23p40. Since the cytokine profile of DCs influences the outcome of the ensuing immune response and thus may prove valuable in intestinal immunity, a careful choice is necessary when β-glucans are used as dietary supplement.

Topics: Animals; beta-Glucans; Cell Differentiation; Cytokines; Dendritic Cells; Glucans; Humans; Interleukins; Lymphocyte Activation; Mice; Monocytes; Polysaccharides; Swine; T-Lymphocytes; Zymosan

Human phagocyte-specific chitotriosidase is part of innate immunity and shows anti-fungal activity towards chitin-containing fungi. We investigated the effect of stimulation of the C-type lectin receptor dectin-1 by beta-1,3-glucan (curdlan) on chitotriosidase expression and release by human phagocytes. We observed that curdlan triggers chitotriosidase release from human neutrophils. In addition, we show that curdlan impairs chitotriosidase induction in monocytes. Finally, curdlan temporarily induces chitotriosidase in enzyme-expressing monocyte-derived macrophages, followed by reduction of chitotriosidase expression after prolonged stimulation. These data on regulation of phagocyte-specific chitotriosidase following curdlan recognition support an important role of chitotriosidase in the elimination of chitin-containing pathogens.

Topics: beta-Glucans; Cell Differentiation; Chitin; Hexosaminidases; Humans; Immunity, Innate; Lectins, C-Type; Macrophages; Membrane Proteins; Monocytes; Nerve Tissue Proteins; Neutrophils; Phagocytes

Curdlan, an extracellular bacterial polysaccharide, is a linear beta-1,3-glucan. Previously, we developed Curdlan-oligo (CRDO). We investigated its effect on the production of cytokines in leukocytes from mice, and compared its activity with that of SCG, a 6-branched 1,3-beta-glucan.. Splenocytes from DBA/2 mice were cultured with CRDO or SCG (0, 1, 10 or 100 microg/ml) in vitro, and then the supernatants were collected to measure cytokines. Bone marrow-derived dendritic cells (BMDCs) were cultured with CRDO (0, 1, 10 or 100 ng/ml) in vitro, and then the supernatant was collected to measure cytokines.. SCG stimulated splenocytes in DBA/2 mice to produce GM-CSF, IFN-gamma and TNF-alpha. CRDO induced production of GM-CSF and IFN-gamma, but not TNF-alpha. The amounts of GM-CSF and IFN-gamma were small compared with those produced in response to SCG. The effect of SCG on TNF-alpha production was partially inhibited by CRDO. In bone marrow-derived dendritic cells, CRDO induced production of TNF-alpha and IL-6.. Taken together, these results suggest that CRDO stimulated mouse leukocytes to induce the production of cytokines, and the mechanism of the effect of CRDO on leukocytes is different from that of SCG.

Topics: Animals; beta-Glucans; Bone Marrow Cells; Cells, Cultured; Cytokines; Dendritic Cells; Humans; Leukocytes; Male; Mice; Mice, Inbred DBA; Polysaccharides, Bacterial; Spleen

A critical component of host innate immunity is recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Dectin-1 is the primary PRR for exogenous beta-glucan, a component of fungal and bacterial cell walls. A previous study conducted in our laboratory demonstrated that administration of beta-glucan as a feed additive resulted in increased innate immune function of neonatal chickens, suggesting that chickens possess a Dectin-1-like beta-glucan receptor. In the present study, we demonstrated that heterophils and peripheral blood mononuclear cells (PBMCs) from day-old chicks had a significant increase in the generation of reactive oxygen species (ROS) following stimulation with the Dectin-1 specific agonist, curdlan. Pretreatment of heterophils and PBMCs with laminarin, a beta-glucan receptor blocking agent and specific inhibitor of Dectin-1 activity, significantly reduced the curdlan-induced ROS production. Together these data provide evidence for the first time of the presence of a functional Dectin-1-like beta-glucan receptor in chicken heterophils and PBMCs.

Topics: Animals; Animals, Newborn; beta-Glucans; Chickens; Glucans; Granulocytes; Immunity, Innate; In Vitro Techniques; Lectins, C-Type; Leukocytes, Mononuclear; Membrane Proteins; Nerve Tissue Proteins; Polysaccharides; Reactive Oxygen Species; Receptors, Immunologic; Respiratory Burst

In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.

