Hypolipidemic effect of mannans from C. albicans serotypes a and B in acute hyperlipidemia in mice.

Mannans, which are biological macromolecules of polysaccharide origin and function as immunomodulators, have been shown to stimulate macrophages in vivo by interaction with the mannose receptor. Thus, they can be used to stimulate macrophages in order to effectively remove circulating atherogenic lipoproteins. Our primary aim was to evaluate the hypolipidemic potential of mannans from C. albicans serotype A (mannan A) and serotype B (mannan B) in a murine model of hyperlipidemia. Mannan A and mannan B were shown to significantly (p<0.05) stimulate both the proliferation (p <0.05) and nitric oxide production of murine peritoneal macrophages in vitro. Pre-treatment of CBA/Lac mice with mannan A prior to induction of hyperlipidemia significantly (p<0.001) reduced serum atherogenic LDL-cholesterol, total cholesterol, and triglycerides. Mannan B exhibited a similar, but more potent, hypolipidemic effect. Electron microscopic analysis of liver revealed a significant (p<0.001) decrease in the volume of lipid droplets when hyperlipidemic mice were pretreated by both mannans. In conclusion, our findings would suggest that both polysaccharide-based biological macromolecules evaluated in the present study, specifically, the natural immunomodulators (mannans A and B), appeared to function as effective lipid-lowering macromolecules, which could potentially serve as adjunct therapy to more conventional hypolipidemic medications such as a statin drug.

Serum cystatin C and chitotriosidase in acute P-407 induced dyslipidemia: Can they serve as potential early biomarkers for atherosclerosis?

In an attempt to better understand potential biomarkers for, and the role of macrophages in, the development of atherosclerosis, the toxicologic, and any therapeutic pharmacologic effects of carboxymethylated ß-glucan, gadolinium chloride, and poloxamer 407 were studied in mice for their capacity to perturb serum lipids, cystatin C, and chitotriosidase-1. Gadolinium and carboxymethylated ß-glucan dosed separately to control mice had no effect on serum lipids, whereas carboxymethylated ß-glucan, but not gadolinium, exerted a significant (p<0.01) and unexpected hypolipidemic effect in poloxamer 407-induced hyperlipidemic mice. An acute hyperlipidemic state (∼4 days), induced with poloxamer 407 administration alone, resulted in a significant (p<0.01) time-dependent decrease and increase in serum cystatin C and chitotriosidase, respectively. Carboxymethylated ß-glucan administration to hyperlipidemic mice significantly (p<0.05) increased the serum concentration of cystatin C, but significantly (p<0.01) decreased chitotriosidase activity, when each was compared to mice treated with poloxamer 407 only. Gadolinium administration caused a significant decrease in serum chitotriosidase activity in both controls (p<0.01) and poloxamer 407-induced hyperlipidemic (p<0.001) mice, but had no effect on the concentration of cystatin C in either controls or poloxamer 407-induced hyperlipidemic mice. Gadolinium administration resulted in both morphological and functional changes to liver macrophages, which included incorporation of excess lipids, especially when simultaneously administered with poloxamer 407. It is suggested that serum cystatin C and chitotriosidase may represent potential early biomarkers for eventual atherosclerosis in the poloxamer 407-induced mouse model of atherogenesis, and that two compounds known to either increase (carboxymethylated ß-glucan) or decrease (gadolinium chloride) the number of macrophages in vivo were able to modulate serum chitotriosidase activity, This, in turn, would appear to support the premise that serum chitotriosidase activity may be a more sensitive indicator of macrophage involvement than cystatin C in the context of future atherosclerosis.

Scavenging and antioxidant activities of immunomodulating polysaccharides isolated from Salvia officinalis L.

Crude polysaccharides, isolated from the aerial parts of sage (Salvia officinalis L.) by sequential extraction with water (A), hot ammonium oxalate (B), dimethyl sulfoxide (C), 1M (D) and 4M (E) potassium hydroxide solutions, and six ion-exchange fractions of A were examined for their ability to inhibit peroxidation of liposome lipid by hydroxyl radicals and to reduced DPPH radical content. The highest inhibition of liposome lipid peroxidation was found with crude polysaccharides A, B and D, antioxidant activities reached approximately 37%. The purified fractions A1 and A2 inhibited the liposome peroxidation to approximately 35%. However, the radical scavenging abilities of the most active crude polysaccharides A, B and C on DPPH radicals were found in the range 80-90%, while the most active purified fractions A3-A6 in three or fourfold doses achieved 75-92%. The least effective tested polysaccharides succeeded 20% inhibition using both methods.

Importance and prospects for design of selective muscarinic agonists.

