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Variation in microbial and biochemical components of human four-day plaque was studied in seven subjects who were maintained on a high-sucrose diet during a four-week period. Significant changes in populations of lactobacilli dextranase-producing organisms, Streptococcus mutans, and S sanguis occurred during this period.
S mutans strain MT703 from an active carious lesion in the tooth of a child had type e specificity and showed a cross-reaction with the Lancefield group E cell wall streptococcal polysaccharide antigen. Heat-killed cells MT703 adhered to a glass surface in the presence of CGT MT703 and sucrose. Pretreatment of the cells with anti-MT703 whole cell serums inhibited adherecne. The removal of glycerol teichoic acid antibody and group E antibody from the MT703 serum did not result in a loss of inhibitory activity. Antiserum with or without adsorption significantly inhibited glucan synthesis by CGT from sucrose. Antibodies specific for the polyglycerol phosphate of teichoic acid did not inhibit adherence. Anti-group E serum and serums specific for other types of S mutans, did not show adherence inhibitory activity except for an occasional type c specific antiserum. Antibody specific for the type e antigen produced significant inhibition of the binding of CGT to the MT703 cell wall, and adherence of these cells did not occur. Antibody to CGT inhibited glucan synthesis. Treatment of the cells with dextranase, dextran antibody, or trypsin caused a significant reduction in adherence. The results suggest that the type antigen and dextran on the surface of the S mutans type e cell are functional in adherence, and that these polymers are associated with cell wall protein.
A rapid quantitative in vitro assay measured the effects of antiplaque agents and antiserum on growth and sucrose-mediated adherence of radio-labeled S. mutans 6715. Acid production was measured by change in pH. In this assay the primary effect of chlorhexidine and fluoride was bacteriostasis and inhibition of acid production, respectively; the primary effect of dextran, dextranase, and specific antiserum was inhibition of sucrose-mediated adherence.
GlucosyltransferaseB, GtfC, and GtfD were purified by hydroxyapatite column chromatography, followed by ultrafiltration from the culture supernatant fluids of three Streptococcus milleri constructs (gift from Dr. H.K. Kuramitsu) which harbored individual gtf genes of Streptococcus mutans GS5. GtfB, GtfC, and GtfD were enzymatically active both in solution and in an experimental pellicle (HA-CWS-Gtf) formed by adsorbing Gtf onto the surface of clarified human whole saliva (CWS)-coated hydroxyapatite (HA). The Km values for sucrose for all three enzymes were lower when the enzyme was adsorbed to a surface, compared with when it was in solution. In solution phase assays, and in the absence of primer dextran, glucan production was enhanced 75% when both GtfB and GtfD were present in the reaction mixture, compared with the sum of the individual enzyme activities (p < 0.005). This enhancement did not occur when GtfC was additionally present, or when the GtfB+GtfD enzyme pair was adsorbed onto HA-CWS. In additional experiments, glucan formed by GtfB or GtfC, but not by GtfD, on a HA-CWS-Gtf surface increased adherence of Streptococcus mutans GS5 and Streptococcus sobrinus 6715 by seven- to nine-fold compared with adherence when no glucan was present on the pellicle surface (p < 0.001). Further, treatment of the HA-CWS-GtfB-glucan or HA-CWS-GtfC-glucan pellicle with alpha-1,6 dextranase significantly reduced adherence of both streptococcal strains (p < 0.001). These results show that GtfB, GtfC, and GtfD are enzymatically active in an adsorbed state and that the nature of their product glucan can influence the adherence of cariogenic oral streptococci to an experimental pellicle.