Topics: Animals; beta-Glucans; Binding Sites; Cellulase; Cellvibrio; Conserved Sequence; Endo-1,4-beta Xylanases; Evolution, Molecular; Glucans; Horseshoe Crabs; Lectins; Polysaccharides; Proteoglycans

The main cytokine induced by the interaction of oral epithelial cells with C. glabrata is granulocyte monocyte colony-stimulating factor (GM-CSF); however, the mechanisms regulating this response are unknown. Based on previously published information on the interactions of C. albicans with oral epithelial cells, we hypothesized that interaction with viable C. glabrata triggers GM-CSF synthesis via NF-kappaB activation. We found that C. glabrata-induced GM-CSF synthesis was adhesion-dependent, enhanced by endocytosis, and required fungal viability. NF-kappaB activation was noted during interaction of epithelial cells with C. glabrata, and pre-treatment with an NF-kappaB inhibitor partly inhibited GM-CSF synthesis. Blocking TLR4 with anti-TLR4 antibody did not inhibit GM-CSF production. In contrast, an anti-CDw17 antibody triggered significant inhibition of NF-kappaB activation and GM-CSF synthesis. beta-glucans did not stimulate GM-CSF synthesis, suggesting that the CDw17/NF-kappaB/GM-CSF pathway may be beta-glucan-independent. This study provides new insights into the mechanism of GM-CSF induction by C. glabrata.

Topics: Antibodies; Antigens, CD; beta-Glucans; Candida glabrata; Cell Line; Cell Line, Tumor; Cytochalasin D; Endocytosis; Epithelial Cells; Glucans; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Lactosylceramides; Mouth Mucosa; NF-kappa B; Nucleic Acid Synthesis Inhibitors; Polysaccharides; Polysaccharides, Bacterial; Toll-Like Receptor 4; Zymosan

(1-->3)-beta-D-glucan is found in cell walls of some fungi, bacteria and plants. It plays a crucial role in bioaerosol-induced inflammatory reactions. To estimate the level of airborne (1-->3)-beta-D-glucan exposure, a monoclonal antibody-based two-site enzyme immunoassay (mAb-EIA) was developed. The results obtained with the mAb-EIA were compared with the results of a Limulus amoebocyte lysate-based assay for (1-->3)-beta-D-glucan. Three mAbs produced by mouse immunization with bovine serum albumin-conjugated laminarin were enriched by in vitro production in a modular mini-fermenter and affinity purified. Two mAbs were selected for the development of a two-site EIA specific for (1-->3)-beta-D-glucan. Different polysaccharides, fungal and plant seed extracts, and airborne inhalable dust from workplaces (poultry farms, pig stables, grain storage houses, and a laboratory animal facility) were sampled with portable pumps and measured with both the mAb-EIA and Glucatell assay. Using carboxymethylated curdlan as a standard, the mAb-EIA gave a steep dose-response curve for concentrations between 0.36-15 ng/ml. The mAb-EIA was specific for (1-->3)-beta-D-glucan and was sufficiently sensitive to detect (1-->3)-beta-D-glucan in airborne dust samples. In comparing the EIA results to the values obtained with the Glucatell assay, the correlation was found to be high (coefficient of correlation r(2)=0.91), and the mean ratio of the values was 1.7. Depending on the dust source, either the Glucatell assay or the mAb-EIA gave higher results. The mAb-EIA is sensitive enough to detect (1-->3)-beta-D-glucan in airborne dust samples collected with portable pumps. Thus, the assay is suited for the investigation of the health effects induced by exposure to this class of biologically active molecules.

Topics: Animals; Antibodies, Monoclonal; beta-Glucans; Dose-Response Relationship, Immunologic; Environmental Monitoring; Enzyme-Linked Immunosorbent Assay; Glucans; Humans; Hybridomas; Inhalation Exposure; Limulus Test; Mice; Mice, Inbred BALB C; Occupational Exposure; Particulate Matter; Polysaccharides; Proteoglycans; Reproducibility of Results

A cationic polysaccharide bearing a beta-1,3-glucan main-chain structure (CUR-N(+)) forms a complex with a hetero-sequence oligonucleotide, that is, a CpG ODN, and facilitates the transportation of the resultant complex into a murine macrophage-like cell J774.A1, which induces an efficient secretion of a cytokine (IL-12) as compared with that induced by conventional carriers such as poly(ethyleneimine) (PEI) and poly(L-lysine) (PLL).

