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C F Schachtele

Publications and source records attributed to C F Schachtele.

At least 37 records · Page 2Linked to original sources
PubMed1982

Comparison of methods for monitoring changes in the pH of human dental plaque.

Changes in human dental plaque pH can be used to obtain estimates of the acidogenic potential of ingested foods. The presence of acid in plaque is influenced by a large number of host, microbial, and substrate factors. Several useful methods have been developed for monitoring changes in plaque pH. Plaque sampling involves repeated removal of small samples of plaque from a number of teeth at intervals after food ingestion, dispersion of the sample, and in vitro measurement of pH. Touch electrode methods utilize glass or antimony microelectrodes, which are placed onto plaque in situ where direct readings can be obtained. Telemetry methods involve placement of glass microelectrodes or ion-sensitive field effect transistors within the dentition. Plaque is allowed to accumulate, and pH changes can subsequently be transmitted with radio or wire. Each of the methods has clear advantages and limitations. The methods have been simultaneously compared in human volunteers using solutions of fermentable carbohydrate. Inter-method differences in response were observed depending upon the site of measurement. Data obtained from caries-prone surfaces via telemetry showed lower pH minima and retarded returns to resting pH levels. The technology is available for controlled comparative plaque pH studies, with the method of choice depending upon the goals of the investigation. It is essential that the results be compared to data obtained with other models designed to evaluate the cariogenic potential of foods.

Acids↗
PubMed1980

Streptococcus mutans dextransucrase: effect of cerulenin on lipid synthesis and enzyme production.

The effect of the fatty acid synthesis inhibitor cerulenin on growth and dextransucrase (EC 2.4.1.5) production by Streptococcus mutans 6715 was analyzed. Growth was markedly inhibited by less than 1 microgram of the antibiotic per ml. Under conditions where cerulenin did not inhibit amino acid incorporation into protein but did block acetate metabolism into lipid, the production of extracellular dextransucrase was suppressed. Inhibition was not due to a direct effect of the antibiotic on the enzyme or the lack of release of enzyme from the bacterial cell surface. Gel column chromatography demonstrated that enzyme produced in the presence of cerulenin was highly aggregated, similar to the control enzyme. Although the addition of ysophosphatidylcholine to enzyme which had been synthesized in the presence of cerulenin stimulated glucan formation from sucrose, the increase was not greater than that produced with the control enzyme. The differential inhibition of dextransucrase production by cerulenin indicates that enzyme secretion requires the production of lipid and may reflect the mechanism by whch this enzyme is transported from the bacterial cell.

Antifungal Agents↗
PubMed1979

Modification of human parotid saliva proteins by oral streptococcus sanguis.

Polyacrylamide gel electrophoresis was used to analyze the effect of Streptococcus sanguis on the anionic proteins in human parotid gland saliva. Cell-free culture supernatants and washed-cell preparations from 23 strains of S. sanguis caused marked modification of various salivary proteins. Control studies showed that the alterations in protein profiles by the bacteria were not due to attachment of protein to the cells. Protease inhibitors were used to confirm that proteolysis by distinct enzymatic activities was responsible for most of the modifications. There was no discernible relationship between the degradation patterns and the various immunologic or genetic groups of S. sanguis. Proteins which contained high concentrations of proline were extensively degraded by S. sanguis. This effect could be blocked with the protease inhibitor phenylmethyl-sulphonyl fluoride.

Electrophoresis, Polyacrylamide Gel↗
PubMed1978

Streptococcus mutans dextransucrase: stimulation of glucan formation by phosphoglycerides.

Lysophosphatidylcholine (LPC) and other phosphoglycerides stimulated water-insoluble and water-soluble glucan production by the Streptococcus mutans 6715 dextransucrase (EC 2.4.1.5). LPC stimulated crude extracellular dextransucrase 1.7-fold, the water-insoluble glucan-producing alpha form of the enzyme 6.5-fold, the water-soluble glucan-producing beta form of the enzyme 2.1-fold, and the cell-associated dextransucrase 2.0-fold. Kinetic studies demonstrated that LPC did not change the K(m) for sucrose of alpha or beta but increased the maximum velocity of the enzymes. The K(m) for LPC of the alpha enzyme was 10(-5) M. LPC from various sources and synthetic preparations of lauroyl-LPC, myristoyl-LPC, and palmitoyl-LPC all stimulated glucan formation. Portions of phosphoglyceride molecules including fatty acids, phosphatidic acid, glycerophosphoric acid, glycerophos-phorylcholine, and choline, when tested individually or in combinations, did not enhance dextransucrase activity. The increased rates of glucan production caused by LPC and primer dextran were additive. Enzyme incubated with LPC before addition of sucrose was stimulated by dextran primer, and, conversely, enzyme treated with dextran was stimulated by addition of LPC with the sucrose substrate. Thus, dextransucrase can be activated by binding of intact phosphoglyceride molecules to a site on the enzyme that is distinct from either the glucosyl donor or glucosyl acceptor (primer) binding sites. Interactions between the S. mutans dextransucrase and amphipathic phosphoglycerides may explain properties of this enzyme which contribute to the cariogenicity of S. mutans.

