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Determination, by carbon-13 nuclear magnetic resonance spectroscopy, of the composition of glucans synthesized by enzymes of the cariogenic organism Streptococcus mutans.

The 13C-n.m.r. spectra of water-soluble and -insoluble glucans synthesized by enzymes isolated from six strains of Streptococcus mutans are interpreted. The glucans are shown to be composed primarily of alpha-(1 linked to 3)- and alpha-(1 linked to 6)-linked glucosyl residues, and the relative abundance of each linkage is estimated from peak areas. Treatment of water-insoluble glucans with dextranase is found to result in water-soluble and -insoluble products, the former enriched in alpha-(1 linked to 6)-linkages and the latter in alpha-(1 linked to 3)-linkages. The structural conclusions arrived at by 13C-n.m.r. spectroscopy are consistent with data from methylation analysis and 1H-n.m.r. spectroscopy.

Chemical Phenomena↗

Identification of mutans streptococcal species by the PCR products of the dex genes.

A pair of polymerase chain reaction (PCR) primers was designed on the basis of the nucleotide sequence homology of dextranase genes (dex) of Streptococcus mutans, S. sobrinus and S. downei. The primer pair amplified a 530-bp DNA fragment on the dex genes of mutans streptococcal species: S. mutans, S. sobrinus, S. downei, S. rattus and S. cricetus. HaeIII digestion of the 530-bp fragments generated species-specific subfragments, which were easily distinguishable from each other by agarose gel electrophoresis. These results suggest that the PCR-amplification of the dex gene followed by the HaeIII digestion is useful for rapid identification of the five species of mutans streptococci.

Animals↗

Lauroyldextran and crosslinked galactomannan as coating materials for site-specific drug delivery to the colon.

Lauroyldextran (LD) and crosslinked galactomannan (XGM) were investigated as microbiologically degradable film coating materials for site-specific drug delivery to the colon. LD was used with degrees of substitution between 0.12 and 0.40, and swelling in aqueous media between 195 and 50%, XGM-batches showed swelling between 309 and 520%. Theophylline tablets were coated in a Hüttlin Kugelcoater with coating quantities of 4-17 mg/cm2. Sprayable coating formulations were obtained with 4% aqueous dispersions of XGM or 4% dispersions of LD in a 1:1 mixture of 1-propanol and water with 10% glycerol (based on the polymer) as a plasticizer. Theophylline dissolution was monitored in a USP XXIII paddle dissolution apparatus with buffer pH 5.5. After 4 h, which is an average small intestine transit time, colon conditions were simulated by adding galactomannanase or dextranase, respectively. Results showed similar dissolution rates for all XGMs and high-swelling LDs during the first 4 h and a relatively quick disintegration after enzyme addition. Both parameters decreased with increasing coating quantities. Dissolution from low-swelling lauroyldextrans was very low but no disintegration was observed after enzyme addition. The disintegration rate was found to be proportional to the square root of the enzyme activity. All swollen materials exhibited low mechanical stability. XGM coatings, especially at higher coating quantities, showed small transient ruptures at the edges not caused by enzyme addition. This behaviour was explained by internal stress due to the high degree of swelling. In principle, materials of both types proved to be suitable as degradable coating materials. The ideal zero-dissolution before and quick disintegration after enzyme addition, however, was not realized with the present materials.

Biodegradation, Environmental↗

Biosensor based on enzyme-catalysed degradation of thin polymer films.

A biosensor based on the enzyme-catalysed dissolution of biodegradable polymer films has been developed. Three polymer-enzyme systems were investigated for use in the sensor: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Dissolution of the polymer films was monitored by Surface Plasmon Resonance (SPR). The rate of degradation was directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive over a concentration range from 4 x 10(-11) to 4 x 10(-7) mol l(-1) of enzyme. The rate of degradation was shown to be independent of the thickness of the poly(ester amide) films. The dextran hydrogel/dextranase couple was less sensitive than the poly(ester amide)/alpha-chymotrypsin couple but showed greater degradation rates at low enzyme concentrations. Enzyme concentrations as low as 2 x 10(-11) mol l(-1) were detected in less than 20 min. Potential fields of application of such a sensor system are the detection of enzyme concentrations and the construction of disposable enzyme based immunosensors, which employ the polymer-degrading enzyme as an enzyme label.