The synthesis of extracellular glucan is an integral component of the sucrose-dependent colonization of tooth surfaces by species of the mutans streptococci. In investigators' attempts to understand the mechanisms of plaque biofilm development, several glucan-binding proteins (GBPs) have been discovered. Some of these, the glucosyltransferases, catalyze the synthesis of glucan, whereas others, designated only as glucan-binding proteins, have affinities for different forms of glucan and contribute to aspects of the biology of their host organisms. The functions of these latter glucan-binding proteins include dextran-dependent aggregation, dextranase inhibition, plaque cohesion, and perhaps cell wall synthesis. In some instances, their glucan-binding domains share common features, whereas in others the mechanism for glucan binding remains unknown. Recent studies indicate that at least some of the glucan-binding proteins modulate virulence and some can act as protective immunogens within animal models. Overall, the multiplicity of GBPs and their aforementioned properties are testimonies to their importance. Future studies will greatly advance the understanding of the distribution, function, and regulation of the GBPs and place into perspective the facets of their contributions to the biology of the oral streptococci.
Bloodstream (BSF) and culture forms (CF) of Trypanosoma lewisi were specifically agglutinated with the plant lectins concanavalin A (Con A), soybean agglutinin (SBA), wheat germ agglutinin (WGA), and fucose-binding protein (FBP). Lectin-mediated cell agglutination was inhibited, and reversed in the presence of specific lectin-binding saccharides. Cells were agglutinated randomly with all lectins suggesting a uniform distribution in the trypanosome cell surface of the lectin-binding saccharide ligands. The BSF and CF were not agglutinated with phytohaemagglutinin-M, phytohaemagglutinin-P, or influenza virions. Living trypsinized BSF, which lacked a surface coat, gave agglutination results with the lectins identical to those obtained with living intact BSF. Glutaraldehyde- or formalin-fixed intact and trypsinized BSF gave results similar to those obtained with living cells and SBA, WGA, and FBP. However, intact, fixed BSF gave much lower agglutination levels with Con A than trypsinized-fixed, living intact, or living trypsinized BSF cells. Intact and trypsinized living and fixed CF gave identical agglutination results with each of the lectins. Living and fixed cells treated extensively with the glycoside hydrolases alpha-amylase, dextranase, and neuraminidase gave results with the lectins identical to those obtained with untreated cells. Con A bound at the cell surface was visualized with an iron-dextran (Fe-Dex) conjugate. Dense iron marker particles were distributed randomly in the intact BSF surface coat. The Con A-bound Fe-Dex marker was present on the pellicular and flagellar membrane outer lamina of trypsinized BSF and intact CF cells. Horseradish peroxidase (HRPO)-diaminobenzidine (DAB) coupled reactions also were used to visualize surface-bound Con A. Dense Con A-HRPO-DAB deposits were present uniformly in the BSF surface coat, and on the membranes of trypsinized BSF and intact CF trypanosomes. SBA and WGA were conjugated to HRPO and these used in DAB-coupled reactions at the ultrastructure level. Results obtained with the HRPO-conjugated lectins were similar in surface localization and distribution to those obtained with the Con A-HRPO-DAB preparations. Treatment of BSF and CF with the several glycoside hydrolases produced no apparent enhanced or reduced reactivity for the lectins in any of the fine-structure cytochemistry experiments. The cumulative results indicate that ligands similar or identical to alpha-D-mannose, N-acetylgalactosamine, and N-acetylglucosamine, and alpha-L-fucose are constituents in the extracellular surface coat matrix of T. lewisi BSF. Similar conclusions also pertain to the pellicular and flagellar membrane ligands of the BSF and CF cells. Moreover, results obtained with the glycoside hydrolases and influenza virions suggest that the T. lewisi cell surface ligands are not associated directly with repetitively bonded alpha-I,4- and alpha-I,6-D-glucans or sialic acid moieties.
