Biofilm Journal Biofilm, Volume 1, Paper 2 (BF96002) September 20th 1996 Online Journal, URL -

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1 JOURNALS REPORTS NEWSLETTERS BOOKS registration prices about help Biofilm Journal Biofilm, Volume 1, Paper 2 (BF96002) September 20th 1996 Online Journal, URL - Effect of Some Antiplaque Agents on the Activity of Glucosyltransferases of Streptococcus mutans Adsorbed onto Saliva-Coated Hydroxyapatite and in Solution Anne M. Vacca-Smith^1 and William H. Bowen Department of Dental Research, University of Rochester, 601 Elmwood Avenue, Box 611, Rochester, New York USA Tel:1 (716) Fax:1 (716) avsm@medinfo.rochester.edu ^To whom correspondence should be addressed: Code Number: BF96002 Size of Files: Text: 35.8K No associated graphics files SUMMARY [Tables at end of text] The bulk of dental plaque is composed of bacterial-derived extracellular polysaccharide known as glucan, which is synthesized by streptococcal glucosyltransferase (Gtf) enzymes. At least three Gtf gene products of Streptococcus (1 of 19) [8/1/ :05:15 AM]

2 mutans have been identified. GtfB synthesizes a polymer of insoluble alpha1,3-linked glucan, GtfC produces a mixture of insoluble alpha1,3-linked glucan and soluble alpha1,6-linked glucan, and GtfD synthesizes an alpha1,6- linked soluble glucan. Polysaccharides are important in the development of plaque and in the pathogenesis of dental caries, so we therefore decided to explore the effects of putative antiplaque agents on the activities of streptococcal Gtf enzymes in solution and on the surface of parotid-saliva-coated hydroxyapatite (SHA) beads. Most of the agents tested, particularly cetylpyridinium chloride, hexylresorcinol, alexidine dihydrochloride and triclosan, inhibited the activity of GtfB in solution and on the surface of SHA beads. The agents moderately reduced the glucan-forming activity of GtfD in solution and on the surface of SHA beads, and had no effect on the activity of GtfC in solution or on the SHA surfaces. Several commercial mouthrinses were also evaluated for their effects on reducing Gtf activity; only Plax(R) (European formulation) significantly reduced Gtf activity, and its effect was limited to GtfB in solution. These data suggest that some mouthrinses are highly inhibitory towards GtfB enzymes only, and are with low effect on GtfC and GtfD. This lack of effect may in part explain the comparative poor clinical efficacy of some antiplaque agents. Keywords: Anti-plaque, mouthrinse, glucosyltransferase, glucan, streptococci, plaque. INTRODUCTION The matrix of dental plaque is composed mainly of bacterial- derived polysaccharide termed glucan (Critchley, 1969; Critchley, 1971; Critchley et al., 1967; Critchley et al., 1968; Hamada and Slade, 1980; Wood and Critchley, 1968). Streptococcus mutans has been implicated as a prime microbial agent in the pathogenesis of dental caries and plaque formation (Critchley et al., 1967; Hamada and Slade, 1980; Loesche, 1986; Wood and Critchley, 1968; Yamashita et al., 1993). The virulence of this organism can be attributed, in part, to glucosyltransferases [Gtf, EC , (Critchley, 1971; DeStoppelaar et al., 1971)]. Gtf enzymes catalyze the formation of glucan from sucrose. These enzymes are important particularly in the development of smooth surface carious lesions (Yamashita et al., 1993). Gtfs have been identified in human whole saliva and in salivary pellicles derived in vitro and in vivo (Rolla et al., 1983; Scheie et al., 1987). Gtfs can adsorb onto saliva- coated hydroxyapatite (SHA) beads and synthesize glucan from sucrose in situ (Schilling and Bowen, 1988). This glucan has been shown to serve as an attachment base for colonization by oral streptococci including Streptococcus mutans and Streptococcus sobrinus (McCabe and Donkersloot, 1977; Schilling et al., 1989; Schilling and Bowen, 1992) and contributes to the bulk of the (2 of 19) [8/1/ :05:15 AM]

