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PubMed2001

Development and functions of seed transfer cells.

In secretion or absorption processes, solutes are transported across the plasmalemma between the symplastic and apoplastic compartments. For this purpose, certain plant cells have developed a specialised transfer cell morphology characterised by wall ingrowths, which amplify the associated plasmalemma surface area up to 20-fold. Detailed studies on the function and development of transfer cells in the context of seed filling have been carried out mainly in cereal endosperm, and for the cotyledon and seed coat cells of legumes. The major solutes transferred are amino acids, sucrose and monosaccharides. The contributions of recently identified symporter proteins to solute transfer are reviewed here, as is the role of apoplastic invertases in promoting solute assimilation. Expression of invertase and monosaccharide transporters early in both cereal and legume seed development orchestrates the distribution of free sugars which play an important role in regulating transfer cell function and determining final endosperm or embryo cell number. Transfer cell differentiation is subject to developmental control, and may also be modulated by sugar levels. The most abundant genes specifically expressed in the transfer layer of maize endosperm encode small antipathogenic proteins, pointing to a role for these cells in protecting the developing endosperm against pathogen ingress. The functional characterisation of the corresponding transfer layer-specific promoters has provided a tool for dissecting transfer cell functions. Transfer cells are highly polar in their organisation, the characteristic cell wall ingrowths developing on one face only. The presence of cytoskeletal components bordering wall ingrowths is documented, but their role in establishing transfer cell morphology remains to be established.

Journal Article↗
PubMed1999

Sulfolipo-cyclodextrin in squalane-in-water as a novel and safe vaccine adjuvant.

Previously, we described synergistic adjuvanticity of combinations of synthetic sulfolipo(SL)-derivatives of polysaccharide (SL-polysaccharides) and squalane-in-water emulsions (squalane/W). In this paper, effects of type of polysaccharide and nature of oil on adjuvanticity, reactogenicity and stability are described. SL-derivatives of the following polysaccharides were synthesised: synthetic polysucroses with weight-average molecular weight (MW) of 400,000 (Ficoll400), 70,000 (Ficoll70) and 39,000 Da (Ficoll39), polyfructose of 5,000 Da (inulin), linear polyglucose of 1,200 Da (maltodextrin) and cyclic polyglucose of 1,135 Da (beta-cyclodextrin). The number of sulphate groups per monosaccharide of the different SL-polysaccharides varied between 0.15 and 0.23 and the number of lipid groups per monosaccharide between 1.15 and 1.29. Adjuvant formulations were prepared by incorporating these SL-polysaccharides into oil-in-water emulsions of either squalane, hexadecane, soya oil or mineral oil. Adjuvanticity of the formulations obtained for humoral responses to inactivated pseudorabies virus (PRV) and inactivated influenza virus strains A/Swine (A/Swine) and MRC-11 (MRC-11) in pigs and MRC-11 and ovalbumin (OVA) in mice depended on the type of oil (squalane = mineral oil > hexadecane = soya oil) but not on the type of polysaccharide backbone of the SL-derivative. Reactogenicity assessed by local swelling in mice decreased with decreasing MW (SL-Ficoll400 = Ficoll70 = Ficoll39 > SL-inulin = SL-maltodextrin > SL-cyclodextrin) when combined with squalane and decreased with the type of oil in the following order: squalane > mineral oil > hexadecane > soya oil when combined with SL-Ficoll400. Stability of the SL-polysaccharide/squalane/W emulsions at elevated temperature increased with decreasing MW of the SL-polysaccharide (SL-Ficoll400 < SL-Ficoll70 = SL-Ficoll39 < SL-inulin = SL-maltodextrin = SL-cyclodextrin). SL-cyclodextrin/squalane/W remained stable for > 2.5 years at 4 degrees C, > 18 weeks at 37 degrees C and > 10 days at 60 degrees C. We concluded that reactogenicity and stability but not adjuvanticity of SL-polysaccharide/squalane/W formulations depended on the MW of SL-polysaccharide and that SL-cyclodextrin/squalane/W is a promising non-mineral oil adjuvant as it combines strong adjuvanticity (i.e. better than the mineral oil-based adjuvant presently applied) with low reactogenicity and good stability.