In glycoside hydrolase family 66 (see http://afmb.cnrs-mrs.fr/CAZY/), cyclodextran glucanotransferase (CITase) is the only transglycosylation enzyme, all the other family 66 enzymes being dextranases. To analyze the catalytic amino acids of CITase, we modified CITase chemically from the T-3040 strain of Bacillus circulans with 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). EDC inactivated the enzyme by following pseudo-first order kinetics. In addition, the substrates of an isomaltooligosaccharide and a cyclodextran inhibited EDC-induced enzyme inactivation, implicating the carboxyl groups of CITase as the catalytic amino acids of the enzyme. When two conserved aspartic acid residues, Asp145 and Asp270, were replaced with Asn in T-3040 mature CITase, CIT-D270N was completely inactive, and CIT-D145N had reduced activity. The V(max) of CIT-D145N was 1% of that of wild-type CITase, whereas the K(m) of CIT-D145N was about the same as that of the wild-type enzyme. These findings indicate that Asp145 and Asp270 play an important role in the enzymatic reaction of T-3040 CITase.
An affinity matrix of soluble starch gel was prepared by cross-linking catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA) indicated that the amount of enzyme bound to the starch gel column increased with increases in the ammonium sulfate (AmS) concentration in the equilibrating buffer. TAA had an affinity for the gels with a starch structure, and desorbed from the column with the buffer containing no AmS. Bound TAA was also eluted with starch and cyclodextrin solution. The AmS stimulative effect was partially replaced by polyethylene glycol and surfactants. Besides TAA, various, other amylases bound satisfactorily to the starch gel. Moreover, affinity purifications of dextranase, cellulase, and pectinase were done by gels with dextran, cellulose, and pectin structures, respectively. By the aid of forced effects of AmS, various carbohydrases could be purified by the affinity gels of polysaccharide linked by epichlorohydrin.
A novel glucanhydrolase from a mutant of Lipomyces starkeyi ATCC 74054 was purified. The single protein (100 kDa) showed either dextranolytic or amylolytic activity. We referred to the glucanhydrolase as a DXAMase. The DXAMase was produced in a starch medium and it was 3.75-fold more active for hydrolysis of the purified insoluble-glucan of Streptococcus mutans than Penicillium funiculosum dextranase. Aggregation of S. mutans cells with dextran and adherence to glass were eliminated by incubating with the DXAMase. The addition of DXAMase (0.1 IU/ml) to the mutansucrase reaction digest with sucrose reduced the formation of insoluble-glucan about 80%. Also the DXAMase (0.5 IU/ml) removed 80% of the pre-formed sucrose-dependent adherent film. These in vitro properties of L. starkeyi KSM 22 DXAMase are desirable for its application as a dental plaque control agent.
This review deals with the pharmacokinetics of dextrans and hydroxyethylstarch, the most commonly used plasma expanders. The complex composition of these colloidal agents (broad range of molecular weight distribution in vitro and in vivo) confounds their specific assay and meaningful pharmacokinetic analysis. In addition, the time-dependent decline of plasma concentrations of the plasma expanders is at least biphasic, and in some clinical studies the time period for plasma concentration monitoring has been inadequate to characterise the terminal elimination phase. According to their average molecular weight, dextrans can be differentiated into dextran 1, dextran 40, dextran 60 and dextran 70. Metabolism of dextrans by dextranases and extrarenal excretion account for only 2 to 10% of the overall drug loss from the body. Persistence of dextrans in the systemic circulation and elimination by the renal route are dependent on the size of dextrans and their molecular weight distribution. Dextran species with a molecular weight below 15,000 daltons are filtered unrestricted, and consequently the elimination half-life of dextran 1 is relatively short (2 hours) and that of dextran 40 (10 hours) or dextran 60 (42 hours) much longer. In patients with renal insufficiency elimination is impaired in parallel to the reduction in glomerular filtration rate, and smaller doses are advisable in these patients. Dosage reduction might be also indicated if multiple infusions of dextrans are used, since dextran 40 accumulates considerably during long term use (particularly the fractions with higher molecular weights). As only about 50 to 70% of a single dose could be recovered within 48 hours in the urine, the remainder of the dose is probably stored somewhere in the body. Disposition of hydroxyethylstarch is dependent on 2 major factors. As with dextrans, the molecular weight distribution affects the rate of renal elimination. In addition, the degree of substitution with hydroxyethyl groups mainly determines the metabolism of hydroxyethylstarch by alpha-amylase, and thus the overall elimination rate. A higher molecular weight range (e.g. hydroxyethylstarch 450,000 vs 200,000) and a more extensive degree of substitution (e.g. 0.7 vs 0.5) result in a slower elimination, as can be seen by comparing the half-life values of hydroxyethylstarch 450/0.7 (48 days) and hydroxyethylstarch 200/0.5 (20 days). Since only 40 to 65% of an infused dose could be recovered in the urine in humans, the remainder of the dose may be stored in the body. Animal experiments suggest that certain fractions of hydroxyethylstarch might be stored in some tissues.(ABSTRACT TRUNCATED AT 400 WORDS)
Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.
