General methods for the synthesis of glycopyranosyluronic acid azides

Per-O-acetylated D-glycopyranoses derived from both mono- and disaccharides were first converted to glycosyl iodides and subsequently reacted with an azide source to achieve the stereoselective synthesis of beta-D-glycosyl azides after deacetylation. Low-temperature (4 degrees C) TEMPO oxidation of the monosaccharides provided the corresponding uronic acids, which were purified as the free acids. Oxidation of the lactosyl- and cellobiosyl azides resulted in diacid formation. However, 4',6'-O-benzylidene protection enabled selective oxidation of the C-6 hydroxyl. 2-Acetamido-2-deoxy-D-glycopyranosyl azides were also prepared and converted to uronic acids completing the library synthesis.     

The uronic acids assay: a method for the determination of chemical activity on biofilm EPS

In this work, the uronic acids assay was evaluated for its potential to function as a bioassay to screen for antagonistic activity against the production of microbial biofilm exopolysaccharide (EPS). The assay was first applied to biofilms produced in the presence of two universal disinfectants (sodium hypochlorite and sodium dodecyl sulfate) known to inhibit microbial growth and biofilm formation. The performance of the assay was then characterized through statistical assessment of threshold concentrations for disinfection efficiency and consistency relative to values reported in the literature. The assay was then evaluated for its utility in screening for enzymatic or chemical inhibitors of biofilm formation (eg glycosidases, halogenated furanones, and semi-crude fractions extracted from minimally fouled marine plants) and its ability to distinguish between true anti-biofilm activity and simple disinfection. Activity was characterized as (i) no effect, (ii) a true positive effect (ie increased biofilm EPS), (iii) anti-bacterial activity (ie decreased biofilm EPS and analogous decrease in planktonic growth), and (iv) anti-biofilm EPS activity (ie decreased biofilm EPS, without analogous decrease in planktonic growth). Results demonstrate that the uronic acids assay can augment existing biofilm characterization methods by providing a quantitative measure of biofilm EPS.     

Differential gas-liquid chromatography method for determination of uronic acids in carbohydrate mixtures

An integrated gas-liquid chromatography method is described for the quantitation of mixtures containing simple monosaccharides in addition to mannuronic, glucuronic, and/or galacturonic acids. A hydrolyzed sample is divided into two portions. One portion is analyzed by the standard aldononitrile method. Glucuronic, galacturonic, and mannuronic acids are converted into compounds that do not chromatograph in the region of the standard aldononitrile acetates. Thus, this analysis gives an accurate estimation of the neutral monosaccharide content. The second portion is analyzed by a modified alditol acetate procedure. The reduction step is repeated three times to convert mannuronic, galacturonic, and glucuronic acids to their corresponding alditols via their intermediate lactones. The results of this gas-liquid chromatography analysis reflect the sum of the monosaccharides present plus their corresponding uronic acids. The difference between the values obtained by the aldononitrile acetate method and the modified alditol acetate method, therefore, is a measure of the uronic acid(s) present.     

Estimations of Uronic Acids as Quantitative Measures of Extracellular and Cell Wall Polysaccharide Polymers from Environmental Samples

The extracellular polysaccharide polymers can bind microbes to surfaces and can cause physical modification of the microenvironment. Since uronic acids appear to be the components of these extracellular films that are most concentrated in a location outside the cell membrane, a quantitative assay for uronic acids was developed. Polymers containing uronic acids are resistant to quantitative hydrolysis, and the uronic acids, once released, form lactones irreproducibly and are difficult to separate from the neutral sugars. These problems were obviated by the methylation of the uronic acids and their subsequent reduction with sodium borodeuteride to the corresponding alcohol while they were in the polymer and could not form lactones. This caused the polymers to lose the ability to adhere to their substrates, so they could be quantitatively recovered. The hydrolysis of the dideuterated sugars was reproducible and could be performed under conditions that were mild enough that other cellular and extracellular polymers were not affected. The resulting neutral sugars were readily derivatized and then were separated and assayed by glass capillary gas-liquid chromatography. The dideuterated portion of each pentose, hexose, or heptose, identified by combined capillary gas-liquid chromatography and mass spectrometry, accurately provided the proportion of each uronic acid in each carbohydrate of the polymer. Examples of the applications of this methodology include the composition of extracellular polymers in marine bacteria, invertebrate feeding tubes and fecal structures, and the microfouling films formed on titanium and aluminum surfaces exposed to seawater.     

1H-NMR analysis of water mobility in cultured phototrophic biofilms

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the (1)H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.     