Adjuvants, Immunologic↗
PubMed1996

The sensing of plant signal molecules by Agrobacterium: genetic evidence for direct recognition of phenolic inducers by the VirA protein.

The virulence (vir) genes of Agrobacterium tumefaciens are induced by low-molecular-weight phenolic compounds and monosaccharides through a two-component regulatory system consisting of the VirA and VirG proteins. Although it is clear that the monosaccharides require binding to a periplasmic binding protein before they can interact with the sensor VirA protein, it is not certain whether the phenolic compounds also interact with a binding protein or directly interact with the sensor protein. To shed light on this question, we tested the vir-inducing abilities of several different phenolic compounds using two wild-type strains of A. tumefaciens, KU12 and A6. We found that several compounds such as 4-hydroxyacetophone and p-coumaric acid induced the vir of KU12, but not A6. On the other hand, acetosyringone and several other phenolic compounds induced the vir of A6, but not KU12. By transferring different Ti plasmids into isogenic chromosomal backgrounds, we showed that the phenolic sensing determinant is associated with the Ti plasmid. Subcloning of the Ti plasmid indicated that the virA locus determines which phenolic compounds can function as vir inducers. These results suggest that VirA directly senses the phenolic compounds for vir activation.

Agrobacterium tumefaciens↗
PubMed1997

Endo-N-acetyl-beta-D-glucosaminidases and their potential substrates: structure/function relationships.

Endo-N-acetyl-beta-D-glucosaminidases (ENGases) have been defined as the enzymes that hydrolyse the glycosidic bond between an N-acetyl-beta-D-glucosamine residue and the adjacent (partner) monosaccharide within an oligosaccharide chain. Three types of enzymes have been distinguished according to this definition: ENGases acting on murein (type I), those acting on chitin (type II) and, finally, those acting on N-glycans (type III). Considering that N-acetylmuramic acid is a derivative of N-acetylglucosamine (3-O-substituted by a lactyl group), only ENGases acting between two N-acetylglucosamine residues are actually known despite the fact that other possibilities of partner monosaccharides for N-acetyl-beta-D-glucosamine are reported. Similarities in the amino acid sequences were found to occur only between chitin-ENGases and N-glycan-ENGases, but the substrate specificities of these two types of enzymes are different. However, it is possible that certain enzymes are able to cleave more than one type of substrate, and this could in particular explain why the N-glycan-ENGases are largely produced by bacteria in which no potential substrate for this type of enzymes was identified. Further study in this area is expected.

Chitin↗
PubMed1998

Aldose reductase: a window to the treatment of diabetic complications?

Kinetic studies on the aldose reductase protein (AR2) have shown that it does not behave as a classical enzyme in relation to ring aldose sugars. These results have been confirmed by X-ray crystallography studies, which have pinpointed binding sites for pharmacological "aklose reductase inhibitors" (ARIs). As with non-enzymic glycation reactions, there is probably a free-radical element involved derived from monosaccharide autoxidation. In the case of AR2, there is free radical oxidation of NADPH by autoxidising monosaccharides, enhanced in the presence of the NADPH-binding protein. Whatever the behaviour of AR2, many studies have showed that sorbitol production is not an initiating aetiological factor in the development of diabetic complications in humans. Vitamin E (alpha-tocopherol), other antioxidants and high fat diets can delay or prevent cataract in diabetic animals even though sorbitol and fructose levels are not modified; vitamin C acts as an AR1 in humans. Protein post-translational modification by glyc-oxidation or other events is probably the key factor in the aetiology of diabetic complications. There is now no need to invoke AR2 in xylitol biosynthesis. Xylitol can be produced in the lens from glucose, via a pathway involving the enzymes myo-inositol-oxygen oxidoreductase, D-glucuronate reductase. L-gulonate NAD(+)-3-oxidoreductase and L-iditol-NAD(+)-5-oxidoreductase, all of which have recently been found in bovine and rat lens. This chapter investigates the molecular events underlying AR2 and its binding and kinetics. Induction of the protein by osmotic response elements is discussed, with detailed analysis of recent in vitro and in vivo experiments on numerous ARIs. These have a number of actions in the cell which are not specific, and which do not involve them binding to AR2. These include peroxy-radical scavenging and recently discovered effects of metal ion chelation. In controlled experiments, it has been found that incubation of rat lens homogenate with glucose and the copper chelator o-phenanthroline abolishes production of sorbitol. Taken together, these results suggest AR2 is a vestigial NADPH-binding protein, perhaps similar in function to a number of non-mammalian crystallins which have been recruited into the lens. There is mounting evidence for the binding of reactive aldehyde moieties to the protein, and the involvement of AR2 either as a 'housekeeping' protein, or in a free-radial-mediated 'catalytic' role. Interfering with the NADPH binding and flux levels--possibly involving free radicals and metal ions--has a deleterious effect. We have yet to determine whether aldose reductase is the black sheep of the aldehyde reductase family, or whether it is a skeleton in the cupboard, waiting to be clothed in the flesh of new revelations in the interactions between proteins, metal ions and redox metabolites.