Topics: Adjuvants, Immunologic; Animals; beta-Glucans; Cells, Cultured; Circular Dichroism; Cytokines; Drug Carriers; Macrophages; Mice; Microscopy, Confocal; Models, Biological; Oligodeoxyribonucleotides; Polysaccharides; Quaternary Ammonium Compounds

We synthesized a semiartificial beta-1,3-glucan, curdlan with dialkylaniline groups (CUR-DA), that bears chromophoric aromatic groups at its peripheral positions. Spectroscopic studies as well as microscopic observations indicate that CUR-DA adopts a triple-stranded helical structure in water- or methanol-rich solutions of dimethyl sulfoxide (DMSO). This triple-stranded helical structure exhibits high thermal stability and resistance to base, attributes that are similar to those of the triple-stranded helical structure of native beta-1,3-glucans such as schizophyllan. Moreover, we found that the stability of the triple-stranded helical structure can be easily modulated by solvent composition and metal-ion (Zn2+) binding. As beta-1,3-glucan polysaccharides are known to serve as "polymeric" hosts, including certain DNA molecules, carbon nanotubes, and conjugated polymers, and complexation occurs only with the single-stranded structure, this information is very useful for the creation of these attractive polymeric composites, the controlled release of DNA, and so on.

Topics: beta-Glucans; Circular Dichroism; Dimethyl Sulfoxide; Ions; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Molecular Conformation; Spectrophotometry, Ultraviolet; Zinc

(1-->3)-beta-D-Glucans, a cell wall component in most microfungi, are suggested to play a role in the development of respiratory and general symptoms in organic dust-related diseases. The mechanisms by which they induce these effects are, however, not clear. In the present study, mediator release and its potentiation by the (1-->3)-beta-D-glucan as well as by the (1-->6)-beta-D-glucan found in yeast and other fungi were therefore examined. Blood leucocytes from healthy volunteers and from patients allergic to house dust mite were incubated with (1-->3)-beta-D-glucans with increasing 1,6-branchings: curdlan [a linear (1-->3)-beta-D-glucan], laminarin and scleroglucan, and furthermore with pustulan, a linear (1-->6)-beta-D-glucan. Histamine release was not observed on exposure to the glucans only, but in the presence of anti-immunoglobulin E (IgE) antibody or specific antigens, all the glucans investigated led to an enhancement of the IgE-mediated histamine release. The glucans induced a significant potentiation of the mediator release when present at concentrations in the range of 2-5 x 10(-5) M. These results suggest that (1-->3)-beta-D-glucan as well as (1-->6)-beta-D-glucan aggravates IgE-mediated histamine release. Knowledge concerning the effects of glucans on immune responses may be of importance for understanding and treating inflammatory and allergic diseases.

Topics: Adult; Air Pollution, Indoor; Allergens; beta-Glucans; Cell Wall; Dust; Environmental Exposure; Fungi; Glucans; Histamine; Humans; Immunoglobulin E; In Vitro Techniques; Leukocytes; Middle Aged; Polysaccharides; Respiratory Hypersensitivity

Lipoproteins and molecules for pattern recognition are centrally important in the innate immune response of both vertebrates and invertebrates. Mammalian apolipoproteins such as apolipoprotein E (apoE) are involved in LPS detoxification, phagocytosis, and possibly pattern recognition. The multifunctional insect protein, apolipophorin III (apoLp-III), is homologous to apoE. In this study we describe novel roles for apoLp-III in pattern recognition and multicellular encapsulation reactions in the innate immune response, which may be of direct relevance to mammalian systems. It is known that apoLp-III stimulates antimicrobial peptide production in insect blood, enhances phagocytosis by insect blood cells (hemocytes), and binds and detoxifies LPS and lipoteichoic acid. In the present study we show that apoLp-III from the greater wax moth, Galleria mellonella, also binds to fungal conidia and beta-1,3-glucan and therefore may act as a pattern recognition molecule for multiple microbial and parasitic invaders. This protein also stimulates increases in cellular encapsulation of nonself particles by the blood cells and exerts shorter term, time-dependent, modulatory effects on cell attachment and spreading. All these responses are dose dependent, occur within physiological levels, and, with the notable exception of beta-glucan binding, are only observed with the lipid-associated form of apoLp-III. Preliminary studies also established a beneficial role for apoLp-III in the in vivo response to an entomopathogenic fungus. These data suggest a wide range of immune functions for a multiple specificity pattern recognition molecule and may provide a useful model for identifying further potential roles for homologous proteins in mammalian immunology, particularly in terms of fungal infections, pneumoconiosis, and granulomatous reactions.