Dextrans↗
PubMed1978

Streptococcus mutans dextransucrase: stimulation by phospholipids from human sera and oral fluids.

Serum, gingival crevicular fluid, and parotid, submandibular, and labial minor gland saliva from four individuals stimulated glucan formation from sucrose by the Streptococcus mutans strain 6715 dextransucrase (EC 2.4.1.5). At final dilutions of 1:10 all of the fluids stimulated crude enzyme preparations approximately 1.8-fold. The fluids stimulated the purified water-insoluble glucan-synthesizing form of the dextransucrase approximately 3.2-fold and the water-soluble glucan-producing form of the enzyme approximately 2.4-fold. The fluids all contained concentrations of stimulatory material that could be reduced to undetectable levels only after dilutions of greater than 1:1,000. The increased rates of glucan formation caused by the fluids and dextran were additive, indicating that stimulation by the fluids was primarily due to interactions with entities other than glucan primer molecules. In contrast, the elevated levels of glucan formation in the presence of the fluids was not further enhanced by the addition of lysophosphatidylcholine. Lysophosphatidylcholine purified from parotid and submandibular saliva by solvent extraction and thin-layer chromatography stimulated the dextransucrase as effectively as egg yolk lysophosphatidylcholine. Thus, phospholipids normally found in human oral fluids can enhance the activity of an enzyme believed to be directly associated with the cariogenic potential of S. mutans.

Blood↗
PubMed1977

Streptococcus mutans dextransucrase: functioning of primer dextran and endogenous dextranase in water-soluble and water-insoluble glucan synthesis.

The extracellular enzyme activities of Streptococcus mutans 6715 that synthesize glucans from sucrose were concentrated and partially purified by ammonium sulfate precipitation and gel permeation column chromatography. Polyacrylamide gel analysis demonstrated that all of the major proteins precipitated by ammonium sulfate were quantitatively recovered in the high-molecular-weight, enzyme-containing aggregates found in the void volume of the gel column. Anion-exchange column chromatography was used to fractionate the aggregates into preparations, alpha and beta, which produced water-insoluble and water-soluble glucans, respectively. Polyacrylamide gel analysis showed that alpha and beta contained unique proteins and dextransucrase (EC 2.4.1.5) activities. Studies on the time course of glucan synthesis by alpha demonstrated that this enzyme preparation contained dextranase activity, which partially degraded nascent alcohol-insoluble glucan into alcohol-soluble products that were subsequently reincorporated into insoluble product. The beta enzyme preparation contained no detectable dextranase activity. Mixing experiments in the absence of primer dextran demonstrated that the dextranase activity present in alpha could modify glucan production by beta. CsCl density gradient analysis of product glucans demonstrated that exogenous primer dextrans were used as acceptor molecules by both the alpha and beta enzyme preparations, and that water-soluble glucans synthesized by beta could be converted into water-insoluble glucans by alpha. It is proposed that the structural heterogeneity of the native glucans produced from sucrose by S. mutans is a result of the concerted action of glucan-forming dextransucrases and endohydrolytic dextranase activity.

Chromatography, Gel↗
PubMed1977

Postdoctoral research training in cariology: an opportunity initiated by the National Caries Program at the University of Minnesota School of Dentistry.

In response to the need for development of measures to effectively control human dental caries, a postdocroral research training program in cariology has been established at the University of Minnesota School of Dentistry. This article discusses the concept of cariology, the background leading to the development of the program, and the goals and structure of the program. The recruiting and training of individuals to perform and direct multidisciplinary basic and applied research in cariology are explained. A didactic and clinical program is presented which gives a comprehensive introduction to cariology to trainees with a Ph.D degree and little previous knowledge of the multifactorial nature of dental caries.

Curriculum↗
PubMed1976

Streptococcus mutans dextransucrase: mode of interaction with high-molecular-weight dextran and role in cellular aggregation.

