Molecular heterogeneity of arabinogalactan-protein from Coffea arabica instant coffee.

Arabinogalactan-protein complex (AGP), isolated from freeze-dried instant coffee powder of Coffea arabica beans, was subjected to ion-exchange chromatography. Stepwise elution with water and solutions of sodium chloride with increasing ionic strength afforded eight fractions (F1-F8). Chemical analyses have shown that compositions of individual conjugates varied in carbohydrate and protein contents, molecular mass and slightly in monosaccharide composition. Protein content was increasing by increasing ionic strength of the eluent and it was confirmed also by FT-IR spectra. NMR study has shown that carbohydrate moieties in individual ion exchange fractions differed in the degree of backbone and side chains branching. Performed study has confirmed a molecular heterogeneity of arabinogalactan-protein complex present in a commercial instant coffee.

An arabino(glucurono)xylan isolated from immunomodulatory active hemicellulose fraction of Salvia officinalis L.

From the aerial parts of sage (Salvia officinalis L.) an arabino-(4-O-methyl-glucurono)-xylan (AGX) was isolated by alkaline extraction followed by precipitation with barium hydroxide solution. Polymer was isolated from sage as a light brown polysaccharide material of molecular mass (Mp) 84,000. Compositional analyses of sage AGX revealed xylose (81%), arabinose (10%), glucuronic acid (8%) and small amounts of hexoses (1%). Linkage sugar analyses showed the (1→4)-linked xylopyranosyl backbone with low degree of substitution (9-10%) at O-2 and O-3. Arabinofuranose residues were found as the terminal, 1,3-, 1,5- and 1,3,5-linked. NMR structural analyses of acidic oligomers, generated by partial acidic hydrolysis of AGX, confirmed a substitution of xylose residues by glucuronic acid and its 4-O-methyl derivate at O-2 at an average on every fourteenth xylose residue. NMR and FT-IR measurements, as well as a high negative optical rotation confirmed the ß configuration of glycosidic linkages in AGX backbone.

Vaccination of chickens with Salmonella Pathogenicity Island (SPI) 1 and SPI2 defective mutants of Salmonella enterica serovar Enteritidis.

In this study we were interested in the vaccine potential of two attenuated mutants of Salmonella enterica serovar Enteritidis for poultry. The first mutant was attenuated by the removal of the whole Salmonella Pathogenicity Island 1 (SPI1) and the second mutant was devoid of the whole SPI2. These 2 mutants were used for oral vaccination of 2 chicken lines; Lohmann Brown and ISA Brown. Chickens were vaccinated orally on day 1 of life, revaccinated on day 21 and challenged on day 42. The challenge was performed either orally or intravenously. Despite a slightly different response between the two chicken lines, both the mutants gave protection to poultry against S. Enteritidis challenge as documented by findings such as the bacterial counts in tissues, spleen weight, antibody production and cytokine response (namely IL-17 and IL-22). When the 2 mutants were compared, vaccination with the SPI1 mutant proved to be more effective in the protection of poultry against S. Enteritidis challenge than the vaccination with the SPI2 mutant. On the other hand, vaccination with the SPI2 mutant stimulated a slightly higher antibody production and such a mutant might therefore be a better choice if Salmonella is used as a vector for the delivery of heterologous antigens with a desired stimulation of the humoral part of the immune system.

Extended-spectrum beta-lactamase-producing Escherichia coli in turkey meat production farms in the Czech Republic: national survey reveals widespread isolates with bla(SHV-12) genes on IncFII plasmids.

