epiglucan and curdlan

Curdlan is widely applied in the food and pharmaceutical industries. This review focuses on the biosynthetic pathways, regulatory mechanisms and metabolic engineering strategies for curdlan production. Firstly, curdlan biosynthesis is discussed. Furthermore, various strategies to increase curdlan production are summarized from four aspects, including the overexpression of genes for curdlan biosynthesis, weakening/knockdown of genes from competing pathways, increasing the supply of curdlan precursors, and optimization of fermentation conditions. Moreover, the emerging and advanced applications of curdlan are introduced. Finally, the challenges that are frequently encountered during curdlan biosynthesis are noted with a discussion of directions for curdlan production.

Topics: Animals; Bacteria; beta-Glucans; Biosynthetic Pathways; Fermentation; Food Additives; Humans; Metabolic Engineering

Aerogels are promisingly intended for the use in describing lighter solid materials with huge porous structures. The outcome of aerogels is of potential interest in biomedical purposes owing to many features such as high surface area, low density and porous structure, and so forth. There are numerous inorganic and organic materials employed in the preparation of aerogels. Many drying techniques are a fundamental part of their preparation such as supercritical, freeze-drying, vacuum, ambient pressure and microwave which have been utilized for drying the wet-gel via substitute the liquid inside the wet-gel pores with air. Three common lighter solid materials (i.e. aerogel, cryogel and xerogel) could be synthesized depending on the drying technique applied. This review focuses on aerogel definition, the steps for the preparation of aerogel, techniques used for drying the wet-gel platforms. Further it highlights the pros and cons of each drying technique for synthesizing a demanded material's properties. As polysaccharide considered as one of the most prominent biocompatible and environmentally friendly polymers used for their preparation, thus we will present some examples (e.g.; cellulose, chitosan, starch, alginate, carrageenan and curdlan) and finally the potential biomedical applications of polysaccharides-based aerogel are briefly emphasized.

Topics: Alginates; beta-Glucans; Biomedical Technology; Carrageenan; Cellulose; Chitosan; Cryogels; Desiccation; Drug Delivery Systems; Freeze Drying; Gels; Pectins; Polysaccharides; Porosity; Starch; Tissue Engineering

Curdlan - a homopolysaccharide is comprised of glucose using β-1,3-glycosidic bond and produced by different types of microorganisms as exopolysaccharide. Curdlan gel is stable during freezing and thawing processes which find several applications in food and pharmaceutical industries. It acts as a prebiotic, stabilizer and water-holding, viscosifying and texturing agent. Additionally, curdlan gel is used as a food factor to develop the new products e.g. milk fat substitute, non-fat whipped cream, retorting (freeze-drying) process of Tofu, low-fat sausage, and low-fat hamburger. However, a great variation exists among different countries regarding the regulatory aspects of curdlan as food additives, dietary components or prebiotic substances. Therefore, the present review paper aims to discuss safety issues and the establishment of common guidelines and legislation globally, focusing on the use the applications of curdlan in the food sector including the development of noodles, meat-based products, and fat-free dairy products. This review analyzes and describes in detail the potential of curdlan as a sustainable alternative additive in health and food industries, emphasizing on the chemical composition, production, properties, and potential applications.

Topics: beta-Glucans; Food Additives; Food Industry; Prebiotics

Progress in genomic analysis has resulted in the proposal that the intestinal microbiota is a crucial environmental factor in the development of multifactorial diseases, such as obesity, diabetes, rheumatoid arthritis, and inflammatory bowel diseases represented by Crohn's disease and ulcerative colitis. Dysregulated gut microbiome contributes to the pathogenesis of such disorders; however, there are few effective treatments for controlling only disease-mediating bacteria. Here, we review current knowledge about the intestinal microbiome in health and disease, and discuss a regulatory strategy using a parenteral vaccine with emulsified curdlan and CpG oligodeoxynucleotides, which we have recently developed. Unlike other conventional injectable immunizations, our vaccine contributes to the induction of antigen-specific systemic and mucosal immunity. This vaccine strategy can prevent infectious diseases such as

Topics: Administration, Mucosal; Arthritis, Rheumatoid; Bacterial Vaccines; beta-Glucans; Diabetes Mellitus, Type 2; Dysbiosis; Gastrointestinal Microbiome; Humans; Immunity, Mucosal; Immunization Schedule; Immunization, Secondary; Immunoglobulin A; Inflammatory Bowel Diseases; Injections, Intramuscular; Intestinal Mucosa; Obesity; Oligodeoxyribonucleotides; Polysaccharides, Bacterial; Vaccines, Synthetic

Topics: Adjuvants, Immunologic; beta-Glucans; Glycolipids; Humans; Immune System; Immunity, Innate; Infant, Newborn; Inulin; Vaccination; Vaccines

Exopolysaccharides are synthesized by bacteria and secreted into the external environment and they may be homopolymeric or heteropolymeric in configuration. They are believed to protect bacterial cells from heavy metals, desiccation or other environmental effect. EPS exhibit antitumor, anti-HIV, emulsion stabilization capacity, shear-thinning activity, suspension ability, high viscosities, excellent biocompatibility, high biodegradability and immunomodulatory properties. They are widely used in herbicides, functional food, nutraceuticals, cosmeceuticals, pharmaceuticals, insecticides, immunomodulation and anticoagulants. This review shed light on the properties and versatile applications of xanthan, curdlan, hyaluronic acid and dextran blends and composites with natural and synthetic polymers.

Topics: Animals; Anti-HIV Agents; Antineoplastic Agents; beta-Glucans; Fungal Polysaccharides; Humans; Polysaccharides, Bacterial

Agrobacterium is a genus of gram-negative bacteria that can produce several typical exopolysaccharides with commercial uses in the food and pharmaceutical fields. In particular, succinoglycan and curdlan, due to their good quality in high yield, have been employed on an industrial scale comparatively early. Exopolysaccharide biosynthesis is a multiple-step process controlled by different functional genes, and various environmental factors cause changes in exopolysaccharide biosynthesis through regulatory mechanisms. In this mini-review, we focus on the genetic control and regulatory mechanisms of succinoglycan and curdlan produced by Agrobacterium. Some key functional genes and regulatory mechanisms for exopolysaccharide biosynthesis are described, possessing a high potential for application in metabolic engineering to modify exopolysaccharide production and physicochemical properties. This review may contribute to the understanding of exopolysaccharide biosynthesis and exopolysaccharide modification by metabolic engineering methods in Agrobacterium.

Topics: Agrobacterium; beta-Glucans; Gene Expression Regulation, Bacterial; Genes, Bacterial; Metabolic Engineering; Polysaccharides, Bacterial

Curdlan is a bacterial polysaccharide that has been of significant recent interest due to its interesting and valuable rheological properties and its inherent bioactivity. The simple (1→3)-β-glucan homopolymeric, unbranched structure of curdlan is conducive to enhanced solubility relative to many other abundant natural polysaccharides, thus, providing alternatives for processing the polymer into desired shapes and formulations. At the same time, this relatively good solubility enables chemical modification under mild conditions, leading to a growing body of literature on derivative chemistry, structure-property relationships, and the potential for regioselective modification. Structure, properties, biosynthesis, modification chemistries, and key applications are the foci for this review of the curdlan literature.

Topics: beta-Glucans; Carbohydrate Sequence; Click Chemistry; Cyclic N-Oxides; Food Industry; Gels; Molecular Sequence Data; Phosphorylation; Polysaccharides, Bacterial; Solubility

Curdlan is a water-insoluble β-(1,3)-glucan produced by Agrobacterium species under nitrogen-limited condition. Its heat-induced gelling properties render curdlan to be very useful in the food industry initially. Recent advances in the understanding of the role curdlan plays in both innate and adaptive immunity lead to its growing applications in biomedicine. Our review focuses on the recent advances on curdlan biosynthesis and the improvements of curdlan fermentation production both from our laboratory and many others as well as the latest advances on the new applications of curdlan and its derivatives particularly in their immunological functions in biomedicine.

Topics: Agrobacterium; beta-Glucans; Biological Products; Biotechnology; Immunologic Factors

There is considerable interest in exploiting the novel physical and biological properties of microbial exopolysaccharides in industry and medicine. For economic and scientific reasons, large scale production under carefully monitored and controlled conditions is required. Producing exopolysaccharides in industrial fermenters poses several complex bioengineering and microbiological challenges relating primarily to the very high viscosities of such culture media, which are often exacerbated by the producing organism's morphology. What these problems are, and the strategies for dealing with them are discussed critically in this review, using pullulan, curdlan, xanthan, and fungal β-glucans as examples of industrially produced microbial exopolysaccharides. The role of fermenter configuration in their production is also examined.

Topics: beta-Glucans; Bioreactors; Culture Media; Fermentation; Fungi; Glucans; Industrial Microbiology; Polysaccharides, Bacterial; Rheology; Viscosity

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

Experimental autoimmune encephalomyelitis (EAE), an experimental model for multiple sclerosis, can be induced through inoculation with several different central nervous system (CNS) proteins or peptides. Modulation of EAE, resulting in either protection from EAE or enhancement of EAE, can also be accomplished through either vaccination or DNA immunization with molecular mimics of self-CNS proteins. Previously published data on this method of EAE modulation will be reviewed. New data is presented, which demonstrates that EAE can also be modulated through the administration of the beta-(1,3)-D-glucan, curdlan. Dendritic cells stimulated by curdlan are involved in the differentiation of the interleukin-17 producing subset of CD4(+) T cells that are recognized effector cells in EAE. Using two different systems to study the effects of curdlan on EAE, it was found that curdlan increased the incidence of EAE and/or the severity of the disease course.

Topics: Animals; beta-Glucans; Encephalomyelitis, Autoimmune, Experimental; Humans; Nerve Tissue Proteins; Neuropeptides; Neuroprotective Agents

Three structural classes of (1-->3)-beta-D-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (1-->3)-beta-glucans and side-chain-branched (1-->3,1-->2)-beta-glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (1-->3,1-->6)-beta-glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (1-->3)-beta-glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (1-->3)-beta-glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.

Topics: Anti-Infective Agents; Antineoplastic Agents; Bacteria; Bacterial Capsules; Bacterial Physiological Phenomena; beta-Glucans; Biotechnology; Fermentation; Food Industry; Immunologic Factors; Polymers; Polysaccharides, Bacterial; Rhizobium

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

Alcaligenes faecalis var. myxogenes 10C3, which we isolated from soil, produces a water-soluble and an insoluble extracellular polysaccharide. The former (succinoglycan) is composed of glucose, galactose, pyruvic acid and succinic acid (molar proportions 7:1:1:1) with (beta 1-3)-, (beta 1-4)- and (beta 1-6)-glucosidic linkages. The latter (curdlan) is composed entirely of (beta 1-3)-linked D-glucose and forms a resilient firm gel when heated in suspension. The organism also produces extracellularly a repeating-unit octasaccharide of succinoglycan and cyclic (beta 1-2)-D-glucan. These polymers or oligomers are also produced by many strains of Agrobacterium and Rhizobium. Spontaneous mutation in ability to produce these polysaccharides or oligosaccharides occurs in these strains. The structures of succinoglycan, and similar polymers containing riburonic acid or galactose as the end residue of the side chain, were elucidated by successive fragmentation with two special enzymes obtained from Cytophaga arvensicola followed by methylation analysis. It is interesting that the unit compound in the biosynthesis of succinoglycan is identical with the unit compound in the enzymic decomposition of the polymer. Studies on curdlan gel by X-ray, 13C n.m.r. and electron-microscopic analysis and other physicochemical methods showed that the molecular structure of curdlan changes from a single helix to a triple stranded helix on heat treatment at high temperature. Curdlan seems to be useful for making new types of jelly products and may also be useful in new procedures for food production. Interestingly, curdlan possesses marked antitumour activity. Extracellular isoamylase (EC 3.2.1.68; glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB 15, which we isolated from soil, is very useful for elucidation of the structure of amylopectin and glycogen, and also for the commercial production of amylose or maltose alone or in combination with beta-amylase. Maltose is useful as a sugar for injection, being better than glucose. Maltitol is easily produced from maltose by chemical reduction and is used as a low-calorie sweetener. The isoamylase is also effective for enhancing the production of glucose from starch by the action of glucoamylase.

Topics: Alcaligenes; beta-Glucans; Carbohydrate Conformation; Glucans; Glucose; Glycoside Hydrolases; Isoamylase; Maltose; Microscopy, Electron; Polysaccharides, Bacterial; Starch; Viscosity

The phase behavior in protein-polysaccharide blended systems is the main factor affecting the physical properties of composite gels; however, the phase behavior at room temperature and during heated gelation is lacking discussion. In this research, extracted grouper myofibrillar protein (MP) and curdlan (CUR) were used as models for the MP-CUR blended system. The phase behavior of the MP-CUR blended system was analyzed using rheology and microstructure analysis, and the accuracy of the phase behavior analysis was verified by measuring the physical indices such as gel properties of the MP-CUR composite gels. At room temperature, MP and CUR showed good co-solubility, so the blended system with 0.8% CUR content obtained the best apparent viscosity, structural recoverability, and other rheological properties. After heating gelation, MP and CUR had strong thermodynamic unaffinity leading to phase separation, and the best storage modulus was obtained for the MP-CUR blended system with 0.6% CUR content. Therefore, it is concluded that 0.6% CUR content is the critical concentration for the MP-CUR blended system. The results were also confirmed by the best gel properties of 0.6% CUR composite gel when the physical properties of the composite gel were determined. The phase behavior evaluation was used to determine the appropriate polysaccharide concentrations as a means to improve the physicochemical properties of the composite gels and to exploit the value of polysaccharides in protein-based food applications.

Topics: beta-Glucans; Gels; Muscle Proteins; Rheology

To resolve the major obstacles of poor water solubility and few functional derivatives in the practical utilization of curdlan, we prepared highly soluble and thermal-responsively alkylated curdlan with different degrees of substitution (DS) via homogeneous reaction. The properties of these derivatives were investigated by rheology, inline attenuated total reflective-Fourier transform infrared spectroscopy, Raman and fluorescence spectroscopy, micro-differential scanning calorimetry, dynamic light scattering, and in vitro cytotoxicity assay. The newly developed methylated and ethylated curdlan can form thermal reversible heat-set hydrogels accompanied by volume shrinkage due to syneresis. Their gelation temperatures depend strongly on the DS, size of hydrophobic groups, and the order of the Hofmeister series salts added. Experimental results indicated that the gel network was induced by the enhanced hydrophobic interaction among macromolecular chains and weakened hydrogen bonding between the polymer and water on heating, leading to phase separation and gelation.

Topics: beta-Glucans; Ethers; Hydrogels; Rheology; Spectroscopy, Fourier Transform Infrared; Water

Curdlan, a linear β-1,3-glucan, was reacted with glutaric anhydride and heptanoyl chloride to afford thermoplastic curdlan esters (CrdE(HepGlu)) with a carboxylic acid side chain. CrdE(HepGlu) with a degree of substitution of the glutaric acid monoester moiety (DS

Topics: beta-Glucans; Carboxylic Acids; Esters; Glucans

Topics: Agrobacterium; beta-Glucans; Biological Transport; Fermentation

Chronic wounds, among others, are mainly characterized by prolonged inflammation associated with the overproduction of reactive oxygen species and pro-inflammatory cytokines by immune cells. As a consequence, this phenomenon hinders or even precludes the regeneration process. It is known that biomaterials composed of biopolymers can significantly promote the process of wound healing and regeneration. The aim of this study was to establish whether curdlan-based biomaterials modified with hop compounds can be considered as promising candidates for the promotion of skin wound healing. The resultant biomaterials were subjected to an evaluation of their structural, physicochemical, and biological in vitro and in vivo properties. The conducted physicochemical analyses confirmed the incorporation of bioactive compounds (crude extract or xanthohumol) into the curdlan matrix. It was found that the curdlan-based biomaterials improved with low concentrations of hop compounds possessing satisfactory hydrophilicity, wettability, porosity, and absorption capacities. In vitro, tests showed that these biomaterials were non-cytotoxic, did not inhibit the proliferation of skin fibroblasts, and had the ability to inhibit the production of pro-inflammatory interleukin-6 by human macrophages stimulated with lipopolysaccharide. Moreover, in vivo studies showed that these biomaterials were biocompatible and could promote the regeneration process after injury (study on

Topics: beta-Glucans; Biocompatible Materials; Biopolymers; Humans; Hydrogels; Skin; Wound Healing

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

Nanoparticles (NPs) that can activate macrophages infected with the tuberculosis causative pathogen Mycobacterium tuberculosis, could be an effective host directed therapy for the disease. In this study, curdlan was conjugated to poly(lactic-co-glycolic acid) (PLGA) to produce immunotherapeutic NPs. Various physicochemical characterizations were used to evaluate the curdlan-PLGA copolymer and the NPs. Molecular dynamics and simulation studies were used to characterize the interaction between curdlan, on the polymer and on NPs, with the Dectin-1 macrophage receptor. NPs with varying curdlan densities were evaluated for their effects on the production of pro- and anti-inflammatory cytokines in M. tuberculosis infected RAW264.7 macrophages. The killing efficacy of the NPs against intracellular M. tuberculosis was assessed. Physicochemical characterization of the curdlan-PLGA copolymer and NPs indicated successful formation of curdlan-PLGA copolymer and NPs of varying curdlan density (0-8% w/w) had sizes between 330 and 453 nm. Modelling studies showed curdlan to have a strong affinity for Dectin-1. Cytotoxicity assays showed the NPs to be non-toxic over 72 h. The proinflammatory cytokine TNF-α was found to be significantly upregulated by the NPs. The NPs reduced intracellular M. tuberculosis burden over 72 h. These NPs are a promising host directed therapy for intracellular eradication of M. tuberculosis.

Topics: beta-Glucans; Drug Carriers; Glycols; Lactic Acid; Mycobacterium tuberculosis; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer

Topics: Animals; beta-Glucans; Cations; Mice; Nanoparticles; Polyethylene Glycols; RNA, Small Interfering; Transfection

Spondyloarthritis (SpA) is a systemic inflammatory arthritis mediated mainly by interleukin (IL)-17. The vitronectin-derived bioactive peptide, VnP-16, exerts an anti-osteoporotic effect via β1 and αvβ3 integrin signaling. SpA is associated with an increased risk of osteoporosis, and we investigated the effect of VnP-16 in mice with SpA.. SpA was induced by curdlan in SKG ZAP-70W163C mice, which were treated with vehicle, celecoxib, VnP-16, or VnP-16+celecoxib. The clinical score, arthritis score, spondylitis score, and proinflammatory cytokine expression of the spine were evaluated by immunohistochemical staining. Type 17 helper T cell (Th17) and regulatory T cell (Treg) differentiation in the spleen was evaluated by flow cytometry and in the spine by confocal staining. Splenocyte expression of signal transducer and activator of transcription (STAT) 3 and pSTAT3 was evaluated by in vitro Western blotting.. The clinical score was significantly reduced in the VnP16+celecoxib group. The arthritis and spondylitis scores were significantly lower in the VnP-16 and VnP16+celecoxib groups than the vehicle group. In the spine, the levels of IL-1β, IL-6, tumor necrosis factor-α, and IL-17 expression were reduced and Th17/Treg imbalance was regulated in the VnP-16 alone and VnP-16+celecoxib groups. Flow cytometry of splenocytes showed increased polarization of Tregs in the VnP-16+celecoxib group. In vitro, VnP-16 suppressed pSTAT3.. VnP-16 plus celecoxib prevented SpA progression in a mouse model by regulating the Th17/Treg imbalance and suppressing the expression of proinflammatory cytokines.

Topics: Animals; beta-Glucans; Celecoxib; Cytokines; Disease Models, Animal; Disease Progression; Female; Gene Expression Regulation; Humans; Integrin alphaVbeta3; Integrin beta1; Mice; Peptides; Signal Transduction; Spleen; Spondylarthritis; STAT3 Transcription Factor; T-Lymphocytes, Regulatory; Th17 Cells; Vitronectin

The purpose of this pilot study was to establish whether a novel freeze-dried curdlan/whey protein isolate-based biomaterial may be taken into consideration as a potential scaffold for matrix-associated autologous chondrocyte transplantation. For this reason, this biomaterial was initially characterized by the visualization of its micro- and macrostructures as well as evaluation of its mechanical stability, and its ability to undergo enzymatic degradation in vitro. Subsequently, the cytocompatibility of the biomaterial towards human chondrocytes (isolated from an orthopaedic patient) was assessed. It was demonstrated that the novel freeze-dried curdlan/whey protein isolate-based biomaterial possessed a porous structure and a Young's modulus close to those of the superficial and middle zones of cartilage. It also exhibited controllable degradability in collagenase II solution over nine weeks. Most importantly, this biomaterial supported the viability and proliferation of human chondrocytes, which maintained their characteristic phenotype. Moreover, quantitative reverse transcription PCR analysis and confocal microscope observations revealed that the biomaterial may protect chondrocytes from dedifferentiation towards fibroblast-like cells during 12-day culture. Thus, in conclusion, this pilot study demonstrated that novel freeze-dried curdlan/whey protein isolate-based biomaterial may be considered as a potential scaffold for matrix-associated autologous chondrocyte transplantation.

Topics: beta-Glucans; Biocompatible Materials; Biomarkers; Cartilage, Articular; Cell Proliferation; Cell Survival; Cells, Cultured; Chondrocytes; Elastic Modulus; Extracellular Matrix; Freeze Drying; Gene Expression Regulation; Humans; Pilot Projects; Tissue Scaffolds; Transplantation, Autologous; Whey Proteins

Curdlan is a neutral, water-insoluble, unbranched, linear β-(1,3)-glucan. This study explored the roles of exoR and exoX in curdlan biosynthesis in Agrobacterium sp. ATCC 31749. The microcapsule biosynthesis of ΔexoR strain was reduced, and the motility of this strain increased remarkably compared with the wild-type (WT) strain during the cell growth phase. The curdlan yields of ΔexoR and ΔexoX strains enhanced by 19% and 17%, and the glucose utilization increased by 12% and 11%, respectively, compared with the WT strain during batch fermentation. By contrast, the curdlan yields of exoR and exoX overexpression strains decreased by 28% and 33%, respectively. The gel strength produced by ΔexoR and exoX overexpression strains decreased compared with the WT strain. RT-qPCR analysis at the transcriptional level revealed that key genes in exopolysaccharide synthesis and central metabolic pathways were up-regulated in ΔexoX and ΔexoR strains during gel production. Metabolomics analysis of ΔexoR and ΔexoX mutants proved the rates of central metabolic and electron transport chain were accelerated.

Topics: Agrobacterium; beta-Glucans; Fermentation

Due to the crucial role of gluten network in maintaining the tensile properties of frozen-cooked noodles (FCNs), the underlying mechanism of protective effect of curdlan on FCNs quality during frozen storage was explored from the perspective of aggregation behavior and structure of gluten in this study. The results showed that curdlan weakened the depolymerization behavior of gluten proteins through inhibiting the disruption of disulfide bonds; Curdlan stabilized the secondary structure of gluten proteins by restraining the transformation of compact α-helices to other secondary structures; Atomic force microscope results implied that curdlan inhibited the aggregation of gluten chains; Confocal laser scanning microscopy observation analyzed by AngioTool software indicated that the connectivity and uniformity of gluten network were enhanced because of curdlan. This study may provide more comprehensive theories for the strengthening effect of curdlan on FCNs quality from the perspective of gluten structure and contribute to the quality improvement of FCN in the food technology field.

Topics: beta-Glucans; Cooking; Flour; Glutens

Multiple sclerosis (MS) is an immune-mediated disease characterized by inflammatory demyelination and axonal degeneration in the central nervous system (CNS). Bacterial and fungal infections have been associated with the development of MS; microbial components that are present in several microbes could contribute to MS pathogenesis. Among such components, curdlan is a microbial 1,3-β-glucan that can stimulate dendritic cells, and enhances T helper (Th) 17 responses. We determined whether curdlan administration could affect two animal models for MS: an autoimmune model, experimental autoimmune encephalomyelitis (EAE), and a viral model, Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD). We induced relapsing-remitting EAE by sensitizing SJL/J mice with the myelin proteolipid protein (PLP)

Topics: Animals; beta-Glucans; Disease Models, Animal; Mice; Multiple Sclerosis; Theilovirus

To obtain an analogue of pork backfat (PBF), we combined emulsion and gel to fabricate emulsion gel, which was prepared by using soybean protein isolate (SPI) and curdlan (CL) through a facile heat-treatment method in this paper. The microstructures, rheology properties, water holding capacity and freeze-thawing stability of the emulsion gel were investigated. The results suggested that the SPI/CL-stabilized emulsion gel was thermal-irreversible, and SPI was the emulsifying agent of the emulsion gel. Oil contents significantly affect the water holding capacity and freeze-thawing stability of emulsion gel. Subsequently, the TPA, gel strength and color of emulsion gels with different oil contents were compared with PBF. The hardness, chewiness, springiness, and gel strength of emulsion gel with 10 wt% oil contents were no significant differences from that of PBF (P > 0.05). Hence, this SPI/CL based emulsion gel can be used as an analogue to PBF, providing an alternative ingredient for the development of plant-based low-fat meat products.

Topics: Animals; beta-Glucans; Emulsions; Gels; Pork Meat; Red Meat; Soybean Proteins; Swine; Water

Manufacturing facile and low-cost wound dressings that simultaneously meet the needs of the entire repair process remains the major challenge of effective wound healing. Herein, a series of curdlan-tannic acid hybrid hydrogels were successfully fabricated through the annealing technique. Notedly, when the mixing weigh ratio was 1:1, the hydrogel exhibited excellent physicochemical properties, including swellability, degradability, water retention, porosity, and rheology. Additionally, the hydrogel did not display significant cytotoxicity to fibroblasts and the hemolysis rate at 12 h was 3%. Interestingly, the hybrid hydrogel showed multifunctional properties, including remarkable antioxidant, antibacterial, and rapid hemostasis effects reduce blood loss by 0.35 g, that were achieved through the temperature-dependent release of tannic acid. Moreover, a full-thickness skin defect animal model was used to verify that the multifunctional hydrogel could accelerate wound healing in vivo. These results suggest that this hybrid hydrogel is a promising candidate for the clinical treatment of full-thickness wounds.

Topics: Animals; Anti-Bacterial Agents; Antioxidants; beta-Glucans; Hemostasis; Hemostatics; Hydrogels; Hydrogen; Tannins; Wound Healing

Topics: Agrobacterium; beta-Amylase; beta-Glucans; Carbon; Maltose; Manihot; Spectroscopy, Fourier Transform Infrared; Starch

With the increasing attention to food preservation and environmental safety, there is great pressing demand to explore novel edible and environment-friendly food packaging films. In the present study, a new kind natural curdlan (CD) film was developed with the addition of bacterial cellulose (BC) and cinnamon essential oil (CEO) at 2% and 10% (w/w) amounts, with regard to improve mechanical properties and investigate potential food applications. Our results showed that the tensile strength, the crystallinity and the thermal stability of the CD/BC blending film were improved, while the water vapor permeability, moisture content and the lightness were reduced. Moreover, the CEO addition to the CD/BC film further increased the barrier properties and also mechanical properties. The results of FTIR and XRD were applied for analyzing the potential interactions of the film matrix. Finally, addition of CEO endowed the blending films with good antibacterial activity and antioxidant capacity, which could effectively inhibit the bacterial growth and the lipid oxidation of chilled chicken during the preservative period. Thus, this work demonstrates that the novel CD/BC/CEO blending film with improved mechanical and barrier properties can be of great potential for developing food packaging material for promising applications.

Topics: Bacteria; beta-Glucans; Cellulose; Cinnamomum zeylanicum; Food Packaging; Oils, Volatile; Permeability; Tensile Strength

In this study, carboxylic curdlan (Cur-48) and negatively charged ferulic acid (FA)-grafted carboxylic curdlan (Cur-48-g-FA) were separately used to fabricate polyelectrolyte nanoparticles (PNPs: PNPs-CQ and PNPs-CFQ) with positively charged quaternized curdlan (Qcurd) for curcumin delivery. Results showed that curcumin-loaded PNPs-CQ and PNPs-CFQ had particle sizes of 338.1 and 301.3 nm, zeta potentials of -19.07 and -24.10 mV, and encapsulation efficiencies of 76.32% and 83.54%, respectively. Curcumin was properly encapsulated inside the two PNPs through electrostatic interactions and hydrogen bonds. Compared with free curcumin, entrapped curcumin in the two PNPs exhibited better redispersion performance, thermo- and photostability, and sustained release property. Furthermore, FA molecules surrounding the surface of PNPs-CFQ were conductive to the entrapped curcumin's particulate characteristics, stability, release behavior, and antioxidant potentials. Therefore, our findings indicated that PNPs formulated via Cur-48-g-FA and Qcurd can provide a novel delivery platform for encapsulation of hydrophobic nutrients, including curcumin, in functional foods.

Topics: beta-Glucans; Curcumin; Drug Carriers; Drug Delivery Systems; Nanoparticles; Particle Size; Polysaccharides

In the present study, clove essential oil (CEO) Pickering emulsions were stabilized by octadecylamine-modified carboxymethyl curdlan (CMCD-ODA) at different pH values. The droplet size and negatively charged zeta potential of the CMCD-ODA emulsions decreased as the pH increased from 3.0 to 11.0. Rheology results indicated that the CMCD-ODA polymer/emulsion prepared at pH 5.0 showed higher apparent viscosity and viscoelasticity than other pH conditions, which might prevent droplets from flocculating. The Pickering emulsions obtained at pH 5.0 were spherical droplets with a uniform size distribution and a mean diameter of 9.54 μm, and they exhibited excellent stability during 28 days of storage. The morphological structures of the emulsions investigated by confocal laser scanning microscopy and scanning electron microscopy indicated that the CMCD-ODA Pickering emulsion obtained at pH 5.0 was stabilized by loading amphiphilic CMCD-ODA polymer around the spherical oil droplets and forming a weak gel network structure. The CEO-loaded CMCD-ODA emulsions had higher antioxidant capacity than free CEO after 28 days of storage at pH 5.0. Given the good emulsion stability, antioxidant activity, and great antibacterial effect, the CEO-loaded carboxymethyl curdlan Pickering emulsion has promising applications in food, cosmetic, and biomedicine industries.

Topics: beta-Glucans; Biological Availability; Clove Oil; Emulsions; Oils, Volatile; Particle Size; Polymers; Syzygium

Topics: Anti-Bacterial Agents; Bandages; beta-Glucans; Biocompatible Materials; Durapatite; Gentamicins; Humans; Pseudomonas aeruginosa; Staphylococcus aureus; Surgical Wound Infection; Zinc

Osteochondral defects remain a huge problem in medicine today. Biomimetic bi- or multi-phasic scaffolds constitute a very promising alternative to osteochondral autografts and allografts. In this study, a new curdlan-based scaffold was designed for osteochondral tissue engineering applications. To achieve biomimetic properties, it was enriched with a protein component - whey protein isolate as well as a ceramic ingredient - hydroxyapatite granules. The scaffold was fabricated via a simple and cost-efficient method, which represents a significant advantage. Importantly, this technique allowed generation of a scaffold with two distinct, but integrated phases. Scanning electron microcopy and optical profilometry observations demonstrated that phases of biomaterial possessed different structural properties. The top layer of the biomaterial (mimicking the cartilage) was smoother than the bottom one (mimicking the subchondral bone), which is beneficial from a biological point of view because unlike bone, cartilage is a smooth tissue. Moreover, mechanical testing showed that the top layer of the biomaterial had mechanical properties close to those of natural cartilage. Although the mechanical properties of the bottom layer of scaffold were lower than those of the subchondral bone, it was still higher than in many analogous systems. Most importantly, cell culture experiments indicated that the biomaterial possessed high cytocompatibility towards adipose tissue-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells in vitro. Both phases of the scaffold enhanced cell adhesion, proliferation, and chondrogenic differentiation of stem cells (revealing its chondroinductive properties in vitro) as well as osteogenic differentiation of these cells (revealing its osteoinductive properties in vitro). Given all features of the novel curdlan-based scaffold, it is worth noting that it may be considered as promising candidate for osteochondral tissue engineering applications.

Topics: beta-Glucans; Biocompatible Materials; Biomimetics; Mesenchymal Stem Cells; Osteogenesis; Tissue Engineering; Tissue Scaffolds

Many biomaterials for bone regeneration have recently been produced using thermally gelled curdlan (1,3-β-d-glucan) as a binder for bioceramics. As the human organism does not produce enzymes having the ability to degrade curdlan, it is not clear what is the fate of curdlan gel after its implantation in the bone. To clarify this point, in this research osteoclasts were cultured on the curdlan gel to show its degradation by acidic hydrolysis. The studies clearly demonstrated microstructural (AFM and SEM imaging) and chemical changes (Raman spectroscopy) on the curdlan surface caused by osteoclast culture. Moreover, degradation test in a cell-free system using HCl solution (pH = 4.5), mimicking environment in the resorption lacuna, showed great weight loss of the sample, release of glucose, and chemical changes typical of curdlan degradation. Thus, the presented research for the first time provides a strong evidence of osteoclast-mediated acidic hydrolysis of thermally obtained curdlan gel.

Topics: beta-Glucans; Gels; Humans; Hydrolysis; Osteoclasts

Curdlan (β-1,3-glucan), as a biodegradable polymer, is still an underestimated but potentially attractive matrix for the production of dressing materials. However, due to its lack of susceptibility to functionalization, its use is limited. The proposed curdlan modification, using a functional polycatecholamine layer, enables the immobilization of selected oxidoreductases (laccase and peroxidase) on curdlan hydrogel. The following significant changes of biological and mechanical properties of polycatecholamines + oxidoreductases-modified matrices were observed: reduced response of human monocytes in contact with the hydrogels, modulated reaction of human blood, in terms of hemolysis and clot formation, and changed mechanical properties. The lack of toxicity towards human fibroblasts and the suppression of cytokines released by human monocytes in comparison to pristine curdlan hydrogel, seems to make the application of such modifications attractive for biomedical purposes. The obtained results could also be useful for construction of a wide range of biomaterials based on other polymer hydrogels.

Topics: beta-Glucans; Glucans; Humans; Hydrogels; Oxidoreductases

Hydroxypropyl methylcellulose (HPMC) and curdlan (CL) were used to prepare uniform films. The influence of the composition ratios and drying temperature on the microstructures, compatibility and physical performance of HPMC/CL films were studied. The crystalline peaks corresponding to CL component of HPMC/CL films increased with the increasing CL content. Increasing CL content resulted in increased hydrogen bonds in HPMC/CL film, reduced transmittance at 500 nm, oxygen permeability and water solubility of the HPMC/CL films. Higher drying temperature led to increased phase separation and decreased physical properties of pure HPMC film, and led to increased compatibility, cross-section smoothness, oxygen barrier property and mechanical properties of pure CL and blending films.

Topics: beta-Glucans; Desiccation; Hypromellose Derivatives; Oxygen; Temperature

The immunostimulatory activity of polysaccharides can improve human immunity, but their activity is low and prompting the activity is a great challenge. Curdlan, is a linear beta-1,3-glucan and has the potential to induce immune responses. However, owing to its tight triple helix structure and insolubility in water, its immunostimulatory activity is weakened. The keyway to promote its immunostimulatory activity is to relax its tight triple helix structure. In this work, methoxypolyethylene glycol (mPEG) was grafted onto curdlan (curdlan-g-mPEG) to unwind its triple helix structure. With its grafting mPEG, the water solubility of curdlan was enhanced. Moreover, with curdlan-g-mPEG treatment, macrophages secreted more tumor necrosis factor (TNF)-alpha and interleukin (IL)-6 and exhibited favorable phagocytosis of bacteria (Staphylococcus aureus and Pseudomonas aeruginosa). These results reveal that curdlan-g-mPEG as an immunostimulant has potential applications in immunology and antibiotics.

Topics: beta-Glucans; Humans; Interleukin-6; Polyethylene Glycols; Tumor Necrosis Factor-alpha; Water

Ankylosing spondylitis (AS) is a type of rheumatic disease characterized by chronic inflammation and pathological osteogenesis in the entheses. Previously, we demonstrated that enhanced osteogenic differentiation of MSC from AS patients (AS-MSC) resulted in pathological osteogenesis, and that during the enhanced osteogenic differentiation course, AS-MSC induced TNF-α-mediated local inflammation. However, whether TNF-α in turn affects AS-MSC remains unknown. Herein, we further demonstrate that a high-concentration TNF-α treatment triggers enhanced directional migration of AS-MSC in vitro and in vivo, which enforces AS pathogenesis. Mechanistically, TNF-α leads to increased expression of ELMO1 in AS-MSC, which is mediated by a METTL14 dependent m

Topics: Adaptor Proteins, Signal Transducing; Adenosine; Animals; beta-Glucans; Biopsy; Bone Marrow; Case-Control Studies; Cell Differentiation; Cell Movement; Disease Models, Animal; DNA Methylation; Epigenesis, Genetic; Female; Healthy Volunteers; HEK293 Cells; Humans; Male; Mesenchymal Stem Cells; Mice; Osteogenesis; Primary Cell Culture; Spondylitis, Ankylosing; Tumor Necrosis Factor-alpha; X-Ray Microtomography

Induction of cellular immunity is important for effective cancer immunotherapy. Although various antigen carriers for cancer immunotherapy have been developed to date, balancing efficient antigen delivery to antigen presenting cells (APCs) and their activation

Topics: Animals; Antigen-Presenting Cells; beta-Glucans; Biocompatible Materials; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Cytokines; Female; Hydrogen-Ion Concentration; Immunity, Cellular; Immunotherapy; Liposomes; Macrophage Activation; Mice; Mice, Inbred C57BL; Neoplasms; Ovalbumin; Tumor Microenvironment

Chitooligosaccharides (CHOS) with multiple biological activities are usually produced through enzymatic hydrolysis of chitosan or chitin. However, purification and recycling of the enzyme have largely limited the advancement of CHOS bioproduction. Here, we engineered a novel enzyme by fusing the native chitosanase Csn75 with a carbohydrate-binding module (CBM) that can specifically bind to curdlan. The recombinase Csn75-CBM was successfully expressed by Pichia pastoris and allowed one-step purification and immobilization in the chitosanase immobilized curdlan packed-bed reactor (CICPR), where a maximum adsorption capacity of 39.59 mg enzyme/g curdlan was achieved. CHOS with degrees of polymerization of 2-5 (a hydrolysis yield of 97.75%), 3-6 (75.45%), and 3-7 (73.2%) were continuously produced by adjusting the ratio of enzyme and chitosan or the flow rate of chitosan. Moreover, the CICPR exhibited good stability and reusability after several cycles. The recombinase Csn75-CBM has greatly improved the efficiency of the bioproduction of CHOS.

Topics: Aspergillus fumigatus; Bacillus; Bacterial Proteins; beta-Glucans; Chitosan; Enzymes, Immobilized; Fungal Proteins; Glucan 1,3-beta-Glucosidase; Glycoside Hydrolases; Mutation; Oligosaccharides; Peptide Fragments; Protein Domains; Protein Engineering; Recombinant Fusion Proteins

Receptor-mediated endocytosis has been used for tissue targeted delivery of short interfering RNA (siRNA) drugs. Herein, we investigated adenosine receptor (AR) as a candidate for receptor-mediated siRNA internalization. We synthesized adenosine functionalized cationic curdlan derivatives (denote CuAMP polymers). One of these polymers, CuAMP4, efficiently delivered siRNA to breast cancer cells expressing high level of A

Topics: Adenosine Monophosphate; Animals; beta-Glucans; Cell Line, Tumor; Humans; Mice; Nanoparticles; Receptor, Adenosine A2B; RNA, Small Interfering

Bioactive dressings are usually produced using natural or synthetic polymers. Recently, special attention has been paid to β-glucans that act as immunomodulators and have pro-healing properties. The aim of this research was to use β-1,3-glucan (curdlan) as a base for the production of bioactive dressing materials (curdlan/agarose and curdlan/chitosan) that were additionally enriched with vitamin C and/or hydrocortisone to improve healing of chronic and burn wounds. The secondary goal of the study was to compressively evaluate biological properties of the biomaterials. In this work, it was shown that vitamin C/hydrocortisone-enriched biomaterials exhibited faster vitamin C release profile than hydrocortisone. Consecutive release of the drugs is a desired phenomenon since it protects wounds against accumulation of high and toxic concentrations of the bioactive molecules. Moreover, biomaterials showed gradual release of low doses of the hydrocortisone, which is beneficial during management of burn wounds with hypergranulation tissue. Among all tested variants of biomaterials, dressing materials enriched with hydrocortisone and a mixture of vitamin C/hydrocortisone showed the best therapeutic potential since they had the ability to significantly reduce MMP-2 synthesis by macrophages and increase TGF-β1 release by skin cells. Moreover, materials containing hydrocortisone and its blend with vitamin C stimulated type I collagen deposition by fibroblasts and positively affected their migration and proliferation. Results of the experiments clearly showed that the developed biomaterials enriched with bioactive agents may be promising dressings for the management of non-healing chronic and burn wounds.

Topics: Anti-Inflammatory Agents; Antioxidants; Ascorbic Acid; Bandages; beta-Glucans; Burns; Collagen Type I; Drug Therapy, Combination; Fibroblasts; Humans; Hydrocortisone; Keratinocytes; Sepharose; Wound Healing

Hydrogel, as a three-dimensional material with high water content, has unique physicochemical and variable mechanical properties. Natural polysaccharide-based composite hydrogels are very popular within medical industry as these viscoelastic materials are non-toxic, biodegradable, bioabsorbable, and biocompatible. This research investigates the engineering of novel composite hydrogels from natural polysaccharides salecan and curdlan without any structural modification and chemical crosslinking. The scanning electron microscopy, Fourier transform infrared spectroscopy and various rheological methods were employed to investigate the morphology, molecular interaction, and flow behavior of the samples respectively. The key rheological parameters were compared using the Power Law, Herschel-Bulkley and Arrhenius models. This is the first study reporting a novel composite hydrogel made from Salecan and Curdlan with ideal elasticity, enhanced thermostability, good injectability, self-recovery and other rheological properties that will pave the way for application in different fields.

Topics: beta-Glucans; Biocompatible Materials; Elasticity; Hydrogels; Hydrogen-Ion Concentration; Microscopy, Electron, Scanning; Rheology; Spectroscopy, Fourier Transform Infrared; Water

Dietary supplementation of fish with β-glucans has been commonly associated with immunomodulation and generally accepted as beneficial for fish health. However, to date the exact mechanisms of immunomodulation by β-glucan supplementation in fish have remained elusive. In mammals, a clear relation between high-fibre diets, such as those including β-glucans, and diet-induced immunomodulation

Topics: Animal Feed; Animals; beta-Glucans; Carps; Fatty Acids, Volatile; Gastrointestinal Microbiome; Immunomodulation

Anti-cancer T-cell responses are often halted due to the immune-suppressive micro-environment, in part related to tumor-associated macrophages. In the current study, we assessed indigestible β-glucans (oatβG, curdlan, grifolan, schizophyllan, lentinan, yeast whole glucan particles (yWGP), zymosan and two additional yeast-derived β-glucans a and b) for their physicochemical properties as well as their effects on the plasticity of human monocyte-derived macrophages that were polarized with IL-4 to immune-suppressive macrophages. Beta-glucans were LPS/LTA free, and tested for solubility, molecular masses, protein and monosaccharide contents. Curdlan, yeast-b and zymosan re-polarized M(IL-4) macrophages towards an M1-like phenotype, in particular showing enhanced gene expression of CCR7, ICAM1 and CD80, and secretion of TNF-α and IL-6. Notably, differential gene expression, pathway analysis as well as protein expressions demonstrated that M(IL-4) macrophages treated with curdlan, yeast-b or zymosan demonstrated enhanced production of chemo-attractants, such as CCL3, CCL4, and CXCL8, which contribute to recruitment of monocytes and neutrophils. The secretion of chemo-attractants was confirmed when using patient-derived melanoma-infiltrating immune cells. Taken together, the bacterial-derived curdlan as well as the yeast-derived β-glucans yeast-b and zymosan have the unique ability to preferentially skew macrophages towards a chemo-attractant-producing phenotype that may aid in anti-cancer immune responses.

Topics: beta-Glucans; Chemotactic Factors; Humans; Tumor-Associated Macrophages; Zymosan

A biopolymer-polyphenol conjugate-stabilized oil-in-water emulsion system was established to improve the chemical stability and bioaccessibility of β-carotene (BC). In this study, the emulsifying properties and contribution of a ferulic acid-grafted curdlan conjugate (Cur-D-g-FA) to the chemical stability of BC were investigated. Results showed that the emulsification ability of emulsions stabilized by Cur-D-g-FA remarkably increased with an increasing concentration from 0.05% to 0.8% (w/v) along with decreasing average droplet sizes, negatively charged zeta potentials, and uniform size distributions. The emulsions stabilized by 0.8% Cur-D-g-FA exhibited pronounced shear thinning and solid-like elastic properties as well as satisfactory oxidation stability. The emulsions stabilized by 0.8% Cur-D-g-FA had excellent ability to improve the chemical stability of BC when exposed to different environmental stresses and resulted in the favorable bioaccessibility of BC in vitro. The results prove that Cur-D-g-FA as a promising stabilizer has great potential to protect liposoluble nutrients in food-grade emulsion-delivery systems.

Topics: beta Carotene; beta-Glucans; Biological Availability; Corn Oil; Coumaric Acids; Emulsifying Agents; Emulsions; Food Storage; Hydrogen-Ion Concentration; Osmolar Concentration; Oxidation-Reduction

In this study, we report a rapid statistical approach used in determining the caprolactam (CPL) content in curdlan packaging films, which is based on the spectral data observed in the near-infrared (NIR) and Mid-infrared (MIR) regions. At the first stage of the study, the CPL content was added into the curdlan films prepared by controlling the concentration, and then the effect of the CPL concentration on the measured mechanical properties of the produced films were evaluated. At the next stage, the NIR and MIR spectra of the curdlan films with different CPL concentrations were recorded by using the FT-NIR and FT-IR spectroscopy technique, and the spectral data to be used in the regression models in our quantitative analyses were carefully selected. It was observed that the curdlan film with 5% CPL exhibited the best mechanical properties. The obtained best correlation parameters which are used in evaluation of CPL content through the observed NIR and MIR spectral data are Rp = 0.9552, RMSEP = 1.2506 (NIR); Rp = 0.9092 and RMSEP = 1.9136 (MIR), respectively. These optimal values support the expectation that our statistical approach based on NIR and MIR data can provide a rapid, accurate and nondestructive way of determining CPL content in curdlan packaging films.

Topics: beta-Glucans; Caprolactam; Food Packaging; Spectroscopy, Fourier Transform Infrared; Spectroscopy, Near-Infrared

The impact of preparation conditions including heating temperature (from 60 °C to 90 °C) and drying temperatures (from 25 °C to 90 °C) on the properties of pure curdlan film and konjac glucomannan (KGM) and curdlan blend films were analyzed. Microstructure analysis indicated the KGM addition could significantly improve the relatively poor film-forming property of curdlan. FTIR and X-ray analysis showed that at high heating temperature 90 °C, molecular interaction might be enhanced in the films due to the stretched structure of curdlan and dissociation of curdlan bundles or triple-helix structure. This was supported by the changes in the mechanical property, surface hydrophobicity, moisture barrier, and moisture tolerance property. The impacts of drying temperature were some different for the curdlan film and KGM/curdlan blend film, and were explained from the molecular hydrophilicity-hydrophobicity, compactness of the films, curdlan conformation, and molecular interaction. This work guided biodegradable film production especially with curdlan added.

Topics: beta-Glucans; Desiccation; Heating; Hydrophobic and Hydrophilic Interactions; Mannans; Mechanical Tests; Microscopy, Electron, Scanning; Permeability; Spectroscopy, Fourier Transform Infrared; Surface Properties; Temperature; Tensile Strength; Water; X-Ray Diffraction

In this study, to improve the mechanical and thermal properties of curdlan film, a curdlan/nanocellulose (NC) blended film was prepared and characterized for the first time. NC was successfully prepared from microcrystalline cellulose (MCC) with NaOH/urea treatment. The particle size of NC was observed to be 70-140 nm by cryo-electron microscope (cryo-EM). The blended film was prepared by adding the NC to curdlan solution. The tensile strength (TS) of the blended film reached the maximum value of 38.6 MPa, and the elongation at break (EB) was 40%. The DSC curve showed that the heat absorption peak of the film was 240 °C, indicating that the blended film has good temperature stability. Additionally, some other film properties were also improved, including gas barrier properties and transparency. Obvious morphological and molecular differences between the blended film and the pure curdlan film were discovered by SEM and FTIR analysis. Finally, the blended film was used for the preservation of chilled meat and extended the storage time of meat to 12 days. These results provided a theoretical basis for future application and development of biodegradable film.

Topics: beta-Glucans; Food Packaging; Meat; Tensile Strength

AS is a rheumatic disease characterized by chronic inflammation and bony ankylosis. This study was to evaluate whether a signal transducer and activator of transcription 3 phosphorylation inhibitor (stat3-p Inh) could treat both chronic inflammation and bone formation in AS.. Primary AS osteoprogenitor cells and spinal entheseal cells were examined for osteogenic differentiation. SF mononuclear cells (SFMCs) and lamina propria mononuclear cells (LPMCs) were obtained from AS patients. Inflammatory cytokine-producing cells were analysed using flow cytometry and ELISA. Female SKG mice were treated with stat3-p Inh, IL-17A blocker or vehicle. Inflammation and new bone formation were evaluated using immunohistochemistry, PET and micro-CT.. In the SKG mouse model, stat3-p Inh significantly suppressed arthritis, enthesitis, spondylitis and ileitis. In experiments culturing SFMCs and LPMCs, the frequencies of IFN-γ-, IL-17A- and TNF-α-producing cells were significantly decreased after stat3-p Inh treatment. When comparing current treatments for AS, stat3-p Inh showed a comparable suppression effect on osteogenesis to Janus kinase inhibitor or IL-17A blocker in AS-osteoprogenitor cells. Stat3-p Inh suppressed differentiation and mineralization of AS-osteoprogenitor cells and entheseal cells toward osteoblasts. Micro-CT analysis of hind paws revealed less new bone formation in stat3-p Inh-treated mice than vehicle-treated mice (P = 0.005). Hind paw and spinal new bone formation were similar between stat3-p Inh- and anti-IL-17A-treated SKG mice (P = 0.874 and P = 0.117, respectively).. Stat-3p inhibition is a promising treatment for both inflammation and new bone formation in AS.

