Ethanol changes atpB gene expression and proton permeability in Streptococcus mutans / 대한구강보건학회지

OBJECTIVES: As a first step to study the anticaries effect of ethanol alone, we investigated the effects of ethanol on the expression levels of the atpB gene and proton permeability of Streptococcus mutans in suspension cultures. METHODS: S. mutans UA159 was grown in brain heart infusion medium at either pH 4.8 or 6.8. The total extracted RNA was reverse-transcribed into cDNA using a Superscript™ First-Strand Synthesis System. The resulting cDNA and negative controls were amplified by ABI PRISM 7700 real-time PCR system with SYBR Green PCR Master Mix. For proton flux assay, bacterial suspensions were titrated to pH 4.6 with 0.5 M HCl, and then additional 0.5 M HCl was added to decrease the pH values by approximately 0.4 units. The subsequent increase in pH was monitored using a glass electrode. Ten percent (v/v) butanol was added to the suspensions at 80 min to disrupt the cell membrane. RESULTS: In a concentration-dependent manner, ethanol alone not only decreased the growth rate of S. mutans and the expression of the atpB gene but also increased the proton permeability at both pH 4.8 and 6.8. CONCLUSIONS: These findings suggest that ethanol has the potential for an anticaries ingredient. We believe that ethanol may be used together with fluoride and/or other cariostatic agents in order to develop better anticaries toothpastes and/or mouthrinses.

The pH-dependent effects of combining ethanol with fluoride on proton permeability in Streptococcus mutans / 대한구강보건학회지

OBJECTIVES: The aim of this research was to determine the pH-dependent changes in F-ATPase activity and proton fluxes in Streptococcus mutans (S. mutans) as induced by varying the concentration of fluoride ±10 mM (0.058% (v/v)) ethanol. METHODS: S. mutans UA159 was grown in Brain Heart Infusion medium at pH 4.8, 6.8, or 8.8. The F-ATPase assay was initiated by the addition of ATP, and stopped by adding 10% trichloroacetic acid. For the proton flux assay, bacterial suspensions were titrated to pH 4.6 with 0.5 M HCl, and then 0.5 M HCl was added to decrease the pH values in units of approximately 0.4 pH. The subsequent increase in pH was monitored using a glass electrode. To disrupt the cell membrane, 10% (v/v) butanol was added to the suspensions after 80 minutes. RESULTS: At all pH levels, fluoride ±10 mM ethanol not only decreased F-ATPase activity but also increased the proton permeability of S. mutans. The largest effects were observed at pH 4.8. Ethanol enhanced these effects only at pH 4.8. CONCLUSIONS: A very low concentration of ethanol enhanced the action of fluoride on F-ATPase activity and the proton permeability in S. mutans at acidic pH levels. We expect that low concentrations of ethanol may be used together with fluoride and/or other anticaries agents to develop more effective anticaries preparations.

The Cholesterol-Binding Antibiotic Nystatin Induces Expression of Macrophage Inflammatory Protein-1 in Macrophages

Nystatin, a polyene antifungal antibiotic, is a cholesterol sequestering agent. The antifungal agent alters composition of the plasma membrane of eukaryotic cells, whereas its effects on cells are poorly investigated. In the current study, we investigated the question of whether nystatin was able to induce expression of macrophage inflammatory protein-1 (MIP-1). THP-1 cells rarely express MIP-1alpha and MIP-1beta, however, upon exposure to nystatin, significantly elevated expression of MIP-1alpha and MIP-1beta was observed in a dose-dependent fashion at the messenger and protein levels. Cellular factors activated by nystatin as well as involved in nystatin-induced expression of MIP-1 proteins were identified in order to understand the molecular mechanisms of action of the anti-fungal agent. Treatment with nystatin resulted in enhanced phosphorylation of Akt, ERK, p38 MAPK, and JNK. Abrogation or significant attenuation of nystatin-induced expression of MIP-1alpha and MIP-1beta was observed by treatment with Akt inhibitor IV, LY294002, and SP6001250. Inhibition of ERK or p38MAPK using U0126 and SB202190 did not lead to attenuation of MIP-1 expression. In addition, inhibitors of protein kinase C, such as GF109203X and Ro-318220, also attenuated expression of MIP-1. These results indicate that nystatin is able to activate multiple cellular kinases and, among them, Akt and JNK play primary roles in nystatin-induced expression of MIP-1 proteins.

Multiple Signaling Molecules are Involved in Expression of CCL2 and IL-1beta in Response to FSL-1, a Toll-Like Receptor 6 Agonist, in Macrophages

TLR6 forms a heterodimer with TLR2 and TLR4. While proinflammatory roles of TLR2 and TLR4 are well documented, the role of TLR6 in inflammation is poorly understood. In order to understand mechanisms of action of TLR6 in inflammatory responses, we investigated the effects of FSL-1, the TLR6 ligand, on expression of chemokine CCL2 and cytokine IL-1beta and determined cellular factors involved in FSL-1-mediated expression of CCL2 and IL-1beta in mononuclear cells. Exposure of human monocytic leukemia THP-1 cells to FSL-1 resulted not only in enhanced secretion of CCL2 and IL-1beta, but also profound induction of their gene transcripts. Expression of CCL2 was abrogated by treatment with OxPAPC, a TLR-2/4 inhibitor, while treatment with OxPAPC resulted in partially inhibited expression of IL-1beta. Treatment with FSL-1 resulted in enhanced phosphorylation of Akt and mitogen-activated protein kinases and activation of protein kinase C. Treatment with pharmacological inhibitors, including SB202190, SP6001250, U0126, Akt inhibitor IV, LY294002, GF109203X, and RO318220 resulted in significantly attenuated FSL-1-mediated upregulation of CCL2 and IL-1beta. Our results indicate that activation of TLR6 will trigger inflammatory responses by upregulating expression of CCL2 and IL-1beta via TLR-2/4, protein kinase C, PI3K-Akt, and mitogen-activated protein kinases.

Extracellular Nucleotides Can Induce Chemokine (C-C motif) Ligand 2 Expression in Human Vascular Smooth Muscle Cells

To understand the roles of purinergic receptors and cellular molecules below the receptors in the vascular inflammatory response, we determined if extracellular nucleotides up-regulated chemokine expression in vascular smooth muscle cells (VSMCs). Human aortic smooth muscle cells (AoSMCs) abundantly express P2Y1, P2Y6, and P2Y11 receptors, which all respond to extracellular nucleotides. Exposure of human AoSMCs to NAD+ , an agonist of the human P2Y11 receptor, and NADP+ as well as ATP, an agonist for P2Y1 and P2Y11 receptors, caused increase in chemokine (C-C motif) ligand 2 gene (CCL2) transcript and CCL2 release; however, UPT did not affect CCL2 expression. CCL2 release by NAD+ and NADP+ was inhibited by a concentration dependent manner by suramin, an antagonist of P2-purinergic receptors. NAD+ and NADP+ activated protein kinase C and enhanced phosphorylation of mitogen-activated protein kinases and Akt. NAD(+)- and NADP(+)-mediated CCL2 release was significantly attenuated by SP6001250, U0126, LY294002, Akt inhibitor IV, RO318220, GF109203X, and diphenyleneiodium chloride. These results indicate that extracellular nucleotides can promote the proinflammatory VSMC phenotype by up-regulating CCL2 expression, and that multiple cellular elements, including phosphatidylinositol 3-kinase, Akt, protein kinase C, and mitogen-activated protein kinases, are involved in that process.