Glucomannan reduces neutrophil free radical production in vitro and in rats with adjuvant arthritis.

The effect of glucomannan (GM), a natural polysaccharide isolated from the yeast Candida utilis, on reactive oxygen species (ROS) generation in human neutrophils in vitro and in rats with Mycobacterium butyricum induced adjuvant arthritis (AA) was tested by the luminol/isoluminol-enhanced chemiluminescence (CL) method. In vitro, GM (500 microg/ml) significantly decreased spontaneous CL of human whole blood, while PMA (4beta-phorbol-12beta-myristate-alpha13acetate)-stimulated CL was decreased by GM in the concentrations of 100 and 500 microg/ml. To specify the site of action of GM, its effect on extra- and intracellular ROS generation in isolated neutrophils was evaluated. GM significantly decreased spontaneous and PMA-stimulated CL and it was more effective extracellularly than intracellularly. In vivo experiments included healthy animals as controls, arthritic animals without any drug administration, and arthritic animals with GM administration (once daily in the oral dose of 15 mg/kg, over a period of 28 days). On day 28, CL in whole blood, spleen and joint was monitored. Arthritic animals treated with GM showed decrease in spontaneous and PMA-stimulated CL of whole blood as well as CL of the joint, in comparison with untreated animals. The obtained findings demonstrated an antioxidant effect of GM in vitro and in rats with AA, which may be due to its free radical scavenger activity and to interaction with different receptors and/or modulation of postreceptor intracellular signalling pathways. The specific physicochemical parameters, such as structure of GM, its low molecular weight and good water solubility, play an important role in the above effects.

Degradation of high-molar-mass hyaluronan by ascorbate plus cupric ions: effects of D-penicillamine addition.

Pro- and anti-oxidative effects of an anti-rheumatoid drug, D-penicillamine (D-PN), on the kinetics of high-molar-mass hyaluronan (HA) degradation were monitored using the method of rotational viscometry. The degradation of the dissolved HA macromolecules was attained by applying the Weissberger's system comprising ascorbic acid plus cupric ions. Electron paramagnetic resonance (EPR) spectroscopy was used to identify the generated free radicals. The results obtained indicate that the initial anti-oxidative action of D-PN is followed by induction of pro-oxidative conditions due to the generation of reactive free radicals. It is speculated, however, that the latter situation may be considered as an advantageous property of D-PN. Hydroxyl radicals formed in this way may participate in decomposition of proteinases, which are believed to be responsible for the destruction of joint cartilage under rheumatoid arthritic conditions.

Beauvericin decreases cell viability of wheat.

Recently, beauvericin (BEA) has been recognized as an important toxic compound synthesized by several Fusarium strains, infecting maize, wheat, and rice, worldwide. The effects of BEA on mammalian cells have been studied; however, its effects on the function of host plant cells are largely unknown. The purpose of our work was to assess whether BEA can affect the root and leaf cells of wheat cultivar (cv.) 'Arina' seedlings, using a cytotoxicity assay and fluorescence microscopy. Toxigenicity during wheat germination was higher in BEA-treated wheat seedlings than in non-treated seedlings (control). Leaf primordial, situated at the base and the tips of treated leaves, were more affected by BEA compared to the control when assayed in medium for cell viability measured by luminescent equipment. BEA-Treated plant cells secrete adenosine triphosphate (ATP) to the extracellular matrix and invoke more luminescence by luciferase than the non-treated seedlings. Our results were confirmed by fluorescence microscopy following '4',6-diamidino-2-phenylindole' (DAPI) staining and by confocal microscopy. In addition, the bioluminescent protein luciferase was observed in the intracellular space indicating presence of ATP. The incidence of nuclear fragmentation increased significantly in cells of seedlings treated with BEA at 40 microM concentration implying that the intracellular phytotoxin BEA plays an important role, possibly as a mediator in cell-death signalling.

Glucomannan in prevention of oxidative stress and inflammation occurring in adjuvant arthritis.