Topics: Amino Acid Sequence; Animals; Apolipoproteins; beta-Glucans; Cell Adhesion; Cell Movement; Dimyristoylphosphatidylcholine; Glucans; Hemocytes; Hypocreales; Immunity, Innate; Insect Proteins; Larva; Microspheres; Molecular Sequence Data; Moths; Protein Binding; Spores, Fungal

Invertebrates, like vertebrates, utilize pattern recognition proteins for detection of microbes and subsequent activation of innate immune responses. We report structural and functional properties of two domains from a beta-1,3-glucan recognition protein present in the hemolymph of a pyralid moth, Plodia interpunctella. A recombinant protein corresponding to the first 181 amino-terminal residues bound to beta-1,3-glucan, lipopolysaccharide, and lipoteichoic acid, polysaccharides found on cell surfaces of microorganisms, and also activated the prophenoloxidase-activating system, an immune response pathway in insects. The amino-terminal domain consists primarily of an alpha-helical secondary structure with a minor beta-structure. This domain was thermally stable and resisted proteolytic degradation. The 290 residue carboxyl-terminal domain, which is similar in sequence to glucanases, had less affinity for the polysaccharides, did not activate the prophenoloxidase cascade, had a more complicated CD spectrum, and was heat-labile and susceptible to proteinase digestion. The carboxyl-terminal domain bound to laminarin, a beta-1,3-glucan with beta-1,6 branches, but not to curdlan, a beta-1,3-glucan that lacks branching. These results indicate that the two domains of Plodia beta-1,3-glucan recognition protein, separated by a putative linker region, bind microbial polysaccharides with differing specificities and that the amino-terminal domain, which is unique to this class of pattern recognition receptors from invertebrates, is responsible for stimulating prophenoloxidase activation.

Topics: Animals; beta-Glucans; Circular Dichroism; DNA, Complementary; Dose-Response Relationship, Drug; Electrophoresis, Polyacrylamide Gel; Enzyme Activation; Escherichia coli; Gene Deletion; Glucans; Immunity, Innate; Lipopolysaccharides; Moths; Polysaccharides; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Recombinant Proteins; Surface Plasmon Resonance; Teichoic Acids; Temperature; Time Factors

Sponges (phylum Porifera) live in a symbiotic relationship with microorganisms, primarily bacteria. Until now, molecular proof for the capacity of sponges to recognize fungi in the surrounding aqueous milieu has not been available. Here we demonstrate, for the demosponge Suberites domuncula (Porifera, Demospongiae, Hadromerida), a cell surface receptor that recognizes (1-->3)-beta-D-glucans, e.g. curdlan or laminarin. This receptor, the (1-->3)-beta-D-glucan-binding protein, was identified and its cDNA analysed. The gene coding for the 45 kDa protein was found to be upregulated in tissue after incubation with carbohydrate. Simultaneously with the increased expression of this gene, two further genes showed an elevated steady state level of expression; one codes for a fibrinogen-like protein and the other for the epidermal growth factor precursor. Expression of the (1-->3)-beta-D-glucan-binding protein and the fibrinogen-like protein occurred in cells on the sponge surface, in the pinacoderm. By Western blotting, the product of the fibrinogen-like protein gene was identified, the recombinant protein isolated, and antibodies raised to this protein. Their application revealed that a 5 kDa factor is produced, which is apparently processed from the 77 kDa epidermal growth factor precursor. Finally, we provided evidence that a tyrosine kinase pathway is initiated in response to exposure to D-glucan; its phosphorylation activity could be blocked by aeroplysinin. In turn, the increased expression of the downstream genes was suppressed. We conclude that sponges possess a molecular mechanism for recognizing fungi via the d-glucan carbohydrates on their surfaces.