There are five subtypes of muscarinic receptors that serve various important physiological functions in the central nervous system and the periphery. Mental functions like attention, learning, and memory are attributed to the muscarinic M1 subtype. These functions decline during natural aging and an early deficit is typical for Alzheimer s disease. In addition, stimulation of the M1 receptor increases non-amyloidogenic processing of the amyloid precursor protein and thus prevents accumulation of noxious beta-amyloid fragments. The selectivity of classical muscarinic agonists among receptor subtypes is very low due to the highly conserved nature of the orthosteric binding site among receptor subtypes. Herein we summarize some recent studies with the functionally-selective M1 agonist xanomeline that indicate complex pharmacological profile of this drug that includes interactions with and activation of receptor from both orthosteric and ectopic binding sites, and the time-dependent changes of ligand binding and receptor activation. These findings point to potential profitability of exploitation of ectopic ligands in the search for truly selective muscarinic receptor agonists.

Impairment of muscarinic transmission in transgenic APPswe/PS1dE9 mice.

We assessed the integrity of cholinergic neurotransmission in parietal cortex of young adult (7 months) and aged (17 months) transgenic APPswe/PS1dE9 female mice compared to littermate controls. Choline acetyltransferase and acetylcholinesterase activity declined age-dependently in both genotypes, whereas both age- and genotype-dependent decline was found in butyrylcholinesterase activity, vesicular acetylcholine transporter density, muscarinic receptors and carbachol stimulated binding of GTP gamma S in membranes as a functional indicator of muscarinic receptor coupling to G-proteins. Notably, vesicular acetylcholine transporter levels and muscarinic receptor-G-protein coupling were impaired in transgenic mice already at the age of 7 months compared to wild type littermates. Thus, brain amyloid accumulation in this mouse model is accompanied by a serious deterioration of muscarinic transmission already before the mice manifest significant cognitive deficits.

Carboxymethylated (1 --> 3)-beta-D-glucan protects liposomes against ultraviolet light-induced lipid peroxidation.

In this study we have analyzed antioxidant capabilities of the carboxymethylated (1 --> 3)-beta-D-glucan (M(w) = 5.88 x 10(5)) against lipid peroxidation induced by ultraviolet (UV) radiation--UVA (320-400 nm), which is known to produce mainly singlet oxygen, (1)O(2) . Lipid peroxidation was monitored by measuring the absorption spectra of the conjugated dienes and quantified by Klein oxidation index. The results imply that the (1 --> 3)-beta-D-glucan derivative studied is an antioxidant with the scavenging ability lying between alpha-tocopherol and hyaluronic acid. Thus, glucan as a potential safe and effective dietary supplement may be used for a prolonged time for a systemic photoprotection of humans.

Wash-resistantly bound xanomeline inhibits acetylcholine release by persistent activation of presynaptic M(2) and M(4) muscarinic receptors in rat brain.

We studied the effects of 3-[3-hexyloxy-1,2,5-thiadiazo-4-yl]-1,2,5,6-tetrahydro-1-methylpyridine (xanomeline) wash-resistant binding on presynaptic muscarinic regulation of electrically evoked [(3)H]acetylcholine (ACh) release from rat brain slices. In both cortical and striatal tissues that possess M(2) and M(4) autoreceptors, respectively, immediate application of 10 microM xanomeline had no effect on evoked [(3)H]ACh release or its inhibition by 10 microM carbachol. In contrast, preincubation with 1, 10, or 100 microM xanomeline for 15 min decreased evoked release of ACh measured after 53 min of washing in xanomeline-free medium in a concentration-dependent manner. The maximal inhibitory effect equaled the immediate effect of the muscarinic full agonist carbachol, and it was completely (at 1 and 10 microM xanomeline) or partially (at 100 microM xanomeline) blocked by 1 microM N-methylscopolamine. Neither presence of N-methylscopolamine during 100 microM xanomeline treatment nor previous irreversible inactivation of the classical receptor binding site using propylbenzylcholine mustard in cortical slices prevented the inhibitory effect of wash-resistantly bound xanomeline. Treatment of cortical slices with xanomeline slightly decreased the number of muscarinic binding sites, and it markedly decreased affinity for N-methylscopolamine. When applied as in acetylcholine release experiments, xanomeline did not impair presynaptic alpha(2)-adrenoceptor-mediated regulation of noradrenaline release. The functional studies in brain tissue reported in this work demonstrate that xanomeline can function as a wash-resistant agonist of native presynaptic muscarinic M(2) and M(4) receptors with both competitive and allosteric components of action.

Antioxidant activity of medicinal plant polysaccharides.