3 extracellular matrix of dental plaque (Critchley et al., 1967). S. mutans produces at least three Gtfs (Hamada et al., 1984; Hanada and Kuramitsu, 1988, 1989; Kenney and Cole, 1983; Loesche, 1986; Mukasa et al., 1985). GtfB produces a polymer of mostly insoluble alpha1,3-liked glucan, GtfC produces a mixture of insoluble alpha1,3-linked glucan and soluble alpha1,6-linked glucan, and GtfD produces an alpha1,6-linked soluble glucan (Aoki et al., 1986; Hanada and Kuramitsu, 1988,1989). Many compounds reduce plaque formation in vivo (Gjermo et al., 1970; Gjermo et al., 1973; Lobene and Soparker, 1973) and inhibit the activity of glucosyltransferase enzymes in solution (Ciardi et al., Kawabata et al., 1993; Thaniyavarn et al., 1981). These compounds include: quaternary ammonium salts, bis-biguanides, phenolic compounds and aliphatic amines. These earlier studies were performed with mixtures of Gtf enzymes in undetermined ratios with inconsistent results. The effects of the antiplaque agents on SHA-adsorbed Gtf have not been evaluated. These evaluations are necessary, because glucan formed on the tooth surface provides a binding site for oral microorganisms (Hamada and Slade, 1980; Schilling et al., 1989; Schilling and Bowen, 1992) and in addition, tooth pellicle could serve as a reservoir for antiplaque agents. Furthermore, plaque formation results from interactions which occur on a surface. The purpose of the study presented here was to re-evaluate the effects of some previously studied Gtf inhibitors on glucan formation by individual, purified Gtf enzymes both in solution and on the surface of saliva-coated hydroxyapatite beads. In addition, the ability of some common mouthrinses to decrease glucan-formation by the Gtfs was also explored. Results from this study could help to clarify the role of individual Gtf enzymes in plaque formation and point the way towards the further development of effective antiplaque agents. MATERIALS AND METHODS Collection of Parotid Saliva Parotid saliva was collected on ice from one donor using a Lashley cup (Lashley, 1916). The saliva was diluted 1:1 with 25.0 mmol/l imidazole buffer, ph=6.5, supplemented with sodium azide (0.02%, final concentration) and the protease inhibitor phenylmethylsulfonylfluoride (PMSF, 1.0 mmol/l, final concentration). Hydroxyapatite Beads In each sample, 10.0 mg of hydroxyapatite (HA) beads were used (Integration Separation Systems, Hyde Park, (3 of 19) [8/1/ :05:15 AM]

4 MA). The surface area of the beads was 0.24m^2, and the particle size was microns. Glucosyltransferase enzymes Glucosyltransferase enzymes were prepared from constructs of Streptococcus milleri (Fukushima et al., 1992) which contained the genes for Gtfs of S. mutans GS-5. The strains used were S. milleri KSB8, which harbors the gene for GtfB, S. milleri KSC43, which has the gene for GtfC, and S. milleri NH5, which contains the gene for GtfD. The strains were gifts from Dr. H.K. Kuramitsu, SUNY Buffalo, NY. The enzymes were purified by hydroxyapatite column chromatography as previously described (Venkitaraman et al., 1995). The enzymes were stored at C, in imidazole buffer supplemented with 10.0% glycerol. Protein concentration was determined with the Quantigold Protein Assay kit, Diversified Biotech, Boston, MA, using bovine serum albumin as a standard (Sigma Chemical Co., St. Louis, MO), and Gtf activity was assayed as described previously (Schilling and Bowen, 1988). Purification of GtfB resulted in 1.22 mg/ml (specific activity umol of glucose incorporated into glucan/min./ug protein), purification of GtfC yielded 2.66 ug/ml (specific activity umol of glucose incorporated into glucan/min./ug protein) and purification of GtfD produced 800 ug/ml (specific activity umol of glucose incorporated into glucan/min./ug protein). One unit of enzyme was defined as the amount of enzyme needed to incorporate 1 umol of glucose into glucan over a two-hour time period. Test Agents Cetylpyridinium chloride (CPC), hexylresorcinol, alexidine dihydrochloride, mixedalkyltrimethylammoniumbromide (MTAB) and chlorhexidine digluconate were purchased from Sigma. Triclosan (2,4,4'-trichloro-2'-hydroxyphenylether) was obtained from the Ciba-Geigy Corp. Greensboro, NC. Hexylresorcinol and triclosan were prepared in a 40% ethanol solution (40% ethanol, 60% 25.0 mmol/l imidazole buffer, ph=6.5). CPC and chlorhexidine digluconate were prepared in imidazole buffer, and MTAB was provided at a concentration of 5% in a 1 mol/l sodium citrate buffer, ph=6.0. Mouthrinses in the study included Plax(R), United States formulation (Plax(R)USA, Pfizer Inc., New York, NY); Plax(R), European formulation (Plax(R)E, Colgate-Palmolive, Guildford, Surrey, England); Scope(R), The Procter and Gamble Co., Cincinnati. OH; and Biotene(R), Laclede Professional Products, Gardena, CA. The mouthrinses were purchased at local supermarkets or in Europe. (4 of 19) [8/1/ :05:15 AM]