Topics: Adult; Animals; beta-Glucans; Cell Differentiation; Disease Models, Animal; Female; Humans; Ileitis; Inflammation; Male; Mice; Middle Aged; Osteoblasts; Osteogenesis; Phosphorylation; Positron-Emission Tomography; Spondylitis, Ankylosing; STAT3 Transcription Factor; Stem Cells; Thiophenes; X-Ray Microtomography; Young Adult

The ternary HAp/curdlan/nanomagnetite hybrids with ceramic and polymer phase incorporation of magnetite nanoparticles (MNPs) were fabricated to study their heating ability under action of the alternating magnetic field (AMF), 808 nm near infrared laser radiation (NIR) and their synergic stimulation. The energy conversion was evaluated in terms of the specific absorption rate (SAR) as a function of the MNPs concentration in composites and to estimate their potential in temperature-controlled regenerative processes and hyperthermia. Measurements were carried out on dry and Ringer's solution soaked composite materials in order to mimic in situ conditions. It was found that the MNPs release during prolonged experiment is limited and has no significant effect on energy conversion emphasizing stability of the hybrids. Incorporation of the MNPs in polymer phase of the hybrid can additionally limit particle leaking as well as plays a role as insulating layer for the heat dissipation lowering the risk of sample overheating. In general, it was shown that maximum temperature of hybrid can be achieved in a relatively short time of exposure to stimulating factors whereas its control can be done through optimization of experiment conditions. MNPs incorporation into the curdlan (polymer phase) lead to strengthening of the mechanical properties of the whole network.

Topics: beta-Glucans; Durapatite; Hot Temperature; Hyperthermia, Induced; Magnetite Nanoparticles; Temperature

The cbm6e gene from Saccharophagus degradans 2-40

Topics: beta-Glucans; Biocatalysis; Cellulase; Cloning, Molecular; Escherichia coli; Gammaproteobacteria; Glycoside Hydrolases; Hydrogen-Ion Concentration; Hydrolysis; Oligosaccharides; Polysaccharides, Bacterial; Substrate Specificity; Temperature

Topics: beta-Glucans; Chemokines; Cytokines; Fluorescence Resonance Energy Transfer; Gene Expression Regulation; Humans; Hydrogen; Immunomodulation; Macrophages; Nanoparticles; Polyglutamic Acid

RNA interference technology is a powerful tool with substantially clinical prospects for carcinoma therapy, in which efficiency and specificity of delivery of dsRNA remains a critical issue. Herein, aiming at delivery of dsRNA in efficient and safe way, we constructed targeting delivery platform (CTL-PEG-FA) by grafting curdlan with trilysine through click reaction, then modifying with PEG linked folic acid. The CTL-PEG-FA vector exhibited excellent gene binding capacity to condense siRNA and dramatically reduced cytotoxicity. Increased cell uptake of CTL-PEG-FA/Bcl-2 siRNA was achieved by the synergism of folate mediated endocytosis and charge interaction, and further causing severe HepG2 cells injury through apoptosis mechanism after down-regulation of Bcl-2 protein. In vivo experiments, CTL-PEG-FA/Bcl-2 siRNA complex distinctly accumulated in tumor site and significantly inhibited the growth of tumor, while no obvious toxicity was observed. Therefore, well-performed CTL-PEG-FA with excellent biocompatibility, has the potential to be the candidate of gene therapy for clinical applications.

Topics: beta-Glucans; Carcinoma; Cell Line, Tumor; Folic Acid; Humans; Polyethylene Glycols; RNA, Small Interfering

Permanent orthopedic/dental implants should reveal good osseointegration, which is defined as an ability of the biomaterial to form a direct connection with the surrounding host bone tissue after its implantation into the living organism. Currently, biomaterial osseointegration is confirmed exclusively with the use of in vivo animal tests. This study presents for the first time ex vivo determination of osseointegration process using human trabecular bone explant that was drilled and filled with the chitosan/curdlan/hydroxyapatite biomaterial, followed by its long-term culture under in vitro conditions. Within this study, it was clearly proved that tested biomaterial allows for the formation of the connection with bone explant since osteoblasts, having ability to produce bone extracellular matrix (type I collagen, fibronectin), were detected at a bone-implant interface by confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). Importantly, in this research it was demonstrated by Live/Dead staining and CLSM imaging that human bone explants may stay alive for a long period of time (at least approx. 50 days) during their culture under in vitro conditions. Therefore, ex vivo bone explant, which is a heterogeneous tissue containing many different cell types, may serve as an excellent model to test biomaterial osseointegration during comparative and preliminary studies, reducing animal tests which is compatible with the principles of '3Rs', aiming to Replace, Reduce and Refine the use of animals wherever possible.

Topics: Animals; beta-Glucans; Biocompatible Materials; Bone and Bones; Cancellous Bone; Chitosan; Dental Implants; Durapatite; Humans; Materials Testing; Microscopy, Electron, Scanning; Osseointegration; Surface Properties; Titanium

Curdlan has been applied to weaken the quality deterioration of frozen cooked noodles (FCN) during frozen storage. However, the underlying mechanism is still unclear. In this paper, an A/LKB-F probe was used for texture profile analysis and mercury intrusion was firstly used for analyzing ice crystals state in three dimensions. Meanwhile, a systematic study on the water state was conducted, as well as the freeze-thawed stability of FCN under curdlan intervention during frozen storage. The results showed that 0.5% curdlan significantly (P < 0.05) alleviated the decrement in hardness, chewiness and extension, and enhanced the freeze-thawed stability of FCN. This was closely associated with the fact that the addition of curdlan minimized freezable water content, inhibited water mobility and migration, and raised the homogeneity of ice crystals in FCN. This study provides more comprehensive theories for the strengthening effect of curdlan on FCN quality from the perspective of water state.

Topics: beta-Glucans; Cooking; Food Storage; Freezing; Hardness; Mercury; Water

Curdlan hydrogel obtained after thermal gelling exhibits elasticity and high water-absorbing capacity. However, its modifications leading to the increase of biofunctionality usually alter its solubility and reduce mechanical parameters. Therefore, curdlan hydrogel was modified by deposition of polydopamine to improve its capacity to bind biologically active molecules with free amino groups. It exhibited the unchanged structure, mechanical properties and increased soaking capacity. Aminoglycoside antibiotic (gentamicin) as a model molecule was effectively immobilized to such modified curdlan via quinone moiety (but not amino groups) of polydopamine. Approximately 50 % of the immobilized drug was released following Fickian diffusion and inhibited the bacterial growth in matrix-surrounding medium in prolonged manner. The remaining drug amount was stably attached and prevented the hydrogel against bacterial adhesion even when all the mobile drug has been released. Therefore, polydopamine-modified curdlan hydrogel shows the potential for fabrication of functional materials for different purposes, including drug-loaded biomaterials.

Topics: Anti-Bacterial Agents; Bacterial Adhesion; beta-Glucans; Coated Materials, Biocompatible; Drug Carriers; Drug Compounding; Drug Liberation; Elasticity; Escherichia coli; Gentamicins; Humans; Hydrogels; Indoles; Kinetics; Microbial Sensitivity Tests; Polymers; Solubility; Staphylococcus aureus; Staphylococcus epidermidis; Wettability

In this work, an innovative composite hydrogel composed of curdlan (CD)/polyvinyl alcohol (PVA) hydrogels with a 3-d network structure was successfully prepared by freeze-thaw processing. The presence of interactions, changes in crystallinity, and thermal behaviour were investigated by Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), and thermogravimetry (TGA and DTG), respectively. The morphology of the hydrogels was investigated by scanning electron microscopy (SEM). With the increase of PVA concentration, the composite hydrogel had a greater mechanical strength while remaining remarkably ductile as evinced by tensile test results. PVA content affects the swelling and water retention of CD/PVA hydrogels. The results of CCK-8 assay showed that CD/PVA hydrogels have no cytotoxic effect on the mouse fibroblast L929 cells. The AO/EB double-staining experiment further proved that the cells in the composite hydrogels had good cytocompatibility. The porous biohydrogels developed in the present work can provide an ideal cell growth environment as a scaffold. CD/PVA hydrogels highlight the value of this system for cell adhesion and proliferation, and further soft tissue engineering application.

Topics: Animals; beta-Glucans; Biocompatible Materials; Cell Line; Freezing; Hydrogels; Mice; Microscopy, Electron, Scanning; Polyvinyl Alcohol; Porosity; Spectroscopy, Fourier Transform Infrared; Tensile Strength; Thermogravimetry; Tissue Engineering; Tissue Scaffolds; X-Ray Diffraction

Grafting copolymerization of phenolic acids onto polysaccharides is an important strategy to improve their biological activities. In this study, ferulic acid (FA)-grafted carboxylic curdlan conjugates, namely, Cur-8-g-FA, Cur-24-g-FA, and Cur-48-g-FA, were synthesized by free radical-induced grafting. Results showed that FA was covalently grafted onto carboxylic curdlans via ester bonds. The grafting ratios of Cur-8-g-FA, Cur-24-g-FA, and Cur-48-g-FA were 223.03 ± 12.63, 115.63 ± 5.96, and 152.30 ± 4.57 mg FA/g, respectively, which were related with the carboxylate contents, molecular weights, and chain conformations of carboxylic curdlans. Compared with carboxylic curdlans, the FA-grafted carboxylic curdlan conjugates had lower thermal stability, molecular weight, and rheological property and looser surface morphology but had more prominent antioxidant benefits in vitro, which were proportional to their grafting ratios. Moreover, good storage stability against chemical degradation was exhibited by the β-carotene in Pickering emulsions stabilized by Cur-8-g-FA with a high grafting ratio and molecular weight.

Topics: Antioxidants; beta Carotene; beta-Glucans; Carboxylic Acids; Coumaric Acids; Emulsions; Free Radicals; Hydroxybenzoates; Molecular Weight

In this study, curdlan sulphate - chitosan nanoparticles were prepared through polyelectrolyte complexing at a mass ratio of 2:1 respectively. The curdlan was produced by fermentation with Agrobacterium sp. ATCC 31750, which was then sulphated to form the polyanionic polymer. A first-line tuberculosis drug, Rifampicin and a phytochemical, DdPinitol, were encapsulated into Curdlan Sulphate (CS) - Chitosan Nanoparticles (C) (CSC NPs) of size 205.41 ± 7.24 nm. The drug release kinetics followed a Weibull model with initial burst release (48 % Rifampicin and 27 % d-Pinitol within 6 h), followed by a sustained release. The prepared CSC: d-PIN + RIF NPs was cytocompatible and entered the M.smegmatis infected macrophages through multiple endocytic pathways including clathrin, caveolae and macropinocytosis. They showed superior bactericidal activity (2.4-2.7 fold) within 4 h when compared to free drug Rifampicin (1.6 fold). The drug encapsulated CSC: RIF suppressed the pro-inflammatory gene (TNF-α by 3.66 ± 0.19 fold) and CSC: d-PIN + RIF increased expression of the anti-inflammatory gene (IL-10 by 13.09 ± 0.47 fold). Expression of TGF- β1 gene also increased when treated with CSC: d-PIN + RIF (13.00 ± 0.19 fold) which provided the immunomodulatory activity of the encapsulated CSC NPs. Thus, curdlan sulphate - chitosan polyelectrolyte complex can be a potential nanocarrier matrix for intracellular delivery of multiple drugs.

Topics: Animals; beta-Glucans; Cell Survival; Chitosan; Drug Carriers; Drug Delivery Systems; Drug Liberation; Endocytosis; Hydrogen-Ion Concentration; Inflammation; Kinetics; Macrophages; Mice; Mycobacterium Infections, Nontuberculous; Mycobacterium smegmatis; Nanoparticles; Polyelectrolytes; Polymers; RAW 264.7 Cells; Rifampin

Beta-glucans are naturally occurring polysaccharides present in cell walls of fungi, yeast, bacteria, cereals, seaweed, and algae. These microbe-associated molecular patterns (MAMPs) possess immunomodulatory properties. In human, it has been suggested that NK cells can be activated by β-glucans. Here, we aimed to elucidate whether β-glucans modulate porcine NK cell responses

Topics: Animals; beta-Glucans; Coculture Techniques; Cytotoxicity, Immunologic; Humans; Interleukin-10; K562 Cells; Killer Cells, Natural; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Monocytes; Paracrine Communication; Secretory Pathway; Sus scrofa

Curdlan (CN)-doped montmorillonite/poly(N-isopropylacrylamide-co-N,N'-methylene-bis-acrylamide) [CN/MT/P(NIPA-co-MBA)] smart nanocomposites (NCs) were developed for efficient erlotinib HCl (ERL) delivery to lung cancer cells. The placebo NCs demonstrated excellent biodegradability, pH/thermo-responsive swelling profiles and declined molar mass (M¯c) between the crosslinks with increasing temperature. The XRD, FTIR, DSC, TGA, and SEM analyses revealed the architectural chemistry of these NC scaffolds. The NCs loaded with ERL (F-1-F-3) displayed acceptable diameter (734-1120 nm) and zeta potential (+1.16 to -11.17 mV), outstanding drug entrapping capability (DEE, 78-99%) and sustained biphasic ERL elution patterns (Q

Topics: A549 Cells; Acrylamides; Adsorption; Bentonite; beta-Glucans; Cell Survival; Drug Liberation; Endocytosis; Erlotinib Hydrochloride; Humans; Hydrogen-Ion Concentration; Kinetics; Mucins; Nanocomposites; Particle Size; Spectroscopy, Fourier Transform Infrared; Staining and Labeling; Static Electricity; Temperature; Thermogravimetry; X-Ray Diffraction

Elucidating the structure-activity relationship of curdlan is hampered by a lack of characterization with unique specific conformations (i.e., single- or triple-helix). In this study, single-helical curdlan is generated in dilute NaOH solutions at 35-50 °C, and characterized with NMR, SAXS, and GPC. The conformational transition from coil to single-helix and the intramolecular hydrogen bond interaction are explored using NMR. It is found that the two aforementioned types of curdlan interact with Congo Red in very different ways. Single-helical curdlan can encapsulate Congo Red to form a stable, supramolecular dye assembly, which is demonstrated by the shortest distance between the H3 of curdlan and the phenyl groups of Congo Red, and also the same self-diffusion coefficients of Congo Red and curdlan. In contrast, random-coil curdlan interacts weakly with Congo Red and cannot enwrap it. This study offers insight into the specific structure-activity relationship of beta-(1,3)-glucans.

Topics: beta-Glucans; Chromatography, Gel; Congo Red; Hydrogen Bonding; Magnetic Resonance Spectroscopy; Molecular Conformation; Scattering, Small Angle; Sodium Compounds; Spectrophotometry, Ultraviolet; Structure-Activity Relationship; Temperature

Curdlan activates dendritic cells (DCs) and enhances DC-based antitumor immunity. However, hydrophobicity and heterogeneity of curdlan particulates hinder perfect binding of curdlan to dectin-1 receptor, resulting in the reduced activation of antigen presenting cells and limited antitumor effects. Herein, we synthesized partially oxidized curdlan derivative (β-1,3-polyglucuronic acid, denote PGA). PGA-45 polymer, the reaction product prepared from curdlan by oxidation with 4-acetamido-TEMPO/NaClO/NaClO

Topics: Adjuvants, Immunologic; Animals; Antineoplastic Agents; beta-Glucans; Cell Line, Tumor; Cell Proliferation; Cytokines; Dendritic Cells; Humans; Immunity; Immunotherapy; Lectins, C-Type; Mice; Mice, Inbred C57BL; Neoplasms; Oxidation-Reduction; Polymers; T-Lymphocytes; Toll-Like Receptor 4

β-glucan consumption is known for its beneficial health effects, but the mode of action is unclear. While humans and mice lack the required enzymes to digest β-glucans, certain intestinal microbes can digest β-glucans, triggering gut microbial changes. Curdlan, a particulate β-glucan isolated from

Topics: Animals; beta-Glucans; Bifidobacterium; Colitis; Colon; Dextran Sulfate; Diet; Gastrointestinal Microbiome; Humans; Mice

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

Topics: Bandages; beta-Glucans; Colloids; Exudates and Transudates

The production of curdlan oligosaccharides, a multifunctional and valuable carbohydrate, by hydrolyzing polysaccharides is of great interest. The endo-β-1,3-glucanase derived from Trichoderma harzianum was expressed in Pichia pastoris with three commonly used promoters (AOX1, GAP and FLD1). The purified recombinant endo-β-1,3-glucanase expressed by Pichia pastoris with GAP promoter displayed high specific activity at pH 5.5 and 50 °C. Thereafter, a co-culture system of Pichia pastoris GS115 (GAP promoter) and Agrobacterium sp. was constructed in which Agrobacterium sp.-metabolized curdlan can be directly hydrolyzed by Pichia pastoris-secreted endo-β-1,3-glucanase to produce functional curdlan oligosaccharides. The co-culture conditions were optimized and the process was carried out in a 7-L bioreactor. The maximum yield of curdlan oligosaccharides reached 18.77 g/L with 3-10 degrees of polymerization. This study presents a novel and easy curdlan oligosaccharide production strategy that can replace traditional sophisticated production procedures and could potentially be implemented for production of other oligosaccharides.

Topics: Agrobacterium; beta-Glucans; Cellulase; Coculture Techniques; Oligosaccharides; Pichia; Saccharomycetales

In this study, curdlan-based and calcium ion (Ca

Topics: beta-Glucans; Hypocreales; Magnetic Phenomena; Microspheres

Curdlan is a water-insoluble exopolysaccharide produced by Agrobacterium species under nitrogen starvation. The curdlan production in the ΔmdeA, ΔmetA, ΔmetH, and ΔmetZ mutants of methionine biosynthesis pathway of Agrobacterium sp. CGMCC 11546 were significantly impaired. Fermentation profiles of four mutants showed that the consumption of ammonia and sucrose was impaired. Transcriptome analysis of the ΔmetH and ΔmetZ mutants showed that numerous differentially expressed genes involved in the electron transfer chain (ETC) were significantly down-regulated, suggesting that methionine biosynthesis pathway affected the production of energy ATP during the curdlan biosynthesis. Furthermore, metabolomics analysis of the ΔmetH and ΔmetZ mutants showed that ADP and FAD were significantly accumulated, while acetyl-CoA was diminished, suggesting that the impaired curdlan production in the ΔmetH and ΔmetZ mutants might be caused by the insufficient supply of energy ATP. Finally, the addition of both dibasic sodium succinate as a substrate of FAD recycling and methionine significantly restored the curdlan production of four mutants. In conclusion, methionine biosynthesis pathway plays an important role in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546, which affected the sufficient supply of energy ATP from the ETC during the curdlan biosynthesis.

Topics: Adenosine Triphosphate; Agrobacterium; Bacterial Proteins; beta-Glucans; Biosynthetic Pathways; Fermentation; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Metabolomics; Methionine; Mutation; Nitrogen

The epithelium-associated cytokine thymic stromal lymphopoietin (TSLP) can induce OX40L and CCL17 expression by myeloid dendritic cells (mDCs), which contributes to aberrant Th2-type immune responses. Herein, we report that such TSLP-induced Th2-type immune response can be effectively controlled by Dectin-1, a C-type lectin receptor expressed by mDCs. Dectin-1 stimulation induced STAT3 activation and decreased the transcriptional activity of p50-RelB, both of which resulted in reduced OX40L expression on TSLP-activated mDCs. Dectin-1 stimulation also suppressed TSLP-induced STAT6 activation, resulting in decreased expression of the Th2 chemoattractant CCL17. We further demonstrated that Dectin-1 activation was capable of suppressing ragweed allergen (Amb a 1)-specific Th2-type T cell response in allergy patients

Topics: Adult; Allergens; Animals; Antigens, Dermatophagoides; Antigens, Plant; beta-Glucans; Case-Control Studies; Cytokines; Dendritic Cells; Dermatophagoides farinae; Disease Models, Animal; Female; HEK293 Cells; Humans; Hypersensitivity; Immunity; Lectins, C-Type; Macaca mulatta; Male; Middle Aged; NF-kappa B p50 Subunit; OX40 Ligand; Plant Proteins; Signal Transduction; STAT3 Transcription Factor; STAT6 Transcription Factor; Th2 Cells; Thymic Stromal Lymphopoietin; Transcription Factor RelB

Topics: Animals; Anti-Bacterial Agents; Anti-Inflammatory Agents; beta-Glucans; Carbohydrate Conformation; Escherichia coli; Hydrogels; Male; Metal Nanoparticles; Mice; Mice, Inbred BALB C; Nanofibers; NIH 3T3 Cells; Silver; Skin; Staphylococcal Skin Infections; Staphylococcus aureus; Tensile Strength; Wound Healing

The chain conformational change in curdlan during carboxymethylation was investigated using nuclear magnetic resonance (NMR), circular dichroism (CD) spectroscopy, and atomic force microscopy (AFM). The distributions of carboxymethyl substituents within anhydroglucose unit (AGU) of CMCD were found to follow the order of OH (6) > OH (4) > OH (2) for CMCD with a low DS and OH (6) > OH (2) > OH (4) for CMCD with relatively high DS. The increased carboxymethylation level induced the chain conformation transition of curdlan from triple helix to random coil in water. The DS of 0.25 was the critical value of chain conformation transition, below which CMCD chains were triple helices. For DS larger than 0.25, CMCD existed in the state of random coils. The intermolecular hydrogen bonding between C2 hydroxyls in AGU sustained the triple helical conformation and stiffness of the polymer chain, which weakened with the increase in DS.

Topics: beta-Glucans; Chemical Phenomena; Circular Dichroism; Hydrogen Bonding; Macromolecular Substances; Magnetic Resonance Spectroscopy; Methylation; Microscopy, Atomic Force; Molecular Conformation; Polymers; Water

Developing nucleic acid-based tools to control disease-relevant gene expression in human disorders, such as siRNAs, opens up potential opportunities for therapeutics. Because of their high molecular weight and polyanionic nature, synthetic siRNAs fail to cross biological membranes by passive diffusion and therefore, generally require transmembrane siRNA delivery technologies to access the cytoplasm of target cells. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan derivative 6AC-100 in a regioselective manner to introduce different ratios of imidazole rings in the amino units (denoted as Curimi) and evaluated their siRNA binding ability, cytotoxicity, endosome buffering capacity and siRNA transfection efficiency. The novel curdlan based Curimi polymers formed nanoparticles with siRNA at pH 7.4 in range of 85-105 nm and their size distribution increased along with decreasing pH condition. The zeta potential increased by lowering pH value as well. Curimi polymers showed lower toxicity and higher buffering capacity compared to 6AC-100, and efficiently delivered siRNA against to PLK1 into cancer cells, and subsequently, significantly inhibited target mRNA level. Our result suggested that novel curdlan based Curimi polymers may be used as efficient siRNA carrier for cancer therapy.

Topics: beta-Glucans; Buffers; Endosomes; Gene Transfer Techniques; HeLa Cells; Hep G2 Cells; Humans; Hydrogen-Ion Concentration; Imidazoles; Lysosomes; Nanoparticles; Particle Size; Polyelectrolytes; Polymers; Polysaccharides, Bacterial; RNA, Small Interfering; Transfection

Cationic curdlan derivatives are a class of promising carriers for nucleic acid delivery including short interfering RNA (siRNA). While our previous studies demonstrated the siRNA delivery efficiency of aminated curdlan derivatives, the associated cytotoxicity issue remained unsolved. To investigate the effects of alkylation on the toxicity as well as the transfection efficiency, we conjugated short alkyl chains to 6-amino-6-deoxy-curdlan (6AC-100). The cytotoxicity of alkylated 6AC-100 derivatives (denote CuVa polymers) decreased with the increase of the degree of substitution (DS). CuVa3, with the highest DS, showed a 50% decreased cytotoxicity compared to 6AC-100 to 6AC-100 at a concentration of 140 μg/mL. The CuVa polymers readily complexed with siRNA to form nanoparticles, and induced significant knockdown of a disease related gene (STAT3) in mouse melanoma cell line B16. However, B16 cells transfected with siSTAT3 complexed to CuVa3 showed the highest phenotypic changes. These findings suggest that CuVa polymers have significantly enhanced biocompatibility and may be a promising delivery system for delivery of therapeutic siRNAs.

Topics: Alkylation; Animals; beta-Glucans; Cations; Cell Line, Tumor; Gene Transfer Techniques; Humans; Magnetic Resonance Spectroscopy; Mice; Nanoparticles; Particle Size; RNA, Small Interfering; Transfection

In this study, the effects of the addition of various polysaccharides (konjac gum, gellan gum, and curdlan gum) on the rheological and textural properties of calcium sulfate-induced soy protein isolate gels were investigated. The incorporation of konjac gum and curdlan gum at 0.3 and 0.5% (w/v) concentrations and gellan gum at 0.5% concentration significantly enhanced (P < 0.05) the hardness and water-holding capacity of the resultant gels. The increased elastic moduli during and after gelation, reinforced fracture stress, and lowered onset gelling temperature indicated that the addition of the abovementioned polysaccharides strengthened gel structures and accelerated gelation. Confocal laser scanning microscopy analysis revealed that the polysaccharides also improved gel microstructures, with the gels containing konjac gum displaying the highest homogeneity. The findings of this study may provide important information for the development of innovative soy protein isolate-based gel products with improved texture.

Topics: Amorphophallus; beta-Glucans; Calcium Sulfate; Elastic Modulus; Gels; Polysaccharides; Polysaccharides, Bacterial; Rheology; Soybean Proteins; Temperature; Water

This study investigated the gelling and structural properties of composite gels prepared with myofibrillar protein (MP) and different concentrations of thermo-reversible curdlan gels (TRC) and thermo-irreversible curdlan gels (TIRC). The gel strength, water holding capacity, and whiteness of MP gels were significantly increased with increasing TRC and TIRC content (P < 0.05). Dynamic rheological testing upon temperature sweeping indicated that the G' and G″ values of MP were significantly enhanced with added TRC or TIRC, which were positively related to the lower particle size of composite MP sols. Moreover, the presence of TRC or TIRC changed the secondary structure of MP gels, as well as altered the tertiary structure of MP sols. The micrographs of MP gels indicated that TRC prompted the formation of more dense and stable protein matrix. Our results revealed that TRC or TIRC had distinctive effects on the gelling properties and secondary structure of MP.

Topics: Animals; beta-Glucans; Gels; Muscle Proteins; Rheology; Swine; Temperature; Water

Curdlan, an insoluble and neutral polysaccharide, was produced from Agrobacterium sp. ATCC 31750 and chemically modified with dimethylaminoethyl (DMAE) group to introduce gene binding ability. The resulting DMAE-curdlan was crosslinked with curdlan nanoparticles using epichlorohydrin. The prepared nanoparticles are spherical with an average diameter of 523 ± 195 nm, stable and are highly biocompatible with differentiated THP-1 macrophages with viability of above 90%. They are taken up more efficiently by RAW 264.7 macrophage cells than by L929 fibroblast cells. They increase the expression of M1 macrophage marker genes, TNFα and CXCL10, and decrease the expression of M2 marker, CD206, indicating their ability to activate M1 phenotype and aid in tumor regression. They are also capable of delivering siRNA to human macrophage-like cells efficiently and inhibit ~59% of the expression of target MMP-9 protein. These results indicate that this modified curdlan-based nanoparticle is a promising vehicle for the delivery of siRNAs to macrophages, which could open up treatment strategies for a range of diseases.

Topics: Animals; beta-Glucans; Biomarkers; Cell Death; Endocytosis; Ethylamines; Fibroblasts; Gene Transfer Techniques; Humans; Macrophages; Matrix Metalloproteinase 9; Matrix Metalloproteinase Inhibitors; Mice; Nanoparticles; Particle Size; RAW 264.7 Cells; RNA, Messenger; RNA, Small Interfering; Static Electricity; THP-1 Cells; Transfection

The unique conformation transition from a triple helix to single coils for the triple helical β-d-glucans has paved the way to fabricate various functional nanocomposites through the denaturing-renaturing process. This study firstly reports a novel kind of naturally derived supramolecular polymer micelle consisting of single-stranded chains of curdlan (CUR) and β-CDs. It is proposed that β-CDs as the host molecules were threaded onto single β-glucan chains (denatured triplex CUR) via the host-guest interaction, thereby forming supramolecular micelles. The results from the 1H NMR, FT-IR, XRD and 2D 1H NOESY NMR studies confirmed the formation of the inclusion complex and the existence of the core-shell structure of the supramolecular assembly. TEM images and DLS revealed that the self-organized micelles displayed a regular spherical shape with an average diameter of ∼27 nm. Furthermore, the hydrophobic anticancer drug camptothecin (CPT) was selected as a model drug and successfully encapsulated into the CUR/β-CD micelles. The drug-loaded micelles exhibited a steady sustained-release pattern regardless of the environmental pH. The flow cytometry and confocal laser scanning microscopy measurements confirmed that the CPT-loaded micelles could be well internalized into HepG 2 cells and continuously release the drug molecules inside the tumor cells. Meanwhile, the in vivo experiments demonstrated that CPT-loaded micelles could effectively inhibit tumor growth in comparison to free drugs. This concept will give a favorable platform to construct intelligent drug delivery systems for potential use.

Topics: Animals; Antineoplastic Agents, Phytogenic; beta-Glucans; Camptothecin; Cell Proliferation; Cell Survival; Delayed-Action Preparations; Drug Compounding; Drug Delivery Systems; Hep G2 Cells; Humans; Liver Neoplasms; Mice; Micelles; Microscopy, Confocal; Models, Molecular; Protein Conformation, alpha-Helical; Spectroscopy, Fourier Transform Infrared; Xenograft Model Antitumor Assays

Topics: Animals; beta-Glucans; Cell Physiological Phenomena; Cells, Cultured; Disease Models, Animal; Drug Delivery Systems; Gene Knockdown Techniques; Infarction, Middle Cerebral Artery; Mice; Microglia; Nanoparticles; Neuroprotective Agents; RNA Interference; RNA, Small Interfering; Transcription Factor RelA

Hydrogels composed of food gums have gained attention for future biomedical applications, such as targeted delivery and tissue engineering. For their translation to clinical utilization, reliable biocompatibility, sufficient mechanical performance, and tunable structure of polysaccharide hydrogels are required aspects. In this work, we report a unique hybrid polysaccharide hydrogel composed of salecan and curdlan, in which the former is a thickening agent and the latter serves as a network matrix. The physicochemical properties, such as mechanical strength, thermal stability, swelling, and morphology, of the developed composite hydrogel can be accurately modulated by varying the polysaccharide content. Importantly, cytotoxicity assays show the non-toxicity of this hybrid hydrogel. Furthermore, this hydrogel system can support cell proliferation, migration, and function. Altogether, our work proposes a new strategy to build a polysaccharide-constructed hydrogel scaffold, which holds much promise for tissue engineering in terms of cell engraftment, survival, proliferation, and function.

Topics: Animals; beta-Glucans; Biocompatible Materials; Cell Culture Techniques; Cell Line; Cell Proliferation; Cell Survival; Food Additives; Hydrogels; Materials Testing; Mice; Tissue Engineering; Tissue Scaffolds

Mast cells (MCs) are engaged in host defense against various pathogens as they are equipped with pattern recognition receptors (PRRs). Among PRRs expressed on MCs, there are also molecules recognizing components of the fungal cell wall, which are able to induce cellular activation and response. However, little information is available concerning the MC activation by various fungal-derived components. The aim of the study was to determine whether curdlan, a model fungal particle of β-(1,3)-glucan, can directly stimulate tissue MCs. We demonstrated that curdlan triggers MCs to initiate pro-inflammatory response as it activates these cells to synthesize essential pro-inflammatory and/or immunoregulatory factors. We also showed that curdlan serves as a potent chemoattractant for MCs and stimulates those cells to generate reactive oxygen species (ROS). Finally, we documented that curdlan induces MC response via Dectin-1. Our observations support the idea that MCs serve as important sentinels modulating immune response during fungal infection.

Topics: Animals; beta-Glucans; Cell Degranulation; Chemotaxis, Leukocyte; Female; Lectins, C-Type; Mast Cells; Rats; Rats, Wistar; Reactive Oxygen Species

Ankylosing spondylitis (AS) is characteristically male-predominant, and progressive spinal ankylosis affects male patients more severely; however, the hormonal effects in males with AS are poorly understood.. In the present study, the regulatory effects of dutasteride, a 5-α reductase inhibitor that blocks the conversion of testosterone to dihydrotestosterone (DHT), were examined in curdlan-administered male SKG mice to determine spinal bone formation, bone metabolism-related markers, and interleukin (IL)-17A cytokine and T cell populations. In addition, the effects of DHT on primary osteoprogenitors from the facet joints of AS patients were assessed based on osteoblast-related parameters. DHT level was measured, and the correlation with modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS) was analyzed in AS patients.. In curdlan-administered SKG mice, dutasteride treatment resulted in an increased accumulation of hydroxyapatite in the spine which was positively correlated with serum IL-17A levels. In the analysis of bone metabolism-related molecules, a decrease in sclerostin levels was observed in the sera in the dutasteride group. Continuous exposure to DHT resulted in fewer calcium deposits in AS osteoprogenitors during osteoblast differentiation. DHT-treated AS osteoprogenitors showed decreased osteocalcin and increased DKK1 and SOST1 mRNA expression, supporting the results of the in vivo experiments. Treatment with dutasteride upregulated bone formation in the spine of curdlan-administered SKG mice and DHT treatment downregulated osteoblast differentiation in vitro.. Treatment with dutasteride affected the bone formation in the spine of curdlan-treated SKG mice, and DHT treatment attenuated osteoblast differentiation in vitro. Therefore, contrary to what could be expected if osteoblasts contributed to spinal ankylosis, DHT inhibition might increase rather than decrease the progression of spinal ankylosis despite the higher levels of DHT observed in many AS patients.

Topics: Animals; beta-Glucans; Dihydrotestosterone; Humans; Male; Mice; Osteoblasts; Spondylitis, Ankylosing

In this study, we prepared a series of edible blend films of xanthan and curdlan by mixing different ratios of these two biopolymers. Characterization techniques like FTIR, XRD, TGA and SEM analysis were applied to investigate the newly formed films. Moreover, mechanical properties, moisture absorbance properties and water solubility of these films were also determined. The obtained results demonstrated that the strong intermolecular hydrogen bonding was observed between xanthan and curdlan at pH 5. At this pH, the xanthan-curdlan hydrogel retained the original structure of xanthan and sustained self-aggregation of xanthan chains via hydrogen bonding, which led to strong intermolecular bonding between xanthan and curdlan. Furthermore, the 5:5 and 4:6 ratios of xanthan and curdlan showed greater interaction in the blend films that resulted in their excellent miscibility. Moreover, highest tensile strength of 28.13 and 26.45 MPa were also found in the same rational of XG/CG blend films. In addition, it was observed that the curdlan incorporation improved the water solubility properties of XG/CG blend films. Conclusively, this xanthan/curdlan nexus with excellent mechanical and moisture barrier properties confirm its potential application and prospective use as food packaging material.

Topics: beta-Glucans; Biopolymers; Food Packaging; Hydrogels; Hydrogen Bonding; Hydrogen-Ion Concentration; Microscopy, Electron, Scanning; Permeability; Polysaccharides, Bacterial; Solubility; Spectroscopy, Fourier Transform Infrared; Temperature; Tensile Strength; Water; X-Ray Diffraction

An important comorbidity of chronic inflammation is anemia, which may be related to dysregulated activity of hematopoietic stem and progenitor cells (HSPCs) in the bone marrow (BM). Among HSPCs, we found that the receptor for IL-33, ST2, is expressed preferentially and highly on erythroid progenitors. Induction of inflammatory spondyloarthritis in mice increased IL-33 in BM plasma, and IL-33 was required for inflammation-dependent suppression of erythropoiesis in BM. Conversely, administration of IL-33 in healthy mice suppressed erythropoiesis, decreased hemoglobin expression, and caused anemia. Using purified erythroid progenitors in vitro, we show that IL-33 directly inhibited terminal maturation. This effect was dependent on NF-κB activation and associated with altered signaling events downstream of the erythropoietin receptor. Accordingly, IL-33 also suppressed erythropoietin-accelerated erythropoiesis in vivo. These results reveal a role for IL-33 in pathogenesis of anemia during inflammatory disease and define a new target for its treatment.

Topics: Anemia; Animals; Annexin A5; beta-Glucans; Bone Marrow; Cell Differentiation; Chronic Disease; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Hematopoiesis; Inflammation; Injections; Interleukin-1 Receptor-Like 1 Protein; Interleukin-33; Ki-67 Antigen; Mice, Inbred BALB C; Mice, Inbred C57BL; Models, Biological; Myelopoiesis; NF-kappa B; Phosphorylation; Receptors, Erythropoietin; Signal Transduction; Spondylarthritis

Curdlan, a bacteria-derived polysaccharide resource, possesses substantial potential for periodontal antimicrobial delivery. Here, the facile engineering of a functionalized curdlan/polydopamine (PDA) composite hydrogels was reported. The physiochemical evaluations of composite hydrogels proved their tunable properties associated with concentration of PDA including pore size, rheological property and swelling behavior. We have systematically assessed biocompatibility in vitro and found these hydrogels toxicity-free. Moreover, photothermal performance upon near infrared light (NIR) exposure was conducted and eventually indicated the best matches for antibacterial application. The acetate chlorhexidine (CHX) was chosen as a model antimicrobial and the release profiles demonstrated the entrapped CHX could be triggered and nicely controlled by NIR. The optimized bacteriostatic rate reached 99.9 %. Overall, we aimed to provide new curdlan-based hydrogels for periodontal antibacterial treatment by combining photothermal effect and antimicrobial simultaneously.

Topics: Anti-Bacterial Agents; beta-Glucans; Biocompatible Materials; Cell Survival; Cells, Cultured; Chlorhexidine; Drug Liberation; Drug Synergism; Escherichia coli; Humans; Hydrogels; Indoles; Infrared Rays; Microbial Viability; Periodontal Ligament; Periodontitis; Photochemical Processes; Polymers; Polysaccharides, Bacterial; Porosity; Rheology; Staphylococcus aureus

The heterogeneous sulfoethylation of cellulose, xylan, α-1,3-glucan, glucomannan, pullulan, curdlan, galactoglucomannan, and agarose was studied using sodium vinylsulfonate (NaVS) as reagent in presence of sodium hydroxide and iso-propanol (i-PrOH) as slurry medium. The influence of the concentration of polymer, water, and NaOH (solid or aqueous solution) on the degree of substitution (DS) was investigated. The sulfoethylation rendered the polysaccharides studied water-soluble. Sulfoethylation of heteropolysaccharides yielded products with higher DS compared to the conversion of homopolysaccharides. Structure characterization was carried out by means of

Topics: 2-Propanol; beta-Glucans; Carbon-13 Magnetic Resonance Spectroscopy; Cellulose; Dimethyl Sulfoxide; Glucans; Mannans; Sepharose; Sodium Hydroxide; Solubility; Water; Xylans

Eugenol is hepatotoxic and potentially hazardous to human health. This paper reports on a rapid non-destructive quantitative method for the determination of eugenol concentration in curdlan (CD) biofilms by electronic nose (E-nose) combined with gas chromatography-mass spectrometry (GC-MS). Different concentrations of eugenol were added to the film-forming solution to form a series of biofilms by casting method, and the actual eugenol concentration in the biofilm was determined. Analysis of the odor collected on the biofilms was carried out by GC-MS and an E-nose. The E-nose data was subjected to principal component analysis (PCA) and linear discriminant analysis (LDA) in order to establish a discriminant model for determining eugenol concentrations in the biofilms. Further analyses involving the application of all sensors and featured sensors, the prediction model-based partial least squares (PLS) and support vector machines (SVM) were carried out to determine eugenol concentration in the CD biofilms. The results showed that the optimal prediction model for eugenol concentration was obtained by PLS at R

Topics: beta-Glucans; Biofilms; Electronic Nose; Eugenol; Gas Chromatography-Mass Spectrometry; Humans; Odorants

With the growing interest in food safety and in environmental protection, it is more attractive to develop novel biodegradable packaging films. In this regard, one new blending film was prepared with curdlan (CD)/polyvinyl alcohol (PVA)/thyme essential oil. Our results demonstrated that the mechanical properties of the blending film were the best when the ratio of the CD and PVA was 4:1. Further, the barrier properties of the film were optimized by incorporating with thyme essential oil. It was proved that not only water vapor permeability was lower, but also the elongation at break was improved, when 2% (w/w) thyme essential oil used. The potential interactions of the film matrix were analyzed by FTIR, XRD and Cryo-scanning electron microscopy. Importantly, both the antioxidant activity and antibacterial activity were improved. Finally, the blending film was employed for the preservation of chilled meat, while the shelf life was extended up to 10 days.

Topics: Anti-Bacterial Agents; Antioxidants; beta-Glucans; Escherichia coli; Food Packaging; Food Preservation; Meat; Oils, Volatile; Polyvinyl Alcohol; Thymus Plant

In order to determine the effect of different gelation temperatures (80 °C and 90 °C) on the structural arrangements in 1,3-β-d-glucan (curdlan) matrices, spectroscopic and microscopic approaches were chosen. Attenuated total reflection Fourier transform infrared spectroscopy (ATR FT-IR) and Raman spectroscopy are well-established techniques that enable the identification of functional groups in organic molecules based on their vibration modes. X-ray photoelectron spectroscopy (XPS) is a quantitative analytical method utilized in the surface study, which provided information about the elemental and chemical composition with high surface sensitivity. Contact angle goniometer was applied to evaluate surface wettability and surface free energy of the matrices. In turn, the surface topography characterization was obtained with the use of atomic force microscopy (AFM) and scanning electron microscopy (SEM). Described techniques may facilitate the optimization, modification, and design of manufacturing processes (such as the temperature of gelation in the case of the studied 1,3-β-d-glucan) of the organic polysaccharide matrices so as to obtain biomaterials with desired characteristics and wide range of biomedical applications, e.g., entrapment of drugs or production of biomaterials for tissue regeneration. This study shows that the 1,3-β-d-glucan polymer sample gelled at 80 °C has a distinctly different structure than the matrix gelled at 90 °C.

Topics: beta-Glucans; Drug Carriers; Microscopy, Atomic Force; Microscopy, Electron, Scanning; Molecular Structure; Photoelectron Spectroscopy; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Surface Properties; Temperature; Wettability

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

Curdlan is a neutral linear exopolysaccharide produced by Agrobacterium spp. under nitrogen-limiting conditions. In this study, we explored the role of glnA in curdlan biosynthesis in Agrobacterium sp. CGMCC 11546. The curdlan production of the ΔglnA strain was impaired, decreasing by 93% compared with that of the wild-type strain after 96 h fermentation. Analysis of fermentation profiles revealed that cell growth and utilization of carbon and nitrogen sources were impaired in the ΔglnA strain. Transcriptome analysis indicated that various of genes involved in curdlan biosynthesis were downregulated after 24 h fermentation in the ΔglnA strain, particularly genes involved in heme synthesis and the electron transport chain, which are essential for energy generation. Metabolomics analysis revealed flavin adenine dinucleotide (FAD) and adenosine diphosphate (ADP) accumulation in the ΔglnA strain, suggesting insufficient energy supply. Furthermore, glnA overexpression led to an 18% increase in the curdlan yield of the ΔglnA mutant compared with that of the wild-type strain after 96 h fermentation. Taken together, the findings demonstrate that glnA plays a vital role in curdlan biosynthesis by supplying ATP via regulating the expression of genes involved in heme synthesis and the electron transport chain.

Topics: Agrobacterium; Bacterial Proteins; beta-Glucans; Glutamate-Ammonia Ligase; Mutation

This study provides important information about the impacts of various levels of oat (OBG) and bacterial (curdlan) β-glucan and fat contents in milk on survivability and metabolism of yogurt starter cultures. The results show that addition of β-glucans in the concentration higher than 0.25% reduced starter bacterial counts during storage and prolonged the milk acidification process. A significant increase in lactose consumption by starter cultures was noted in the yogurt samples with OBG addition up to 0.75%. The highest (by 567% on average) increase in lactic acid content was noted in the control yogurts. Whereas the lowest (by 351%) increase in lactic acid content was noted in yogurts with OBG. After 28-day storage, the acetic aldehyde content was significantly influenced by fat content, type and addition level of polysaccharide. A higher increase in acetoin content was noted in samples with 0.25% than in samples with 1% of polysaccharides. In turn, significantly lower increases in diacetyl and 2,3-pentanedione contents were observed in the yogurt samples with OBG than in these with curdlan, with diacetyl production increase along with the higher concentration of the polysaccharide. The addition of OBG and curdlan to milk contributed to differences in the starter culture metabolism, consequently, in the milk acidification dynamics.

Topics: Acids; Animals; Avena; Bacteria; beta-Glucans; Lactobacillales; Microbial Viability; Milk; Sugars; Volatile Organic Compounds; Yogurt

A hybrid small-caliber artificial vascular graft based on bilayer porcine small intestinal submucosa (SIS) with curdlan and dipyridamole mixture film serving as the so-called sandwich filler was developed for biological performance evaluation. We evaluated the performance of the graft and filler.. SIS was coated with heparin by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Curdlan acted as the carrier of dipyridamole. Three types of graft tubes (2 mm internal diameter and 20 mm in length) were manufactured: bilayer SIS with 10% curdlan + 10% dipyridamole mixture film (SCD), bilayer SIS with 10% curdlan film (SC), and monolayer SIS (S). The remodeling characteristics of the grafts were evaluated by implanting them as bypass in rabbit carotid arteries for 2 and 3 months. Each group contained 16 rabbits, and 16 nonsurgical rabbits served as the control group.. Eight rabbits of each group, including the graft occluded group, were killed at 2 months and the others were killed at 3 months. Follow-up showed that all 8 grafts in SCD group were patent at 2 months. Six of 16 grafts in the SC group and 5 of 16 grafts in the S group were occluded at 2 months. One of 8 SCD grafts were occluded at 3 months and the patent showed a confluent endothelium without intimal hyperplasia. The neointima layer was composed of circumferentially aligned vascular smooth muscle cells. At 3 months, SC and S group grafts showed incomplete endothelialization and varying degrees of mural thrombus, accompanied by occlusion in the SC group (3 of 8) and S group (2 of 8).. The novel hybrid small caliber artificial vascular graft exhibited an improvement in revascularization resulting in high patency rate.

Topics: Animals; beta-Glucans; Biocompatible Materials; Bioprosthesis; Carotid Arteries; Heparin; Intestine, Small; Rabbits; Swine; Vascular Grafting; Vascular Patency

Water-soluble glycyrrhiza polysaccharides (GPs), extracted from the root and stem of licorice, were utilized in the photo-induced synthesis of Ag nanoparticles. The size and size distribution, morphology and structures of the Ag nanoparticles were characterized by UV-vis spectroscopy (UV), X-ray diffraction (XRD), transmission electron microscopy (TEM), dynamic light scattering (DLS), and Fourier transform infrared spectroscopy (FT-IR). The results showed that GPs played an essential role in the synthesis and stabilization of Ag particles. The prepared Ag nanoparticles varied in size and shape, dependent on the concentrations of GPs and silver nitrate. The obtained GP-stabilized Ag nanoparticles were incorporated into a biopolymeric film of curdlan as a novel source of potential antibacterial biomaterials. The resulting nanocomposite films exhibited an obvious antibacterial property with uniformly distributed Ag nanoparticles of 20-50 nm. Our results suggest that these nanocomposite films are promising templates for the design of novel antibacterial biomaterials.

Topics: Anti-Bacterial Agents; beta-Glucans; Glycyrrhiza; Membranes, Artificial; Metal Nanoparticles; Nanocomposites; Silver

Collagen is the most abundant structural protein in soft tissues, and the duplication of its structure and mechanics represents a key challenge to nanotechnology. Here we report a fibrous supramolecular network that can mimic nearly all of the aspects of collagen from dynamic hierarchical architecture to nonlinear mechanical behavior. This complex self-assembly system is solely based on a glucose polymer: curdlan, which is synthesized by bacteria and can form a similar triple helix as collagen. Triggered by solvent and temperature cues, free curdlan chains wind into superhelical trimers, and the trimers then bundle hexagonally into nanofibers of 20-40 nm in diameter. The fibers are interconnected in a water-rich 3D network structure. The network is highly dynamic and stress-responsive, which can shift from isotropic to anisotropic organization by the winding/unwinding of stress-induced interfiber triple helical net-points. Mechanical tests show that these nanofiber networks exhibit similar nonlinear elasticity as collagenous tissues including skin and tendon. The supramolecular networks also display a very wide range of tensile strength from ∼60 KPa to ∼50 MPa depending on the specific network organization. These biomimetic and dynamic supernetworks may have applications in tissue engineering, drug delivery systems, artificial skin, and soft robotics.

Topics: Anisotropy; beta-Glucans; Mechanical Phenomena; Nanofibers; Stress, Mechanical; Tensile Strength; Tissue Engineering

A long-anticipated cancer therapy would deliver the right type of therapeutic agents to the target in control with minimal systemic toxicity. The purpose of this study was to prepare lactosylated curdlan-triornithine nanocarriers (CTOLs), and target deliver gene to hepatoma cells. Structures and biophysical properties had been elucidated with physical and chemical methods. The results revealed that those functionalized polymers can completely condense the gene into spherical nanoparticles. Cytotoxicity assay, GFP-pDNA and siRNA transfection in vitro were implemented successively. Observations showed that CTOL 20% with the highest lactose acid substitution degree targeted delivered gene into HepG2 cells over expressing ASGPR receptors and had pretty gene knockdown efficiency over 70%. Meanwhile, the carriers showed excellent biocompatibility. Our studies demonstrated that CTOLs with lower toxicity and higher gene binding capacity may serve as a potential valuable platform that can be tailored to target the liver cancer cells for therapeutic gene.

Topics: beta-Glucans; DNA; Drug Carriers; Drug Delivery Systems; Gene Targeting; Gene Transfer Techniques; Genetic Therapy; Hep G2 Cells; Humans; Nanoparticles; Neoplasms; RNA, Small Interfering

The purpose of this study was to determine whether dialysis method allows for efficient protein entrapment in curdlan-based hydrogel. Thus, bovine serum albumin, a model of bioactive protein, was incorporated into curdlan matrix using ion-exchanging dialysis method against two concentrations of CaCl

Topics: Animals; beta-Glucans; Cattle; Cell Death; Cell Line; Circular Dichroism; Dialysis; Humans; Hydrogels; Ion Exchange; Serum Albumin, Bovine; Spectroscopy, Fourier Transform Infrared; Swine

To prevent spinal progression in ankylosing spondylitis, initiating TNF-inhibitor treatment as early as possible is suggested. However, the outcomes are inconsistent in previous clinical studies. Here, we investigated the effect of TNF inhibition alone on spinal progression when used during arthritis development in a murine model. We injected 8-week-old SKG mice with curdlan (curdlan group). We injected adalimumab at 3 and 9 weeks after the first curdlan injection (ADA group). The clinical scores of peripheral arthritis decreased in the ADA group at 3 weeks after first adalimumab injection. Using positron emission tomography-magnetic resonance imaging and histologic examination, spinal inflammation was observed in the curdlan group, and was significantly deceased in the ADA group. However, spinal osteoblast activities by imaging using OsteoSense 680 EX and bone metabolism-related cytokines such as receptor activator of nuclear factor-kappa B ligand, osteoprotegerin, Dickkopf-1, and sclerostin levels except IL-17A level were not different between the two groups. We conclude that treating TNF inhibitor alone reduced peripheral arthritis score and spinal inflammation in curdlan-injected SKG mice but did not decrease the spinal osteoblast activity, suggesting little effect on spinal ankylosis.

