Interspecies dynamics among bacteria associated with canine periodontal disease.

The etiology and pathogenic mechanisms associated with canine periodontal disease are less well understood than the disease in humans. In this study we have reconstructed defined consortia biofilms in vitro of microorganisms identified as prevalent in a same-breed cohort of dogs with or without periodontal disease. Frederiksenia canicola and Neisseria canis were selected as potential early colonizers of salivary pellicle, and Fusobacterium nucleatum and Porphyromonas gulae were included as high incidence canine oral bacteria. N. canis formed a biofilm substratum under aerobic conditions, but was unable to tolerate anaerobic conditions. Fr. canicola exhibited synergistic biofilm growth with Po. gulae under anaerobic conditions, but displayed an antagonistic relationship with Fu. nucleatum. However, strong co-adhesion between Fu. nucleatum and Po. gulae was able to overcome the inhibitory effects of Fr. canicola to facilitate three-species biofilm formation. Parvimonas micra, an anaerobic, asaccharolytic Gram-positive coccus found only under disease conditions in vivo, was able to form biofilms in conjunction with Fr. canicola and Po. gulae. Furthermore, the specific proteolytic activities of biofilms containing Fr. canicola and Po. gulae or Fu. nucleatum and Po. gulae were increased several-fold upon the addition of Pa. micra. This suggests that anaerobic cocci such as Pa. micra might provide a catalyst for progressive tissue destruction, inflammation and alveolar bone loss in canine periodontal disease, in keeping with the keystone-pathogen hypothesis.

HMSH2 gene alterations associated with recurrence of oral squamous cell carcinoma.

Oral cancer, one of the ten most widespread cancers in Thailand, is a major public health problem. The aim of the study was to assess hMSH2 and hMLH1 gene mutations, microsatellite DNA alterations, and investigate the association between these alterations and clinicopathological features of oral squamous cell carcinomas (SCC) in a sample of Thai patients. Microsatellite alterations at D2S391, D3S647, D17S513, and D17S520 were detected at a frequency of 40.6%. Among these alterations, 12.5% exhibited loss of heterozygosity (LOH) at D3S647 and D17S513, while 34.4% exhibited microsatellite instability (MI) at D2S391, D17S513, and D17S520. Polymorphic change in the intronic region of hMSH2 at IVS 1 nt 211+9, c-->g was observed in 50% of cases. Significant correlation was observed between IVS 1 nt 211+9 polymorphism and the recurrence status of the patients (p = 0.030, OR = 10.67). This study demonstrated that the polymorphism of hMSH2 at IVS 1 nt 211+9 (c-->g) was associated with oral cancer recurrence status and could be used as a biomarker for prognosis and follow-up treatment of oral cancer.

The effect of different milk formulas on dental plaque pH.

OBJECTIVES: The purposes of this study were (1) to investigate the effect of different milk formulas on dental plaque pH after rinsing with these three categories, type of protein-based formulas (milk-based, soy-based, protein hydrolysate), type of sugar (only lactose, lactose and other sugars, only non-milk extrinsic sugars), and casein ratio (high and low casein), and (2) to observe organic acids formed by different milk formulas. METHODS: Baseline plaque pH and plaque pH at 2, 5, 10, 15, 20, 25, 30, and 60 min after rinsing with milk formulas were recorded by a combination electrode in 14 healthy subjects. Deionized water and 10% sucrose were used as a negative and positive control. The plaque sample was also analysed to identify and quantify the organic acids using a high-performance liquid chromatography. Parameters including minimum pH, maximum pH drop, and area under curve were compared by RMANOVA and paired t-test. RESULTS: The minimum pH was not significantly different among different protein-based formulas, whereas, the maximum plaque pH drop of soy-based and milk-based formula was significantly higher than that produced by protein hydrolysate formula (P=0.022 and 0.03, respectively). Area under curve produced by soy-based and milk-based formulas was significantly larger than that created by protein hydrolysate formula (P=0.025 and<0.001, respectively). Milk formulas containing only lactose caused significantly less plaque pH change in minimum pH (P<0.001), maximum pH drop (P=0.003), and area under curve (P<0.001) when compared with formulas containing lactose and other sugar but not with special formulas containing only non-milk extrinsic sugar. Similarly, special formulas containing non-milk extrinsic sugar produced significantly lower minimum pH and smaller area under curve than formulas containing lactose and other sugar did (P=0.044 and 0.009, respectively). No different results were found between high and low casein follow-on formulas. Lactic acid was produced more by rinsing with formulas containing lactose and other sugars than that produced by formulas containing only lactose. CONCLUSIONS: This study suggests that milk formulas containing added other sugars tend to cause a decrease in plaque pH.