Aldehyde Reductase↗
PubMed2004

Izumoring: a novel and complete strategy for bioproduction of rare sugars.

Starch, whey or hemicellulosic waste can be used as a raw material for the industrial production of rare sugars. D-glucose from starch, whey and hemicellulose, D-galactose from whey, and D-xylose from hemicellulose are the main starting monosaccharides for production of rare sugars. We can produce all monosaccharides; tetroses, pentoses and hexoses, from these raw materials. This is achieved by using D-tagatose 3-epimerase, aldose isomerase, aldose reductase, and oxidoreductase enzymes or whole cells as biocatalysts. Bioproduction strategies for all rare sugars are illustrated using ring form structures given the name Izumoring.

Journal Article↗
PubMed2003

Determination of 1-phenyl-3-methyl-5-pyrazolone-labeled carbohydrates by liquid chromatography and micellar electrokinetic chromatography.

In this paper, the method for the derivatization of carbohydrates with 1-phenyl-3-methyl-5-pyrazolone (PMP) was simplified. One-third of the derivatization time was saved. Five monosaccharide derivatives have been well separated by MEKC and HPLC under optimized conditions. Good reproducibility could be obtained with relative standard deviation (RSD) values of the migration times within 5.0 and 2.3%, respectively. Furthermore, the developed methods have been successfully applied to the analysis of carbohydrates in Aloe powder and food. These methods are quite useful for routine analysis of monosaccharides and oligosaccharides in real samples.

Antipyrine↗
PubMed2003

Multiple sugar binding sites in alpha-glucosidase.

Twenty-five analogs of D-glucose were examined as reversible inhibitors of yeast alpha-glucosidase (EC 3.2.1.20). The K(i) values range from 0.38 mM for 6-deoxy-D-glucose (quinovose) to 1.0 M for D-lyxose at pH=6.3 (0.1 M NaCl, 25 degrees ). All the monosaccharides and the three disaccharides (maltose, isomaltose and alpha,alpha-trehalose) were found to be linear competitive inhibitors with respect to alpha-p-nitrophenyl glucoside (pNPG) hydrolysis. Multiple inhibition analysis reveals that there are at least three monosaccharide binding sites on the enzyme. One of these can be occupied by glucose [K(i)=1.8(+/-0.1) mM], one by D-galactose [K(i)=164(+/-11) mM] and one by D-mannose [K(i)=120(+/-9) mM]. The pH dependence for glucose binding closely follows that of V/K [pK(a1)=5.55(+/-0.15), pK(a2)=6.79(+/-0.15)], but the binding of mannose does not. Although the glucose subsite can be occupied simultaneously with the mannose or galactose subsites in the enzyme-product complex, no transglucosylation can be detected between pNPG and either mannose or galactose. This suggests that neither of these nonglucose subsites can be occupied in a productive manner in the covalent glucosyl-enzyme intermediate.

Binding Sites↗
PubMed2000

Chromatographic characterization of ovine kappa-casein macropeptide.

