An open-label dosing study to evaluate the safety and effects of a dietary plant-derived polysaccharide supplement on the N-glycosylation status of serum glycoproteins in healthy subjects.

BACKGROUND: The functional role of dietary carbohydrates in nutrition is one of the most complex and at times controversial areas in nutritional science. In-vitro and in-vivo studies suggest that certain dietary saccharide biopolymers can have bifidogenic and or immunomodulatory effects, and that some could represent preferential substrates or precursors that can impact cellular glycosylation. OBJECTIVE: Examine the impact of oral ingestion of a standardized dietary plant-derived polydisperse polysaccharide supplement (Advanced Ambrotose powder (AA)) on the N-glycosylation status of serum glycoproteins in a cohort of healthy individuals. DESIGN: An open-label study was carried out. This study was in two phases: pilot study (n=6 individuals) to assess safety and dose, and a larger study (n=12) to evaluate specific glycosylation changes. Serum N-glycosylation profiles, using mass spectrometry, were monitored at weekly intervals, for 7 weeks, to evaluate baseline levels and normal fluctuations. The individuals were then monitored for a further 7 weeks, during which time increasing doses of AA were ingested (1.3-5.2 g/day). RESULTS: No adverse events were encountered. AA supplementation resulted in distinct changes in the relative intensities of seven biantennary N-glycans (P<0.001), and a significant overall shift towards increased sialylation. Regression analysis revealed a dose-dependent decrease in mono- and di-galactosylated structures (coefficient -0.130 decrease/week: P=0.02 and -0.690: P=0.005), and a concomitant increase in disialylated glycans ( × 1.083: P<0.05). CONCLUSIONS: Supplementation with the dietary plant-derived polysaccharides in AA resulted in significant changes in serum protein N-glycosylation in healthy individuals. How this occurs and whether it has biological significance remains to be evaluated.

Intra-oral acid production associated with eating whole or pulped raw fruits.

The hypotheses that raw fruits, whether whole or pulped, were cleared rapidly from the mouth and that the sugars in the whole and pulped fruits are fermented with equal efficiency to acids by the oral microflora were tested in this study. Groups of 7-9 adult subjects chewed 10 g of raw, whole or pulped fruit (apple, banana, orange, pear and pineapple) for 1 min and whole, unstimulated saliva samples were collected during the following 60-min interval. Each saliva sample was assayed for the concentrations of fruit-derived sugars (glucose, fructose and sucrose), fruit-derived acids (malic and citric) and acids which may be produced as a result of bacterial fermentation (acetic, lactic, formic and succinic). We found the fruit-derived sugars were rapidly cleared from the mouth (within 5 min). The major bacterially produced acids were lactic and succinic, which reached maximum concentrations in the 5-min sample. There was no significant difference, within a fruit, in the salivary levels of any of the sugars or acids between the raw whole or raw pulped forms. In light of these findings it seems unwise to assume that fruits may be consumed without consideration of their acidogenic potential.

Dental caries, oral hygiene, and oral clearance in children with craniofacial disorders.

The reason that children with cleft palates tend to have a greater prevalence of tooth decay than normal children is unclear. We hypothesized that children with cleft palates would have increased oral clearance times for foods and, consequently, higher levels of caries and caries-associated micro-organisms than control children. Children aged 6-16 yrs, with (n = 81) or without (n = 61) cleft palates, were studied. Children with cleft palates had DMFT and dmft scores greater (p < 0.01) than those of the control group. The number of caries-associated organisms was greater in the saliva of the cleft palate children (all p < 0.001). The oral hygiene, plaque and gingival index scores were greater (p < 0.0001), oral clearance was longer (p < 0.01), and levels of sucrose and starch-derived saccharides higher (p < 0.01) in the cleft palate group. However, salivary concentrations of organic acids were lower in the children with craniofacial disorders, probably reflecting the altered physiology of the more mature dental biofilm. The longer oral clearance times of foods and the consequent generation of fermentable sugars from starches may contribute to the higher caries prevalence observed in children with cleft palates.

Ammonia cleaves polypeptides at asparagine proline bonds.