Biodegradation, Environmental↗

A transducer based on enzyme-induced degradation of thin polymer films monitored by surface plasmon resonance.

A novel transducer based on the dissolution of biodegradable polymer films as a direct result of enzymatic reaction has been developed. Three polymers were investigated for use in the transducer: a poly(ester amide), which is degraded by the proteolytic enzyme alpha-chymotrypsin; a dextran hydrogel, which is degraded by dextranase; and poly(trimethylene) succinate, which is degraded by a lipase. Degradation of the polymer films was monitored by surface plasmon resonance (SPR) and impedance measurements. SPR was shown to be suitable for a greater variety of materials, since it does not require the polymer film to be electrically insulating. Rate of degradation was shown to be directly related to enzyme concentration for each polymer/enzyme couple. The poly(ester amide)/alpha-chymotrypsin couple proved to be the most sensitive. Degradation of the films was complete in less than 20 min for enzyme concentrations greater than 9 x 10(-9) mol dm-3. Enzyme concentrations as low as 4 x 10(-11) mol dm-3 were detected in less than 30 min. The transducer has great potential for the detection of enzyme concentrations as well as for use in immunosensing where the enzyme degrading the polymer would be the enzyme label.

Chymotrypsin↗

A DNA probe specific to Streptococcus sobrinus.

Three DNA fragments (SSB-1, -2 and -3) in the dextranase gene (dex) of Streptococcus sobrinus were amplified by polymerase chain reaction and used as DNA probes. The probes were examined for the specificity and the sensitivity of hybridization with DNA of oral streptococcal species. While probes SSB-1 and SSB-2 were specific to both S. sobrinus and Streptococcus downei, SSB-3 was specific only to S. sobrinus. SSB-3 was able to detect 5 ng of chromosomal DNA purified from S. sobrinus NIDR6715 and DNA extracted from 1 x 10(5) cells of the strain. In addition, SSB-3 could differentiate clinical isolates of S. sobrinus from Streptococcus mutans. These results suggest that SSB-3 is an effective DNA-probe to detect and to identify S. sobrinus.

Bacterial Proteins↗

Drug release from core-shell type nanoparticles of poly(DL-lactide-co-glycolide)-grafted dextran.

This study prepared core-shell type nanoparticles of a poly(DL-lactide-co-glycolide) (PLGA) grafted-dextran. The synthesis of the PLGA-dextran conjugate was confirmed by Fourier transform-infrared (FT-IR) spectroscopy. The PLGA grafted-dextran was able to form nanoparticles in water by self-assembly and their particle size was 245.3 +/- 95.1 nm. From fluorescence probe study using pyrene as a hydrophobic probe, critical association concentration (CAC) values were determined from the fluorescence excitation spectra and were found to be 0.006 g l(-1). Morphological observations using a scanning electron microscope (SEM) showed that the polymeric nanoparticles of the PLGA-dextran conjugate have uniformly spherical shapes. Their size and morphology provide them with acceptable properties for use as a drug-targeting carriers. Drug release from core-shell type nanoparticles was faster in the presence of dextranase, indicating that core-shell type nanoparticles of PLGA grafted-dextran can be used as an oral drug carriers.

Administration, Oral↗

Structural aspects of glucans formed in solution and on the surface of hydroxyapatite.

Streptococcus mutans glucosyltransferases (GtfB, -C, and -D) and their products formed from sucrose, glucans, play an essential role in the pathogenesis of dental caries. Enzymatically active Gtf is found in whole human saliva (solution), and incorporated into the salivary pellicle that is formed on teeth in vivo (surface). GtfB glucans are predominantly 1,3-linked; however, surface-formed glucans from GtfB contain greater amounts of 3-linked glucose than glucans formed in solution. In contrast, the major linkage of glucans formed on the surface by GtfB in the presence of sucrose and starch hydrolysates in 4-linked glucose. GtfC-derived glucans in solution have a major linkage of 6-linked glucose, while surface-formed glucans from the same enzyme have 3-linked glucose as the major linkage. GtfD glucans formed either in solution or on the surface are predominantly 1,6-linked; however, surface-formed glucans contain more 6-linked glucose than solution-formed glucans. Digestion with the glucanohydrolases mutanase and dextranase shows differences in susceptibility among glucans formed either in solution or on the surface by each of the Gtf enzymes, and differences are also seen in the soluble end products from these digestions. Our results show that the same Gtf enzyme can form structurally distinct glucans in solution and on a surface. These observations are important in the study of naturally occurring microbial films.

