Vladimír Pätoprstý

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.

Structural characterization and antitussive activity of a glucuronoxylan from Mahonia aquifolium (Pursh) Nutt.

Abstract From the stems of Mahonia aquifolium (Pursh) Nutt. a water-soluble (4-O-methyl-α- d -glucurono)- d -xylan was isolated by alkaline extraction and fractionation of the crude hemicellulose, employing ion-exchange chromatography and gel filtration. The results of compositional and linkage analysis, supported by NMR spectral data of the polysaccharide, showed the (1→4)-linked β- d -xylopyranosyl backbone with about 15% of 4-O-methyl- d -glucuronic acid attached to O-2 of the xylose residues. The distribution pattern of uronic acid units along the xylan chain was determined by the method based on interpretation of the activity coefficient of the calcium counter-ions, estimated in a molecular-disperse solution of calcium salt of the polysaccharide. The results evidenced that the branching of the xylan molecule is not regular. When tested for antitussive activity on mechanically induced coughing in cats, the glucuronoxylan exhibited a much greater effect in comparison to the drugs used in clinical practice to treat coughing.

A comparative study of lipopolysaccharides from two Coxiella burnetii strains considered to be associated with acute and chronic Q fever

Abstract Coxiella burnetii strains Nine Mile and Priscilla, considered to be associated with acute and chronic forms of Q fever, were investigated for variation in composition of their lipopolysaccharides. Though SDS-PAGE profiles of the lipopolysaccharides were distinct, chemical analyses showed only small differences in their overall composition. Further studies on lipid A-deprived O-polysaccharide fractions of both lipopolysaccharides, obtained by steric-exclusion chromatography, revealed noticeable differences in distribution and chemical composition of the O-polysaccharide chains. It is likely that C. burnetii strains are capable of synthesizing chemically distinct subclasses of O-specific polysaccharide molecules differing in their antigenic reactivities. The results provide suggestive evidence that virulence of C. burnetii may be modulated through lipopolysaccharide composition and structure.

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).

Off-line FIA monitoring of D-sorbitol consumption during L-sorbose production using a sorbitol biosensor.

A ferricyanide mediated amperometric biosensor system implementing D-sorbitol dehydrogenase together with diaphorase for sensitive detection of D-sorbitol was used. The biosensor system was successfully integrated into an off-line FIA system with a throughput of detection of 10 h(-1). The device exhibited limit of detection of 20 microM with an average relative standard deviation of analysis of samples of 2.2%. The signal of the biosensor was linear up to 1.1 mM for D-sorbitol with sensitivity of (72 +/- 2) nA mM(-1), while a dynamic range was much wider up to 18 mM. The sorbitol biosensor gave reliable results even in the presence of a high molar excess of L-sorbose, a product of the biotransformation process, as judged from an excellent agreement with HPLC and GC.

Production of oligosaccharides and cellobionic acid by Fibrobacter succinogenes S85 growing on sugars, cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.

Carbohydrate polymers from underground parts of Cistanche deserticola

Abstract The subterraneous parts of the holoparasite, Cistanche deserticola, was extracted with methanol and the easily extractable polysaccharides were isolated from the insoluble residue using in succession cold water, hot water, 0.5 M NaOH, and 0.01 M EDTA. Based on the data of sugar composition analysis, methylation analysis and NMR spectroscopy, it was shown that the bulk of the easily extractable polysaccharides was composed of starch-like α-1,4- d -glucan, α- l -arabino-3,6-β- d -galactan, pectic polysaccharides and 4-O- methyl- d -glucurono- d -xylan .

Flow calorimetry--a useful tool for determination of immobilized cis-epoxysuccinate hydrolase activity from Nocardia tartaricans.

Bacterial cells Nocardia tartaricans with cis-epoxysuccinate hydrolase activity were entrapped in hardened calcium pectate gel by a commercial high performance encapsulator. This enzyme (in a single step reaction with no formation of side products) was used to hydrolyze disodium cis-epoxysuccinate to a pure enantiomer—disodium l-(+)-tartrate. Activities of this enzyme were determined using flow calorimetry. The validity of this method was corroborated by HPLC and isotachophoresis. The immobilized biocatalyst has activity (75.8 U/mgdry) able to convert disodium cis-epoxysuccinate to disodium tartrate at 94% yield in 5.5 h. Immobilization of N. tartaricans in hardened calcium pectate gel beads had a positive effect on the activity of cis-epoxysuccinate hydrolase, storage stability, yield, and time of bioconversion.

Enzymatic Oxidation and Separation of Various Saccharides with Immobilized Glucose Oxidase

Glucose oxidase from Aspergillus niger, the specific enzyme for β-d-glucose oxidation, can also oxidize other related saccharides at very slow or negligible rates. The present study aimed to compare the kinetics of d-glucose oxidation using immobilized glucose oxidase on bead cellulose for the oxidation of related saccharides using the same biocatalyst. The significant differences were observed between the reaction rates for d-glucose and other saccharides examined. As a result, kcat/KM ratio for d-glucose was determined to be 42 times higher than d-mannose, 61.6 times higher than d-galactose, 279 times higher than d-xylose, and 254 times higher than for d-fructose and d-cellobiose. On the basis of these differences, the ability of immobilized glucose oxidase to remove d-glucose from d-cellobiose, d-glucose from d-xylose, and d-xylose from d-lyxose was examined. Immobilized catalase on Eupergit and mixed with immobilized glucose oxidase on bead cellulose or co-immobilized with glucose oxidase on bead cellulose was used for elimination of hydrogen peroxide from the reaction mixture. The accelerated elimination of d-glucose and d-xylose in the presence of co-immobilized catalase was observed. The co-immobilized glucose oxidase and catalase were able to decrease d-glucose or d-xylose content to 0–0.005% of their initial concentrations, while a minimum decrease of low oxidized saccharides d-xylose, d-cellobiose, and d-lyxose, respectively, was observed.

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.