Peter Bystrický

Effect of coenzyme Q10 and vitamin E on brain energy metabolism in the animal model of Huntington's disease.

The neuropathological and clinical symptoms of Huntingtons disease (HD) can be simulated in animal model with systemic administration of 3-nitropropionic acid (3-NP). Energy defects in HD could be ameliorated by administration of coenzyme Q(10) (CoQ(10)), creatine, or nicotinamid. We studied the activity of creatine kinase (CK) and the function of mitochondrial respiratory chain in the brain of aged rats administered with 3-NP with and without previous application of antioxidants CoQ(10)+vitamin E. We used dynamic and steady-state methods of in vivo phosphorus magnetic resonance spectroscopy ((31)P MRS) for determination of the pseudo-first order rate constant (k(for)) of the forward CK reaction, the phosphocreatine (PCr) to adenosinetriphosphate (ATP) ratio, intracellular pH(i) and Mg(i)(2+) content in the brain. The respiratory chain function of isolated mitochondria was assessed polarographically; the concentration of CoQ(10) and alpha-tocopherol by HPLC. We found significant elevation of k(for) in brains of 3-NP rats, reflecting increased rate of CK reaction in cytosol. The function of respiratory chain in the presence of succinate was severely diminished. The activity of cytochromeoxidase and mitochondrial concentration of CoQ(10) was unaltered; tissue content of CoQ(10) was decreased in 3-NP rats. Antioxidants CoQ(10)+vitamin E prevented increase of k(for) and the decrease of CoQ(10) content in brain tissue, but were ineffective to prevent the decline of respiratory chain function. We suppose that increased activity of CK system could be compensatory to decreased mitochondrial ATP production, and CoQ(10)+vitamin E could prevent the increase of k(for) after 3-NP treatment likely by activity of CoQ(10) outside the mitochondria. Results of our experiments contributed to elucidation of mechanism of beneficial effect of CoQ(10) administration in HD and showed that the rate constant of CK is a sensitive indicator of brain energy disorder reflecting therapeutic effect of drugs that could be used as a new in vivo biomarker of neurodegenerative diseases.

Exopolysaccharides of Lactobacillus reuteri: Their influence on adherence of E. coli to epithelial cells and inflammatory response.

The aim of the study was to characterize exopolysaccharides (EPS) originated from Lactobacillus reuteri strain DSM 17938 (EPS-DSM17938) and L. reuteri strain L26 Biocenol™ (EPS-L26) and evaluate their influence on adherence of enterotoxigenic Escherichia coli (ETEC) to IPEC-1 cells and proinflammatory gene expression. Both EPS were d-glucan polysaccharides with higher molecular weight (Mw), but differing in spatial conformation and elicited variable cytokine profile. EPS-DSM17938, relatively linear polysaccharide with (1→4) and (1→6) glycosidic linkages, increased IL-1β gene expression (0.1mg/mL; P<0.05), while EPS-L26, more branched polysaccharide with (1→3) and (1→6) glycosidic linkages, exerted slight but statistically significant up-regulation of NF-κB, TNF-α and IL-6 mRNA (P<0.05). The most significant finding is that preincubation of IPEC-1 cells with both EPS followed by ETEC infection inhibit ETEC adhesion on IPEC-1 cells (P<0.01) and ETEC-induced gene expression of proinflammatory cytokine IL-1β and IL-6 (P<0.01).

Preparation and characterization of carboxymethyl derivatives of yeast mannans in aqueous solutions.

Novel carboxymethyl derivatives of yeast mannans of different degrees of substitution (DS) were prepared by optimized reaction of concentrated polysaccharides in alkaline aqueous solution. Mannans from various yeasts differing in size and degree of branching show similar reactivity. Strong alkaline conditions during carboxymethylation caused degradation of the polysaccharides. The degree of substitution (DS) of Candida albicans mannan and dextran were proportional to the amount of monochloroacetate added. However, degrees of carboxymethylation of Candida albicans mannan (0.30, 0.41, 0.73) were lower than those of dextran (DS=0.33, 0.6, 1.1) using the same amounts of monochloroacetate. Evidently the resulted polyanionic derivatives have higher hydrodynamic sizes than the original polysaccharides. Non-uniform, variable position of substitutions results to non-proportional change of optical rotation and increase of complexity of NMR spectra. Basic physico-chemical characteristics of novel carboxymethyl mannans obtained by potentiometric titration, FT-IR, UV, HPLC, 1H NMR and optical rotation measurements are presented here.

Effect of carboxymethylation on antioxidant properties and radical degradation of mannans and glucans.

Carboxymethyl derivatives (CM-derivatives) of α,β-mannans from yeasts, Saccharomyces cerevisiae β-glucan and dextran (α-glucan) were found to possess strong antioxidant activities against reactive hydroxyl radicals (OH*) compared to underivatized polysaccharides. When CM-derivatives having similar DS (0.41-0.45) were compared, the antioxidant activity decreased in order CM-mannan>CM-β-glucan>CM-dextran. Moreover, the antioxidant activities against OH* increased with increasing degree of substitution (DS) of polysaccharides. The CM-mannan and CM-dextran with the highest DS (0.73 and 1.1, respectively) were the strongest antioxidants and their degradation by OH* decreased with increased carboxymethylation. The scavenging abilities of CM-polysaccharides against stable DPPH radical (DPPH) were lower than those of original underivatized ones. Also this scavenging property against DPPH was lower compared to antioxidant effect against OH*.

