• Restorative Dentistry and Endodontics
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• v.24 no.3
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• pp.447-452
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• 1999

The role of bacteria in root canals and periapical infections is well known and established. In these bacteria, black-pigmented bacteria(BPH) play important role in endodontic infection. BPB are Gram negative anaerobic rods which are closely related 50 clinical symptoms such as pain, percussion, tenderness, foul odor, etc. In America and Europe, many studies on BPB have been done and are continued. But, relatively few studies have been done in Korea, especially its prevalence in Korean population is not yet studied. The purpose of this study is to establish prevalence of BPB in infected root canals and periapical abscesses in Korean people. Microbial samples were collected from the root canals of 34 intact tooth with periapical rarefactions of endodontic origin and 3 periapical abscesses. All samples were incubated in an anaerobic chamber(Coy, Model No. 77. Ann Arbor, Michigan, USA.). Identification of In microorganism was based on its growth in the anaerobic chamber, colonial pigmentation, colonial morphology, Gram stain, and Rapid ID32A(BioMericux SA/69280 Marcy-l'Etoile/France) results. In addition, the polyme ase chain reaction using specific primers for 16S rRNA genes were used differentiate Prevotella nigrescens for Prevotella intermedia. The results were as follows : 1. In this study, thirteen (35%) of thirty seven samples were positive for the growth of BPB. In thirteen samples, sixteen strains of BPR were found. 2. The most frequently identified BPB in root canals and abscesses in Korean were P. nigrescens 5/37(14%) and P. intermedia 5/37(14%). Porphyromonas gingivalis 3/37(8%), Porphyromonas endodontalis 2/37(5%) and Prevotella loecheii 1/37(3%) were also found. 3. In this study, no significant differences were found between the prevalence of BPB in Korean and that of American and European.

• Journal of Periodontal and Implant Science
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• v.43 no.4
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• pp.191-197
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• 2013

Purpose: Nitric oxide (NO) is a short-lived bioactive molecule that is known to play an important role in the pathogenesis of periodontal disease. In the current study, we investigated the effect of the flavonoid quercetin on the production of NO in murine macrophages activated with lipopolysaccharide (LPS) from Prevotella intermedia, a pathogen related to inflammatory periodontal disease, and tried to elucidate the underlying mechanisms of action. Methods: LPS was isolated from P. intermedia ATCC 25611 cells by the standard hot phenol-water method. The concentration of NO in cell culture supernatants was determined by measuring the accumulation of nitrite. Inducible NO synthase (iNOS) and heme oxygenase-1 (HO-1) protein expression, phosphorylation of c-Jun N-terminal kinase (JNK) and p38, inhibitory ${\kappa}B$ $(I{\kappa}B)-{\alpha}$ degradation, and signal transducer and activator of transcription 1 (STAT1) phosphorylation were analyzed via immunoblotting. Results: Quercetin significantly attenuated iNOS-derived NO production in RAW246.7 cells activated by P. intermedia LPS. In addition, quercetin induced HO-1 protein expression in cells activated with P. intermedia LPS. Tin protoporphyrin IX (SnPP), a competitive inhibitor of HO-1, abolished the inhibitory effect of quercetin on LPS-induced NO production. Quercetin did not affect the phosphorylation of JNK and p38 induced by P. intermedia LPS. The degradation of $I{\kappa}B-{\alpha}$ induced by P. intermedia LPS was inhibited when the cells were treated with quercetin. Quercetin also inhibited LPS-induced STAT1 signaling. Conclusions: Quercetin significantly inhibits iNOS-derived NO production in murine macrophages activated by P. intermedia LPS via anti-inflammatory HO-1 induction and inhibition of the nuclear factor-${\kappa}B$ and STAT1 signaling pathways. Our study suggests that quercetin may contribute to the modulation of host-destructive responses mediated by NO and appears to have potential as a novel therapeutic agent for treating inflammatory periodontal disease.