Ovine casein macropeptide (CMP) was characterized by anion-exchange FPLC and reversed-phase (RP) HPLC. To study heterogeneity (the degree of glycosylation and phosphorylation), CMP was desialylated with neuraminidase and dephosphorylated with acid phosphatase. Following RP-HPLC, the main CMP components were identified using either on-line or off-line mass spectrometry. The most abundant ovine CMP component was a diphosphorylated carbohydrate-free form, followed by one or two monophosphorylated and a non-phosphorylated asialo-aglyco species. Aglyco non-phosphorylated, monophosphorylated and diphosphorylated forms were in the ratio 3:20:77. Only approximately 30% of ovine CMP was glycosylated. Assuming that the monosaccharide fraction of ovine CMP is composed of N-acetylgalactosamine, galactose and N-glycolylneuraminic acid, molecular masses consistent with the presence of CMP containing tetra-, tri-, di- and monosaccharide were identified.

Acid Phosphatase↗
PubMed2001

Heterogeneity of caprine kappa-casein macropeptide.

The heterogeneity of caprine caseinmacropeptide (CMP) was determined by means of treatments with neuraminidase and acid phosphatase and analyses by anion exchange FPLC and reversed-phase (RP)-HPLC, with on-line and off-line electrospray ionization mass spectrometry. The main CMP components were two non-glycosylated and di-phosphorylated forms, as well as two other mono-phosphorylated species, each corresponding to a genetic variant of caprine kappa-casein due to the silent substitution Ile/Val at position 119. Asialo-aglyco mono- and di-phosphorylated forms were found in the ratios 8-14% and 86-92%, respectively. Approximately 36% of caprine CMP was glycosylated. Based on the obtained molecular masses, the occurrence of tri-, di- and monosaccharide-containing di-phosphorylated CMP are reported, assuming that N-acetylgalactosamine, galactose, N-acetyl and N-glycolylneuraminic acids would constitute the main monosaccharides of caprine CMP. CMP microheterogeneity due to the genetic polymorphism was also observed in the glycosylated forms.

Acid Phosphatase↗
PubMed2002

Effects of macrocyclic lactone anthelmintics on feeding and pharyngeal pumping in Trichostrongylus colubriformis in vitro.

The effects of macrocyclic lactone anthelmintics (MLs) on feeding by Trichostrongylus colubriformis nematodes in vitro were examined using inulin uptake as a measure of ingestion and electropharyngeograms as a record of the electrical events associated with pharyngeal pumping. Inulin uptake was inhibited by the 4 MLs tested (EC50s 0.045-4.57 nM), with an order of potency of eprinomectin (most potent), ivermectin, ivermectin monosaccharide, and ivermectin aglycone. The MLs caused both the frequency and amplitude of pharyngeal electrical events to decrease. In individual worms the inhibition of pump frequency preceded the inhibition of pump amplitude. The order of potency of the MLs as inhibitors of frequency was ivermectin aglycone, ivermectin, ivermectin monosaccharide and eprinomectin. The difference compared with the inulin assay results are probably due to the dynamics of drug uptake in the two systems. It was possible that the nematodes in the electrophysiology experiments were effectively orally ligated by enclosure of the worm's head in the recording pipette which contained no drug. Despite this difference in relative potencies, both the ingestion assays and the electrical events indicate that MLs are potent inhibitors of the pharynx in T. colubriformis in vitro. The sequence of effects on pharyngeal electrical activity suggests that ML action involves an initial inhibitory effect on the rate of pharyngeal contractions, followed by a decrease in the amplitude of the potentials associated with pharyngeal pumping events.

Animals↗
PubMed2001

Improved analysis of dissolved carbohydrates in stream water with HPLC-PAD.

This paper describes improvements made to the determination of dissolved carbohydrates in stream water with high-performance liquid chromatography and pulsed amperometric detection (HPLC-PAD). We eliminated interference from dissolved oxygen, separated xylose and mannose along with other molecules, reduced the chromatographic peak shift associated with carbonate accumulation in the column to less than 1% for all samples, and achieved an 8% increase in recovery of hydrolyzed carbohydrates by replacing ion-exchange desalting cartridges with evaporative removal of HCl under N2. These modifications lowered detection limits to less than or equal to 0.4 nM for 10 different monosaccharides and enhanced our ability to follow the dynamics of these molecules in stream ecosystems. In the determination of dissolved free monosaccharides, average relative precision was 1.3% and recovery ranged from 92 to 109%. For dissolved total saccharides, average relative precision was 3.3%. Concentrations were stable when filtered (0.2 microm) stream water was stored for 1 day at 22 degrees C, 1 week at 4 degrees C, or 1.5 months at -20 degrees C.