AIM: The occurrence and epidemiology of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in the environment of turkey farms in the Czech Republic were studied. METHODS AND RESULTS: Extended-spectrum beta-lactamase-producing E. coli isolates were found on 8 (20%) of 40 turkey farms surveyed. A total of 200 environmental smears were examined, and a total of 25 ESBL-producing E. coli were isolated. These isolates were analysed using XbaI pulsed-field gel electrophoresis and divided into nine pulsotypes. Most of the isolates harboured the gene bla(SHV-12) on a 40-kb plasmid of the IncFII group with an identical EcoRV restriction profile. Indistinguishable or clonally related SHV-12-producing isolates belonging to the same pulsotypes were found at some unrelated farms. CONCLUSIONS: Widespread occurrence of ESBL-producing E. coli isolates with bla(SHV-12) carried on IncFII plasmids in meat production flocks in the Czech Republic was demonstrated. SIGNIFICANCE AND IMPACT OF THE STUDY: Results indicate vertical transmission of ESBL-producing E. coli within the turkey production pyramid. The study shows the risk of multiresistant ESBL-producing bacteria and antibiotic-resistance genes being transmitted to humans via the food chain.

Antitussive and immunomodulating activities of instant coffee arabinogalactan-protein.

A low molecular mass arabinogalactan-protein (AGP) composed of galactose and arabinose with a low protein content, isolated from the instant coffee powder of Coffea arabica beans, has been tested on antitussive (in vivo) and immunomodulating (ex vivo) activities. The results of antitussive tests revealed a significant dose dependant cough-suppressive effect of coffee AGP. It was observed 30 or 60 min after AGP administration and its efficacy lasted during the entire experiment course. Immunological tests showed that AGP affected some mediators of immunocompetent cells of immune system as TNF-α, IFN-γ and IL-2 cytokines. It seems that coffee AGP is a good inductor of both pro-inflammatory cytokines TNF-α and IFN-γ, however, less potent in TNF-α induction in comparison with that of ß-D-glucan. Evident induction of TNF-α, IL-2 and IFN-γ cytokines, pro-TH1 polarization supports our conclusion about bio-immunological efficacy of AGP with an emphasis on the cellular immunity.

Dietary fibre degradation and fermentation by two xylanolytic bacteria Bacteroides xylanisolvens XB1A and Roseburia intestinalis XB6B4 from the human intestine.

AIMS: To characterize fibre degradation, colonization and fermentation, and xylanase activity of two xylanolytic bacteria Bacteroides xylanisolvens XB1A(T) and Roseburia intestinalis XB6B4 from the human colon. METHODS AND RESULTS: The bacteria grew well on all the substrates chosen to represent dietary fibres: wheat and corn bran, pea, cabbage and leek fibres, and also on purified xylans. Roseburia intestinalis colonized the substrates more efficiently than Bact. xylanisolvens. For the two bacteria, 80-99% of the total xylanase activity was associated with the cells whatever the substrate and time of growth. Optimal specific activities of cells were obtained on oat spelt xylan; they were higher than those previously measured for xylanolytic bacteria from the human gut. Roseburia intestinalis produced high molecular mass xylanases (100-70 kDa), while Bact. xylanisolvens produced lower molecular mass enzymes, including a cell-associated xylanase of 37 kDa. CONCLUSIONS: The two bacteria display very high xylanolytic activity on the different substrates. Differences were observed on substrate attachment and enzyme systems, suggesting that the two species occupy different niches within the gut microbiota. SIGNIFICANCE AND IMPACT OF THE STUDY: This study characterizes xylan degradation by two major species of the human intestine.

The extracellular proteoglycan produced by Rhodella grisea.

Highly viscous extracellular proteoglycan (EPG) has been isolated from culture medium of the unicellular red alga Rhodella grisea (Rhodophyceae) by ethanol precipitation. EPG was composed of xylose (29.3%), 3-O-methyl-xylose (26.0%), uronic acids (17.1%), rhamnose (14.4%), galactose (7.5%), glucose (3.9%), arabinose (1.4%) and mannose (0.4%), and traces of fucose, 4-O-methyl-xylose and 2,3-di-O-methyl-rhamnose or fucose. In addition, the polymer contained proteins (13.1%), sulphates and 13C-CP MAS spectra indicated the presence of acetyl and succinyl groups. The molecular mass was estimated to be 136,000. Ion-exchange chromatography afforded five fractions differing in composition of neutral sugars, uronic acids, and protein content indicating thus the complex structure of the EPG.

An extracellular galactoxylomannan of acapsular Cryptococcus laurentii mutant.