The interaction between Streptococcus mutans dextransucrase (EC 2.4.1.5) and high-molecular-weight dextran was studied in both the presence and absence of substrate sucrose. Equivalent weight-percent solutions of primer dextrans that differed 200-fold in molecular weight were found to be equally efficient in priming new dextran synthesis. Sodium borohydride reduction of dextran had no effect on its priming ability. These results suggest that dextran synthesis proceeds by addition of glucosyl residues to nonreducing termini of primer dextrans and that several enzyme molecules simultaneously bind to single high-molecular-weight dextran molecules. Kinetic data suggested that dextransucrase contains only one dextran binding site per enzyme molecule. The nature of the commonly observed highly aggregated state of dextransucrase was also studied. Two types of enzyme aggregates were distinguished: (i) oligomeric enzyme aggregates that formed in the absence of dextran and were dissociated by 1 M KCl; and (ii) dextran-induced enzyme aggregates that were stable to 3 M salt. Oligomeric enzyme aggregates were obtained from supernatants of fructose-grown cultures, whereas dextran-induced enzyme aggregates appeared to be present in glucose-grown cultures. The molecular weight of the smallest species of dextran-free detransucrase observed in solutions of 1 M KCl was estimated to be 40,000 by gel column chromatography. Addition of dextran to primer-dependent dextransucrase resulted in formation of complexes that were stable in CsCl density gradients and exhibited a buoyant density of 1.382 g/cm3 as compared with a buoyant density of 1.302 g/cm3 exhibited by dextransucrase. The enzyme-dextran complexes observed in CsCl density gradients contained about 25% dextran. This corresponded to 150 enzyme molecules (molecular weight, 40,000) per dextran molecule (molecular weight, 2 X 10(6)). The implication of these results to the mechanism of sucrose- and dextran-induced aggregation of S. mutans is discussed.

Binding Sites↗
PubMed1976

Streptococcus mutans dextransucrase: availability of disaggregated enzyme after growth in a chemically defined medium.

The soluble dextransucrase (EC 2.4.1.5) activity produced by Streptococcus mutans strain 6715 during growth on a chemically defined synthetic medium (FMS) was compared to enzyme from glucose broth cultures (TSB). Growth on the two media was similar. The specific activity of ammonium sulfate-precipitated FMC enzyme was 17 times greater than similar TSB enzyme preparations. The FMC enzyme was stimulated 11-fold, whereas the TSB enzyme was stimulated 1.2-fold by the addition of exogenous primer dextran. In contrast to the TSB enzyme, the FMC activity could be disaggregated to a low-molecular-weight form by 1 M salt. Thus, low-molecular-weight S. mutans dextransucrase activity free of contaminating primer glucan may be readily obtained after growth of the bacterium in a chemically defined sucrose-free medium.

Culture Media↗
PubMed1976

Streptoccus mutans dextransucrase: purification, properties, and requirement for primer dextran.

We attempted to purify dextransucrase from S mutans strain 6715 to investigate its properties and determine if multiple species of the enzyme existed. It was concluded that the properties of this enzyme such as the pH (5.5), temperature (37 C) optimum, and Km for sucrose (3 mM) are very similar to those reported for S sanguis, S bovis, S mutans strain OMZ-176 isozymes, S mutans strain GS-5, and the single dextransucrase purified from S mutans strain HS-6. The IEF enzyme preparation consisted of two enzyme species, possibly differing in their ability to synthesize different dextran linkages. The minor enzyme activity demonstrated a strict primer dependency. Similarly, primer dependency has been reported for dextransucrases from S mutans, S sanguis, and L mesenteroides. S mutans strain 6715 dextransucrase also showed both the insertion and stepwise mechanisms for dextran synthesis. Sucrose was the sole glucose donor, whereas dextran was a specific, highly efficient glucose acceptor. The complex primer kinetics are not fully understood at this time and require further investigation. Without linkage analysis of the products of our enzymes, we can only postulate that each enzyme has a different function in the synthesis of interresidue and interchain alpha1-3 and alpha1-6 bonds. Insoluble dextran synthesis may involve a special enzyme mechanism characteristic of S mutans. This synthesis would require both enzymes, possibly in some aggregated form, with one enzyme synthesizing endogenous primer dextran. This endogeneous primer or some cell wall polysaccharide could stimulate both enzymes to rapidly synthesize heterogeneously linked insoluble dextran.

Chemical Phenomena↗
PubMed1975

Dextranases from oral bacteria: inhibition of water-insoluble glucan production and adherence to smooth surfaces by Streptococcus mutans.