Topics: Amino Acid Sequence; Animals; beta-Glucans; Carrier Proteins; Epidermal Growth Factor; Fibrinogen; Gene Expression; Glucans; Lectins; Molecular Sequence Data; Phosphorylation; Phylogeny; Porifera; Protein Precursors; Recombinant Proteins; Sequence Alignment

The gelation of aqueous suspensions of the polysaccharide curdlan has been studied by dynamic rheological measurements, differential scanning calorimetry, and low-resolution time-domain 1H-NMR. Gel formation from several samples, each originating from a curdlan fraction of differing molecular weight, has been observed in order to further clarify the nature of observed phenomena by monitoring their dependence on degree of polymerisation. The results from the complementary techniques described here, in addition to those in existing literature, both for curdlan and for other ss-(1,3) glucans, have been used to build up a consistent framework for the interpretation of results. Broadly, this involves the plasticisation and dissolution of dried material on heating, the time-dependent annealing of native (as biosynthesised) structures, and the trapping of imperfectly formed pseudo-equilibrium states on re-cooling, in concert with the creation of microfibrils and network formation.

Topics: beta-Glucans; Calorimetry, Differential Scanning; Elasticity; Gels; Glucans; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Weight; Polysaccharides, Bacterial; Rheology; Viscosity; Water

The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.

Topics: beta-Glucans; Carbohydrate Conformation; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glycosylation; Gram-Negative Aerobic Bacteria; Hordeum; Hydrolysis; Magnetic Resonance Spectroscopy; Polysaccharides; Substrate Specificity

Low-level (subanomole) determination of amino acids in samples of naturally derived polymeric carbohydrates has been demonstrated using vapor-phase acid hydrolysis and subsequent precolumn derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Application has been demonstrated for neutral polysaccharide polymers such as laminarin (beta-1,3; branched), curdland (beta-1,3; unbranched), pullulan (alpha-1, 6-maltotriose), glycogen (alpha-1,4-glucan), and inulin (polyfructose). Successful determination (acceptable recovery and lack of interferences) was possible in samples which also contained up to roughly 50 micrograms amino sugars (e.g., chitosan or glucans with copurified glucosamine oligomers), although optimum utility is for samples containing up to ca. 10 micrograms total amines. The limit of quantification was roughly 20 ng protein/ mg sample, based on analysis of reagent blanks, although the limit of detection was much lower (ca. 0.1 ng protein/mg sample). Incorporation of a relatively rapid hydrolysis (150 degrees C for 1.5 h) gave similar results to use of 110 degrees C for 24 h and allowed for relatively rapid processing. The method has shown good sensitivity, linearity, ruggedness, and ease. Recovery has been optimized, although yield varied somewhat depending on polymer composition.

Topics: Amino Acids; Aminoquinolines; beta-Glucans; Carbamates; Carbohydrates; Chromatography, High Pressure Liquid; Glucans; Glucosamine; Inulin; Laminaria; Polymers; Polysaccharides

An endo-1,3-beta-glucanase was purified from a cell wall autolysate of Aspergillus fumigatus. This beta-glucanase activity was associated with a glycosylated 74-kDa protein. Using a sensitive colorimetric assay and a high-performance anion-exchange chromatography with a pulsed electrochemical detector for product analysis, it was shown that the endoglucanase hydrolysed exclusively linear 1,3-beta-glucan chains, had an optimum pH of 7.0 and an optimum temperature of 60 degrees C. A substrate kinetic study gave a Km value of 0.3 mg/ml for soluble (laminarin and laminari-oligosaccharides) and 1.18 mg/ml for insoluble (curdlan) 1,3-beta-glucan. Laminari-oligosaccharide degradation, analysed by HPLC, showed that the endoglucanase bind to the subtrate at several positions and suggested that the active site of the enzyme recognized five glucose units linked by a 1,3-beta bond. The association of the present endo-1,3-beta-glucanase with the cell wall of A. fumigatus suggests a putative role for this enzyme during cell-wall morphogenesis.

