Remineralization and acid resistance of enamel lesions after chewing gum containing fluoride extracted from green tea.

BACKGROUND: The aim of this study was to evaluate enamel remineralization and the acquisition of acid resistance by using sugar-free chewing gum containing fluoride extracted from green tea. METHODS: Forty-five volunteers participated in a crossover, double-blind study and wore intraoral appliances with human demineralized enamel. Subjects chewed fluoride chewing gum (FCG: 50 µg fluoride) or placebo gum. Remineralization and acid resistance were evaluated using the mineral change value (ΔZ, in vol%·µm). Fluoride concentrations in saliva and remineralized enamel were analysed. RESULTS: The peak salivary fluoride concentration was 3.93 ± 1.28 ppm (mean ± SD). The elevated salivary fluoride concentration resulted in a higher fluoride concentration of 656 ± 95 ppm in the remineralized region versus 159 ± 26 ppm for placebo gum (p < 0.001). After remineralization, the ΔZ of the FCG group was higher than that of the placebo gum group. After an acid challenge, ΔZ of the FCG group was lower than the placebo gum group. Both ΔZ were statistically significant. CONCLUSIONS: FCG produced a superior level of remineralization and acid resistance, as compared to the placebo gum. The in situ results suggest that regular use of FCG is useful for preventing dental caries.

Logistic regression analysis of pregnancy rate following transfer of Bos indicus embryos into Bos indicus x Bos taurus heifers.

Factors affecting pregnancy rate of 5627 Zebu embryos in crossbred females with unknown proportions of Holstein and Zebu breeding were examined. After evaluation for developmental stage, quality, and viability, embryos were immediately transferred to recipients. Pregnancy diagnosis was conducted approximately 53 d after transfer; pregnancy rate was coded as a binomial event and analyzed using logistic regression models. Maximum likelihood methodology and the likelihood ratio statistic were used to estimate regression coefficients and test hypotheses. Explanatory variables were year of transfer (1992-1999), season of transfer (summer, autumn, winter and spring), breed of the embryo (Guzerat, Gyr or Nellore), stage of the embryo (morula, early blastocyst, blastocyst, expanded blastocyst, and hatching blastocyst), quality of the embryo (excellent, good or regular), and donor-recipient synchrony (estrus in the recipient occurred 2-3 d before, 1 d before, the day of, 1 d after, or 2-3 d after estrus in the donor). Average pregnancy rate was 63.7%. Pregnancy rates were not significantly affected by breed of embryo. The best multiple-logistic model to explain the pregnancy result included the effects of year and season of transfer, embryo stage and quality, and estrous synchrony between donor and recipient (P

Mechanism of action of hammerhead ribozymes and their applications in vivo: rapid identification of functional genes in the post-genome era by novel hybrid ribozyme libraries.

A hammerhead ribozyme was demonstrated to be a metalloenzyme. By controlling the metal-binding ability of the hammerhead ribozyme in the presence or absence of a specific sequence of interest, we engineered an allosterically controllable ribozyme, designated the maxizyme. Hybrid ribozymes were then constructed by coupling the site-specific cleavage activity of a hammerhead ribozyme with the unwinding activity of an endogenous RNA helicase. This leads to extremely efficient cleavage of target mRNA, not only in vitro, but also in vivo, and eliminates one of the major problems arising in the application of ribozymes for cleavage of mRNA in vivo : that many target sites on the RNA were previously inaccessible to cleavage owing to secondary and/or tertiary structure formation. Since hybrid ribozymes can efficiently attack target sites within mRNA, libraries were made of hybrid ribozymes with randomized binding arms, which were then introduced into cells. This procedure made it possible to readily identify the relevant genes associated with a specific phenotype, such as in apoptosis and cancer metastasis pathways. This application of a randomized library of hybrid ribozymes represents a simple, yet powerful, method for the identification of genes associated with specific phenotypes in the post-genome era. Moreover, vector-based siRNA (short-interfering RNA for RNA interference, RNAi) can also be used for the creation of the libraries and for the subsequent confirmation of the identified genes, relevant in the examined phenotype.

Characterization of pro-apoptotic gene Bak using hammerhead ribozymes.

Bak gene encodes a 23kDa-protein homologue of apoptosis inhibitor Bcl-2. Unlike Bcl-2, Bak stimulates several apoptosis pathways, and by interacting with Bcl-2 or Bcl-XL, it counteracts the anti-cell death effect. Enforced expression of Bak induces rapid and extensive apoptosis of serum-deprived fibroblasts. In contrast, the mutation of the Bak gene was observed in several cancer cells, suggesting a deficiency of the Bak cellular function can cause a cancerous malignancy. Although many studies of Bak have been performed, the mechanisms by which Bak functions in mammalian cells remain unclear. In this study, we constructed a hammerhead ribozyme specifically targeted to the Bak mRNA for inhibition of the intracellular function of Bak. This Bak-ribozyme should be useful for a detailed functional analysis of Bak.