Eleven polysaccharides have been isolated from the leaves of Arctium lappa var. herkules, Aloe barbadensis, Althaea officinalis var. robusta, Plantago lanceolata var. libor, aerial parts and roots of Rudbeckia fulgida var. sullivantii, stems of Mahonia aquifolium, and peach-tree (Prunus persica) gum exudates. The polysaccharides were investigated for their ability to inhibit peroxidation of soyabean lecithin liposomes by OH radicals. The highest inhibition was found with glucuronoxylans of A. officinalis var. robusta and P. lanceolata var. libor, aerial parts. Their antioxidant activity accounted for approximately 69% of the activity of the reference compound alpha-tocopherol. The activity of eight polysaccharides ranged from 20 to 45%, while the fructofuranan from P. lanceolata var. libor roots was practically inactive.

Immunological properties of complex conjugates based on Vibrio cholerae O1 Ogawa lipopolysaccharide antigen.

Host protection by humoral immunity against Vibrio cholerae O1 confers lipopolysaccharide (LPS)-specific vibriocidal antibodies. Levels of relevant specific antibodies are closely related to complement-mediated inactivation of the vibrios inoculum, especially on the mucosal surface of intestine. We have tested complex V. cholerae O1 Ogawa-detoxified lipopolysaccharide (dLPS) conjugates. The first conjugate contained glucan both as the immunomodulator and the matrix; the second conjugate contained immunologically inert amylose as matrix. Both d-LPS conjugates contain multiply attached dLPS antigen. These conjugates elicited a statistically significant increase of antigen-specific IgG levels in mice (P<0.001 and P<0.05, respectively). The specific anti-conjugate IgG and IgA response after the second (booster) dose were significantly higher compared to pre-immune and whole-cell response. The most effective vibriocidal activity was observed in the case of conjugate, with glucan as the matrix. The highest correlation was found between vibriocidal activity and specific IgG2b (r=0.765) and IgA (r=0.887) sera levels. The determination of specific IgG subclasses and IgG2a + 2b/IgG1 ratio revealed a dominant T(H)1 cell response crucial for effective vaccine candidate.

Antioxidant properties of carboxymethyl glucan: comparative analysis.

Antioxidative capabilities of carboxymethylated (1 --> 3)-beta-D-glucan from Saccharomyces cerevisiae cell wall, alpha-tocopherol, and mannitol against lipid peroxidation in phosphatidylcholine liposomes induced by OH. radicals produced with Fenton's reagent (H(2)O(2)/Fe(2+)) were studied using absorption ultraviolet-visible spectrophotometry. It was found that (1 --> 3)-beta-D-glucan is an antioxidant with the scavenging ability lying between that of alpha-tocopherol, which is known to be incorporated in the lipid bilayer, and the water-soluble antioxidant, mannitol.

Effect of gluco-monosaccharides and different conditions on digestion of hyaluronan by testicular hyaluronidase.

The changes of molecular size of hyaluronan during enzymatic reaction of bovine testicular hyaluronidase at different conditions are monitored by size exclusion high performance liquid chromatography. The effect of glucuronate, galacturonate, glucosamines and pyridoxin as potential inhibitors of hydrolysis is evaluated. The most effective of all tested inhibitors was the presence of glucuronate which not only inhibited the hydrolysis, but also initiated enzymatic reconstruction by transglycosylation reaction at pH 7.0 and absence of any buffer or salt. That effect was not found in the presence of a salt or with any other of the compounds tested.

Site-specific in vivo targeting of magnetoliposomes using externally applied magnetic field.

Human serum albumin labeled with technetium-99m was encapsulated together with magnetite particles into phosphatidylcholine/cholesterol liposomes. In order to investigate the stability of this complex and its ability to be used for magnetic drug targeting, the in-vivo distribution after intravenous administration in rats was estimated. For in-vivo targeting an SmCo permanent magnet with intensity approximately 0.35 T was attached near the right kidney. Difference between the relative radioactivity in the magnetically targeted right kidney (25.92+/-5.84%) and non-targeted left kidney (0.93+/-0.05%) is sufficiently high for relevant clinical applications.

Sonication of chitin-glucan, preparation of water-soluble fractions and characterization by HPLC.

A water-insoluble chitin-glucan complex, isolated from the mycelium of Aspergillus niger, was swollen in various aqueous media and treated subsequently by high-energy sonication. The concentration of the resulting water-soluble polysaccharide fractions was dependent on the swelling medium, the amount of the chitin-glucan complex in the suspension, and on the time of sonication. The yields of water-soluble chitin-glucan were within the range 13.6 to 24.4% relative to the mass of the original chitin-glucan. The nitrogen content obtained for the samples of water-soluble chitin-glucan indicated a higher content of chitin (3.45% of nitrogen in high-molecular fraction) than in the original water-insoluble chitin-glucan sample (1.8%). The distribution of the molecular weights of the water-soluble fractions prepared was determined by high-performance liquid chromatography.