The effect of dextranases (EC 3.2.1.11) from the oral isolates Actinomyces israelii and Bacteroides ochraceus on water-insoluble glucan production by the Streptococcus mutans dextransucrase (EC 2.4.1.5) and sucrose-dependent adherence to smooth glass surfaces by S. mutans was studied. Collection on membrane filters of water-insoluble polysaccharides synthesized from radioactive sucrose was used to demonstrate the marked sensitivity of insoluble glucan formation to the presence of dextranase. Concentrations of A. israelii dextranase as low as 0.002 U/ml inhibited insoluble glucan formation by 60%. Similar results were obtained the the B. ochraceus enzyme. An assay for sucrose-stimulated adherence of S. mutans to smooth surfaces involved attachment of radioactively labeled nongrowing cells to the bottom of glass scintillation vials. This facile and sensitive assay was utilized to demonstrate that sucrose-dependent adherence was affected by low levels of dextranase from either A. israelii or B. ochraceus. Enzyme at 0.005 U/ml reduced adherence of S. mutans by 80%. Treatment of S. mutans cells previously attached to glass with low concentrations of the dextranases resulted in removal of 50% to 60% of the bacteria. The results indicate that dextranase-producing oral bacteria may affect sucrose-dependent colonization of S. mutans on the tooth surface and offer a possible explanation for both the difficulties involved in implanting this bacterium into the human mouth and the limited intraoral transmission of S. mutans from one tooth surface to another.

Actinomyces↗
PubMed1975

Characterization of a dextranase produced by an oral strain of Actinomyces israelii.

A dextranase-producing, gram-positive, anaerobic, rod-shaped bacterium isolated from human dental plaque was identified as Actinomyces israeli. Although the extracellular dextranase (EC 3.2.1.11) formed by this microbe appeared to be constitutively produced, the bacterium did not utilize the reaction products as a carbon source during growth. A striking feature of the dextranase was the formation of two distinct groups of oligosaccharide end products. The two groups presumably correspond to the limit dextran and the released reaction product which appeared to be cleaved from the end(s) of larger dextran molecules. Low levels of dextranase activity were measured by [3H]NaBH4 reduction and alcohol fixation of the large, tritiated end products on filter paper disks. Of the carbohydrate substrates tested, only alpha-1,6-linked glucans were cleaved. The enzyme did not exhibit any metal ion requirements, and its pH optimum was 6.3. It is suggested that the A. israelii dextranase may function as a regulatory factor during extracellular in vivo glucan synthesis from sucrose by various plaque microbes.

Actinomyces↗
PubMed1975

Production of elevated levels of dextransucrase by a mutant of Streptococcus mutans.

A mutant (S19) of Sreptococcus mutans strain 6715 which produces elevated levels of dextransucrase (EC 2.4.1.5) was isolated. Soluble enzyme in culture supernatant solutions from S19 polymerized the glucosyl moiety of sucrose into alcohol-insoluble and water-insoluble glucans at a rate three to six times greater than that of the parent strain. Washed-cell suspensions of S19 also contained increased amounts of cell-associated enzyme. Adherence of S19 to glass in the presence of sucrose occurred at twice the rate of strain 6715. The Km values for sucrose and primer dextran were similar for the mutant and parent enzymes. Mutant S19 should facilitate studies on the mechanism of adherence of S. Mutans and the control of dextransucrase production by this bacterium.

Glucosyltransferases↗
PubMed1974

Evaluation of dextranase production by the cariogenic bacterium Streptococcus mutans.

A recently developed blue dextran-agar assay was utilized to detect dextranase production by a large number of strains of Streptococcus mutans. An extract obtained from S. mutans strain B2 hydrolyzed high-molecular-weight radioactive dextran to oligosaccharides. There are several possible roles for this enzyme activity in the metabolism of S. mutans dextrans.

Chromatography, Gel↗
PubMed1974

Purification and properties of dextransucrase from Streptococcus mutans.

The dextransucrase (EC 2.4.1.5) activity from cell-free culture supernatants of Streptococcus mutans strain 6715 has been purified approximately 1,500-fold by ammonium sulfate precipitation, hydroxylapatite chromatography, and isoelectric focusing. The enzyme was eluted as a single peak of activity from hydroxylapatite, and isoelectric focusing of the resulting preparation gave a single band of dextransucrase activity which focused at a pH of 4.0. The final enzyme preparation contained two distinct, enzymatically active proteins as judged by assay in situ after polyacrylamide gel electrophoresis. One of the proteins represented 90% of the total dextransucrase activity and 53% of the total protein. The molecular weight of the enzyme was estimated by gel filtration to be 94,000. The temperature optimum of the enzyme was broad (34 to 42 C) and its pH range was rather narrow, with optimal activity at pH 5.5. The K(m) for sucrose was 3 mM, and fructose competitively inhibited the enzyme reaction with a K(i) of 27 mM.

Ammonium Sulfate↗