Topics: Adalimumab; Animals; Arthritis, Experimental; beta-Glucans; Disease Progression; Female; Humans; Injections, Intraperitoneal; Mice; Severity of Illness Index; Spine; Spondylitis, Ankylosing; Tomography, X-Ray Computed; Tumor Necrosis Factor-alpha

Natural carbohydrate polymer-based nanoparticles have great biocompatibility that is required for the safe delivery of various drugs including nucleic acid therapeutics. Herein, we designed curdlan-based nanoparticles for cancer cell targeted delivery of short interfering RNA (siRNA). iRGD peptide conjugated 6-amino-6-deoxy curdlan specifically delivered siRNA to integrin expressing cancer cells. Incubation of cancer cells with free iRGD peptide competitively blocked cellular uptake of the iRGD functionalized curdlan nanoparticles. Chloroquine but not nystatin inhibited cellular uptake of iRGD functionalized curdlan nanoparticles, indicating that the iRGD peptide conjugated curdlan nanoparticles were internalized through the receptor (clathrin)-mediated endocytosis. Moreover, a disease related gene Plk1 was substantially knocked down by siRNA carried by 6AC-iRGD nanoparticles in HepG2 cells. Our data suggested that iRGD functionalized curdlan may provide a biocompatible carrier for siRNA delivery.

Topics: beta-Glucans; Endocytosis; Gene Transfer Techniques; Hep G2 Cells; Humans; Nanoparticles; Oligopeptides; Receptors, Cell Surface; RNA, Small Interfering

This study evaluated the immunostimulatory activity of curdlan oligosaccharides (GOS) in cyclophosphamide (CTX)-induced immunosuppressed mice and in RAW264.7 cells. GOS was able to stimulate the release of nitric oxide (NO), cytokines (IL-1β, IL-6 and TNF-α) and improve the phagocytic rate of peritoneal macrophages and RAW264.7 cells. It further enhanced immunoglobulins (Ig) release (IgG by 50.6%-74.7%, IgA by 31.3%-34.9%, IgM by 28.3%-66.7%), splenic lymphocyte proliferation (by 74.8%-91.3%), nature killer cells cytotoxicity (by 32.0%-49.6%), immunophenotypes of splenic lymphocytes (from 1.7 to 2.4, 2.2 and 2.7) in immunosuppressed mice. Compared with curdlan, higher immunostimulatory activity of GOS was found in CTX-treated mice. Moreover, GOS could activate nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways through toll-like receptor 2 (TLR2) and complement receptor 3 (CR3). These results indicated that GOS may be a favorable candidate of functional food in regulating immune responses.

Topics: Adjuvants, Immunologic; Administration, Oral; Alcaligenes; Animals; beta-Glucans; Body Weight; Cyclophosphamide; Cytokines; Immune System Diseases; Immunity, Humoral; Immunosuppression Therapy; Killer Cells, Natural; Macrophages; Male; MAP Kinase Signaling System; Mice; Mice, Inbred BALB C; Oligosaccharides; RAW 264.7 Cells; T-Lymphocytes

Topics: Ammonium Compounds; beta-Glucans; Carbohydrate Conformation; Chromatography, Gel; Dynamic Light Scattering; Microscopy, Atomic Force; Molecular Weight; Rheology; Sodium Chloride; Solutions; Temperature; Viscosity; Water

The secretomes of the strain Cellulosimicrobium cellulans F16 grown on different carbon sources were analyzed by zymography, and the subcellular surface structures were extensively studied by electron microscope. The exo-cellulase and xylanase systems were sparse when cells were grown on soluble oligosaccharides, but were significantly increased when grown on complex and water-insoluble polysaccharides, such as Avicel, corn cob, and birchwood xylan. The cellulosome-like protuberant structures were clearly observed on the cell surfaces of strain F16 grown on cellulose, with diameters of 15-20 nm. Fibrous structures that connected the adjacent cells can be seen under microscope. Moreover, protuberances that adsorbed the cell to cellulose were also observed. As the adhesion of Cellulosimicrobium cellulans cells onto cellulose surfaces occurs via thick bacterial curdlan-type exopolysaccharides (EPS), such surface layer is potentially important in the digestion of insoluble substrates such as cellulose or hemicellulose, and the previously reported xylanosomes are part of such complex glycocalyx layer on the surface of the bacterial cell.

Topics: Actinobacteria; Bacterial Adhesion; beta-Glucans; beta-Glucosidase; Carbon; Cellulose; Cellulosomes; Glycocalyx; Polysaccharides, Bacterial; Xylosidases

Certain gut bacterial families, including Bacteroidaceae, Porphyromonadaceae and Prevotellaceae, are increased in people suffering from spondyloarthropathy (SpA), a disease group associated with. We treated SKG mice weekly with anti-IL-23 or isotype mAb for 3 weeks, rested them for 3 weeks, then administered curdlan or saline. We collected faecal samples longitudinally, assessed arthritis, spondylitis, psoriasis and ileitis histologically, and analysed the microbiota community profiles using next-generation sequencing. We used multivariate sparse partial least squares discriminant analysis to identify operational taxonomic unit (OTU) signatures best classifying treatment groups and linear regression to develop a predictive model of disease severity.. IL-23p19 inhibition in naïve SKG mice decreased Bacteroidaceae, Porphyromonadaceae and Prevotellaceae. Abundance of Clostridiaceae and Lachnospiraceae families concomitantly increased, and curdlan-mediated SpA development decreased. Abundance of Enterobacteriaceae and Porphyromonadaceae family and reduction in Lachnospiraceae. Dysbiosis in SKG mice reflects human SpA and is IL-23p19 dependent. In genetically susceptible hosts, IL-23p19 favours outgrowth of SpA-associated pathobionts and reduces support for homeostatic-inducing microbiota. The relative abundance of specific pathobionts is associated with disease severity.

Topics: Animals; Bacteria; beta-Glucans; Dysbiosis; Feces; Female; Gastrointestinal Microbiome; Homeostasis; Host-Pathogen Interactions; Interleukin-23 Subunit p19; Mice, Mutant Strains; Severity of Illness Index; Spondylarthritis

In this work, a novel green solvent based on the ionic liquid (IL) 1-ethyl-3-methylimidazolium acetate (EmimAc) with a superior dissolving ability to biomacromolecules was utilized to boost solubility of water-insoluble curdlan. Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), circular dichroism (CD), and nuclear magnetic resonance (NMR) were used to characterize the structural changes of curdlan before and after regeneration. Thermal decomposition property of curdlan was also investigated using thermal gravimetric analysis (TGA). The results indicated after EmimAc treatment, the water-solubility of regenerated curdlan (RC) achieved 74.41 ± 0.63%. In addition, the hydrogen bonds in curdlan and its native triple helix structure were partially broken. In the meantime, new hydrogen bonds between EmimAc and curdlan formed. Moreover, the disruption of curdlan's original structure made it decreased thermostability and easier to dissolve in water. Therefore, this research can provide a feasible and effective approach for improving solubility of water-insoluble curdlan to enlarge its food and biomedical applications.

Topics: beta-Glucans; Imidazoles; Ionic Liquids; Solubility; Solvents; Spectrum Analysis; Structure-Activity Relationship; Thermogravimetry

Enzymatic curdlan hydrolysis is gaining more attention for the value of oligo-β-glucans in many aspects. Currently, the triple-helical conformation of curdlan fiber was imposed to the structure of β-1,3-glucanase as its substrate without experimental evidence. Here, solution conformation of differently treated curdlan and each hydrolysis rate by a variety of β-1,3-glucanases were systematically examined. Results showed that different enzymes exhibited preferences over the trajectories of pH change that curdlan solution went through, and all enzymes hydrolyzed heat treated curdlan solution at their maximum rates where most of the higher ordered helices were diminished. Combined with molecular docking studies, a multi-step hydrolysis process was proposed. Recognition of triple-helical curdlan by their ancillary region of β-1,3-glucanase occurred before its unwinding into single- and double-helical forms, and the later ones fitted better to the catalytic cavity of the enzyme where the polysaccharides chain eventually got hydrolyzed into oligo-β-glucans.

Topics: beta-Glucans; Glucan 1,3-beta-Glucosidase; Hydrolysis; Protein Conformation

Immune-modulatory effects of β-glucans are generally considered beneficial to fish health. Despite the frequent application of β-glucans in aquaculture practice, the exact receptors and downstream signalling remains to be described for fish. In mammals, Dectin-1 is a member of the C-type lectin receptor (CLR) family and the best-described receptor for β-glucans. In fish genomes, no clear homologue of Dectin-1 could be identified so far. Yet, in previous studies we could activate carp macrophages with curdlan, considered a Dectin-1-specific β-(1,3)-glucan ligand in mammals. It was therefore proposed that immune-modulatory effects of β-glucan in carp macrophages could be triggered by a member of the CLR family activating the classical CLR signalling pathway, different from Dectin-1. In the current study, we used primary macrophages of common carp to examine immune modulation by β-glucans using transcriptome analysis of RNA isolated 6 h after stimulation with two different β-glucan preparations. Pathway analysis of differentially expressed genes (DEGs) showed that both β-glucans regulate a comparable signalling pathway typical of CLR activation. Carp genome analysis identified 239 genes encoding for proteins with at least one C-type Lectin Domains (CTLD). Narrowing the search for candidate β-glucan receptors, based on the presence of a conserved glucan-binding motif, identified 13 genes encoding a WxH sugar-binding motif in their CTLD. These genes, however, were not expressed in macrophages. Instead, among the β-glucan-stimulated DEGs, a total of six CTLD-encoding genes were significantly regulated, all of which were down-regulated in carp macrophages. Several candidates had a protein architecture similar to Dectin-1, therefore potential conservation of synteny of the mammalian

Topics: Animals; beta-Glucans; Carps; Cells, Cultured; Fish Proteins; Gene Expression Profiling; Gene Ontology; Lectins, C-Type; Macrophages; Phylogeny; Signal Transduction; Synteny; Transcriptome; Zebrafish

The influence of different addition levels (0.1% to 0.5%) of thermo-reversible curdlan gels (TRC) and thermo-irreversible curdlan gels (TIRC) on the physicochemical and textural characteristics of frankfurters, as well as dynamic rheological properties of meat batters, was investigated. Increased percentages of TRC and TIRC were associated with lower cooking loss and quicker relaxation times, as well as superior emulsion stability, and higher L

Topics: beta-Glucans; Cooking; Emulsions; Gels; Meat Products; Rheology

The semi-artificial branched-polysaccharides, amylose-grafted curdlans, were synthesized utilizing an enzymatic polymerization. Both a curdlan main chain and amylose side chains on the polysaccharides maintain the original helical structure as well as the molecular binding ability. Thanks to the difference in their molecular recognition properties between β-1,3-glucan chain and α-1,4-glucan chain, the amylose-grafted curdlans can provide two different orthogonal binding sites within one polymeric system. When a water-soluble polythiophene was mixed with the amylose-grafted curdlan, the polythiophene was twisted in two different modes and therein, fluorescence energy of the polythiophene wrapped by the amylose side chains was successfully transferred to the polythiophene wrapped by the curdlan main chain. We thus concluded that in the dendritic superstructure of this polysaccharide, a self-organized "Janus-type FRET system" was successfully constructed.

Topics: Amylose; beta-Glucans; Binding Sites; Fluorescence Resonance Energy Transfer; Macromolecular Substances; Polymerization

In this study, a positively charged quaternized curdlan (Qcurd) was used to fabricate polyelectrolyte complex nanoparticles (PEC NPs) with a negatively charged pectin via electrostatic complexation. Results showed that the Qcurd/pectin PEC NPs prepared with 0.5 mg/mL Qcurd and pectin solutions, 1:2 pectin/Qcurd mass ratio, and pH 4.0 in the absence of NaCl were characterized by a spherical morphology in nanoscale, an average particle size of 68 nm, and good dispersibility in aqueous solutions. Curcumin was encapsulated in the Qcurd/pectin PEC NPs through hydrogen bonding with an encapsulation efficiency of ∼82%, a loading content of 13%, and a pH-dependent controlled release. Curcumin-loaded PEC NPs exhibited a significantly enhanced water solubility, excellent free radical scavenging ability and antioxidant capacity in vitro as compared with those of free curcumin.

Topics: beta-Glucans; Biocompatible Materials; Curcumin; Drug Carriers; Nanoparticles; Particle Size; Pectins; Polyelectrolytes; Solubility

β-glucan is an abundant cell wall component of fungi and yeast. Dectin-1, a β-glucan receptor, plays an important regulatory role in the natural immunity. In the present study, we investigated the effect of β-glucan on mouse macrophages that had been invaded by the periodontopathic bacterium, Aggregatibacter actinomycetemcomitans. Exposure to curdlan, a type of β-glucan, suppressed cell death and led to the accumulation of a sub-G1-phase population upon A. actinomycetemcomitans invasion under conditions of constitutive expression of dectin-1. Members of the nucleotide-binding domain leucine-rich repeat-containing (NLR) protein family, such as NLR protein 3 (NLRP3), NLR family apoptosis inhibitory protein (NAIP), and NLR family CARD domain-containing protein 4 (NLRC4), as well as an associated protein, caspase-11, were clearly detected in A. actinomycetemcomitans-invaded control RAW cells (c-RAW cells; negative control). Interestingly, NAIP expression was upregulated and caspase-11 expression was downregulated by dectin-1 activity in A. actinomycetemcomitans-invaded dectin-1 overexpressing RAW 264.7 cells (d-RAW cells), suggesting that dectin-1 in macrophages regulates cell death upon A. actinomycetemcomitans invasion. These results support a potential correlation between dectin-1 and regulation of cell death in macrophages.

Topics: Aggregatibacter actinomycetemcomitans; Animals; beta-Glucans; Caspases, Initiator; Cell Death; Cell Survival; Down-Regulation; Gene Expression Regulation; Lectins, C-Type; Macrophages; Mice; NLR Family, Pyrin Domain-Containing 3 Protein; RAW 264.7 Cells; Signal Transduction

Chitooligosaccharide has been reported to possess diverse bioactivities. The development of novel strategies for obtaining optimum degree of polymerization (DP) chitooligosaccharides has become increasingly important. In this study, two glycoside hydrolase family 46 chitosanases were studied for immobilization on curdlan (insoluble β-1,3-glucan) using a novel carbohydrate binding module (CBM) family 56 domain from a β-1,3-glucanase. The CBM56 domain provided a spontaneous and specific sorption of the fusion proteins onto a curdlan carrier, and two fusion enzymes showed increased enzyme stability in comparison with native enzymes. Furthermore, a continuous packed-bed reactor was constructed with chitosanase immobilized on a curdlan carrier to control the enzymatic hydrolysis of chitosan. Three chitooligosaccharide products with different molecular weights were prepared in optimized reaction conditions. This study provides a novel CBM tag for the stabilization and immobilization of enzymes. The controllable hydrolysis strategy offers potential for the industrial-scale preparation of chitooligosaccharides with different desired DPs.

Topics: Bacterial Proteins; beta-Glucans; Biocatalysis; Carbohydrates; Enzyme Stability; Enzymes, Immobilized; Glycoside Hydrolases; Multigene Family; Oligosaccharides; Paenibacillus; Protein Domains

In tuberculosis, macrophages serve as a host for Mycobacterium tuberculosis and hence targeting them with nanoparticles-based drug delivery could be the best strategy to achieve high therapeutic efficacy. Two tuberculosis drugs, namely rifampicin and levofloxacin, which have different mechanism of action on the bacteria, were complexed with cyclodextrin and conjugated to curdlan nanoparticles, to achieve simultaneous sustained release of both the drugs over a prolonged period of time. They are non-cytotoxic to both RAW 264.7 and L929 cells. They are taken up ˜1.8 times more by the macrophage cells through dectin-1 receptor than the fibroblast cells. They are also able to kill more than 95% of Mycobacterium smegmatis residing within the macrophages in 4 h. These results demonstrate that curdlan-CD nanoparticles can be a promising system for the loading and intracellular release of hydrophobic drugs into macrophages for various therapeutic applications.

Topics: Animals; Antitubercular Agents; beta-Cyclodextrins; beta-Glucans; Cell Line; Drug Carriers; Drug Liberation; Levofloxacin; Macrophages; Mice; Microbial Sensitivity Tests; Mycobacterium smegmatis; Nanoparticles; Rifampin

Immobilization of enzymes is an essential strategy with outstanding prospects in biocatalytic processes. Nontoxic, inexpensive immobilized enzyme approach is especially important for food enzymes. We here demonstrate that a carbohydrate-binding module family 56 domain (CBM56-Tag) mediates the immobilization of fusion enzymes with the curdlan (β-1,3-glucan) particle support, thereby enabling the one-step immobilization-purification of target enzymes. CBM56-Tag exhibits an immunoglobulin-like β-sandwich fold, which can be adsorbed by curdlan via hydrogen bond-mediated binding. The maximum adsorption capacity of a fusion chitosanase (CBM56-GsCsn46A) on curdlan is 50.72 mg/g. The immobilized enzyme could be directly used in the packed-bed reactor. This immobilization strategy utilizes a natural polysaccharide without any treatment, avoiding the negative environmental effects. Moreover, the one step immobilization-purification simplifies the purification step, which reduces the use of chemicals. Our study provides a nontoxic and inexpensive immobilization strategy for the biocatalytic reaction in food industry.

Topics: beta-Glucans; Biocatalysis; Enzymes, Immobilized; Glycoside Hydrolases; Hydrogen Bonding; Receptors, Cell Surface; Recombinant Fusion Proteins

A novel strategy was used to produce inulin fructotransferase from Arthrobacter aurescens (Aa-IFTase) embedded in curdlan-based mesoporous silica microspheres (CMSiM-Aa-IFTase). The CMSiM-Aa-IFTase was constructed by co-entrapping cross-linked Aa-IFTase aggregates and curdlan into biomemitic silica, and the curdlan was subsequently removed by digestion with endo-β-1,3-glucanase. During this process, the curdlan served as an agent to introduce pores in silica microspheres. The resulting CMSiM-Aa-IFTase showed higher stability and activity than free Aa-IFTase and mCLEAs-Aa-IFTase (modified cross-linked enzyme aggregates with Aa-IFTase). Furthermore, the CMSiM-Aa-IFTase displayed good reusability and excellent storage stability. The excellent catalytic performances were due to the combinational structure from the cross-linked enzyme aggregates and hard shell of mesoporous silica microspheres, which might decrease the negative interaction between support and enzyme, and improve the mechanical properties. The CMSiM-Aa-IFTase was applicable for efficient production of Difructose Anhydride III (DFA III), and this approach should be highly valuable for preparing various mesoporous composites for catalysis.

Topics: Arthrobacter; beta-Glucans; Catalysis; Disaccharides; Enzyme Stability; Enzymes, Immobilized; Hexosyltransferases; Microspheres; Silicon Dioxide

In this study, water-soluble curdlan products (Cur and Cur-D) were prepared by an alkali-neutralization treatment process, after which ferulic acid (FA)-grafted Cur conjugates (Cur-g-FA and Cur-D-g-FA) were fabricated in the presence and absence of salt by adopting an approach involving free-radicals generated by the ascorbic acid/hydrogen peroxide redox pair under an inert atmosphere. Results showed that FA was successfully grafted onto the C-6 and C-4 positions of the Cur chains through covalent linkages and that the presence of salt exerted minor influences on the grafting ratios and structural characterizations of the products. Cur-g-FA and Cur-D-g-FA showed decreased crystallinity, thermal stability, and rheological properties, as well as a distinct surface morphology, when compared with those of native Cur. However, Cur-g-FA and Cur-D-g-FA also exhibited remarkably enhanced free-radical scavenging ability and antioxidant capacity in vitro. These results indicate that FA-grafted Cur conjugates have great potential application in the field of functional foods.

Topics: Antioxidants; Ascorbic Acid; beta-Glucans; Coumaric Acids; Free Radicals; Hydrogen Peroxide; Oxidation-Reduction; Rheology; Solubility; Surface Properties; Water

A C6-carboxylated curdlan (C6-Cc) obtained from 4-acetamido-TEMPO-mediated oxidation of curdlan was used both as a reducing and stabilizing agent for green synthesis of pH-responsive AuNPs, which was carried out by controlling the pH of the C6-Cc solution at a high temperature (100°C). C6-Cc presented a semi-flexible random coil chain in the aqueous medium at pH 5.5 and became more expanded and rigid in alkaline conditions (pH 7.1-12.0), though the primary chemical structure of C6-Cc was virtually unchanged with the pH variation. The AuNPs prepared with C6-Cc at various pHs were characterized by various instrumental measurements. The shapes and sizes of AuNPs were found to be strongly dependent on the pH of the C6-Cc solution. The C6-Cc-decorated AuNPs exhibited a more well-dispersed spherical morphology with smaller particle sizes under alkaline conditions (pH 7.1-12.0). Through this study, a facile, simple, and green method has been demonstrated for preparation of stimuli-sensitive AuNPs using biocompatible polyanionic polysaccharides.

Topics: beta-Glucans; Cyclic N-Oxides; Gold; Green Chemistry Technology; Hot Temperature; Hydrogen-Ion Concentration; Metal Nanoparticles; Oxidation-Reduction; Particle Size

In order to explore the mechanism by which Tween-80 enhances the production of curdlan produced by Agrobacterium sp., the effects of Tween-80 on the production and structure of curdlan and Agrobacterium sp. were evaluated. Maximum curdlan production (51.94g/L) was achieved when 16g/L Tween-80 was added at the beginning of the cell growth stage. The addition of Tween-80 at higher concentration inhibited cell growth. However, the addition of 16g/L Tween-80 enhanced the production of curdlan with a looser ultrastructure, significantly weakened the envelopment of curdlan on Agrobacterium sp., altered the fine structure of cell membrane, and increased the cell membrane permeability. Moreover, the efficiency of oxygen and mass transport, respiration intensity, UTP regeneration, ATP regeneration, activity of curdlan synthetase, capacity of stress response and energy supply of Agrobacterium sp. were all greatly improved by the addition of Tween-80. These findings demonstrate the mechanisms by which Tween-80 enhances curdlan production and provide a cheap and feasible approach to weaken the envelopment of water-insoluble polysaccharides on bacteria.

Topics: Adenosine Triphosphate; Agrobacterium; beta-Glucans; Cell Membrane; Cell Membrane Permeability; Enzyme Activation; Fermentation; Glucosyltransferases; Kinetics; Oxygen; Polysaccharides, Bacterial; Polysorbates; Uridine Triphosphate; Water

In this study, negatively charged carboxylic curdlan (Cc) bearing a β-1,3-polyglucuronic acid structure was employed to fabricate nanosized polyelectrolyte complexes (PECs) with positively charged chitosan (CS) in aqueous solution as potential carriers for 5-fluorouracil (5Fu) delivery. Nanosized CS/Cc PECs were formed by the addition of 0.5mg/mL solutions of CS and Cc with a mixing ratio of 1:1 (w/w) at pH 3.0. Under optimized conditions, the prepared CS/Cc PECs showed spherical morphology with an average size of about 180nm and a zeta potential of around 41mV. The 5Fu drug was incorporated into the nanosized CS/Cc PECs and showed excellent encapsulation efficiency (86.47%) and loading content (10.81%). The drug release data in vitro indicated that the nanosized CS/Cc PECs are promising carriers for the sustained release of 5Fu with an anomalous transport mechanism following the Ritger-Peppas model. Besides, the CS/Cc PECs exhibited low cytotoxic activity against SPCA-1 and HeLa cell lines in vitro. This finding suggested that the development of the nanosized CS/Cc PECs offered great promise as an antitumor drug platform.

Topics: beta-Glucans; Chitosan; Drug Carriers; Fluorouracil; HeLa Cells; Humans; Nanoparticles; Neoplasms; Polyelectrolytes

The nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome has developed as an important bridge between innate immune and infection recently, and has the ability to drive proteolytic procaspase-1 into bioactive caspase-1, then responsible for proteolytic processing of inflammatory cytokines IL-1β and IL-18. Fungal β-glucan, a major component of fungal cell wall, triggers inflammatory response in multiple immune cells, but rarely described in epithelial cells. Also, the relationship between fungal β-glucan and NLRP3 inflammasome is not clear yet. In this study, we first identified that curdlan, a large particulate β-glucan, could activate the NLRP3 inflammasome in LPS-primed human bronchial epithelial cells (HBECs). RT-PCR and Western Blot showed that curdlan upregulate the mRNA as well as intracellular protein expression of NLRP3 and IL-1β in HBECs, along with the activity of caspase-1, and the level of mature IL-1β in cell supernatants was higher by ELISA detection. Further studies demonstrated that the activation of NLRP3 inflammasome could be attenuated by NAC, an inhibitor of ROS. Thus, it indicated curdlan activate NLRP3 inflammasome through a pathway requiring ROS production in HBECs. These findings may provide a new therapeutic target, NLRP3 inflammasome, in invasive pulmonary fungal infections.

Topics: Antioxidants; beta-Glucans; Bronchi; Caspase 1; Cell Line; Dose-Response Relationship, Drug; Epithelial Cells; Humans; Interleukin-1beta; NLR Family, Pyrin Domain-Containing 3 Protein; Oxidative Stress; Reactive Oxygen Species; Signal Transduction; Up-Regulation

A novel β-1,3-glucanase gene (PaBglu50A) from Pseudomonas aeruginosa CAU 342A was cloned and expressed in Escherichia coli. The deduced amino acid sequence of PaBglu50A showed the highest identity of 34% with the β-agarase belonging to glycoside hydrolase (GH) family 50. The purified PaBglu50A had maximal activity at pH 5.5 and 45°C, respectively. It was stable in the range of pH 4.0-8.0 and at temperatures below 40°C. The K

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Glucans; Genes, Bacterial; Glucan Endo-1,3-beta-D-Glucosidase; Hydrolysis; Kinetics; Phylogeny; Pseudomonas aeruginosa; Recombinant Proteins; Sequence Homology, Amino Acid; Substrate Specificity

In this study, carboxylic curdlans (Cur-4, Cur-8, and Cur-24) with different molecular properties and chain conformations were used as stabilizer and capping agent to fabricate stable and water-dispersible selenium nanoparticles (SeNPs). Results showed that molecular properties and chain conformations of carboxylic curdlans remarkably influenced the size, morphology, structure, and stability of SeNPs and the carboxylic curdlan was ligated to SeNPs via OH⋯Se interaction. The as-prepared SeNPs was amorphous and showed homogeneous and monodisperse spherical structure with size of ∼50-90nm. The Cur-8-decorated SeNPs (SeNPs@Cur-8) exhibited smaller particle size (∼56nm) and greater stability than those of the others. The carboxylic curdlan-stabilized SeNPs exhibited excellent antioxidant capacities compared to the control SeNPs. Specifically, SeNPs@Cur-8 with smaller particle size possessed strong antioxidant efficacy. SeNPs@Cur-8 also exhibited low cytotoxic activity against SPCA-1 and HeLa cell lines in vitro.

Topics: Antioxidants; Ascorbic Acid; beta-Glucans; Biocompatible Materials; Cell Survival; HeLa Cells; Humans; Molecular Weight; Nanoparticles; Particle Size; Sodium Selenite; Water

The current avenues for prevention and/or control of Mycoplasma bovis infection in cattle involve antibiotic treatment of affected animals, herd management practices including separation and or culling infected animals, and the use of commercial vaccines, which offer limited protection. Some bacterin vaccines may cause negative reactions; therefore a different approach is needed, such as the use of recombinant vaccines based on protective antigens formulated with effective adjuvants. The role of Th-17 immune responses in protection against bacterial infections has been investigated for several pathogens. In this study, our goal was to identify M. bovis antigens that may elicit Th-17 protective responses. We tested a vaccine containing M. bovis proteins formulated with Montanide ISA61™ VG and curdlan. After vaccination, the animals were challenged using a BHV-1/M. bovis co-infection model. We detected IL-17 and other cytokines in supernatants of PBMCs incubated with the recall antigens. In addition, we detected antibody and PBMC proliferative responses to the antigens. Despite observing slight decreases in the proportion of the lung lesions and in weight loss in the vaccinated group, we concluded that Th-17 responses to the antigens used here were not protective.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Bacterial; Antigens, Bacterial; Bacterial Proteins; Bacterial Vaccines; beta-Glucans; Cattle; Cattle Diseases; Mycoplasma bovis; Mycoplasma Infections; Th17 Cells; Vaccination; Vaccines, Synthetic

Prebiotic effects of curdlan (1 → 3)-β-d-glucan oligosaccharides (GOS) were examined. GOS was tolerant against simulated gastrointestinal digestion, as well as low pH, thermal, and Maillard reaction conditions likely occurred during food processing. Growth of tested Lactobacillus (L.) strains was improved by GOS except L. brevis NRRL B-4527. E. coli did not grow on GOS as the only carbon source. In vitro batch fermentation using human faecal microbiota showed that GOS significantly increased the population of Lactobacillus sp. followed by Bifidobacterium sp. and Bacteroides sp. Growth of L. strains on GOS produced lactic acid, acetic, and propionic acid with decreased culture medium pH. Utilization pattern of GOS by representative L. strains was strain dependent. GOS with degree of polymerization (DP) of 2 and 3 were readily consumed. Findings here indicated that curdlan GOS (DP = 2 and 3) are promising physiologically active prebiotics for improvement of human intestinal health.

Topics: beta-Glucans; Bifidobacterium; Escherichia coli; Lactobacillus; Microbiota; Oligosaccharides; Prebiotics

Interleukin-33 (IL-33) is a potent contributor to antiviral immune responses and antitumor immunity. We recently discovered that IL-33 is overexpressed in dectin-1-activated dendritic cells (DCs). However, mechanisms of dectin-1-induced IL-33 expression in DCs remain elusive. Curdlan, an agonist of dectin-1, was used to mature DCs in this study. We found that dectin-1-induced IL-33 expression in DCs relies on Syk and Raf-1 pathways. By using nuclear factor (NF)-κB inhibitors, we also found that dectin-1-induced IL-33 expression relies on NF-κB signaling. Furthermore, through Syk/Raf-1-NF-κB pathway, dectin-1 signaling stimulates DCs to overexpress interferon regulatory factor 4 (IRF4), which directly upregulates the expression of IL-33 in dectin-1-activated DCs. Thus, our study provides new insights into the mechanisms of dectin-1-induced IL-33 expression in DCs and may provide new targets for improving DC-based cancer immunotherapy.

Topics: Animals; beta-Glucans; Dendritic Cells; Interferon Regulatory Factors; Interleukin-33; Lectins, C-Type; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; NF-kappa B; Proto-Oncogene Proteins c-raf; Signal Transduction; Syk Kinase

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

Topics: Bacillus cereus; beta-Glucans; Culture Media; Fabaceae; Fermentation; Humans; Industrial Microbiology; Polysaccharides, Bacterial; Rhizosphere

Topics: Alcaligenes faecalis; beta-Glucans; Carbon; Fermentation; Fructose; Glucose; Kinetics; Maltose; Sucrose

Topics: Animals; beta-Glucans; Fish Proteins; Gene Expression; Lipopolysaccharides; Macrophages; NAD; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; Respiratory Burst; Salmo salar

6-Amino-6-deoxy-curdlan is a promising nucleic acid carrier that efficiently delivers plasmid DNA as well as short interfering RNA (siRNA) to various cell lines. The highly reactive C6-NH

Topics: Animals; beta-Glucans; Macrophages; Mannose; Mice; Nanoparticles; RNA, Small Interfering

Induction of a robust and long-lived mucosal immune response during vaccination is critical to achieve protection against numerous pathogens. However, traditional injected vaccines are generally poor inducers of mucosal immunity. One of the effective strategies to improve vaccine efficacy is incorporation of adjuvant molecules that enhance and polarize adaptive immune reactions. Effects of Syk-coupled lectin receptor agonists as adjuvants to induce mucosal immune reactions during parenteral immunization are not fully studied. We now report that the agonists trehalose-6,6-dibehenate (TDB), curdlan, and furfurman, which stimulate Dectin-1, Dectin-2, and Mincle, respectively, activate transcription factors (NF-

Topics: Adjuvants, Immunologic; Animals; beta-Glucans; Cell Differentiation; Dendritic Cells; Female; Humans; Immunity, Mucosal; Immunization; Immunoglobulin A; Infusions, Parenteral; Intestinal Mucosa; Lectins, C-Type; Membrane Proteins; Mice; Mice, Inbred C57BL; RAW 264.7 Cells; Receptors, Mitogen; Syk Kinase; Vaccination

Fungal β-glucan is a potent immunological stimulator, and that it activates both the innate immune system and adaptive immunity. Curdlan is (1→3)-β-glucan, a linear form of β-glucan with a high molecular weight; it modulates the immune response. However, its role in bone tissue is controversial, and the effects of curdlan on bone tissues are unknown. Toll-like receptors (TLRs) play critical roles in innate immunity, and various ligands for TLRs are thought to regulate the host defense mechanisms against pathogens. TLR2 is known to form heterodimers with TLR6, and the TLR2-TLR6 heterodimer (TLR2/6) recognizes diacylated lipopeptides from Gram-positive bacteria. In the present study, we prepared low molecular-weight curdlan, (1→3)-β-D-glucan, and examined its effects on bone resorption induced by TLR2/6 signaling. In co-cultures of bone marrow cells and osteoblasts, low molecular-weight curdlan suppressed the osteoclast formation induced by TLR2/6 ligand, and attenuated bone resorption in mouse calvarial organ cultures. Curdlan acted on mouse osteoblasts and suppressed the expression of receptor activator of nuclear factor-kappa B (NF-κB) ligand (RANKL), a key molecule for osteoclastogenesis. Curdlan also acted on mouse bone marrow macrophages and suppressed RANKL-dependent osteoclast differentiation from osteoclast precursor cells. The present study indicates that low molecular-weight curdlan attenuated TLR2-induced inflammatory bone resorption. Curdlan, (1→3)-β-glucan may be a natural agent with beneficial effects on bone health in humans.

Topics: Animals; beta-Glucans; Bone Marrow Cells; Bone Resorption; Cells, Cultured; Coculture Techniques; Mice; Osteoblasts; Osteoclasts; Osteoprotegerin; RANK Ligand; RNA, Messenger; Skull; Toll-Like Receptor 2

Curdlan is a water-insoluble microbial exo-polysaccharide that is hardly degraded. The gene CcGluE encoding an endo-β-1→3-glucanase consisting of 412 amino acids (44 kDa) from Cellulosimicrobium cellulans E4-5 was cloned and expressed in Escherichia coli. The recombinant CcGluE hydrolysed curdlan powder effectively. CcGluE shows high endo-β-1→3 glucanase activity and low β-1,4 and β-1,6 glucanase activities with broad substrate specificity for glucan, including curdlan, laminarin and β-1→3/1→6-glucan, and the highest catalytic activity for curdlan. Moreover, the hydrolytic products of curdlan were glucan oligosaccharides with degrees of polymerisation of 2-13, and the main products were glucobiose and glucotriose. Degradation mode analysis indicated that CcGluE is more likely to hydrolyse glucopentaose and revealed that CcGluE was an endo-glucanase. Furthermore, upon combination with a homogenising pre-treatment method with curdlan, the degradation efficiency of CcGluE for curdlan powder was greatly improved 7.1-fold, which laid a good foundation for the utilisation of curdlan.

Topics: Actinobacteria; Bacterial Proteins; beta-Glucans; Catalysis; Cellulase; Escherichia coli; Recombinant Proteins

To optimize the physicochemical properties of phthalocyanine (PC), we examined its behavior in particles of triple helix glucan curdlan (CUR). CUR was denatured and renatured in DMSO, in the presence of PC. Infrared spectroscopy and transmission electron microscopy (TEM) showed that PC and CUR formed an inclusion complex, in which PC was trapped inside CUR molecules. This redshifted the absorption peak of PC, which would improve its usefulness as a photosensitizer, because infrared light can penetrate more deeply into human tissues. The conductivity of the solution of CUR-PC was higher than the conductivities of either a CUR solution or a PC dispersion, indicating that CUR-PC is more water soluble than PC. In addition, CUR-PC was highly stable in water. Thus, the use of CUR as a carrier of PC improves several of its physical properties. PC is used as a photosensitizer for killing cancer cells, but its use is hampered by its low solubility. Further, its absorption range limits its use to a depth of 1⁻3 mm in tissues. CUR-PC, with its high solubility and infrared absorption peak, was highly effective as a photosensitizer. It killed 84% of HeLa cells under 15 min of long wavelength radiation and had little cytotoxicity in the absence of light. These results demonstrate that CUR-PC has promise as a photosensitizer, as well as provide theoretical support for a wide range of applications for PC and CUR.

Topics: beta-Glucans; HeLa Cells; Humans; Indoles; Isoindoles; Microscopy, Electron, Transmission; Molecular Structure; Particle Size; Photosensitizing Agents; Solubility; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared

This work evaluated the improvement of curdlan production of Agrobacterium sp. ATCC 31749 by using culture medium containing juice of discarded bottom part of green Asparagus spear (MJDA). Curdlan production was carried out using Agrobacterium sp. ATCC 31749 in flasks with different volumes of MJDA and its non-juice-adding control (CK) incubated in shaker at 30 °C, 200 rpm rotation for 168 h.. All MJDA media increased Agrobacterium sp. ATCC 31749 cell mass and enhanced the cells' ability to utilise sucrose, the carbon source for curdlan biosynthesis, and thereby produced higher concentration of curdlan than CK which is used for commercial production of curdlan. After 168 h of fermentation, 10% MJDA produced 40.2 g/l of curdlan whiles CK produced 21.1 g/l. Curdlan production was increased by 90.4% higher in 10% MJDA than CK. Curdlan produced by 10% MJDA contains 1.2 and 1.5 µg/ml of Asparagus flavonoids and saponins respectively as additives which have wide range of health benefits. The mass of sucrose needed to produce 1.0 g curdlan by Agrobacterium sp. ATCC 31749 in CK is 1.7-fold more than in 10% MJDA.. The results strongly revealed that 5-10% MJDA is a good curdlan fermentation media which increase curdlan production yield with cheaper cost of production and simultaneously reduce environmental waste resulting from the large scaled discarded bottom parts of green Asparagus spear during Asparagus production.

Topics: Agrobacterium; Asparagus Plant; beta-Glucans; Biomass

The development of environmentally responsive drug delivery systems for the treatment of cancer has attracted particular interest in recent years. However, the enhancement of drug loading capacity and realization of pH-responsive drug delivery remain challenging. Herein, we employ carboxymethyl curdlan as a hydrophilic carrier to wrap doxorubicin (DOX) directly via electrostatic interaction. The sizes of the formed nanoparticles can be simply tuned by changing their feeding ratios. In particular, the nanoparticles are highly stable in aqueous solution without size variation. In vitro drug release and cytotoxicity assays illustrate that this delivery system can release DOX differentially under various environmental conditions and transport it into cell nuclei efficiently, with comparable therapeutic effect to the free drug. These results suggest that the carrying of antitumor drugs by polysaccharide via electrostatic interaction is a simple but effective way to construct a pH-dependent drug delivery platform.

Topics: beta-Glucans; Delayed-Action Preparations; Doxorubicin; HeLa Cells; Humans; Hydrogen-Ion Concentration; Neoplasms; Static Electricity

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

Cationic polymers are powerful carriers for intracellular delivery of therapeutic nucleic acids. However, the positively charged macromolecules have considerable cytotoxicity and often induce irreversible damages to the cells and tissues, which greatly negate the clinical application of such materials as drug delivery system. Herein, we report the synthesis of novel amphoteric polymers based on curdlan, and the evaluation of their cytotoxicity as well as the nucleic acid delivery efficiency. β-(1,3)-polyglucuronic acid, a TEMPO-oxidized derivative of curdlan, was chemically modified by conjugation of tetraethylenepentamine. The resulting amphoteric polymers, denoted tetraethylenepentamine-curdlan (TEPAC) polymers have the degree of substitution (DS) ranging from 25% to 48%. The result of MTT assay indicated that TEPAC polymers have negligible cytotoxicity on HeLa cells and A549 cells. The novel amphoteric polymers efficiently bound with plasmid DNA and delivered pcDNA-eGFP plasmid to 293T cells and induced expression of GFP 48h after the transfection. Moreover, TEPAC polymers delivered siRNA to HeLa cells and HepG2 cells in high efficiency, and induced significant RNAi for the expression of an endogenous gene. Collectively, our data demonstrate that the novel curdlan-based amphoteric polymers are biocompatible and may provide a highly efficient system for the delivery of therapeutic nucleic acids.

Topics: A549 Cells; beta-Glucans; Drug Delivery Systems; HEK293 Cells; HeLa Cells; Humans; Nucleic Acids; Plasmids; Polymers; RNA, Small Interfering; Transfection

Curdlan was grafted to poly(vinyl alcohol) (PVA) to form a porous scaffold. The grafted PVA-curdlan 3D scaffold was then examined by Fourier transform infrared spectroscopy (FT-IR). Grafting increased the water absorbency of the scaffold by 280%. Scanning electron microscopic (SEM) observations of the material revealed that the 3D scaffold was highly porous when it was fabricated using a homogenizer at 300rpm. Compression testing revealed that, increasing the amount of curdlan increased the strength of the 3D scaffold to 8-16×10

Topics: beta-Glucans; Biocompatible Materials; Materials Testing; Polyvinyl Alcohol; Porosity; Spectroscopy, Fourier Transform Infrared; Tissue Engineering; Tissue Scaffolds

A coupled fermentation system of Agrobacterium sp. ATCC 31749 and Trichoderma harzianum GIM 3.442 (AT-CFS) with wheat bran as the optimal nitrogen source was established for producing low-molecular-weight curdlan with high production, which can potentially reduce the cost of low-molecular-weight curdlan biosynthesis. The initial inoculate ratio, pH and the fermentation time were optimized. Compared with the curdlan from the single fermentation system of Agrobacterium sp. ATCC 31749 (A-SFS), the molecular weight (Mw) of the curdlan produced from AT-CFS decreased by 34.01% (from 110.85kDa to 73.15kDa), and the curdlan production (47.9g/L) and conversion rate of glucose to curdlan (0.60gg

Topics: Agrobacterium; beta-Glucans; Fermentation; Industrial Microbiology; Trichoderma

Mast cells are sentinel cells with a tissue-specific localization in the interface between the host and the external environment. Their quick and selective response upon encountering pathogens is part of the innate host response and typically initiates the following adaptive immune response. Among several pattern recognition receptors (PRRs) involved in the recognition of pathogens by mast cells, the C-type lectin receptor Dectin-1 has been associated with the recognition of fungi. Our previous studies have shown that mast cells are the predominant cell type expressing Dectin-1 in human skin, and they also recognize and respond to Malassezia sympodialis by producing cytokines connected to the innate host response and upregulating the expression of Dectin-1. In the present study, we investigated mast cell responses to Curdlan, a β-glucan that acts as an agonist for the fungi receptor Dectin-1, and found a unique response pattern with induced degranulation, but surprisingly without synthesis of Leukotriene C

Topics: Animals; beta-Glucans; Cell Degranulation; Chemokine CCL2; Exocytosis; Interleukin-6; Leukotriene C4; Mast Cells; Mice; Receptors, IgE

Curdlan is a linear polysaccharide considered a dietary fiber and with gelation properties. This study evaluated the structure, morphology and the physicochemical and technological properties of curdlan produced by Agrobacterium sp. IFO 13140 recovered by pre-gelation and precipitation methods. Commercial curdlan submitted or otherwise to the pre-gelation process was also evaluated. The data obtained from structural analysis revealed a similarity between the curdlan produced by Agrobacterium sp. IFO 13140 (recovered by both methods) and the commercial curdlans. The results showed that the curdlans evaluated differed significantly in terms of dispersibility and gelation, and only the pre-gelled ones had significant potential for food application, because this method influence on the size of the particles and in the presence of NaCl. In terms of technological properties, the curdlan produced by Agrobacterium sp. IFO 13140 (pre-gelation method) had a greater water and oil holding capacity (64% and 98% greater, respectively) and a greater thickening capacity than the pre-gelled commercial curdlan. The pre-gelled commercial curdlan displayed a greater gelling capacity at 95°C than the others. When applied to food, only the pre-gelled curdlans improved the texture parameters of yogurts and reduced syneresis. The curdlan gels, which are rigid and stable in structure, demonstrated potential for improving the texture of food products, with potential industrial use.

Topics: Agrobacterium; beta-Glucans; Culture Media; Dietary Carbohydrates; Food Technology; Gels; Polysaccharides, Bacterial; Rheology; Sodium; Solvents; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Temperature; Viscosity; Water; Yogurt

A cationic group has been quantitatively and selectively introduced into C6 position of each glucose units of Curdlan by "Click Chemistry" successfully. The resulting cationic Curdlan-Imidazole-lysine polymers (Cur-6-100Lys) exhibit excellent water solubility. Structure of the Cur-6-100Lys complexes was verified by FTIR and NMR spectroscopic measurements, and analysis of Cur-6-100Lys by GPC, DLS and SEM revealed that they have stoichiometric, nanosized spheroidal structures. Cytotoxicity measurement, electrophoretic mobility shift assay and EGFP-pDNA transfection have been carried out respectively. The results clearly show that Cur-6-100Lys nanocarriers have bound to dsDNA promptly, are less cytotoxic to both 7901 cells and HeLa cells, and are readily able to transport EGFP-pDNA into HepG2 cells. Our studies indicated that Cur-6-100Lys can potentially be used as a versatile nano platform for efficient gene delivery in living cells.

Topics: beta-Glucans; Cations; Cell Survival; Click Chemistry; Genetic Vectors; HeLa Cells; Humans; Nanoparticles; Plasmids; Transfection

The effect of nitrogen source concentration on the production of the polysaccharide curdlan by the bacterium Agrobacterium sp. ATCC 31749 from hydrolysates of prairie cordgrass was examined. The highest curdlan concentrations were produced by ATCC 31749 when grown on a medium containing a solids-only hydrolysate and the nitrogen source ammonium phosphate (2.2 mM) or on a medium containing a complete hydrolysate and 3.3 mM ammonium phosphate. The latter medium sustained a higher level of bacterial curdlan production than the former medium after 144 hr. Biomass production by ATCC 31749 was highest after 144 hr when grown on a medium containing a solids-only hydrolysate and 2.2 or 8.7 mM ammonium phosphate. On the medium containing the complete hydrolysate, biomass production by ATCC 31749 was highest after 144 hr when 3.3 mM ammonium phosphate was present. Bacterial biomass production after 144 hr was greater on the complete hydrolysate medium compared to the solids-only hydrolysate medium. Curdlan yield produced by ATCC 31749 after 144 hr from the complete hydrolysate medium containing 3.3 mM ammonium phosphate was higher than from the solids-only hydrolysate medium containing 2.2 mM ammonium phosphate.

Topics: Agrobacterium; beta-Glucans; Biomass; Hydrolysis; Nitrogen; Poaceae

Agrobacterium sp. IFO 13140 cells were immobilized on a loofa sponge and used to produce curdlan over five successive cycles. The interaction between microbial cells and the loofa sponge as well as the produced curdlan were characterized by Fourier transform infrared-attenuated total reflectance (FTIR-ATR) spectrometry. The purity of the curdlan was also evaluated. The storage stability of the immobilized cells was assessed and the produced curdlan was used in a functional yogurt formulation.. The average curdlan production by immobilized cells was 17.84 g L(-1) . The presence of the microorganism in the sponge was confirmed and did not cause alterations in the matrix, and the chemical structure of the curdlan was the same as that of commercial curdlan. The purity of both was similar. The immobilized cells remained active after 300 days of storage at -18 °C. The use of the produced curdlan in a functional yogurt resulted in a product with lower syneresis.. A large number of cells physically adhered to the surface of loofa sponge fibers, and its use as an immobilization matrix to produce curdlan was effective. The use of the produced curdlan in yogurt allowed the development of a more stable product. © 2015 Society of Chemical Industry.

Topics: Agrobacterium; beta-Glucans; Biopolymers; Cells, Immobilized; Food Handling; Luffa; Yogurt

A β-1,3-glucanase (LpGluA) of deep subseafloor Laceyella putida JAM FM3001 was purified to homogeneity from culture broth. The molecular mass of the enzyme was around 36 kDa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). LpGluA hydrolyzed curdlan optimally at pH 4.2 and 80 °C. In spite of the high optimum temperature, LpGluA showed relatively low thermostability, which was stabilized by adding laminarin, xylan, colloidal chitin, pectin, and its related polysaccharides. The gene for LpGluA cloned by using degenerate primers was composed of 1236 bp encoding 411 amino acids. Production of both LpGluA and a chitinase (LpChiA; Shibasaki et al. Appl Microbiol Biotechnol 98, 7845-7853, 2014) was induced by adding N-acetylglucosamine (GluNAc) to a culture medium of strain JAM FM3001. Construction of expression vectors containing the gene for LpGluA and its flanking regions showed the existence of a putative repressor protein.

Topics: Acetylglucosamine; Amino Acid Sequence; Bacillales; Base Sequence; beta-Glucans; Cloning, Molecular; Culture Media; Electrophoresis, Polyacrylamide Gel; Enzyme Stability; Geologic Sediments; Glucan 1,3-beta-Glucosidase; Hydrogen-Ion Concentration; Hydrolysis; Molecular Sequence Data; Molecular Weight; Temperature; Transcriptional Activation

Curdlan, a bioactive β-1,3-glucan, is of intense interest for pharmaceutical and biomedical applications. Cationic derivatives of curdlan and other polysaccharides are especially attractive for their potential to interact in controlled fashion with proteins, among many other possible applications, but relatively few methods exist for their synthesis. Herein we report a regioselective method for preparation of cationic, water-soluble 6-(N,N,N-trialkylammonio)-6-deoxycurdlan salts by reaction of 6-bromo-6-deoxycurdlan and its 2,4-O-diesters with trialkylamines. Dimethyl sulfoxide was identified as the optimal solvent for this nucleophilic displacement to produce cationic curdlan derivatives (80 °C, 24h), providing maximum degree of triethylammonium substitution (DS) of 0.89, exclusively at the C-6 position. 6-Bromo-6-deoxycurdlan was also reacted with heterocyclic amines such as pyridine and imidazole, providing ammonium-substituted curdlan derivatives with substantial DS (0.66 and 0.86, respectively). The new combination of regioselective Furuhata bromination and bromine displacement under optimized conditions with tertiary amines provides access to quaternized curdlan derivatives that possess high, permanent positive charge and are readily water-soluble, properties that indicate potential application promise including for mucoadhesion, permeation enhancement, and delivery of genes and anionic drugs. Regioselectivity and DS of those curdlan ammonium derivatives were quantified by means of (1)H NMR, (13)C NMR and FTIR spectroscopic methods and by elemental analysis.