Journal Article↗
PubMed1989

Improved resolution of glycoproteins by chromatography with concanavalin A immobilized on microparticulate silica via temperature-programmed elution.

The ability of the column temperature to control elution in the affinity chromatography of glycoproteins (e.g., ovalbumin and horseradish peroxidase) on silica immobilized concanavalin A has been studied. Column temperature programs can be achieved by placing a small HPLC column within a commercial mobile phase preheater assembly. It is shown that elution of adsorbed proteins can be initiated by changing the column temperature without altering the chemical composition of the mobile phase. Further, due to the enhancement in the rate of dissociation of the sample from the ligand, the peaks are narrowed. The resolution can be controlled by changing the initial temperature, dwell time at the initial temperature, and the rate of change of the temperature program. Addition of a competitive binding agent to the mobile phase decreases the temperature needed to elute strongly retained proteins. The effect of heating the column through many thermal cycles is assessed by periodically measuring the retention of a small monosaccharide that binds to the immobilized concanavalin A. The effect of two different immobilization procedures (glutaraldehyde and carbonyldiimidazole), as well as the effect of including a monosaccharide in the mobile phase, on the stability of the column is easily monitored by thermal elution chromatography. The effect of column temperature on the above glycoproteins has been assessed through studies of enzyme activities and anion exchange and isoelectric focusing patterns before and subsequent to temperature-programmed elution affinity chromatography.

Chromatography, Affinity↗
PubMed2002

Detection and identification of aqueous saccharides by using surface-enhanced Raman spectroscopy.

The sensitive detection and characterization of carbohydrates by means of a strategy based on surface-enhanced Raman spectroscopy is demonstrated. Spectra are obtained after injecting a small amount of saccharide solution onto a roughened silver substrate, with subsequent deposition of silver colloid. The sensitivity achieved by this two-step approach enables high-quality Raman spectra to be obtained for small amounts of aqueous saccharides (5 microL of a 10(-2) M solution) utilizing minimal laser power and small signal acquisition times (a few seconds). Spectral "fingerprints" obtained for seven structurally similar monosaccharides demonstrate clearly an effective means by which each sugar can be identified. The application to more complex analyses is demonstrated for monosaccharide mixtures and a disaccharide, whereby the SERS fingerprints aid in the determination of components.

Carbohydrates↗
PubMed1999

Catalog-library approach for the rapid and sensitive structural elucidation of oligosaccharides.

We obtained the nearly complete structural elucidation of oligosaccharide components, including sequence, linkage, and even stereochemistry in the picomolar levels. The "catalog-library" approach is used for elucidating the structures of minor components in a mixture of oligosaccharides. Oligosaccharides released from a family of glycoproteins are often composed of a small finite set of monosaccharides. In this regard, the numerous oligosaccharide species are analogous to the products found in syntheses involving combinatorial libraries. The great structural diversity in the library is the result of the nearly infinite combinations in which even a small number of monosaccharides can be arranged. Fortunately, structural similarities exist between different oligosaccharides, as specific substructural motifs are preserved among different compounds. We propose that a catalog of substructural motifs can be identified and characterized by collision-induced dissociation mass spectrometry. The catalog is constructed from a set of known compounds that have been fully structurally elucidated by, for example, nuclear magnetic resonance. The catalog consists of the characteristic fragmentation patterns belonging to a set of specific substructural motifs. Collision-induced dissociation is used to determine the presence of these motifs and reconstruct the structures of less abundant components.

Carbohydrate Conformation↗
PubMed2000

Combined partial acid hydrolysis and electrospray ionization-mass spectrometry for the structural determination of oligosaccharides.