A conjugate with specific binding to the epidermal growth factor receptor, EGFR, and of interest for clinical tests was prepared using mouse epidermal growth factor, mEGF, and dextran. The mEGF was first coupled to dextran by reductive amination in which the free amino group on the N-terminal of mEGF was reacted with the aldehyde group on the reductive end of the dextran chain. The end-end coupled intermediate was further activated by the cyanopyridinium agent CDAP and tyrosines introduced to the dextran part of the conjugate. The mEGF-dextran-tyrosine conjugate was, with high efficiency, iodinated with the chloramine-T method. Approximately 25-35% of the radioactivity could be removed from the conjugate after exposure to protease K while 65-75% of the radioactivity could be removed after exposure to dextranase. Thus, the largest amount of the iodine was on the dextran part of the conjugate. The iodinated mEGF-dextran-tyrosine had EGFR specific binding since the binding to an EGFR rich human glioma cell line could be displaced by an excess of non-radioactive mEGF. The conjugate was to a large extent internalized in these cells and the administrated radioactivity was thereby retained inside the cells for at least up to 50 h.
OBJECTIVE: To establish a kind of molecular biology clinical detective method to cariogenic S. mutans. METHODS: Using the coamplification of target and reference genes. One pair of specific primers were designed according to a portion of the dextranase (dexA) gene of S. mutans. The reference gene was plasmid pET23b DNA. The saliva samples of 196 children were quantitative detected. The PCR method was compared with the routine culture method. RESULTS: The rate of S. mutans counts >/= 10(8) CFU/L (colony-forming unit per millilitre) saliva by quantitative PCR was 91.3%. The results of coincidence rate between the new method and the routine way was 94.9%. CONCLUSIONS: The new quantitative detective method is fast and provides with high scoincidence rate and high specificity, so have extensive clinical practice foreground.
In previous studies we could show that the third component of complement (C3) is bound from normal serum to cross-linked dextran (Sephadex) after activation via an alternate pathway. Data presented in this paper demonstrate that a subpopulation of human lymphocytes adheries to C3 coated Sephadex particles. The adhering subpopulation of lymphocytes is identical with or overlaps extensively with lymphocytes bearing easily detectable membrane Ig. This phenomenon can be used for the fractionation of lymphocyte subpopulations. After a single passage of tonsillar lymphocytes through C3 coated Sephadex bead columns, no membrane Ig positive lymphocytes were detectable in the effluent. 97-100% of effluent cells formed rosettes with untreated sheep red blood cells, thus resembling pure T cells. Apart from that, the use of C3 coated dextran gel particles allows also the recovery of adherent cells after dextranase digestion of the dextran gel particles. An investigation of retained cells showed enrichment of B cells, but also T cells were demonstrable. The retention of these T cells is probably due to nonspecific interactions.