Polypeptides that contain the sequence Asn-Pro undergo complete cleavage at this amide bond with ammonia. One cleavage product possesses Pro as the new amino terminus and the other Asn or isoAsn as the new C-terminus, the formation of the latter probably arising by way of a cyclic succinimide intermediate. Other Asn-X bonds where X = Tyr, Gln, Ile, Glu, Ala, Gly, Asn or Phe did not exhibit any peptide bond cleavage, whereas when X = Leu, Thr and Ser partial cleavage was observed. Asn residues not involved in chain-cleavage underwent deamidation to Asp as shown by MALDI-ToF mass spectrometry (MS) analysis. The partial conversion of in-chain Asp residues to isoAsp under the reaction conditions was inferred from RP-HPLC and MS analysis of reaction mixtures.

Distribution of endo-beta-N-acetylglucosaminidase amongst enterococci.

Enterococci are becoming increasingly important nosocomial pathogens, a fact mainly attributed to their antimicrobial resistance profiles. However, the enzymic activities required for these organisms to proliferate in vivo have received little attention. Enterococcus faecalis has been shown previously to produce an endo-beta-N-acetylglucosaminidase activity which cleaves high mannose-type glycans in glycoproteins between the N-acetylglucosamine residues of the pentasaccharide core. This study investigated the distribution of this endoglycosidase activity amongst the other enterococcal species. Ribonuclease B, a high mannose-type glycoprotein, was used as a substrate and endoglycosidase activity was demonstrated by a combination of matrix-assisted laser desorption ionisation time-of-flight mass spectrometry and high pH anion-exchange chromatography. Endo-beta-N-acetylglucosaminidase activity was present in 10 of the 18 enterococcal species isolated from both human and animal sources, including all E. faecalis strains. The most notable exception was the lack of this activity in all E. faecium isolates tested. All enterococcal species possessing endoglycosidase activity utilised the liberated glycans to support bacterial growth.

Mannosidase production by viridans group streptococci.

The production of mannosidase activity by all currently recognized species of human viridans group streptococci was determined using an assay in which bacterial growth was dependent on the degradation of the high-mannose-type glycans of RNase B and subsequent utilization of released mannose. RNase B is an excellent substrate for the demonstration of mannosidase activity since it is a glycoprotein with a single glycosylation site which is occupied by high-mannose-type glycoforms containing five to nine mannose residues. Mannosidase activity was produced only by some members of the mitis group (Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus cristatus, Streptococcus infantis, Streptococcus parasanguinis, and Streptococcus pneumoniae) and Streptococcus intermedius of the anginosus group. None of the other species within the salivarius and mutans groups or Streptococcus peroris and Streptococcus sanguinis produced mannosidase activity. Using matrix-assisted laser desorption ionization time-of-flight mass spectrometry, it was demonstrated that the Man(5) glycan alone was degraded while Man(6) to Man(9), which contain terminal alpha(1-->2) mannose residues in addition to the alpha(1-->3), alpha(1-->6), and beta(1-->4) residues present in Man(5), remained intact. Investigations on mannosidase production using synthetic (4-methylumbelliferone- or p-nitrophenol-linked) alpha- or beta-mannosides as substrates indicated that there was no correlation between degradation of these substrates and degradation of the Man(5) glycan of RNase B. No species degraded these alpha-linked mannosides, while degradation of the beta-linked synthetic substrates was restricted to strains within the Streptococcus anginosus, S. gordonii, and S. intermedius species. The data generated using a native glycoprotein as the substrate demonstrate that mannosidase production within the viridans group streptococci is more widely distributed than had previously been considered.

Detecting mannosidase activities using ribonuclease B and matrix-assisted laser desorption/ionization-time of flight mass spectrometry.

Ribonuclease (RNase) B incubated with purified enzymes, whole bacterial cultures, or their separated components-cells and supernates-have been directly analyzed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-ToF) to detect exomannosidases and to evaluate their specificities and location. Enzymatic cleavage was monitored by observing changes in RNase B glycoform population. Thus a nonspecific alpha-(1 --> 2)-mannosidase activity converts the glycoprotein to its Man(5) form, identifiable by its mass of 14,899 [M + H](+); this species subsequently is converted, by the actions of alpha-(1 --> 3) and alpha-(1 --> 6)-mannosidases, to the Man(1) form via Man(4), Man(3), and Man(2). The Man(1) glycoform (which is readily isolated) has then similarly been used for identifying beta-(1 --> 4)-mannosidase and the derived Man(0) form has served in turn as a natural substrate for beta-(1 --> 4) N-acetylglucosaminidase producing a species possessing a single asparagine-linked GlcNAc residue (mass 13,886). Mannose liberated from the actions of mannosidases can, if desired, be quantified by, for example, chromatography. The actions and specificities of endoglycosidases such as a peptide-N-glycosidase F (PNGase F) and of endo-N-acetlyglucosaminidases (e.g., endo-F and endo-H), which respectively cleave between the GlcNAc&bond;Asn and GlcNAc&bond;GlcNAc bonds of N-linked glycoproteins, are also demonstrable by MALDI-ToF analysis of RNase B (and derived products). From these digests the completely deglycosylated polypeptide corresponding to RNase A in which Asn has been converted to Asp (mass 13,684) and a species corresponding to RNase A + GlcNAc (mass 13,886) are produced, together with their corresponding free oligosaccharides which are amenable to analysis by both MALDI-ToF and by HPLC.