Topics: beta-Glucans; Chitosan; Drug Carriers; Quaternary Ammonium Compounds

Herein, we present a new approach for the synthesis of gold nanoparticles (AuNPs) individually and as bimetallic core-shell nanoparticles (AgNPs-AuNPs). The novelty of the approach is further maximized by using curdlan (CRD) biopolymer to perform the dual role of reducing and capping agents and microwave-aided technology for affecting the said nanoparticles with varying concentrations in addition to those affected by precursor concentrations. Thus, for preparation of AuNPs, curdlan was solubilized in alkali solution followed by an addition of tetrachloroauric acid (HAuCl4). The curdlan solution containing HAuCl4 was then subjected to microwave radiation for up to 10 min. The optimum conditions obtained with the synthesis of AuNPs were employed for preparation of core-shell silver-gold nanoparticles by replacing definite portion of HAuCl4 with an equivalent portion of silver nitrate (AgNO3). The portion of AgNO3 was added initially and allowed to be reduced by virtue of the dual role of curdlan under microwave radiation. The corresponding portion of HAuCl4 was then added and allowed to complete the reaction. Characterization of AuNPs and AgNPs-AuNPs core-shell were made using UV-vis spectra, TEM, FTIR, XRD, zeta potential, and AFM analysis. Accordingly, strong peaks of the colloidal particles show surface plasmon resonance (SPR) at maximum wavelength of 540 nm, proving the formation of well-stabilized gold nanoparticles. TEM investigations reveal that the major size of AuNPs formed at different Au(+3)concentration lie below 20 nm with narrow size distribution. Whilst, the SPR bands of AgNPs-AuNPs core-shell differ than those obtained from original AgNPs (420 nm) and AuNPs (540 nm). Such shifting due to SPR of Au nanoshell deposited onto AgNPs core was significantly affected by the variation of bimetallic ratios applied. TEM micrographs show variation in contrast between dark silver core and the lighter gold shell. Increasing the ratio of silver ions leads to significant decrease in zeta potential of the formed bimetallic core-shell. FT-IR discloses the interaction between CRD and metal nanoparticles, which could be the question of reducing and stabilizing metal and bimetallic nanoparticles. XRD patterns assume insufficient difference for the AuNPs and AgNPs-AuNPs core-shell samples due to close lattice constants of Ag and Au. Based on AFM, AuNPs and AgNPs-AuNPs core-shell exhibited good monodispersity with spherical particles possessing different sizes in

Topics: beta-Glucans; Chemistry Techniques, Synthetic; Gold; Green Chemistry Technology; Kinetics; Metal Nanoparticles; Microwaves; Models, Molecular; Molecular Conformation; Nanotechnology; Silver

To investigate the expression and function of the mannose receptor (MR) and to explore its interaction with dectin-1 in human corneal epithelial cells (HCECs) exposed to Aspergillus fumigatus.. HCECs were stimulated with A. fumigatus for 0, 4, 8, 12, 16, and 24 hours. MR expression was tested by the polymerase chain reaction, Western blot, and immunohistochemistry. HCECs were pretreated with 2 μg/mL MR-blocking antibody. The expressions of p38, phosphorylated p38 (p-p38), and the downstream cytokines (TNF-α and IL-1β) and dectin-1 were tested by the polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay. HCECs were pretreated with dectin-1 agonists (curdlan, 100 μg/mL) and inhibitors (laminarin, 10 μg/mL), and the expression of the MR was tested.. MR expression was upregulated after stimulation with A. fumigatus. MR mRNA and protein levels began to rise at 8 and 16 hours, respectively. Stronger immunostaining of the MR was observed in fungal-infected corneal epithelium than in normal corneal epithelium. Aspergillus fumigatus increased the production of TNF-α (11-fold, 4-fold of the control), IL-1β (4.7-fold, 3-fold of the control), p-p38 (2.1-fold of the control), and dectin-1 (2.3-fold, 2-fold of the control) in mRNA and protein levels. The MR antibody significantly suppressed the expression of TNF-α (28%, 50% reduction), IL-1β (38%, 42% reduction), p-p38 (38% reduction), and dectin-1 (48%, 47% reduction). Curdlan increased the production of the MR (1.5-fold, 1.9-fold of the control), whereas laminarin decreased the expression of the MR (50%, 60% reduction) induced by A. fumigatus.. HCECs express the MR, and A. fumigatus infection can increase MR expression. A. fumigatus induces the expression of inflammatory cytokines through the MR and p38 MAPK pathway. The expression of dectin-1 and the MR had mutual influence.

Topics: Aspergillosis; Aspergillus fumigatus; beta-Glucans; Blotting, Western; Corneal Ulcer; Cytokines; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Epithelium, Corneal; Eye Infections, Fungal; Humans; Lectins, C-Type; Mannose Receptor; Mannose-Binding Lectins; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Polysaccharides; Real-Time Polymerase Chain Reaction; Receptors, Cell Surface; Tissue Donors; Up-Regulation

Tumor-elicited immunosuppression is one of the essential mechanisms for tumor evasion of immune surveillance. It is widely thought to be one of the main reasons for the failure of tumor immunotherapy. Myeloid-derived suppressor cells (MDSCs) comprise a heterogeneous population of cells that play an important role in tumor-induced immunosuppression. These cells expand in tumor-bearing individuals and suppress T cell responses via various mechanisms. Curdlan, the linear (1 → 3)-β-glucan from Agrobacterium, has been applied in the food industry and other sectors. The anti-tumor property of curdlan has been recognized for a long time although the underlying mechanism still needs to be explored. In this study, we investigated the effect of curdlan on MDSCs and found that curdlan could promote MDSCs to differentiate into a more mature state and then significantly reduce the suppressive function of MDSCs, decrease the MDSCs in vivo and down-regulate the suppression in tumor-bearing mice, thus leading to enhanced anti-tumor immune responses. We, therefore, increase the understanding of further mechanisms by which curdlan achieves anti-tumor effects.

Topics: Agrobacterium; Animals; Antineoplastic Agents; beta-Glucans; Carcinoma, Lewis Lung; Cell Differentiation; Immunity; Immunosuppression Therapy; Immunotherapy; Lung Neoplasms; Male; Mice; Mice, Inbred C57BL; Myeloid-Derived Suppressor Cells; Neoplasms, Experimental; T-Lymphocytes; Tumor Burden; Tumor Escape

RNA interference (RNAi) is an evolutionarily conserved gene-silencing phenomenon that shows great promise for developing new therapies. However, the development of small interfering RNA (siRNA)-based therapies need to establish efficient delivery system that silences target genes with siRNA doses that is clinically feasible in humans. Here we report synthesis and in vivo study of a novel PEGylated curdlan-based nanoparticle, designated as 6AC-100PEG, obtained by conjugation of mPEG 2000 to 6-amino-6-deoxy-curdlan. The complex of siRNA/6AC-100PEG showed homogenous nanoparticles with an average diameter of 200nm. MTT assay indicated that 6AC-100PEG does not have apparent cytotoxicity. Systemic administration of a complex of siapoB/6AC-100PEG significantly reduced the level of apoB mRNA in mouse liver, indicating that 6AC-100PEG can efficiently deliver siRNA to mouse liver and induce RNAi. Administration of siRNA/6AC-100PEG to mouse did not elevate liver enzyme level in the serum, indicating that 6AC-100PEG nanoparticle is a promising in vivo siRNA delivery agent.

Topics: Animals; Apolipoproteins B; beta-Glucans; Hep G2 Cells; Humans; Liver; Male; Mice; Mice, Inbred BALB C; Nanocapsules; Polyethylene Glycols; RNA, Small Interfering; Tissue Distribution

Curdlan is a polysaccharide that consists of β-1,3-linked glucose residues. A polysaccharide-producing bacterium isolated from soil samples was identified as Pseudomonas sp. QL212. The polysaccharide was purified to homogeneity via sequential ethanol precipitation, deproteinization, CM ion-exchange, and gel chromatography sequentially. Analysis of the purified polysaccharide revealed that it consisted of many glucosyl residues, and its molecular weight was only 6.18×10(5)Da. This low molecular weight endowed it with excellent solubility. Infrared and nuclear magnetic resonance spectral analysis confirmed that the polysaccharide was curdlan. Single-factor and Response surface methodology experiments were used to optimize the culture medium and conditions. The optimal culture conditions consisted of seed culture age of 12h, and an incubation temperature of 30°C, with 10% inoculum and a total fluid volume of 75mL in a 250-mL Erlenmeyer flask. The maximum curdlan yield of about 5.92gL(-1) was achieved with an optimal medium consisting of 30.11gL(-1) of sucrose, 5.94gL(-1) of yeast extract, and an initial pH of 8.03. To our best knowledge, this is the highest reported yield of curdlan produced by Pseudomonas sp., and the curdlan production medium components were much simpler than those in previous reports.

Topics: Analysis of Variance; beta-Glucans; Carbon; Cell Culture Techniques; Chromatography, High Pressure Liquid; Gas Chromatography-Mass Spectrometry; Hydrogen-Ion Concentration; Hydrolysis; Nitrogen; Phylogeny; Proton Magnetic Resonance Spectroscopy; Pseudomonas; Spectroscopy, Fourier Transform Infrared

Biologically active β-(1,3)-glucan oligosaccharides were prepared from curdlan using GH64 enzyme (KfGH64). KfGH64 showed low activity toward native curdlan; thereby pretreatment conditions of curdlan were evaluated. KfGH64 showed the highest activity toward curdlan with heat treatment. The most efficient pretreatment (90°C for 0.5h) converted approximately 60% of curdlan into soluble saccharides under the optimized enzyme reaction conditions (pH 5.5, 37°C, 100rpm mixing speed, 24h, and 10μg of KfGH64/1g of curdlan). The resulting products were predominantly laminaripentaose and a small amount of β-(1,3)-glucans with an average degree of polymerization (DP) of 13 and 130. The products did not contain small oligosaccharides (DP<5), indicating that the hydrolysis of heat-treated curdlan by KfGH64 is a suitable method for the production of biologically active β-(1,3)-glucan oligosaccharides.

Topics: Actinobacteria; Bacterial Proteins; beta-Glucans; Glycoside Hydrolases; Hot Temperature; Hydrolysis; Oligosaccharides; Solubility

Curdlan is a secondary metabolite synthesized by Agrobacterium sp. and some other bacteria. A newly isolated exopolysaccharide-producing strain was identified to be Rhizobium radiobacter CGMCC 12099. The polysaccharide product was confirmed to be curdlan with a molecule weight of 1.4×10(5)Da, and its molecular structure was determined by HPLC and infrared spectrum. Although nitrogen source is necessary for cell reproduction, curdlan production is largely dependent on nitrogen limitation, as well as cell vitality. Here, a nitrogen feeding strategy was investigated to elevate the curdlan production by R. radiobacter. The optimal concentration and addition time of (NH4)2HPO4 were investigated. The results showed that the enhanced cell density was correlated to the amount of (NH4)2HPO4 added. Also, nitrogen addition in earlier fermentation stage was beneficial to the cell growth and curdlan production. Furthermore, continuously feeding strategy was employed by feeding (NH4)2HPO4 at a constant rate of 1.24g/h at 35(th)h of fermentation for 9h, achieving a final curdlan production of 65.27g/L, productivity of 0.544g/L/h and glucose conversion rate of 38.89%. The curdlan production was improved by 2.1 times compared with that without nitrogen addition. This study provides a feasible and cheap nitrogen feeding strategy to enhance curdlan production.

Topics: beta-Glucans; Biomass; Cell Proliferation; Fermentation; Phosphates; Rhizobium

Spondyloarthritis (SpA) usually manifests as arthritis of the axial and peripheral joints but can also result in extra-articular manifestations such as inflammatory bowel disease. Proinflammatory cytokine interleukin-17 (IL-17) plays a crucial role in the pathogenesis of SpA. Rebamipide inhibits signal transducer and activator of transcription 3 that controls IL-17 production and Th17 cell differentiation. This study examined the effect of rebamipide on SpA development.. SKG ZAP-70(W163C) mice were immunized with curdlan to induce SpA features. The mice were then intraperitoneally injected with rebamipide or vehicle 3 times a week for 14 weeks and their clinical scores were evaluated. Histological scores of the paw and spine and the length of the gut were measured at sacrifice. Immunohistochemical staining of IL-17 and tumor necrosis factor-α (TNF-α) was performed using tissue samples isolated from the axial joints, peripheral joints, and gut. Spleen tissue samples were isolated from both rebamipide- or vehicle-treated mice with SpA at 14 weeks after curdlan injection to determine the effect of rebamipide on Th17 and regulatory T (Treg) cell differentiation.. Rebamipide decreased the clinical and histological scores of the peripheral joints. The total length of the gut was preserved in rebamipide-treated mice. IL-17 and TNF-α expression in the spine, peripheral joints, and gut was lower in rebamipide-treated mice than in control mice. Th17 cell differentiation was suppressed whereas Treg cell differentiation was upregulated in the spleen of rebamipide-treated mice.. Rebamipide exerted beneficial effects in mice with SpA by preventing peripheral arthritis and intestinal inflammation and by regulating Th17/Treg cell imbalance, suggesting that it can be used as a potential therapeutic agent for treating arthritis to SpA patients.

Topics: Alanine; Animals; Arthritis, Experimental; beta-Glucans; Cytokines; Inflammation; Inflammation Mediators; Interleukin-17; Intestines; Joints; Mice, Inbred BALB C; Quinolones; Spine; Spondylarthritis; T-Lymphocytes, Regulatory; Th17 Cells; Tumor Necrosis Factor-alpha

Curdlan is a commercial polysaccharide made by fermentation of Agrobacterium sp. Its anticipated expansion to larger volume markets demands improvement in its production efficiency. Metabolic engineering for strain improvement has so far been limited due to the lack of genetic tools. This research aimed to identify strong promoters and to engineer a strain that converts cellobiose efficiently to curdlan. Three strong promoters were identified and were used to install an energy-efficient cellobiose phosphorolysis mechanism in a curdlan-producing strain. The engineered strains were shown with enhanced ability to utilize cellobiose, resulting in a 2.5-fold increase in titer. The availability of metabolically engineered strain capable of producing β-glucan from cellobiose paves the way for its production from cellulose. The identified native promoters from Agrobacterium open up opportunities for further metabolic engineering for improved production of curdlan and other products. The success shown here marks the first such metabolic engineering effort in this microbe.

Topics: Agrobacterium; beta-Glucans; Cellobiose; Fermentation; Metabolic Engineering; Promoter Regions, Genetic

Curdlan, a β-1,3-glucan isolated from Alcaligenes faecalis, is an agonist of dectin-1 in various immune cells, including dendritic cells (DCs). However, whether curdlan also activates DCs through other receptors remains unknown. In this study, we found that curdlan activates DCs through dectin-1 and toll-like receptor 4 (TLR4). Curdlan increased the expression levels of surface molecules (CD40, CD80, CD86, and MHC-I/II), the production of cytokines (IL-12, IL-1β, TNF-α, and IFN-β), migration toward MIP-3β, and allogeneic T cell stimulation activity of DCs. Curdlan increased the phosphorylation of Syk, Raf-1, Akt, MAPKs, IKK, and NF-κB p65 in DCs. However, curdlan only slightly activated DCs transfected with small interfering RNAs against dectin-1 or TLR4 and C3H/HeJ DCs, which have non-functional TLR4, in comparison with control DCs. Curdlan increased antitumor activity of DCs in a syngeneic tumor model. In summary, our data show that curdlan activates DCs through dectin-1 and TLR4 signaling and the combination of curdlan and DCs efficiently inhibit tumor growth in mice.

Topics: Alcaligenes faecalis; Animals; Antineoplastic Agents; beta-Glucans; Cell Differentiation; Cell Movement; Cytokines; Dendritic Cells; Inflammation Mediators; Lectins, C-Type; Lymphocyte Activation; Melanoma, Experimental; Mice; Mice, Inbred C3H; Mice, Inbred C57BL; Mice, Knockout; RNA, Small Interfering; Skin Neoplasms; T-Lymphocytes; Toll-Like Receptor 4

Dectin-1 signalling in dendritic cells (DCs) has an important role in triggering protective antifungal Th17 responses. However, whether dectin-1 directs DCs to prime antitumour Th9 cells remains unclear. Here, we show that DCs activated by dectin-1 agonists potently promote naive CD4(+) T cells to differentiate into Th9 cells. Abrogation of dectin-1 in DCs completely abolishes their Th9-polarizing capability in response to dectin-1 agonist curdlan. Notably, dectin-1 stimulation of DCs upregulates TNFSF15 and OX40L, which are essential for dectin-1-activated DC-induced Th9 cell priming. Mechanistically, dectin-1 activates Syk, Raf1 and NF-κB signalling pathways, resulting in increased p50 and RelB nuclear translocation and TNFSF15 and OX40L expression. Furthermore, immunization of tumour-bearing mice with dectin-1-activated DCs induces potent antitumour response that depends on Th9 cells and IL-9 induced by dectin-1-activated DCs in vivo. Our results identify dectin-1-activated DCs as a powerful inducer of Th9 cells and antitumour immunity and may have important clinical implications.

Topics: Animals; beta-Glucans; Cell Differentiation; Chemokines; Cross-Priming; Dendritic Cells; Humans; Immunity; Lectins, C-Type; Mice, Inbred C57BL; Neoplasms; NF-kappa B; OX40 Ligand; Proto-Oncogene Proteins c-raf; Signal Transduction; Syk Kinase; T-Lymphocytes, Helper-Inducer; Tumor Necrosis Factor Ligand Superfamily Member 15

Curdlan (CURD) and polyethylene oxide were used to synthesize nanofibers as carriers of hydro soluble tetracycline hydrochloride (TCH). The viscosity, surface tension and conductivity of the precursor multicomponent aqueous solutions were determined and adjusted to produce defect-free fiber webs. Except for a slight increase in diameter, the addition of TCH did not affect the original morphology of the CURD/PEO nanofibers, as determined by FE-SEM imaging. However, the thermal stability of the system was enhanced (TGA and DSC). Moreover, water resistance, as measured with 24-h immersion tests, was observed upon crosslinking with glutaraldehyde. In-vitro activity measurements indicated a sustained and controlled TCH time-release pattern and excellent antibacterial activity against E. coli, as assessed by UV-vis spectroscopy and viable cell counting, respectively. Overall, we propose nanofibers based on CURD as promising platforms for scaffolds for wound dressing and drug delivery.

Topics: Anti-Infective Agents; beta-Glucans; Calorimetry, Differential Scanning; Cross-Linking Reagents; Delayed-Action Preparations; Drug Carriers; Escherichia coli; Glutaral; Microscopy, Electron, Scanning; Nanofibers; Polyethylene Glycols; Rheology; Solubility; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Surface Properties; Tetracycline

The objective of this study is to observe whether cyclosporine A (CsA) inhibits the expression of dectin-1 in human corneal epithelial cells infected with Aspergillus fumigatus (A. fumigatus) and to investigate the molecular mechanisms of the inhibition.. Immortalized human corneal epithelial cells (HCECs) were pretreated with 1,25(OH)2D3 and VDR inhibitor for 1 h, and then they were pretreated with CsA for 12h. After these pretreatments, the HCECs were stimulated with A. fumigatus and curdlan respectively, and the expression of dectin-1 and proinflammatory cytokines (IL-1β and TNF-α) were detected by RT-PCR, western blot and ELISA.. Dectin-1 mRNA and dectin-1 protein expression increased when HCECs were stimulated with A. fumigatus or curdlan, and CsA inhibited the dectin-1 expression both in mRNA and protein levels specifically. Dectin-1 and proinflammatory cytokine expression levels were higher when HCECs were pretreated with VDR inhibitor and CsA compared to pretreatment with CsA alone, while dectin-1 and proinflammatory cytokine levels were lower when HCECs were pretreated with 1,25(OH)2D3 and CsA compared to pretreatment with CsA alone.. These data provide evidence that CsA can inhibit the expression of dectin-1 and proinflammatory cytokines through dectin-1 when HCECs are stimulated by A. fumigatus or curdlan. The active form of vitamin D, 1,25(OH)2D3, and VDR signaling pathway regulate the inhibition of CsA. The inhibition is enhanced by 1,25(OH)2D3, and the VDR inhibitor suppresses the inhibition.

Topics: Antigens, Bacterial; Aspergillus fumigatus; beta-Glucans; Calcitriol; Cells, Cultured; Cyclosporine; Enzyme-Linked Immunosorbent Assay; Epithelium, Corneal; Gene Expression; Humans; Interleukin-1beta; Lectins, C-Type; Receptors, Calcitriol; RNA, Messenger; Signal Transduction; Tumor Necrosis Factor-alpha

In this study, a new non-toxic, biodegradable, biocompatible and water-soluble carboxylic curdlan bearing the dissociable COOH group in 100% purity, which was prepared by 4-acetamido-TEMPO-mediated oxidation, was hydrophobically modified by deoxycholic acid (DOCA) to attain novel amphiphilic curdlan derivatives (CCDs) for the preparation of nano-carriers for antitumor drug doxorubicin (DOX). Under the effect of ultrasonication, the carboxylic curdlan derivatives in water were self-aggregated into spherical nanoparticles with diameters ranging from 214 nm to 380 nm. The critical aggregation concentrations decreased from 0.047 mg/mL to 0.016 mg/mL with increasing DS of DOCA. DOX-loaded CCD nanoparticles were prepared in an aqueous medium with dialysis method. The DOX-CCD nanoparticles exhibited pH- and dose-dependent drug release profiles during in vitro release experiments. Moreover, the drug transport mechanism was Fickian diffusion according to the Ritger-Peppas model. The CCD nanoparticles might be explored as potential carriers for hydrophobic drugs with controlled release and delivery functions.

Topics: Antineoplastic Agents; beta-Glucans; Cell Line, Tumor; Chitosan; Deoxycholic Acid; Doxorubicin; Drug Carriers; Humans; Hydrophobic and Hydrophilic Interactions; Nanoparticles; Neoplasms

RNA interference (RNAi) down-regulates gene expression post-transcriptionally, which is a therapeutically significant phenomenon that could potentially reduce the level of disease related proteins that are undruggable by conventional small molecular approaches. However, clinical application of small interference RNAs (siRNAs) requires design of potent siRNA sequences and development of safe and efficient delivery systems. To create a biocompatible siRNA delivery agent, we chemically modified natural polysaccharide curdlan in a regioselective manner to introduce amino group in the glucose units. The resulting 6-amino-curdlan (6AC) is water soluble and forms nanoparticles upon complexing with siRNAs. The novel curdlan-based nanoparticles efficiently delivered siRNAs to human cancer cells and mouse primary cells, and reduced 70-90% of target mRNA level. Moreover, 6AC nanoparticles delivered siRNA targeting eGFP to mouse embryonic stem (mES) cells stably expressing eGFP, and produced substantial reductions of GFP protein level. The novel curdlan-based nanoparticle is a promising vehicle for delivery of short RNAs to knock down endogenous mRNAs.

Topics: Animals; beta-Glucans; Cell Survival; Cells, Cultured; Drug Carriers; Embryonic Stem Cells; HCT116 Cells; HeLa Cells; Humans; Mice; Nanoparticles; RNA Interference; RNA, Small Interfering

This study demonstrates a facile, green strategy for the preparation of gold nanoparticles (AuNPs) from chloroauric acid (HAuCl4) using carboxylic curdlan (Cc) as both reducing and stabilizing agent. The as-prepared AuNPs are characterized by UV-vis spectroscopy, high resolution transmission electron microscopy, X-ray diffraction, energy dispersive X-ray spectrometry and Fourier transform infrared spectroscopy. The results indicated that the particle size of the AuNPs changes with variations in the reaction time and concentrations of Cc and HAuCl4. The spherical AuNPs are well dispersed, exhibiting high stability even after six months storage. The carboxylic groups (COO(-)) in the Cc molecules tend to adsorb and stabilize the surface of the AuNPs. The interaction between BSA and the Cc-capped AuNPs was investigated using fluorescence and circular dichroism spectroscopies. The results indicated that the BSA molecules adsorb on the surface of the AuNPs, without significant change in its helical structure even after conjugation with the AuNPs.

Topics: Animals; beta-Glucans; Carboxylic Acids; Cattle; Chemistry Techniques, Synthetic; Chlorides; Gold; Gold Compounds; Green Chemistry Technology; Kinetics; Metal Nanoparticles; Serum Albumin, Bovine

The effects of curdlan in combination with microbial transglutaminase on the gelling properties of hairtail muscle protein were investigated. When curdlan of 4g/100g paste was combined with transglutaminase at a concentration of 0.4units/g meat paste, the gel strength, water holding capacity and the whiteness of the heated gel were improved. Textural profiles, such as hardness, springiness, cohesiveness, guminess and chewiness, reached their peaks as well. The increased band intensity of cross-linked proteins, accompanied by weakened myosin heavy chain, was observed from the SDS-PAGE pattern, indicating that curdlan might activate the formation of more ε-(γ-glutamyl) lysine cross-links induced by transglutaminase, especially at the level of 0.4units/g paste, leading to a denser gel matrix.

Topics: Animals; beta-Glucans; Electrophoresis, Polyacrylamide Gel; Fish Proteins; Food Handling; Muscle Proteins; Perciformes; Seafood; Transglutaminases

Bioactive compounds were orally administered to the native European oyster Ostrea edulis to evaluate the immune response and the progression of infection of the protozoan parasite Bonamia ostreae. The immunostimulants lipopolysaccharide and zymosan directly administrated to the water column induced an increase in lysozyme activity and the percentage of granulocytes in naïve oysters over a period of 7 days. In another set of experiments, zymosan and curdlan were microencapsulated in alginate and also administered to the water column to naïve and B. ostreae infected O. edulis. Oyster mortality, prevalence and intensity of infection and several immune parameters were evaluated up to 28 days post-administration. Lysozyme activity, nitric oxide production and the expression of galectin, lysozyme and superoxide dismutase increased after 24 h in both infected and uninfected oysters. Zymosan immunostimulated oysters displayed a decrease in the prevalence of B. ostreae infection not attributed to mortalities but which could be associated to the enhanced ability of immunostimulants to evoke an enhanced immune response in the oysters and reduce infection.

Topics: Adjuvants, Immunologic; Administration, Oral; Alginates; Animals; beta-Glucans; Haplosporida; Host-Parasite Interactions; Immunity, Innate; Ostrea; Zymosan

DCs are crucial for sensing pathogens and triggering immune response. Upon activation by pathogen-associated molecular pattern (PAMP) ligands, GM-CSF myeloid DCs (GM-DCs) secrete several cytokines, including IL-2. DC IL-2 has been shown to be important for innate and adaptive immune responses; however, IL-2 importance in DC physiology has never been demonstrated. Here, we show that autocrine IL-2 signaling is functional in murine GM-DCs in an early time window after PAMPs stimulation. IL-2 signaling selectively activates the JAK/STAT5 pathway by assembling holo-receptor complexes at the cell surface. Using the sensitivity of targeted mass spectrometry, we show conclusively that GM-DCs express CD122, the IL-2 receptor β-chain, at steady state. In myeloid DCs, this cytokine pathway inhibits survival of PAMP-matured GM-DCs which is crucial for maintaining immune tolerance and preventing autoimmunity. Our findings suggest that immune regulation by this novel autocrine signaling pathway can potentially be used in DC immunotherapy.

Topics: Animals; Autocrine Communication; beta-Glucans; Cell Differentiation; Cell Survival; Dendritic Cells; Granulocyte-Macrophage Colony-Stimulating Factor; Interleukin-2; Interleukin-2 Receptor beta Subunit; Janus Kinases; Ligands; Mice; Mice, Knockout; Protein Subunits; Receptors, Interleukin-2; Receptors, Pattern Recognition; STAT5 Transcription Factor; Up-Regulation

In this study, high-intensity ultrasound (20 kHz), a simple, effective and without any additive method, was used to the degradation of carboxylic curdlan (Cc) produced by 4-acetamido-TEMPO-mediated oxidation. The effects of ultrasound on molecular properties, structure and chain conformations of Cc were investigated by viscometry, size-exclusion chromatography with multiangle laser-light scattering (SEC-MALLS) analysis, as well as FTIR and NMR spectroscopies. The results indicated that the intrinsic viscosity [η] and the weight-average molecular weight (Mw) of Cc decreased obviously after ultrasound, and a uniform and narrow distribution of degradation product was obtained. The z-average radius of gyrations (Rg) firstly increased and then decreased as the sonication time prolonged. Ultrasound destroyed the hydrogen bonds resulting in the transition from compact random coil conformation to more flexible and even shorter extended chains. Ultrasonic treatment could not alter the primary chemical structure of Cc molecules according to the structural analysis by FTIR and NMR spectroscopies. Degradation kinetics based on Schmid model was applied to estimate the degradation rate constant k. It was found that the k value of Cc decreased with increasing the polymer concentration from 0.05 to 0.2% (w/v).

Topics: beta-Glucans; Carbohydrate Conformation; Cyclic N-Oxides; Kinetics; Molecular Weight; Oxidation-Reduction; Sonication; Viscosity

Innate immunity can facilitate nervous system regeneration, yet the underlying cellular and molecular mechanisms are not well understood. Here we show that intraocular injection of lipopolysaccharide (LPS), a bacterial cell wall component, or the fungal cell wall extract zymosan both lead to rapid and comparable intravitreal accumulation of blood-derived myeloid cells. However, when combined with retro-orbital optic nerve crush injury, lengthy growth of severed retinal ganglion cell (RGC) axons occurs only in zymosan-injected mice, and not in LPS-injected mice. In mice deficient for the pattern recognition receptor dectin-1 but not Toll-like receptor-2 (TLR2), zymosan-mediated RGC regeneration is greatly reduced. The combined loss of dectin-1 and TLR2 completely blocks the proregenerative effects of zymosan. In the retina, dectin-1 is expressed by microglia and dendritic cells, but not by RGCs. Dectin-1 is also present on blood-derived myeloid cells that accumulate in the vitreous. Intraocular injection of the dectin-1 ligand curdlan [a particulate form of β(1, 3)-glucan] promotes optic nerve regeneration comparable to zymosan in WT mice, but not in dectin-1(-/-) mice. Particulate β(1, 3)-glucan leads to increased Erk1/2 MAP-kinase signaling and cAMP response element-binding protein (CREB) activation in myeloid cells in vivo. Loss of the dectin-1 downstream effector caspase recruitment domain 9 (CARD9) blocks CREB activation and attenuates the axon-regenerative effects of β(1, 3)-glucan. Studies with dectin-1(-/-)/WT reciprocal bone marrow chimeric mice revealed a requirement for dectin-1 in both retina-resident immune cells and bone marrow-derived cells for β(1, 3)-glucan-elicited optic nerve regeneration. Collectively, these studies identify a molecular framework of how innate immunity enables repair of injured central nervous system neurons.

Topics: Animals; Axons; beta-Glucans; CARD Signaling Adaptor Proteins; Central Nervous System; Cyclic AMP Response Element-Binding Protein; Inflammation; Lectins, C-Type; Lipopolysaccharides; Mice, Inbred C57BL; Microglia; Myeloid Cells; Myeloid Differentiation Factor 88; Nerve Regeneration; Phagocytosis; Radiation Tolerance; Retina; Signal Transduction; Toll-Like Receptor 2; Zymosan

The influence of curdlan at different levels, as well as the method of addition, on the viscoelastic characteristics of ribbonfish meat gel was investigated. From a small amplitude oscillatory shear analysis (SAOA), a variety of viscoelastic parameters were established and identified to measure the intensity of the interactions between curdlan and protein in the fish meat gel network structure. The results of water holding capacity, texture, sensory property and microstructure analyses were strongly in agreement with the rheology data, suggesting that SAOA might be an appropriate method for the industrial assessment of the quality of fish meat gel. Additionally, the recombination mechanism of the complex system formed by the fish protein and curdlan was also clarified. Compared with the irreversible curdlan gel samples, the addition of reversible curdlan gel to the fish meat gel formed a much denser cross-linked interpenetrating structure, which led to a more stable and ordered three-dimensional gel complex.

Topics: Animals; beta-Glucans; Fish Products; Fishes; Gels; Rheology

There is significant interest in the application of nanoparticles to deliver immunostimulatory signals to cells. We hypothesized that curdlan (immune stimulating polymer) could be conjugated to PLGA and nanoparticles from this copolymer would possess immunostimulatory activity, be non-cytotoxic and function as an effective sustained drug release system.. Carbodiimide chemistry was employed to conjugate curdlan to PLGA. The conjugate (C-PLGA) was characterized using (1)H and (13)C NMR, FTIR, DSC and TGA. Nanoparticles were synthesized using an emulsion-solvent evaporation technique. Immunostimulatory activity was characterized in THP-1 derived macrophages. MTT assay and real-time impedance measurements were used to characterize polymer and nanoparticle toxicity and uptake in macrophages. Drug delivery capability was assessed across Caco-2 cells using rifampicin as a model drug.. Spectral characterization confirmed successful synthesis of C-PLGA. C-PLGA nanoparticles enhanced phosphorylated ERK production in macrophages indicating cell stimulation. Nanoparticles provided slow release of rifampicin across Caco-2 cells. Polymers but not nanoparticles altered the adhesion profiles of the macrophages. Impedance measurements suggested Ca(2+) dependent uptake of nanoparticles by the macrophages.. PLGA nanoparticles with macrophage stimulating and sustained drug delivery capabilities have been prepared. These nanoparticles can be used to stimulate macrophages and concurrently deliver drug in infectious disease therapy.

Topics: Antitubercular Agents; beta-Glucans; Biological Transport, Active; Caco-2 Cells; Carbohydrate Sequence; Cell Membrane Permeability; Cell Survival; Chemistry, Pharmaceutical; Drug Delivery Systems; Excipients; Humans; Intestinal Absorption; Lactic Acid; Macrophages; Molecular Sequence Data; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; Rifampin; Stimulation, Chemical

Water-solubility can often enhance the utility of polysaccharide derivatives, for example in pharmaceutical and biomedical applications. Synthesis of water-soluble aminopolysaccharides, particularly those bearing other sensitive functional groups, can be a challenging endeavor. Curdlan is a bioactive β-1,3-glucan with considerable promise for biomedical applications. Aminocurdlans are intriguing target molecules for study of, for example, their interactions with the proteins that form tight junctions between enterocytes. Herein we report the preparation of two water-soluble 6-aminocurdlans starting from 6-bromo-6-deoxycurdlan. The 6-bromide was first displaced by nucleophilic substitution with sodium azide in dimethyl sulfoxide. The O-2 groups were acylated with hydrophilic oligo (ethylene oxide) esters, so as to enhance aqueous solubility. The resultant 6-azido-6-deoxy-2,4-di-O-trioxadecanoylcurdlan was then treated with excess sodium borohydride to reduce the azide; unexpectedly, the water-soluble product proved to be the amide, 6-trioxadecanamido-6-deoxycurdlan. Regioselectivity and degree of substitution (DS) of those derivatives were characterized by means of (1)H NMR, (13)C NMR and FTIR-spectroscopy, elemental analysis, and titration. Alternatively, direct borohydride reduction of the parent 6-azido-6-deoxycurdlan afforded 6-amino-6-deoxycurdlan that was also water-soluble.

Topics: Azides; beta-Glucans; Borohydrides; Oxidation-Reduction; Solubility; Stereoisomerism; Water

A β-glucan produced by Aureobasidium pullulans (AP-PG) is consisting of a β-(1,3)-linked main chain with β-(1,6)-linked glucose side residues. Various β-glucans consisting of β-(1,3)-linked main chain including AP-PG are believed to exhibit anti-tumor activities, and actually, anti-tumor activities of AP-PG in mice have been demonstrated. In this study, we demonstrate that stimulation with AP-PG induces TRAIL expression in mouse and human macrophage-like cell lines. TRAIL is known to be a cytokine which specifically induces apoptosis in transformed cells, but not in untransformed cells. The expression of TRAIL mRNA after stimulation with AP-PG was increased in RAW264.7 cells, Mono Mac 6 cells, and macrophage-differentiated THP-1 cells. The mRNA expression of TNF-α and FasL is only weakly increased after stimulation with AP-PG. The induction activity of TRAIL by curdlan, a bacterial β-glucan, was very similar to that by AP-PG in RAW264.7 cells, but weaker in macrophage-differentiated THP-1 cells. Activation of caspases was found in HeLa cells after treatment with the supernatant of cultured medium from AP-PG-stimulated Mono Mac 6 cells, and was inhibited by the anti-TRAIL neutralizing antibody. These findings suggest that the stimulation with AP-PG effectively induces TRAIL in macrophages, and that it may be related to apoptosis induction of tumor cells.

Topics: Animals; Apoptosis; Ascomycota; beta-Glucans; Caspases; Cells, Cultured; Humans; Macrophages; Mice; Polysaccharides, Bacterial; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha

Agrobacterium sp. ATCC31749 is an efficient curdlan producer at low pH and under nitrogen starvation. The helix-turn-helix transcriptional regulatory protein (crdR) essential for curdlan production has been analyzed, but whether crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC) is uncertain. To elucidate the molecular function of crdR in curdlan biosynthesis, we constructed a crdR knockout mutant along with pBQcrdR and pBQNcrdR vectors with crdR expression driven by a T5 promoter and crdR native promoter, respectively. Also, we constructed a pAG with the green fluorescent protein (GFP) gene driven by a curdlan biosynthetic operon promoter (crdP) to measure the effects of crdR expression on curdlan biosynthesis.. Compared with wild-type (WT) strain biomass production, the biomass of the crdR knockout mutant was not significantly different in either exponential or stationary phases of growth. Mutant cells were non-capsulated and planktonic and produced significantly less curdlan. WT cells were curdlan-capsulated and aggregated in the stationery phase. pBQcrdR transformed to the WT strain had a 38% greater curdlan yield and pBQcrdR and pBQNcrdR transformed to the crdR mutant strain recovered 18% and 105% curdlan titers of the WT ATCC31749 strain, respectively. Consistent with its function of promoting curdlan biosynthesis, curdlan biosynthetic operon promoter (crdP) controlled GFP expression caused the transgenic strain to have higher GFP relative fluorescence in the WT strain, and no color change was observed with low GFP relative fluorescence in the crdR mutant strain as evidenced by fluorescent microscopy and spectrometric assay. q-RT-PCR revealed that crdR expression in the stationary phase was greater than in the exponential phase, and crdR overexpression in the WT strain increased crdA, crdS, and crdC expression. We also confirmed that purified crdR protein can specifically bind to the crd operon promoter region, and we inferred that crdR directly acts to cause expression of the curdlan biosynthesis operon (crdASC).. CrdR is a positive transcriptional regulator of the crd operon for promoting curdlan biosynthesis in ATCC31749. The potential binding region of crdR is located within the -98 bp fragment upstream from the crdA start codon.

Topics: Agrobacterium; Bacterial Proteins; beta-Glucans; Binding Sites; Codon; Gene Expression Regulation, Bacterial; Gene Knockout Techniques; Genes, Reporter; Genetic Vectors; Green Fluorescent Proteins; Hydrogen-Ion Concentration; Nitrogen; Operon; Promoter Regions, Genetic; Protein Binding; Transcription, Genetic

Curdlan production by Agrobacterium sp. IFO13140 immobilized on loofa sponge, alginate and loofa sponge with alginate was investigated. There was no statistically-significant difference in curdlan production when the microorganism was immobilized in different matrices. The loofa sponge was chosen because of its practical application and economy and because it provides a high stability through its continued use. The best conditions for immobilization on loofa sponge were 50 mg of cell, 200 rpm and 72 h of incubation, which provided a curdlan production 1.50-times higher than that obtained by free cells. The higher volumetric productivity was achieved by immobilized cells (0.09 g/L/h) at 150 rpm. The operating stability was evaluated, and until the fourth cycle, immobilized cells retained 87.40% of the production of the first cycle. The immobilized cells remained active after 300 days of storage at 4 °C. The results of this study demonstrate success in immobilizing cells for curdlan biosynthesis, making the process potentially suitable for industrial scale-up. Additional studies may show a possible contribution to the reduction of operating costs.

Topics: Agrobacterium; Animals; beta-Glucans; Cells, Immobilized; Luffa; Porifera; Temperature

Experiments in which the effect of combined manipulations is compared with the effects of their pure constituents have received a great deal of attention. Examples include the study of combination therapies and the comparison of double and single knockout model organisms. Often the effect of the combined manipulation is not a mere addition of the effects of its constituents, with quite different forms of interplay between the constituents being possible. Yet, a well-formalized taxonomy of possible forms of interplay is lacking, let alone a statistical methodology to test for their presence in empirical data.. Starting from a taxonomy of a broad range of forms of interplay between constituents of a combined manipulation, we propose a sound statistical hypothesis testing framework to test for the presence of each particular form of interplay. We illustrate the framework with analyses of public gene expression data on the combined treatment of dendritic cells with curdlan and GM-CSF and show that these lead to valuable insights into the mode of action of the constituent treatments and their combination.. R code implementing the statistical testing procedure for microarray gene expression data is available as supplementary material. The data are available from the Gene Expression Omnibus with accession number GSE32986.

Topics: Animals; beta-Glucans; Cells, Cultured; Computer Simulation; Databases, Genetic; Dendritic Cells; Drug Therapy, Combination; Gene Expression Profiling; Gene Expression Regulation; Granulocyte-Macrophage Colony-Stimulating Factor; Mice; Models, Statistical; Oligonucleotide Array Sequence Analysis; Pattern Recognition, Automated

The effect of adding xanthan-curdlan hydrogel complex (XCHC) at 2 concentrations (0.25 and 0.5% w/w) on the freeze-thaw stability of heat-induced whey protein isolate (WPI) gel was investigated. Samples were stored at 4 °C for 24 h before subjected to 5 freeze-thaw cycles alternating between -16 °C (18 h) and 25 °C (6 h). Adding XCHC to the WPI solution resulted in the reduction of a significant amount of syneresis up to 5 repeated freeze-thaw cycles. Addition of XCHC decreased the amount of syneresis from 45% in the control sample (pure WPI gel) to 31.82% and 5.44% in the samples containing 0.25% and 0.5% gum, respectively, after the 5th freeze-thaw cycle. XCHC increased the storage modulus (G') of the gels and minimized the changes of the G' values over the 5 freeze-thaw cycles, indicating improvement of the stability of the system. Furthermore, the minimum protein concentration for gel formation decreased in the presence of the XCHC. Scanning electron microscopy (SEM) images showed that addition of XCHC resulted in the formation of a well-structured gel with numerous small pores in the network, which consequently improved the water retention ability during the temperature abuses up to 5 freeze-thaw cycles. These results have important implications for using XCHC in the formulation of the frozen WPI-based products with improved freeze-thaw stability and rheological properties.. Application of XCHC in the formulation of frozen dairy-based food products has the potential to enhance freeze-thaw stability and minimize moisture migration caused by temperature abuses of the products during distribution and consumer application.

Topics: beta-Glucans; Food Handling; Freezing; Frozen Foods; Hydrogels; Microscopy, Electron, Scanning; Polysaccharides, Bacterial; Rheology; Temperature; Whey Proteins

Topics: beta-Glucans; Caco-2 Cells; Cell Line, Tumor; Drug Delivery Systems; Humans; Lactic Acid; Macrophages; Nanoparticles; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer

The ultimate goal of this work was to examine the effect of xanthan-curdlan hydrogel complex (XCHC) on the rheology of whey protein isolate (WPI) within the pH range of 4-7 upon heating and cooling. Dynamic rheological properties of WPI and XCHC were studied individually and in combination, as a function of time or temperature. For pure WPI, gels were pH-dependent, and in all pH values except 7, gels formed upon first heating from 40 to 90 °C. At pH 7, WPI did not form gel upon first heating, and the storage modulus (G') started to increase during the holding time at 90 °C. The onset of gelation temperature of WPI was lower in acidic pH ranges compared to the neutral pH. In mixed gels, the presence of XCHC increased the G' of the gels. The rheological behaviour was pH-dependent and initially was controlled by XCHC; however, after the consolidation of WPI network, the behaviour was led by the whey protein isolate. Results showed that XCHC had a synergistic effect on enhancing the elastic modulus of the gels after the consolidation of WPI network. Based on the results of this study, it is possible to use these biopolymers in the formulation of frozen dairy-based products and enable food manufactures to improve the textural and physicochemical properties, and as a result the consumer acceptance of the food product.

Topics: beta-Glucans; Gels; Hydrogen-Ion Concentration; Materials Testing; Polysaccharides, Bacterial; Rheology; Temperature; Whey Proteins

Systemic rheumatic conditions are often accompanied by intraocular inflammatory disease (termed uveitis). Despite the frequent manifestation of uveitis with arthritis, very little is understood of the underlying mechanisms that mediate the eye's susceptibility to disease. The genetically susceptible SKG mouse strain develops arthritis that arises from an inherent mutation that disrupts T-cell antigen receptor signal transduction and thymic selection. The ensuing T-cell-mediated disease is further modulated through exposure to microbial triggers. The purpose of this study was to elucidate how a genetically determined shift in the T-cell repertoire toward self-reactive T cells that drive arthritis influences uveitis in SKG mice.. SKG mice (BALB/c mice that harbor the W163C point mutation in zeta-chain-associated protein kinase 70 [i.e., ZAP-70]) were housed under arthritis-resistant, specific pathogen-free conditions. Arthritis was induced by intraperitoneal injection with fungal glucans (zymosan or curdlan). Arthritis onset and severity were evaluated by clinical scoring, histopathology and infrared imaging within the joints. Periocular traits involving blepharoconjunctivitis were evaluated by clinical scoring and histology. Eyes were evaluated for signs of anterior uveitis using intravital videomicroscopy to document cell-trafficking responses within the iris vasculature and stroma and by histology to detect inflammatory infiltrate and tissue damage within the anterior and posterior eye segments.. Exposure to zymosan resulted in the predicted arthritic, sexually dimorphic phenotype in SKG mice. The eyes of SKG mice exhibited episodic intravascular cellular responses to zymosan or curdlan as indicated by significant increases in leukocyte-endothelium interactions akin to ocular vasculitis. However, despite the significant increase in early cell-trafficking responses, cellular infiltration into the iris stroma was not observed and histopathological signs indicative of a sustained uveitis were absent. Instead, eyes of SKG mice developed blepharoconjunctivitis that coincided with arthritis and exhibited sexual dimorphism.. This study underscores the complexity surrounding the pathogenesis of uveitis and its relationship with arthritis. The findings suggest that distinct mechanisms exist by which pathogenic autoimmune T cells target the eyes versus joints, which likely involves the environmental context but nonetheless should be taken into account in the identification and development of effective therapies for each organ.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; beta-Glucans; Disease Models, Animal; Disease Progression; Genetic Predisposition to Disease; Humans; Mice, Inbred BALB C; Mice, Knockout; Mutation, Missense; Severity of Illness Index; T-Lymphocytes; Time Factors; Uveitis; ZAP-70 Protein-Tyrosine Kinase; Zymosan

A strain Agrobacterium HX1126 was isolated from soil sample near the canal in Wuxi. α-lactose was used as the sole carbon source for the production of an exopolysaccharide which was named PLHX. The highest production of PLHX (21.4g/L) was obtained under nitrogen depletion. PLHX composed mainly of glucose, with lower amounts of galactose and aminogalactose. The structure of the product was confirmed by NMR and FTIR and was identified as curdlan. This exopolysaccharide formed a gel when 30g/L was put in boiling water for 10min, with an achieved gel strength of 831g/cm(2). Moreover, a hypothesis for higher gel strength production is proposed. The gel forming property makes this exopolysaccaride a good potential application in the food, pharmaceutical and cosmetic industries.

Topics: Agrobacterium; Base Sequence; beta-Glucans; Biomass; Calorimetry, Differential Scanning; Fermentation; Lactose; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Phylogeny; RNA, Ribosomal, 16S; Spectroscopy, Fourier Transform Infrared

Tissue-specific delivery of therapeutic RNAi has great potential for clinical applications. Receptor-mediated endocytosis plays a crucial role in targeted delivery of biotherapeutics including short interfering RNA (siRNA). Previously we reported a novel Curdlan-based nanoparticle for intracellular delivery of siRNA. Here we designed a nanoparticle based on ligand-functionalized Curdlan. Disaccharides were site-specifically conjugated to 6-deoxy-6-amino Curdlan, and the cell line specificity, cellular uptake, cytotoxicity, and siRNA delivery efficiency of the corresponding disaccharide-modified 6-deoxy-6-amino-Curdlan were investigated. Observation by fluorescence microscopy as well as flow cytometry showed that galactose-containing Curdlan derivatives delivered fluorescently labeled short nucleic acid to HepG2 cells expressing ASGPR receptor but not in other cells lacking surface ASGPR protein. Moreover, highly galactose-substituted Curdlan derivatives delivered siRNA specifically to ASGPR-expressing cells and induced RNAi activities, silencing endogenous GAPDH gene expression. Our data demonstrated that galactose-functionalized 6-deoxy-6-amino-Curdlan is a promising carrier for short therapeutic nucleic acids for clinical applications.

Topics: Asialoglycoprotein Receptor; beta-Glucans; Buffers; Carbon-13 Magnetic Resonance Spectroscopy; Cell Death; Electrophoretic Mobility Shift Assay; Flow Cytometry; Gene Transfer Techniques; Hep G2 Cells; Humans; Ligands; Microscopy, Fluorescence; Nanoparticles; Particle Size; RNA Interference; RNA, Small Interfering; Static Electricity

Bacterial cellulose pellicle produced by Gluconacetobacter xylinus (G. xylinus) is one of the best biobased materials having a unique supernetwork structure with remarkable physiochemical properties for a wide range of medical and tissue-engineering applications. It is still necessary to modify them to obtain materials suitable for biomedical use with satisfactory mechanical strength, biodegradability, and bioactivity. The aim of this research was to develop a gene-transformation route for the production of bacterial cellulose/Curdlan (β-1,3-glucan) nanocomposites by separate but simultaneous in vivo synthesis of cellulose and Curdlan. Modification of the cellulose-nanofiber-producing system of G. xylinus enabled Curdlan to be synthesized simultaneously with cellulose nanofibers in vivo, resulting in biopreparation of nanocomposites. The obtained Curdlan/cellulose composites were characterized, and their properties were compared with those of normal bacterial cellulose pellicles, indicating that Curdlan mixed with the cellulose nanofibers at the nanoscale without disruption of the nanofiber network structure in the pellicle.