OBJECTIVES: The aim of this study was to evaluate the effects of a biological response modifier, glucomannan (GM), isolated from Candida utilis, on the progress of adjuvant arthritis in Lewis rats. METHODS: Adjuvant arthrithis was induced in Lewis rats by a single intradermal injection of Mycobacterium butyricum. GM was administered in two different doses of 5 and 7.5 mg/kg b.w. The treatment involved daily oral or intraperitoneal administration of the substance from day 0, i.e. the day of immunization to the end of the experiment - day 28. Cyclosporin A was used as a therapeutic standard in daily oral dose of 2.5 mg/kg b.w. The following parameters were monitored: hind paw volume, total antioxidant status, protein carbonyl groups, activity of N-acetyl-beta-D-glucosaminidase in plasma, lysozyme and peroxidase activity of peritoneal macrophages and activity of gamma-glutamyltransferase in homogenates of spleen, hind paw muscle and hind paw joint. RESULTS: Beneficial action of GM was revealed mainly in hind paw volume decrease. Further decrease of the activity of the enzyme gamma glutamyltransferase (GGT) in the spleen, hind paw joint and muscle tissue homogenates, decrease of the plasmatic activity of N-acetyl-beta-D-glucosaminidase (NAGA), and finally suppression of lysozyme and peroxidase activity assessed in peritoneal macrophages were observed in arthritic animals treated with GM. All these findings speak in favor of the anti-inflammatory activity of glucomannan. Moreover, a significant improvement of the arthritis induced suppression of total antioxidant status and decrease of the level of the arthritis-associated protein carbonyls in plasma were detected. CONCLUSIONS: The important characteristics of GM isolated from Candida utilis, such as good water solubility and relatively small molecular weight, along with the observed in vivo anti-inflammatory and antioxidant effects, appear to be promising features for its prospective use as a natural agent in prevention and supplementary therapy of rheumatoid arthritis.

Enhanced antioxidant formula based on a selenium-supplemented carotenoid-producing yeast biomass.

Carotenoid-producing yeast species such as Rhodotorula glutinis and Sporobolomyces roseus efficiently accumulated selenium from the growth medium. It was observed that incorporation of selenium into yeast cells during the growth inhibited production of beta-carotenoid and other carotenoid precursors (torularhodin and torulene). The yeasts with high content of the carotenoid pigments and selenium may be used for the preparation of a new type of antioxidant formula that could be directly applied for various human and animal diets. We have demonstrated that such a formula can only be produced by separate processes of the cultivation of red yeasts and a subsequent sorption of selenium into the cells.

The many ways to cleave hyaluronan.

Hyaluronan is being used increasingly as a component of artificial matrices and in bioengineering for tissue scaffolding. The length of hyaluronan polymer chains is now recognized as informational, involving a wide variety of size-specific functions. Inadvertent scission of hyaluronan can occur during the process of preparation. On the other hand, certain size-specific hyaluronan fragments may be desirable, endowing the finished bioengineered product with specific properties. In this review, the vast arrays of reactions that cause scission of hyaluronan polymers is presented, including those on an enzymatic, free radical, and chemical basis.

Solution properties of high-molar-mass hyaluronans: the biopolymer degradation by ascorbate.

An accurate molecular characterization, molar mass and size distributions, of 10 hyaluronan (HA) samples was performed by using a multi-angle light scattering detector connected on-line to a size exclusion chromatographic system. The dynamic viscosity eta of the HA solutions was investigated using a rotational viscometer. On monitoring the sample dynamic viscosity for up to 5h, a small however constant increase of the eta value was observed, indicating rheopectic behavior of all 10 HA solutions. Addition of ascorbic acid to the HA solutions caused significant changes in the rheological properties of the samples investigated. The change of eta values in the course of time was explained by the redox reactions (caused by the added ascorbate) that occur during the dynamic viscosity monitoring.

Antioxidant capacity changes and phenolic profile of Echinacea purpurea, nettle (Urtica dioica L.), and dandelion (Taraxacum officinale) after application of polyamine and phenolic biosynthesis regulators.