Isolation and characterisation of sialidase from a strain of Streptococcus oralis.

Streptococcus oralis, the most virulent of the viridans streptococci, produces a sialidase and this exo-glycosidase has been implicated in the disease process of a number of pathogens. The sialidase of S. oralis strain AR3 was purified in order to understand the characteristics of this putative virulence determinant. The enzyme isolated as a high mol. wt aggregate (c. 325 kDa) was purified 4520-fold from late exponential phase cultures by a combination of ultrafiltration, ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. The sialidase component had a mol.wt of 144 kDa as determined by SDS-PAGE analysis. The purified sialidase released N-acetylneuraminic acid from a range of sialoglycoconjugates including human alpha1-acid glycoprotein, bovine submaxillary mucin, colominic acid and sialyl-alpha2,3- and sialyl-alpha2,6-lactose. Also, N-glycolylneuraminic acid was cleaved from bovine submaxillary mucin. The sialidase had a Km of 11.8 microM for alpha1-acid glycoprotein, was active over a broad pH range with a pH optimum of 6.0 and cleaved alpha2,3-, alpha2,6- and alpha2-8-sialyl glycosidic linkages with a marked preference for alpha2,3-linkages. The enzyme was competitively inhibited by the sialic acid derivative, 2,3-dehydro-N-acetylneuraminic acid, with a K(IC) of 1.2 microM. The characteristics of the purified sialidase would support a nutritional role for this enzyme that may be significant in the proliferation of this organism in the oral cavity and at extra-oral sites in association with life-threatening infections.

Production of an endo-beta-N-acetylglucosaminidase activity mediates growth of Enterococcus faecalis on a high-mannose-type glycoprotein.

Enterococcus faecalis is associated with a high proportion of nosocomial infections; however, little is known of the ability of this organism to proliferate in vivo. The ability of RNase B, a model glycoprotein with a single N-glycosylation site occupied by a family of high-mannose-type glycans (Man(5)- to Man(9)-GlcNAc(2)), to support growth of E. faecalis was investigated. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of RNase B demonstrated a reduction in the molecular mass of this glycoprotein during bacterial growth. Further analysis by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry revealed that this mass shift was due to the degradation of all high-mannose-type glycoforms to a single N-linked N-acetylglucosamine residue. High-pH anion-exchange chromatography analysis during exponential growth demonstrated the presence of RNase B-derived glycans in the culture supernatant, indicating the presence of an endoglycosidase activity. The free glycans were eluted with the same retention times as those generated by the action of Streptomyces plicatus endo-beta-N-acetylglucosaminidase H on RNase B. The cleavage specificity was confirmed by MALDI-TOF analysis of the free glycans, which showed glycan species containing only one N-acetylglucosamine residue. No free glycans were detectable after 5 h of bacterial growth, and we have subsequently demonstrated the presence of mannosidase activity in E. faecalis, which releases free mannose from RNase B-derived glycans. We propose that this deglycosylation of glycoproteins containing high-mannose-type glycans and the subsequent degradation of the released glycans by E. faecalis may play a role in the survival and persistence of this nosocomial pathogen in vivo.

N-acetylneuraminic acid transport by Streptococcus oralis strain AR3.