Partial biochemical characterisation of collagen in carious dentin of human primary teeth.

The process of dentin caries is a complex event involving demineralisation and matrix degradation. The objective of this study was to biochemically characterise collagen, the major dentin matrix component, by amino acid and cross-link analyses in two distinct carious dentin regions of human primary teeth. Twenty-seven carious primary teeth were obtained from 3 to 11-year-old patients and three layers of dentin, i.e. outer, inner carious dentin and normal dentin, were identified by a 1% acid red solution and dissected from each tooth. The samples were pulverised, the respective layers obtained from three to five teeth were pooled and six samples per layer were analysed. Aliquots of the dried dentin powder were hydrolysed with 6N HCl at 110 degrees C for 24h and subjected to amino acid analyses. Other aliquots were demineralised, reduced with standardised NaB3H4, hydrolysed and subjected to quantitative collagen cross-link analyses. The results demonstrated that in the outer carious layer the collagen-associated amino acids were significantly lower and the reducible cross-links were markedly diminished when compared to the other two groups. There was no significant difference in these parameters between the inner carious and the normal layers. The data indicate that while the collagen in the outer carious dentin is significantly altered and degraded, the one in the inner carious dentin is relatively unaffected in primary teeth.

Fluoride in water consumed by children in remote areas of Thailand.

The objective of this study was to analyze fluoride content in water for drinking and for use in remote areas of Thailand. Water was sampled from schools and villages along the border by Multiple Stratified Cluster Random Sampling. Fluoride levels of 214 water samples from 48 schools and 48 villages were assessed in triplicate by fluoride ion electrode. The fluoride content in different regions and types of water were statistically analyzed by Kruskal-Wallis test at a significance level of 0.05. Results showed that fluoride in drinking water and water for use from the schools and villages were 0.01-0.37 ppm, 0.01-0.19 ppm, 0.01-0.87 ppm and 0.01-0.92 ppm, respectively. There was no difference in fluoride content in drinking water from various regions (p=0.23). However, there was a statistical difference in fluoride level in water for use (p=0.04, p=0.01) in various regions. The highest fluoride content was found in samples from the central and eastern region (0.19+/-0.24 ppm and 0.29+/-0.28 ppm respectively). When comparing types of water, ie ground water, surface water and rain water, there were differences in fluoride content (p=0.0). Underground water had the highest fluoride content (0.31+/-0.23 ppm).

Dietary fluoride intake of children aged 3-7 years in remote areas of Thailand.

The objectives of this study were to measure dietary fluoride intake in children aged 3-7 years, to correlate dietary fluoride and fluoride content in water for use in schools and to estimate fluoride gained from the daily diet. Fifty food samples were collected in 45 schools under the jurisdiction of the Border Patrol Police Department. The schools were sampled by multiple stratified cluster random sampling. The food samples were weighed, then measured for fluoride content by a microdiffusion method. Statistical analysis was conducted using the Kruskal-Wallis test. Dietary fluoride in each age group was compared by Student's t test. Analysis for the relationship between dietary fluoride and fluoride content in water was done using Kendall's tau-b. Our results showed that the mean of dietary fluoride in lunch was 0.08 +/- 0.1 ppm. There were no differences when comparing dietary fluoride between different regions of Thailand (p = 0.07). No correlation was found between dietary fluoride and fluoride content in water used in different schools (r(tau) = 0.017, p = 0.85). The daily dietary fluoride intake in children aged 3-6 years was 0.002-0.004 mgF/kg bw/day, in children aged 7 years was 0.003-0.004 mgF/kg bw/day in boys and 0.002-0.004 mgF/kg bw/day in girls.