Analysis of bacterial exopolysaccharides

Extracellular polysaccharides have been isolated from cultures of freshwater and marine bacteria originally isolated from material adhering to surfaces and underivatized hydrolysates have been analyzed by high-performance liquid chromatography methods. A scheme has been developed whereby the uronic acids can be identified on strong anion-exchange columns, while neutral monosaccharides can be separated and identified using aminobonded columns or cation-exchange adsorbent loaded with a heavy metal ion. The methods permit rapid and accurate comparison of polysaccharides with differing chemotype. The strains studied show a range of different chemotypes, all containing a uronic acid and several neutral monosaccharides. Some of the polysaccharides isolated from marine bacteria possessed a very high acetyl content.     

Measurement of uronic acids without interference from neutral sugars

Replacement of carbazole with meta-hydroxydiphenyl greatly improves the determination of uronic acids in the presence of neutral sugars by preventing substantially, but not completely, the browning that occurs during the heating of sugars in concentrated sulfuric acid and avoiding the formation of additional interference by the carbazole reagent (Blumenkrantz, N., and Asboe-Hansen, G. (1973) Anal. Biochem. 54, 484-489). However, interference is still substantial when uronic acids are determined in the presence of excess neutral sugar, particularly because of the browning that occurs during the first heating before addition of the diphenyl reagent. The browning can be essentially eliminated by addition of sulfamate to the reaction mixture (Galambos, J. T. (1967) Anal. Biochem. 19, 119-132). Although others have reported that sulfamate and the diphenyl reagent were incompatible, we find that a small amount of sulfamate suppresses color production by a 20-fold excess of some neutral sugars without substantial sacrifice of the sensitive detection of uronic acids by the diphenyl reagent. Sodium tetraborate is required for the detection of D-mannuronic acid and enhances color production by D-glucuronic acid. We propose this modified sulfamate/m-hydroxydiphenyl assay as a rapid and reliable means for the assay of uronic acids, particularly when present in much smaller amounts than neutral sugars.     

Characterization and comparison of raft-like membranes isolated by two different methods from rat submandibular gland cells

Lipid rafts are defined as cholesterol and sphingolipid enriched domains in biological membranes. Their role in signalling and other cellular processes is widely accepted but the methodology used for their biochemical isolation and characterization remains controversial. Raft-like membranes from rat submandibular glands were isolated by two different protocols commonly described in the literature; one protocol was based on selective solubilization by Triton X-100 at low temperature and the other protocol consisted in extensive sonication. In both cases a low density vesicular fraction was obtained after ultracentrifugation in a sucrose density gradient. These fractions contained about 20% of total cholesterol but less than 8% of total proteins, and were more rigid than bulk membranes. Fatty acid analyses revealed a similar composition of raft-like membranes isolated by the two different methods, which was characterized by an enrichment in saturated fatty acids in detriment of polyunsaturated acids when compared with the whole cell membranes. Protein profile of detergent resistant membranes or raft-like membranes prepared by sonication was assessed by silver staining after SDS-PAGE and by MALDI-TOF. Both analyses provided evidence of a different protein composition of the Triton X-100 and sonication preparations. Immunoblot experiments revealed that raft-like membranes prepared by detergent extraction or sonication were free of Golgi apparatus or endoplasmic reticulum protein markers (β-COP and calnexin, respectively) and that they were not substantially contaminated by transferrin receptor (a non-raft protein). While caveolin-1 was highly enriched in raft-like membranes prepared by the two methods, the P2X₇ receptor was enriched in raft-like membrane fractions prepared by sonication, but almost undetectable in the detergent resistant membranes. It can be concluded that both methods can be used to obtain raft-like membranes, but that detergent may affect protein interactions responsible for their association with different membrane domains.     

1H-NMR analysis of water mobility in cultured phototrophic biofilms

The present work reports on the first attempt to study water mobility in phototrophic biofilms, applying the ¹H-NMR relaxometry technique to closely monitored microbial communities grown in a microcosm under controlled ambient conditions. Longitudinal water proton relaxation times exhibited a bi-exponential behavior in all biofilm samples, indicating two types of water molecules with diverging dynamic properties, confined to different compartments of the biofilm. The fast-relaxing component can be attributed to water molecules tightly bound to the intracellular matrix, while the slow-relaxing component could reflect the behavior of water embedded in the biopolymer matrix, confined into matrix pores and channels. The results are discussed with respect to a possible key role of exopolysaccharides and uronic acids in water binding in phototrophic biofilms.     