An extracellular galactoxylomannan (GalXM) composed of D-Gal (34.0%), D-Xyl (26.6%) and D-Man (31.0%), and a small amount of L-Ara (4.0%) and D-Glc (4.4%) has been isolated from culture medium of acapsulated mutant of Cryptococcus laurentii by ethanol precipitation and gel filtration. Phosphorylated polymer of Mw approximately 75,000 contained 90% carbohydrates, 3.9% phosphorus and 5.3% proteins. Results of chemical and spectroscopic studies showed a highly branched structure of GalXM with a 1,6-linked mannopyranosyl-galactopyranosyl backbone (approximately 44%) branched predominantly at C-2 and C-3 of mannosyl, and C-3 of galactosyl residues by side chains terminated mainly by xylosyl and mannosyl residues, and to a less extent by arabinosyl and glucosyl ones.

Degradation of wheat straw by Fibrobacter succinogenes S85: a liquid- and solid-state nuclear magnetic resonance study.

Wheat straw degradation by Fibrobacter succinogenes was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with 13C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of 13C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of F. succinogenes S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than alpha-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.

[The relation of GERD, bronchial asthma and the upper respiratory tract]. / Vztah refluxní choroby jícnu k astmatu bronchiale a horním cestám dýchacím.

UNLABELLED: Gastroesophageal reflux disease (GERD) is one of the most common diseases affecting upper gastrointestinal tract. It is a chronic disease, whith stadily growing incidence and prevalence in west countries during last 30 years. GERD is caused by pathologic gastroesophageal reflux (GER). GERD includes endoscopically positive, endoscopically negative and extraesophageal reflux disease. Extraesophageal symptoms of GERD have been of a growing attention and discussion during last few years. The most discussed topics are the relation of GERD and bronchial asthma (BA), chronic cough and symptomatology from ear, nose and throught (ENT) regions, but also non - cardial chest pain and many others. AIM: In our clinic we ran a 5 years study which aim was to evaluate the presence of GERD in patients with bronchial asthma, chronic cough and affections from ENT regions. To assess if 3 months GERD treatment would improve lung function, subjective complaints (cough) and GERD control in asthmatics; if this treatment would allow to step - down with antiasthma medication. To assess if 3 months GERD treatment can improve objective and subjective assessments in patients with chronic cough and findings in ENT regions. As for GERD, we evaluated the improvement of pH and subjective complaints (pyrosis). METHODS: We examined 86 patients with different severity of bronchial asthma, 54 patients with chronic cough and 31 patients with ENT symptoms. All patients underwent 24 hour esophageal pH metry, spirometry with lung function evaluation and objective ENT examination by flexible laryngoscopy. In case of pathologic finding on 24 hour pH-metry 3 months full antireflux treatment with proton pump inhibitors (PPI) and prokinetics was introduced. After 3 months of GERD treatment we performed control 24 hour esophageal pH metry, control spirometry and ENT examination by flexible laryngoscopy. Patients were asked to make their subjective symptoms assessments. RESULTS: We found that GERD prevalence in patients with respiratory symptoms was very high. Three months GERD treatment improved lung function (FEV1) with statistical significance (p = 0.0319), and so improved GERD control (in 60.7% of patients with high statistical significance p = 0.0009). Subjective complaints (cough) also improved in most patients. 3 months GERD treatment did not allow to step down with maintenance BA therapy according to GINA guidelines, but it enabled to decrease the rescue medications in 50% of patients. Patients with chronic cough can benefit from GERD treatment as cough improved in 75.8% of patients. CONCLUSION: Objective findings as well as subjective complaints improved in 75% of patients with ENT symptomatology. GERD control (DeMeester score and pyrosis if present) was highly statistically significant in all three groups of patients. It is necessary to mention, that there is a high presence of nocturnal acid breakthrough (NAB) in patients with respiratory symptoms: 30.3 % in patients with bronchial asthma, 63.6 % in patients with chronic cough and 45 % of patients with ENT manifestations.

Concurrent maltodextrin and cellodextrin synthesis by Fibrobacter succinogenes S85 as identified by 2D NMR spectroscopy.