Streptococcus oralis has emerged as one of the most important organisms of the viridans streptococcus group in terms of infections and is recognised as an agent of infective endocarditis and, in immunocompromised patients, septicaemia. The mechanisms by which this organism proliferates in vivo are unknown. However, host-derived sialic acids -- including N-acetylneuraminic acid (NeuNAc) which is present in serum and cell-associated glycoproteins -- are a potential source of fermentable carbohydrate for bacterial proliferation, especially for sialidase-producing bacteria, including S. oralis. To further elucidate the role of NeuNAc in supporting growth, this study determined the ability of S. oralis strain AR3 (isolated from a patient with infective endocarditis) to transport NeuNAc and characterised the transport system. The transport of [14C]-labelled NeuNAc into S. oralis was monitored and this transport system was induced by growth of the bacteria in the presence of the N-acetylated sugars NeuNAc, N-acetylglucosamine and N-acetylmannosamine. The transport system followed typical Michaelis-Menten kinetics, with a Km of 21.0 microM and a Vmax of 2.65 nmoles of NeuNAc transported/min/mg of dry cell mass. NeuNAc transport was inhibited by the presence of exogenous N-glycolylneuraminic acid, a related sialic acid. Chlorhexidine, NaF and 2,4-dinitrophenol were potent inhibitors of the transport system, suggesting that the uptake of NeuNAc occurs via a proton motive force-dependent permease system. This is the first report of the mechanism by which NeuNAc transport occurs in pathogenic streptococci. This transport process may have relevance to the acquisition of a source of fermentable carbohydrate and thus bacterial proliferation in vivo.

Growth of Viridans streptococci on human serum alpha1-acid glycoprotein.

Viridans streptococci have emerged as major opportunistic pathogens. We suggest that for these bacteria to proliferate in vivo and cause disease, they must utilize host tissue components. We have therefore examined the ability of all recognized species of viridans streptococci to liberate and utilize the constituent sugars of the glycans of the extensively sialylated human serum alpha1-acid glycoprotein (AGP) as the sole source of carbohydrate to support in vitro growth. Analysis of residual glycans following bacterial growth was performed by high-pH anion exchange chromatography with pulsed amperometric detection and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Only those species which produced sialidase-namely, Streptococcus oralis, S. intermedius, and S. defectivus--grew on AGP. The extent of degradation of glycans was dependent on the particular glycosidases produced by the bacteria. S. defectivus produced only a sialidase which released the terminal N-acetylneuraminic acid residues of the glycans, and the liberated sugar was utilized. S. intermedius also produced beta-galactosidase and beta-N-acetylglucosaminidase, which removed galactose and N-acetylglucosamine from desialylated glycans, all of which again were utilized by the organism. S. oralis produced beta-galactosidase, beta-N-acetylglucosaminidase, and alpha-fucosidase and novel alpha- and beta-mannosidases which were apparent only from the analysis of the residual sugars of AGP. S. oralis cleaved all the sugars from AGP except for 22% of the N-acetylglucosamine. The residual N-acetylglucosamine residues remaining were those linked to the asparagine of the peptide backbone. All the monosaccharides released by S. oralis from AGP, with the exception of fucose, were utilized. Sialidase production may be a key factor for growth of these species of viridans streptococci on glycoproteins in vivo, since they are commonly associated with extra-oral diseases, with S. oralis emerging as an important pathogen.

Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis.

Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo.

Evidence for mannosidase activities in Streptococcus oralis when grown on glycoproteins as carbohydrate source.

Streptococcus oralis when cultured using ribonuclease B as the sole source of carbohydrate, selectively uses the sugars of the Man5 glycoform as shown by HPAEC and MALDI-TOF mass spectrometric analyses. The organism is able to do this by producing novel alpha-(1-->3), alpha-(1-->6) and beta-(1-->4) mannosidase activities and these act in a concerted manner in what appears as a single-step process. The selective utilisation of Man5 is explained by the absence of an alpha-(1-->2) mannosidase which is required to initiate breakdown of the glycan chains present in the other glycoforms which are components of the glycoprotein.

Recombinant human albumin as a stabilizer for biological materials and for the preparation of international reference reagents.

Recombinant human albumin expressed in Saccharomyces cerevisiae was compared with native human serum albumin in its physicochemical properties and in its use as a stabilizer in lyophilized preparations of thyroid-stimulating hormone (TSH), interleukin 15 (IL-15) and granulocyte colony-stimulating factor (G-CSF). Advantages of recombinant albumin include its lack of potential human contaminants and infectious agents. When used at concentrations of 0.1-0.2% (w/v), recombinant albumin was equivalent to native serum albumin in its capacity to protect immunological, biological and biochemical properties of TSH, IL-15 and G-CSF. Physicochemical characteristics of the two forms of albumin including their binding to fatty acids were also similar. The recombinant form of albumin used in this study should be considered as a suitable stabilizer in the preparation of lyophilized products and reference reagents.

Direct preparation of cyclodextrin monophosphates.

