New assay for enzymatic phosphate release: Application to aspartate transcarbamylase and other enzymes

BackgroundThe association of specific bioactive flavonoids and terpenoids with fluoride can modulate the development of cariogenic biofilms by simultaneously affecting the synthesis of exopolysaccharides (EPS) and acid production by Streptococcus mutans, which enhanced the cariostatic effectiveness of fluoride in vivo. In the present study, we further investigated whether the biological actions of combinations of myricetin (flavonoid), tt-farnesol (terpenoid) and fluoride can influence the expression of specific genes of S. mutans within biofilms and their structural organization using real-time PCR and confocal fluorescence microscopy.ResultsTwice-daily treatment (one-minute exposure) during biofilm formation affected the gene expression by S. mutans both at early (49-h) and later (97-h) stages of biofilm development. Biofilms treated with combination of agents displayed lower mRNA levels for gtfB and gtfD (associated with exopolysaccharides synthesis) and aguD (associated with S. mutans acid tolerance) than those treated with vehicle-control (p < 0.05). Furthermore, treatment with combination of agents markedly affected the structure-architecture of S. mutans biofilms by reducing the biovolume (biomass) and proportions of both EPS and bacterial cells across the biofilm depth, especially in the middle and outer layers (vs. vehicle-control, p < 0.05). The biofilms treated with combination of agents were also less acidogenic, and had reduced amounts of extracellular insoluble glucans and intracellular polysaccharides than vehicle-treated biofilms (p < 0.05).ConclusionThe data show that the combination of naturally-occurring agents with fluoride effectively disrupted the expression of specific virulence genes, structural organization and accumulation of S. mutans biofilms, which may explain the enhanced cariostatic effect of our chemotherapeutic approach.

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“…The released phosphate (over the 10-min reaction time) was determined by the method of Bencini et al . [30]. …”

Section: Methodsmentioning

confidence: 99%

Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

et al. 2009

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“…The F-ATPase activity was measured in terms of the release of phosphate in the following reaction mixture: 75.0 m M of Tris-maleate buffer (pH 7.0) containing 5.0 m M ATP, 10.0 m M MgCl 2 , permeabilized biofilm cells, and each concentration of fluoride. The amount of phosphate released (30 min reaction time) was determined using the method reported by Bencini et al [1983]. In the F-ATPase activity assay, 1,000 and 2,000 ppm F -were not tested since false-positive response was found at the concentrations (data not shown).…”

Section: Biochemical Analysismentioning

confidence: 99%

Relationship between Fluoride Concentration and Activity against Virulence Factors and Viability of a Cariogenic Biofilm: in vitro Study

et al. 2013

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Despite widespread use of various concentrations of fluoride for the prevention of dental caries, the relationship between fluoride concentration and activity against cariogenic biofilms has not been much studied. Herein we investigated the relationship between fluoride concentration and activity against virulence factors and viability of Streptococcus mutans biofilms. S. mutans biofilms were formed on saliva-coated hydroxyapatite discs. The 70-hour-old biofilms were exposed to 0, 1, 3, 10, 30, 100, 300, 1,000 or 2,000 ppm F^-. The changes of virulence factors and viability of the biofilms were analyzed using biochemical methods and laser scanning confocal fluorescence microscopy. At 1-2,000 ppm F^-, the activity of fluoride against acid production, acid tolerance, and extracellular polysaccharide formation of S. mutans biofilms accurately followed a sigmoidal pattern of concentration dependence (R² = 0.94-0.99), with EC₅₀ values ranging from 3.07 to 24.7 ppm F^-. Generally, the activity of fluoride against the virulence factors was concentration-dependently augmented in 10-100 ppm F^- and did not increase further at concentrations higher than 100 ppm F^-. However, fluoride did not alter glucosyltransferase activity and viability of S. mutans biofilm cells in all concentrations tested. These results can provide a basis for the selection of appropriate fluoride concentrations that reduce the physiological ability of cariogenic biofilms.

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“…The amount of phosphate released over the 30-min reaction time was determined using a previous method (Bencini, Shanley, Wild, & O'Donovan, 1983).…”

Section: Effects On Acidogenicity and Aciduricity Of S Mutans Biofilmsmentioning

confidence: 99%

Separation of an effective fraction from turmeric against Streptococcus mutans biofilms by the comparison of curcuminoid content and anti-acidogenic activity

Pandit

Kim

et al. 2011

Food Chemistry

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New assay for enzymatic phosphate release: Application to aspartate transcarbamylase and other enzymes

Cited by 40 publications

References 20 publications

Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

Influences of naturally occurring agents in combination with fluoride on gene expression and structural organization of Streptococcus mutans in biofilms

Relationship between Fluoride Concentration and Activity against Virulence Factors and Viability of a Cariogenic Biofilm: in vitro Study

Separation of an effective fraction from turmeric against Streptococcus mutans biofilms by the comparison of curcuminoid content and anti-acidogenic activity

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