5 Solution Assays GtfB, -C and -D (0.5 Units/200.0 ul final volume) were mixed with 200ul of either Plax(R)USA, Plax(R)E, Scope(R), Biotene(R), CPC (1.25 mmol/l, final concentration), hexylresorcinol (1.25 mmol/l, final concentration), triclosan (1.25 mmol/l, final concentration), alexidine dihydrochloride (1.25 mmol/l, final concentration), MTAB (1.25%, final concentration), chlorhexidine digluconate (1.25 mmol/l, final concentration), or appropriate buffer and incubated with ^14C-(glucosyl)-sucrose substrate (200.0 mmol/l sucrose, 40.0 umol/l dextran 9,000, 1.0 mmol/l PMSF, 0.02% sodium azide in 50 mmol/l imidazole buffer, ph=6.5) to reach a final concentration of mmol/l sucrose. The specific activity of the substrate was 300,000 cpm/ml-0.2 uci/ml. The samples were incubated 37 C with rocking for 2 hrs., and ice cold ethanol (1.0 ml) was added to each sample to precipitate glucans. The glucans were collected on glass-fiber filters and quantitated as previously described (Schilling and Bowen, 1988). The amount of glucans formed was expressed as umoles of glucose incorporated into glucan. Surface Assays HA beads were coated with ul of ductal parotid saliva (which does not contain Gtf) solution for 30 minutes, 37 C. This amount of saliva was sufficient for saturation of HA surface (unpublished data). The parotid-salivacoated hydroxyapatite (SHA) beads were washed three times with imidazole buffer and exposed to 0.5 Units (200.0 ul final volume) of each Gtf enzyme for 30 minutes, 37 C (this concentration of enzyme and time of incubation were sufficient for saturation of the SHA surface, unpublished data). After the incubation, the beads were washed and exposed to ul of the test agents at concentrations described above for 30 minutes. The beads were washed and exposed to radioactive sucrose (100.0 mmol/l sucrose, final concentration) for 2 hrs., and glucans were formed and quantitated as described above. Desorption Assays We also explored the possibility that some of the agents which inhibited surface-associated Gtf enzymes agents actually desorbed the enzymes from the surface. SHA beads were incubated with either imidazole buffer or 0.5 Units (200.0 ul final volume) of GtfB or GtfD for 30 minutes, 37 C. (GtfC was not tested because none of the agents inhibited surface-associated GtfC). After the incubations, the beads were washed with buffer and were exposed to selected inhibitors for 30 minutes, 37 C. The resulting supernatant fluids (which would contain any enzyme desorbed from the SHA beads) were then removed. Twenty microliters of each supernatant fluid was mixed with (5 of 19) [8/1/ :05:15 AM]