Characterization of a C-terminal-type kinesin-related protein from Dictyostelium discoideum.

We have determined the full sequence of K2, a kinesin-related protein (KRP) in Dictyostelium discoideum. Sequence homology and domain organization placed K2 in the ncd/Kar3 subfamily of the C-terminal-type KRPs. Bacterially expressed, truncated K2 showed ATP-dependent binding to microtubules and microtubule-stimulated ATPase activity. K2-null cells grew and developed normally, suggesting overlapping functions of K2 with other microtubule motor(s). Overexpression of K2 caused partial mitotic arrest. Green fluorescent protein-tagged full-length K2 localized in the nucleus at the interphase and on the mitotic spindle during mitosis. These results suggest that K2 is a microtubule-dependent motor which may play some roles in mitotic spindles.

In vitro characterization of chondrogenic cells isolated from chick embryonic muscle using peanut agglutinin affinity chromatography.

Specific binding to the lectin, peanut agglutinin (PNA), has been reported in embryonic precartilage tissues, including the condensing limb bud blastema and the caudal half of the developing somite. The present study aimed to test the hypothesis that PNA-binding may be a surface characteristic of chondroprogenitor cells residing within noncartilage tissues, such as muscle, which have the potential of being induced to form cartilage, e.g., in the presence of bone matrix-derived factors. Day-14 chick embryonic pectoral muscle, which contained histochemically detectable PNA-binding cells, was dissociated into single cells (TM cells) and fractionated by PNA affinity chromatography into PNA-binding (PNA+) and nonbinding (PNA-) cells by PNA-Sepharose 6 MB affinity chromatography. The differentiation potential of the PNA-affinity fractionated cells in vitro was analyzed as a function of culture plating cell density. Immunohistochemistry of a number of cell-type-specific differentiation markers, including sarcomeric actin, collagen type II, and aggrecan core protein, demonstrated that PNA+ cells, when cultured as a micromass at high density (20 x 10(6) cells/ml), exhibited a chondrocyte-like phenotype, whereas the PNA-cells remained myogenic; however, both PNA+ and PNA- monolayer cultures (4 x 10(4) cells/ml) behaved as myoblastic cells. The expression of collagen type II mRNA was also confirmed by coupled reverse transcription/polymerase chain reaction analysis. These observations suggest that PNA binding, i.e., the presence of specific galactose-containing cell surface moieties, is likely to be one of the characteristics of chondrogenic cells residing in mesenchymally derived embryonic tissues.

Developmental expression and vitamin D regulation of calbindin-D28K in chick embryonic yolk sac endoderm.

The yolk is an important calcium source for the developing chick embryo. The epithelial yolk sac endodermal cells lie in direct contact with the yolk and are the principal nutrient-transporting cell type. We previously reported that vitamin D treatment stimulated yolk calcium mobilization and that the vitamin D-dependent Ca2+-binding protein, calbindin-D28K, is present in the yolk sac. We report here the developmental expression and regulation of calbindin-D28K in the yolk sac. Calbindin-D28K is expressed as early as incubation d 3 and is found exclusively within the cytoplasm of endodermal cells. Comparative protein and mRNA analyses of yolk sac and dissociated yolk sac endodermal cells as a function of development and treatment with calcitriol (1,25-dihydroxyvitamin D3) in vitro and in vivo showed a development-specific and vitamin D-inducible expression of calbindin-D28K. Northern analysis revealed the expression of vitamin D receptor mRNA in the yolk sac, beginning as early as d 3, strongly indicating that the extraembryonic yolk sac is an early vitamin D target tissue. Cultured yolk sac endodermal cells should serve as a useful in vitro cell model for analyzing the cellular and molecular mechanisms of vitamin D action.

Conversion of rat urinary prokallikrein to its active form.

A peptide derived from rat urinary prokallikrein by trypsin treatment comprised 7 amino acids, the sequence (Ala-Pro-Pro-Val-Gln-Ser-Arg) of which was identical with that of the N-terminal region in prokallikrein. Thus, with trypsin treatment, rat urinary prokallikrein is converted to the active form with the release of the N-terminal propeptide consisting of 7 amino acids. An Arg-1-Val+1 bond in the prokallikrein was found to be the site of proteolytic cleavage of the propeptide.