Topics: beta-Glucans; Biocompatible Materials; Cellulose; Gluconacetobacter xylinus; Microscopy, Electron, Scanning; Microscopy, Fluorescence; Nanocomposites; Tissue Engineering; X-Ray Diffraction

Indoleamine 2,3-dioxygenase (IDO), which is mainly expressed in activated dendritic cells, is known as a regulator of immune responses. However, the role of IDO in immune responses against fungal corneal infection has not been investigated. To evaluate the regulatory mechanisms of IDO in fungal inflammation, we resorted to human corneal epithelial cells (HCECs), known as the first barrier of cornea against pathogenic microorganisms. We found that IDO was significantly up-regulated in corneal epithelium infected with Aspergillus fumigatus (A. fumigatus) and HCECs incubated with spores of A. fumigatus. Furthermore, IDO inhibitor (1-methyltryptophan, 1-MT) enhanced inflammatory cytokines IL-1β and IL-6 expression which were up-regulated by A. fumigatus spores infection. Dectin-1, as one of the important C-type lectin receptors, can identify β-glucan, and mediate fungal innate immune responses. In the present study, pre-treatment with curdlan, a Dectin-1 agonist, further enhanced IDO expression compared with A. fumigatus stimulation. While laminarin, the Dectin-1 specific inhibitor, partially inhibited IDO expression stimulated by A. fumigatus. Further studies demonstrated inhibition of IDO activity amplified the expressions of inflammatory cytokines IL-1β and IL-6 induced by activation of Dectin-1. These results suggested that IDO was involved in the immune responses of fungal keratitis. The activation of Dectin-1 may contribute to A. fumigatus spores-induced up-regulation of IDO.

Topics: Animals; Aspergillus fumigatus; beta-Glucans; Cytokines; Disease Models, Animal; Epithelium, Corneal; Female; Gene Expression; Humans; Indoleamine-Pyrrole 2,3,-Dioxygenase; Inflammation Mediators; Keratitis; Lectins, C-Type; Mice; RNA, Messenger

In order to find an efficient enzymatic tool for curdlan degradation to produce (1 → 3)-linked β-D-glucan oligosaccharides, strain E4-5 (registration number JN089883, Genbank) was isolated from seaside soil. The 16S rRNA gene sequencing classified it as Cellulosimicrobium cellulans. It was the first reported microorganism that succeeded in degrading high-set heated curdlan blocks. The ferments of strain E4-5 also showed good degradation effects on laminaran and alkali-neutralized curdlan. Due to the products with less amount of glucose, it was assumed that endo-1,3-β-glucanases of strain E4-5 had a greater hydrolyzing effect than exo-1,3-β-glucanases. This indicated that strain E4-5 was a promising microorganism to hydrolyze (1 → 3)-linked β-D-glucan. Moreover, alkali-neutralization pretreatment was effective for promoting a more diversified degree of polymerization (DP) of (1 → 3)-linked β-D-glucan oligosaccharides under enzymatic hydrolysis and will pave the way for making full use of curdlan for production of glucan oligosaccharides.

Topics: Actinobacteria; beta-Glucans; Glucan Endo-1,3-beta-D-Glucosidase; Hydrogen-Ion Concentration; Hydrolysis; Oligosaccharides

Immune responses during fungal infections are predominately mediated by 5/15-lipoxygenases (LO)- or cyclooxygenase (COX)-2-catalysed bioactive eicosanoid metabolites like leukotrienes, lipoxins and prostaglandins. Although few host mediators of fungi-triggered eicosanoid production have been established, the molecular mechanism of expression and regulation of 5-LO, 15-LO and COX-2 are not well-defined. Here, we demonstrate that, macrophages infected with representative fungi Candida albicans, Aspergillus flavus or Aspergillus fumigatus or those treated with Curdlan, a selective agonist of pattern recognition receptor for fungi Dectin-1, displays increased expression of 5-LO, 15-LO and COX-2. Interestingly, Dectin-1-responsive Syk pathway activates mTOR-sonic hedgehog (SHH) signaling cascade to stimulate the expression of these lipid metabolizing enzymes. Loss-of-function analysis of the identified intermediaries indicates that while Syk-mTOR-SHH pathway-induced 5-LO and 15-LO suppressed the Dectin-1-responsive pro-inflammatory signature cytokines like TNF-α, IL-1β and IL-12, Syk-mTOR-SHH-induced COX-2 positively regulated these cytokines. Dectin-1-stimulated IL-6, however, is dependent on 5-LO, 15-LO and COX-2 activity. Together, the current study establishes Dectin-1-arbitrated host mediators that direct the differential regulation of immune responses during fungal infections and thus are potential candidates of therapeutic intervention.

Topics: Animals; Arachidonate 15-Lipoxygenase; Arachidonate 5-Lipoxygenase; Aspergillus flavus; Aspergillus fumigatus; beta-Glucans; Candida albicans; Cell Line; Cyclooxygenase 2; Gene Expression Regulation; Hedgehog Proteins; Host-Pathogen Interactions; Interleukin-12; Interleukin-1beta; Intracellular Signaling Peptides and Proteins; Lectins, C-Type; Macrophages, Peritoneal; Mice; Primary Cell Culture; Protein-Tyrosine Kinases; Signal Transduction; Syk Kinase; TOR Serine-Threonine Kinases; Tumor Necrosis Factor-alpha

β-Glucans have beneficial health effects due to their immune modulatory properties. Oral administration of β-glucans affects tumour growth, microbial infection, sepsis, and wound healing. We hypothesized that pre-treatment with orally delivered soluble and particulate β-glucans could ameliorate the development of aggravate dextran sulfate sodium (DSS) induced intestinal inflammation. To study this, mice were orally pre-treated with β-glucans for 14 days. We tested curdlan (a particulate β-(1,3)-glucan), glucan phosphate (a soluble β-(1,3)-glucan), and zymosan (a particle made from Saccharomyces cerevisiae, which contains around 55% β-glucans). Weight loss, colon weight, and feces score did not differ between β-glucan and vehicle treated groups. However, histology scores indicated that β-glucan-treated mice had increased inflammation at a microscopic level suggesting that β-glucan treatment worsened intestinal inflammation. Furthermore, curdlan and zymosan treatment led to increased colonic levels of inflammatory cytokines and chemokines, compared to vehicle. Glucan phosphate treatment did not significantly affect cytokine and chemokine levels. These data suggest that particulate and soluble β-glucans differentially affect the intestinal immune responses. However, no significant differences in other clinical colitis scores between soluble and particulate β-glucans were found in this study. In summary, β-glucans aggravate the course of dextran sulfate sodium (DSS)-induced intestinal inflammation at the level of the mucosa.

Topics: Administration, Oral; Animals; beta-Glucans; Chemokines; Colitis; Colon; Cytokines; Dextran Sulfate; Glucans; Inflammation; Intestinal Mucosa; Mice, Inbred C57BL; Zymosan

Curdlan is produced by Agrobacterium sp. ATCC 31749 under nitrogen limiting condition. The biosynthesis of crudlan is a typical aerobic bioprocess, and the production of curdlan would be severely restricted under micro-aerobic and anoxic conditions. Proteomic analysis of Agrobacterium sp. was conducted to investigate the effect of dissolved oxygen on the crucial enzymes involved in curdlan biosynthesis.. Two-dimensional gel electrophoresis was performed to separate and visualize the differential expression of the intracellular proteins extracted from Agrobacterium sp. ATCC 31749 cultured under various dissolved oxygen levels (75%, 50%, 25% and 5%). In addition, a comparative proteomic analysis of the intracellular proteins expression level under various dissolved oxygen levels was done. Significant differently expressed proteins were identified by MALDI-TOF/TOF.. Finally, we identified 15 differently expressed proteins involved in polysaccharide synthesis, fatty acid synthesis, amino acid synthesis pathway. Among these proteins, phosphoglucomutase and orotidine 5-phosphate decarboxylase were the key metabolic enzymes directing curdlan biosynthesis.. Oxygen could affect the expression of the proteins taking charge of curdlan synthesis significantly.

Topics: Agrobacterium; Bacterial Proteins; beta-Glucans; Electrophoresis, Gel, Two-Dimensional; Molecular Sequence Data; Oxygen; Proteomics

Regioselective oxidation was applied to commercial curdlan for the preparation of its water-soluble derivatives with improved antioxidant activities, using a 4-acetamido-2,2, 6,6-tetramethylpiperidine-1-oxyl radical/NaClO/NaClO2 system at pH 4.8 and 40°C. The structural features, molecular properties, and chain conformations of the oxidised curdlans were determined using Fourier transform (FT) infrared and FT Raman spectroscopy, carbon nuclear magnetic resonance, X-ray diffraction, and size-exclusion chromatography with multi-angle laser-light scattering analyses. The C6 primary hydroxyls of curdlan were successfully oxidised into carboxylate groups with less depolymerization, and no aldehyde groups were formed during oxidation. The crystalline region of curdlan was destroyed after oxidation. The oxidised curdlans formed random coils in aqueous solution, and the chain became more flexible and expanded with increasing carboxylate contents from 2.07mmol/g to 4.87mmol/g. The high polyglucuronic acid derivative (Cur-24) showed the best antioxidant activity in TEAC and FRAP assays, thus it could be explored as novel potential antioxidants for dietary and therapeutic applications.

Topics: Antioxidants; beta-Glucans; Carbohydrate Conformation; Cyclic N-Oxides; Multigene Family; Oxidation-Reduction; Spectroscopy, Fourier Transform Infrared; X-Ray Diffraction

Pure (1→3)-β-polyglucuronic acid sodium salt was prepared from curdlan by oxidation with 4-acetamido-TEMPO/NaClO/NaClO₂ in water at pH 4.7 and 35°C. The oxidation conditions, including the reaction time and amounts of reagents added, were optimized for the preparation of (1→3)-β-polyglucuronic acids with high molecular weights. The primary C6 hydroxyl groups of curdlan were completely oxidized to the corresponding C6-carboxylates using a one- or two-step reaction process by controlling the oxidation conditions, thus providing pure (1→3)-β-polyglucuronic acids consisting only of D-glucuronosyl units. Unfortunately, however, the increased amounts of reagents and long reaction time led to significant depolymerization of the curdlan during the oxidation process, and the resulting (1→3)-β-polyglucuronic acids had weight-average degrees of polymerization of 340-360. The (13)C and (1)H NMR chemical shifts of the products were successfully assigned using pure (1→3)-β-polyglucuronic acid.

Topics: beta-Glucans; Carboxylic Acids; Catalysis; Chlorides; Cyclic N-Oxides; Glucuronic Acid; Oxidation-Reduction; Polymerization; Sodium Hypochlorite

Amphoteric β-1,3-glucans possessing both amino groups and carboxylic acid groups on the C6 positions of glucose units were designed and synthesized from naturally produced curdlan. The amphoteric polysaccharides showed the isoelectric point and the pH responsive interconversion between the original triple helix and single-stranded random structures. Since the pH dependences are comparable to the typical properties of proteins, the polysaccharides can be considered as a new class of giant amino acids. Utilizing the pH responsiveness, pH-controlled catch-and-release has been realized for cationic peptides or anionic DNA. We believe that the amphoteric polysaccharide can act as a new potential polymer to construct stimuli-responsive smart materials on the basis of the polysaccharide scaffold.

Topics: Amino Acids; beta-Glucans; Biocompatible Materials; Drug Design; Hydrogen-Ion Concentration; Proteins

Production of the commercially available polysaccharide curdlan by Agrobacterium sp. strain ECP-1, isolated as a mutant strain from ATCC 31749, on a medium containing a hydrolysate of the plant prairie cordgrass with selected ammonium phosphate concentrations was investigated for a period of 144 h. Although several ammonium phosphate concentrations supported curdlan production by the strain, the optimal concentration after 120 or 144 h was 3.3 mmol·L⁻¹. Only ammonium phosphate concentrations of 1.1 or 8.7 mmol·L⁻¹ failed to support curdlan production by the strain after 120 or 144 h. Biomass production by strain ECP-1 on the hydrolysate-containing medium after 120 or 144 h was comparable, independent of the ammonium phosphate concentration present. The curdlan yield from the cordgrass hydrolysate indicated that the grass was an effective plant biomass substrate for polysaccharide production.

Topics: Agrobacterium; beta-Glucans; Biomass; Industrial Microbiology; Poaceae; Polysaccharides, Bacterial

Type B T cells recognize peptide-MHC class II (pMHCII) isoforms that are structurally distinct from those recognized by conventional type A T cells. These alternative type B conformers result from peptide loading in the absence of HLA-DM. Type A conformers are more stable than type B pMHCII conformers but bind the same peptide in the same register. Here, we show that interaction of Salmonella Typhimurium with bone marrow derived dendritic cells (BMDCs) isolated from C3H/HeNCr1 mice results in enhanced presentation of peptide Ag to type B T cells. The effect could be mimicked by purified PAMPs, the most potent of which were curdlan and zymosan, β-(1,3)-glucan-containing polymers that are recognized by Dectin-1. Blocking of Dectin-1 with Ab and laminarin inhibited the induction of the type B T-cell response by BMDCs, confirming its role as a PRR for S. Typhimurium. Splenic DCs (sDCs) expressed Dectin-1 but were refractive to the induction of type B responses by S. Typhimurium and curdlan. Type B T cells have been shown to escape thymic tolerance and to transfer pathology in an autoimmune disease model. The induction of type B responses by gram-negative bacteria provides a mechanism by which autoreactive T cells may be produced during infection.

Topics: Animals; Antigen Presentation; Antigens, Bacterial; B7-1 Antigen; beta-Glucans; Bone Marrow Cells; Cells, Cultured; Dendritic Cells; Flow Cytometry; Histocompatibility Antigens Class II; Host-Pathogen Interactions; Lectins, C-Type; Lipopolysaccharides; Mice; Mice, Inbred C3H; Peptides; Salmonella typhimurium; T-Lymphocyte Subsets

A series of ester derivatives of curdlan, which is a β-(1 → 3)-D-glucan extracellularly produced by microorganism, with varying alkyl chain lengths (C2-C12) were synthesized by the heterogeneous reaction using trifluoroacetic anhydride. As a result, high-molecular-weight (Mw ≥ 6 × 10(5)) and fully-acylated curdlan was obtained with relatively high yield (>70%). Thermal stability of curdlan was greatly improved by esterification. Crystallization was observed for curdlan esters with C2-C6 side chains. Both Tg (170 → 50 °C) and Tm (290 → 170 °C) of curdlan esters decreased with increasing the side-chain length. By the increase in the side-chain carbon number, curdlan esters showed lower Young's modulus and tensile strength, and larger elongation at break. Thus, material properties of curdlan esters can be controlled by changing the side-chain length. It was found that the increase of the side-chain length resulted in the decrease of crystallinity and the change of crystal structures.

Topics: beta-Glucans; Esters; Molecular Structure

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

There has been growing interest in aminopolysaccharide synthesis over the last two decades due to the critical natural functions of aminopolysaccharides, and their potential in biomedical applications. Regioselective introduction of amino groups into polysaccharide backbones is a challenge. Natural curdlan is a linear β-(1→3)-glucan that is of interest both for its physical properties and its biomedical applications. Aminated curdlan derivatives were synthesized in three steps. First, curdlan was regioselectively brominated at the C-6 position in lithium bromide-N,N-dimethylacetamide (DMAc/LiBr). Second, the bromide of the product 6-bromo-6-deoxycurdlan was displaced by nucleophilic substitution with sodium azide (NaN3) in dimethyl sulfoxide (DMSO). Third, O-acylated 6-amido-6-deoxycurdlan was produced by a one-pot method. 6-Azido-6-deoxycurdlan was subjected to Staudinger reduction, followed by reaction in situ with excess carboxylic anhydride, without isolation of the 6-amino-6-deoxycurdlan intermediate. Regioselectivity and degree of substitution (DS) of these derivatives were confirmed by (1)H and (13)C NMR spectroscopy, FTIR spectroscopy, and elemental analysis.

Topics: Acetylation; Alcaligenes; Amination; beta-Glucans; Halogenation; Polysaccharides, Bacterial; Stereoisomerism

Our studies showed that tumor-infiltrating dendritic cells (DC) in breast cancer drive inflammatory Th2 (iTh2) cells and protumor inflammation. Here, we show that intratumoral delivery of the β-glucan curdlan, a ligand of dectin-1, blocks the generation of iTh2 cells and prevents breast cancer progression in vivo. Curdlan reprograms tumor-infiltrating DCs via the ligation of dectin-1, enabling the DCs to become resistant to cancer-derived thymic stromal lymphopoietin (TSLP), to produce IL-12p70, and to favor the generation of Th1 cells. DCs activated via dectin-1, but not those activated with TLR-7/8 ligand or poly I:C, induce CD8+ T cells to express CD103 (αE integrin), a ligand for cancer cells, E-cadherin. Generation of these mucosal CD8+ T cells is regulated by DC-derived integrin αvβ8 and TGF-β activation in a dectin-1-dependent fashion. These CD103+ CD8+ mucosal T cells accumulate in the tumors, thereby increasing cancer necrosis and inhibiting cancer progression in vivo in a humanized mouse model of breast cancer. Importantly, CD103+ CD8+ mucosal T cells elicited by reprogrammed DCs can reject established cancer. Thus, reprogramming tumor-infiltrating DCs represents a new strategy for cancer rejection.

Topics: Animals; beta-Glucans; Breast Neoplasms; CD8-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Cell Transformation, Neoplastic; Dendritic Cells; Disease Models, Animal; Female; Humans; Lectins, C-Type; Mice; Mucous Membrane; Signal Transduction; T-Lymphocyte Subsets; Th2 Cells; Transforming Growth Factor beta

Activation of the innate immune system before the invasion of pathogens is a promising way to improve the resistance of plant against infection while reducing the use of agricultural chemicals. Although several elicitors were used to induce the resistance of potato plant to microbial pathogen infection, the role of curdlan oligosaccharide (CurdO) has not been established. In the current study, the defense responses were investigated at biochemical and proteomic levels to elucidate the elicitation effect of CurdOs in foliar tissues of potato (Solanum tuberosum L. cv. McCain G1). The results indicate that the CurdOs exhibit activation effect on the early- and late-defense responses in potato leaves. In addition, glucopentaose was proved to be the shortest active curdlan molecule based on the accumulation of H₂O₂ and salicylic acid and the activities of phenylalanine amino-lyase, β-1,3-glucanase and chitinase. The 2D-PAGE analysis reveals that CurdOs activate the integrated response reactions in potato cells, as a number of proteins with various functions are up-regulated including disease/defense, metabolism, transcription, and cell structure. The pathogenesis assay shows that the ratio of lesion area of potato leaf decreased from 15.82%±5.44% to 7.79%±3.03% when the plants were treated with CurdOs 1 day before the infection of Phytophthora infestans. Furthermore, the results on potato yield and induction reactions indicate that the defense responses induced by CurdOs lasted for short period of time but disappeared gradually.

Topics: Analysis of Variance; beta-Glucans; Chitinases; Electrophoresis, Gel, Two-Dimensional; Gene Expression Regulation, Plant; Hydrogen Peroxide; Immunity, Innate; Phytophthora infestans; Plant Diseases; Plant Leaves; Salicylic Acid; Solanum tuberosum; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Several immune system cell surface receptors are reported to be associated with osteoclastogenesis. Dectin 1, a lectin receptor for β-glucan, is found predominantly on cells of the myeloid lineage. In this study, we examined the effect of the dectin 1 agonist curdlan on osteoclastogenesis. In mouse bone marrow cells and dectin 1-overexpressing RAW 264.7 cells (d-RAWs), curdlan suppressed receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation, bone resorption, and actin ring formation in a dose-dependent manner. This was achieved within non-growth inhibitory concentrations at the early stage. Conversely, curdlan had no effect on macrophage colony-stimulating factor-induced differentiation. Furthermore, curdlan inhibited RANKL-induced nuclear factor of activated T cell cytoplasmic 1 (NFATc1) expression, thereby decreasing osteoclastogenesis-related marker gene expression, including tartrate-resistant acid phosphatase, osteoclast stimulatory transmembrane protein, cathepsin K, and matrix metallopeptidase 9. Curdlan inhibited RANKL-induced c-fos expression, followed by suppression of NFATc1 autoamplification, without significantly affecting the NF-κB signaling pathway. We also observed that curdlan treatment decreased Syk protein in d-RAWs. Inhibition of the dectin 1-Syk kinase pathway by Syk-specific siRNA or chemical inhibitors suppressed osteoclast formation and NFATc1 expression stimulated by RANKL. In conclusion, our results demonstrate that curdlan potentially inhibits osteoclast differentiation, especially NFATc1 expression, and that Syk kinase plays a crucial role in the transcriptional pathways. This suggests that the activation of dectin 1-Syk kinase interaction critically regulates the genes required for osteoclastogenesis.

Topics: Active Transport, Cell Nucleus; Animals; beta-Glucans; Bone Marrow Cells; Cell Line; Cell Nucleus; Dose-Response Relationship, Drug; Enzyme Activation; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Lectins, C-Type; Male; Mice; NFATC Transcription Factors; Osteoclasts; Protein-Tyrosine Kinases; Proteolysis; RANK Ligand; Syk Kinase

Paenibacillus polymyxa ATCC 21830 was used for the production of curdlan gum for first time. A Box-Behnken experimental design was applied to optimize six variables of batch fermentation culture each at three levels. Statistical analyses were employed to investigate the direct and interactive effects of variables on curdlan production. Optimum cultural conditions were temperature (50°C), pH (7), fermentation time (96 h), glucose (100 g/L), yeast extract (3 g/L) and agitation speed (150 rpm). The yield of curdlan production was 6.89 g/L at optimum condition medium. Response surface methodology (RSM) and artificial neural network (ANN) were used to model cultural conditions of curdlan production. The maximum yield of curdlan production were predicted to be 6.68 and 6.85 g/L by RSM and ANN at optimum condition. The prediction capabilities of RSM and ANN were then statistically compared. The results showed that the ANN model is much more accurate in prediction as compared to the RSM. The infrared (IR) and NMR spectra, the thermogram of DSC and pattern of X-ray diffraction for the curdlan of the present study were almost identical to those of the commercial curdlan sample. The average molecular weight of the purified curdlan was determined to be 170 kDa by gel permeation chromatography.

Topics: beta-Glucans; Culture Media; Fermentation; Hydrogen-Ion Concentration; Kinetics; Neural Networks, Computer; Paenibacillus; Temperature

Crohn's disease (CD) is characterized by loss of tolerance to intestinal microorganisms. This is reflected by serological responses to fungal glycans such as mannan and β-glucans. Fungal glycans have various effects on immune cells. However, the evidence for their effects in CD is vague. This study aimed to assess the effects of fungal cell wall glycans on human peripheral blood mononuclear cells (PBMCs) from CD and control patients.. Human PBMCs from CD and control patients were stimulated by fungal cell wall glycans. Cytokine secretion was detected by ELISA and glycan receptor expression by flow cytometry.. Mannan, β-glucans (curdlan), chitosan, and zymosan induced the secretion of interleukin (IL)-1β, IL-6, IL-23, IL-10, and tumor necrosis factor-α by PBMCs. Spleen tyrosin kinase and Src tyrosine kinase were involved in the response to mannan and β-glucans. Mannan and whole yeast cells induced a significantly higher pro-inflammatory cytokine response in CD compared with control patients.. The results may suggest that CD is characterized by hyperresponsiveness to fungal glycans. Thus, glycans may potentially be triggering or perpetuating inflammation.

Topics: Adult; Aged; beta-Glucans; Chitosan; Crohn Disease; Female; Humans; Interleukin-10; Interleukin-1beta; Interleukin-23; Interleukin-6; Intracellular Signaling Peptides and Proteins; Leukocytes, Mononuclear; Male; Mannans; Middle Aged; Protein-Tyrosine Kinases; src-Family Kinases; Syk Kinase; Tumor Necrosis Factor-alpha; Zymosan

A Gram-stain-negative, aerobic, non-spore-forming, non-flagellated and rod-shaped bacterial strain, designated DMCK3-4(T), was isolated from the zone where the ocean and a freshwater spring meet at Jeju island, South Korea. Strain DMCK3-4(T) grew optimally at 30 °C, at pH 7.0-8.0 and in the presence of 2.0% (w/v) NaCl. Phylogenetic trees based on 16S rRNA gene sequences revealed that strain DMCK3-4(T) clustered with the strains of three members of the genus Simiduia, with which it exhibited 97.0-99.0% sequence similarity. Sequence similarities to the type strains of the other species with validly published names were less than 92.2%. Strain DMCK3-4(T) contained Q-8 as the predominant ubiquinone and summed feature 3 (C(16:1)ω7c and/or C(16:1)ω6c), C(17:1)ω8c, C(16:0), C(17:0) and C(18:1)ω7c as the major fatty acids. The major polar lipids of strain DMCK3-4(T) were phosphatidylethanolamine, phosphatidylglycerol, two unidentified glycolipids, one unidentified lipid and one unidentified aminolipid. The DNA G+C content of strain DMCK3-4(T) was 51.8 mol% and its mean DNA-DNA relatedness values with Simiduia agarivorans KCTC 23176(T), Simiduia areninigrae KCTC 23293(T) and Simiduia litorea NRIC 0917(T) were 23-34%, respectively. The differential phenotypic properties, together with the phylogenetic and genetic distinctiveness, revealed that strain DMCK3-4(T) is distinct from other species of the genus Simiduia. On the basis of the data presented, strain DMCK3-4(T) is considered to represent a novel species of the genus Simiduia, for which the name Simiduia curdlanivorans sp. nov. is proposed. The type strain is DMCK3-4(T) ( = KCTC 42075(T) =CECT 8570(T)). An emended description of the genus Simiduia is also proposed.

Topics: Bacterial Typing Techniques; Base Composition; beta-Glucans; DNA, Bacterial; Fatty Acids; Fresh Water; Gammaproteobacteria; Molecular Sequence Data; Nucleic Acid Hybridization; Phylogeny; Republic of Korea; RNA, Ribosomal, 16S; Seawater; Sequence Analysis, DNA; Ubiquinone

Trehalose-6,6-dimycolate (TDM), the mycobacterial cord factor, and its synthetic analog Trehalose-6,6-dibehenate (TDB) bind to the C-type lectin receptors macrophage-inducible C-type lectin (Mincle) and Mcl to activate macrophages. Genetically, the transcriptional response to TDB/TDM has been defined to require FcRγ-Syk-Card9 signaling. However, TDB/TDM-triggered kinase activation has not been studied well, and it is largely unknown which transcriptional regulators bring about inflammatory gene expression. In this article, we report that TDB/TDM caused only weak Syk-phosphorylation in resting macrophages, consistent with low basal Mincle expression. However, LPS-priming caused MYD88-dependent upregulation of Mincle, resulting in enhanced TDB/TDM-induced kinase activation and more rapid inflammatory gene expression. TLR-induced Mincle expression partially circumvented the requirement for Mcl in the response to TDB/TDM. To dissect transcriptional responses to TDB/TDM, we mined microarray data and identified early growth response (Egr) family transcription factors as direct Mincle target genes, whereas upregulation of Cebpb and Hif1a required new protein synthesis. Macrophages and dendritic cells lacking C/EBPβ showed nearly complete abrogation of TDB/TDM responsiveness, but also failed to upregulate Mincle. Retroviral rescue of Mincle expression in Cebpb-deficient cells restored induction of Egr1, but not of G-CSF. This pattern of C/EBPβ dependence was also observed after stimulation with the Dectin-1 ligand Curdlan. Inducible expression of hypoxia-inducible factor 1α (HIF1α) also required C/EBPβ. In turn, HIF1α was not required for Mincle expression, kinase activation, and Egr1 or Csf3 expression, but critically contributed to NO production. Taken together, we identify C/EBPβ as central hub in Mincle expression and inflammatory gene induction, whereas HIF1α controls Nos2 expression. C/EBPβ also connects TLR signals to cord factor responsiveness through MYD88-dependent upregulation of Mincle.

Topics: Animals; beta-Glucans; CCAAT-Enhancer-Binding Protein-beta; Cord Factors; Dendritic Cells; Early Growth Response Protein 1; Enzyme Activation; Granulocyte Colony-Stimulating Factor; Hypoxia-Inducible Factor 1, alpha Subunit; Intracellular Signaling Peptides and Proteins; Lectins, C-Type; Macrophages; Membrane Proteins; Mice; Mice, Knockout; Mycobacterium tuberculosis; Myeloid Differentiation Factor 88; Nitric Oxide; Nitric Oxide Synthase Type II; Phosphorylation; Protein Biosynthesis; Protein-Tyrosine Kinases; Syk Kinase; Toll-Like Receptors; Up-Regulation

Emerging evidence indicates that innate immunodeficiency syndromes are linked to mutations in innate receptors and to specific infections. X-linked lymphoproliferative syndrome type-2 (XLP-2) is associated with deficiency in X-linked inhibitor of apoptosis protein (XIAP), with poorly understood molecular mechanisms. Here we showed that XIAP deficiency selectively impaired B-cell chronic lymphocytic leukemia/lymphoma 10 (BCL10)-mediated innate responses to dectin-1 ligands but did not affect responses to various Toll-like receptor agonists. Consequently, Xiap(-/-) mice became highly vulnerable on Candida albicans infection. The compromised early innate responses led to the persistent presence of C albicans and inflammatory cytokines in Xiap(-/-) mice. Furthermore, priming of Xiap(-/-) mice with the dectin-1 ligand curdlan alone resulted in XLP-2-like syndromes. Restoration of dectin-1-induced Rac1 activation and phagocytosis by resolvin D1, but not up-regulation of nuclear factor-κB, rescued Xiap(-/-) mice from C albicans lethal infection. Therefore, development of XLP-2 in XIAP-deficient patients could be partly due to sustained inflammation as a consequence of defective BCL10-dependent innate immunity toward specific pathogens. Importantly, our results suggest the potential therapeutic value of resolvin D1 in the treatment of XLP-2 and innate immunodeficiency syndromes.

Topics: Adaptor Proteins, Signal Transducing; Animals; B-Cell CLL-Lymphoma 10 Protein; beta-Glucans; Candida albicans; Candidiasis; ErbB Receptors; Genetic Diseases, X-Linked; Humans; Imidazoles; Immunity, Innate; Inhibitor of Apoptosis Proteins; Lectins, C-Type; Lipopeptides; Lipopolysaccharides; Lymphoproliferative Disorders; Lysine; Lysophospholipids; Macrophages; Mice; NF-kappa B; Phagocytosis; Poly I-C; Protein Binding; Receptors, Antigen, T-Cell; Toll-Like Receptors; Tumor Necrosis Factor-alpha; Ubiquitination

Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity.

Topics: Animals; Antifungal Agents; beta-Glucans; Cell Line, Tumor; Cytokines; Dendritic Cells; Gene Expression Profiling; Gene Expression Regulation, Enzymologic; Immunity, Innate; Intracellular Signaling Peptides and Proteins; Lectins, C-Type; Macrophages; Mast Cells; Mice; Mycoses; Phosphorylation; Protein-Tyrosine Kinases; Rats; Signal Transduction; Syk Kinase; Tyrosine

Low molecular weight curdlan (LMWC) was prepared by hydrolysis of curdlan with commercial α-amylase. The hydrolysis reaction was conducted using 31.94 mg α-amylase per 500 mL reaction mixture, which contained 5 g curdlan. The hydrolysis was performed at pH 5.98 and 55.92 °C for 10 min. The molecular weight and structure of LMWC were characterized by high-performance liquid chromatography and Fourier transform infrared spectroscopy, respectively. Generally, LMWC showed lower gel strength than high molecular weight curdlan (HMWC). Unlike HMWC, LMWC could form into a gel at 50 °C. By contrast, HMWC could form into a gel at pH 11, but LMWC gel failed to form at this pH level. The strength of LMWC and HMWC gels increased with increasing temperature and decreased with increasing pH level.

Topics: alpha-Amylases; beta-Glucans; Gels; Hydrolysis; Molecular Weight

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

People living in damp buildings are typically exposed to spore and mycelial fragments of the fungi that grow on damp building materials. There is experimental evidence that this exposure to triple-helical (1, 3)-β-D glucan and low molecular weight toxins may be associated with non-atopic asthma observed in damp and moldy buildings. However, the mechanisms underlying this response are only partially resolved. Using the pure (1, 3)-β-D glucan, curdlan, and the murine macrophage cell line, RAW 264.7, there were two objectives of this study. The first was to determine whether signal transduction pathways activating asthma-associated cell signaling pathways were stimulated using mouse transduction Pathway Finder(®) arrays and quantitative real-time (QRT) PCR. The second objective was to evaluate the dose and temporal responses associated with transcriptional changes in asthma-associated cytokines, the signal transduction receptor gene Dectin-1, and various transcription factor genes related to the induction of asthma using customized RT-PCR-based arrays. Compared to controls, the 10(-7) M curdlan treatment induced significant changes in gene transcription predominately in the NFkB, TGF-β, p53, JAK/STAT, P13/AKT, phospholipase C, and stress signaling pathways. The 10(-8) M curdlan treatment mainly induced NFkB and TGF-β pathways. Compared to controls, curdlan exposures also induced significant dose- and time-dependent changes in the gene translations. We found that that curdlan as a non-allergenic potentiator modulates a network of transduction signaling pathways not only associated with TH-1, TH-2, and TH-3 cell responses including asthma potentiation, but a variety of other cell responses in RAW 264.7 cells. These results help provide mechanistic basis for some of the phenotypic changes associated with asthma that have been observed in in vitro, in vivo, and human studies and open up a hypothesis-building process that could explain the rise of non-atopic asthma associated with fungi.

Topics: Animals; Asthma; beta-Glucans; Cell Line; Cytokines; Dose-Response Relationship, Drug; Lectins, C-Type; Macrophages; Mice; NF-kappa B; Real-Time Polymerase Chain Reaction; Signal Transduction; Time Factors; Transcription, Genetic; Transforming Growth Factor beta

Curdlan derivative with anionic phosphate groups was used for the first time to obtain hydrogel microspheres. The chemical cross-linking of the phosphorylated curdlan was performed with epichlorohydrin using the water-in-oil inverse emulsion technique. The optical and scanning electron microscopies were used to analyze the morphology of the microgels, whereas the FTIR spectroscopy was used to investigate their chemical structure. The main characteristics such as the swelling degree, the exchange capacity, and the thermal resistance were also studied. These new anionic microgels could be used as potential carriers for controlled release of opposite charged drugs retained through electrostatic forces. Diphenhydramine, a cationic model drug, was used to investigate the loading and the release processes in various pH media simulating physiological fluids. Several mathematical models were applied to evaluate the drug transport processes and to calculate the drug diffusion coefficients. The synthesized microspheres presented an excellent biocompatibility.

Topics: beta-Glucans; Diffusion; Gels; Hydrogen-Ion Concentration; Microspheres; Models, Molecular; Pharmaceutical Preparations; Phosphorylation; Temperature; Water

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

2,2,6,6-Tetramethylpiperidine-1-oxyl radical (TEMPO)-mediated oxidation was applied to curdlan to prepare water-soluble sodium (1→3)-β-polyglucuronate, and its biodegradation behavior was then investigated. A bacterial strain (EH621) having the ability to degrade (1→3)-β-polyglucuronate was isolated from soil, and identified as Paenibacillus sp. Strain EH621 cultured with (1→3)-β-polyglucuronate decreased the initial total carbon in the supernatant by approximately 60% within 3 days, showing that (1→3)-β-polyglucuronate can be readily degraded and metabolized by microorganisms present in the natural environment. Hydrolytic enzyme activity was detected in the cell-free extract of EH621, which was highly specific to (1→3)-β-polyglucuronate. Analyses of the enzymatic degradation products revealed that (1→3)-β-polyglucuronate was endolytically degraded to its monomeric unit, glucuronate, via some oligomers and dimers.

Topics: Base Sequence; beta-Glucans; Biodegradation, Environmental; Cyclic N-Oxides; DNA, Bacterial; Glucuronates; Molecular Sequence Data; Oxidation-Reduction; Paenibacillus; RNA, Ribosomal, 16S; Sequence Analysis, DNA; Soil Microbiology

Biologically active β-1,3-oligosaccharides with rapidly growing biomedical applications are produced from hydrolysis of curdlan polysaccharide. The water-insoluble curdlan impedes its hydrolysis efficiency which is enhanced by our newly developed alkali-neutralization treatment process to increase the stability of curdlan suspension to more than 20 days, while the untreated control settled within 5 min. A putative double-layer structure model comprising of a compact core and a hydrated outer layer was proposed to describe the treated curdlan particles based on sedimentation and scanning electron microscopy observation. This model was verified by single- and two-step acid hydrolysis, indicative of the reduced susceptibility to hydrolysis when close to the compact core. Electrospray ionization-mass spectrometry, thin-layer chromatography analyses, and effective HPLC procedure led to the development of improved process to produce purified individual β-1,3-oligosaccharides with degrees of polymerization from 2 to 10 and potential for biomedical applications from curdlan hydrolyzate. Our new curdlan oligosaccharide production process offers an even better alternative to the previously published processes.

Topics: Acids; Alkalies; beta-Glucans; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Hydrolysis; Microscopy, Electron, Scanning; Oligosaccharides; Spectrometry, Mass, Electrospray Ionization; Water

A facile, simple, and eco-friendly method using 4-acetamido-2,2,6,6-tetramethypiperidine-1-oxyl radical-oxidized curdlan (Oc) as both reducing and stabilizing agents was developed for the fabrication of silver nanoparticles (AgNPs) from silver nitrate (AgNO₃). The structure, morphology, and particle size of the as-prepared AgNPs were investigated by ultraviolet-visible spectroscopy, transmission electron microscopy, energy dispersive X-ray spectrometry, X-ray diffraction, and dynamic laser light scattering. The well-dispersed AgNPs were sphere like with a mean diameter of 15 nm. Their formation was dependent on reaction duration, reaction temperature, Oc concentration, and AgNO₃ concentration. Fourier transform-infrared and Raman spectra demonstrated that the as-prepared AgNPs can readily bind covalently with the carboxylate groups of Oc through the strong monodentate interaction in the reaction medium.

Topics: Acetamides; beta-Glucans; Chromatography, Gel; Cyclic N-Oxides; Green Chemistry Technology; Light; Metal Nanoparticles; Molecular Conformation; Particle Size; Scattering, Radiation; Silver; Silver Nitrate; Spectrometry, X-Ray Emission; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared; Spectrum Analysis, Raman; Temperature; X-Ray Diffraction

Galectin-3 is a β-galactoside-binding animal lectin with diverse functions, including regulation of T helper (Th) 1 and Th2 responses. Current data indicate that galectin-3 expressed in dendritic cells (DCs) may be contributory. Th17 cells have emerged as critical inducers of tissue inflammation in autoimmune disease and important mediators of host defense against fungal pathogens, although little is known about galectin-3 involvement in Th17 development. We investigated the role of galectin-3 in the induction of Th17 immunity in galectin-3-deficient (gal3(-/-)) and gal3(+/+) mouse bone marrow-derived DCs. We demonstrate that intracellular galectin-3 negatively regulates Th17 polarization in response to the dectin-1 agonist curdlan (a β-glucan present on the cell wall of fungal species) and lipopolysaccharide, agents that prime DCs for Th17 differentiation. On activation of dectin-1, gal3(-/-) DCs secreted higher levels of the Th17-axis cytokine IL-23 compared with gal3(+/+) DCs and contained higher levels of activated c-Rel, an NF-κB subunit that promotes IL-23 expression. Levels of active Raf-1, a kinase that participates in downstream inhibition of c-Rel binding to the IL23A promoter, were impaired in gal3(-/-) DCs. Modulation of Th17 by galectin-3 in DCs also occurred in vivo because adoptive transfer of gal3(-/-) DCs exposed to Candida albicans conferred higher Th17 responses and protection against fungal infection. We conclude that galectin-3 suppresses Th17 responses by regulating DC cytokine production.

Topics: Adoptive Transfer; Animals; beta-Glucans; Candida albicans; Candidiasis; Cell Polarity; Chickens; Cytokines; Dendritic Cells; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Galectin 3; Immunity; Interleukin-23; Lectins, C-Type; Lipopolysaccharides; Mice; Mice, Inbred C57BL; Models, Biological; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins c-rel; Signal Transduction; Th17 Cells

This study aims to investigate the effects of Tween 80 on curdlan production, cell growth, and glucosyltransferase activity. The addition of Tween 80 to the culture medium increased curdlan production. However, curdlan production did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. The addition of Tween 80 to the culture medium did not affect cell growth. The glucosyltransferase activity involved in the curdlan synthesis increased with the increase of Tween 80 concentration. The glucosyltransferase activity did not increase further when excessive Tween 80 (>0.3% Tween 80) was added to the culture medium. Maximum curdlan was observed at day 5 and then levelled off. The biomass continued to increase until the end of the experimental period (6d). Maximum glucosyltransferase activity was also observed at day 5 and decreased thereafter. The results indicate that the enhanced curdlan production by Tween 80 is highly correlated with glucosyltransferase activity.

Topics: Alcaligenes; beta-Glucans; Biomass; Cell Proliferation; Dose-Response Relationship, Drug; Fermentation; Glucosyltransferases; Kinetics; Polysorbates

A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-d-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.

Topics: Agrobacterium; beta-Glucans; Catalysis; Cell-Free System; Escherichia coli; Glucose; Glucosyltransferases; Liposomes; Microscopy, Electron, Transmission; Nanoparticles; Nanotechnology; Peptides; Plasmids; Protein Biosynthesis; Proteins; RNA, Messenger; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Surface-Active Agents; Trypsin; Uridine Diphosphate

This study aims to investigate the effect of nitrogen source on curdlan production by Alcaligenes faecalis ATCC 31749. Curdlan production fell when excess nitrogen source was present, while biomass accumulation increased as the level of nitrogen source raised. Curdlan production and biomass accumulation were greater with urea compared with those with other nitrogen sources. The highest production of curdlan and biomass accumulation by A. faecalis ATCC 31749 was 28.16 g L(-1) and 9.58 g L(-1), respectively, with urea, whereas those with NH(4)Cl were 15.17 g L(-1) and 6.25 g L(-1), respectively. The optimum fermentation time for curdlan production was also affected by the nitrogen source in the medium.

Topics: Alcaligenes faecalis; beta-Glucans; Biomass; Nitrogen

The aim of this study was to evaluate and characterize the therapeutic potential of curdlan, a naturally occurring β-glucan immunomodulator, against visceral leishmaniasis, a fatal parasitic disease. Curdlan eliminated the liver and spleen parasite burden in a 45-day BALB/c mouse model of visceral leishmaniasis at a dosage of 10 mg/kg/day as determined by Giemsa-stained organ impression smears. Curdlan was associated with production of the disease-resolving T-helper (Th) 1 and Th17-inducing cytokines interleukin (IL)-6, IL-1β, and IL-23, as well as with production of Th17 cytokines IL-17 and IL-22, as determined by enzyme-linked immunosorbent assay (ELISA) and real time polymerase chain reaction (RT-PCR). Reversal of curdlan-mediated protection by anti-IL-17 and anti-IL-23 monoclonal antibodies showed the importance of Th17 cytokines. Significantly decreased production of both IL-17 and IL-22 by mice that received anti-IL-23 antibody suggested the essential role of IL-23 in Th17 differentiation. Although administration of recombinant IL-17 or IL-23 caused significant suppression of the organ parasite burden, with marked generation of interferon γ and nitric oxide (NO), effects were much faster for IL-17. These results documented that although both IL-23 and IL-17 play major roles in the antileishmanial effect of curdlan, the effect of IL-23 may occur indirectly, through the induction of IL-17 production.

Topics: Animals; Antibodies, Monoclonal; beta-Glucans; Disease Models, Animal; Immunologic Factors; Interferon-gamma; Interleukin-1; Interleukin-17; Interleukin-1beta; Interleukin-22; Interleukin-23; Interleukins; Leishmania donovani; Leishmaniasis, Visceral; Macrophages; Mice; Mice, Inbred BALB C; Nitric Oxide; Th1 Cells; Th17 Cells

Epicutaneous sensitization with protein allergen that induces predominant Th2 responses is an important sensitization route in atopic dermatitis. Fungal components have been shown to modulate Th cell differentiation. However, the effects of fungal components on epicutaneous sensitization are unclear.. In this study, we showed that co-administration of curdlan, a dectin-1 agonist, during epicutaneous ovalbumin sensitization of BALB/c mice decreased the IL-5 and IL-13 levels in supernatants of lymph node cell ovalbumin reactivation cultures. Mechanistically, curdlan co-administration decreased IL-4 and IL-1β expressions in draining lymph nodes. Curdlan co-administration also lower the migration of langerin+ CD103- epidermal Langerhans cells into draining lymph nodes at 96 hours post-sensitization which might be attributed to decreased expressions of IL-18 and IL-1β in patched skin. Moreover, adoptive transfer of CFSE-labeled transgenic CD4 T cells confirmed that curdlan co-administration decreased the proliferation and IL-4-production of ovalbumin -specific T cells primed by epidermal Langerhans cells.. These results indicated that concurrent exposure to a dectin-1 agonist suppresses the epicutaneously induced Th2 response by modulating the cytokine expression profiles in draining LNs and the migration of epidermal Langerhans cells. These results highlight the effects of fungal components on epicutaneous allergen sensitization in atopic diseases.

Topics: Administration, Cutaneous; Allergens; Animals; Antigens, Surface; beta-Glucans; CD4-Positive T-Lymphocytes; Cell Movement; Dermatitis, Atopic; Interleukin-13; Interleukin-5; Lectins, C-Type; Lymph Nodes; Mannose-Binding Lectins; Mice; Mice, Inbred BALB C; Ovalbumin; Th2 Cells

A series of novel edible blend films of konjac glucomannan (KGM) and curdlan were prepared by a solvent-casting technique with different blending ratios of the two polymers. The Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), differential scanning calorimetry (DSC), scanning electron microscopy (SEM), etc. were used to characterize the change of structure and properties of blend films. The results showed that the strong intermolecular hydrogen bonds took place between KGM and curdlan. And the interaction of the blend film was much greater than that of the others when the KGM content in the blend films was around 70 wt% (KC7), resulting in excellent miscibility. The conclusion of the electron tensile testing analysis indicated that the blend film KC7 showed the maximum tensile strength (42.93±1.92 MPa). In addition, the blend films displayed excellent moisture barrier properties, which had a potential application in the food field.

Topics: beta-Glucans; Calorimetry, Differential Scanning; Food Packaging; Hydrogen Bonding; Mannans; Microscopy, Electron, Scanning; Permeability; Spectroscopy, Fourier Transform Infrared; Tensile Strength; Water; X-Ray Diffraction

Carboxymethyl curdlan (CMc), a β-D-glucan derivative, was used in the photoinduced synthesis of Ag nanoparticles. The size, size distribution, morphology and structure of the as-prepared Ag nanoparticles were investigated with UV-vis spectroscopy, transmission electron microscopy (TEM), energy-dispersive X-ray spectrometry (EDX), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectroscopy. The experimental results indicated that the particle size increased and the size distribution became broader with increasing the concentrations of both AgNO3 and CMc, and the effect of the latter was more pronounced. With the CMc concentration increasing, the diversity of morphology was obtained as a result of the plasmon excitation and the role of CMc. It was found that CMc played an important role in the synthesis and stabilization of Ag nanoparticles through a series of contrastive experiments. The enhancement effect of the produced Ag nanoparticles in surface enhanced Raman scattering (SERS) was also investigated.

Topics: beta-Glucans; Green Chemistry Technology; Metal Nanoparticles; Particle Size; Silver; Spectrum Analysis, Raman

A significant problem in scale-down cultures, rarely studied for metabolic characterization and curdlan-producing Agrobacterium sp. ATCC 31749, is the presence of dissolved oxygen (DO) gradients combined with pH control. Constant DO, between 5% and 75%, was maintained during batch fermentations by manipulating the agitation with PID system. Fermentation, metabolic and kinetic characterization studies were conducted in a scale-down system. The curdlan yield, intracellular nucleotide levels and glucose conversion efficiency into curdlan were significantly affected by DO concentrations. The optimum DO concentrations for curdlan production were 45-60%. The average curdlan yield, curdlan productivity and glucose conversion efficiency into curdlan were enhanced by 80%, 66% and 32%, respectively, compared to that at 15% DO. No apparent difference in the gel strength of the resulting curdlan was detected. The comparison of curdlan biosynthesis and cellular nucleotide levels showed that curdlan production had positive relationship with intracellular levels of UTP, ADP, AMP, NAD(+), NADH and UDP-glucose. The curdlan productivity under 45% DO and 60% DO was different during 20-50 h. However, after 60 h curdlan productivity of both conditions was similar. On that basis, a simple and reproducible two-stage DO control process for curdlan production was developed. Curdlan production yield reached 42.8 g/l, an increase of 30% compared to that of the single agitation speed control process.

Topics: Agrobacterium; beta-Glucans; Culture Media; Fermentation; Glucose; Hydrogen-Ion Concentration; Nucleotides; Oxygen; Time Factors

The microbial degradation of cellulose contributes greatly to the cycling of carbon in terrestrial environments and feedbacks to the atmosphere, a process that is highly responsive to nitrogen inputs. Yet how key groups of cellulolytic microorganisms adaptively respond to the global conditions of nitrogen limitation and/or anthropogenic or climate nitrogen inputs is poorly understood. The actinobacterial genus Cellulomonas is of special interest because it incorporates the only species known to degrade cellulose aerobically and anaerobically. Furthermore, despite their inability to fix nitrogen, they are active decomposers in nitrogen-limited environments. Here we show that nitrogen limitation induced biofilm formation in Cellulomonas spp., a process that was coupled to carbon sequestration and storage in a curdlan-type biofilm matrix. The response was reversible and the curdlan matrix was solubilized and used as a carbon and energy source for biofilm dispersal once nitrogen sources became available. The biofilms attached strongly to cellulosic surfaces and, despite the growth limitation, produced cellulases and degraded cellulose more efficiently. The results show that biofilm formation is a competitive strategy for carbon and nitrogen acquisition and provide valuable insights linking nitrogen inputs to carbon sequestration and remobilization in terrestrial environments.

