Drying enhances immunoactivity of spent brewer's yeast cell wall ß-D-glucans.

Due to immunological activity, microbial cell wall polysaccharides are defined as 'biological response modifiers' (BRM). Cell walls of spent brewer's yeast also have some BRM activity. However, up to date there is no consensus on the use of spent brewer's yeast D-glucan as specific BRM in humans or animals. The aim of this paper is to demonstrate the potential of spent brewer's yeast ß-D-glucans as BRM, and drying as an efficient pretreatment to increase ß-D-glucan's immunogenic activity. Our results revealed that drying does not change spent brewer's yeast biomass carbohydrate content as well as the chemical structure of purified ß-D-glucan. However, drying increased purified ß-D-glucan TNF-α induction activity in the murine macrophage model. We presume drying pretreatment enhances purity of extracted ß-D-glucan. This is corroborated with FT-IR analyses of the ß-D-glucan spectra. Based on our results, we suggest that dry spent brewer's yeast biomass can be used as a cheap source for high-quality ß-D-glucan extraction. Drying in combination with carboxylmethylation (CM), endows spent brewer's yeast ß-D-glucan with the immunoactivity similar or exceeding that of a well-characterized fungal BRM pleuran.

Protective effects of manganese(II) chloride on hyaluronan degradation by oxidative system ascorbate plus cupric chloride.

The degradation of several high-molar-mass hyaluronan samples was investigated in the presence of ascorbic acid itself and further by an oxidative system composed of ascorbic acid plus transition metal ions, i.e. Fe(II) or Cu(II) ions. The latter oxidative system imitates conditions in a joint synovial fluid during early phase of acute joint inflammation and can be used as a model for monitoring oxidative degradation of hyaluronan under pathophysiological conditions. The system Cu(II) plus ascorbate (the Weissberger oxidative system) resulted in a more significant decrease of hyaluronan molar mass compared to the oxidative system Fe(II) plus ascorbate. Addition of manganese(II) chloride was found to decrease the rate of the oxidative damage of hyaluronan initiated by ascorbate itself and by the Weissberger system.

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 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.

Characterization of the novel chemically modified fungal polysaccharides as the macrophage stimulators.

By means of carboxymethylation, a novel water-soluble carboxymethyl chitin-glucan (CM-CG) was prepared from the mycelium of Aspergillus niger, and its ability to stimulate macrophages was assessed and compared to that of the previously studied carboxymethylated glucan (CMG) from the yeast Saccharomyces cerevisiae. It was demonstrated that single intraperitoneal (i.p.) administration of CMG and CM-CG to the CBA mice led to a significant increase of leukocyte number. At the same time, the number of monocytes in the bone marrow was increased to more than two-fold. Application of both polysaccharides also resulted in the augmented number of liver macrophages and to the rise of their content of the secondary lysosomes. A markedly enhanced carbon clearance was observed as well as the increased release of tumor necrosis factor-alpha by the peritoneal macrophages indicating their amplified phagocytic activity. The effect of CM-CG in these experiments was ca. 1.7 times higher than that of CMG. Administration of both polysaccharides also led to the elevated level of free acid phosphatase in liver homogenate, implying labilization of the lysosomes. Increased serum chitotriosidase also indicated increased macrophage activity. The results obtained indicate similar in vivo macrophage stimulation activity of both applied fungal polysaccharides and suggest their potential clinical use as non-toxic natural compounds.

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.

Regulation of the phenolic profile of berries can increase their antioxidant activity.

The changes of the antioxidant activities (AOA), antiradical activities (ARA), low-density lipoprotein (LDL) oxidation, and total contents of phenolics, anthocyanins, flavonols, hydroxybenzoic acids, and hydroxycinnamic acids in black currant and black chokeberry, after treatment with ornithine decarboxylase inhibitor, a polyamine inhibitor (O-phosphoethanolamine, KF), and a phenol biosynthesis stimulator (carboxymethyl chitin glucan, CCHG), were analyzed spectrophotometrically. Gallic acid, hydroxycinnamic acids, and selected flavonol contents was analyzed by RP-HPLC. Both regulators increased the AOA measured as inhibition of peroxidation (IP) in black chokeberry, 1.71-fold after treatment with KF(1) and 1.74-fold after treatment with CCHG. In black currant IP was elevated after CCHG application only in lower dose (CCHG(1) 63.36% vs control 53.23%). In black chokeberry the total phenolics content was elevated 1.49-fold after KF(1) application and 1.31-fold after CCHG(2) application. The regulators had the lower effect on the phenolic accumulation in black currant. There was a strong relationship between the total phenolics in the both crops and anthocyanins, hydroxybenzoic acids, and hydroxycinnamic acids contents, respectively. Both regulators significantly changed the ratio of conjugated (rutin) to free (quercetin) flavonol mainly in black chokeberry. The antioxidant activities compared using LDL in vitro oxidation assay were increased more expressively after treatment with KF(2) in both crops.