Fluoride analysis of human milk in remote areas of Thailand.

The objective of this study was to measure the fluoride content in human milk collected from mothers living in remote areas of Thailand and to correlate it with fluoride concentrations in drinking water and water for domestic use. Four to five ml of breast milk were sampled from mothers living in villages where schools under the jurisdiction of the Department of Border Patrol Police were located. The schools were sampled by Multiple Stratified Cluster Random Sampling. Fluoride was determined by microdiffusion method. Statistical analysis were made by ANOVA and LSD test. Correlation between fluoride content in milk and water was assessed by Kendall's tau-b. The mean fluoride concentration in breast milk was 0.017+/-0.02 ppm. There was no difference in breast milk fluoride concentration between regions (p=0.6). No correlation was found between breast milk fluoride content and fluoride concentrations in either drinking water or water for domestic use (r(tau) = -0.09, p = 0.32, r(tau) = -0.04, p = 0.65 respectively).

Interleukin-1beta level in gingival crevicular fluid of patients with active periodontitis.

Previous studies revealed that interleukin-1beta (IL-1beta) was detectable in gingival crevicular fluid (GCF) of patients with periodontitis, and the level was increased in level in gingival tissue extracts of active periodontal disease sites (defined as attachment loss > or = 2.5 mm over the preceding 2 months) compared to inactive sites or healthy sites. The present study evaluated the relationship of IL-1beta level in GCF and periodontal disease status. GCF was collected with Periopaper strips from 34 disease-active and 45 disease-inactive teeth in 11 untreated periodontitis patients and from 60 teeth in 15 healthy control subjects. Disease activity was defined as attachment loss of > or = 2.5 mm in at least one site of a tooth as determined by sequential probing. The absorbed GCF volume was determined using a Periotron 6000 and the crevicular IL-1beta level was determined using IL-1beta monoclonal antibody (Otsuka Pharmaceutical, Japan). IL-1beta was below the detection level of the assay (6 pg/ml) in the healthy control group but was detected in most teeth of the periodontitis group. However, disease-active teeth had higher IL-1beta level (Mann-Whitney U-test, p < 0.05) than disease-inactive teeth (mean total IL-1beta of 5.89 +/- 7.88 pg/tooth and 1.72 +/- 2.28 pg/tooth; mean concentration of 1.6 +/- 2.5 ng/ml and 0.6 +/- 0.83 ng/ml, respectively). The level of IL-1beta showed no correlation with probing depth, but had significant correlation (p < 0.05) with the extent of attachment loss. This study suggests that the level of IL-1beta in GCF may have a predictive value for determining active and inactive periodontal status.

Plasma amino acid analyses in two cases of maple syrup urine disease.

Maple syrup urine disease is a rare inborn error of metabolism, characterized by elevated plasma levels of branched chain amino acids and urinary excretion of branched chain keto acids. Plasma amino acid levels in two subjects were followed by deproteinizing plasma, derivatizing the free amino acids with phenylisothiocyanate, and analysis by HPLC. The results indicate that valine, leucine and isoleucine are elevated in Maple syrup urine disease, and that leucine remains high even after dietary treatment.

Isolation and characterization of an enzyme with beta-glucosidase and beta-fucosidase activities from Dalbergia cochinchinensis Pierre.

