Effects of missense mutations in sortase A gene on enzyme activity in Streptococcus mutans.

BACKGROUND: Streptococcus mutans (S. mutans) is the major aetiological agent of dental caries, and the transpeptidase Sortase A (SrtA) plays a major role in cariogenicity. The T168G and G470A missense mutations in the srtA gene may be linked to caries susceptibility, as demonstrated in our previous studies. This study aimed to investigate the effects of these missense mutations of the srtA gene on SrtA enzyme activity in S. mutans. METHODS: The point mutated recombinant S.mutans T168G and G470A sortases were expressed in expression plasmid pET32a. S. mutans UA159 sortase coding gene srtA was used as the template for point mutation. Enzymatic activity was assessed by quantifying increases in the fluorescence intensity generated when a substrate Dabcyl-QALPNTGEE-Edans was cleaved by SrtA. The kinetic constants were calculated based on the curve fit for the Michaelis-Menten equation. RESULTS: SrtA△N40(UA159) and the mutant enzymes, SrtA△N40(D56E) and SrtA△N40(R157H), were expressed and purified. A kinetic analysis showed that the affinity of SrtA△N40(D56E) and SrtA△N40(R157H) remained approximately equal to the affinity of SrtA△N40(UA159), as determined by the Michaelis constant (K m ). However, the catalytic rate constant (k cat ) and catalytic efficiency (k cat /K m ) of SrtA△N40(D56E) were reduced compared with those of SrtA△N40(R157H) and SrtA△N40(UA159), whereas the k cat and k cat /K m values of SrtA△N40(R157H) were slightly lower than those of SrtA△N40(UA159). CONCLUSIONS: The findings of this study indicate that the T168G missense mutation of the srtA gene results in a significant reduction in enzymatic activity compared with S. mutans UA159, suggesting that the T168G missense mutation of the srtA gene may be related to low cariogenicity.

Impact of early childhood caries on oral health-related quality of life of preschool children.

AIM: Child oral health-related quality of life (COHRQoL) has been assessed in developed areas; however, it remains unstudied in mainland China. Studies on COHRQoL would benefit a large number of children in China suffering from oral health problems such as dental caries. This study explored the relationship between COHRQoL and early childhood caries, adjusted by socioeconomic factors, in 3- to 4-year-old children in a region of southern China. MATERIALS AND METHODS: In this study, 1062 children aged 3-4 years were recruited by cluster sampling and their oral health statuses were examined by a trained dentist. The Chinese version of the Early Childhood Oral Health Impact Scale (ECOHIS) and questions about the children's socioeconomic conditions were completed by the children's parents. A negative binomial regression analysis was used to assess the prevalence of early childhood caries among the children and its influence on COHRQoL. RESULTS: The total ECOHIS scores of the returned scale sets ranged from 0 to 31, and their average scores was 3.1±5.1. The negative binomial analysis showed that the dmfs indices were significantly associated with the ECOHIS score and subscale scores (P<0.05). The multivariate adjusted model showed that a higher dmft index was associated with greater negative impact on COHRQoL (RR = 1.10; 95% CI = 1.07, 1.13; P < 0.05). However, demographic and socioeconomic factors were not associated with COHRQoL (P>0.05). CONCLUSION: The severity of early childhood caries has a negative impact on the oral health-related quality of life of preschool children and their parents.

[Complete revascularization of coronary artery].

25 cases of complete revascularization for coronary artery disease by sequential aorto-coronary saphenous bypass operation were performed from April, 1990 to July, 1993. All the patients suffered from three vessels diseases. Left ventricular ejection fraction ranged from 22-60%. Left ventricular end diastolic pressure ranged from 1.3-4.0 kPa. They received grafts from 2-5, 3.6 grafts for a patient in an average. There were no perioperative myocardial infarction and operative death. And they recovered smoothly postoperatively. Angina has been remarkably improved. The paper briefly discussed the technique of this type of the operation and the advantages of the sequential graft bypass operation.

An in vitro study of silver and fluoride ions on remineralization of demineralized enamel and dentine.

BACKGROUND: The purpose of this study was to compare the effect of silver fluoride, silver nitrate and potassium fluoride on remineralization of demineralized enamel and dentine in vitro. METHODS: Forty premolars were cut into cuboidal blocks. Acid-resistant varnish was painted onto each block to cover all surfaces, except two windows, one in enamel and one in dentine. The tooth blocks were placed in demineralizing solution for 96 hours. They were then randomly divided into four groups of 10 blocks each and immersed in solutions of AgF, AgNO(3), KF or water for 3 minutes. Afterwards, they were immersed in a remineralizing solution for 108 hours. Micro CT scanning was conducted before and after remineralization. RESULTS: The increase in linear attentuation coefficient (LAC) for the enamel lesions after remineralization was 1.08/cm, 0.95/cm, 0.86/cm and 0.60/cm in the AgF, AgNO(3), KF and control groups, respectively (ANOVA, p < 0.001; AgF, AgNO(3), KF > control; AgF > KF). The increase in LAC for the dentine lesions was 1.01/cm, 0.92/cm, 0.88/cm and 0.53/cm, respectively (ANOVA, p < 0.001; AgF, AgNO(3), KF > control). CONCLUSIONS: Topical application of silver or fluoride ions can increase the mineral density of demineralized enamel and dentine lesions during remineralization. The synergistic effect of silver and fluoride ions is relatively small.

