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The fructan of Streptococcus mutans OMZ 176 was shown to have a levan structure by comparing the chromatographic mobilities of the saccharides produced by partial acid hydrolysis of ghe fructan and known levan and inulin. This was confirmed by using concanavalin A as a lectin in a double-diffusion gel technique.
Fructose has recently received much attention due to renewed interest in natural sweeteners. In addition, fructose has some advantages to sucrose in sweetness, solubility, viscosity, and dental health characteristics. Fructose is deposited as storage fructans of the inulin (beta-1,2) type in tubers and rhizomes of the Compositae family. The utilization of the Jerusalem artichoke (Helianthus tuberosus) tuber as a source of fructose syrup is discussed. This plant has the potential to produce more sugar per acre than corn or sugar beets. In addition, the artichoke has higher frost resistance and lower heat unit requirements than corn and is somewhat more tolerant to low moisture conditions than sugar beets. A high quality fructose syrup can be produced from artichoke tubers. The extraction step was found to be particularly important since development of adverse colors and flavors must be prevented. The fructans may be acid or enzyme hydrolyzed but the latter method gave a higher quality syrup. Ion-exchange resins and activated charcoal were effective in removing coloring and flavoring materials, and also reduced other noncarbohydrate constituents. Since the enzymatic hydrolysis of the fructans is an attractive alternative to acid hydrolysis, a process was developed for producing and purifying a special beta-fructofuranosidase (inulase) from Saccharomyces fragilis. Inulase has a much higher specificity for fructans than commerically available beta-fructofuranosidase (invertase).
Streptococcus salivarius synthesizes extracellular polysaccharides (EPS) from saccharose under aerobic and anaerobic conditions. Under aerobic conditions primarily soluble fructanes are formed. There are no differences between the EPS synthesized by r- and s-forms under aerobic conditions; anaerobically the s-forms produce more EPS and more fructane than the r-forms. As compared to streptococcus mutans, the streptococcus salivarius produces considerably more fructane and insoluble glucane under the conditions used.
Two dextran-specific (PC 3858 and PC 3936) and one levan-specific (PC 3660) NZB myeloma proteins were studied by quantitative precipitin and precipitin-inhibition assays. Both myeloma antidextrans were alphaD-(1 leads to 6) specific and precipitated strongly with a synthetic, linear dextran, molecular weight 35,500, and with other dextrans. The two myeloma antidextrans differed with respect to their relative reactivities with dextrans containing various proportions of alpha-D-(1 leads to 6), alpha-D-(1 leads to 4)-like, and alpha-D-(1 leads to 3)-like linkages. In inhibition assays, the two antidextran myeloma proteins behaved differently from each other, from alpha-D-(1 leads to 6)-specific BALB/c myeloma antidextrans, and from the human antidextrans previously studied. Isomalto-oligosaccharides IM3, IM4, and IM5 were all equal in inhibitory power but were only about 60% as potent as IM6 and IM7, which also inhibited equally on a molar basis. Although precipitation with linear dextran suggests that both may have groove-type sites, as previously inferred for QUPC 52, the size of their combining sites is uncertain. It is not clear whether the sites are only as big as three glucose residues with the increased inhibition by six and seven glucose residues being attributable to partial bivalence and to their ability to combine in several ways along the chain, or whether the site is as big as six glucose residues with the increment in binding by the fourth and fifth glucose residues being minimal and the sixth contributing considerable additional binding-energy. The fructan-specific myeloma protein did not react with inulin, but reacted with many levans and with perennial rye-grass levan containing only beta-D-(2 leads to 6) links. The levan-antilevan reaction was not inhibited by beta-D-(2 leads to 1)- linked oligosaccharides. The findings suggest that PC 3660 has a specificity for (2 leads to 6)-linked chains.
The glycosyltransferases of S. mutans strain Ingbritt have been resolved by SDS-polyacrylamide gel electrophoresis, followed by incubation in the presence of non-ionic detergent to restore enzyme activity. A group of high molecular weight proteins synthesizing glucans has been identified, as well as three distinct fructan-synthesizing activities. The glucan-forming enzymes have been purified by affinity chromatography on insoluble glucan, followed by gel chromatography in SDS, and antiserum to the purified enzymes has shown that they are antigenically identical within serotypes c, e and f, and cross-react strongly with serotype b.