Topics: Aspergillus fumigatus; beta-Glucans; Cell Wall; Fungal Proteins; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Glycosylation; Hydrogen-Ion Concentration; Membrane Glycoproteins; Molecular Weight; Polysaccharides; Substrate Specificity; Temperature

A method to detect peptidoglycan and (1-->3) beta-D-glucan with silkworm larvae plasma (SLP) derived from the hemolymph of the silkworm, Bombyx mori was developed. SLP contains all of the factors of the pro-phenol oxidase cascade, an important self-defense mechanism of insects. Peptidoglycan or (1-->3)-beta-D-glucan initiates the cascade, in which pro-phenol oxidase is finally activated to phenol oxidase. The phenol oxidase activity was colorimetrically or visually detected with 3,4-dihydroxyphenylalanine as a substrate. SLP displayed high reactivity with peptidoglycan and polysaccharides containing 1,3-beta-glucosidic linkages, but not with endotoxins. SLP is useful for the detection of microbial contamination because peptidoglycan and (1-->3)-beta-D-glucan are cell wall components of bacteria and fungi, respectively.

Topics: Animals; beta-Glucans; Biological Assay; Bombyx; Catechol Oxidase; Cell Wall; Enzyme Precursors; Glucans; Gram-Positive Bacteria; Hemolymph; Larva; Lipopolysaccharides; Monophenol Monooxygenase; Peptidoglycan

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

Topics: Actinomycetales; Amino Acid Sequence; Bacillus subtilis; Base Sequence; beta-Glucans; beta-Glucosidase; Cloning, Molecular; DNA Primers; DNA, Bacterial; Genes, Bacterial; Glucan 1,3-beta-Glucosidase; Glucans; Hydrogen-Ion Concentration; Isoelectric Point; Isoenzymes; Molecular Sequence Data; Polysaccharides; Recombinant Proteins; Sequence Homology, Amino Acid; Sequence Homology, Nucleic Acid; Substrate Specificity

A 1,3-beta-glucanase purified from rice grain is a 33 kDa monomer with a pI of > or = 10.4. The enzyme was determined to be an endo-1,3-beta-glucanase (EC 3.2.1.39) by end product analysis using Laminaria digitata laminarin as substrate. Its amino-terminal amino acid sequence revealed strong homology to an endo-1,3-beta-glucanase from barley.

Topics: Amino Acid Sequence; beta-Glucans; Glucan Endo-1,3-beta-D-Glucosidase; Glucans; Molecular Sequence Data; Oryza; Polysaccharides; Substrate Specificity

The coagulation of Limulus amebocyte lysate (LAL) can be activated through two pathways, one initiated by endotoxin and the other by beta-glucans. The two pathways join at the step of activation of the proclotting enzyme. We report here that the endotoxin-activated pathway can be differentially inhibited by two methods in a Limulus enzyme-linked immunosorbent assay (ELISA), either by the combined use of dimethyl sulfoxide and polymyxin B or by a monoclonal antibody against Limulus factor C. LAL reactivities to 10 different endotoxin preparations could be inhibited by the former method by a factor of 10(4) to 10(6) and could be blocked almost totally by the latter method, irrespective of the source of endotoxin. The sensitivity of the assay was approximately 50 pg/ml both for curdlan from Alcaligenes faecalis and for laminarin from Laminaria digitata. We also found that the beta-glucan-activated pathway could be totally blocked by laminarin (> 1 microgram/ml) without affecting the endotoxin-activated pathway, allowing endotoxin to be quantitated specifically by the Limulus ELISA with a detection limit of 0.005 endotoxin unit per ml. The use of uninhibited and differentially inhibited ELISAs demonstrated that different LAL preparations showed much greater variation in assaying beta-glucans than in assaying endotoxins. The LAL reactivity of normal human plasma was found to be due to the activation of the beta-glucan pathway, but not the endotoxin pathway, of LAL.

Topics: Animals; Antibodies, Monoclonal; Arthropod Proteins; beta-Glucans; Blood Cells; Cell Extracts; Dimethyl Sulfoxide; Endotoxins; Enzyme Activation; Enzyme Precursors; Enzyme-Linked Immunosorbent Assay; False Positive Reactions; Glucans; Hemolymph; Horseshoe Crabs; Limulus Test; Lipopolysaccharides; Mice; Polymyxin B; Polysaccharides; Sensitivity and Specificity; Serine Endopeptidases

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.