Lectinomics II. A highway to biomedical/clinical diagnostics.

The review assesses current status and attempts to forecast trends in the development of lectin biorecognition technology. The progressive trend is characterized scientometrically and reflects the current transient situation, when standard low-throughput lectin-based techniques are being replaced by a novel microarray-based techniques offering high-throughput of detection. The technology is still in its infancy (validation phase), but already shows promise as an efficient tool to decipher the enormous complexity of the glycocode that influences physiological status of the cell. Further enhancement in robustness and flexibility of lectin microarrays is predicted by using recombinant and artificial lectins that will render production of lectin microarrays cost-effective and more affordable. Mass spectrometry is expected to play an important role to characterize the binding profile of new lectins. Differences in glycan recognition by lectins and anti-carbohydrate antibodies are given on a molecular basis, and strong and weak points of both biorecognition molecules in diagnosis are briefly discussed.

Catabolism of hyaluronan: involvement of transition metals.

One of the very complex structures in the vertebrates is the joint. The main component of the joint is the synovial fluid with its high-molar-mass glycosaminoglycan hyaluronan, which turnover is approximately twelve hours. Since the synovial fluid does not contain any hyaluronidases, the fast hyaluronan catabolism is caused primarily by reductive-oxidative processes.Eight transition metals - V(23), Mn(25), Fe(26), Co(27), Ni(28), Cu(29), Zn(30), and Mo(42) - naturally occurring in living organism are essential for the control of various metabolic and signaling pathways. They are also the key elements in catabolism of hyaluronan in the joint.In this overview, the role of these metals in physiological and pathophysiological catabolism of hyaluronan is described. The participation of these metals in the initiation and propagation of the radical degradation hyaluronan is critically reviewed.

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.

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.

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.

Antibody response in mice infected with Mesocestoides vogae (syn. Mesocestoides corti) tetrathyridia after treatment with praziquantel and liposomised glucan.

The therapeutic effect of praziquantel (PZQ) involves synergy with the humoral immune response during helminth infections, which is modulated by parasitic antigens. During experimental murine infections with the larval stage of cestoda Mesocestoides vogae (syn. M. corti), dynamic changes in the IgG and IgM antibody serum levels to both soluble somatic and secretory larval antigens were investigated after administration of PZQ alone and after its co-administration with the immunomodulator (l-->3)-beta-D-glucan entrapped in liposomes (lip.glucan). During the 2 weeks of follow-up after termination of therapy, specific IgG and IgM serum levels to the somatic antigens (enzyme-linked immunosorbent assay test) significantly decreased, whereas concentrations of the antibodies to the secretory antigens moderately increased, both in comparison with the control. Moreover, the number of immunogenic larval antigens (analysed by Western blot) was higher after combined therapy in comparison with single drug administration, which correlated with the intensity of reduction of the larval counts in the liver and peritoneal cavity of mice. Our data showed that administration of PZQ alone and in combination with lip.glucan resulted in marked changes in the dynamics of IgG and IgM antibodies to the somatic larval antigens, which were probably induced by the newly exposed antigens. In this respect, glucan can enhance chemotherapeutic activity of PZQ against larval cestodes by means of stimulation of the macrophage/monocyte effector functions, which seemed to contribute to the more intense larval damage.

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.

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.

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.

Effects of dietary intake of a fungal beta-D-glucan derivative on the level of DNA damage induced in primary rat hepatocytes by various carcinogens.

Water-soluble derivative of chitin-glucan complex used in our study, carboxymethyl chitin-glucan (CM-CG), enables oral administration without harmful side-effects, which can occur upon parenteral administration of the insoluble fungal beta-D-glucans. The aim of this study was to determine in ex vivo experiments the effects of dietary CM-CG on the level of DNA lesions in primary rat hepatocytes induced by various indirectly acting carcinogens. Multiorgan carcinogen benzo[a]pyrene (BaP); two hepatocarcinogens, dimethyldibenzocarbazole (diMeDBC) and N-nitrosomorpholine (NMOR); as well as a complex mixture of organic compounds adsorbed on ambient air particles (TP-S) were used for this purpose. The amount of DNA lesions was assessed using the comet assay and the micronucleus test. In addition, the mitotic indexes and the frequencies of necrotic and apoptotic cells were evaluated as well. Our results showed that the diet enriched with CM-CG (200 mg/kg of body weight) during 21 days did not induce any negative effect on DNA nor did the mitotic indexes and the frequencies of necrotic and apoptotic cells differ statistically from the controls. On the other hand, the hepatocytes isolated from CM-CG fed animals were more resistant to the action of all genotoxins used in our study [BaP (5-20 microM), diMeDBC (0.2-2 microM), NMOR (3.4-10.2 mM), TP-S (5-20 microM)]. We can conclude that in addition to the known immunopotentiating activity of beta-D-glucans, they can efficiently inhibit the genotoxicity of carcinogens requiring metabolic activation in rat heptocytes.

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.