Ultrasonic and free-radical degradation of mannan from Candida albicans.

Mannan from pathogenic Candida albicans serotype A was degraded by means of ultrasound and/or OH generated in situ by Fenton reaction. The kinetics of degradation was monitored by HPLC analysis and the weight-average molecular weights (Mw) and index of polydispersity (PDI) were compared. A well-defined low-molecular-weight mannan (∼ 30 kDa) with narrowed PDI of 1.8 was obtained after 120 min of ultrasonication. Similar or even lower Mw (up to 16 kDa) was achieved upon free-radical exposure depending on Fe(2+) concentration used; however, this was accompanied by overall broadening of PDI and distinct changes in polymer structure as indicated by NMR analysis.

Comparative ESI FT-MS and MALDI-TOF structural analyses of representative human N -linked glycans

Modern glycan analysis is primarily based on mass spectrometry, where instruments based on electrospray or matrix-assisted laser desorption ionization are currently the most frequently used. In the present study, electrospray ionization (ESI) coupled with a high-resolution Fourier transform mass spectrometer (LTQ Orbitrap) and matrix-assisted laser desorption/ionization (MALDI) coupled with a time-of-flight (TOF/TOF) detector were used to analyze two N-glycan standards with intact free reducing ends (disialo biantennary and asialo triantennary) and representative PA-labeled human serum N-glycan structures isolated by hydrophilic interaction anion-exchange chromatography (HIAX), confirmed by 1H NMR analysis and consequently compared with the ProteinScape Glycome database. Different combinations of ion sources with fragmentation devices results in various fragmentation patterns and adducts. Also, the effect of sample derivatization on the acquired signals is discussed. Compared to the MALDI technique, free glycans did not lose labile sialic acids easily in the ESI source. On the other hand, fluorescent PA-labeling leads to improved core fragmentation and signal intensities; linkage-specific ethyl esterification leads to reduced adduct and fragment formation and enhanced stability of sialic acids in the MALDI ion source. Thereby, both methods have their advantages and disadvantages in terms of detection, fragmentation and robustness.

NMR characteristics of α-D-Man-(1→2)-D-Man and α-D-Man-(1→3)-D-Man mannobioses related to Candida albicans yeast mannan structures

Candida albicans mannans are highly perspective polysaccharides for pharmaceutical and biomedical industry. However, they have not been fully characterized. Generally, the larger, acid-stable part of these complex polymers mostly contain α- (and a few β-) linked mannoses. According to this statement all 1H–13C NMR crosspeaks of α-(1→2) and α-(1→3) mannobioses in d2-water as model disaccharides were assigned (and in d6-DMSO—partial assignment). It is clearly shown that it is possible to differentiate the type, configuration and position of the glycosidic linkage i.e. α-(1→2) or α-(1→3) by one bond heteronuclear correlated spectroscopy methodology. Subsequently we compared the reference NMR data and isolated dimer fraction from Candida albicans and concluded that it is exclusively composed of α-(1→2) mannobiose. Notably α-(1→2) linkages as the branching points in the mannan polysaccharide structure imply rather spatially rigid orientation of its sidechains.

One-pot preparation of labelled mannan–peptide conjugate, model for immune cell processing

An efficient method for preparation of fluorescently labelled mannan–peptide glycoconjugates has been developed. After selective Dess–Martin periodinane oxidation of mannan, it was conjugated to the fluorescent label alone and a peptide with the label via reductive amination. Prepared glycoconjugates were characterised by HPSEC, FTIR-ATR and UV-VIS spectroscopy. Finally, the fluorescently labelled mannan and mannan–peptide conjugate were used for microscopic visualization of their accumulation in intracellular organelles of RAW 264.7 cells.

NMR comparison of hyphal and yeast Candida albicans serotype B mannans

A change from a globular to a filamentous hyphal form is an important feature in the pathogenicity of yeasts. Such a dimorphism while infecting a host organism is thought to be also accompanied in the case of Candida albicans spp. by a structural rearrangement of surface mannan antigen. The presented work brings new insights into the molecular structural changes of mannan C. albicans serotype B based on NMR experimental data. 1H and 13C signal identification of the anomeric region and the assignment of their linkage type is presented here. 2D deconvolution of the HSQC spectra facilitated accurate integration of all anomeric cross-peaks. Analysis of the differences in the integrals led to the proposal that C. albicans serotype B hyphal mannan side chains have the shortened structural moieties: Manα1-2Manα1- and Manα1-3 [Manα1-6] Manα1-2Manα1-. These represent the dominant structures important for construction of a saccharide-based prospective anti-candida vaccine.