Production and applications of carbohydrate-derived sugar acids as generic biobased chemicals

This review considers the chemical and biotechnological synthesis of acids that are obtained by direct oxidation of mono- or oligosaccharide, referred to as sugar acids. It focuses on sugar acids which can be readily derived from plant biomass sources and their current and future applications. The three main classes of sugar acids are aldonic, aldaric and uronic acids. Interest in organic acids derived from sugars has recently increased, as part of the interest to develop biorefineries which produce not only biofuels, but also chemicals to replace those currently derived from petroleum. More than half of the most desirable biologically produced platform chemicals are organic acids. Currently, the only sugar acid with high commercial production is d-gluconic acid. However, other sugar acids such as d-glucaric and meso-galactaric acids are being produced at a lower scale. The sugar acids have application as sequestering agents and binders, corrosion inhibitors, biodegradable chelators for pharmaceuticals and pH regulators. There is also considerable interest in the use of these molecules in the production of synthetic polymers, including polyamides, polyesters and hydrogels. Further development of these sugar acids will lead to higher volume production of the appropriate sugar acids and will help support the next generation of biorefineries.     

Role of uronic acids present in phytopathogenic fungi as inducers of polygalacturonases during autolysis

The presence of uronic acids in the culture fluid and mycelium of the fungi: Alternaria alternata, Botrytis cinerea, Drechslera halodes, Fusarium culmorum, Fusarium oxysporum, Monilinia fructigena, Mucor mucedo, Rhizopus stolonifer and Trichoderma hamatum was detected and quantified. In these fungi the concentration of uronic acids increased during the growth phase and the maximal concentrations were found at the end of the growth phase or onset of autolysis both in the mycelium as well as in the culture fluid. The uronic acids were metabolized during the first days of autolysis decreasing to constant levels until the end of the autolytic period studied.The variations in the activity of polygalacturonase and polymethylgalacturonase present in the culture fluid were determined at the onset and during autolysis in these fungi. These enzymic activities were found in the culture fluid of these fungi, with exception of M. rouxii, and they showed an increasing activity in the first days of autolysis and later a slight increase or decrease was observed. The presence of uronic acids in these phytopathogenic or saprophytic fungi and the low levels detected during autolysis could be related to the induction of pectic enzymes and the pathogenicity of these fungi.     

Chemical composition and 13C NMR spectroscopic characterisation of ulvans from Ulva (Ulvales, Chlorophyta)

The chemical composition and structures of several ulvan extracts isolated from various Ulva species were studied. They were all composed mainly of rhamnose, glucuronic acid, xylose, glucose and sulphate with smaller amounts of iduronic acid and traces of galactose. Proteins were also present, most likely as contaminants. Precise quantification of the uronic acid content by chemical-enzymatic hydrolysis coupled to HPAEC-PAD analysis and by colorimetry was not achieved, most likely due to the incomplete hydrolysis of glucuronan segments, inadequate HPAEC-pulsed-amperometric response factor for iduronic acid and to a possible differential colorimetric response of the two uronic acids. ¹³C NMR spectroscopic investigation of different ulvans demonstrated that they were all based on ulvanobiuronic acid 3-sulphate A and B repeating units [β-D-Glc pA-(1->4)-α-L-Rhap3S and α-L-IdopA-(1->4)-α-L-Rha p3S, respectively] as well as contiguous β 1->4 linked D-glucuronic acids possibly occurring either in ulvan or as a separate glucuronan. Marked variations in the content of the repeating structures were seen among the different samples. However, due to the limited number of samples studied, no conclusion was reached concerning the effects of species and ecophysiological conditions on the chemistry of ulvan. This revised version was published online in June 2006 with corrections to the Cover Date.     

Isolation, purification and characterization of hyaluronan from human umbilical cord residues

The main objective of this paper is to discuss new procedures of the isolation of Hyaluronan. Hyaluronic acid can be obtained from human umbilical cord residual, which is obtained from other biopharmaceutical productions. The route involves treatment of human umbilical cord residuals with sodium chloride solution, followed by ammonium quaternary salt solution precipitation; the solid is re-suspended in calcium chloride solution in order to dissociate the hyaluronan ammonium quaternary salt complex followed by ethanol-induced precipitation to give a product. The product was purified four times by chloroform extraction, and characterized by chemical methods such as the Blumenkrantz and Asboe-Hansen uronic technique for uronic acid determination, Elson Morgan qualitative tests for hexosamines, intrinsic viscosity, ion-exchange chromatography, and ¹³C NMR spectroscopy. The results showed that the product might be used in the formulation of ointment, lotion and cream for the treatment of skin diseases.     