Carbohydrate Conformation↗

Effect of dextran and ammonium sulphate on the reaction catalysed by a glucosyltransferase complex from Streptococcus mutans.

The highly aggregated proteins precipitated by (NH4)2SO4 from the culture fluid of three strains of Streptococcus mutans gradually released less aggregated glucosyltransferase activities - dextransucrase and mutansucrase - which catalysed the synthesis of water-soluble and insoluble glucans from sucrose. Mutansucrase was eluted from a column of Sepharose 6B before dextransucrase. This activity was lost during subsequent dialysis and gel filtration, but there was a corresponding increase in dextransucrase activity which catalysed the formation of soluble glucan when incubated with sucrose alone, and insoluble glucan when incubated with sucrose and 1.55 M-(NH4)2SO4. Relative rates of synthesis of soluble and insoluble glucan in the presence of 1.55 M-(MH4)2SO4 were dependent upon the enzyme concentration: high concentrations favoured insoluble glucan synthesis. Insoluble glucans synthesized by mutansucrase or by dextransucrase in the presence of 1.55 M-(NH4)2SO4 were more sensitive to hydrolysis by mutanase than by dextranse, but soluble glucans were more extensively hydrolysed by dextranase than by mutanase. Partially purified dextransucrase sedimented through glycerol density gradients as a single symmetrical peak with an apparent molecular weight in the range 100000 to 110000. In the presence of 1.55 M-(NH4)2SO4, part of the activity sedimented rapidly as a high molecular weight aggregate. The results strongly suggest that soluble and insoluble glucans are synthesized by interconvertible forms of the same glucosyltransferase. The aggregated form, mutansucrase, preferentially catalyses (1 leads to 3)-alpha bond formation but dissociates during gel filtration to the dextransucrase form which catalyses (1 leads to 6)-alpha bond formation.

Ammonium Sulfate↗

Subcellular localization of D-glucanases in Bacteroides oralis Ig4a.

Three D-glucan-hydrolysing enzymes from Bacteroides oralis Ig4a have been isolated. Two of them are dextranases which hydrolyse (1----6) but not (1----3) linked alpha-D-glucans; one (EC 3.2.1.11, 1,6-alpha-D-glucan 6-glucanohydrolase) is localized in the periplasm, and the other, which is an exo-enzyme (EC 3.2.1.70, 1,6-alpha-D-glucan glucohydrolase), in the cytoplasm. The third is a mutanase (EC 3.2.1.59, 1,3-(1,3;1,4)-alpha-D-glucan 3-glucanohydrolase) that hydrolyses (1----3) but not (1----6) linked alpha-D-glucans, and is present only in the cytoplasm.

Bacteroides↗

Multilevel control of extracellular sucrose metabolism in Streptococcus salivarius by sucrose.

Standardized experimental conditions were established to test the role of sucrose in the regulation of control of its metabolism in Streptococcus salivarius. A fresh isolate of S. salivarius was used. The extracellular dextranase activity of cells grown on sucrose was 10-fold higher than that of cells grown on glucose, fructose or galactose. This activity increased in less than 5 min following the addition of sucrose to galactose-grown cells, a phenomenon which was affected by neither rifampicin nor chloramphenicol which inhibit transcription and translation, respectively. Extracellular fructanase activity was 2-fold higher when cells were grown on sucrose than when they were grown on the other sugars. This increase also occurred within 5 min, but was diminished by transcriptional and translational inhibitors. De novo synthesis was required for the production of extracellular glucosyltransferase (GTF) activity which, upon the addition of sucrose, became associated with the cell surface. Conversely, cell-associated fructosyltransferase (FTF) activity appeared to require genetic induction for its production and cell-surface association, but required sucrose for its release from the surface framework. Versatility in the control mechanisms of this complex set of enzymes allows their expression and function to be regulated at several widely separated stages in the life histories of these proteins.