Aqueous solutions of cyclodextrins and inorganic metaphosphate at pH 4, upon drying and subsequent warming, produce mixtures of isomeric monophosphate esters which are amenable to separation by anion-exchange chromatography. The products are characterised by enzymatic, mass, and NMR spectroscopic analysis. The methodology provides a route to these derivatives by a single reaction.

Fibrin degradation product (FnDP) assays: analysis of standardization issues and target antigens in plasma.

The in vivo formation of fibrin and its subsequent secondary fibrinolytic digestion yields a variety of crosslinked fibrin degradation products (XL/FnDP). One of these is known as D-dimer and a variety of ELISA-type commercial kits using monoclonal antibodies to D-dimer have been developed. We have investigated the possibility of establishing a standard such that these various kits might indicate the same levels of D-dimer in the same samples. We have concluded that because it seems that each individual monoclonal antibody to D-dimer targets a unique and distinct epitope in the FnDP fraction the notion of introducing a standard D-dimer which will respond uniformly to each D-dimer monoclonal antibody is not feasible. Using various monoclonal and polyclonal antibodies in conjunction with gel exclusion chromatography we have examined human plasma derived from patients with disseminated intravascular coagulation (DIC) which contained high levels of fibrin degradation products (FnDP). The data suggested that the FnDP fraction in plasma contained mostly high molecular weight (> 2 x 10(6) daltons) crosslinked fragments which contain high levels of fibrinopeptide A. Many of these crosslinked fragments crossreact with antibodies to D-dimer because they each contain D-dimer as a structural component. On the basis of this data, a novel sequence of events is proposed which may occur during the aggregation of fibrin in vivo.

Iron-binding affinity of bacterial vaccine polysaccharides which contain phosphodiester linkages as part of the polymer chain and of other polyphosphates, including DNA.

The interaction of iron (II) with bacterial polysaccharides, possessing phosphodiester bonds as part of their polymer chain, has been studied by equilibrium binding dialysis using atomic absorption spectrophotometry. Ferrous ions were found to bind with a stoichiometry of one per two phosphates and with a binding constant of about 2.5 x 10(3) M-1. Similar results, but with larger (ca 1 x 10(4) M-1) binding constants were observed with DNA. This interaction helps explain the depolymerization of polyphosphates which has been observed in the presence of iron salts, and highlights the need to avoid iron contamination of vaccines (and other substances) which contain phosphodiester bonds. The interaction may also be a means of iron sequestration in bacteria which possess these cell-surface polyphosphates.

Lysine vasopressin undergoes rapid glycation in the presence of reducing sugars.

Lysine vasopressin (LVP) readily reacts with reducing saccharides both in lyophilized preparations and in aqueous solution. Incubation of LVP with, for example, lactose over a pH range of 3.0-8.5 in phosphate buffer or simply in water, gives rise to a number of reaction products, some of which form rapidly (in a matter of hours) even in the frozen state. Reaction mixtures were analysed by reversed-phase HPLC and the structures of the products were deduced from the amino-acid composition of isolated components, by comparison with product profiles obtained with analogues under similar conditions and by FAB mass-spectral analysis of derivatives isolated after reduction with cyanoborohydride. The primary products arise from the formation of Schiff's bases at one or both of the two amino functions. The alpha-amino group of the N-terminal cystine is considerably more reactive than is the epsilon-amino group of lysine and it is the N-terminal adduct which rapidly forms even at -20 degrees C. It is concluded that caution must be shown in using reducing sugars in formulations containing peptides and proteins, particularly the vasopressins and oxytocin.

Drying from phosphate-buffered solutions can result in the phosphorylation of primary and secondary alcohol groups of saccharides, hydroxylated amino acids, proteins, and glycoproteins.

Drying (e.g., freeze-drying) aqueous phosphate-buffered solutions of organic compounds containing primary and/or secondary alcohol groups promotes esterification, producing orthophosphate esters. The reaction is accelerated by heat and by pH values < 7 and is affected by residual moisture content. The same preparations as solutions, similarly treated, produced little or no phosphorylated materials. The promotion of esterification in the dry state can, in part, be rationalized in terms of a mass-action effect because of the resulting low-water activity in dried preparations. When metaphosphates are used, esterification proceeds at a greatly increased rate and to a greater extent. Because of the relatively facile interconversion of phosphorus oxyacid salts, it is possible that a "metaphosphate species" may be responsible for the esterification reaction in all cases. We conclude that care should be exercised when hydroxylated organic compounds are dried from phosphate buffers so as to avoid the formation of phosphorylated artifacts.