1D and 2D NMR experiments were used to analyse the synthesis of various metabolites by resting cells of Fibrobacter succinogenes S85 when incubated with [1-(13)C]glucose, in both extracellular and cellular media. Besides the expected glycogen, succinate, acetate, glucose-1-P and glucose-6-P, maltodextrins and cellodextrins were detected. Maltodextrins were excreted into the external medium. They were found to have linear structures with a maximum degree of polymerization (DP) of about 6 or 7 units. Cellodextrins were located in the cells (cytoplasm and/or periplasm), and their DP was < or = 4. Both labelled (1-(13)C and 6-(13)C) and unlabelled maltodextrins and cellodextrins were detected, showing the contribution of carbohydrate cycling in F. succinogenes, including the reversal of glycolysis and the futile cycle of glycogen. The mechanisms of these oligosaccharide syntheses are discussed.

13C and 1H NMR study of cellulose metabolism by Fibrobacter succinogenes S85.

Fibrobacter succinogenes S85, a cellulolytic rumen bacterium, is very efficient in degrading lignocellulosic substrates and could be used to develop a biotechnological process for the treatment of wastes. In this work, the metabolism of cellulose by F. succinogenes S85 was investigated using in vivo 13C NMR and 13C-filtered spin-echo difference 1H NMR spectroscopy. The degradation of unlabelled cellulose synthesised by Acetobacter xylinum was studied indirectly, in the presence of [1-13C]glucose, by estimating the isotopic dilution of the final bacterial fermentation products (glycogen, succinate, acetate). During the pre-incubation period of F. succinogenes cells with cellulose fibres, some cells ('non-adherent') did not attach to the solid material. Results for 'adherent' cells showed that about one fourth of the glucose units entering F. succinogenes metabolism originated from cellulose degradation. A huge reversal of succinate metabolism pathway and production of large amounts of unlabelled acetate which was observed during incubation with glucose only, was found to be much decreased in the presence of solid substrate. The synthesis of glucose 6-phophate was slightly increased in the presence of cellulose. Results clearly showed that 'non-adherent' cells were able to metabolise glucose very efficiently; consequently the metabolic state of these cells was not responsible for their 'non-adherence' to cellulose fibre.

(4-O-Methyl-alpha-D-glucurono)-D-xylan from Rudbeckia fulgida, var. sullivantii (Boynton et Beadle).

From the medicinal plant Rudbeckia fulgida, var. sillivantii (Boynton et Beadle) a low-molecular-mass (4-O-methyl-alpha-D-glucurono)-D-xylan was isolated by alkaline extraction, followed by ethanol precipitation, ion-exchange chromatography and gel filtration. The results of compositional and linkage analyses, supported by those of 1H and 13C NMR measurements of oligomers generated on partial acid hydrolysis, showed the (1-->4)-linked beta-D-xylopyranosyl backbone with about 18% of 4-O-methyl-D-glucuronic acid attached to O-2 of the xylose residues. From the mean distance of adjacent carboxyl groups, obtained from experimentally determined single-ion activity coefficients of calcium counterions, it followed that the uronic acid units are separated and distributed regularly along the xylan chain, i.e. approximately each sixth D-xylose unit bears a 4-O-methyl-D-glucuronic acid residue.

A nitro sugar derivative route to 2-thioepisophorose and 2-thiosophorose and their remarkable facile epimerization.

The addition of 1-thio-D-glucose sodium salt to per-O-acetylated 1,2-dideoxy-1-nitro-D-arabino-hex-1-enitol, readily available from D-arabinose, afforded the corresponding 2-S-glycosylated 1-deoxy-1-nitro-D-mannitol and -D-glucitol peracetates. These, after deacetylation, were transformed by the Nef reaction to 2-thioepisophorose and 2-thiosophorose, respectively. The 2-thiodisaccharides easily epimerize in aqueous sodium bicarbonate at ambient temperature to a 1:4 equilibrium mixture. The predominant 2-thiosophorose was obtained crystalline. A 1H NMR study of the epimerization in deuterium oxide showed that the reaction involves an H-2 proton exchange mechanism.