A general oligosaccharide acid hydrolysis method, amenable to electrospray ionization mass spectrometry (ESI-MS), is described that allows for hydrolysis of glycosidic bonds for both hexose- and N-acetylhexosamine-containing oligosaccharides. The partial acid hydrolysis of oligosaccharides is obtained by using an acid-exchange resin as the acid catalyst. A ladder sequence of the glycan is produced in solution that is directly analyzed by ESI tandem mass spectrometry, employing both ion trap and Fourier transform ion cyclotron resonance mass spectrometers, to provide sequence and linkage information. Unlike traditional acid hydrolysis procedures, there is minimal degradation of monosaccharide residues or deacetylation of N-acetylhexosamines by employing this technique. It is further demonstrated that the stereochemistry of the released monosaccharides and the anomeric configuration within disaccharides is determined by direct derivatization of the hydrolysate with Zn(dien)-Cl2 followed by ESI-MS/MS.

Carbohydrate Sequence↗
PubMed1993

Further structural definition of a new family of glycopeptidolipids from Mycobacterium xenopi.

The highly antigenic surface glycolipids of serovars of the Mycobacterium avium complex are glycopeptidolipids of the general structure [formula: see text] However, it has recently been shown [Rivière, M., & Puzo, G. (1991) J. Biol. Chem. 266, 9057-9063; Rivière, M., & Puzo, G. (1992) Biochemistry 31, 3575-3580] that the characteristic glycopeptidolipid of a strain of Mycobacterium xenopi is of the structure [formula: see text] [monosaccharide, 3-O-CH3-6-deoxy-alpha-L-talopyranose; tetrasaccharide, 2,3,4-tri-O-CH3-alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L- rhamnopyranose(1-->3)2,4-di-O-acetyl/lauryl-6-deoxy-alpha-L-glucopyranos e]. The type of analyses previously applied to the glycopeptidolipids of M. avium combined with fast atom bombardment mass spectrometry allowed the recognition of other variants of this structure in other strains of M. xenopi: [formula: see text] in which the monosaccharide is 2-O-acetyl-3-O-CH3-6-deoxy-alpha-L-talopyranose or 3-O-CH3-6-deoxy-alpha-L-talopyranose, and the disaccharide is 4-O-octanoyl(or decanoyl)-alpha-L- rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose or alpha-L-rhamnopyranose(1-->3)2-O-lauryl-alpha-L-rhamnopyranose. Recognition of serine-containing glycopeptidolipids substituted in one case with a tetrasaccharide and in another case with a disaccharide unit implies that M. xenopi, an important opportunistic pathogen, exists as a serocomplex in nature.

Amino Acid Sequence↗
PubMed2000

O-glycan analysis of natural human neutrophil gelatinase B using a combination of normal phase-HPLC and online tandem mass spectrometry: implications for the domain organization of the enzyme.

Gelatinase B is a matrix metalloproteinase (MMP-9) expressed under strict control by many cell types including neutrophils, monocytes, macrophages, and tumor cells. MMP-9 is a key mediator in the physiological maintenance of the extracellular matrix both in tissue remodeling and development, while uncontrolled enzyme activity contributes to pathologies such as cancer and inflammation. Neutrophils release MMP-9 from granules in response to IL-8 stimulation. Human MMP-9 has three potential N-linked glycosylation sites and contains a Ser/Pro/Thr rich domain, known as the type V collagen-like domain, which is expected to be heavily O-glycosylated. Indeed, approximately 85% of the total sugars on human neutrophil MMP-9 are O-linked. This paper presents the detailed analysis of picomole amounts of these O-glycans using a novel HPLC-based strategy for O-glycan analysis that provides linkage and arm specific information in addition to monosaccharide sequence. The initial structural assignments were confirmed using HPLC with online MS/MS fragmentation analysis. Twelve sugars were identified that contained from two to nine monosaccharide residues. Most of these contained type 2 core structures with Galbeta1-4GlcNAc (N-acetyl lactosamine) extensions, with or without sialic acid or fucose. The O-glycans were modeled using the oligosaccharide structural database. On the basis of the structure of gelatinase A (MMP-2), a model of MMP-9 suggests that the type V collagen-like domain in gelatinase B is located on a loop remote from the active site. Fourteen potential O-glycosylation sites are multiply presented on this loop of 52 amino acids. Many of the O-glycans identified contain terminal galactose residues that may provide recognition epitopes. Importantly, heavy glycosylation of this loop region, absent in gelatinase A, has considerable implications for the domain organization of MMP-9.

Carbohydrate Conformation↗