A glycosidase enzyme with both beta-glucosidase and beta-fucosidase activities has been purified from the seeds of Dalbergia cochinchinensis Pierre (Thai Rosewood) by ammonium sulfate fractionation, preparative isoelectric focusing, and Sephadex G-150 chromatography. The enzyme has molecular weights of 330,000 in the native state and 66,000 in the denatured state. Hydrolysis of p-NP-beta-D-glucoside and p-NP-beta-D -fucoside showed pH optimum at pH 5.0 and was inhibited by delta-gluconolactone, HgCl2, and p-chloromercuribenzoate. The Km and kcat values of the purified enzyme were 5.4 mM and 307 s-1 for p-NP-beta-D-glucoside and 0.54 mM and 151 s-1 for p-NP-beta-D-fucoside, so that the latter had by far the higher kcat/Km ratio. p-NP-beta-D-galactoside, p-NP-beta-D-xyloside, and p-NP-alpha-L-arabinoside were hydrolyzed more slowly. Hydrolysis of sophorose, laminaribiose, and gentiobiose were also rather slow, and hydrolysis of cellobiose was even slower. No hydrolysis of the cyanogenic glucosides linamarin or prunasin, but some hydrolysis of amygdalin and salicin was found. Further studies are required to identify the natural substrates of the enzyme. However, high yields, ease of purification, and storage stability of the enzyme make it a useful candidate for various applications, such as study of oligosaccharide synthesis by reversal of hydrolysis.

Effect of an anticalculus toothpaste containing pyrophosphate on pyrophosphatase activity and the state of calcium phosphate in saliva.

Experiments were performed to determine if a pyrophosphate-containing anticalculus toothpaste could affect certain salivary constituents involved in the mineralization of dental plaque. The findings indicated no obvious changes in acid and alkaline pyrophosphatases, ionized calcium, total calcium, inorganic phosphate or pH in saliva for 1-135 min after brushing the teeth with the anticalculus toothpaste. Data obtained by brushing with the toothpaste three times daily for two weeks also confirmed these results. Our findings clearly indicate that pyrophosphate-stabilizing agents in the anticalculus toothpaste are not fully effective in the oral cavity. In addition, the pyrophosphate-containing toothpaste has no influence on the state of calcium and phosphate in saliva.

Evidence for a glycosidic linkage between chitin and glucan in the cell wall of Candida albicans.

The alkali-insoluble glucan was isolated from regenerating spheroplasts and intact cells of Candida albicans. Sequential enzymic hydrolysis of this fraction by Zymolyase 100T and purified chitinase and subsequent gel filtration produced a fraction which was enriched in glycosaminoglycans. This fraction was analysed by partial acid hydrolysis, TLC and GLC-MS. The GLC-MS peaks identified included 2,3,4,6-tetra-O-methylglucitol acetate and 2,3,4-tri-O-methylglucitol acetate of beta-1,6-glucan and the 3,6-di-O-methyl-2-N-methylglucosaminitol acetate of chitin. In addition, 3-O-methyl-2-N-methylglucosaminitol acetate was identified, which indicated a branch point in chitin. These data provide evidence for a covalent linkage between chitin and beta-(1,6)-glucan through a glycosidic linkage at position 6 of N-acetylglucosamine and position 1 of the glucose in the glucan.

The effects of azole and polyene antifungals on the plasma membrane enzymes of Candida albicans.

The two clinically important classes of antimycotic drugs, the polyenes and azoles, act on the plasma membrane of the cell. The primary modes of action are believed to be through interaction with sterols (polyenes) and alteration in sterol composition of the membrane (azoles). In this report we show that, at growth inhibitory concentrations, the polyenes (nystatin and amphotericin) and azoles (miconazole and ketoconazole) also inhibit plasma membrane enzymes. There was extensive (greater than 75%) inhibition of the Candida albicans plasma membrane enzymes ATPase, glucan synthase, adenyl cyclase and 5'-nucleotidase, when assayed in situ. The antifungals papulacandin and echinocandin, which inhibit glucan synthesis, also inhibited plasma membrane enzymes in situ; glucan synthase (greater than 90%), 5'-nucleotidase (greater than 80%) and ATPase (70-80%). Purified plasma membrane was prepared from yeast cells of C. albicans by two different techniques: concanavalin A stabilization and coating of spheroplasts with silica microbeads. In the purified plasma membrane vesicles prepared from concanavalin A the adenyl cyclase and phosphodiesterase were extensively (greater than 90%) inhibited by the three different classes of antifungal drugs; variable inhibition was observed with ATPase (70-100%). The 3',5'-cyclic phosphodiesterase of the plasma membrane purified by the microbeads method was almost completely inhibited by all of the antifungals tested and there was partial inhibition of ATPase (20-85%) and adenyl cyclase (30-90%).