6 equal volumes of SDS-PAGE sample buffer (Laemmli, 1970) and was loaded onto a 7.5% polyacrylamide gel. The samples were separated by electrophoresis (Laemmli, 1970) and were then transferred to an Immobilon-P membrane (Millipore, Bedford, MA) (Towbin et al., 1979). After incubating the blots in blocking buffer (phosphatebuffered saline/0.1% Tween-20 [Sigma]), the blots were probed with an antibody (1:100 dilution in blocking buffer) raised against a mixture of GtfC, GtfB and GtfD adsorbed onto HA beads, followed by exposure to a secondary antibody, goat anti-rabbit IgG- alkaline phosphatase conjugate (1:1000 dilution, blocking buffer). The blots were developed with the BCIP and NBT substrate kit (Gibco BRL/Life Technologies Inc., Gaithersburg, MD) according to the manufacturer's instructions. As a positive control, enzymes were desorbed from the SHA beads coated with the individual enzymes by exposing them to SDS-PAGE sample buffer prior to electrophoresis and western blot analyses. RESULTS Solution Assays The results of the solution assays are shown in Tables 1, 2 and 3. The activity of GtfB in solution was decreased by many of the test agents (Table 1). Cetylpyridinium chloride (CPC), hexylresorcinol, triclosan, alexidine dihydrochloride and Plax(R)E greatly reduced the activity of GtfB in solution (by 75-99%). Plax(R)USA decreased glucan formation by 40+/-18%, MTAB increased glucan-formation by GtfB two-fold and chlorhexidine digluconate was without effect on glucan-formation by GtfB in solution. Scope(R) and Biotene(R) enhanced glucan production by GtfB in solution. The glucan-forming activity of GtfC was unaffected by most of the test agents, with Plax(R)USA and Plax(R)E enhancing activity several-fold (Table 2). Chlorhexidine digluconate was the only test agent able to inhibit the activity of GtfC in solution (64+/-8%; Table 2). Hexylresorcinol, alexidine dihydrochloride and chlorhexidine digluconate decreased the activity of GtfD in solution by 50-85% (Table 3). Plax(R)E, Scope(R), Biotene(R) and CPC were mildly inhibitory on the activity of GtfD (decreasing activity by 25-35%, Table 3). Plax(R)USA and MTAB increased activity of GtfD in solution several-fold (Table 3). Surface Assays The results of the surface assays are shown in Tables 1, 2 and 3. GtfB, when adsorbed onto the surface of SHA, was inhibited up to 35-80% by Plax(R)E, Plax(R)USA, CPC, hexylresorcinol, alexidine dihydrochloride, MTAB and triclosan (Table 1) and was unaffected by Scope and chlorhexidine digluconate (Table 1). Biotene(R) increased the (6 of 19) [8/1/ :05:15 AM]

7 activity of SHA-adsorbed GtfB. Only a few of the test agents decreased glucan formation by surface- bound GtfD (Table 3). CPC, alexidine hydrochloride, MTAB and Plax(R)E decreased the activity of surface-bound GtfD by 50-55% (Table 3). Most interestingly, GtfC was unaffected by any of the agents when adsorbed onto the SHA beads (Table 2). Desorption Assays Some of the test agents reduced the activities of GtfB and GtfD adsorbed onto SHA beads, thereby giving the impression that they were inhibitory. Therefore, we determined whether the agents desorbed the Gtf enzymes from the surface of the beads. The results from western blots indicated that GtfB and GtfD were not desorbed from the SHA beads by the test agents (data not shown). However, the enzymes were desorbed from the surfaces in positive control samples exposed to SDS-PAGE sample buffer (data not shown). DISCUSSION Glucan contributes to the bulk of the extracellular matrix of plaque (Critchley, 1969; Critchley, 1971; Critchley et al., 1967; Critchley et al., 1968) and has been shown to be essential for the expression of virulence by S. mutans (Yamashita et al., 1993). Therefore, it appears that Gtfs should be prime targets for antiplaque agents. Almost all mouthrinses contain surface active agents and have a moderate effect on plaque formation (Hull, 1980; Korman, 1986; Mandel, 1988; Marsh, 1991; Muhlemann, 1974; van der Ouderaa and Cummins, 1989; Schroeder, 1969). Agents incorporated into mouthrinse and dentifrice formulations include: bis-biguanides, quaternary ammonium salts, anionic compounds and enzymes (Mandel, 1988). In this study, we tested the effects of various surface-active agents on the glucan-forming activities of individual, purified Gtf enzymes in solution and adsorbed onto the surface of saliva- coated hydroyxapatite beads. It is evident by the results obtained in this study that some of the test agents behave differently towards Gtf enzymes adsorbed onto SHA beads and in solution. Plaque formation results from a surface related phenomenon, so clearly the action of the test substances on surface might be more relevant than are actions occurring solely in solution. The effects of the quaternary ammonium salts cetylpyridinium chloride and mixed alkyltrimethylammonium bromide on Gtf activities were also tested. CPC is highly effective against glucan synthesis by a mixture of Gtf enzymes in solution (Ciardi et al., 1978; Kawabata et al., 1993). However, these agents have little effect on plaque formation in vivo (Bonesvoll and Gjermo, 1978; Gjermo et al., 1970). It can be inferred from the results obtained (7 of 19) [8/1/ :05:15 AM]

