CACNA1S mutation-associated dental anomalies: A calcium channelopathy.

OBJECTIVES: To identify the molecular etiology of distinct dental anomalies found in eight Thai patients and explore the mutational effects on cellular functions. MATERIALS AND METHODS: Clinical and radiographic examinations were performed for eight patients. Whole exome sequencing, mutant protein modelling, qPCR, western blot analysis, scratch assays, immunofluorescence, confocal analysis, in situ hybridization, and scanning electron micrography of teeth were done. RESULTS: All patients had molars with multiple supernumerary cusps, single-cusped premolars, and a reduction in root number. Mutation analysis highlighted a heterozygous c.865A>G; p.Ile289Val mutation in CACNA1S in the patients. CACNA1S is a component of the slowly inactivating L-type voltage-dependent calcium channel. Mutant protein modeling suggested that the mutation might allow leakage of Ca2+ or other cations, or a tightening, to restrict calcium flow. Immunohistochemistry analysis showed expression of Cacna1s in the developing murine tooth epithelium during stages of crown and root morphogenesis. In cell culture, the mutation resulted in abnormal cell migration of transfected CHO cells compared to wildtype CACNA1S, with changes to the cytoskeleton and markers of focal adhesion. CONCLUSIONS: The malformations observed in our patients suggest a role for calcium signaling in organization of both cusps and roots, affecting cell dynamics within the dental epithelium.

Activity of azithromycin or erythromycin in combination with antimalarial drugs against multidrug-resistant Plasmodium falciparum in vitro.

Azithromycin, an azalide analog of erythromycin was assayed for its in vitro activity against multidrug-resistant Plasmodium falciparum K1 strain by measuring the 3H-hypoxanthine incorporation. Azithromycin caused inhibitory effects on the parasite growth with IC50 and IC90 values of 8.4+/-1.2 microM and 26.0+/-0.9 microM, respectively. Erythromycin inhibited growth of P. falciparum with IC50 and IC90 values of 58.2+/-7.7 microM and 104.0+/-10.8 microM, respectively. The activity of antimalarial drugs in combination with azithromycin or erythromycin against P. falciparum K1 were compared. Combinations of chloroquine with azithromycin or erythromycin showed synergistic effects against parasite growth in vitro. Combinations of quinine-azithromycin and quinine-erythromycin showed potentiation. Additive effects were observed in mefloquine-azithromycin and mefloquine-erythromycin combinations. Similar results were also produced by pyronaridine in combination with azithromycin or erythromycin. However, artesunate-azithromycin and artesunate-erythromycin combinations had antagonistic effects. The in vitro data suggest that azithromycin and erythromycin will have clinical utility in combination with chloroquine and quinine. The worldwide spread of chloroquine-resistant P. falciparum might inhibit the ability to treat malaria patients with chloroquine-azithromycin and chloroquine-erythromycin in areas of drug-resistant. The best drug combinations against multidrug-resistant P. falciparum are quinine-azithromycin and quinine-erythromycin.

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).

The effect of an anticalculus dentifrice on calculus formation and gingival recession in Thai children and teenagers: one-year study. Study #2. An anticalculus dentifrice containing 1.3% soluble pyrophosphate and 1.5% of a copolymer.

A one-year, double-blind clinical study was conducted on a population of Thai children and teenagers to determine the effect of the twice daily use of an anticalculus dentifrice on supragingival calculus formation and gingival recession, as compared to the use of a placebo dentifrice and the continuation of customary oral hygiene procedures. The subjects were initially evaluated for calculus using the Volpe-Manhold procedure and for gingival recession using the GRT procedure of Volpe, Triratana, and Rustogi. All subjects received an oral prophylaxis and were assigned to the use of the following: (1) a placebo dentifrice containing 0.243% sodium fluoride in a silica base, (2) an anticalculus dentifrice containing 1.3% soluble pyrophosphate and 1.5% Gantrez copolymer in a 0.243% sodium fluoride/silica base, or (3) continuation of customary oral hygiene procedures. After six months, the subjects using the anticalculus dentifrice had 35.93% less supragingival calculus than the placebo dentifrice subjects, and 52.74% less supragingival calculus than the subjects who continued their regular/customary oral hygiene procedures. After one year, the subjects using the anticalculus dentifrice had 39.76% less supragingival calculus and 18.33% less gingival recession than the subjects using the placebo dentifrice. Also, after one year, the subjects using the anticalculus dentifrice had 57.86% less supragingival calculus and 16.55% less gingival recession than the subjects who continued their customary oral hygiene procedures. All reductions were statistically significant at the 99% level of confidence.

Toxicological study of pyridoxal isonicotinoyl hydrazone: acute and subchronic toxicity.