Topics: Animals; Anticoagulants; beta-Glucans; Blood Proteins; Glucans; Horseshoe Crabs; Kinetics; Lipopolysaccharides; Oligosaccharides; Polysaccharides; Polysaccharides, Bacterial; Structure-Activity Relationship

Activation of the alternative (APC) and classical (CPC) pathways of complement by fungal (1----3)-beta-D-glucans having different degrees of branching (DB) and different conformations were examined by using human serum and plasma. The glucans used in this study were curdlan (no branch; 0/1), grifolan (one branch in every third main chain unit; 1/3), schizophyllan (1/3), SSG (1/2), and OL-2(2/3). Triple or single helix conformer of these glucans were prepared by heating at 150 degrees C or dissolution in sodium hydroxide. Activation of APC by these glucans were dependent on incubation time, concentration, molecular weight, and DB. Interestingly, the triple helix conformer of all glucans tested activated APC stronger than a single helix one. The activity of branched glucans in plasma was weaker than those in serum. On the other hand, in the case of CPC, a single helix conformer activated CPC stronger than a triple helix one, and the activity was dependent on DB. Activation of CPC by a single helix conformer was thought to be dependent on the binding of beta-glucan to immunoglobulin in serum, because the complex was clearly detected by gel permeation chromatography only in the case of single helix one. From these results, it appears that the different conformers were recognized by the host complement systems in different ways. (1----3)-beta-D-Glucan is one of the major constituents of fungal cell wall and is thought to be clearly recognized by the host immune systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Adjuvants, Immunologic; beta-Glucans; Blood Proteins; Complement Activation; Glucans; Humans; Molecular Conformation; Molecular Weight; Protein Binding; Sizofiran; Structure-Activity Relationship

By separating Limulus amebocyte lysate by cation-exchange chromatography with an SP-Toyopearl 650C column, a fraction insensitive to endotoxin, yet specifically sensitive to beta-glucan, was successfully obtained in the unadsorbed portion. This fraction showed beta-glucan dose-dependent clotting enzyme activity, although no sensitivity to endotoxin. This beta-glucan-dependent reaction showed no interference in the presence of endotoxin, with the fraction also showing sensitivity towards various kinds of beta-glucan, i.e. curdlan, pachyman, laminaran and lichenan. The sensitivity towards curdlan was approximately 10(-10) g/ml.

Topics: Animals; Bacterial Toxins; beta-Glucans; Chromatography, Liquid; Electrophoresis, Polyacrylamide Gel; Glucans; Horseshoe Crabs; Polysaccharides; Polysaccharides, Bacterial; Sensitivity and Specificity; Spectrophotometry, Ultraviolet

We developed a simple new endotoxin-specific assay method that uses Limulus amebocyte lysate (LAL) containing a sufficient amount of a water-soluble (1----3)-beta-D-glucan derivative as a blocker of the (1----3)-beta-D-glucan-mediated coagulation pathway. The addition of 0.1 mg/ml or more of carboxymethylated (1----3)-beta-D-glucan completely blocked the activation of LAL by (1----3)-beta-D-glucan itself. The assay of endotoxin was unaffected by the presence of 1 mg/ml carboxymethylated (1----3)-beta-D-glucan. Spiked endotoxin was recovered well from beta-glucans by the turbidimetric kinetic method with LAL containing 1 mg/ml of carboxymethylated (1----3)-beta-D-glucan. Besides, this new LAL formulation was applied for an endotoxin-specific assay by the conventional gel-clot method or the chromogenic method. Gram-negative bacteria were specifically detected by the turbidimetric kinetic method with the LAL formulation. This LAL formulation may be used for an endotoxin-specific assay not only in pharmacology but also in clinical microbiology.

Topics: Bacteria; beta-Glucans; Endotoxins; Fungi; Glucans; Limulus Test

Mouse macrophages were cultured on chemically modified plastic dishes. On dishes covered with immobilized glycans, the macrophages were stimulated as judged by increased 14C-glucosamine incorporation, increased cytostatic and cytolytic capacities and by morphology as seen by scanning electron microscopy. The corresponding soluble glycans did not have the capacity to stimulate macrophages as measured by these criteria. Plastic surfaces covered with polyethylenimine showed stimulation of the macrophages with regard to some of the parameters measured. These results may indicate that the stimulation is a multistep process and that, contrary to earlier findings, it is not a prerequisite for stimulation that the glycan be intracellular. The results support the idea that a fixed steric arrangement of glycans is necessary for the stimulation of macrophages in vitro.

Topics: Amylose; Animals; beta-Glucans; Cell Line; Cytotoxicity, Immunologic; Glucans; Macrophage Activation; Macrophages; Mice; Polysaccharides; Stereoisomerism; Structure-Activity Relationship