Bacterial Proteins↗

Deletion in sortase gene of Streptococcus mutans Ingbritt.

Our previous studies on Streptococcus mutans have demonstrated that surface proteins containing a C-terminal sorting signal, such as surface protein antigen (PAc), glucan-binding protein C (GbpC) and dextranase (Dex), are anchored to the cell wall by a sortase (SrtA). In this study we found that, unlike other strains of S. mutans, strain Ingbritt did not exhibit cell wall-anchoring of PAc, GbpC and Dex. It is speculated that the SrtA of strain Ingbritt did not function in the cell wall-anchoring process of these surface proteins. Sequence analysis revealed a deletion of an 11-bp nucleotide sequence in the srtA gene of strain Ingbritt, resulting in the generation of a new termination codon, resulting in production of an incomplete SrtA enzyme protein. As a result, strain Ingbritt showed a localization change of PAc, GbpC and Dex in the cell, implying that strain Ingbritt loses the biological functions mediated by the cell surface-associated proteins of S. mutans. These results suggest that strain Ingbritt could be less cariogenic than other strains of S. mutans.

Aminoacyltransferases↗

Rapid identification of mutans streptococcal species.

Mutans streptococci are considered the predominant pathogens in dental caries. Three methods, i.e. dot blot hybridization analysis, PCR analysis and SDS-blue dextran-PAGE, were examined for identifying mutans streptococcal species. In dot blot hybridization, DNA probe derived from the dextranse gene (dexA) of Streptococcus mutans hybridized with different intensities under the condition of low stringency against each species of mutans streptococci although the dexA probe was specific for S. mutans under the condition of high stringency. Oligonucleotide primers for polymerase chain reaction (PCR) were designed on the basis of the dexA DNA sequence. The primers amplified species-specific PCR products in the reference species (15 strains of 5 species) of mutants streptococci. An electrophoretic profile of dextranases from the mutans streptococci on SDS-blue dextran-PAGE also showed species-specific behavior. These results suggest that the three identification methods examined here are useful for distinguishing the species of mutans streptococci and also indicate that PCR analysis is suitable for simple, rapid and reliable identification of mutans streptococcal species.

Bacterial Typing Techniques↗

A thymus-independent IgG response against dextran B512 can be induced in C57BL but not in CBA mice, even though both strains possess a VHdex gene.

CBA and C57BL mice both possess a VHdex gene coding for antibodies against the alpha 1-6 epitope of dextran B512. After immunization with dextran, CBA mice produce IgM plaque-forming cells (PFC) only, which show regular cyclical fluctuations. The second PFC peak disappeared after injection of dextranase, indicating that it is antigen-dependent. The anti-dextran response in C57BL mice is characterized by only one IgM peak, followed 1 day later by an IgG peak that may exceed the IgM response by a factor of 10. The IgG anti-dextran response in C57BL mice was thymus-independent. CBA mice gave an IgG response to the hapten fluorescein isothiocyanate (FITC) after immunization with FITC-dextran, indicating that dextran can function as a carrier for an IgG response in this strain. Attempts to induce IgG PFC against dextran by immunizing CBA mice with thymus-dependent dextran-protein conjugates consistently failed, althouth the conjugates induced IgG fc in C57BL mice. Spleen cells from CBA mice failed to produce IgG antibodies against dextran after transfer into lethally irradiated C57BL mice, whereas the C57BL spleen cells produced IgG PFC after transfer into CBA mice. The lack of IgG synthesis against the alpha 1-6 epitope of dextran in CBA mice appears to be regulated exclusively at the B cell level and is restricted specifically to the VHdex gene product.

Animals↗

Chemical inhibition of plaque.