Oxygen-derived free radical (ODFR) action on hyaluronan (HA), on two HA ester derivatives, and on the metabolism of articular chondrocytes.

Oxygen-derived free radicals (ODFR) appear to be involved in the pathogenesis of arthritic disorders. In order to gain new insight on their role in the phenomenon and as a basis for a therapeutic approach, the effect of ODFR (produced by the xanthine oxidase-hypoxantine system) on hyaluronic acid, on two HA ester derivatives, and on pig articular chondrocytes was investigated. High M(r) HA (1.1 x 10(6)) and low M(r) HA (16 x 10(4)) were depolymerized by ODFR but the methyl and hydrocortisone esters of HA (HYAFF 2P50 and HYC13) turned out to be nearly unaffected. When articular chondrocytes were treated with ODFR, a rapid nucleoside triphosphate (NTP) depletion, a transient appearance of pyrophosphate (PPi), and an increase of phosphomonoester and diphosphodiester concentrations have been observed. The NTP depletion and the DPDE increase are related to the concentration of free radicals. Glyceraldehyde-3-phosphate accumulation during ODFR treatment suggests that ATP depletion can occur as a consequence of the blockage of glycolysis at the level of glyceraldehyde-3-P dehydrogenase. The hypothesis is presented that PPi can be produced from the pathway of the FAD-NAD (DPDE) biosynthesis and then either hydrolyzed by endogenous pyrophosphatases or precipitated in the form of insoluble calcium salts. Long-term treatment (16 h) with ODFR causes a loss of chondrocyte membrane integrity which can be revealed both by an increased free LDH activity and by the characteristic signal of free phospholipids in the 31P-NMR spectra. While high M(r) HA shows a significant protective activity for chondrocytes against ODFR action, low M(r) HA and ester derivatives do not. It is suggested that the therapeutic activity of HA ester derivatives can be ascribed to their in vivo hydrolysis products.

NMR analysis of succinoglycans from different microbial sources: partial assignment of their 1H and 13C NMR spectra and location of the succinate and the acetate groups.

In order to obtain information on the location of succinate and acetate groups, comparative NMR analyses were carried out on succinoglycans from different microbial sources by using conventional and advanced NMR techniques. In particular, one-dimensional, 1H and 13C NMR spectra were recorded for qualitative and quantitative analysis on native high-molecular-weight succinoglycans (both in the Na+ salt and free-acid forms) from Pseudomonas sp. NCIB 11592, Agrobacterium radiobacter A201-25, Rhizobium meliloti YE-2, and Rhizobium sp. isolated from Vicia faba and compared with those of the deacylated and deacylated-depyruvated, partially depolymerised exopolysaccharides from Rhizobium meliloti YE-2. Moreover, a series of two-dimensional experiments was performed on all the exopolysaccharides aiming at the partial assignment of the NMR spectra. The NMR data showed that succinate is located on O-6 of either one or both of the two side chain 3-linked beta-D-Glc residues, whereas the acetate (when it is present) is located on one of the O-6 of backbone 4-linked beta-D-Glc units, but the specific site could not be determined. In addition, the spectral features of the succinate substituent were found to be sensitive to pH changes.

Immobilisation of beta-D-galactosidase from Escherichia coli on cellulose beads and its use for the synthesis of disaccharide derivatives.

beta-D-Galactosidase, isolated from cloned E. coli, was immobilised on cellulose beads via oxidation with sodium periodate, activation by cyanuric chloride, or diazotisation. beta-D-Galactosidase immobilised via azo bonds showed the highest relative activity and thermostability, and was used for synthesis of disaccharide methyl glycosides.

Antimicrobial effect of 4-nitrophenylhydrazones, isonicotinoylhydrazones and N-4-nitrophenylglycosylamines of D- and L-aldoses.

The antibacterial effect of 19 4-nitrophenylhydrazones, isonicotinoylhydrazones, and N-4-nitrophenylhydrazones, of 9 mono- and 2 disaccharides was tested with Micrococcus luteus, Bacillus licheniformis and Escherichia coli.