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Streptococcus mutans의 양성자 투과성에 미치는 불소와 에탄올의 병용효과
이새아 ( Sae-a Lee ),정승일 ( Seung-il Jung ),김진범 ( Jin-bom Kim ),강정숙 ( Jung Sook Kang ) 대한구강보건학회 2016 大韓口腔保健學會誌 Vol.40 No.4
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 FATPase 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.
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펩티도글라이칸에 의한 인터루킨-1 베타 발현 기전 연구
서현철(Hyun-Cheol Seo),김선미(Sun-Mi Kim),이새아(Sae-A Lee),임병용(Byung-Yong Rhim),김관회(Koanhoi Kim) 한국생명과학회 2012 생명과학회지 Vol.22 No.12
본 연구에서는 IL-1β 발현에 PG의 영향을 조사하였고, 단핵세포에서 PG에 의한 IL-1β 상향조절에 포함된 세포인자를 밝혔다. PG에 사람의 THP-1 세포를 노출시키면 IL-1β 분비 증가뿐만 아니라 IL-1β 유전자 전사를 유도하는 결과를 가져왔고, TLR-2/4의 억제제인 OxPAPC에 의해 저해되었다. U0126, SP6001250, Akti IV, rapamycin, DPI 같은 약리학적 저해제도 PG에 의한 IL-1β의 상향조절을 상당히 약화시켰다. 그러나 polymyxin B는 IL-1β 발현에 영향을 미치지 않았다. 본 연구는 PG는 TLR-2, Akt, mTOR, MAPKs, ROS를 통하여 IL-1β의 발현을 상향시킴을 확인하였다. This study investigated the effects of PG on IL-1β expression and determined cellular factors involved in PG-mediated IL-1β up-regulation in mononuclear cells in order to understand the molecular mechanisms underlying inflammatory responses associated with bacterial pathogen-associated molecular patterns in the diseased artery. Exposure of human onocytic leukemia THP-1 cells to PG resulted in enhanced secretion of IL-1β and also profound induction of the IL-1β gene transcript. These effects were abrogated by OxPAPC, an inhibitor of TLR-2/4. Pharmacological inhibitors such as U0126, SP6001250, Akti IV, rapamycin, and DPI also significantly attenuated PG-mediated IL-1β up-regulation. However, polymyxin B did not influence the IL-1β expression. This study indicates that PG contributes to vascular inflammation in atherosclerotic plaques by up-regulating expression of IL-1β via TLR-2, Akt, mTOR, MAPKs, and ROS.
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에탄올이 Streptococcus mutans의 atpB 유전자 발현 및 양성자 투과성에 미치는 영향
조철민 ( Chul Min Cho ),박용진 ( Yong Jin Park ),이새아 ( Sae A Lee ),김진범 ( Jin Bom Kim ),강정숙 ( Jung Sook Kang ) 대한예방치과·구강보건학회 2018 大韓口腔保健學會誌 Vol.42 No.4
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 SuperscriptTM 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.
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pH Stress Alters Cytoplasmic Membrane Fluidity and atpB Gene Expression in Streptococcus mutans
Chul Min Cho(조철민),Seung Il Jung(정승일),Myung Sup Kim(김명섭),Sae A Lee(이새아),Jung Sook Kang(강정숙) 한국생명과학회 2017 생명과학회지 Vol.27 No.1
치아우식의 주원인균인 Streptococcus mutans (S. mutans)는 산 생성 뿐 아니라 산에 대한 탁월한 저항성을 나타낸다. 본 연구에서는 S. mutans가 pH stress에 노출될 때 형질막 유동성, F-ATPase 활성과 발현 및 양성자 투과성 변화와 그 상관관계를 규명하였다. S. mutans로부터 형질막을 분리한 후 1,6-diphenyl-1,3,5-hexatriene을 사용하여 pH stress가 형질막 유동성 변화에 미치는 영향을 측정하였다. pH 4.8과 pH 8.8에서 배양한 S. mutans는 pH 6.8에서 배양한 S. mutans에 비하여 형질막 유동성이 감소되었다. F-ATPase 활성과 발현은 pH 4.8에서 가장 높았고, pH 8.8에서 가장 낮았다. 양성자 투과성은 pH 4.8과 pH 8.8에서 모두 감소되었으며, 특히 pH 4.8에서의 감소가 컸다. F-ATPase 활성만으로 양성자 투과성이 결정된다면 pH 8.8에서 가장 높아야 하나 pH 6.8보다 감소하는 것은 형질막 유동성 감소에 기인된 양성자 세포내 유입 감소와 관련된 것으로 추정한다. 또한 pH 4.8에서 양성자 투과성이 아주 낮은 것은 높은 F-ATPase 활성에 의한 양성자 세포외 유출 증가 뿐 아니라 형질막 유동성 감소에 의한 양성자 세포내 유입 감소에 기인된 것으로 추정한다. 따라서 pH stress에 의한 형질막 유동성 감소는 S. mutans가 세포내 pH 를 유지하는데 중요한 역할을 하는 것으로 생각되며 에탄올을 포함하여 비특이적으로 세포막 유동성을 증가시키는 약물들은 항우식제에 활용될 수 있을 것으로 추정한다. Streptococcus mutans (S. mutans), which plays a major role in the etiology of human dental caries, is able to tolerate exposure to acid shock in addition to its acidogenicity. We investigated the effects of pH stress on membrane fluidity, activities and expression levels of F-ATPase, and proton permeability in S. mutans. Using 1,6-diphenyl-1,3,5-hexatriene, we observed membrane ordering at pH 4.8 and pH 8.8. The ordering effects were larger at pH 4.8 in cytoplasmic membranes isolated from S. mutans (CMSM). Increasing pH resulted in a decrease in the activities and expression levels of F-ATPase. The proton permeability was decreased at both acidic and alkaline pHs, and the lowest permeability was observed at pH 4.8. The lower permeability at pH 8.8 than pH 6.8 is likely to be caused by the decreased proton influx due to the decreased CMSM fluidity. In addition, it seems to be evident that extremely low permeability at pH 4.8 was caused by the decreased proton influx due to the decreased CMSM fluidity as well as the increased proton efflux due to the increased activity and expression level of F-ATPase. It is likely that CMSM fluidity and F-ATPase activity are two major key factors that determine proton permeability in S. mutans. We suggest that CMSM fluidity plays an important role in the determination of proton permeability, which sheds light on the possibility of using nonspecific membrane fluidizers, e.g., ethanol, for anti-caries purposes.
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