8 with the effects of CPC on GtfD that the conformation of GtfD differs in solution when compared with that on a surface. MTAB increased the activity of GtfD in solution and decreased activity of GtfD on the SHA beads, providing further evidence of a conformational change in the GtfD enzyme upon immobilization onto SHA beads. Available evidence suggests that the dominant Gtf in pellicle (Vacca-Smith et al., 1996) has characteristics of GtfC. The observation that CPC and MTAB reduced the activities of SHA- associated GtfB and GtfD only, may explain, in part, the poor clinical efficacy of these antiplaque agents. CPC may also be a comparatively ineffective antiplaque agent because it reacts with salivary mucin-glycoproteins (Carlson et al., 1973). We also tested the effects of an additional phenol-derivative, triclosan, (2,4,4'-trichloro-2'-hydroxyphenylether) against the activities of the individual Gtf enzymes. Perhaps the inability of triclosan to reduce glucan formation by GtfC in solution and on SHA beads could explain its moderate effects on plaque formation (Jenkins et al., 1989 a,b; Marsh, 1991; Saxton, 1986; Saxton et al., 1987). Plax(R), (European formulation), contains triclosan and sodium fluoride and was inhibitory against GtfB and GtfD in solution and on the surface of SHA beads. Triclosan alone, however, did not inhibit the activity of GtfD on SHA beads. Therefore, inhibition of GtfD on SHA beads by Plax(R)E is probably due to an ingredient other than triclosan. Plax(R), United States formulation, does not contain triclosan or sodium fluoride and was moderately effective in reducing the activity of GtfB in solution and on the SHA surface. Plax(R)USA, however, was virtually ineffective against reducing the activities of GtfC or GtfD in either the free or adsorbed state. Many potential antiplaque agents may be ineffective when incorporated into mouthrinse formulations, because they are incompatible with other ingredients (Garcia-Gody, 1989). We, therefore, tested the effects of some mouthrinses that contained some of our selected agents on glucan-forming activities of the Gtf enzymes in solution and on the SHA surface. Scope(R), which contains CPC, was without effect on reducing glucan-formation by any of the Gtf enzymes. The ineffectiveness of Scope(R) could be due to the inactivation of the CPC once in mouthrinse formulation. Alternatively, the ineffectiveness of Scope(R) could be due to an alteration of the Gtf enzymes by other ingredients of the mouthrinse. The latter observation is quite plausible since Scope(R) doubled the activity of GtfB in solution, suggesting an interaction between components of Scope(R) and GtfB. Biotene(R) does not contain any of the selected test agents of this study but does contain lactoperoxidase, lysozyme, lactoferrin and glucose oxidase. Some of these components are antibacterial factors found in saliva and are believed to be protective in the oral cavity (Mandel, 1987). Biotene(R) did not reduce glucan-formation by the Gtf enzymes. Biotene(R) stimulated the activity of GtfB in solution and on the SHA surface. (8 of 19) [8/1/ :05:15 AM]