Topics: beta-Glucans; Biodegradation, Environmental; Biofilms; Carbon; Cellulomonas; Cellulose; Climate; Nitrogen

Autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) syndrome is a complex immunologic disease caused by mutation of the autoimmune regulator (AIRE) gene. Autoimmunity in patients with APECED syndrome has been shown to result from deficiency of AIRE function in transcriptional regulation of thymic peripheral tissue antigens, which leads to defective T-cell negative selection. Candidal susceptibility in patients with APECED syndrome is thought to result from aberrant adaptive immunity.. To determine whether AIRE could function in anticandidal innate immune signaling, we investigated an extrathymic role for AIRE in the immune recognition of β-glucan through the Dectin-1 pathway, which is required for defense against Candida species.. Innate immune signaling through the Dectin-1 pathway was assessed in both PBMCs from patients with APECED syndrome and a monocytic cell line. Subcellular localization of AIRE was assessed by using confocal microscopy.. PBMCs from patients with APECED syndrome had reduced TNF-α responses after Dectin-1 ligation but in part used a Raf-1-mediated pathway to preserve function. In the THP-1 human monocytic cell line, reducing AIRE expression resulted in significantly decreased TNF-α release after Dectin-1 ligation. AIRE formed a transient complex with the known Dectin-1 pathway components phosphorylated spleen tyrosine kinase and caspase recruitment domain-containing protein 9 after receptor ligation and localized with Dectin-1 at the cell membrane.. AIRE can participate in the Dectin-1 signaling pathway, indicating a novel extrathymic role for AIRE and a defect that likely contributes to fungal susceptibility in patients with APECED syndrome.

Topics: AIRE Protein; beta-Glucans; CARD Signaling Adaptor Proteins; Cell Line; Humans; Immunity, Innate; Intracellular Signaling Peptides and Proteins; Lectins, C-Type; Leukocytes, Mononuclear; Microscopy, Confocal; Polyendocrinopathies, Autoimmune; Protein-Tyrosine Kinases; Syk Kinase; Transcription Factors; Transduction, Genetic; Tumor Necrosis Factor-alpha

A simultaneous engagement of different pathogen recognition receptors provides a tailor-made adaptive immunity for an efficient defense against distinct pathogens. For example, cross-talk of TLR and C-type lectin signaling effectively shapes distinct gene expression patterns by integrating the signals at the level of NF-κB. In this study, we extend this principle to a strong synergism between the dectin-1 agonist curdlan and an inflammatory growth factor, GM-CSF. Both together act in synergy in inducing a strong inflammatory signature that converts immature dendritic cells (DCs) to potent effector DCs. A variety of cytokines (IL-1β, IL-6, TNF-α, IL-2, and IL-12p70), costimulatory molecules (CD80, CD86, CD40, and CD70), chemokines (CXCL1, CXCL2, CXCL3, CCL12, CCL17), as well as receptors and molecules involved in fugal recognition and immunity such as Mincle, dectin-1, dectin-2, and pentraxin 3 are strongly upregulated in DC treated simultaneously with curdlan and GM-CSF. The synergistic effect of both stimuli resulted in strong IκBα phosphorylation, its rapid degradation, and enhanced nuclear translocation of all NF-κB subunits. We further identified MAPK ERK as one possible integration site of both signals, because its phosphorylation was clearly augmented when curdlan was coapplied with GM-CSF. Our data demonstrate that the immunomodulatory activity of curdlan requires an additional signal provided by GM-CSF to successfully initiate a robust β-glucan-specific cytokine and chemokine response. The integration of both signals clearly prime and tailor a more effective innate and adaptive response against invading microbes and fungi.

Topics: Animals; Antigens, CD; beta-Glucans; Cell Differentiation; Chemokines; Cytokines; Dendritic Cells; Drug Synergism; Granulocyte-Macrophage Colony-Stimulating Factor; I-kappa B Proteins; Immunologic Factors; Lectins, C-Type; Mice; Mice, Inbred C57BL; Mitogen-Activated Protein Kinases; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Polysaccharides, Bacterial; Signal Transduction

The ability to synthesize exopolysaccharides (EPS) is widespread among microorganisms, and microbial EPS play important roles in biofilm formation, pathogen persistence, and applications in the food and medical industries. Although it is well established that EPS synthesis is invariably in response to environmental cues, it remains largely unknown how various environmental signals trigger activation of the biochemical synthesis machinery.. We report here the transcriptome profiling of Agrobacterium sp. ATCC 31749, a microorganism that produces large amounts of a glucose polymer known as curdlan under nitrogen starvation. Transcriptome analysis revealed a nearly 100-fold upregulation of the curdlan synthesis operon upon transition to nitrogen starvation, thus establishing the prominent role that transcriptional regulation plays in the EPS synthesis. In addition to known mechanisms of EPS regulation such as activation by c-di-GMP, we identify novel mechanisms of regulation in ATCC 31749, including RpoN-independent NtrC regulation and intracellular pH regulation by acidocalcisomes. Furthermore, we show evidence that curdlan synthesis is also regulated by conserved cell stress responses, including polyphosphate accumulation and the stringent response. In fact, the stringent response signal, pppGpp, appears to be indispensible for transcriptional activation of curdlan biosynthesis.. This study identifies several mechanisms regulating the synthesis of curdlan, an EPS with numerous applications. These mechanisms are potential metabolic engineering targets for improving the industrial production of curdlan from Agrobacterium sp. ATCC 31749. Furthermore, many of the genes identified in this study are highly conserved across microbial genomes, and we propose that the molecular elements identified in this study may serve as universal regulators of microbial EPS synthesis.

Topics: Agrobacterium; Bacterial Proteins; beta-Glucans; Cyclic GMP; Gene Expression Profiling; Gene Expression Regulation, Bacterial; Genes, Bacterial; Guanosine Pentaphosphate; Metabolic Engineering; Nitrogen; Polyphosphates; Up-Regulation

The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17-dependent inflammatory arthritis developed after dectin 1-mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process.. SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti-IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies.. After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell- and IL-23-dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies.. Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.

Topics: Animals; Arthritis, Experimental; Arthritis, Rheumatoid; Autoantibodies; Autoimmune Diseases; Autoimmunity; beta-Glucans; Ileitis; Interleukin-17; Joints; Mice; Spondylarthritis; T-Lymphocytes

Recent studies have shown that the ligation of Toll-like receptor 3 (TLR3) or Dectin-1 on human monocyte-derived dendritic cells (MoDC) elicits their maturation, but with a different outcome on immunomodulation. Therefore the aim of this work was to study the response of MoDC to the combined effect of polyinosinic:polycytydilic acid [Poly (I:C)] and curdlan, selective TLR3 and Dectin-1 agonists, respectively.. Immature MoDC, generated from human monocytes, were treated with Poly (I:C), curdlan or their combination for 2 days. Phenotypic characteristics of MoDC were determined by flow cytometry, and cytokine production was measured by enzyme-linked immunosorbent assay (ELISA) and FlowCytomix, while the stimulatory capability of MoDC was tested using a mixed leukocyte reaction assay.. The combination of Poly (I:C) and curdlan induced phenotypic maturation of MoDC with the capability to stimulate an alloreactive response. Such treated MoDC up-regulated the production of interleukin (IL)-12, IL-23 and IL-10, compared with the effect of Poly (I:C) alone. Curdlan-treated MoDC stimulated the production of IL-17 by alloreactive CD4 (+) T cells more strongly than Poly (I:C)-treated MoDC. The opposite effect was observed for interferon(IFN)-γ production. When combined, these agonists primed MoDC to increase further the production of IFN-γ by CD4 (+) T cells in co-culture, especially those of naive (CD45RA (+)) phenotype, and IL-17 by memory (CD45RO (+)) CD4 (+) T cells.. Ligation of TLR3 and Dectin-1 receptor up-regulates T-helper (Th) 1 and Th17 immune responses compared with single agonists. These findings may have therapeutic implications for the use of MoDC in immunotherapy.

Topics: beta-Glucans; CD4-Positive T-Lymphocytes; Cytokines; Dendritic Cells; Drug Synergism; Humans; Immunity, Cellular; Lectins, C-Type; Monocytes; Poly I; Signal Transduction; Th1 Cells; Th17 Cells; Toll-Like Receptor 3

Curdlan is a microbial polysaccharide composed exclusively of β-(1,3)-linked glucose residues. Until now only bacteria belonging to the Alcaligenes and Agrobacterium species have been reported to produce Curdlan. In this study, a bacterium capable of producing extracellular Curdlan, identified as Pseudomonas sp. on the basis of 16S rDNA gene sequencing, was isolated from soil samples. From the HPLC, permethylation linkage analysis, (13)C NMR, and FT-IR analytical data, the polysaccharide consisted exclusively of glucose; the most prominent sugar was 1,3-linked glucose, and most glycosidic bonds joining these sugar residues were of the β-type. This also supported that the exopolysaccharide produced by Pseudomonas sp. was actually Curdlan. In addition, the Pseudomonas sp. was studied for the production of Curdlan by conventional "one-factor-at-a-time technique" and response surface methodology (RSM). It was observed that glucose and yeast extract were the most suitable carbon source and nitrogen source for Curdlan production, respectively. By using RSM, Curdlan production was increased significantly by 188%, from 1.25 to 2.35 g/L, when the strain was cultivated in the optimal condition developed by RSM, and the highest Curdlan production rate of 0.81 g/(L h) was obtained. To the best of the authors' knowledge, this is the first report on Curdlan production by Pseudomonas sp.

Topics: beta-Glucans; Extracellular Space; Molecular Sequence Data; Molecular Structure; Phylogeny; Pseudomonas; Soil Microbiology; Solubility

The mushroom known as Reishi (Ganoderma lucidum) has been used as an herbal medicine for tumor treatment and immune system activation. Because its effects on the differentiation of effector T helper cells have not yet been fully understood, we investigated the effects of Reishi and those of its principal ingredient, β-glucan, on the activation of dendritic cells and the differentiation of Th17 cells. Reishi extracts as well as purified β-glucan (Curdran) activated DCs and caused them to produce large amounts of IL-23. β-glucan also enhanced and sustained the transcription of IL-23p19. The MEK-ERK signaling pathway positively regulates IL-23p19 transcription in β-glucan-stimulated DCs. In a mixed leukocyte reaction, Reishi-stimulated DCs preferentially induced Th17 cells. Furthermore, orally-administrated Reishi increased the percentages of Th17 cells and the transcription levels of antimicrobial peptides. Our results show that Reishi and β-glucan activate DCs to produce large amounts of IL-23, which induces Th17 differentiation both in vitro and in vivo.

Topics: Animals; Antimicrobial Cationic Peptides; beta-Glucans; Dendritic Cells; Galactans; Interleukin-12; Interleukin-23; Interleukin-23 Subunit p19; Lymphocyte Activation; MAP Kinase Signaling System; Mice; Mice, Inbred C57BL; Reishi; Th17 Cells; Transcription, Genetic

In silkworm larvae, the mature form of paralytic peptide (PP), an insect cytokine, is produced from pro-PP in association with activation of innate immune responses, resulting in slow muscle contraction. We utilized this reaction, muscle contraction in silkworms coupled with innate immunity stimulation, to quantitatively measure the innate immune stimulating activity of various natural polysaccharides. β-Glucan of Gyrophora esculenta (GE-3), fucoidan from sporophyll of Undaria pinnatifida, and curldan induced silkworm muscle contraction. We further demonstrated that GE-3 had therapeutic effects on silkworms infected by baculovirus. Based on these findings, we propose that the silkworm muscle contraction assay is useful for screening substances that stimulate innate immunity before evaluating therapeutic effectiveness in mammals.

Topics: Animals; beta-Glucans; Bombyx; Drug Evaluation, Preclinical; Hemolymph; Immunity, Innate; Larva; Muscle Contraction; Neuropeptides; Nucleopolyhedroviruses; Polysaccharides

Silkworm β-1,3-glucan recognition protein (βGRP) tightly and specifically associates with β-1,3-glucan. We report here an affinity purification system named the 'GRP system', which uses the association between the β-1,3-glucan recognition domain of βGRP (GRP-tag), as an affinity tag, and curdlan beads. Curdlan is a water-insoluble β-1,3-glucan reagent, the low cost of which (about 100 JPY/g) allows the economical preparation of beads. Curdlan beads can be readily prepared by solubilization in an alkaline solution, followed by neutralization, sonication and centrifugation. We applied the GRP system to preparation of several proteins and revealed that the expression levels of the GRP-tagged proteins in soluble fractions were two or three times higher than those of the glutathione S-transferase (GST)-tagged proteins. The purity of the GRP-tagged proteins on the curdlan beads was comparable to that of the GST-tagged proteins on glutathione beads. The chemical stability of the GRP system was more robust than conventional affinity systems under various conditions, including low pH (4-6). Biochemical and structural analyses revealed that proteins produced using the GRP system were structurally and functionally active. Thus, the GRP system is suitable for both the large- and small-scale preparation of recombinant proteins for functional and structural analyses.

Topics: Amino Acid Sequence; Animals; Base Sequence; beta-Glucans; Bombyx; Carrier Proteins; Chromatography, Affinity; DEAD Box Protein 58; DEAD-box RNA Helicases; Escherichia coli; Glutathione Transferase; Humans; Insect Proteins; Molecular Sequence Data; Protein Stability; Receptors, Immunologic; Recombinant Fusion Proteins

Curdlan, a linear glucan interconnected by β-(1→3) linkages, is soluble in alkaline solutions but not in water, which limits its wide application, particularly in the food industry. In this study, curdlan was subjected to oxidative degradation using hydrogen peroxide (H(2)O(2)). The optimal hydrolysis conditions were determined, and the results were as follows: reaction time, 40 min; temperature, 60°C; H(2)O(2) concentration, 1.5% (v/v); and NaOH concentration, 2.5M. Under these optimised conditions, the maximum dextrose equivalent value (13.49%) was obtained. The composition and the structure of the hydrolysates were characterised by high-performance liquid chromatography and Fourier-transform infra-red spectroscopy, respectively. The hydrolysates were filtered, neutralised with HCl, concentrated to ∼12% (w/v), desalted, and freeze dried to yield a water-soluble, white powder. The (1→3)-β-d-glucan oligosaccharide content of the product was 98.6% and the yield was 91.4% (w/w).

Topics: beta-Glucans; Hot Temperature; Hydrogen Peroxide; Hydrolysis; Kinetics; Molecular Structure

No one single pathogen has been identified as the causative agent of multiple sclerosis (MS). Alternately, the likelihood of an autoimmune event may be nonspecifically enhanced by different infectious agents. In a novel animal model of MS, SJL/J mice primed through infection with a recombinant vaccinia virus (VV) encoding myelin proteolipid protein (PLP) (VV(PLP)) were susceptible to a central nervous system (CNS) inflammatory disease following administration of a nonspecific immunostimulant [complete Freund's adjuvant (CFA) plus Bordetella pertussis (BP)]. Mononuclear cells isolated from the brains, but not the spleens, of VV(PLP)-primed CFA/BP challenged mice produced interleukin (IL)-17 and interferon-γ and transferred a CNS inflammatory disease to naïve SJL/J mice. Administration of curdlan, a T helper 17 cell inducer, unexpectedly resulted in less severe clinical and histological signs of disease, compared to CFA/BP challenged mice, despite the induction of IL-17 in the periphery. Further examination of the VV(PLP)-prime CFA/BP challenge model may suggest new mechanisms for how different pathogens associated with MS can protect or enhance disease.

Topics: Adjuvants, Immunologic; Animals; beta-Glucans; Brain; Disease Models, Animal; Female; Genetic Vectors; Immunomodulation; Interferon-gamma; Interleukin-17; Leukocytes, Mononuclear; Mice; Multiple Sclerosis; Myelin Proteolipid Protein; Organ Specificity; Polysaccharides, Bacterial; Rats; Recombinant Proteins; Severity of Illness Index; Spleen; Vaccinia virus

T helper 1 and T helper 17 cells play important roles in immunity against foreign invaders. Differentiation of these Th subsets is affected by state of maturation and cytokines that are produced by dendritic cells (DCs). Curdlan is a linear (1→3)-β-glucan and has shown activity against tumors and infectious agents.. This study aims to investigate whether curdlan plays its role through affecting the maturation and cytokine production by DCs.. DCs were isolated from the spleen of BALB/c mice by MACS method. After an overnight culture of DCs in the presence of curdlan, the expression levels of CD40, CD86, and MHC-II molecules were determined by flow cytometry. The production of cytokines involved in Th1 and Th17 cell differentiation (IL-12 and IL-6, respectively) was also evaluated by ELISA. Lipopolysaccharide (LPS) treated and untreated cells were considered as positive and negative controls, respectively.. The results of this study did not show a significant difference in the levels of surface expression of CD40 (p=0.82), CD86 (p=0.79), and MHC class II (p=0.84) molecules upon exposure to curdlan. However, LPS increased the intensity of CD40 expression on dendritic cells (p=0.04). In addition, it was revealed that curdlan-exposed DCs are not able to produce a significant amount of IL-6 and IL-12 cytokines. Conversely, LPS-treated DCs were able to make a significant amount of IL-12 (p=0.005).. The results of the present study suggest that curdlan has no effect on Th1 or Th17 differentiation while LPS may induce Th1 deviation by induction of CD40 expression and IL-12 production.

Topics: Animals; B7-2 Antigen; beta-Glucans; CD40 Antigens; Cell Differentiation; Cells, Cultured; Dendritic Cells; Female; Histocompatibility Antigens Class II; Immunity, Cellular; Immunologic Factors; Immunomodulation; Interleukin-12; Interleukin-6; Mice; Mice, Inbred BALB C; Spleen

A number of studies have suggested a correlation between a decreased incidence in infectious diseases and an increased incidence of allergic diseases, including asthma. Although several pathogen-derived products have been shown to possess therapeutic potential for allergic diseases, it remains largely unknown whether β-glucan, a cell wall component of a variety of fungi, yeasts, and bacteria, has a regulatory potential for allergic diseases. In this study, we examined the effect of curdlan, a linear β-(1-3)-glucan, on the development of allergic airway inflammation. We found that i.p. injection of curdlan significantly inhibited Ag-induced eosinophil recruitment and Th2 cytokine production in the airways. The activation of CD4(+) T cells in the presence of curdlan induced IL-10-producing CD4(+) T cells with high levels of c-Maf expression. Curdlan-induced development of IL-10-producing CD4(+) T cells required the presence of APCs and ICOS/ICOS ligand interaction. Curdlan-induced development of IL-10-producing CD4(+) T cells also required intrinsic expression of STAT6. Furthermore, the transfer of Ag-specific CD4(+) T cells that were stimulated in the presence of curdlan inhibited Ag-induced eosinophil recruitment into the airways. Taken together, these results suggest that curdlan is capable of inducing IL-10-producing CD4(+) T cells and inhibiting the development of eosinohilic airway inflammation, underscoring the therapeutic potential of curdlan for allergic diseases.

Topics: Animals; beta-Glucans; Bronchial Hyperreactivity; CD4-Positive T-Lymphocytes; Cells, Cultured; Eosinophilia; Inflammation Mediators; Injections, Intraperitoneal; Interleukin-10; Mice; Mice, Inbred BALB C; Mice, Transgenic; Respiratory Hypersensitivity

Agrobacterium sp. ATCC 31749 (formerly named Alcaligenes faecalis var. myxogenes) is a non-pathogenic aerobic soil bacterium used in large scale biotechnological production of curdlan. However, little is known about its genomic information. DNA partial sequence of electron transport chains (ETCs) protein genes were obtained in order to understand the components of ETC and genomic-specificity in Agrobacterium sp. ATCC 31749. Degenerate primers were designed according to ETC conserved sequences in other reported species. DNA partial sequences of ETC genes in Agrobacterium sp. ATCC 31749 were cloned by the PCR method using degenerate primers. Based on comparative genomic analysis, nine electron transport elements were ascertained, including NADH ubiquinone oxidoreductase, succinate dehydrogenase complex II, complex III, cytochrome c, ubiquinone biosynthesis protein ubiB, cytochrome d terminal oxidase, cytochrome bo terminal oxidase, cytochrome cbb (3)-type terminal oxidase and cytochrome caa (3)-type terminal oxidase. Similarity and phylogenetic analyses of these genes revealed that among fully sequenced Agrobacterium species, Agrobacterium sp. ATCC 31749 is closest to Agrobacterium tumefaciens C58. Based on these results a comprehensive ETC model for Agrobacterium sp. ATCC 31749 is proposed.

Topics: beta-Glucans; Cytochrome d Group; Electron Transport; Electron Transport Complex IV; Genes, Bacterial; Genome, Bacterial; Genomics; Oxidoreductases; Phylogeny; Rhizobium; Succinate Dehydrogenase

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

An in situ hybrid complex of Curdlan with water-soluble polythiophene functioned as a highly sensitive and selective saccharide chemosensor in aqueous media, enabling us to discriminate tetrasaccharide acarbose at 1 μM from 24 mono-, di-, tri-, tetra-, and pentasaccharides.

Topics: beta-Glucans; Chemistry Techniques, Analytical; Oligosaccharides; Polymers; Solubility; Thiophenes; Water

Cellulomonas flavigena strain KU (ATCC 53703) is a cellulolytic, Gram-positive bacterium which produces large quantities of an insoluble exopolysaccharide (EPS) when grown in minimal media with a high carbon-to-nitrogen (C/N) ratio. Earlier studies proved the EPS is structurally identical to the linear β-1,3-glucan known as curdlan and provided evidence that the EPS functions as a carbon and energy reserve compound. We now report that C. flavigena KU also accumulates two intracellular, glucose-storage carbohydrates under conditions of carbon and energy excess. These carbohydrates were partially purified and identified as the disaccharide trehalose and a glycogen/amylopectin-type polysaccharide. A novel method is described for the sequential fractionation and quantitative determination of all three carbohydrates from culture samples. This fractionation protocol was used to examine the effects of C/N ratio and osmolarity on the accumulation of cellular carbohydrates in batch culture. Increasing the C/N of the growth medium caused a significant accumulation of curdlan and glycogen but had a relatively minor effect on accumulation of trehalose. In contrast, trehalose levels increased in response to increasing osmolarity, while curdlan levels declined and glycogen levels were generally unaffected. During starvation for an exogenous source of carbon and energy, only curdlan and glycogen showed substantial degradation within the first 24 h. These results support the conclusion that extracellular curdlan and intracellular glycogen can both serve as short-term reserve compounds for C. flavigena KU and that trehalose appears to accumulate as a compatible solute in response to osmotic stress.

Topics: beta-Glucans; Cellulomonas; Chromatography, Thin Layer; Gene Expression Regulation, Bacterial; Glycogen; Trehalose

We have examined the potential to generate bona fide macrophages (MØ) from conditionally immortalised murine bone marrow precursors. MØ can be derived from Hoxb8 conditionally immortalised macrophage precursor cell lines (MØP) using either M-CSF or GM-CSF. When differentiated in GM-CSF (GM-MØP) the resultant cells resemble GM-CSF bone marrow-derived dendritic cells (BMDC) in morphological phenotype, antigen phenotype and functional responses to microbial stimuli. In spite of this high similarity between the two cell types and the ability of GM-MØP to effectively present antigen to a T-cell hybridoma, these cells are comparatively poor at priming the expansion of IFN-γ responses from naïve CD4(+) T cells. The generation of MØP from transgenic or genetically aberrant mice provides an excellent opportunity to study the inflammatory role of GM-MØP, and reduces the need for mouse colonies in many studies. Hence differentiation of conditionally immortalised MØPs in GM-CSF represents a unique in vitro model of inflammatory monocyte-like cells, with important differences from bone marrow-derived dendritic cells, which will facilitate functional studies relating to the many 'sub-phenotypes' of inflammatory monocytes.

Topics: Animals; Antigen Presentation; Antigens, Surface; beta-Glucans; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Transformed; Cytokines; Dendritic Cells; Granulocyte-Macrophage Colony-Stimulating Factor; Homeodomain Proteins; Interferon-gamma; Interleukin-2; Lectins, C-Type; Lipopeptides; Lipopolysaccharides; Macrophage Colony-Stimulating Factor; Macrophages; Membrane Proteins; Mice; Mice, 129 Strain; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Knockout; Monocyte-Macrophage Precursor Cells; Nerve Tissue Proteins; Nitric Oxide; Ovalbumin; Transduction, Genetic; Zymosan

The inflammatory potential and molecular mechanisms underscoring inflammatory responses of lung cells to compounds from fungi that grow on damp building materials is poorly understood in vitro. In this study we evaluated the effect of pure fungal compounds on potentiating acute inflammatory response in primary mouse alveolar macrophages (AMs) and tested the hypothesis that AM responses to low molecular weight fungal compounds exhibit temporal and compound specificity that mimic that observed in the whole lung. Transcriptional responses of 13 inflammation/respiratory burst-associated genes (KC=Cxcl1, Cxcl2, Cxcl5, Cxcl10, Ccl3, Ccl112, Ccl20, IL-1β, Il-6, ifi27 Tnfα, iNOS and Blvrb) were evaluated in mouse AMs exposed to a 1ml (10(-8)mol) dose of either pure atranone C, brevianimide, cladosporin, curdlan, LPS, neoechinulin A & B, sterigmatocystin or TMC-120A for 2h, 4h and 12h PE using customized reverse transcription (RT)-PCR based arrays. Multianalyte ELISA was used to measure expression of 6 pro-inflammatory cytokines common to the transcriptional assays (Cxcl1, Cxcl10, Ccl3, IL1β, Ifn-λ and Tnf-α) to determine whether gene expression corresponded to the transcription data. Compared to controls, all of these compounds induced significant (≥2.5-fold or ≤-2.5-fold change at p≤0.05) time- and compound-specific transcriptional gene alterations in treatment AMs. The highest number of transcribed genes were in LPS treatment AMs at 12h PE (12/13) followed by neoechinulin B at 4h PE (11/13). Highest fold change values (>30) were associated with KC, Cxcl2, Cxcl5 and IL1β genes in cells exposed to LPS. Compound exposures also induced significant (p≤0.05) time- and compound-specific pro-inflammatory responses manifest as differentially elevated Cxcl1, Cxcl10, Ccl3, Ifn-λ and Tnf-α concentrations in culture supernatant of treatment AMs. Dissimilarity in transcriptional responses in AMs and our in vivo model of lung disease is likely attributable to whole lung vs. isolated cell responsive and dose differences between the two studies. The results not only indicate that low molecular weight compounds from fungi that grow in damp built environments are potently pro-inflammatory in vitro, it further highlights the important role AMs play in innate lung defence, and against exposure to low molecular weight fungal compounds. These observations further support our position that exposure to low molecular weight compounds from indoor-associated fungi may provoke some of the

Topics: Alkaloids; Animals; Benzofurans; beta-Glucans; Cells, Cultured; Cluster Analysis; Cytokines; Fungi; Gene Expression Regulation; Indole Alkaloids; Inflammation Mediators; Isocoumarins; Isoquinolines; Lipopolysaccharides; Macrophages, Alveolar; Male; Mice; Molecular Weight; Piperazines; Spiro Compounds; Sterigmatocystin; Transcription, Genetic

Curdlan, a bacterial polysaccharide, can form different types of thermogels, having the very same chemical composition, but whose structures depend on the incubation temperature. Structural characterization of 10% (w/v) low-set and high-set curdlan gels was carried out by Fourier transformed infrared (FT-IR) imaging and environmental scanning electron microscopy (eSEM) in the hydrated state. Considerable differences were observed between the two gels, the high-set one being overall more homogeneous. The self-diffusion coefficients of a series of analytes of different sizes (water, phosphate, glucose-6-phosphate, polyphosphate, polyethylene glycol, and dextran labelled with rhodamine B) were measured in aqueous solution (D(s)(sln)) and in both types of curdlan gels (D(s)(gel)) using (1)H and (31)P pulsed field gradient nuclear magnetic resonance (PFG NMR) spectroscopy. The mutual-diffusion coefficients (D(m)(gel)) of dextran in the curdlan gels were determined from release experiments based on fluorescence spectroscopy. The dependence of the relative diffusion coefficient (D(s)(gel)D(s)(sln)) on the size of the analyte, expressed by its hydrodynamic radius (R(h)), could be expressed by D(s)(gel)D(s)(sln) ∝ exp(-R(h)(0.46)), valid for both types of gels. The self-diffusion measurements for the largest investigated analytes were not compatible with a single diffusion coefficient and, therefore, were analysed using an approach based on a normal distribution of self-diffusion coefficients. In the hydrogels, broadening of the self-diffusion coefficient distribution increased as a function of the analyte size. This phenomenon was associated with the limited distance travelled by the analytes during the measurements, and it is inferred that the distribution of diffusion coefficients is representative of the distribution of local environments of the individual analyte. It was found that the structural differences observed between both types of curdlan gels are not correlated with the gel transport properties, highlighting the complexity of the relationship between structural details and transport properties in gels.

Topics: beta-Glucans; Hydrogels; Magnetic Resonance Spectroscopy; Microscopy, Electron, Scanning; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spectroscopy, Fourier Transform Infrared

Activation of pattern recognition receptors (PRR) may contribute to arthritis. Here, we elucidated the role of NOD2, a genetic cause of inflammatory arthritis, and several other PRR in a murine model of inflammatory arthritis.. The roles of CR3, TLR2, MyD88, NOD1, NOD2, Dectin-1 and Dectin-2 were tested in vivo in arthritis elicited by intra-articular injections of zymosan, the fungal cell wall components curdlan, laminarin and mannan, and the bacterial cell wall peptidoglycan.. Dectin-1, and to a lesser extent Dectin-2, contributed to arthritis. TLR2, MyD88 and CR3 played non-essential roles. Observations based on injection of curdlan, laminarin or mannan supported the dominant role of the Dectin-1 pathway in the joint. We demonstrated differential roles for NOD1 and NOD2 and identified NOD2 as a novel and essential mediator of zymosan-induced arthritis.. Together, Dectin-1 and NOD2 are critical, sentinel receptors in the arthritogenic effects of zymosan. Our data identify a novel role for NOD2 during inflammatory responses within joints.

Topics: Animals; Arthritis, Experimental; beta-Glucans; Cathepsins; Disease Models, Animal; Immunity, Innate; Joints; Lectins, C-Type; Membrane Proteins; Mice; Mice, Knockout; Nerve Tissue Proteins; Nod2 Signaling Adaptor Protein; Signal Transduction; Toll-Like Receptors; Zymosan

Expression at the mRNA level of ten selected genes in Agrobacterium sp. ATCC 31749 under various dissolved oxygen (DO) levels during curdlan fermentation related to electron transfer chain (ETC), tricarboxylic acid (TCA) cycle, peptidoglycan/lipopolysaccharide biosynthesis, and uridine diphosphate (UDP)-glucose biosynthesis were determined by qRT-PCR. Experiments were performed at DO levels of 30%, 50%, and 75%, as well as under low-oxygen conditions. The effect of high cell density on transcriptional response of the above genes under low oxygen was also studied. Besides cytochrome d (cyd A), the transcription levels of all the other genes were increased at higher DO and reached maximum at 50% DO. Under 75% DO, the transcriptional levels of all the genes were repressed. In addition, transcription levels of icd, sdh, cyo A, and fix N genes did not exhibit significant fluctuation with high cell density culture under low oxygen. These results suggested a mechanism for DO regulation of curdlan synthesis through regulation of transcriptional levels of ETCs, TCA, and UDP-glucose synthesis genes during curdlan fermentation. To our knowledge, this is the first report that DO concentration apparently regulates curdlan biosynthesis in Agrobacterium sp. ATCC 31749 providing essential lead for the optimization of the fermentation at the industrial scale.

Topics: Bacterial Proteins; beta-Glucans; Gene Expression Regulation, Bacterial; Molecular Sequence Data; Oxygen; Rhizobium; Transcription, Genetic

N-Acetyl-d-glucosamine branches were incorporated at the C-6 position of curdlan, a linear β-1,3-d-glucan, and the resulting nonnatural branched polysaccharides were evaluated in terms of the immunomodulation activities in comparison with lentinan, a β-1,3-d-glucan having d-glucose branches at C-6. To incorporate the amino sugar branches, we conducted a series of regioselective protection-deprotections of curdlan involving triphenylmethylation at C-6, phenylcarbamoylation at C-2 and C-4, and detriphenylmethylation. Subsequent glycosylation with a d-glucosamine-derived oxazoline, followed by deprotection gave rise to the branched curdlans with various substitution degrees. The products exhibited remarkable solubility in both organic solvents and water. Their immunomodulation activities were determined using mouse macrophagelike cells, and the secretions of both the tumor necrosis factor and nitric oxide proved to be significantly higher than those with lentinan. These results conclude that the amino sugar/curdlan hybrid materials are promising as a new type of polysaccharide immunoadjuvants useful for cancer chemotherapy.

Topics: Acetylglucosamine; Adjuvants, Immunologic; Animals; beta-Glucans; Biomimetic Materials; Carbohydrate Sequence; Cell Line; Glucose; Glycosylation; Immunologic Factors; Lentinan; Macrophage Activation; Macrophages; Magnetic Resonance Spectroscopy; Methylation; Mice; Molecular Mimicry; Molecular Sequence Data; Nitric Oxide; Solubility; Tumor Necrosis Factor-alpha

The regulatory function of global regulator NtrC on curdlan biosynthesis and nitrogen consumption under nitrogen-limited condition in Agrobacterium sp. ATCC 31749 was investigated. The ntrC mutant of Agrobacterium sp. was constructed by homologous recombination. The ability to utilize NH4Cl and KNO3 was impaired in the mutant. Other nitrogenous compounds, such as glutamic acid and glutamine, were utilized normally. Curdlan production capability was impaired severely in the mutant. Curdlan production was 5-fold lower than the wild type strain in batch fermentation with NH4Cl as the sole nitrogen source. However, up to 6.5 g l(-1) of a newly found alkali-insoluble biopolymer was produced by the ntrC mutant when glutamic acid was used as nitrogen source. The new biopolymer had glycosidic bond and hydroxyl group but no β-configuration absorption peak on IR spectrum was found as different from curdlan. In addition, the mutant exhibited a rapid morphological change from the dot to rod form. These results deduced that the global regulator NtrC was involved in curdlan and other biopolymer biosynthesis in Agrobacterium sp. ATCC 31749 in response to nitrogen-limited condition.

Topics: Bacterial Proteins; beta-Glucans; Mutation; Nitrogen; Nitrogen Compounds; Rhizobium

A thermo- and light-responsive system consisting of single-walled carbon nanotube and helical polysaccharide modified with poly(N-isopropylacrylamide) side-chains has been developed through supramolecular polymer wrapping. Coagulation of the complex can be induced by the external stimuli, which leads to a catch-and-release action of a porphyrin derivative.

Topics: Acrylamides; Acrylic Resins; beta-Glucans; Hot Temperature; Light; Nanotubes, Carbon; Photochemotherapy; Polymers; Porphyrins

Agrobacterium sp. ATCC 31749 is an industrial strain for the commercial production of curdlan, an important exopolysaccharide with food and medical applications. Here we report the genome sequence of the curdlan-producing strain ATCC 31749. Genome sequencing is the first step toward the understanding of regulation of curdlan biosynthesis.

Topics: beta-Glucans; Gene Expression Regulation, Bacterial; Genome, Bacterial; Molecular Sequence Data; Rhizobium

Laminaripentaose-producing β-1,3-glucanase (LPHase) from Streptomyces matensis DIC-108 uniquely catalyzes the hydrolysis of β-1,3-glucan to release laminaripentaose as the predominant product. For studying this novel enzyme, the gene of LPHase was reconstructed with polymerase chain reaction and over-expressed in Escherichia coli. The recombinant wild-type enzyme and various mutants were further purified to >90% homogeneity on an ion-exchange chromatograph. The catalysis of the recombinant LPHase is confirmed to follow a one-step single-displacement mechanism with (1)H-NMR spectrometry. To determine the amino-acid residues essential for the catalysis, more than ten residues, including five highly conserved residues--Asp(143), Glu(154), Asp(170), Asp(376) and Asp(377), were mutated. Among the mutants, E154Q, E154G, D174N and D174G significantly lost catalytic activity. Further investigation with chemical rescue using sodium azide on E154G and D174G confirmed that Glu(154) functions as the general acid whereas Asp(170) serves as the general base in a catalytic turnover. This work is the first report that provides direct information for the identification of the essential residues of GH-64 through kinetic examination.

Topics: Amino Acid Sequence; Bacterial Proteins; beta-Glucans; Chromatography, Ion Exchange; Circular Dichroism; Escherichia coli; Glucan 1,3-beta-Glucosidase; Kinetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Nuclear Magnetic Resonance, Biomolecular; Oligosaccharides; Recombinant Proteins; Sequence Alignment; Sodium Azide; Streptomyces

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

Xanthan-curdlan hydrogel complex (XCHC) has been shown capable of retaining moisture up to 5 freeze-thaw cycles (FTCs); however, moisture distribution in the complex in relation to the hydrogel composition and structure remains uncharacterized. In the present study, magnetic resonance imaging (MRI), nuclear magnetic resonance (NMR) relaxometry, rheology, and scanning electron microscopy (SEM) were used to examine the effect of water distribution and interaction with 2.0% aqueous solutions of xanthan, curdlan, and XCHC consisting of equal amounts of both polysaccharides. A gel structure with an indication of syneresis was clearly seen in the MR image of curdlan alone, whereas the distribution of protons throughout xanthan and XCHC samples remained homogeneous and showed no detectable syneresis. The three-dimensional network, indicated by frequency sweeps, of curdlan was responsible for curdlan's gel structure. The frequency sweep and slope of the storage modulus (G') of XCHC was significantly closer to curdlan with higher elasticity and less dependency upon angular frequency than xanthan alone. The reduction in XCHC dynamic moduli (G' and G″) compared to curdlan could be attributed to the formation of wavy layers instead of a fully cured three-dimensional structure. Addition of xanthan to curdlan restricted XCHC spin-spin relaxation time (T₂) to intermediate and slower exchange regimes, namely approximately 110 and 342 ms, respectively, promoting the polymer's interaction with water while inhibiting interpolymer interactions found in curdlan. A 3rd proton pool with the slowest T₂ seen in curdlan was not found in XCHC, correlating to the absence of syneresis.. The combination of texture measurements and discrete noninvasive techniques was found capable of providing insightful understanding of water distribution in a gel system. These techniques may be applied to other hydrogel complexes. The XCHC system investigated has the potential to enhance freeze-thaw stability in frozen food products by minimizing syneresis due to undesirable temperature fluctuations during distribution and consumer application.

Topics: beta-Glucans; Carbohydrate Conformation; Chemical Phenomena; Elastic Modulus; Emulsifying Agents; Food Storage; Food, Formulated; Freeze Drying; Frozen Foods; Hydrogels; Imaging, Three-Dimensional; Magnetic Resonance Imaging; Magnetic Resonance Spectroscopy; Mechanical Phenomena; Microscopy, Electron, Scanning; Polysaccharides, Bacterial; Reproducibility of Results; Rheology; Surface Properties; Water

Complexes of curdlan and genistein were prepared using four different methods. The total amount of genistein in curdlan-genistein complexes prepared at 40 degrees C (system I) was significantly higher (2.3 mg/100 mg dry matter) than that in other systems studied: curdlan-genistein complexes prepared at 60 degrees C (system II; 1.8 mg/100 mg dry matter); curdlan-genistein gel complexes (system III; 1.0 mg/100 mg dry matter); and curdlan-genistein dimethyl sulfoxide complexes (system IV; 1.8 mg/100 mg dry matter). x-ray diffraction results indicate that complexation of curdlan with genistein changes the crystalline nature of the pure components. Particle size analysis, atomic force microscopy and scanning electron microscopy imaging of curdlan-genistein complexes showed strong difference in particle size, surface and distribution in comparison with pure curdlan, confirming our assumption of a molecular interaction between curdlan and genistein and the formation of a new structure, which was revealed at the nanoscale level. All the curdlan-genistein complexes showed prolonged genistein release of up to 72 h, enhanced upon enzymatic digestion of curdlan by lyticase, thus opening the possibility of release regulation by the incorporation of lyticase in the delivery system. It is therefore suggested that the complexes could be used as a delivery system for the protection and slow release of genistein in the digestive tract.

Topics: beta-Glucans; Drug Delivery Systems; Food; Genistein; Microscopy, Atomic Force; Particle Size; X-Ray Diffraction

The form of (1-3)-beta-D glucan found in the cell walls of the anamorphic Trichocomaceae that grow on damp building materials is considered to have potent toxic and inflammatory effects on cells of the respiratory system. It is also considered to have a potential role in the development of non-allergenic respiratory health effects. While human studies involving experimental exposures all point to the inflammatory potential of pure curdlan, a linear (1-3)-beta-D glucan in a triple helix configuration, animal experiments result in conflicting conclusions concerning the inflammatory potency of this glucan. However, because mice appear to be a better model than guinea pigs for exploring the respiratory effects of curdlan and because molecular mechanisms associated with this glucan remain largely unknown, we conducted further work to clarify the role of curdlan on the inflammatory response using our mouse model of lung disease. This study used in situ hybridization (ISH) to probe dectin-1 mRNA transcription with a digoxigenin-labeled cDNA probe, with reverse transcription (RT)-PCR based arrays used to measure inflammation gene and receptor transcriptional responses. Also, immunohistochemistry (IHC) was used to probe dectin-1 as well as anti-mouse Ccl3, Il1-alpha, and TNF-alpha expression to evaluate dose and time-course (4 and 12 h) postexposure (PE) response patterns in the lungs of intratracheally instilled mice exposed to a single 50 mul dose of curdlan at 10(-7), 10(-8), 10(-9), and 10(-10) M/animal (=4 mug to 4 ng curdlan/kg lung wt). Dectin-1 mRNA transcription and expression was observed in bronchiolar epithelium, alveolar macrophages (AMs), and alveolar type II cells (ATIIs) of lungs exposed to 4 mug to 40 ng curdlan/kg lung wt, at both time points. Compared to controls, array analysis revealed that 54 of 83 genes assayed were significantly modulated by curdlan. mRNA transcription patterns showed both dose and time dependency, with highest transcription levels in 10(-7) and 10(-8) M treatment animals, especially at 4-h PE. Nine gene mRNA transcripts (Ccl3, Ccl11, Ccl17, Ifng, Il1alpha, Il-20, TNF-alpha, Tnfrsf1b, and CD40lg) were significantly expressed at all doses suggesting they may have a central role in immunomodulating curdlan exposures. IHC revealed Ccl3, Il1-alpha, and TNF-alpha expression in bronchiolar epithelium, AMs and ATIIs illustrate the important immunomodulatory role that these cells have in the recognition of, and response to glucan.

Topics: Animals; beta-Glucans; Chemokines; In Situ Hybridization; Intubation, Intratracheal; Lectins, C-Type; Lung; Male; Membrane Proteins; Mice; Nerve Tissue Proteins; Polysaccharides, Bacterial; RNA, Messenger; Specific Pathogen-Free Organisms; Transcription, Genetic

This study carried out a novel sulfation process on curdlan. Results document that sulfation of the polysaccharide was more effective with the assistance of ultrasonication. Ultrasonication significantly increased the DS value of samples by almost fourfold (from about 0.030 to 0.115). Evaluation of the extent of sulfation has been made using FTIR, (1)H NMR, (13)C NMR and SEC. Both FTIR and (1)H NMR spectra confirmed the occurrence of sulfation. The combined effect of ultrasonication and acid resulted in relatively small molecular weights (e.g., sample 360_360: MW approximately 49.0 kDa), as opposed to curdlan (MW approximately 807 kDa). This work contributes to better understanding of the molecular mechanism governing the sonochemical processes of polysaccharide modification with improved physicochemical properties, and potential biomedical application.

Topics: beta-Glucans; Chromatography, Gel; Magnetic Resonance Spectroscopy; Molecular Weight; Sonication; Spectroscopy, Fourier Transform Infrared; Sulfur

Th17-inducing activity is carried by certain polysaccharides such as beta-glucan derived from Candia albicans. Our previous studies have shown that Th1- and Th2-inducing activities can be qualitatively evaluated by the expression patterns of Notch ligand isoforms, using human monocyte-derived dendritic cells (Mo-DCs) and some leukemic cell lines such as THP-1. The association of Th17-inducing activities with Notch ligand expression patterns has been unclear.. Mo-DCs from healthy volunteers were co-cultured with HLA-DR-nonshared allogeneic CD4+ naïve T cells to induce a mixed lymphocyte reaction, in the presence of adjuvants, such as curdlan. Culture supernatants were assayed for IFNgamma, IL-5 and IL-17 by an enzyme-linked immunosorbent assay (ELISA). Notch ligand expression on Mo-DCs and THP-1 cells was evaluated by using RT-PCR.. The present study shows that curdlan, one of the beta-glucans, has the ability to induce DC-mediated Th17 differentiation. It is also interesting to note that Jagged1 mRNA in Mo-DCs and THP-1 cells is up-regulated by curdlan. Furthermore, polyclonal anti-Jagged1 antibody inhibited such DC-mediated Th17 differentiation.. This study suggests that curdlan induces human DC-mediated Th17 polarization via Jagged1 activation in DCs.

Topics: Adjuvants, Immunologic; Antibodies, Blocking; beta-Glucans; Calcium-Binding Proteins; Candida albicans; CD4-Positive T-Lymphocytes; Cell Differentiation; Cell Line, Tumor; Dendritic Cells; Enzyme-Linked Immunosorbent Assay; HLA-DR Antigens; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-17; Jagged-1 Protein; Lymphocyte Culture Test, Mixed; Membrane Proteins; Monocytes; Polysaccharides, Bacterial; Serrate-Jagged Proteins; Up-Regulation

DC NK lectin group receptor-1 (DNGR-1, also known as CLEC9A) is a C-type lectin receptor expressed by mouse CD8alpha+ DC and by their putative equivalents in human. DNGR-1 senses necrosis and regulates CD8+ T-cell cross-priming to dead-cell-associated antigens. In addition, DNGR-1 is a target for selective in vivo delivery of antigens to DC and the induction of CD8+ T-cell and Ab responses. In this study, we evaluated whether DNGR-1 targeting can be additionally used to manipulate antigen-specific CD8+ T lymphocytes. Injection of small amounts of antigen-coupled anti-DNGR-1 mAb into mice promoted MHC class II antigen presentation selectively by CD8alpha+ DC. In the steady state, this was sufficient to induce proliferation of antigen-specific naïve CD4+ T cells and to drive their differentiation into Foxp3+ regulatory lymphocytes. Co-administration of adjuvants prevented this induction of tolerance and promoted immunity. Notably, distinct adjuvants allowed qualitative modulation of CD4+ T-cell behavior: poly I:C induced a strong IL-12-independent Th1 response, whereas curdlan led to the priming of Th17 cells. Thus, antigen targeting to DNGR-1 is a versatile approach for inducing functionally distinct CD4+ T-cell responses. Given the restricted pattern of expression of DNGR-1 across species, this strategy could prove useful for developing immunotherapy protocols in humans.

Topics: Adjuvants, Immunologic; Animals; Antibodies, Monoclonal; Antigen Presentation; beta-Glucans; CD4-Positive T-Lymphocytes; CD8 Antigens; CD8-Positive T-Lymphocytes; Dendritic Cells; Epitopes; Forkhead Transcription Factors; Immune Tolerance; Lectins, C-Type; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Ovalbumin; Peptide Fragments; Poly I-C; Receptors, Immunologic; Specific Pathogen-Free Organisms; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Th1 Cells

Patients taking immunosuppressive drugs, like cyclosporine A (CsA), that inhibit calcineurin are highly susceptible to disseminated fungal infections, although it is unclear how these drugs suppress resistance to these opportunistic pathogens. We show that in a mouse model of disseminated Candida albicans infection, CsA-induced susceptibility to fungal infection maps to the innate immune system. To further define the cell types targeted by CsA, we generated mice with a conditional deletion of calcineurin B (CnB) in neutrophils. These mice displayed markedly decreased resistance to infection with C. albicans, and both CnB-deficient and CsA-treated neutrophils showed a defect in the ex vivo killing of C. albicans. In response to the fungal-derived pathogen-associated molecular pattern zymosan, neutrophils lacking CnB displayed impaired up-regulation of genes (IL-10, Cox2, Egr1, and Egr2) regulated by nuclear factor of activated T cells, the best characterized CnB substrate. This activity was Myd88 independent and was reproduced by stimulation with the beta(1,3) glucan curdlan, indicating that dectin-1, rather than toll-like receptors, is the upstream activator of calcineurin. Our results suggest that disseminated fungal infections seen in CsA-treated patients are not just a general consequence of systemic suppression of adaptive immunity but are, rather, a result of the specific blockade of evolutionarily conserved innate pathways for fungal resistance.

Topics: Animals; Antifungal Agents; beta-Glucans; Calcineurin; Candida albicans; Candidiasis; Cyclosporine; Disease Susceptibility; Homeostasis; Humans; Immunity, Innate; Immunosuppressive Agents; Mice; Mycoses; Neutrophils; Polysaccharides, Bacterial; Zymosan

Curdlan, amylodextrin, and regenerated cellulose fiber were subjected to electromediated oxidation with a 4-acetamido-TEMPO catalyst in a buffer at pH 6.8 without NaClO or NaClO(2). More than 90% of the C6 primary hydroxyls of Curdlan and amylodextrin were converted to sodium carboxylate groups by this method. Molecular mass values of the oxidized products were much higher than those prepared by the TEMPO/NaBr/NaClO system at pH 10. When the regenerate cellulose fiber was treated by the TEMPO electromediated oxidation for 45 h, carboxylate and aldehyde groups of 1.1 and 0.6 mmol/g, respectively, were formed in the oxidized cellulose fiber. The original fibrous and fine surface morphologies were maintained, and nearly no weight losses by the oxidation were observed. Thus, the TEMPO electromediated oxidation is a characteristic and environmentally friendly chemical modification for regenerated cellulose fibers, films, and related forming materials, and ion-exchangeable carboxylate and reactive aldehyde groups can be efficiently introduced into regenerated celluloses.