The changes of the antioxidant (AOA) and antiradical activities (ARA) and the total contents of phenolics, anthocyanins, flavonols, and hydroxybenzoic acid in roots and different aerial sections of Echinacea purpurea, nettle, and dandelion, after treatment with ornithine decarboxylase inhibitor, a polyamine inhibitor (O-phosphoethanolamine, KF), and a phenol biosynthesis stimulator (carboxymethyl chitin glucan, CCHG) were analyzed spectrophotometrically; hydroxycinnamic acids content was analyzed by RP-HPLC with UV detection. Both regulators increased the AOA measured as inhibition of peroxidation (IP) in all herb sections, with the exception of Echinacea stems after treatment with KF. In root tissues IP was dramatically elevated mainly after CCHG application: 8.5-fold in Echinacea, 4.14-fold in nettle, and 2.08-fold in dandelion. ARA decrease of Echinacea leaves treated with regulators was in direct relation only with cichoric acid and caftaric acid contents. Both regulators uphold the formation of cinnamic acid conjugates, the most expressive being that of cichoric acid after treatment with CCHG in Echinacea roots from 2.71 to 20.92 mg g(-1). There was a strong relationship between increase of the total phenolics in all sections of Echinacea, as well as in the studied sections of dandelion, and the anthocyanin content.

Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications.

Hyaluronic acid (hyaluronan, HA) is a linear polysaccharide formed from disaccharide units containing N-acetyl-D-glucosamine and glucuronic acid. It has a high molecular mass, usually in the order of millions of Daltons, and interesting viscoelastic properties influenced by its polymeric and polyelectrolyte characteristics. HA is present in almost all biological fluids and tissues. In clinical medicine, it is used as a diagnostic marker for many diseases including cancer, rheumatoid arthritis and liver pathologies, as well as for supplementation of impaired synovial fluid in arthritic patients by means of intra-articular injections. It is also used in certain ophthalmological and otological surgeries and cosmetic regeneration and reconstruction of soft tissue. Herein we present an overview of the occurrence and physiological properties of HA, as well as of the recent advances in production biotechnology and preparation of the HA-based materials for medical application.

Isolation and characterization of chitin from bumblebee (Bombus terrestris).

Insect chitin possessing shell-like structure was prepared from the bumblebee corpses by a consequent treatment with 1M HCl and 1M NaOH. The bumblebee chitin was compared with crustacean (shrimp) chitin by using elemental analysis, Fourier-transform infrared (FT-IR) and solid-state (13)C cross-polarization magic angle spinning nuclear magnetic resonance (CP/MAS)-NMR spectroscopy and confocal microscopy. Both chitins (bumblebee and shrimp) exhibited identical spectra, while the bumblebee chitin had a 5% lower degree of acetylation and was characterized by a fine membrane texture.

Controlling the association of adamantyl-substituted poly{N-[tris(hydroxymethyl)methyl]acrylamide} and a beta-cyclodextrin/epichlorohydrin polymer by a small drug molecule--naproxen.

Two polymeric substances, a poly{N-[tris(hydroxymethyl)methyl]acrylamide} (THMMA) substituted with adamantyl moieties and a beta-cyclodextrin/epichlorohydrin polycondensate, formed a host-guest type complex, which resulted in the gel formation upon mixing of these two compounds at appropriate conditions. Introduction of a drug molecule, i.e., naproxen, that was able to fill the beta-cyclodextrin cavities, thus expulsing adamantyl moieties, led to disruption of such association and inhibition of gel formation. The conditions required for the association of the two polymeric components and formation of the gel, as well as the dynamics of its inhibition by addition of naproxen was established. The procedure of using solutions of two associating polymers and an appropriate drug competitor can be used at targeted viscosupplementation.

Biofilms of clinical strains of Staphylococcus that do not contain polysaccharide intercellular adhesin.

Staphylococcus aureus and coagulase-negative staphylococci, primarily Staphylococcus epidermidis, are recognized as a major cause of nosocomial infections associated with the use of implanted medical devices. The capacity of S. epidermidis to form biofilms, allowing it to evade host immune defence mechanisms and antibiotic therapy, is considered to be crucial in colonizing the surfaces of medical implants and dissemination of infection. It has previously been demonstrated that the biofilm of a model strain S. epidermidis RP62A comprises two carbohydrate-containing moieties, a polysaccharide having a structure of a linear poly-N-acetyl-(1-->6)-beta-D-glucosamine and teichoic acid. In the present paper we show that, unlike this model strain, certain clinical isolates of coagulase-negative staphylococci produce biofilms that do not contain detectable amounts of poly-N-acetyl-(1-->6)-beta-D-glucosamine. In contrast to that of S. epidermidis RP62A, these biofilms are not detached with metaperiodate, while proteinase K causes their partial dispersal.