9 A surprising observation in this study is that glucan-formation by GtfC adsorbed to a surface was not reduced by any of the antiplaque agents or mouthrinses. Chlorhexidine digluconate reduced its activity in solution only. The bisbiguanide chlorhexidine digluconate is an effective antiplaque agent (Emilson, 1981,1994; Kidd. 1991). Chlorhexidine has been shown to reduce glucan formation by a mixture of Gtfs from S. mutans adsorbed onto bare HA beads (Scheie and Kjeilen, 1987). The Gtfs of human whole saliva adsorbed onto HA beads have properties of GtfC (Vacca-Smith et al., 1996). The clinical effectiveness of chlorhexidine could be due to interaction of the antiplaque agent with GtfC prior to adsorption of the enzyme onto tooth and apatitic surfaces, thus inhibiting the enzyme before adsorption onto a tooth surface. This suggestion could be consistent with the staining frequency observed on tooth surfaces of persons who use chlorhexidine (L e and Schiott, 1970) (ie, chlorhexidine interacts with GtfC in solution and is bound onto the tooth surface with GtfC). Alexidine dihydrochloride, another bis-biguanide that has been shown to reduce plaque formation and reduce gingivitis in human trials (Lobene and Soparker, 1973), was highly effective against the activity of GtfB in solution and on the SHA surface. These results are consistent with our earlier results (Ciardi et al., 1978). The test agents used in these and other studies interact primarily with hydrophobic groups of the Gtf enzymes (Ciardi et al., 1978). The three Gtf enzymes used in this study have the same basic structure and contain hydrophobic domains (Russell, 1994): a signal peptide at the amino terminus similar to those of other secreted proteins, followed by a highly variable domain, distinctive for each Gtf enzyme. The C-terminus contains a series of repeat motifs of amino acids, and the patterns of the motifs vary from Gtf to Gtf enzyme. GtfB has 1475 amino acids and is highly hydrophilic (Shiroza et al., 1987). The N-terminal signal sequence of GtfB has two hydrophobic regions. The structure of GtfD is similar to that of GtfB with 1430 amino acids and is highly hydrophilic (Honda et al., 1990). There are also hydrophobic regions at the signal sequence of GtfD. GtfC is also hydrophilic and has hydrophobic domains at the signal sequence (Ueda et al., 1988). However, GtfC also has small hydrophobic domains in the direct repeat units at the C-terminus (Ueda et al., 1988). These hydrophobic regions may confer a conformation on GtfC that is different from the conformations of GtfB and GtfD. The hydrophobic groups of GtfC may not be exposed on the surface of SHA beads and may explain why the test inhibitors are ineffective against GtfC activity. The studies presented here demonstrated the effects of various agents on glucan formation by Gtf enzymes in solution and in the adsorbed state. Inhibition on the surface is not simply due to desorption of the enzymes from the surface because our results show that the test agents did not desorb the enzymes from the SHA beads. Further (9 of 19) [8/1/ :05:15 AM]

10 studies will be performed to identify an inhibitor of GtfC both in solution and surface assays. Evidence from our laboratory shows that GtfB binds avidly to streptococcal surfaces (unpublished data). Therefore, future studies will include investigating the effects of the test agents on glucan-forming activity of bacterial-bound GtfB, and on sucrose-mediated colonization of oral streptococci. Knowledge of this information is important, for formulations could be prepared which contain agents that inhibit activities of all of the Gtf enzymes in solution, on saliva-coated hydroxyapatite surfaces, and on bacterial surfaces. These formulations could represent a major advance in control of plaque formation. ACKNOWLEDGEMENTS This study was supported, in part, by US PHS Grants R37 DEO7907, T32 DEO7165 and P50 DEO7003. REFERENCES Aoki, H., Shiroza, T., Hayakawa, M., Sato, S. and Kuramitsu, H.K Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. Infect. Immun. 53: Bonesvoll, P. and Gjermo, P A comparison between chlorhexidine and some quaternary ammonium compounds with regard to retention, salivary concentrations and plaque-inhibiting effect in the human oral cavity after mouthrinses. Archs. Oral Biol. 23: Carlson, D.M., McGuire, E.M., Jourian, G.W. and Roseman, S The Sialic Acids. XVI. Isolation of a mucin sialyltransferase from sheep submaxillary gland. J. Biol Chem. 248: Ciardi, J.E., Bowen, W.H. and Rolla, G The effect of antibacterial compounds on glucosyltransferase activity from Streptococcus mutans. Archs. Oral Biol. 23: Critchley, P The breakdown of the carbohydrate and protein matrix of dental plaque. Caries Res. 3: Critchley, P The microbiology of dental plaque with special reference to polysaccharide formation. Dtsh Zah Zeitschrift 26: (10 of 19) [8/1/ :05:15 AM]