Topics: beta-Glucans; Catalysis; Cellulose; Cyclic N-Oxides; Dextrins; Electrochemical Techniques; Hydrogen-Ion Concentration; Molecular Structure; Oxidation-Reduction

Rheumatoid Arthritis (RA) is a chronic immune mediated disease associated with deregulation of many cell types. It has been reported that different T cell subsets have opposite effects in disease pathogenesis, in particular Th17 and Treg cells.. We investigated whether non-depleting anti-CD4 monoclonal antibodies, which have been reported as pro-tolerogenic, can lead to protection from chronic autoimmune arthritis in SKG mice--a recently described animal model of RA--by influencing the Th17/Treg balance. We found that non-depleting anti-CD4 prevented the onset of chronic autoimmune arthritis in SKG mice. Moreover, treated mice were protected from the induction of arthritis up to 60 days following anti-CD4 treatment, while remaining able to mount CD4-dependent immune responses to unrelated antigens. The antibody treatment also prevented disease progression in arthritic mice, although without leading to remission. Protection from arthritis was associated with an increased ratio of Foxp3, and decreased IL-17 producing T cells in the synovia. In vitro assays under Th17-polarizing conditions showed CD4-blockade prevents Th17 polarization, while favoring Foxp3 induction.. Non-depleting anti-CD4 can therefore induce long-term protection from chronic autoimmune arthritis in SKG mice through reciprocal changes in the frequency of Treg and Th17 cells in peripheral tissues, thus shifting the balance towards immune tolerance.

Topics: Animals; Antibodies; Arthritis; Autoimmunity; beta-Glucans; CD4 Antigens; Cell Polarity; Chronic Disease; Disease Progression; Forkhead Transcription Factors; Gene Expression Regulation; Immunization; Immunocompetence; Interleukin-17; Lymph Nodes; Mice; Synovial Membrane; T-Lymphocytes, Regulatory; Time Factors

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

A newly synthesized chromophore-modified curdlan functions as a saccharide chemosensor in aqueous solution, enabling us to discriminate tetrasaccharide acarbose from 24 mono-, di-, tri-, and tetrasaccharides.

Topics: Acarbose; beta-Glucans; Oligosaccharides; Polysaccharides, Bacterial; Solutions; Water

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

(1-->3)-Beta-D-Glucans are structural cell wall components of fungi, plants, and some bacteria and have been linked with human respiratory symptoms following aerosol exposure. A clear interpretation of the health impact of (1-->3)-beta-D-glucans is limited by the high cost and uncertainties associated with current glucan quantitation methods. The objective of this research is to develop DNA aptamers for the measurement of (1-->3)-beta-D-glucans. Aptamers are synthetic DNA functional binding molecules that fold into unique conformations, allowing them to bind specifically to their target. Through the in vitro selection process SELEX, we have produced aptamers that are able to bind with sub-micromolar affinity to curdlan, a linear unbranched form of (1-->3)-beta-D-glucans. These aptamers display high selectivity to curdlan and do not bind to non-(1-->3)-beta-D-polysaccharides, suggesting specificity for the beta-(1-->3)-glycosidic linkage. The aptamers produced here will enable the production of more cost-effective, less ambiguous assays for the environmental measurement of (1-->3)-beta-D-glucans.

Topics: Aptamers, Nucleotide; beta-Glucans; DNA; Nucleic Acid Conformation; Proteoglycans; SELEX Aptamer Technique

Curdlan modified polyurethane was created by physically entrapping the former on TecoflexTM surface. ATR-FT-IR, SEM-EDAX and AFM analysis revealed the formation of stable thin curdlan layer on the film. Contact-angle measurements showed that the modified film was highly hydrophilic. Confocal laser scanning microscopy showed the existence of entrapped layer of approximately 20-25 microm in depth. Surface entrapment of curdlan minimized both protein adsorption and mouse L929 fibroblast cell adhesion relative to the control. Surface induced cellular inflammatory response was determined from the expression levels of proinflammatory cytokine TNF-alpha, by measuring their mRNA profiles in the cells using real time polymerase chain reaction (RT-PCR) normalized to the housekeeping gene GAPDH. The inflammatory response was suppressed on the modified substrate as expression of TNF-alpha mRNA was found to be up regulated on TecoflexTM, while it was significantly lower on curdlan substrate. The adhesion of S. aureus decreased by 62% on curdlan modified surface. Using such simple surface entrapment process, it will be possible to develop well-defined surface modifications that promote specific cell interactions and perhaps better performance in the long-term as implant.

Topics: Adsorption; Animals; Bacterial Adhesion; Base Sequence; beta-Glucans; Cattle; Cell Adhesion; Cell Line; Coated Materials, Biocompatible; DNA Primers; Fibrinogen; Gene Expression; In Vitro Techniques; Inflammation; Materials Testing; Mice; Microscopy, Atomic Force; Microscopy, Confocal; Microscopy, Electron, Scanning; Polyurethanes; Proteins; RNA, Messenger; Spectroscopy, Fourier Transform Infrared; Thermodynamics; Tumor Necrosis Factor-alpha

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

Dectin-1 is a C-type lectin that recognizes beta-glucan in the cell walls of fungi and plays an important role in anti-fungal immunity. It signals via tyrosine kinase Syk and adaptor protein Card9 to activate NF-kappaB leading to proinflammatory cytokine production in dendritic cells (DCs). Other than this, not much else is known of the mechanism of Dectin-1 signaling. We demonstrate here that stimulation of DCs with zymosan triggers an intracellular Ca2+ flux that can be attenuated by a blocking anti-Dectin-1 antibody or by pre-treatment of cells with the phospholipase C (PLC) gamma-inhibitor U73122, suggesting that Dectin-1 signals via a PLCgamma pathway to induce Ca2+ flux in DCs. Interestingly, treatment of DCs with particulate curdlan, which specifically engages Dectin-1, results in the phosphorylation of both PLCgamma1 and PLCgamma2. However, we show that PLCgamma2 is the critical enzyme for Dectin-1 signaling in DCs. PLCgamma2-deficient DCs have drastic impairment of Ca2+ signaling and are defective in their secretion of interleukin 2 (IL-2), IL-6, IL-10, IL-12, IL-23, and tumor necrosis factor alpha. PLCgamma2-deficient DCs also exhibit impaired activation of ERK and JNK MAPKs and AP-1 and NFAT transcription factors in response to Dectin-1 stimulation. In addition, PLCgamma2-deficient DCs are also impaired in their activation of NF-kappaB upon Dectin-1 engagement due to defective assembly of the Card9-Bcl10-Malt1 complex and impaired IKKalpha/beta activation and IkappaBalpha degradation. Thus, our data indicate that pattern recognition receptors such as Dectin-1 could elicit Ca2+ signaling and that PLCgamma2 is a critical player in the Dectin-1 signal transduction pathway.

Topics: Adaptor Proteins, Signal Transducing; Animals; B-Cell CLL-Lymphoma 10 Protein; beta-Glucans; Calcium; Calcium Signaling; CARD Signaling Adaptor Proteins; Caspases; Cell Wall; Cytokines; Dendritic Cells; Enzyme Activation; Estrenes; Extracellular Signal-Regulated MAP Kinases; Fungi; I-kappa B Kinase; Lectins, C-Type; Membrane Proteins; Mice; Mice, Knockout; Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; Neoplasm Proteins; Nerve Tissue Proteins; NF-kappa B; NFATC Transcription Factors; Phosphodiesterase Inhibitors; Phospholipase C gamma; Pyrrolidinones; Zymosan

Microparticles of curdlan, synthesized through crosslinking with epichlorohydrin in organic suspension media, were chemically modified with the aim of introducing strongly and/or weakly acidic anionic and palmitoyl hydrophobic groups. Microparticles of both curdlan and curdlan derivatives were physico-chemically characterized. Study of the interaction with enzymes, such as lysozyme, and vaccines, such as tetanus anatoxin, showed a co-operative protein retention effect, induced by electrostatic and hydrophobic forces. The results of the in vitro release studies on support-protein complexes recommend them as potential controlled release systems.

Topics: beta-Glucans; Carbohydrate Conformation; Carbohydrate Sequence; Enzymes, Immobilized; Ion Exchange; Isotonic Solutions; Kinetics; Microscopy, Electron, Scanning; Microspheres; Molecular Sequence Data; Muramidase; Proteins; Temperature; Tetanus Toxin; Time Factors; Vaccines

Solid lipid nanoparticles (SLN) loaded with doxorubicin were prepared by solvent emulsification-diffusion method. Glyceryl caprate (Capmul)MCM C10) was used as lipid core, and curdlan as the shell material. Dimethyl sulfoxide (DMSO) was used to dissolve both lipid and drug. Polyethylene glycol 660 hydroxystearate (Solutol)HS15) was employed as surfactant. Major formulation parameters were optimized to obtain high quality nanoparticles. The mean particle size measured by photon correlation spectroscopy (PCS) was 199nm. The entrapment efficiency (EE) and drug loading capacity (DL), determined with fluorescence spectroscopy, were 67.5+/-2.4% and 2.8+/-0.1%, respectively. The drug release behavior was studied by in vitro method. Cell viability assay showed that properties of SLN remain unchanged during the process of freeze-drying. Stability study revealed that lyophilized SLN were equally effective (p<0.05) after 1 year of storage at 4 degrees C. In conclusion, SLN with small particle size, high EE, and relatively high DL for doxorubicin can be obtained by this method.

Topics: Antibiotics, Antineoplastic; beta-Glucans; Calorimetry, Differential Scanning; Cell Line, Tumor; Cell Survival; Dialysis; Dimethyl Sulfoxide; Doxorubicin; Drug Compounding; Drug Delivery Systems; Drug Stability; Female; Freeze Drying; Humans; Lipids; Nanoparticles; Solubility; Solvents; Spectrometry, Fluorescence

The branched structure properties of hyperbranched polysaccharides (TM3a and TM3b), extracted from sclerotia of Pleurotus tuber-regium, were studied by using laser light scattering and viscometry. The configurational shrinking factor (g) and viscometric shrinking factor (g') of TM3a and TM3b were discussed, where curdlan and pullulan were taken as the linear references for derivation of g and g'. The dependences of g factor, g' factor, and Flory factor (Phi(branched)) on weight average molecular weight (M(w)) were established to be g=1.07x10(2)M(w)(-0.48+/-0.09), g'=3.63x10(1)M(w)(-0.43+/-0.01), and Phi(branched)=7.08x10(20)M(w)(0.39+/-0.1) for TM3a in 0.25M LiCl/DMSO at 25 degrees C, when curdlan acted as the linear reference. A power law relationship g'=2.71x10(-1)g(-0.61+/-0.1) for TM3a was found, and the exponent was approximately same to 0.60 established by Kurata et al. for polystyrene star molecules. The dependence of g factor on M(w) for TM3b was found to be g=1.99x10(2)M(w)(-0.53+/-0.02), when pullulan was used as the linear reference. On the basis of Zimm-Stockmayer equation for tetrafunctional units, molecular weight of branching unit (M(0)) deduced from nonlinear curve fitting of g versus M(w) was 8739+/-564g/mol and 3961+/-1245g/mol for TM3a and TM3b, respectively. The effect of different linear reference curves and polydispersity was discussed. This work gave valuable information on branched structure characterization and insights into the biosynthetic pathways of the hyperbranched polysaccharide from fungus.

Topics: beta-Glucans; Glucans; Linear Models; Pleurotus; Polysaccharides

Self-diffusion and mutual diffusion are two different transport mechanisms experimentally characterized on different length and time scales. NMR spectroscopy is a highly suitable technique to characterize these two phenomena as both mechanisms can be studied on the same system and in the same experimental conditions. Pulsed field gradient (PFG) NMR was used to measure the self-diffusion whereas (31)P NMR profiling provided an approach to determine the mutual diffusion coefficients. We have characterized the diffusion of phosphate, trimetaphosphate, alendronate, and d-glucose-6-phosphate in hydrogels prepared with 10% (w/v) curdlan, a bacterial polysaccharide built of linear (1-->3)-beta-d-glucose repeating units. These solutes are small compared to the average pore size of the hydrogel, as inferred from environmental scanning electron microscopy (eSEM). Our results show that the self- and mutual-diffusion coefficients of small molecules in curdlan hydrogels are similar and are reduced by 30% compared to those measured in aqueous solutions. These observations are validated for the complete series of investigated analytes. It is therefore concluded that, for this system, the analyte diffusion in the gel is essentially reduced because of interactions at the molecular level and that the open structure of this gel has a very limited influence at the mesoscopic length scale. A literature survey indicates that these conditions prevail for the large majority of the systems that have been investigated up to now.

Topics: Bacteria; beta-Glucans; Diffusion; Hydrogels; Magnetic Resonance Spectroscopy; Microscopy, Electron, Scanning; Phosphorus Isotopes; Solutions; Water

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

Curdlan from Agrobacterium sp. was oxidized using 2,2,6,6,-tetramethylpiperidine-1-oxyl radical (TEMPO)-NaBr-NaClO systems at pH 11. The effects of oxidation conditions on degrees of oxidation and polymerization of the products obtained were studied using SEC-MALLS, NMR and IR analyses. Different families of water-soluble beta-(1,3)-polyglucuronic and beta-(1,3)-polyglucoglucuronic acid sodium salts were quantitatively generated with a yield of 80% and without significant loss of their molecular weights. Given that beta-(1,3)-polyglucuronic acids prepared from the regioselective oxidation of curdlan by the TEMPO-NaBr-NaClO systems regularly consist of the glucuronic acid repeating unit; they may open new biotechnological fields for the utilizations of water soluble forms of curdlan.

Topics: beta-Glucans; Carbohydrates; Cyclic N-Oxides; Electrochemistry; Glucuronates; Hydrogen-Ion Concentration; Magnetic Resonance Spectroscopy; Models, Chemical; Molecular Weight; Oxygen; Rhizobium; Sodium Hypochlorite; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Water

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

A mutant strain of the curdlan-producing bacterium Agrobacterium sp. ATCC 31749, isolated by ethylmethane sulfonate mutagenesis and resistance to ampicillin, was capable of elevated curdlan synthesis. Using 2.5% corn syrup, glucose or maltose as a carbon source, the mutant strain was shown to produce a 1.5-fold, 1.5-fold or 1.5-fold higher level of curdlan, respectively, than its parent strain after 120 h of growth. The mutant strain produced higher curdlan levels after 96 or 120 h of growth on glucose or maltose as a carbon source than it did on corn syrup. Biomass production by the mutant strain grown on the carbon sources studied was slightly elevated compared to its parent strain. It was concluded that the elevated curdlan production observed for the mutant strain grown on corn syrup or glucose was not due to an increase in biomass production.

Topics: beta-Glucans; Carbon; Culture Media; Fermentation; Glucose; Maltose; Polysaccharides, Bacterial; Rhizobium

Fungal beta-glucan, such as curdlan, triggers antifungal innate immune responses as well as shaping adaptive immune responses. In this study, we identified a key pathway that couples curdlan to immune responses. Curdlan promoted the production of the proinflammatory cytokine IL-1beta by dendritic cells and macrophages through the NLRP3 inflammasome. Stimulation with Candida albicans and Saccharomyces cerevisiae also triggered the NLRP3 inflammasome-mediated IL-1beta production. In vivo, NLRP3 was required for efficient Ag-specific Ab production when curdlan was used as an adjuvant, whereas it was dispensable for the induction of Th1 and Th17 cell differentiation. Furthermore, stimulation of purified B cells with curdlan-induced CD69 up-regulation and IgM production while stimulation with other NLRP3 inflammasome activators, such as silica and aluminum salt, did not. Notably, this induction required NLRP3 but was independent of Toll-like receptor and IL-1 receptor family signaling, suggesting the presence of NLRP3-dependent and IL-1 receptor family independent mechanisms in B cells responsible for Ab responses. Collectively, these findings reveal a critical role for the NLRP3 inflammasome in the regulation of antifungal innate immune responses as well as B cell activation.

Topics: Adaptive Immunity; Animals; Antibodies; Antibodies, Fungal; B-Lymphocytes; beta-Glucans; Candida albicans; Carrier Proteins; Cells, Cultured; Immunity, Innate; Inflammation; Inflammation Mediators; Lymphocyte Activation; Mice; Mice, Inbred C57BL; Mice, Knockout; NLR Family, Pyrin Domain-Containing 3 Protein; Saccharomyces cerevisiae

The beta-glucan receptor dectin-1 and Toll-like receptors TLR2 and TLR4 are the main receptors for recognition of Candida albicans by the innate immune system. It has been reported that dectin-1 amplifies TLR2-dependent induction of cytokines in mouse models. In the present study we hypothesized that dectin-1 has potent synergistic effects with both TLR2 and TLR4 in human PBMCs and macrophages. Human PBMCs and monocyte-derived macrophages were stimulated with curdlan, a linear beta-1,3-glucan-polymer derived from Alcaligenes faecalis with specific ligand affinity for dectin-1, in combination with the synthetic TLR2 ligand Pam3Cys and the ultrapure TLR4 ligand LPS. TNF-alpha and IL-10 production was measured in the supernatants with ELISA. Curdlan is a specific dectin-1 ligand without TLR2- or TLR4-stimulating properties. Human primary monocytes and macrophages express dectin-1 on the cell membrane. Stimulation of human PBMCs with curdlan in combination with Pam3Cys or LPS leads to synergistic increase in TNF-alpha production that was inhibited by GE2, a neutralizing dectin-1 antibody. Dectin-1-dependent synergy between curdlan and TLR agonists was also apparent in human monocyte-derived macrophages. Conclusively, dectin-1 synergizes with both TLR2 and TLR4 pathways for the production of TNF-alpha in human primary PBMCs and in monocyte-derived macrophages.

Topics: beta-Glucans; Cells, Cultured; Cysteine; Humans; Interleukin-10; Lectins, C-Type; Lipopolysaccharides; Lipoproteins; Macrophages; Membrane Proteins; Monocytes; Nerve Tissue Proteins; Toll-Like Receptor 2; Toll-Like Receptor 4; Tumor Necrosis Factor-alpha

(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

Curdlan is a water insoluble exopolysaccharide produced by Alcaligenes faecalis under nitrogen-limiting conditions. After excretion, the polysaccharide is attached the cell wall. Thus enhancement of biomass production during the cell growth phase is important to curdlan production. A strategy of increasing nitrogen source to improve biomass production was adopted for curdlan production by Alcaligenes faecalis (ATCC 31749). In the batch fermentation of curdlan, a relatively higher NH4Cl level of 3.6 g/L with continuous glucose feeding increased the cell density leading to improvement of curdlan production. However, excessive NH4Cl would inhibit curdlan production and biomass production was not improved significantly. In addition, feeding of ammonia water at the initial phase replaced NaOH solution to control pH at 7.0. Subsequently, feeding of NaOH solution was resumed to control pH at 5.6 for curdlan production after ammonia was consumed. As a result, biomass production and curdlan yield were both enhanced remarkably. Feeding of ammonia water during the first 24 h led to biomass production of 18.8 g/L. However, higher cell density did not lead to increase in curdlan production. The maximum curdlan production (72 g/L) was obtained by feeding ammonia water for the first 14 h, during which the cell density was about 11.9 g/L.

Topics: Alcaligenes faecalis; Ammonium Chloride; beta-Glucans; Cell Culture Techniques; Cell Proliferation; Fermentation

During batch cultivation of Agrobacterium sp. ATCC 31750, proteome analysis in response to a pH downshift from 7.0 to 5.5 was carried out using two-dimensional electrophoresis and matrix-assisted laser desorption-ionization-time of flight mass spectrometry. When the pH of the exponentially growing Agrobacterium sp. culture was downshifted to pH 5.5, the synthesis level of 27 intracellular proteins showed significant changes in level over a prolonged period of time compared with the batch culture controlled at pH 7.0. In particular, the intracellular protein level of the beta-1,3-glucan synthase catalytic subunit, UTP-glucose-1-phosphate uridylyltransferase, and phosphoglucomutase, which are key metabolic enzymes in the curdlan biosynthesis pathway, were more than 10-, 3- and 17-times higher in the low pH culture. On the other hand, the level of orotidine5-phosphate decarboxylase (conversion of OMP to UMP) was significantly up-regulated after pH downshift. The accumulation of UMP may direct the metabolic flow towards the biosynthetic route of UTP, which is a key metabolic precursor for UDP-glucose. Therefore, it is possible that increase of cellular metabolic enzymes during pH downshift culture can enhance the metabolic flux of the biosynthesis of key precursor, such as UTP- and UDP-glucose, resulting in an increase in curdlan biosynthesis.

Topics: beta-Glucans; Electrophoresis, Gel, Two-Dimensional; Hydrogen-Ion Concentration; Proteomics; Rhizobium; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Stress, Physiological

Much attention has been focused on exploiting novel strategies for the creation of hierarchical polymer assemblies by the control of the assembling number or the relative location among neighboring polymers. We here propose a novel strategy toward the creation of "hierarchical" single-walled carbon nanotube (SWNT) architectures by utilizing SWNT composites with cationic or anionic complementary semi-artificial beta-1,3-glucans as "building blocks". These beta-1,3-glucans are known to wrap SWNTs helically, to create one-dimensional superstructural composites. If the cationic composite is neutralized by an anionic composite, a well ordered SWNT-based sheet structure was created. Transmission electron microscopy (TEM) observation revealed that this sheet structure is composed of highly-ordered fibrous assemblies of SWNTs. This suggests that the cationic and anionic composites are tightly packed through electrostatic interactions. Moreover, both of the final assembly structures are readily tunable by adjusting the cation/anion ratio. The self-assembling modulation of functional polymers is associated with the progress in ultimate nanotechnologies, thus enabling us to create numerous functional nanomaterials. We believe, therefore, that the present system will extend the frontier of SWNT research to assembly chemistry including "hierarchical" superstructures.

Topics: beta-Glucans; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Molecular Structure; Nanotubes, Carbon; Proteoglycans; Spectrophotometry, Infrared

The production of the polysaccharide curdlan from the ethanol processing coproduct condensed corn distillers solubles by the bacterium Agrobacterium sp. ATCC 31749 was investigated. It was found that curdlan could be produced by the bacterium using condensed corn distillers solubles as a source of carbon and nitrogen. As the concentration of condensed corn distillers solubles was increased from 50 g l(-1) to 400 g l(-1), the concentration of curdlan increased but not proportionally. The highest curdlan concentration was produced by the strain on 400 g l(-1 )condensed corn distillers solubles after 120 h and its level was higher than was observed for glucose-based curdlan production. Biomass production by ATCC 31749 was also highest after 120 h of growth on 400 g l(-1 )condensed corn distillers solubles and was higher than found for glucose-based biomass production.

Topics: beta-Glucans; Biomass; Culture Media; Ethanol; Fermentation; Industrial Microbiology; Rhizobium; Time Factors; Zea mays

beta-1,3-D-glucans have been isolated from fungi as right-handed 6(1) triple helices. They are categorized by the side chains bound to the main triple helix through beta-(1-->6)-D-glycosyl linkage. Indeed, since a glucose-based side chain is water soluble, the presence and frequency of glucose-based side chains give rise to significant variation in the physical properties of the glucan family. Curdlan has no side chains and self-assembles to form an water-insoluble triple helical structure, while schizophyllan, which has a 1,6-D-glucose side chain on every third glucose unit along the main chain, is completely water soluble. A thermal fluctuation in the optical rotatory dispersion is observed for the side chain, indicating probable co-operative interaction between the side chains and water molecules. This paper documents molecular dynamics simulations in aqueous solution for three models of the beta-1,3-D-glucan series: curdlan (no side chain), schizophyllan (a beta-(1-->6)-D-glycosyl side-chain at every third position), and a hypothetical triple helix with a side chain at every sixth main-chain glucose unit. A decrease was observed in the helical pitch as the population of the side chain increased. Two types of hydrogen bonding via water molecules, the side chain/main chain and the side chain/side chain hydrogen bonding, play an important role in determination of the triple helix conformation. The formation of a one-dimensional cavity of diameter about 3.5 A was observed in the schizophyllan triple helix, while curdlan showed no such cavity. The side chain/side chain hydrogen bonding in schizophyllan and the hypothetical beta-1,3-D-glucan triple helix could cause the tilt of the main-chain glucose residues to the helix.

Topics: beta-Glucans; Carbohydrate Sequence; Hydrogen Bonding; Models, Molecular; Molecular Conformation; Molecular Sequence Data; Proteoglycans; Sizofiran; Solutions; Thermodynamics; Water

Essential aspects of the innate immune response to microbial infection appear to be conserved between insects and mammals. In order to identify new Drosophila melanogaster genes involved in the immune response, we performed gene expression profiling of Drosophila SL2 cells stimulated with bacterial (LPS/PGN) or fungal (curdlan) components using a cDNA microarray that contained 5,405 Drosophila cDNAs. We found that some genes were similarly regulated by LPS/PGN and curdlan. However, a large number, belonging to the functional classes of cell organization, development, signal transduction, morphogenesis, cell cycle, and DNA replication, displayed significant differences in their transcription profiles between the two treatments, demonstrating that bacterial and fungal components induce different immune response even in an in vitro cell system.

Topics: Animals; beta-Glucans; Down-Regulation; Drosophila melanogaster; Gene Expression Profiling; Immunity, Innate; Lipopolysaccharides; Macrophages; Microarray Analysis; Transcription, Genetic; Up-Regulation

We succeeded in the quantitative and selective introduction of an ammonium cationic group into the C6 position of Curdlan (CUR) by "Click Chemistry", and the obtained cationic Curdlan (CUR-N+) showed good solubility in water. ORD studies suggested that CUR-N+ adopts a single-stranded structure, different from a right-handed, triple-stranded helical structure of beta-1,3-glucan polysaccharides in water. It has been revealed that the polymeric complexes of CUR-N+ with polymeric guest molecules, such as polycytidylic acid (poly(C)), permethyldecasilane (PMDS), and single-walled carbon nanotubes (SWNTs), can be easily obtained by just mixing them in water with sonication. The characterization of the resultant CUR-N+-poly(C) complexes by UV-vis, CD spectroscopic measurements, and AFM and TEM observations revealed that they have stoichiometric, nanosized fibrous structures. From these experimental results as well as our precedent studies (e.g., refs 6 and 23), we propose that the complexation would be driven by the cooperative action of (1) the hydrogen-bonding interaction between the OH group at the C2 position and hydrogen-bonding sites of the cytosine ring (ref 6d), (2) the electrostatic interaction between the ammonium cation and the phosphate anion (ref 23), as well as (3) the background hydrophobic interaction. In addition, the complexed polynucleotide chain showed a strong resistance against enzymatic hydrolysis. Likewise, the dispersion of PMDS and SWNTs in water by CUR-N+ and the fibrous structures of the complexes were confirmed by spectroscopic measurements as well as microscopic observations. These binding properties of CUR-N+, which can proceed spontaneously in water, clearly differ from those of schizophyllan (SPG), which inevitably require a denature-renature process corresponding to a conversion of a triple strand to single strands induced by DMSO or base for inclusion of polymeric guest molecules.

Topics: beta-Glucans; Carbohydrate Conformation; Cations; Glucans; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Microscopy, Atomic Force; Microscopy, Electron, Transmission; Models, Molecular; Polynucleotides; Ribonucleases; Solutions; Spectrum Analysis

Infrared spectroscopy was used to probe the hydration and gelation of curdlan, a linear polysaccharide built from repeating units of (1-->3)-beta-D-glucose. The spectra have been recorded using a temperature-controlled attenuated total reflection (ATR) device. Thermal gelation of curdlan could therefore be followed in situ and in real time. The transformation of the low-set gel, mainly formed with single helices, into a high-set gel, associated with a triple helix structure, could be directly observed. The relative intensities and positions of characteristic absorption bands in the C-O region (1200-850 cm-1) were found to be representative of the gel structure, as they are believed to be sensitive to the helical conformation of the polymer chains. Infrared (IR) spectroscopy is shown to be a useful tool for rapid and efficient characterization of curdlan gels.

Topics: beta-Glucans; Crystallization; Crystallography; Gels; Molecular Conformation; Phase Transition; Powders; Solubility; Spectrophotometry, Infrared; Water

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

Beta-1,3-glucans having carbohydrate-appendages (alpha-D-mannoside, N-acetyl-beta-D-glucosaminide and beta-lactoside) at the C6-position of every repeating unit can be readily prepared from curdlan (a linear beta-1,3-glucan) through regioselective bromination/azidation to afford 6-azido-6-deoxycurdlan followed by chemo-selective Cu(i)-catalyzed [3 + 2]-cycloaddition with various carbohydrate modules having a terminal alkyne. The resultant carbohydrate-appended curdlans can interact with polycytosine to form stable macromolecular complexes consistent with two polysaccharide strands and one polycytosine strand. Furthermore, these macromolecular complexes show strong and specific affinity toward carbohydrate-binding proteins (lectins). Therefore, one can utilize these carbohydrate-appended curdlans as a new family of glycoclusters.

Topics: Alkynes; Azides; beta-Glucans; Catalysis; Circular Dichroism; Copper; Cytosine; Glycosylation; Lectins; Magnetic Resonance Spectroscopy; Models, Molecular; Molecular Structure; Oxidation-Reduction; Polynucleotides; Solvents; Surface Plasmon Resonance

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

(1-->3)-beta-D-Glucans having various functional appendages (lactoside, ferrocene, pyrene, and porphyrin) can be prepared in an convenient, quantitative, and regioselective manner through regioselective bromination-azidation of curdlan to afford 6-azido-6-deoxycurdlan followed by chemoselective [3+2]-cycloadditions with various functional modules bearing a terminal alkyne group. The ability to monitor reaction conversions is an additional advantage of this synthetic approach over the conventional direct modifications on polysaccharides; the reaction can be readily monitored based on the intensity of azido peaks in the in situ attenuated total reflection infrared spectra.

Topics: Alkynes; Azides; beta-Glucans; Nuclear Magnetic Resonance, Biomolecular; Proteoglycans; Spectroscopy, Fourier Transform Infrared

Curdlan was carboxymethylated in an aqueous alkaline medium using monochloroacetic acid as the etherifying agent. The structure of carboxymethylated curdlan (CMc) was analyzed by FT-IR and NMR spectroscopy, which revealed that the carboxymethyl group was introduced mainly at the C-6 position as well as at the C-2 and C-4 positions. Furthermore, CMc was compared with the native curdlan by using rheology and DSC methods. It was found that in water, both polysaccharides behaved as pseudoplastic fluids and fit the power law and Herschel-Bulkley rheological models well. Both the storage shear modulus G' and the loss shear modulus G'' of CMc aqueous solutions decreased and became more frequency dependent with decreasing concentration in comparison with the curdlan aqueous suspensions. The modulus-temperature curve also suggested that the gel characteristic of curdlan has been lost after chemical modification, which is consistent with the DSC results. AFM images revealed differences in the conformation of native and carboxymethylated curdlan, which changed from the aggregation of macromolecules to triple helices. All the experimental results suggest that the hydrogen bonds that bind curdlan with interstitial water to form the micelles have been destroyed completely and that the hydrophobic interactions related to the methylene groups at C-6 formed above 55 degrees C disappeared due to the introduction of the hydrophilic carboxymethyl group.

Topics: beta-Glucans; Calorimetry, Differential Scanning; Elasticity; Gels; Hydrophobic and Hydrophilic Interactions; Microscopy, Atomic Force; Nuclear Magnetic Resonance, Biomolecular; Rheology; Solubility; Spectroscopy, Fourier Transform Infrared; Temperature

Pyrimidine base supplementation of the culture medium was found to increase curdlan production by Agrobacterium sp. ATCC 31749. With sucrose as a carbon source, adding uracil or cytosine to the culture medium of strain ATCC 31749 after 48 h of growth resulted in an elevation of curdlan levels by 1.7-fold or 1.5-fold, respectively, after 120 h compared to no addition. Supplementation of the pyrimidine bases thymine, dihydrouracil or orotic acid had no effect upon curdlan production. Biomass production by strain ATCC 31749 after uracil supplementation was noted to increase by 3.0-fold after 120 h of growth compared to the control cultures. The increase in curdlan production by Agrobacterium sp. ATCC 31749 following uracil addition appeared to be related to the observed increase in biomass production.

Topics: beta-Glucans; Biomass; Culture Media; Pyrimidines; Rhizobium

Curdlan-producing Agrobacterium sp. is unique in possessing a highly efficient UDP-glucose regeneration system. A broad-host-range expression strategy was successfully developed to exploit the unique metabolic capability for UDP-galactose regeneration during oligosaccharide synthesis. The engineered Agrobacterium cells functioned as a UDP-galactose regeneration system, allowing galactose-containing disaccharides to be synthesized from glucose or other simple sugars. Unexpectedly, a lag period of 24h preceded the active synthesis, which could be eliminated with rifampicin. An intracellular nucleotide profiling revealed that the UMP level was elevated by 3.8 fold in the presence of rifampicin, suggesting that rifampicin simulated a nitrogen-limitation condition that triggered the metabolic change. Product selectivity was improved nearly 40-fold by using high acceptor concentration and restricting glucose supply. N-acetyllactosamine concentration near 20 mM (7.5 g/l) was obtained, demonstrating the effectiveness of the engineered strain in UDP-galactose regeneration. This organism could be engineered to regenerate other UDP-sugar nucleotides using the same strategy as illustrated here.

Topics: beta-Glucans; Galactose; Genetic Engineering; Oligosaccharides; Recombinant Proteins; Rhizobium; Uridine Diphosphate

Agrobacterium isolated from soil samples produced two extracellular polysaccharides: succinoglycan, an acidic soluble polymer, and curdlan gum, a neutral, insoluble polymer. Maize glucose, cassava glucose, and maize maltose were used in fermentation medium to produce insoluble polysaccharide. Two Agrobacterium sp. strains which were used (ATCC 31749 and IFO 13140) in the production of insoluble exopolysaccharide presented equal or superior yields compared to the literature. The strain ATCC 31749 yielded better production when using maize maltose, whose yield was 85%, whereas strain IFO 13140 produced more when fed maize glucose, producing a yield of 50% (on reducing sugars).

Topics: beta-Glucans; Polysaccharides, Bacterial; Rhizobium; Solubility; Species Specificity; Zea mays

The activation of an immune response to invading microorganisms generally requires recognition by pattern recognition receptors. Beta 1, 3-glucan recognition proteins (GRPs) have specific affinity for beta 1, 3-glucan, a component on the surface of fungi and bacteria. In this study, we show that GRP from Armigeres subalbatus mosquitoes (AsGRP) is able to bind different bacterial species, and that this binding varies from species to species and is independent of Gram type. AsGRP knockdown with double-stranded RNA increases the mortality of mosquitoes to those bacteria that strongly bind AsGRP, but not to bacteria that do not detectably bind AsGRP. This increase in susceptibility is partially evidenced by decreased melanization in Salmonella typhimurium. Furthermore, AsGRP expression is differentially affected by the presence of different species of bacteria. These results demonstrate that AsGRP is selective in its affinity to different bacteria and; therefore, plays a role in the antibacterial immune response of mosquitoes.

Topics: Animals; Bacteria; beta-Glucans; Blotting, Western; Carrier Proteins; Culicidae; Female; Gene Expression Profiling; Green Fluorescent Proteins; Immunity, Innate; Insect Proteins; Melanins; Protein Binding; Receptors, Pattern Recognition; Recombinant Fusion Proteins; RNA Interference; RNA, Double-Stranded; Salmonella typhimurium; Up-Regulation

Solid lipid nanoparticles (SLN) were prepared using cacao butter, as the lipid core, and curdlan, as the shell material. Tween 80 was used as a co-surfactant in order to prevent aggregation and gelling of the curdlan. Mannitol was used as a cryoprotectant in order to prevent aggregation during redispersion. No significant change in the size of the SLN was observed up to a lipid concentration of 1.0%, and the particle size ranged from 140 to 200 nm with a unimodal distribution. When an alternating pH between 7 and 11 was used to test the physical stability of an SLN solution, the change in the particle size remained within a narrow range up to a lipid concentration of 0.5%. Above 0.5%, the particles began to aggregate due to the insufficient amount of the coating material, curdlan and Tween 80. The critical aggregation concentration at pH 7.4 was found to be 6.95 x 10(-4) mg/ml. Pyrene was used as a fluorescence probe. As the temperature increased, pyrene was gradually released from the SLN. The loading efficiency was >75% when the verapamil to lipid ratios were 1:10 and 1:5 and decreased significantly as the ratio became 1:1. The release rate was significantly delayed when verapamil was loaded into the SLN.

Topics: beta-Glucans; Cacao; Nanostructures; Particle Size

Heat-induced gelation of a cold-water insoluble polysaccharide, Curdlan, was investigated using atomic force microscopy (AFM). Curdlan dissolved into NaOH aqueous solutions exhibited a spectral transition around 0.2 mol/L NaOH, which is an indicative of conformational transitions from a single helix at a lower alkali concentration to a disordered chain at a higher concentration. Nevertheless, AFM images of Curdlan solubilized in 0.01 mol/L NaOH revealed the presence of heterogeneous supramolecular assemblies of Curdlan: the majority of the molecules were in the form of microfibrils, the lengths of which were on the order of micrometers and the cross-sectional heights of which were approximately 2-3 nm, whereas single molecular chains, partially dissociated from these microfibrils, were also observed. Heating such a sol resulted in the formation of densely cross-linked microgel networks. Heat-induced gelation of Curdlan appears to be initiated by partial dissociation of single chains from supramolecular microfibrils and followed by cross-linking of microfibrils via hydrophobic interactions among these partially dissociated chains.

Topics: beta-Glucans; Gels; Hot Temperature; Hydrogen-Ion Concentration; Microscopy, Atomic Force; Spectrum Analysis

The genus Cellulomonas is comprised of a group of Gram-positive, soil bacteria capable of utilizing cellulose as their sole source of carbon and energy. Cellulomonas flavigena KU was originally isolated from leaf litter and subsequently shown to produce large quantities of a curdlan-type (beta-1,3-glucan) exopolysaccharide (EPS) when provided with an excess of glucose or other soluble carbon-source. We report here that curdlan EPS is also produced by Cellulomonas flavigena KU when growing on microcrystalline cellulose in mineral salts-yeast extract media. Microscopic examination of such cultures shows an adherent biofilm matrix composed of cells, curdlan EPS, and numerous surface structures resembling cellulosome complexes. Those Cellulomonas species that produce curdlan EPS are all non-motile and adhere to cellulose as it is broken down into soluble sugars. These observations suggest two very different approaches towards the complex process of cellulose degradation within the genus Cellulomonas.

Topics: Bacterial Adhesion; beta-Glucans; Biofilms; Cellulomonas; Cellulose; Cellulosomes; Glycocalyx; Microscopy, Electron, Scanning; Microscopy, Phase-Contrast; Polysaccharides, Bacterial

We have found that single-chain schizophyllan and curdlan (s-SPG and s-curdlan, respectively) can dissolve as-grown and cut single-walled carbon nanotubes (ag-SWNTs and c-SWNTs, respectively) in aqueous solution. The vis-NIR spectra of the composites suggest that c-SWNTs are dissolved as a bundle, whereas ag-SWNTs exist as one or only a few pieces in the tubular hollow constructed by the helical structure inherent to these beta-1,3-glucans. EDX and CLSM measurements and TEM observation established that the distribution map of these polysaccharides overlaps well with the image of SWNTs, indicating that these two components form a composite. Very interestingly, when c-SWNTs were dissolved with the aid of s-SPG or s-curdlan in water, a clear periodical structure with inclined stripes, as detected by AFM, appeared on the fibrous composite surface. Because this periodical structure has never been recognized for the composites with other water-soluble polymers, one can regard that s-SPG or s-curdlan wraps c-SWNTs constructing a helically twined structure. High-resolution TEM observation of an ag-SWNTs/s-SPG composite gave a clearer image in that two s-SPG chains twine one ag-SWNT and the helical motif is right-handed. When this sample was subjected to the AFM measurement, the composite showed the 2-3 nm height. This height implies that one piece of ag-SWNT is included in the s-SPGs helical structure. As a summary, it has been established that beta-1,3-glucans such as s-SPG and s-curdlan not only dissolve SWNTs but also create a novel superstructure on the surface.

Topics: beta-Glucans; Carbohydrate Sequence; Microscopy, Atomic Force; Models, Molecular; Molecular Sequence Data; Nanotubes, Carbon; Sizofiran; Solutions; Spectroscopy, Near-Infrared; Spectrum Analysis, Raman; Water

We already found that beta-1,3-glucan polysaccharides form polymeric complexes with certain polynucleotides, but the parallel vs. anti-parallel orientation in those complexes had remained unsolved. In this paper, this controversial problem has been discussed for curdlan/oligo(dA) complexes utilizing two different energy transfer techniques. The first system consists of a combination of fluorescein-labeled curdlan and 3'-(or 5'-)tetramethyl-rhodamine (TAMRA)-labeled oligo(dA). The second system utilizes gold nanoparticles: that is, two curdlan chains were linked by a disulfide bond and after complexation with oligo(dA), the complex was immobilized on gold nanoparticles. In this system, TAMRA was attached to the 3'(or 5') end of oligo(dA) and the gold particle acted as a fluorescence quencher (energy acceptor). These experiments have led us to conclude that in the curdlan/oligo(dA) complex, parallel orientation is more favourable than anti-parallel orientation. These findings have enabled us to envision a clearer image for the complexation mode between beta-1,3-glucan polysaccharides and polynucleotides.

Topics: beta-Glucans; Fluorescein; Fluorescence Resonance Energy Transfer; Oligodeoxyribonucleotides; Spectrophotometry, Ultraviolet

The effect of initial ammonium chloride level on production of curdlan in Alcaligenesfaecalis was investigated. It was found that ammonium chloride was the limiting substrate for cell growth during the batch fermentation process. However, the cell growth and curdlan production could not be enhanced by solely increasing the initial ammonium chloride level. The pH drop in the broth due to the consumption of ammonium chloride also effected the cell growth and curdlan production. By simultaneously increasing the initial ammonium chloride concentration and implementing an optimal pH control strategy, which is to control pH at 7.0 in the growth phase, and then shift to 5.6 in the production phase, the biomass and curdlan production in batch fermentation were increased markedly. If the initial ammonium chloride concentration was increased from 1.1 g/L to 3.6 g/L, biomass concentration of 7.2 g/L was obtained, and the final curdlan concentration reached 30.5 g/L, which was 51.7% higher than that of the former case. As the cell growth was improved due to the increase of the initial ammonium chloride concentration, the agitation speed and aeration rates must be enhanced to suit the higher oxygen uptake requirement. However, as curdlan molecules is subject to the structural breakage due to the high shear stress at higher agitation speed, an overall optimal condition for both productivity and quality of curdlan should be considered comprehensively.

Topics: Alcaligenes faecalis; Ammonium Chloride; beta-Glucans; Culture Media; Dose-Response Relationship, Drug; Fermentation; Hydrogen-Ion Concentration

We developed the microbial immobilization particle with curdlan and activated carbon, which has great adsorption capacity. The characteristics of porosity and mechanical strength of these supporting particles are dependent on manufacturing method. The supporting particle showed the best performance when the ratio of curdlan and activated carbon was 30 to 6 g/L. Brumauer-Emmett-Teller (specific surface area) and swelling capacity of the carrier were 52.63 m2/g and 17 (w/w), respectively. The immobilization characteristics of iron-oxidizing bacteria on supporting particles were observed using a scanning electron microscope. The concentration of microorganism on the surface of supporting particle was increased with reaction time. As the number of iron oxidation batch cycles increased, the iron oxidation rate increased.

Topics: Bacterial Adhesion; beta-Glucans; Biocompatible Materials; Bioreactors; Cell Culture Techniques; Cell Division; Cells, Immobilized; Charcoal; Compressive Strength; Glucans; Hydrogen-Ion Concentration; Iron; Materials Testing; Oxidation-Reduction; Particle Size; Surface Properties; Thiobacillus

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

Curdlan dissolved in aqueous sodium hydroxide was dialyzed to aqueous calcium chloride to form a gel. Transparent and turbid concentric layers observed in the gel cross section perpendicular to the long axis of the dialysis tube were identified as liquid crystalline gels with refractive index gradient and amorphous gels, respectively. The thickness of each layer was proportional to the diameter of the dialysis tube, and the gelation proceeded in proportion to the root of time. The unique pattern formation was attributed to the change of curdlan conformation and calcium-induced cross-linking resulting from a diffusion of calcium cations and hydroxide anions through the dialysis tube. It is suggested that the orderedness of the curdlan molecules decreases by the increase of the curvature of the concentric liquid crystal layers as the layer comes toward the center of the dialysis tube.

Topics: beta-Glucans; Calcium Chloride; Crystallization; Gels; Liquid Crystals; Refractometry; Sodium Hydroxide; Surface Properties

Callose (1,3-beta-glucan) is a suggested physiological indicator of aluminum (Al) toxicity in crop plants. It is not known if callose serves a similar function in forest trees, because quantitative data on callose formation in tree roots are limited, particularly under controlled conditions. To evaluate callose as a physiological indicator of Al toxicity in tree roots, we quantified callose formation in roots of Norway spruce (Picea abies (L.) Karst.) seedlings. Seedlings were grown in simulated soil solutions in the presence or absence (control) of Al under controlled conditions. In seedlings grown in solutions containing 280 microM Al, callose concentrations in roots were twice as high as control values after 6 h of Al treatment and 5 times higher than control values after 1 day. Thereafter, root callose concentrations gradually decreased and were only twice as high as control values after 7 days. The presence of various Al concentrations in the simulated soil solutions indicated that callose was induced by a relatively low Al concentration (84 microM). We conclude that callose in tree roots is an indicator of Al toxicity.

Topics: Aluminum; beta-Glucans; Glucans; Picea; Plant Roots; Seedlings; Trees

Curdlan and other beta-1,3-D-glucans form right-handed triple helices, and it has been believed that the intermolecular H-bond is present at the center of the helix to maintain the structure. In this H-bond model, three secondary OH groups form an inequilateral hexagonal shape perpendicular to the helix axis. This hexagonal form seems to be characteristic for beta-1,3-D-glucans and is widely accepted. We carried out MOPAC and ab initio calculations for the curdlan helix, and we propose a new intermolecular H-bonding model. In our model, the H-bonds are formed between the O2-atoms on different x-y planes along the curdlan helix, hence the H-bonds are not perpendicular to the helix axis. The new H-bonds are connected along the helix, traversing three curdlan chains to make a left-handed helix. Therefore, the H-bonding array leads to a reverse helix of the main chain. According to our MOPAC calculation, this model is more stable than the previous one. We believe that the continuous H-bonding array is stabilized by cooperative phenomena in the polymeric system.

Topics: beta-Glucans; Carbohydrate Conformation; Hydrogen Bonding; Protein Structure, Secondary

Carboxymethylated chitin, gellan gum, and curdlan gels were soaked in a simulated body fluid (SBF) having ion concentrations nearly equal to those of human blood plasma. Some of the gels had been soaked in a saturated Ca(OH)(2) solution, while others had not. The carboxymethylated chitin and gellan gum gels have carboxyl groups, while the curdlan gel has hydroxyl groups. None of the gels formed apatite on their surfaces in the SBF when they had not been subjected to the Ca(OH)(2) treatment, whereas the carboxymethylated chitin and gellan gum gels formed apatite on their surfaces when they had been subjected to the Ca(OH)(2) treatment. The curdlan gel did not form an apatite deposit even after the Ca(OH)(2) treatment. Apatite formation on the carboxymethylated chitin and gellan gum gels was attributed to the catalytic effect of their carboxyl groups for apatite nucleation, and acceleration of apatite nucleation from released Ca(2+) ions. This result provides a guiding principle for obtaining apatite-organic polymer fiber composites. This composite is expected to have an analogous structure to that of natural bone.

Topics: Apatites; beta-Glucans; Biomimetics; Body Fluids; Bone Substitutes; Calcium Hydroxide; Carbon Dioxide; Chitin; Gels; Glucans; Molecular Conformation; Polymers; Polysaccharides, Bacterial

This paper focuses on the development of MALDI sample preparation protocols for the analysis of a bioactive beta-(1 --> 3) polysaccharide, i.e. Curdlan. The crude Curdlan sample was first separated into a low molecular weight water-soluble portion and a high molecular weight water-insoluble portion. The water-soluble portion was analyzed using a standard MALDI sample preparation method developed for dextran analysis. Two low-mass (<4000 Da) polysaccharide distributions differing by 16 Da were observed. For the analysis of the water-insoluble portion, several sample preparation protocols were evaluated using GPC-fractionated samples. A sample preparation method based on the deposition of the analyte solution with a mixture of 2,5-dihydroxybenzoic acid (DHB) and 3-aminoquinoline (3AQ) matrices in dimethyl sulfoxide (DMSO) at elevated temperature of 70 degrees C was found to reliably produce good MALDI spectra. MALDI analysis of the water-insoluble Curdlan portion gave number-average (Mn) and weight-average (Mw) molecular weights and polydispersity of 8000 Da, 8700 Da, and 1.10, respectively.

Topics: Alcaligenes; Aminoquinolines; beta-Glucans; Dimethyl Sulfoxide; Gentisates; Glucans; Hydroxybenzoates; Indicators and Reagents; Solvents; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Curdlan jelly was prepared by heating an aqueous curdlan suspension at 70 degrees C for 5 min. Theophylline, as a model drug, was entrapped in the jelly network. Curdlan jelly had a hardness comparable with that of commercially available jelly for confectionary. Syneresis was observed for 8 days after the preparation and was not detected during the experimental term from the gel prepared from 10% w/v curdlan suspension. Release of theophylline from the jellies was sustained, and was increasingly delayed with an increase in the curdlan concentration. An aqueous curdlan suspension was studied by means of differential scanning calorimetry (DSC) up to 80 degrees C and down to -25 degrees C, and subsequent re-heating to 30 degrees C. Enthalpy increased with the concentration of curdlan, while the temperature at the onset of the endothermic peak decreased with the concentration of curdlan. The enthalpy due to thermal gelation of 1 mg curdlan was 12.2 mJ. An increase in curdlan concentration decreased the enthalpy and lowered the onset temperature of the endothermic peak during the DSC re-heating scan. The results are due to an increase in the amount of non-freezing water and freezing bound water and a decrease in free water. The number of water molecules entrapped in the curdlan jelly as non-freezing water was 8.1 per glucopyranose residue.

Topics: beta-Glucans; Calorimetry; Chemistry, Pharmaceutical; Delayed-Action Preparations; Glucans; Humans; Polysaccharides, Bacterial; Suspensions; Theophylline

Although (1-->3)-beta-d-glucans, which are one of major fungal cell wall components, are known to activate invertebrate innate immune systems, their activities on mammalian cells remain elusive. Here, we report their activities on mouse macrophages. Among the various (1-->3)-beta-d-glucans, curdlan, a linear (1-->3)-beta-d-glucan, although not branched beta-glucans, exhibits significant activity to stimulate nuclear factor-kappaB in macrophages. The activity of curdlan is dramatically enhanced by pretreatment with sodium hydroxide or dimethyl sulfoxide, which disrupts multiple-stranded helices of (1-->3)-beta-d-glucans, and is dose-dependently inhibited by a (1-->3)-beta-d-glucan-binding protein and by laminarioligosaccharides with (1-->3)-beta-d-glucosidic linkages. Intriguingly, the activity of curdlan is also augmented by incubation with zymolyase, which releases (1-->3)-beta-d-glucans with a single helical structure from the glucan-networks assembled by multiple-stranded helices. The activation of macrophages culminates in the production of inducible nitric-oxide synthase, tumor necrosis factor-alpha, and macrophage inflammatory protein-2. Furthermore, a dominant-negative mutant of MyD88, an adaptor protein mediating signaling through the Toll-like receptor/inerleukin-1 receptor-like (TIR) domain, inhibits the activation of macrophages by curdlan. These results strongly suggest that macrophages respond to linear (1-->3)-beta-d-glucans, possibly released from fungal cell walls, via a receptor(s) harboring the TIR domain, such as a Toll-like receptor, to induce inflammatory reactions.