Carbohydrate-containing components of biofilms produced in vitro by some staphylococcal strains related to orthopaedic prosthesis infections.

The capacity of coagulase-negative staphylococci to colonize implanted medical devices is generally attributed to their ability to produce biofilms. Biofilm of the model strain of Staphylococcus epidermidis RP62A was shown to contain two carbohydrate-containing moieties, a linear poly-beta-(1-->6)-N-acetyl-D-glucosamine (PNAG) and teichoic acid. In the present study, we investigated several biofilm-producing staphylococci isolated from infected orthopaedic implants and characterized the composition of the laboratory-grown biofilms using chemical analysis and 1H nuclear magnetic resonance spectroscopy. Extracellular teichoic acid was produced by all strains studied. Some of the clinical strains were shown to produce biofilms with compositions similar to that of the model strain, containing a varying amount of PNAG. The chemical structure of PNAG of the clinical strains was similar to that previously described for the model strains S. epidermidis RP62A and Staphylococcus aureus MN8m, differing only in the amount of charged groups. Biofilms of the strains producing a substantial amount of PNAG were detached by dispersin B, a PNAG-degrading enzyme, while being unsusceptible to proteinase K treatment. On the other hand, some strains produced biofilms without any detectable amount of PNAG. The biofilms of these strains were dispersed by proteinase K, but not by dispersin B.

Diverse biomodulatory effects of glucomannan from Candida utilis.

Using four experimental model systems, it was demonstrated that glucomannan (GM) isolated from the cell wall of the industrial yeast Candida utilis revealed a broad range of protective activities. This effect depended on the nature and mode of action of the counteracting genotoxic compound as well as on the experimental model system used. In the Saccharomyces bioprotectivity assay, GM increased resistance towards ofloxacin-induced toxicity in the wild type and recombination repair-deficient yeast strains significantly enhancing survival of the cells. In the chromosomal aberration assay, GM exerted anticlastogenic effect against maleic hydrazide induced clastogenicity in Vicia faba L. In the DNA-topology assay, GM protected plasmid DNA from the breaks induced by Fe(2+) ions, but enhanced damage induced by bleomycin and hydrogen peroxide. In the cell-revitalization assay, it enhanced cytotoxic/cytostatic effect of teniposide applied to mouse leukemia cells. Thus, depending on the experimental model, GM acted as antimutagen, anticlastogen, DNA breaks inhibitor or inducer, and as cytotoxic/cytostatic effect enhancer. Several possible mechanisms of bioprotective action underlying the observed activities are suggested including iron chelation and free radical scavenging. The results imply that GM is a polysaccharide with marked biological activities and suggest its potential biomedical application, especially in combination with other bioactive compounds.

Degradation of high-molecular-weight hyaluronan by hydrogen peroxide in the presence of cupric ions.

Dynamic viscosity (eta) of the high-molecular-weight hyaluronan (HA) solution was measured by a Brookfield rotational viscometer equipped with a Teflon cup and spindle of coaxial cylindrical geometry. The decrease of eta of the HA solution, indicating degradation of the biopolymer, was induced by a system containing H2O2 alone or H2O2 plus CuCl2. The reaction system H2O2 plus CuCl2 as investigated by EPR spin-trapping technique revealed the formation of a four-line EPR signal characteristic of a *DMPO-OH spin adduct. Thus, hydroxyl radicals are implicated in degradation of high-molecular-weight HA by the system containing H2O2 and CuCl2.

Hyaluronan degradation by copper(II) chloride and ascorbate: rotational viscometric, EPR spin-trapping, and MALDI-TOF mass spectrometric investigations.