Is uronic acid oxidase involved in the hormonal regulation of abscission in explants of citrus leaves and fruits?

The role of uronic acid oxidase in abscission was studied in explants of citrus ( Citrus sinensis L. Osbeck; var. Shamouti) leaves and fruits. In leaf explants, activity of uronic acid oxidase prior to onset of abscission and the rate of abscission were markedly accelerated by ethylene and delayed by 2,4-dichlorophenoxyacetie acid. Similar results were obtained for uronic acid oxidase activity in the exocellular fraction of young fruit explants. In mature fruit explants, treated with ethylene, an immediate increase in activity was evidenty in the non-active shoot/peduncle abscission zone, whereas in the calyx abscission zone the rise in activity occurred after a prolonged exposure to ethylene, when most of the fruits had already abscised. Whenever ethylene enhanced uronic acid oxidase activity, 2,4-dichlorophenoxyacetic acid delayed it. A gradient of decreasing activity or uronic acid oxidase was recorded from both sides of the abscission zone in leaves and fruits toward the separation line, where activity was the lowest as compared with the activity found in adjacent tissues. It is suggested that uronic acid oxidase is involved in senescence and cell wall degradation. However, it is yet questionable whether this enzyme is directly related to the control mechanism of abscission.     

Characterisation of secreted polysaccharides and (glyco)proteins from suspension cultures of Pyrus communis

High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.     

Features of ionogenic group composition in polymeric matrix of lily pollen wall

The composition of ionogenic groups and ion-exchange capacity were studied in the polymeric matrix of cell walls isolated from the pollen grain and tissues of vegetative organs (leaves and stems) of Lilium longiflorum Thunb. The ion-exchange capacity was evaluated at different pH values and ionic strength of 100 mM. In the two-layered pollen wall and the somatic cell walls four types of ionogenic groups were found: amino groups, two carboxyl groups (represented by residues of uronic and hydroxycinnamic acids), and phenolic OH-groups. The groups of all four types are present in the intine, whereas the exine contains one type of anion-exchange and two types of cation-exchange groups. The contents of each type group and their ionization constants were determined. The qualitative and quantitative compositions of structural polymers of the pollen intine and somatic cell walls are significantly different. It is suggested that hydroxycinnamic acids should be involved in cross-linking of polysaccharide chains in both the intine and somatic cell primary walls, and such cross-links play a crucial role in the structural organization and integrity of the pollen grain wall.     

Nutritional content of fresh, bee-collected and stored pollen of Aloe greatheadii var. davyana (Asphodelaceae)

Aloe greatheadii var. davyana is the most important indigenous South African bee plant. Fresh, bee-collected and stored pollen of this aloe was collected and analysed for its nutritional content, including amino acid and fatty acid composition. Highly significant differences were found between the three types of pollen. Collection and storage by the bees resulted in increased water (13-21% wet weight) and carbohydrate content (35-61% dry weight), with a resultant decrease in crude protein (51-28% dry weight) and lipid content (10-8% dry weight). Essential amino acids were present in equal or higher amounts than the required minimum levels for honeybee development, with the exception of tryptophan. Fatty acids comprised a higher proportion of total lipid in fresh pollen than in bee-collected and stored pollen. This study is the first to compare the changes that occur in pollen of a single species after collection by honeybees.     

Detection of uronic oxidase activity in ripening peaches

Uronic acid oxidase activity was found in an extract from harvested peaches that was incubated with citrus pectin at pH 8.5. The product of this reaction was identified by GC-MS analysis to be galactaric acid. The reaction was linear at 37 degrees C for up to 20 h, and the pH optimum was 8.5. The activity found in firm peaches one day after harvest did not change as the peaches softened over 5 days to eating softness. The incubation conditions were those suitable for monitoring the activity of pectate lyase, but instead of finding an increase in galacturonosyl residue reducing groups due to generation of pectin-derived oligosaccharides, uronic acid oxidase catalyzed the oxidation of the aldehyde reducing functions to carboxyl groups.     

Quantitative analysis of biofilm EPS uronic acid content

The uronic acids assay was evaluated for its ability to measure the amount of uronic acids contained within a biofilm exopolysaccharide matrix. Cytophaga lytica, a marine bacterium isolated from a naturally occurring biofilm, was used to form single-species biofilms for the method assessment. The assay was found to be simple, reproducible, and sensitive to 1 microg levels, suggesting its potential for application as a screening technique for compounds that inhibit the production of microbial biofilm exopolysaccharide containing uronic acids.