The total effect of sucrose-splitting activity from three sucrose metabolizing enzymes has been investigated in "resting" saliva in contact with dental plaque material in 356 military recruits. Invertase effect is defined as the splitting of sucrose into equimolar quantities of glucose and fructose, dextransucrase as a glucosyl-transferase producing glucan and free fructose, and levansucrase as a fructosyl-transferase producing fructan and free glucose. Total monohexose and glucose production as well were determined quantitatively in each subject. Monohexose production was related to specific oral conditions, and a difference in tooth decay significant at a 5% level was found between samples with high and low enzyme content. The production of free glucose was lower than that of free fructose, significant at less than a 1% level. This may indicate that more glucose is bound to form with a high level enzyme content a slight increase in relative effect of dextranscrase was found. This was not statistically significant.
The effect of extracellular products from Streptococcus salivarius on sucrose-dependent adherence to smooth surfaces by other oral bacteria was studied in vitro. Strains of Streptococcus mitis, Streptococcus pyogenes, and Veillonella parvula without innate ability to adhere to a steel wire were able to do so when incubated with sucrose and cell-free culture fluid from S. salivarius strains 9759, 25975, CNII, and MEPI. These culture fluids synthesized more adherent material and water-insoluble glucan than those from Streptococcus mutans C67-1 and seven other S. salivarius strains. Among the S. salivarius strains, glucosyltransferase (GT; dextransucrase, EC 2.4.1.5) activity varied more than 100-fold. Cells of Veillonella and S. mitis S3 that had been incubated in culture fluids from S. salivarius 25975 and 9759, respectively, and then washed adhered upon subsequent incubation with sucrose. This was due to adsorbed GT because (i) the adherence was sensitive to dextranase; (ii) it was observed only with the high-GT culture fluids; (iii) it was dependent on sucrose; and (iv) the washed Veillonella cells synthesized glucan, but not fructan, from sucrose. These results suggest that sucrose-dependent adherence of bacteria without such innate ability can be mediated by (i) entrapment in insoluble glucan synthesized by S. salivarius culture fluids, and (ii) prior adsorption of GT from S. salivarius culture fluids. The possibility that GT formed by high-yield strains of S. salivarius is distributed through the mouth by the action of salivary flow and contributes to sucrose-dependent adherence and plaque formation is considered.
Tolerance induction of IgE antibody-forming cells by dinitrophenylated levan (DNP-LE) conjugates was investigated in CBA mice. Antibody production was measured by passive coutaneous anaphylaxis on rat skin. Specific antibody neutralization was obtained with conjugates of different substitution degrees. Specific inhibition of IgE antibody production was obtained only with highly substituted conjugates. However, the epitope density required for inducing tolerance to antibody synthesis of the IgE class was lower than for other classes of immunoglobulins. The tolerance induced by DNP-LE was shown to affect only the anti-DNP B cells of IgE class without T cell participation. These results demonstrate that B cell precursors of all Ig classes are susceptible to tolerance induction and not to triggering by T-independent antigens.
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Administration of levan to mice and rats inhibited the passage of intravenously injected trypan blue into the area of skin injected with histamine. In the mouse, the optimal dose of levan was about 5 mg/25 g body weight; higher doses, on the other hand, enhanced diffusion. In the rat, the inhibitory effect was directly related to the dose of levan, the optimal dose being five times higher than for mice. Treatment of rats with levan caused a marked reduction in the uptake and transport of colloidal carbon. In normal and histamine- or turpentine-injected cremaster muscles, treatment with levan prevented carbon from being adsorbed and endocytosed by the endothelial cells. Levan itself was frequently attached to the lumenal surface of endothelial cells, or less frequently was enclosed within pinocytic vesicles. Occasionally, transport across the interendothelial junction was also seen, but the impression gained was that the frequency and extent of patent junctions were reduced in animals receiving phlogistic stimuli and treated with levan. Carbon particles were generally aggregated in the central zone of vessels and were seldom seen near the endothelial cells, within endothelial cells or within any open junctions, presumably because of the coating effect of levan. With partly depolymerised levan, carbon particles were found in endothelial cells and intercellular spaces, but at lower concentrations than in non-levanised control animals. It is suggested that levan acts by altering the rheologic patterns in the microcirculation, by modifying the surface of endothelial cells and by influencing their behaviour, and, finally, by changing the constitution of the ground substance and basement lamina.