Factors associated with colonization of Streptococcus mutans in 8- to 32-month-old children: a cohort study.

BACKGROUND: The factors involved in Streptococcus mutans colonization in young children are not clear. The aim of this study was to determine the factors associated with S. mutans colonization in 8- to 32-month-old children. METHODS: A group of 225 caries-free 8-month-old children was recruited for the study. They were examined every six months until they were 32 months old to investigate their environmental factors, host factors and bacterial transmission factors. At baseline and during each examination, their teeth were checked for the presence of dental plaque and developmental defects of enamel, and S. mutans plaque status was assessed using a real-time PCR test. RESULTS: Eight children (3.6%) showed S. mutans colonization by the age of 8 months. The percentages of colonization were 6.0% at 14 months, 16.2% at 20 months, 26.7% at 26 months, and 33.5% at 32 months. The results showed that females (p = 0.006), children with enamel hypoplasia (p = 0.024), children with low birth weights (p = 0.005), those who consume more sweets (p < 0.001), and those with a higher proportion of visible plaque (p = 0.020 and p = 0.041) were more likely to be colonized by S. mutans. CONCLUSIONS: Streptococcus mutans colonization in young children was associated with gender, tooth enamel hypoplasia, low birth weight, frequent consumption of sweets and poor oral hygiene.

Genetic polymorphisms of the sortase A gene and early childhood caries in two-year-old children.

OBJECTIVE: To explore and compare the genetic polymorphisms of the sortase A (srtA) gene found in Streptococcus mutans (S. mutans) infecting two-year-old children suffering early childhood caries to those found in caries-free children through molecular identification methods. METHODS: Clinical S. mutans strains were isolated from the dental plaques of two-year-old children. Fifteen strains of S. mutans from the caries-active group and 15 strains of S. mutans from the caries-free group were collected. Genomic DNA was extracted from the S. mutans isolates. DNA fragments, including the srtA gene, were amplified by PCR. The PCR products were purified, sequenced and analyzed. A chi-square test and BioEdit software were used to analyze the sequencing results. RESULTS: All 30 clinically isolated S. mutans strains had a 741 base pair (bp) srtA gene. There were no nucleotide sequence insertions or deletions observed in the srtA genes. Twenty mutations were identified in the srtA genes that taken from the 30 clinical strains. There were 10 silent point mutations at the 78, 99, 150, 165, 186, 222, 249, 261, 312, and 636bp positions. The other 10 mutations were point mutations resulting in a missense mutation at the 23, 34, 36, 47, 112, 114, 168, 176, 470, and 671bp positions. None of the positions were enzyme-activity sites of srt A. The missense mutation rates of the two groups did not exhibit statistically significant differences. CONCLUSION: There were no genetic polymorphisms of the sortase A gene associated with early childhood caries in two-year-old children.

Comparing two quantitative methods for studying remineralization of artificial caries.

OBJECTIVES: To compare the detection of changes before and after remineralization of artificial enamel and dentin caries by microCT scanning, polarized light microscopy (PLM) and transverse microradiography (TMR). METHODS: Fourteen extracted premolars were cut into tooth blocks and painted with an acid-resistant varnish leaving one enamel and one dentin surface exposed. The tooth blocks were immersed into demineralizing solution for 4 days to produce artificial caries-like lesions and scanned by microCT. Then the 14 tooth blocks were randomly allocated into two groups. Seven tooth blocks in Group I were cut longitudinally through the exposed surface into 100-150 microm thick sections and microradiographs were taken. The other seven tooth blocks in Group II were left intact. All the tooth blocks and sections were then immersed into remineralizing solution for 5 days. PLM and TMR of the tooth sections in Group I were taken again. Depth of the lesion on the TMR was measured. Tooth blocks in Group II were scanned by microCT. RESULTS: Mean lesion depth in Group I reduced by 13.0% and 8.2% after remineralization for enamel and dentin, respectively (paired t-test, P<0.001). In Group II, linear attenuation coefficient (LAC) of the region of interest (ROI) increased by 11.1% and 23.8% after remineralization for enamel and dentin lesions, respectively (paired t-test, P<0.001). CONCLUSION: Both microCT and microradiography are able to detect a change of similar magnitude in the artificial caries lesions after remineralization. MicroCT may be used to substitute TMR and PLM in in vitro studies about caries.