Attempts to control plaque by chemical means using enzymes, antibiotics and antiseptics are reviewed. Enzymes such as mucinase, dehydrated pancreas, enzymes of fungal origin, dextranase and mutanase showed limited clinical success despite promising in vitro and animal studies. Side effects from the use of enzymes were observed. Many antibiotics have been used in attempts to control plaque and several have been successful. However, problems exist from the long-term use of such drugs which precludes their routine use as agents for controlling plaque. The biguanide chlorhexidine is the most widely used and investigated method of chemical plaque control. Many studies have been demonstrated that it will successfully control plaque. No toxic side effects have been reported from its long-term use but local side effects such as staining of the teeth do occur. The quaternary ammonium compounds have at present no advantages over the biguanides and require more frequent usage to achieve the same degree of plaque control as chlorhexidine.

Anti-Bacterial Agents↗

Enzymes as denture cleansers.

It was the purpose of the study to test the efficiency of dextranase, mutanase, and protease in removing denture plaque. The study group comprised 100 denture-wearers with denture stomatitis. The enzymes were dispensed as dissolvent tablets either in pure or in mixed preparations. Placebo tablets and Steradent, a commercial denture cleanser, were used as control tablets. The following parameters were studied: the amount of denture plaque, the degree of palatal erythema, and the concentration of yeast cells and inflammatory cells in mucosal and denture smears. The study was designed and carried out as a double-blind study. The dissolvent tablets containing the enzymes in mixed preparations were more effective than the tablets containing the pure enzymes, the placebo tablets, or the Steradent tablets. The beneficial effect of the mixed enzyme preparations included a significant reduction of the amount of denture plaque and an improvement of the clinical condition of the palatal mucosa.

Clinical Trials as Topic↗

Analysis of growth rate in sucrose-supplemented cultures of Streptococcus mutans.

In the presence of sucrose, Streptococcus mutans grows in large glucan-containing aggregates. Because of reports of linear rather than exponential growth of sucrose-grown cultures, the kinetics of growth of sucrose-grown cultures of S. mutans strain OMZ-176 were compared with those of glucose-grown cultures. Culture turbidity measurements indicated that growth of sucrose cultures was slower, did not follow exponential kinetics, and slowed and stopped at lower absorbance values than did glucose-grown cultures. However, measurements of the rates of accumulation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein using fully equilibrated radioactively labeled precursors of each of these macromolecular species in sucrose and glucose-grown cultures showed that: (i) for glucose cultures the synthesis of each of the three informational molecules occurred at the same exponential rate, which was identical to the rate of turbidity increase; (ii) for sucrose cultures each macromolecular species was synthesized at the same exponential rate and these rates were identical to the rate of increase of turbidity of the glucose-grown culture for periods of up to 7 h. Furthermore, the ratios of DNA to RNA, RNA to protein, and protein to DNA for the sucrose cultures were identical to those for the glucose cultures for up to 10 doublings. From these data it was concluded that in the presence of sucrose S. mutans grows in a balanced fashion at the same exponential rate as it does in glucose. The deviation from an exponential growth model of the absorbance in sucrose cultures was attributed to an optical artifact due to the formation of large glucan-containing aggregates of cells. The addition of dextranase to sucrose cultures resulted in cultures which increased in turbidity at the same exponential rate as glucose-grown cultures, without affecting the rate or extent of macromolecular synthesis.

Carbon Radioisotopes↗

Dextran-mediated interbacterial aggregation between dextran-synthesizing streptococci and Actinomyces viscosus.

Streptococcus sanguis and Streptococcus mutans bind to the surface of Actinomyces viscosus, producing large microbial aggregates. Aggregates form rapidly and are not easily dissociated by vigorous mixing. The binding is mediated by dextran. Glucose-grown streptococci will not aggregate unless they are first mixed with high-molecular-weight dextran. Aggregation is induced with dextrans isolated from Leuconostoc, S. sanguis, or S. mutans. Sucrose-grown streptococci will adhere to A. viscosus without the addition of an exogenous source of dextran. A. viscosus will bind dextran and then bind glucose-grown streptococci. Aggregation occurs over a wide pH range and is dependent on cations. The aggregating activity of A. viscosus is both protease and heat sensitive. The aggregating activity of S. sanguis is heat stable but sensitive to dextranase.

Actinomyces↗