Conjugation of yeast mannans with protein employing cyanopyridinium agent (CDAP)--an effective route of antifungal vaccine preparation.

The possibility of using 1-cyano-4-dimethylaminopyridinium tetrafluoroborate (CDAP) for activation of saccharide hydroxyl groups (instead of hazardous cyanogen bromide) is examined with cell-surface mannans of the yeasts Candida albicans, Candida tropicalis, Candida lambica and galactoglucoxylomannan of Cryptococcus laurentii. Direct conjugation with human serum albumin yielded soluble products with increased molecular size in comparison with the original polysaccharides. Immunodiffusion experiments revealed that conjugation did not affect the immunospecificity of the antigen epitppe.

Enzymatic digestion of liposome-bound polysaccharides: evidence of bridging mechanism.

Laminarin, a water-soluble polysaccharide is known to induce aggregation and fusion of liposomes. Using laminarin specific lysing enzymes for multi-lamellar liposomes their aggregation was found to be fully reversible, in contrast to small unilamellar liposomes for which irreversible fusion is the main process. Moreover, our results indicate that these processes are probably mediated by the formation of polysaccharide cross-bridges between adjacent liposomes.

Protective effect of the yeast glucomannan against cyclophosphamide-induced mutagenicity.

Glucomannan (GM) isolated from Candida utilis with molecular weight 30 kDa was administered either intraperitoneally or orally prior to cyclophosphamide (CP) injection and its effect on the frequency of micronuclei was evaluated in polychromatic erythrocytes of mouse bone marrow. GM administration by either route decreased significantly (p<0.002) the clastogenic effect of CP. The protective effect was concentration-dependent, with a higher decrease achieved by 200 mg/kg than by 100 mg/kg b. wt. (body weight). The fact that GM was effective also at oral administration is indicative of the passage of GM molecules through the wall of the gastrointestinal tract. The important characteristics of GM isolated from C. utilis, such as good water solubility, relatively small molecular weight (30 kDa), and antimutagenic effect exerted also at oral administration, appear to be promising features for its prospective use as a natural protective agent.

Ultrasonic depolymerization of the chitin-glucan complex from Aspergillus niger and antimutagenic activity of its product.

Sonication was effective for the depolymerization of carboxymethylated chitin-glucan complex (CM-CG) isolated from the cell wall of Aspergillus niger. After 10 min of sonication and subsequent gel filtration, two samples (CM-CG(I) and CM CG(II)) with significantly distinct molecular weights (660 kDa and 19 kDa, respectively) and different nitrogen contents (3.02 and 1.69%) were obtained. CM-CG(II) with lower Mw was also effective against cyclophosphamide mutagenicity by oral administration in mice.

Effect of ultrasonic treatment on the molecular weight of carboxymethylated chitin-glucan complex from Aspergillus niger.

Different factors affecting the efficiency of the ultrasonic depolymerization of the high-molecular-weight carboxymethylated chitin-glucan prepared from the fungal mycelium of Aspergillus niger have been investigated. The influence of the following parameters was examined: concentration of the chitin-glucan complex, duration of ultrasonic irradiation, reaction temperature and volume of the ultrasonicated solution. The optimized conditions for the efficient ultrasonic depolymerization include: polysaccharide concentration--0.2 mg ml-1; volume of the sonicated solution--25 ml; duration of the sonication--10 min; and constant cooling of the sonicated sample in an ice-water bath.

Determination of the cross-linking effect of adipic acid dihydrazide on glycoconjugate preparation.

The cross-linking effect of adipic acid dihydrazide (ADH) on polysaccharide derivatization can be evaluated by applying combination of elemental analysis and colorimetric assay. Elemental analysis is used for estimation of total ADH bound to polysaccharide and a colorimetric trinitrobenzene sulfonic acid assay is used to determine the part of ADH not involved in cross-linking. The difference of values expressed as molar ratios (per repeating unit) provides information on the amount of ADH involved in cross-linking the polysaccharides. Carboxymethylated polysaccharides were derivatized with different amounts of ADH to test the procedure. Analytical results showed that excess of ADH in the reaction only slightly decreased the cross-linking. The number of carboxyl groups remained unmodified even at high excess of ADH and high concentration of carbodiimide (EDC) coupling reagent.

Dextran enhances calcium-induced aggregation of phosphatidylserine liposomes: possible implications for exocytosis.

We have studied the calcium-induced aggregation of phosphatidylserine liposomes in the presence of various concentration of a high-molecular water-soluble polysaccharide dextran. It has been shown that threshold concentrations of calcium necessary to induce liposome aggregation in the presence of approximately 1 mM concentration of dextran is about one order lower than in the absence of dextran. Soluble intracellular polymers may thus play an important role in the process of exocytosis.