The degradation of high-molar-mass hyaluronan (HA) by copper(II) chloride and ascorbate was studied by means of rotational viscometry. It was found that even small amounts of CuCl(2) present in the oxidative system led to the pronounced degradation of HA, reflected in a rapid decrease of the dynamic viscosity of the biopolymer solution. Such degradation was induced by free radicals generated in elevated amounts in the presence of copper ions. Electron paramagnetic resonance investigations performed on a model oxidative system containing Cu(II) and ascorbic acid proved the formation of relatively stable ascorbate anion radicals resulting from the reaction of ascorbic acid with hydroxyl radicals. In this way, by scavenging the hydroxyl radicals, ascorbic acid protected HA from their degradative action. Matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry was applied to analyze the degraded HA. The results showed that only regular fragmentation of hyaluronan occurred using the mentioned oxidative system that led to the formation of HA oligomers with unaffected primary chemical structure.

Regulation of activity of cathepsins B, L, and D in murine lymphosarcoma model at a combined treatment with cyclophosphamide and yeast polysaccharide.

Changes in the activity of cysteine (cathepsins B and L) and aspartyl (cathepsin D) proteases were investigated at the development of susceptible and resistant variants of murine lymphosarcoma (LS). It has been demonstrated that the variant resistant to the cyclophosphamide treatment is characterized by a lower activity of all three cathepsins in the tumor tissue. Application of a higher dose of cyclophosphamide led to a more pronounced increase of the studied enzymatic activity in mice with a resistant variant of LS, than in those with a susceptible one. Administration of a yeast polysaccharide derivative - sulfoethyl glucan - enhanced therapeutic effect of cyclophosphamide in mice with both variants of LS, while the most efficient dose was found to be that of 10mg/kg body mass. In the intact mice, usage of both cyclophosphamide and sulfoethyl glucan led to a similar increase of the cathepsins activity in liver and spleen.

Human monocyte scavenger receptors are pattern recognition receptors for (1-->3)-beta-D-glucans.

Glucans are cell wall constituents of fungi and bacteria that bind to pattern recognition receptors and modulate innate immunity, in part, by macrophage activation. We used surface plasmon resonance to examine the binding of glucans, differing in fine structure and charge density, to scavenger receptors on membranes isolated from human monocyte U937 cells. Experiments were performed at 25 degrees C using a biosensor surface with immobilized acetylated low density lipoprotein (AcLDL). Inhibition of the binding by polyinosinic acid, but not polycytidylic acid, confirmed the interaction of scavenger receptors. Competition studies showed that there are at least two AcLDL binding sites on human U937 cells. Glucan phosphate interacts with all sites, and the CM-glucans and laminarin interact with a subset of sites. Polymer charge has a dramatic effect on the affinity of glucans with macrophage scavenger receptors. However, it is also clear that human monocyte scavenger receptors recognize the basic glucan structure independent of charge.

Yeast polysaccharide affects fusaric acid content in maize root rot.

Protective action of sulfoethyl glucan (SEG), a derivative of the cell wall glucan prepared from the baker's yeast Saccharomyces cerevisiae, was investigated in the maize seedlings infected by a plant pathogen Fusarium verticillioides (Sacc.). Several markers were assayed with the SEG addition and in the control experiments. Two evaluations were performed on the 7th and the 14th days. Addition of SEG led to the increased productivity parameters of the infected plants and maintained them at the level of non-infected plants during the 14 days of experiment. After seven days of cultivation, concentration of fusaric acid (=5-butylpyridine-2-carboxylic acid; FA) decreased in all infected plants cultivated in the presence of SEG when compared to that detected in the infected plants grown in the absence of SEG. After 14 days of cultivation, polysaccharide addition resulted in the reduction of FA concentration almost to 75% in comparison to the infected plants grown without polysaccharide addition. In the experiment, when exogenous FA was added to the growth medium, its concentration decreased up to 60% in the presence of SEG. Thus, it is feasible to assume that SEG binds and adsorbs FA, and, in this way, reduces its content and exerts protective action in plants against its toxic effect.

Contribution of oxidative-reductive reactions to high-molecular-weight hyaluronan catabolism.

Since the content of hyaluronan (HA)-degrading enzymes in synovial fluid (SF), if any, is extremely low, the high rate of HA turnover in SF is to result from a cause different from enzymatic catabolism. An alternative and plausible mechanism is that of oxidative-reductive degradation of HA chains by a combined action of oxygen and transition metal cations maintained in a reduced oxidation state by ascorbate.