The isolation of plasma membrane and characterisation of the plasma membrane ATPase from the yeast Candida albicans.

Plasma membrane ghosts were isolated from Candida albicans ATCC 10261 yeast cells following stabilisation of spheroplasts with concanavalin A, osmotic lysis and Percoll density gradient centrifugation. Removal of extrinsic proteins with NaCl and methyl alpha-mannoside gave increased ATPase and chitin synthase specific activities in the resultant plasma membrane fraction. Sonication of this fraction yielded unilamellar plasma membrane vesicles which exhibited ATPase and chitin synthase specific activities of 4.5-fold and 3.0-fold, respectively, over those of the plasma membrane ghosts. ATPase activity in the membrane ghosts was optimal at pH 6.4, showed high substrate specificity (for Mg X ATP) and was inhibited 80% by sodium vanadate but less than 4% by oligomycin and azide. The effects of a range of other inhibitors were also characterised. Temperature effects of ATPase activity were marked, with a maximum at 35 degrees C. Breaks in the Arrhenius plot, at 12.2 degrees C and 28.9 degrees C, coincided with endothermic heat flow peaks detected by differential scanning calorimetry. ATPase was solubilised from the plasma membranes with Zwittergent in the presence of glycerol and phenylmethylsulphonyl fluoride and partially purified by glycerol density gradient centrifugation. The solubilised enzyme hydrolysed Mg X ATP at Vmax = 20 mumol X min-1 X mg-1 in the presence of phospholipids, with optimal activity at pH 6.0--6.5.

Improved purification and fluorescence changes upon activation of human seminal plasma acidic protease proenzyme.

Modifications have been made to the previous purification procedure so that electrophoretically homogeneous acidic protease (EC 3.4.23.-) proenzyme of specific activity 800 units/mg may be isolated from human seminal plasma with a yield of over 50%. The intrinsic fluorescence of the proenzyme shows maximum excitation and emission wavelengths at 280 and 340 nm, respectively, typical of proteins containing tryptophan. Complete activation causes 30-35% decrease in intrinsic fluorescence, accompanied by a shift in gamma max to the blue of 4-6 nm. Time course studies indicate that acidification of proenzyme to pH 3.1 leads to a sudden large decrease in fluorescence that precedes both the appearance of active enzyme band on sodium dodecyl sulphate (SDS) polyacrylamide gels and the generation of enzyme activity as detected by the turbidimetric milk clotting assay. These results that acidification causes a rapid conformational change which promotes the release of the activation peptide from the proenzyme to yield the active enzyme.

Effect of ligand binding on the conformation of human plasma vitamin D binding protein (group-specific component).

Several techniques have been used to demonstrate that the binding of specific ligands to human plasma vitamin D binding protein induces a change in protein conformation. Apoprotein and holoprotein show circular dichroism spectra of similar form in the peptide region with double minima at 207 and 218 nm. The minimum mean residue ellipticity of apoprotein (20.6 X 10(3) degrees.cm2.dmol-1) is decreased by about 8% after vitamin D3 binding, suggesting a small change in the backbone conformation. Spectrofluorimetric studies showed that 25-hydroxycholecalciferol causes a saturable enhancement of intrinsic fluorescence of human vitamin D binding protein and alters the pH profile of protein fluorescence, suggesting that there are alterations in the local environment of tryptophan residue(s) after ligand binding. Furthermore, in the presence of 25-hydroxycholecalciferol, the rate of chemical modification of the amino groups in human vitamin D binding protein is decreased and the susceptibility of intact vitamin D binding protein to proteolytic degradation is reduced, suggesting that some surface sites in the vitamin D binding protein molecule are less accessible to external agents. In addition, although the absorbance of vitamin made if difficult to interpret the ultraviolet spectra of holoprotein and apoprotein, the presence of vitamin D binding protein appears to stabilize the vitamin in an aqueous environment, a phenomenon that may be of physiological importance.