Topics: Adaptor Proteins, Signal Transducing; Animals; Antigens, Differentiation; beta-Glucans; Blotting, Northern; Blotting, Western; Cell Line; Chemokine CXCL2; Dimethyl Sulfoxide; Dose-Response Relationship, Drug; Escherichia coli; Fungal Proteins; Genes, Dominant; Genes, Reporter; Glucans; Hydrolases; Immunity; Macrophage Activation; Macrophages; Mice; Monokines; Myeloid Differentiation Factor 88; NF-kappa B; Nitric Oxide Synthase; Nitric Oxide Synthase Type II; Oligosaccharides; Protein Binding; Protein Structure, Tertiary; Receptors, Immunologic; RNA, Messenger; Signal Transduction; Sodium Hydroxide; Temperature; Time Factors; Tumor Necrosis Factor-alpha

Cellulomonas flavigena KU produces large quantities of an insoluble exopolysaccharide (EPS) under certain growth conditions. The EPS has previously been shown to be a glucose polymer and to have solubility properties similar to curdlan, a beta-1,3-D-glucan produced by Alcaligenes faecalis var. myxogenes 10C3K. Furthermore, EPS purified by alkaline extraction stains with aniline blue, a dye specific for curdlan-type polysaccharides. However, EPS-producing colonies of C. flavigena KU do not stain on aniline blue agar as do those of curdlan-producing bacteria. These facts prompted a more thorough structural analysis of the EPS. Here we report that purified EPS is indeed identical to curdlan in primary structure, but that the native form of the EPS may differ from curdlan in physical conformation.

Topics: Aniline Compounds; beta-Glucans; Carbohydrate Conformation; Cellulomonas; Fluorescent Dyes; Glucans; Magnetic Resonance Spectroscopy; Methylation; Polysaccharides, Bacterial; Solubility; Spectrophotometry, Infrared

A two-stage pH control method was employed in batch fermentation of curdlan by Alcaligenes faecalis WX-C12 where cell-growth stage was constantly controlled at pH 7.0 and stationary stage was controlled at a constant pH as well. The influence of pH control on the curdlan production was investigated. The optimal pH control of batch process for curdlan production was obtained when cell-growth stage was controlled at pH 7.0 and stationary stage was constantly controlled at pH 5.6. Production and productivity of curdlan, QP and YP/S reached 28.19 g/L, 291 mg/(L.h), 132.27 mg/(L.h.g) and 0.659, an improvement of 20.4%, 38.1%, 38.1% and 29.5% compared to a pH uncontrolled operation respectively.

Topics: Alcaligenes; beta-Glucans; Fermentation; Glucans; Hydrogen-Ion Concentration

Inflammatory airway responses to bioaerosols and to their active compounds, such as endotoxin and beta(1 --> 3)-glucan, vary between individuals. These differences may be explained by variation in cytokine responsiveness, which can be assessed by in vitro stimulation tests with isolated blood leukocytes or lung macrophages. In large-scale population studies, ex vivo induced cytokine production may also be tested with a more simple 'whole blood assay' (WBA). However, applicability of a WBA to characterize a subject's responsiveness depends largely on its reproducibility. This study was conducted to: 1) assess the within- and between-subject variability in cytokine production in a WBA after stimulation with endotoxin or beta(1 --> 3)-glucan; and 2) to determine under which conditions this test is most discriminating between subjects and most reproducible within subjects. Blood was collected from 14 healthy volunteers, of whom 10 also participated on a second occasion. Each blood sample was used in two WBA tests; the first WBA was initiated two hours and the second 26 hours after venapuncture. The WBA test itself comprised overnight incubation with serial dilutions of endotoxin [lipopolysaccharide (LPS)] and curdlan (a beta(1 --> 3)-glucan), after which blood cell supernatant was collected. Interleukin(IL)-1beta, IL6, IL8 and tumor necrosis factor alpha (TNFalpha) were determined in the supernatant. In all individuals, a dose-dependent production of cytokines was observed for both LPS and curdlan. For all cytokines, variation between subjects was higher than within subjects, and this was most pronounced for IL1beta and IL6. There was moderate-to-high correlation in the induced release of all four cytokines, and between cytokine release induced by LPS or curdlan. Optimal stimulation concentrations were 6.25 and 12.5 ng/mL for endotoxin and 12500 and 25000 ng/mL for curdlan. Cytokine production in WBA initiated 26 hours after venapuncture showed lower between-subject and larger within-subject variance, thus favoring an early initiation of the assay. In conclusion, measuring endotoxin- or glucan-induced cytokine production in a WBA initiated within two hours after venapuncture appears to be an effective method to determine a person's cytokine responsiveness, at least in healthy naive subjects.

Topics: Adult; Aerosols; Analysis of Variance; beta-Glucans; Cytokines; Dose-Response Relationship, Drug; Environmental Exposure; Female; Glucans; Humans; Lipopolysaccharides; Male

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

A significant reduction was observed for serum and hepatic cholesterol concentrations in the rats fed diet containing a 5% partially hydrolyzed curdlan (PHCD), whereas only the hepatic cholesterol concentration was decreased in the curdlan (CD)-fed rats. The cecal contents in the CD group contained a significantly larger amount of short-chain fatty acids, but not those in the PHCD group. CD, but not PHCD, significantly increased the population of cecal bifidobacteria. From the in vitro fermentation test with cecal contents from cellulose powder (CP) and CD-fed rats, PHCD proved to be easily fermented by both cecal contents; incidentally CD was more susceptible to the cecal contents from CD-fed rats than to those from CP-fed rats. These results suggest that PHCD is involved in the modulation of lipid metabolism and intestinal microflora through a different manner from the native CD in rats.

Topics: Animals; beta-Glucans; Bifidobacterium; Cecum; Cholesterol; Feces; Fermentation; Glucans; Hydrolysis; Liver; Male; Rats; Rats, Sprague-Dawley

Chemically fully sulfated polysaccharides including xylan (-->4Xylbeta-(1-->4)Xylbeta1-->), amylose (-->4Glcalpha-(1-->4)Glcalpha1-->), cellulose (-->4Glcbeta-(1-->4)Glcbeta1-->), curdlan (-->3Glcbeta-(1-->3)Glcbeta1-->) and galactan (-->3Galbeta-(1-->3)Galbeta1-->), which have been isolated from Korean clam, were prepared, and their anticoagulant activity was investigated. The results strongly suggest that the activity might not be depending on anomeric configuration (alpha or beta) or monosaccharide species but on the glycosidic linkage, either (1-->3) or (1-->4). 1H NMR studies of these modified polysaccharides show that the neighboring sulfate groups at the C-2 and C-3 positions might have caused the conformational changes of each monosaccharide from 4C(1) to 1C(4). Furthermore, the effect of 6-sulfate residues on the anticoagulant activity was investigated using a specific desulfated reaction for the chemically fully sulfated polysaccharides. The 6-sulfate group is very important in determining anticoagulant activity of (1-->3)-linked polysaccharides, whereas the activity is not affected by presence or absence of the 6-sulfate group in (1-->4)-linked polysaccharides.

Topics: Alcaligenes; Amylose; Anticoagulants; beta-Glucans; Betula; Carbohydrate Conformation; Cellulose; Glucans; Molecular Structure; Molecular Weight; Nuclear Magnetic Resonance, Biomolecular; Polysaccharides; Solanum tuberosum; Spectrophotometry, Infrared; Sulfates

Genes involved in the production of the extracellular (1-->3)-beta-glucan, curdlan, by Agrobacterium sp. strain ATCC 31749 were described previously (Stasinopoulos et al., Glycobiology 9:31-41, 1999). To identify additional curdlan-related genes whose protein products occur in the cell envelope, the transposon TnphoA was used as a specific genetic probe. One mutant was unable to produce high-molecular-mass curdlan when a previously uncharacterized gene, pss(AG), encoding a 30-kDa, membrane-associated phosphatidylserine synthase was disrupted. The membranes of the mutant lacked phosphatidylethanolamine (PE), whereas the phosphatidylcholine (PC) content was unchanged and that of both phosphatidylglycerol and cardiolipin was increased. In the mutant, the continued appearance of PC revealed that its production by this Agrobacterium strain is not solely dependent on PE in a pathway controlled by the Pss(AG) protein at its first step. Moreover, PC can be produced in a medium lacking choline. When the pss(AG)::TnphoA mutation was complemented by the intact pss(AG) gene, both the curdlan deficiency and the phospholipid profile were restored to wild-type, demonstrating a functional relationship between these two characteristics. The effect of the changed phospholipid profile could occur through an alteration in the overall charge distribution on the membrane or a specific requirement for PE for the folding into or maintenance of an active conformation of any or all of the structural proteins involved in curdlan production or transport.

Topics: Alkaline Phosphatase; Amino Acid Sequence; beta-Glucans; Cardiolipins; CDPdiacylglycerol-Serine O-Phosphatidyltransferase; Choline; Cloning, Molecular; Cyclin-Dependent Kinases; DNA Transposable Elements; Escherichia coli; Escherichia coli Proteins; Gene Deletion; Genetic Vectors; Glucans; Membrane Proteins; Molecular Sequence Data; Molecular Weight; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Polysaccharides, Bacterial; Recombinant Proteins; Rhizobium; Sequence Alignment

The viscoelastic property of curdlan solution in dimethyl sulfoxide (DMSO) was investigated. We discuss the difference in the viscoelastic properties of curdlan solution in DMSO and that in 0.1 N NaOH aqueous solution. The viscoelastic function for curdlan solution in 0.1 N NaOH aqueous solution showed the Newtonian flow at the concentration of curdlan as high as 10 wt %. On the other hand, the Newtonian flow was observed in the concentrations below 7 wt % for curdlan solution in DMSO, and the plateau region appeared beyond this concentration. It was revealed by small angle x-ray diffraction measurements that the difference in the mechanical property would be originated from the difference in the network structure. The overlapping concentration c* was calculated on the basis of the mean field theory, and disagreement between theoretical prediction and experimental result was shown. We clarified that the above disagreement can be explained by the polydispersity of the curdlan sample, assuming adequate distribution functions. The static structure of the gel prepared by adding water to curdlan solution in DMSO was investigated. It was clarified by the dynamic viscoelasticity measurement that the cross-linking density increases with increasing the water content.

Topics: beta-Glucans; Dimethyl Sulfoxide; Elasticity; Glucans; Polysaccharides, Bacterial; Scattering, Radiation; Solutions; Viscosity; Water; X-Ray Diffraction

Significant increases in the amounts of short-chain fatty acids and lactate, and in numbers of bifidobacteria were observed in the cecum of curdlan (CD) -fed rats as compared with those of cellulose-fed ones. The in vitro proliferation of 5 species of bifidobacteria was markedly increased in the cultures containing the supernatant obtained from the cecal contents of CD-fed rats. These findings suggest that bifidus factors have been produced in the cecum of CD-fed rats.

Topics: Animals; beta-Glucans; Bifidobacterium; Cecum; Cell Division; Colony Count, Microbial; Dietary Fiber; Glucans; In Vitro Techniques; Male; Rats; Rats, Sprague-Dawley

Curdlan is a natural beta-1,3-glucan produced by Agrobacterium biovar 1. In this study, the anticoagulant activity of sulfoalkyl derivatives of curdlan was investigated by carrying out activated partial thromboplastin time (APTT) assay and compared with that of o-sulfonated curdlan. Approximately 100-fold higher concentration of o-sulfonated curdlan than heparin was required to obtain the same level of the clotting time. Anticoagulant activity of curdlan derivatives was dependent on the degree of sulfation in prolonging the clotting time. However, the chain length of the substituent did not play a role in prolonging the clotting time. The curdlan derivatives enhanced thrombin inhibition by mediating through antithrombin III. The inhibition of thrombin by o-sulfonated curdlan was found to be approximately 10-fold weaker than that by heparin.

Topics: Anticoagulants; beta-Glucans; Blood Coagulation; Electrophoresis, Agar Gel; Glucans; Humans; In Vitro Techniques; Magnetic Resonance Spectroscopy; Molecular Weight; Prothrombin; Spectrophotometry, Infrared; Spectroscopy, Fourier Transform Infrared; Sulfur

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

Self-assembled hydrogel nanoparticles were synthesized from carboxymethylated (CM)-curdlan, substituted with a sulfonylurea (SU) as a hydrophobic moiety for self-assembly. The degree of SU substitution was 2.4, 5.6, or 7.2 SU groups per hundred anhydroglucose units of curdlan. The physicochemical properties of the self-assembled hydrogel nanoparticles (DS 2.4, DS 5.6, and DS 7.2) in aqueous media were characterized by dynamic light scattering, transmission electron microscopy, and fluorescence spectroscopy. The mean diameter of all samples was less than 300 nm with a unimodal size distribution. The critical aggregation concentrations (CAC) of self-assembled hydrogel nanoparticles in distilled water were 4.2 x 10(-2), 3.1 x 10(-2) and 1.9 x 10(-2) mg/ml for DS 2.4, 5.6 and 7.2, respectively. The loading and release of all-trans retinoic acid (ATRA) was studied. The ATRA loading efficiencies and loading contents of CM-curdlan/SU nanoparticles increased as the degree of SU substitution increased. The ATRA release rate was controlled by the degree of substitution and drug-loading. For specific interaction with a hepatic carcinoma cell line (HepG2), CM-curdlan was additionally conjugated with lactobionic acid (LBA; galactose moiety) (5.5 LBA molecules per hundred glucose units). HepG2 was strongly luminated by ligand-receptor interactions with fluorescence-labeled LBA/CM-curdlan/SU hydrogel nanoparticles. The luminescence was not observed for other control cases. It is concluded that LBA/CM-curdlan/SU hydrogel nanoparticles are a useful drug carrier for the treatment of liver cancer, because of the potential immunological enhancement activities of CM-curdlan in the body, the ligand-receptor mediated specific interactions, and the controlled release of the anti-cancer drug.

Topics: Animals; Antineoplastic Agents; beta-Glucans; Carbohydrate Sequence; Cells, Cultured; Delayed-Action Preparations; Glucans; Hydrogels; Liver Neoplasms, Experimental; Microscopy, Confocal; Microscopy, Electron; Microspheres; Molecular Sequence Data; Polysaccharides, Bacterial; Scattering, Radiation; Spectrometry, Fluorescence; Sulfonylurea Compounds; Tumor Cells, Cultured

Genes essential for the production of a linear, bacterial (1-->3)-beta-glucan, curdlan, have been cloned for the first time from Agrobacterium sp. ATCC31749. The genes occurred in two, nonoverlapping, genomic fragments that complemented different sets of curdlan( crd )-deficient transposon-insertion mutations. These were detected as colonies that failed to stain with aniline blue, a (1-->3)-beta-glucan specific dye. One fragment carried a biosynthetic gene cluster (locus I) containing the putative curdlan synthase gene, crdS, and at least two other crd genes. The second fragment may contain only a single crd gene (locus II). Determination of the DNA sequence adjacent to several locus I mutations revealed homology to known sequences only in the cases of crdS mutations. Complete sequencing of the 1623 bp crdS gene revealed highest similarities between the predicted CrdS protein (540 amino acids) and glycosyl transferases with repetitive action patterns. These include bacterial cellulose synthases (and their homologs), which form (1-->4)-beta-glucans. No similarity was detected with putative (1-->3)-beta-glucan synthases from yeasts and filamentous fungi. Whatever the determinants of the linkage specificity of these beta-glucan synthases might be, these results raise the possibility that (1-->3)-beta-glucans and (1-->4)-beta-glucans are formed by related catalytic polypeptides.

Topics: Amino Acid Sequence; beta-Glucans; Carbohydrate Conformation; Cloning, Molecular; DNA Restriction Enzymes; Escherichia coli; Glucans; Glucosyltransferases; Membrane Proteins; Molecular Sequence Data; Mutagenesis; Rhizobium; Schizosaccharomyces pombe Proteins; Sequence Alignment; Substrate Specificity

Changes of intracellular nucleotide levels and their stimulatory effects on curdlan synthesis in Agrobacterium species were investigated under different culture conditions. Under nitrogen-limited conditions where curdlan synthesis was stimulated, intracellular levels of UMP were as high as 87 and those of AMP were 78 nmol/mg of cellular protein, while those under nitrogen-sufficient conditions were lower than 45 nmol/mg-protein. The levels of other nucleotides such as UDP, UTP, UDP-glucose, ADP, ATP, and ADP-glucose were lower than 30 nmol/mg-protein under both nitrogen-limited and sufficient conditions. The time profiles of curdlan synthesis and cellular nucleotide levels showed that curdlan synthesis had a positive relationship with intracellular levels of UMP and AMP. After the ammonium concentration in the medium fell below 0.1 g/L, intracellular levels of UMP and AMP increased, followed by curdlan synthesis. However, no significant changes in the specific activities of UMP kinase, UDP kinase, and UDP-glucose pyrophosphorylase were observed during cultivation. In vitro enzyme reactions for the synthesis of UDP-glucose, which serve as a precursor for curdlan synthesis, demonstrated that the synthesis of UDP-glucose increased with the increase of UMP concentration. In contrast, AMP had no effect on UDP-glucose synthesis at all. Addition of UMP in the medium increased the curdlan synthesis, whereas curdlan synthesis was inhibited in the presence of AMP. From these results, we concluded that only the higher intracellular UMP levels caused by nitrogen limitation in the medium enhance the metabolic flux of curdlan synthesis by promoting cellular UDP-glucose synthesis.

Topics: Adenosine Monophosphate; beta-Glucans; Culture Media; Enzymes; Glucans; Nitrogen Compounds; Nucleoside-Diphosphate Kinase; Nucleoside-Phosphate Kinase; Polysaccharides, Bacterial; Rhizobium; Time Factors; Uridine Monophosphate; UTP-Glucose-1-Phosphate Uridylyltransferase

Curdlan was approved for use by the FDA in December 1996 as a formulation aid, processing aid, stabilizer and thickener or texturizer for use in food. It has been evaluated for safety by a series of animal studies and in vitro tests including acute, subchronic and chronic toxicity studies and reproduction and carcinogenicity studies. In addition, nutritional studies in rodents and tolerance and metabolic studies in man have been carried out. The only effects seen in these studies were reductions in weight gain at the higher dietary concentrations due to the replacement of part of the diet by curdlan, which is calorifically inert. No evidence of any toxicity or carcinogenicity nor of any effects on reproduction was seen, although there was an effect on body weights of the pups with the 15% diet, which was shown in additional studies to be due to the reduced food availability in the animals at this dose level. There was no evidence of effects on the nutritional status of the animals nor on the absorption of minerals. This reviews the available toxicological data on curdlan.

Topics: Animal Nutritional Physiological Phenomena; Animals; beta-Glucans; Carcinogens; Dogs; Embryonic and Fetal Development; Evaluation Studies as Topic; Female; Food Additives; Glucans; Humans; Male; Mutagenicity Tests; Polysaccharides, Bacterial; Rabbits; Rats; Rats, Sprague-Dawley; Reproduction; Toxicity Tests

The study was conducted to elucidate the effects of orally administered indigestible saccharides (IDS) on immunoresponses of the intestinal tracts, especially secretion and excretion of immunoglobulin A (IgA). Male 4-week-old Sprague-Dawley rats were fed diets containing several kinds of IDS (cellulose, corn husk, glucomannan, curdlan and lactulose) at 5% for three weeks. The results indicated that the proportion of IgA-presenting lymphocytes in the cecal mucosa of the tested IDS groups increased significantly or tended to increase compared with that of the cellulose group. No significant differences among the experimental groups were observed in the CD4(+)- and CD8(+)-presenting lymphocytes and the CD4+/CD8+ ratios in the small intestine, cecum and mesenteric lymph nodes. IgA amounts in the cecal contents increased significantly in the glucomannan and curdlan groups as compared with that in the cellulose group. The inconsistent results were observed in the cecal IgA amounts of the lactulose group. Although IgA excretion into feces increased periodically in the cellulose, hardly any changes were observed in the glucomannan and curdlan groups. These results revealed that IgA secretion from cecal mucosa to contents was promoted, and its excretion to feces was decreased by the oral administration of highly fermentable IDS, respectively, while non- or low-fermentable IDS functioned adversely to IgA responses in the intestinal tract. It is suggested that the response of IgA in the intestinal immune system differs with the type of IDS ingested.

Topics: Administration, Oral; Animals; beta-Glucans; Cathartics; CD4-CD8 Ratio; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cecum; Cellulose; Dietary Fiber; Enzyme-Linked Immunosorbent Assay; Feces; Flow Cytometry; Glucans; Immunoglobulin A, Secretory; In Vitro Techniques; Intestinal Mucosa; Lactulose; Male; Mannans; Rats; Rats, Sprague-Dawley; T-Lymphocyte Subsets; Weight Gain

The effects of curdlan (CD) and gellan gum (GG), bacteria-producing polysaccharides, on lipid concentrations of serum and liver, fecal bile acid composition and intestinal fermentation products were studied in rats fed diets containing cellulose powder (CP), CD or GG at 5% for 4 wk. The cecal weight of the CD group increased significantly as compared to that of the other two groups and the pH of its contents was significantly low. The gastrointestinal transit time in the GG group was significantly shorter than that in the CP and CD groups. No significant inter-group differences were observed in the serum concentrations of total cholesterol and HDL-cholesterol, but a significant decrease was observed in the hepatic total cholesterol concentration of the CD group as compared to that of the CP and GG groups. No significant difference in the total bile acid excretion in feces was observed among the groups, but significantly low values were observed in the proportion of secondary bile acids in the CD group as compared to those of the CP and GG groups. Amounts of short-chain fatty acids (acetic, propionic and butyric acid) and lactic acid in the cecal contents were significantly higher in the CD group than in the other two groups. These results reveal that dietary CD is easily degraded and fermented by intestinal bacteria in the cecum and lowers cholesterol concentration in the liver, while dietary GG shortens the gastrointestinal transit time, suggesting the promotion of evacuation.

Topics: Animals; beta-Glucans; Bile Acids and Salts; Cecum; Cholesterol; Diet; Feces; Fermentation; Gastrointestinal Transit; Glucans; Hydrogen-Ion Concentration; Lipid Metabolism; Lipids; Liver; Male; Polysaccharides, Bacterial; Rats; Rats, Sprague-Dawley

Glucan, a folded high-molecular-weight polysaccharide, has multiple effects in animals when administered intravenously or intraperitoneally, but not when administered by inhalation. The hypotheses tested were whether intratracheal administration of glucan can cause lung damage and whether some of the resulting lung injury is immunologically mediated. There was a dose-response relationship between the amount of intratracheally injected glucan and the extent of pulmonary histologic abnormalities, which consisted of peribronchiolar and intraalveolar infiltration with chronic inflammatory cells. An attempt to adoptively transfer increased susceptibility to glucan induced lung injury was made. Cells cultured with glucan were transferred into naive recipients before intratracheal glucan exposure. The extent of pulmonary inflammation that occurred as a result of intratracheal injection of glucan was not affected by transfer of cultured cells from glucan-treated animals. However, high concentrations of glucan in culture did produce cells with the appearance of lymphoblasts. These data indicate that glucan induces lung injury, but that there is no evidence of cell mediation of pulmonary injury induced by intratracheal exposure to glucan.

Topics: Animals; beta-Glucans; Cells, Cultured; Dose-Response Relationship, Drug; Glucans; Lung; Male; Mice; Mice, Inbred C3H; Pneumonia; Polysaccharides, Bacterial

We reported in the previous paper that rats fed a curdlan diet showed significant increases in the weight of the cecum and its contents, a decrease in fecal wet weight, a retardation in the transit time of the gastrointestinal tract and morphological changes of the ileal and cecal mucosal surface when compared with the rats fed a cellulose diet. In the present study, we intended to learn if the curdlan effects on the morphological structure of intestinal mucosa were reversible. When rats were fed on the curdlan diet for 2 weeks followed by a cellulose diet for another 2 weeks, the cecum and cecal contents were not different from those of the cellulose group. The transit time of the gastrointestinal tract of the curdlan-followed-by-cellulose group was shorter than that of the curdlan group, whereas it was longer than that of the cellulose group. In scanning electron micrographs, the ileal villi of the curdlan-followed-by-cellulose group were normal, as in the cellulose group. However, their ileal and cecal microvilli were similar to those of the curdlan group, that is, the microvilli were crowded and more tightly packed, and some appeared to have been squeezed out. From these results, it was concluded that the effects of the curdlan feeding were only partially reversible, but the effects on the surface structure of intestinal mucosa were still sustained even after curdlan feeding of 2 weeks was discontinued. This might result from response to the high viscosity of the intestinal contents remaining after discontinuation of the curdlan.

Topics: Animals; beta-Glucans; Cecum; Cellulose; Colon; Dietary Fiber; DNA; Feces; Gastrointestinal Transit; Glucans; Ileum; Intestinal Mucosa; Male; Microscopy, Electron, Scanning; Microvilli; Organ Size; Polysaccharides, Bacterial; Proteins; Rats; Rats, Sprague-Dawley

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

Schizophyllan, a water-soluble (1-->3)-beta-D-glucan with a triple-helical conformation, adheres to yeast glucan and curdlan gel. As the molecular weight of schizophyllan decreases, both its adhesion to the water-insoluble glucans and its ability to promote the regeneration of yeast protoplasts are reduced. Therefore, we hypothesize that schizophyllan can surround yeast protoplasts by adhering to a fragment of yeast glucan remaining or/and resynthesized on the cell surface and that this encapsulation allows regeneration of the protoplast cells to occur at very high frequency.

Topics: beta-Glucans; Carbohydrate Conformation; Cell Wall; Glucans; Molecular Weight; Protoplasts; Saccharomyces cerevisiae; Schizophyllum; Sizofiran; Spectrophotometry; X-Ray Diffraction

Novel (1-->3)-beta-D-glucans (GPBCD, GPECD, GP6CD, and GP3CD) having reducing glucose side chains were prepared from a linear (1-->3)-beta-D-glucan (curdlan: CD) with halogeno glucose isopropylidene derivatives in dimethyl sulfoxide containing dimsyl sodium, followed by treatment with 40% trifluoroacetic acid to remove protecting isopropylidene groups. The side chain glucose moiety was linked or directly or through a spacer at various positions except for its anomeric carbon.

Topics: beta-Glucans; Carbohydrate Conformation; Carbohydrate Sequence; Glucans; Glucose; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Polysaccharides, Bacterial

The effects of curdlan and gellan gum on the gastrointestinal function were studied, and the morphological structure of the intestinal mucosal surface was observed by scanning electron microscopy of rats fed curdlan and gellan gum diets for four weeks. The rats fed the curdlan diet showed a significant increase in the weight of the cecum and its contents and a decrease in fecal weight as compared to the rats fed a cellulose diet. On the other hand, the rats fed the gellan gum diet showed a weight loss in cecal contents and weight gain in colonic contents. The transit time of the gastrointestinal tract was extended by curdlan supplementation whereas it was shortened by gellan gum supplementation. The surface structures of the ileal and cecal mucosa were markedly abnormal in the rats fed the curdlan diet: the microvilli were tightly packed and had fallen out at places. In the gellan gum-fed rats, the tops of the ileal and cecal microvilli adhered to one another and were covered with their contents. There was no difference in the surface structure of colonic mucosa among the cellulose, curdlan and gellan gum diet groups.

Topics: Animals; beta-Glucans; Gastrointestinal Contents; Gastrointestinal Transit; Glucans; Intestinal Mucosa; Male; Microscopy, Electron, Scanning; Polysaccharides, Bacterial; Rats; Rats, Sprague-Dawley

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

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

Tablets (300 mg) having two different surface areas were prepared from spray-dried particles of curdlan (100 mg)/theophylline(200 mg). Drug release from the tablets was studied in vitro and in vivo. The in vitro drug release from a tablet with a larger surface area (Tab L) was faster than that with a smaller one (Tab S). The water uptake of Tab L was larger than that of Tab S, probably due to the difference in the tablets' surface areas. However, the water uptake was not a rate-determining step for the drug release from curdlan tablets containing a large amount of theophylline. A straight line was obtained when release % was plotted vs. time. The slope of each curve was calculated as 0.59 for Tab L and 0.58 for Tab S. This indicates that the release mechanism is non-Fickian diffusion controlled. In addition, the curdlan tablets or theophylline powder were administered orally to 5 healthy volunteers, and saliva concentrations of theophylline were determined. Each saliva concentration was converted to plasma concentration using the saliva to plasma ratio of the drug in each subject. The AUC of Tab L was nearly the same as that of powder, while the AUC of Tab S was smaller than that of powder. The mean residence times (MRTs) of theophylline powder, Tab S and Tab L were 11.1 +/- 1.5, 25.4 +/- 6.3 and 17.1 +/- 1.5 h (N = 4-5, mean +/- S.D.), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: beta-Glucans; Biological Availability; Female; Glucans; Humans; Male; Reference Values; Saliva; Tablets; Theophylline

The use of curdlan, a natural beta-1,3-glucan, in the preparation of sustained release suppositories was studied in vitro. To prepare the suppositories, indomethacin, prednisolone or salbutamol sulfate was mixed with curdlan gel. Preparation conditions, including heating time and curdlan concentrations of 5 and 10%, had little effect on the drug release. The tonicity (hypotonic or isotonic) of the media for the suppository preparation and for in vitro drug release study also had little effect on drug release. However, the heating temperature during gel preparation, the drug amount in the suppository and the type of release media did affect drug release. It was found that drug release was sustained and diffusion-controlled in the three drugs. And finally, curdlan can be applicable for use in a sustained release suppository.

Topics: Albuterol; beta-Glucans; Delayed-Action Preparations; Glucans; Hot Temperature; Indomethacin; Prednisolone; Suppositories

Taxonomic characteristics of seven bacterial strains which were isolated from soil and hydrolyze resistant curdlan were studied. These bacteria were aerobic, spore-forming rods, contained menaquinone 7 as a major quinone, contained anteiso-C15:0 and iso-C16:0 as major cellular fatty acids, had guanine-plus-cytosine contents of 50 to 52 mol%, and could be divided into two groups on the basis of physiological and chemotaxonomic characteristics and DNA-DNA hybridization data. We propose the following two new species: Bacillus curdlanolyticus for strains YK9, YK121, YK161, YK201, and YK203, with type strain YK9 (= IFO 15724); and Bacillus kobensis for strains YK205 and YK207, with type strain YK205 (= IFO 15729).

Topics: Aerobiosis; Bacillus; Bacterial Proteins; Base Composition; beta-Glucans; Carbohydrate Metabolism; Culture Media; DNA, Bacterial; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Glucans; Nucleic Acid Hybridization; Polysaccharides, Bacterial; Sequence Homology, Nucleic Acid; Soil Microbiology; Spores

A (1-->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.6), capable of hydrolysing resistant curdlan, was purified chromatographically from the culture supernatant of Bacillus circulans complex YK9 on Toyopearl HW-55F and butyl-Toyopearl 650M columns. The purified enzyme had a specific activity of 190 units mg-1 on regenerated curdlan. The molecular mass was estimated to be about 70 kDa as judged by SDS-PAGE. The enzyme had a pH optimum of approximately pH 6.0. It hydrolysed regenerated and resistant curdlans yielding predominantly laminari-biose, although the rate of hydrolysis of the former was much higher than the latter. This enzyme rapidly hydrolysed laminaran, curdlan and carboxymethyl-curdlan, but did not cleave schizophyllan and screloglucan, which have glucosyl side chains. The enzyme hydrolysed low molecular mass (1-->3)-beta-D-glucans-(mean degree of polymerization, DPn = 131, 49 and 14) and laminari-heptaose more efficiently than curdlan. It also hydrolysed laminari-hexaose and -pentaose effectively, but laminari-tetraose only slightly and it did not hydrolyse laminari-triose or -biose. The enzyme is an exo-hydrolase of curdlan and various oligomers composed of (1-->3)-beta-D-glucosidic linkages, liberating laminari-biose from their non-reducing terminals. The laminari-biose generated was in the alpha-form.

Topics: Bacillus; beta-Glucans; beta-Glucosidase; Disaccharides; Glucan 1,3-beta-Glucosidase; Glucans; Hydrogen-Ion Concentration; Hydrolysis; Molecular Weight; 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

Topics: Administration, Inhalation; Air Pollution, Indoor; Animals; beta-Glucans; Endotoxins; Glucans; Guinea Pigs; Inflammation; Lipopolysaccharides; Lung

Topics: Air Microbiology; Animals; beta-Glucans; Endotoxins; Glucans; Humans; Occupational Exposure; Polysaccharides, Bacterial

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

It has been demonstrated that both linear and branched (6-O-beta-D-glucosyl) (1-->3)-beta-D-glucans taking a single helical conformation are more effective than those taking a triple helical conformation for the activation of factor G from horseshoe crab amebocytes, as revealed by high-resolution solid-state 13C-NMR spectroscopy [Saitô, H. et al. (1991) Carbohydr. Res. 217, 181-190]. Annealing the linear glucan at 180 degrees C was essential to convert the conformation from the single helix to the triple helix. We found that heating of the glucan at such a high temperature resulted in depolymerization of the sample to molecular weight smaller than 10,000, which may influence the conformation and the above-mentioned biological activity. To eliminate ambiguity arising from the depolymerization of the glucan during annealing, we aimed to relate the biological activity to the conformation of samples whose chain lengths are identical, because the potency is known to depend on the molecular weight of the glucans. This molecular weight dependency of the potency, however, was found to be not the dominant factor, provided that the molecular weight is large enough to allow formation of the single helix conformation. Therefore, the single helical conformation of (1-->3)-beta-D-glucans is clearly demonstrated to be the dominant contributor to the activation of limulus coagulation factor G.

Topics: Animals; beta-Glucans; Blood Coagulation Factors; Carbohydrate Conformation; Glucans; Horseshoe Crabs; In Vitro Techniques; Molecular Weight; Structure-Activity Relationship

13C-Labeled curdlans were biosynthesized by Agrobacterium sp. (ATCC 31749) from culture media containing D-(1-13C)glucose, D-(6-13C)glucose, or D-(2-13C)glucose as the carbon source, and their structures were analyzed by 13C NMR spectroscopy. The labeling was mainly found in the original position, that is, C-1, C-6, or C-2, indicating direct polymerization of introduced glucose. In addition, C-3 in curdlan obtained from D-(1-13C)glucose, C-1 in curdlan obtained from D-(6-13C)glucose, and C-1 and C-3 in curdlan obtained from D-(2-13)glucose were labeled. From analysis of this labeling, the biosynthesis of curdlan was interpreted as involving five routes: (1) direct synthesis from glucose; (2) rearrangement (1-13C-->3-13C); and (3) isomerization (6-13C-->1-13C) of cleaved trioses by the Embden-Meyerhof pathway, followed by neogenesis of glucose and formation of curdlan; (4) from fructose 6-phosphate formed in the pentose cycle (2-13C-->1-13C, 3-13C); and (5) neogenesis of glucose from fragments produced in various pathways of glycolysis. The 13C-labeling at C-6 and C-2 in the starting glucoses is well preserved in the C-6 carbon and the C-1 to C-3 carbons, respectively, in the curdlan produced.

Topics: beta-Glucans; Carbon Isotopes; Glucans; Glucose; Magnetic Resonance Spectroscopy; Polysaccharides, Bacterial; Rhizobium

Bacteria capable of utilizing the water-insoluble purified extracellular (1-->3)-beta-D-glucan (curdlan) from Cellulomonas flavigena strain KU by extracellular enzymes, were isolated and characterized. Enrichment cultures from a Winogradsky column were incubated anaerobically at 55 degrees C with curdlan as the sole source of carbon. Colonies surrounded by zones of clearing were selected from subcultures on solid curdlan media. One of the isolates was chosen for further study and identified by conventional methods, API-tests with calculation of similarity coefficients and ID-scores, estimation of mol% (G+C) and DNA-DNA liquid hybridization. The isolate is a facultatively anaerobic, facultatively thermophilic Bacillus sp. Identification at the species-level was not achieved. The isolate was characterized by some rare traits among bacilli, but it remains unresolved whether it defines a new taxon.

Topics: Actinomycetales; Bacillus; Bacteriological Techniques; Base Composition; beta-Glucans; Biodegradation, Environmental; DNA, Bacterial; Glucans; Microscopy, Electron; Soil Microbiology; Solubility

Crystals of methyl 3-O-beta-D-glucopyranosyl-beta-D-glucopyranoside (methyl beta-D-laminarabioside) belong to the orthorhombic system, space group P2(1)2(1)2, with a = 14.548(2), b = 24.252(7), c = 4.938(1) A, and Z = 4. The crystal structure was solved by the direct method and refined by the full-matrix least-squares procedure to an R-value of 0.062 for 1099 observed reflections in the X-ray data. The molecular structure is similar to that of beta-D-laminarabiose. The torsional angles around the glycosidic bonds are influenced by both the existence of an intramolecular hydrogen bond at O-4'... O-5 and the exo-anomeric effect. In the nonreducing residue, the exocyclic O-5-C-5-C-6-O-6 and C-4-C-5-C-6-O-6 torsional angles are (+)gauche and trans, respectively, whereas the corresponding torsional angles in the reducing residue are (-)gauche and (+)gauche. One water molecule cocrystallizes with each disaccharide, and the crystal structure is stabilized mainly by intra- and inter-molecular hydrogen bonds involving water molecules.

Topics: beta-Glucans; Carbohydrate Conformation; Carbohydrate Sequence; Disaccharides; Glucans; Glucosides; Hydrogen Bonding; Models, Molecular; Molecular Sequence Data; Polysaccharides, Bacterial; Water; X-Ray Diffraction

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

Factor G, the coagulation enzyme from limulus amebocytes, is activated by (1-->3)-beta-D-glucan but not by endotoxin. The endotoxin-specific limulus test, which is devoid of factor G, reacts only with endotoxin. However, the conventional limulus test which includes factors G and C, reacts with not only endotoxin but also with (1-->3)-beta-D-glucan. In this study, the culture supernatant of Candida albicans in RPMI medium activated the conventional limulus test, but not the endotoxin-specific limulus test. All five rabbits inoculated intravenously with Candida albicans (1.0 x 10(7) CFU/rabbit) showed increased levels of reactivity with the conventional limulus test, whereas no elevation in the levels of the endotoxin-specific limulus test was observed in the sera of any infected rabbits. Only one serum sample from rabbit No. 5 on day 8 showed a positive Cand-tectest. The difference in the titers of the two limulus tests was suggestive of a diagnosis of Candida infection.

Topics: Amino Acid Sequence; Animals; beta-Glucans; Candida albicans; Candidiasis; Endotoxins; Glucans; Limulus Test; Molecular Sequence Data; Polysaccharides; Rabbits

To study the use of curdlan, a natural beta-1,3-glucan, in drug delivery, in vitro release studies were carried out with curdlan tablets containing theophylline. Tablets were readily prepared by compressing three different curdlan and theophylline mixtures, namely, a physical mixture, spray-dried curdlan particles with theophylline powder, and spray-dried particles of curdlan/theophylline solution. Drug release from the tablets prepared from spray-dried particles of curdlan/theophylline was lowest. The release rate was constant from 1 to 8 hr, and 59% cumulative release was obtained at 8 hr. Drug release from curdlan tablets was unaffected by pH or various ions; these curdlan tablets might also control drug release in vivo after oral administration. Application of Higuchi's equation indicated that drug release from curdlan tablets was diffusion-controlled. The release profiles of the curdlan tablets were compared to those of a commercial theophylline sustained-release tablet.

Topics: beta-Glucans; Chemistry, Pharmaceutical; Delayed-Action Preparations; Glucans; Hot Temperature; Polysaccharides, Bacterial; Tablets; Theophylline

Derivatives of curdlan and lichenan, linear (1----3)-beta-D- and (1----3/1----4)-beta-D-glucans, respectively, have been synthesised having alpha-L-arabinofuranosyl, alpha-L-rhamnosyl, beta-D-glucosyl, and beta-gentiobiosyl side chains attached at positions 6. These water-soluble derivatives, obtained by condensation of the 2,4- and 2,4-/2,3-di-O-phenylcarbamoyl derivatives of curdlan and lichenan, respectively, with appropriate ortho esters followed by saponification, were characterised by methylation analysis, g.p.c., and interaction with Congo Red. The curdlan derivatives and the lichenan derivative with few glucosyl branches were active against the Sarcoma 180.

Topics: Animals; Antineoplastic Agents; beta-Glucans; Female; Glucans; Mice; Sarcoma 180

2-Sulfoethyl, 3-sulfopropyl, and 4-sulfobutyl derivatives of the (1----3)-beta-D-glucan curdlan and the (1----3/1----4)-beta-D-glucan lichenan have been synthesised. The substituents are located mainly at positions 6. The curdlan derivatives strongly inhibited the growth of the Sarcoma 180 tumour, whereas the lichenan derivatives were inactive, indicating that a (1----3)-linked beta-D-glucan backbone is essential for activity.

Topics: Animals; Antineoplastic Agents; beta-Glucans; Female; Glucans; Mice; Sarcoma 180

beta-Laminaribiose octaacetate (2b) was prepared in greater than 50% yield from the microbial polysaccharide curdlan by specific degradation with a yeast cell-wall lytic enzyme preparation, Kitalase,and subsequent acetylation. Acetolysis of curdlan also gave alpha-laminaribiose octaacetate (2a) in 27% yield. The usefulness of these peracetates 2a and 2b as starting materials for organic synthesis was shown by converting 2b into N-acetylhyalobiuronic acid (23), the disaccharide repeating unit of hyaluronic acid. The conversion was carried out via a series of reactions, which included azidonitration of the glucal derivative and selective alkylidenation or direct tritylation to discriminate two primary hydroxyl groups existing in the disaccharide intermediates.

Topics: Acetates; Acetylation; beta-Glucans; Carbohydrate Sequence; Disaccharides; Glucans; Glycoside Hydrolases; Hyaluronic Acid; Molecular Sequence Data; Polysaccharides, Bacterial

A (1-3)-beta-D-glucan, grifolan (GRN), recovered from the peritoneal cavity after 1 d from i.p. injection contained a significant amount of anionic metabolite(s) having lower Mr than the parent GRN. In parallel with this observation, GRN induced peritoneal exudate cells exhibiting a higher level of oxidative burst than the non-stimulated, resident peritoneal cells. Chemical oxidation of GRN by active oxygen species such as (a) ascorbic acid-CuSO4, (b) hydrogen peroxide, (c) hydrogen peroxide-CuSO4, or (d) hypochlorous acid also produced anionic as well as lower Mr degradation products. Under these experimental conditions the structural changes were remarkable and in the order of a less than b less than c less than d. The products formed under the conditions (a) and (b) retained significant antitumor activity but those of (c) and (d) lost the activity. However, oxidation product(s) of curdlan, an antitumor inactive (1-3)-beta-D-glucan having no branch, by condition (d) induced significant antitumor activity. These results suggested that oxidative degradation of (1-3)-beta-D-glucans produced some temporary active metabolites and then gradually changed to the inactive form. However, these active metabolites contribute less than the parent glucan on the whole activation mechanisms of the host by (1-3)-beta-D-glucans.

Topics: Animals; Antibiotics, Antineoplastic; beta-Glucans; beta-Glucosidase; Glucan 1,3-beta-Glucosidase; Glucans; Hydrogen Peroxide; Macrophages; Male; Mice; Mice, Inbred ICR; Neutrophils; Oxidation-Reduction; Oxygen; Peritoneal Cavity; 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

We have recorded high-resolution 13C-NMR spectra of linear (curdlan) and branched (lentinan, HA-beta-glucan and its polyol and aldehyde derivatives) (1----3)-beta-D-glucans in hydrate and gel states, in order to gain insight into their gelation mechanism. Network structure of curdlan turned out to be highly heterogeneous from its motional state, from liquid-like, through intermediate, to solid-like domains. They are studied by a variety of experiments, conventional high-resolution NMR by broad-band decoupling, high-power decoupling with magic angle spinning (MAS), and cross-polarization-magic-angle-spinning (CP-MAS). Nevertheless, we found that conformations of these distinct liquid-like and solid-like domains exhibit an identical single helix conformation with a small proportion of a triple helix form, supporting our previous view as to the gelation mechanism. In contrast, the network structure of branched (1----3)-beta-D-glucans in the gel state arises mainly from the triple helix conformation. This means that gelation of branched (1----3)-beta-D-glucan proceeds from partial association of the triple helical chains, previously proposed for gelation of a linear glucan. Furthermore, we found that conversion from the single chain to the single helix was not achieved readily by hydration of over 8 h at 96% R.H. for branched glucan but the triple helix form is obtained when these samples are hydrated fully as in gel state.

Topics: beta-Glucans; Carbohydrate Conformation; Carbohydrate Sequence; Gels; Glucans; Lentinan; Magnetic Resonance Spectroscopy; Molecular Sequence Data; Polysaccharides, Bacterial

From the results of consumption experiments of guinea pig complement, Curdlan, a (1----3)-beta-D-glucan obtained from Alcaligenes faecalis var. myxogenes IFO13140, has been found to lack the ability to activate complement when unheated or preheated at either 40 degrees C or 50 degrees C. However, Curdlan heated at or above 60 degrees C increased complement consumption. This activation, dependent on the temperature of the Curdlan, was via the alternative complement pathway as assessed by cleavage of factor B into Ba and Bb fragments. These results suggest a substantial change must occur in Curdlan with heat treatment for alternative pathway activation.

Topics: Alcaligenes; Animals; beta-Glucans; Complement Activation; Complement Hemolytic Activity Assay; Complement Pathway, Alternative; Electrophoresis, Polyacrylamide Gel; Glucans; Guinea Pigs; Hot Temperature; Immunoblotting; Protein Denaturation

Topics: Antigens, Viral; beta-Glucans; Cells, Cultured; Cytopathogenic Effect, Viral; Glucans; HIV; Humans; Lentinan; Polysaccharides; Polysaccharides, Bacterial; Sulfates

The macrophage-stimulating properties of some structurally related polysaccharides were studied in vitro. When the polysaccharides were presented to the macrophages in a sterically fixed form, i.e. as microparticles, they induced the release of interleukin 1 (IL-1) from the macrophages. Microparticulate 1.3-beta-glucan (curdlan) induced nonspecific macrophage mediated tumour cell killing while 1.4-alpha-glucan (starch), 1.6-alpha-glucan (dextran), and 1.6-alpha-mannan were without effect. The corresponding soluble polysaccharides did not stimulate the macrophages. Kinetic studies showed that although IL-1 was released immediately after stimulation, the macrophages needed a time lag of several days to develop tumour cytotoxicity. The development of cytotoxicity paralleled binding of tumour cells to the macrophages. Resident and inflammatory peritoneal macrophages showed differences in their responses to the polysaccharides. Stationary, resident peritoneal macrophages stimulated by macroparticles secreted high levels of IL-1 but expressed a low cytotoxic activity, while newly recruited inflammatory macrophages released lower levels of IL-1 but readily killed the tumour cells. The influence of cyclo-oxygenase products on the IL-1 release and macrophage cytotoxicity was also investigated. When cyclo-oxygenase was blocked with indomethacin, a significantly higher release of IL-1, and then an increased cytotoxicity, were obtained with 1.3-beta-glucan stimulated macrophages. The results suggest that microparticulate polysaccharides may be useful for studies on the induction of macrophage differentiation and also for studies on nonspecific cellular immune responses in vitro and in vivo.

Topics: Animals; beta-Glucans; Cytotoxicity, Immunologic; Dose-Response Relationship, Drug; Glucans; Interleukin-1; Macrophage Activation; Macrophages; Male; Mannans; Mice; Mice, Inbred Strains; Microscopy, Electron, Scanning; Polysaccharides; Structure-Activity Relationship

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

The biosynthesis of a thermogelable, extracellular homo-beta-(1 leads to 3)-glucan called "curdlan," has been studied in batch and continuous cultures of Alcaligenes faecalis var. myxogenes. Curdlan production is associated with the poststationary phase of a nitrogen-depleted, aerobic batch culture. Exopolymer is not detected in single-stage, carbon-limited continuous cultures but curdlan can be isolated from the effluent of a nitrogen-limited chemostat operating at a dilution rate (D) of less than 0.1 h-1. A spontaneous variant of strain ATCC 21680 was isolated and found to be compatible with long-term, nitrogen-limited chemostat culture. The specific rate of curdlan production is approximately four times higher in poststationary batch cultures than in single-stage continuous fermentations. The product yield (Yp/S) associated with batch processing (nongrowing cultures) is approximately 0.5 g curdlan/g glucose, with CO2 being the only detectable by-product.

Topics: Alcaligenes; Ammonia; beta-Glucans; Culture Media; Glucans; Microbiological Techniques; Polysaccharides, Bacterial

As a guide to both strain and process improvement and based on certain assumptions concerning both glucose and energy metabolism of the process organism, Alcaligenes faecalis var. myxogenes, the theoretical "maximum" carbon (glucose) substrate to product conversion efficiency (i.e., product yield) has been estimated for "curdlan-type" beta(1 leads to 3)-glucan exopolysaccharide production in batch fermentations. Under nitrogen limitation, which promotes curdlan biosynthesis (mu = 0), the rate of glucose consumption for cellular maintenance energy (grams of glucose per gram of cells per hour) was approximately five times higher than under carbon limitation. The decrease in the theoretical "maximum" curdlan conversion efficiency of 74% to the average value of 50-56% was due primarily to the high maintenance coefficient of the nitrogen-starved culture.

Topics: Alcaligenes; beta-Glucans; Carbon Dioxide; Glucans; Glucose; Polysaccharides, Bacterial

Topics: Amylose; beta-Glucans; Carbohydrate Conformation; Crystallography; Glucans; Models, Molecular; X-Ray Diffraction

Topics: Amylose; beta-Glucans; Carbohydrate Conformation; Crystallization; Glucans; Hydrogen Bonding; X-Ray Diffraction

Topics: Amines; beta-Glucans; Chemical Phenomena; Chemistry; Glucans; Glycosaminoglycans; Heparin; Indicators and Reagents; Magnetic Resonance Spectroscopy