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13 Laemmli, U.K Cleavage of structural proteins during the assembly of the head of Bacteriophage T4. Nature. 227:680. Lashley, K.S Reflex secretion of the parotid gland. J Exp Psychol 1: Lobene, R.R. and Soparker, P.M The effect of an alexidine mouthwash on human plaque and gingivitis. J. Am. Dent. Ass. 87: L e, H. and Schiott, C.R The effect of mouthrinses and topical application of chlorhexidine on the development of dental plaque and gingivitis in man. J. Periodont Res. 5: Loesche, W.J Role of Streptococcus mutans in human dental decay. Microbiol Rev. 50: Mandel, I The functions of saliva. J. Dent Res. 66: (Special Issue). Mandel, I.D Chemotherapeutic agents for controlling plaque and gingivitis. J. Clin. Periodontol. 5: Marsh, P.P Zinc citrate and triclosan in the prevention of plaque and gingivitis. J. Clin Periodontol. 18: McCabe, R.M. and Donkersloot, J.N Adherence of Veillonella sp. mediated by extracellular glucosyltransferase from S. salivarius. Infect. Immun. 18: Muhlemann, H.R Inhibition of dental calculus in the rat by biguanides and ethanolhydroxydiphosphate. Helv. Odont. Acta. 18:17-18 (Supplement 8.) Mukasa, H., Tsumori, H. and Shimamura, A Isolation and characterization of an extracellular glucosyltransferase synthesizing insoluble glucan form Streptococcus mutans serotype c. Infect. Immun. 49: van der Ouderaa, F.J.G. and Cummins, D Delivery system for agents in supra and subgingival plaque control. (13 of 19) [8/1/ :05:15 AM]

14 J. Dent. Res. 68: Rolla, G., Ciardi, J.E. and Bowen, W.H Identification of IgA, IgG, lysozyme, albumin, amylase and glucosyltransferase in the protein layer adsorbed to hydroxyapatite from whole saliva. Scand. J. Dent. Res. 91: Russell, R.R.B The application of molecular genetics to the microbiology of dental caries. Caries Res. 28: Saxton, C.A The effects of a dentifrice containing zinc citrate and 2,4,4 trichloro-2'-dihydroxydiphenyl ether. J. Periodontol. 57: Saxton, C.A., Lane, R.M and van der Ouderaa, F.J.G The effects of a dentifrice containing a zinc salt and noncationic antimicrobial agent on plaque and gingivitis. J. Clin. Periodontol. 14: Scheie, A.A., Eggen, K.H. and R lla, G Glucosyltransferase activity in human in vivo formed enamel pellicle and in whole saliva. Scand. J. Dent. Res. 95: Scheie, A.A. and Kjeilen, J.C The effects of chlorhexidine, NaF and SnF2 on glucan formation by salivary and culture supernatant GTF adsorbed to hydroxyapatite. Scand. J. Dent. Res. 95: Schilling, K.M., Blitzer, M.H. and Bowen, W.H Adherence of Streptococcus mutans to glucans formed in situ in salivary pellicle. J. Dent. Res. 68: (Special Issue.) Schilling, K.M. and Bowen, W.H The activity of glucosyltransferases adsorbed onto saliva-coated hydroxyapatite. J. Dent. Res. 67:2-8. Schilling, K.M. and Bowen, W.H Glucan synthesized in situ in experimental pellicle functions as specific binding sites for Streptococcus mutans. Infect. Immun. 60: Schroeder, H.E. (1969). Formation and inhibition of dental calculus. Beine, HansHuber. (14 of 19) [8/1/ :05:15 AM]

15 Shiroza, T., Ueda, S. and Kuramitsu, H.K Sequence analysis of the gtfb gene from Streptococcus mutans. J. Bacteriol. 169: Thaniyavarn, S., Singh, S., Maynard, C.M., Taylor, K.G. and Doyle, R.J Amino sugars: a new class of inhibitors of dextran sucrase. Carbohydr. Res. 96: Towbin, H.T., Stoehelin, T. and Gordon, J Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedures and some applications. PNAS., USA. 76: Ueda, S., Shiroza, T. and Kuramitsu, H.K Sequence analysis of the gtfc gene from Streptococcus mutans GS- 5. Gene. 69: Vacca-Smith, A.M., Venkitaraman, A.R., Schilling, K.M. and Bowen, W.H. Characterization of glucosyltransferase of human saliva adsorbed onto hydroxyapatite surfaces. Caries Res., in press. Venkitaraman, A.R., Vacca-Smith, A.M., Kopec, L.K. and Bowen, W.H Characterization of glucosyltransferaseb, gtfc, and gtfd in solution and on the surface of hydroxyapatite. J. Dent. Res. 74: Wood, J.M. and Critchley, P The extracellular polysaccharide produced from sucrose by cariogenic streptococcus. Archs. Oral Biol. 11: Yamashita, Y., Bowen, W.H., Burne, R.A. and Kuramitsu, H.K Role of the Streptococcus mutans gtf genes in caries induction on the specific-pathogen-free rat model. Infect. Immun. 61: Table 1. Effect of Selected Agents on the Activity of GtfB^a Activity (% of Control)^b Agent Solution Surface (15 of 19) [8/1/ :05:15 AM]