Pyridoxal isonicotinoyl hydrazone (PIH) is a highly effective iron chelator. Acute toxicity testing was performed in mice and rats of both sexes, with 10 mice or rats/sex/group. The LD50 values of PIH in both species were 5 and 1 g/kg given orally and intraperitoneally, respectively. Microscopic examination of tissues from the highest oral dose (6 g/kg) animals that survived 7 days revealed fatty degeneration in the liver. Subchronic toxicity was tested in rats of both sexes, with 8 males and 8 females/group. PIH doses of 100, 400 and 800 mg/kg were given orally for 90 consecutive days. Water was given to the control group. Hematocrit and blood chemistries were analysed at weeks 3, 6, 10 and 13. There were no changes in hematocrit and BUN. PIH at doses of 800 mg/kg caused a significant increase in serum alkaline phosphatase and transaminase levels at week 13. Microscopic examination showed hepatic degeneration in a dose-related fashion. Vascular congestion of the kidney and spleen was also found. No histopathological changes were detected in sections from the stomach and intestine.

[Dental care of children with hemophilia].

Hemophilia, the most common of the inherited bleeding disorder, is the result of a deficiency of clotting factor. Its characteristic is easy to bleed, prolong bleeding, and difficult to stop bleeding. In order to give dental treatment to the patient with this disease, consultation with the patient's physician is necessary. In addition, dentist should know the prevention and control of hemorrhage after treatment.

Mechanism of enhanced fusion capacity of mouse red cells infected with Plasmodium berghei.

Plasmodium berghei-infected mouse red cells have enhanced fusion capacity as triggered by addition of poly(ethylene glycol) in the presence of Ca2+. The uptake of Ca2+ in P. berghei-infected cells is greater than in normal cells, and the difference in Ca2+ uptake was found to be enhanced in the presence of poly(ethylene glycol). Fusion of normal and P. berghei-infected red cells by poly(ethylene glycol) was significantly inhibited by N-tosyl-L-lysylchloromethyl ketone and phenylmethylsulphonyl fluoride. In addition, ethyleneglycolbis(aminoethylether)tetra-acetate, N-ethylmaleimide, iodoacetamide, cystamine and tetrathionate also prevented fusion in both systems. In contrast, N-tosyl-L-phenylalanylchloromethyl ketone and N-tosyl-L-arginine methyl ester did not inhibit cell fusion. The latter enhanced fusion of infected cells but was without effect on normal cells. These results indicate that a Ca2+-activated thiol proteinase may be involved in membrane fusion in malaria-infected as well as in normal red cells. However, differences in the effect of proteinase inhibitors and substrate on fusion and Ca2+ entry show that the processes leading to fusion may not be identical.

Effects of retinol, fatty acids and glycerol monooleate on the fusion of chick embryo myoblasts in vitro.

Cell fusion of embryonic chick myoblasts has been studied in the presence of fat-soluble agents that induce erythrocytes to fuse. Retinol inhibited myoblast fusion but the cells recovered their ability to fuse within 48 h of removal of the retinol from the medium. Myristic acid, oleic acid, glycerol monooleate, linolenic acid and arachidonic acid similarly prevented fusion in myogenic cultures. By contrast, linoleic acid moderately enhanced the fusion of chick skeletal myoblasts. In addition, stearic acid, which does not fuse erythrocytes, inhibited myoblast fusion whereas the saturated, non-fusogenic fatty acid, arachidic acid, was without effect.

Enhanced fusion capacity of malaria (Plasmodium berghei)-infected mouse red cells.

The capacity of normal and malaria-infected mouse red cells to undergo fusion was investigated by phase contrast microscopy. The fusion of mouse red cells induced by 50% w/w poly(ethylene glycol)-6000 in the presence of Ca+2 is enhanced by P. berghei infection. Cells carrying parasites in the ring form stage and early trophozoite stage show slightly higher fusion induced by dimethyl sulphoxide and Ca+2 than those carrying parasites in trophozoite and schizont stages.

Effects of betel nut and fermented fish on the thiamin status of northeastern Thais.

Thiamin deficiency could result either from inadequate intake of thiamin or consumption of food containing antithiamin factors. Dietary surveys conducted in northeastern Thailand indicated that both thiamin and caloric intake of subjects under studies were sufficient. In Thailand, correlation exists between the consumption of food containing antithiamin factors and the prevalence of thiamin deficiency. Betel nuts and raw fermented fish possess antithiamin activity. Abstention from both betel nut chewing and raw fermented fish consumption resulted in a significant reduction of thiamin pyrophosphate effect. The thiamin pyrophosphate effect again increased significantly when the subjects resume their chewing habits. Cooking of fermented fish destroyed thiaminase, resulted in a significant decrease of thiamin pyrophosphate effect of the subjects. Thiamin supplementation (10 mg/day) could further reduce their thiamin pyrophosphate effect. This amount of thiamin could counteract the effect of raw fermented fish consumption but was not sufficient to neutralize the effect of betel nut chewing.