The delaying effect of continued administration of levan on mouse skin homograft rejection was found to be due mainly to inhibition of thrombotic occlusion of the nutrient blood vessels. Levan administration did not appear to affect epidermal regeneration, but it seemed to inhibit degranulation of mast cells, resorption of plasma extravasated during the operation and growth of granulation tissue. The delayed rejection caused by levan treatment was apparently caused by infiltration of the graft by activated mononuclear cells from the blood stream. The phenomenon is believed to represent a late stage of the graft rejection process, its apperance depending on the survival of the graft beyond the stage of vascular occlusion.
The ability of fresh or cultured subpopulation of adherent peritoneal exudate cells to perform discrete biological functions commonly associated with macrophages, namely antibody dependent cytotoxicity (ADCC) and phagocytosis, has been compared with the ability of the same cells to present TNP coupled to carbohydrate or protein carriers for a subsequent antibody response, or semi-allogeneic cells for T cell cytotoxic responses, in tissue culture. Data are presented to show that ADCC reactivity rapidly disappears on cultures of peritoneal cells, though phagocytic activity is more persistent throughout the 14 days of culture. When ADCC activity reappeared it was no longer restricted to those fractions active initially. Antigen handling of different TNP-protein conjugates was pronounced throughout the culture period and was not easily explained in terms of the ADCC/phagocytic activity of the macrophage subpopulation concerned. In contrast, however, antigen handling of carbohydrate antigens for presentation to T or B lymphocytes by the different macrophage populations varied during the culture period in fashions which were in some degree correlated with the phagocytic activity of the various cells but were also a function of the antigen under consideration. These data are interpreted in terms of a novel role for the macrophage in discrimination of carbohydrate antigens for presentation to lymphocytes.
Polyfructosan has been used as a substitute for inulin in GFR determinations. However, the validity of this substitution in conditions where renal tubular permeability to other substances, such as mannitol, sucrose and other substances, such as mannitol, sucrose and iothalamate, is increased has not been tested. Experiments were performed on 8 rats to compare the clearances of polyfructosan (CPF) and 14C-inulin (CIN) during hydropenia, 3% BW saline expansion, elevation of one ureteral catheter by 30 cm, and following return of increased ureteral pressure to the control level. No significant difference between CPF and CIN could be detected except in the kidney subjected to increased ureteral pressure. However, the magnitude of this difference--which may relate to the different molecular weights of the two compounds--was so small that we conclude that the use of CPF, as an index of GFR, is no less reliable than CIN under the conditions tested.
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High molecular levan, a polyfructoside, has a dose-dependent inhibitory effect on the primary immune response to sheep red cells (SE) in Balb/c mice, when given as from 1--2 days prior to the antigen injection. A slight stimulation of the immune response was observed when levan was given shortly before or 1 day after the antigen.
Actinomyces viscosus produces both a soluble extracellular levansucrase and a cell wall-associated levansucrase. The enzyme from cell walls was solubilized by lysozyme digestion. The soluble extracellular and cell wall-associated forms of the enzyme were compared and appeared to be identical, based on molecular weight estimations, kinetic parameters, and reactions with antisera. The product of both forms of the enzyme was a high molecular weight, branched levan, as shown by its reactivity with myeloma proteins specific for beta(2 leads to 1) and for beta(2 leads to 6) linkages in fructosans. Although levansucrase remained tightly bound to the levan which it synthesized, the enzyme did not bind to exogeneously added levan. Regarding the potential pathogenicity of the levan product, pure levan, produced using purified levansucrase, did weakly activate complement by the alternative pathway. However, the pure levan did not directly cause bone resorption in an in vitro bone resorption assay.
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