16 CPC (1.25mmol/) 15.0(4.00) 25.0(4.00) Hexylresorcinol (1.25mmol/) 7.00(1.00) 30.0(7.00) Alexidine (1.25mmol/) 26.0(7.00) 28.0(3.00) Chlorhexide (1.25mmol/) 100.0(0.00) 100.0(0.00) Triclosan (1.25mmol/) 1.00(1.00) 48.0(11.0) MTAB (1.25%) 2-fold inc.^c 19.0(4.00) Plax(R)USA 61.0(18.0) 66.0(11.0) Plax(R)E 17.0(1.00) 45.0(10.0) Scope(R) 2-fold inc.^c 100.0(0.00) Biotene(R) 2-fold inc.^c 1.5-fold inc.^c ^a The data shown are the mean of the three samples from one experiment. Standard deviations are indicated in parentheses. ^b Results are expressed as activity, % of control. Control=100% activity. ^c Enhanced activity. Table 2. Effect of Selected Agents on the Activity of GtfC^a Activity (% of Control)^b (16 of 19) [8/1/ :05:15 AM]

17 Agent Solution Surface CPC (1.25mmol/) 100.0(0.00) 100.0(0.00) Hexylresorl (1.25mmol/) 100.0(0.00) 100.0(0.00) Alexidine (1.25mmol/) 100.0(0.00) 100.0(0.00) Chlorhexide (1.25mmol/) 36.0(8.00) 100.0(0.00) Triclosan (1.25mmol/) 100.0(0.00) 100.0(0.00) MTAB (1.25%) 100.0(0.00) 100.0(0.00) Plax(R)USA 3-fold inc.^c 100.0(0.00) Plax(R)E 5-fold inc.^c 100.0(0.00) Scope(R) 100.0(0.00) 100.0(0.00) Biotene(R) 100.0(0.00) 100.0(0.00) ^a The data shown are the mean of the three samples from one experiment. Standard deviations are indicated in parentheses. ^b Results are expressed as activity, % of control. Control=100% activity. ^c Enhanced activity. (17 of 19) [8/1/ :05:15 AM]

18 Table 3. of GtfD^a Effect of Selected Agents on the Activity Activity (% of Control)^b Agent Solution Surface CPC (1.25mmol/) 75.0(17.0) 47.0(3.00) Hexylresorl (1.25mmol/) 36.0(0.50) 75.0(14.0) Alexidine (1.25mmol/) 50.0(6.00) 49.0(13.0) Chlorhexide (1.25mmol/) 15.0(5.00) 100.0(0.00) Triclosan (1.25mmol/) 76.0(7.00) 100.0(0.00) MTAB (1.25%) 4-fold inc.^c 45.0(7.00) Plax(R)USA 3-fold inc.^c 85.0(9.00) Plax(R)E 68.0(6.00) 45.0(6.00) Scope(R) 71.0(7.00) 91.0(6.00) Biotene(R) 66.0(2.00) 100.0(0.00) ^a The data shown are the mean of the three samples from one experiment. Standard deviations are indicated in parentheses. (18 of 19) [8/1/ :05:15 AM]

19 ^b Results are expressed as activity, % of control. Control=100% activity ^c Enhanced activity. Copyright held by the authors Published by Bioline Publications Editorial Office: biofilm@biostrat.demon.co.uk The following images related to this document are available: Videos [bf96002.mov] There is no charge for this document. FREE PAPERS SUPPLIERS FORUM BULLETIN BOARD NEWS BIOLINE MAIL HOME (19 of 19) [8/1/ :05:15 AM]