Dextran sulfate sodium (DSS) is a strong negatively charged heparin-like polysaccharide and has anti-immunodeficiency virus, anti-carcinogenesis, or occasionally tumor-promotion effects. The biological metabolism of DSS, however, remains unclear. In a previous study, we reported a novel method for the separation and quantification of DSS, using fluorometric labeling with 2-aminopyridine and a combination of size-exclusion and reverse phase high-performance liquid chromatography. In the present study, we have applied this method for analyses of in vitro chemical or enzymatic depolymerization of pyridylamino-DSS (PA-DSS). PA-DSS was depolymerized by specific enzymes such as alpha-amylase and alpha-glycosidase, but not by dextranase or heparinase. Unknown enzymes derived from cultured intestinal cells also strongly depolymerized PA-DSS as did alkaline substances. On the other hand, we have established a novel detection system using a post-column reaction. This method utilizes the spectrophotometrically metachromatic reaction of toluidine blue solution with DSS. This novel detection system may be specific and may potentially provide useful information in the analyses of sulfated polysaccharides, which are present in environmental and biological materials.
Amino acid sequence analysis of alpha-galactosidases and other proteins of glycoside hydrolase family 27 (GH27) allowed isolation of three major subfamilies, 27a-27c. Unique isomalto-dextranase of Arthrobacter globiformis clustered separately. Eukaryotic proteins formed five clusters on a phylogenetic tree of the family. Bacterial GH27 proteins, which are relatively few, did not form stable clusters. A monophyletic origin of the GH27 family was demonstrated with the use of related proteins of the GH36 family. The structure of the active center and evolution of alpha-galactosidases are discussed.
OBJECTIVE: To prepare enamel matrix proteins (EMPs) loaded dextran-based hydrogel microspheres (EMPs-dex-MPs), and to evaluate their EMPs controlled release property and their biological effects on the proliferation and differentiation of human periodontal ligament cells (PDLCs) in vitro. METHODS: Using dimethylbenzene as the oil phase, EMPs-dex-MPs were achieved by emulsion-chemical crosslinking technique. The process of the recombination preparation was optimized by orthogonal factorization method. The configuration and size of EMPs-dex-MPs were determined by scanning electron microscope. The EMPs loading content and encapsulation rate of EMPs-dex-MPs, and their biodegradation characteristic were studied by routine analysis methods. Dynamic dialysis method was used to determine the release characteristic of EMPs-dex-MPs in vitro and its influencing factors. The proliferation of cultured PDLCs was measured by MTF method and the differentiation of PDLCs was measured by their alkaline phosphatase (ALP) activities. RESULTS: The results showed that EMPs-dex-MPs were homogenous and stable with the average diameter 25 microm, and the EMPs loading content was (32.8 +/- 1.2)%, the encapsulation rate was (78.9 +/- 1.0)%. Under 9% physiological saline solution contained a very thimbleful quantity of dextranase EMPs-dex-MPs could be biodegraded completely during about 40 days. The in vitro experiments showed that about 80% of EMPs could be released out in 20 days. Using EMPs-dex-MPs could enhance the proliferation responses and ALP activities of PDLCs more than 12 days. CONCLUSION: As a new sustained release system of growth factors, the dex-MPs is stable, workable and biodegradable. EMPs-dex-MPs, whose drug release can be controlled by preparation technique, may be more effective in promoting periodontal tissue regeneration.
Initial velocities of ketoprofen formation from ketoprofen-dextran ester prodrugs incubated in homogenates of various segments of the pig GI-tract were determined. Enzyme-mediated drug release was found in caecum and colon homogenates with their contents, whereas release rates in the stomach, duodenum, jejunum and ileum homogenates were comparable to those determined in pure buffer solutions of identical pH. In colon homogenates adjusted to various pH values between 6.0 and 7.9, little variation in release rates was observed. However, the contribution of enzyme-catalyzed drug regeneration to the overall initial velocity of ketoprofen formation increased significantly as a function of decreasing pH. The presence of several antibiotics and betamethasone in colon homogenates did not affect the drug activation process, whereas the addition of various enzyme inhibitors slowed down the ketoprofen release rates. During incubation in colon homogenates the average molecular weight of the dextran esters decreased. The drug release may therefore involve an initial fragmentation of the drug-liganded dextran chains carried out by dextranases secreted from the microflora which reside in the pig's large bowel.