• Journal of Microbiology and Biotechnology
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• v.13 no.2
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• pp.282-286
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• 2003

The purpose of this study was to isolate a specific DNA probe for the strain ATTC 25611 of the species Prevotella intermedia by using a new rapid screening mothod. The whole-genomic DNA of P. intermedia ATCC 25611 was isolated and purified. The HindIII-digested genomic DNAs from the strain were cloned by the random cloning method. To screen the strain-specific DNA probe, inverted dot blot hybridization tests were performed. In this assay, 20 ng of recombinant plasmids containing the HindIII-digested genomic DNA fragment were boiled and blotted onto a nylon membrane, and hybridized with digoxigenin-dUTP labeled genomic DNAs in a concentration of 100 ng/ml. Southern blot analysis was performed in order to confirm the results of the inverted dot blot hybridization tests. The data showed that a Pi34 probe (2.1 kbp; 1 out of 32 probes) was specific for P. intermedia strain ATCC 25611 and could be useful for the detection and identification of the strain, particularly in epidemiological studies of periodontal disease.

• Korean Journal of Microbiology
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• v.44 no.3
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• pp.212-220
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• 2008

A Pn10 DNA probe was introduced as a Prevotella nigrescens ATCC $33563^T$-specific DNA probe. In that study, the specificity of the Pn10 was tested with only type or reference strains of 5 oral bacterial species. The purpose of this study is to evaluate the specificity of the Pn10 using the wild type strains of P. nigrescens and is to develop the P. nigrescens ATCC $33563^T$-specific PCR primers based on the nucleotide sequence of the Pn10. The specificity of the Pn10 DNA probe was determined by Southern blot analysis. The nucleotide sequence of Pn10 DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pn10 DNA probe were hybridized with the genomic DNAs from P. nigrescens ATCC $33563^T$ and KB6. The Pn10 homologous region, KB6-Pn10, of P. nigrescens KB6 was cloned by PCR and sequenced. The Pn10 and KB6-Pn10 DNA fragments were consisted of 1,875 bp and 1,873 bp, respectively. The percent identity of the two was 98.8% and the divergence of them was 0.6%. The two primer sets (Pn10-F-AC/ Pn10-R-AC and Pn10-F-A/ Pn10-R-A), designed base on the nucleotide sequences of Pn10 DNA probe, were specific to the P. nigrescens ATCC $33563^T$. The two PCR primer sets could detect as little as 4 pg of genomic DNA of P. nigrescens ATCC $33563^T$. These results indicate that the two PCR primer sets have proven useful for the identification of P. nigrescens ATCC $33563^T$, especially with regard to the maintenance of the strain.

• Journal of Periodontal and Implant Science
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• v.37 no.sup2
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• pp.409-426
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• 2007

세균성 치태는 치은의 염증과 치주 조직 파괴를 동반하는 치주염의 주요한 인자로서 치주 조직 건강을 유지하기 위하여 적절한 치태 조절이 필요하다. 본 논문의 목적은 12주 동안 만성 초기 및 중등도 치주염 환자에서 치은염에 대한 임상 지수의 감소, interleukin-6 (IL-6) 농도와 치주질환 원인균인 Prevotella intermedia (P. intermedia), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans)에 대한 소니케어 전동 칫솔의 효과를 일반 칫솔과 비교해 보고자 하는 데 있다. 총 82명의 환자를 선택하였으며, 30명은 일반 칫솔, 52명은 소니케어 전동 칫솔 군으로 분류하여 칫솔질 교육을 실시하였다. 전악을 전치부와 구치부로 나누어 초진, 1, 4, 12주에서의 치태, 치은 지수 및 탐침 시 출혈 여부를 조사하였으며, 가장 깊은 치주낭 탐침을 보이는 치아 3개를 선택하여 탐침 깊이와 부착 정도를 측정하였고, 선택된 치아에서 초진, 1, 12주에 채취된 샘플을 통해 치은열구액 내의 IL-6 농도와 P. intermedia, A. actinomycetemcomitans의 CT값을 추가적으로 조사하여 다음과 같은 결과를 얻었다. 1 소니케어 전동 칫솔과 일반 칫솔군 모두 치은 염증을 나타내는 임상 지수 (치태지수, 치은지수, 탐침 시 출혈)는 12주 기간 동안 유의한 감소 (p<0.05)를 보였으나, 전동 칫솔 군에서 통계학적으로 더욱 유의하게 (p<0.05) 나타났다. 2. 전치부를 제외한 구치부 치아에서 소니케어 전동 칫솔군은 12주 기간 동안 탐침 시 출혈의 감소가 통계학적으로 유의하게 (p<0.05) 나타났다. 3. 가장 깊은 치주낭 탐침 깊이를 보이는 3개의 선택된 치아에서 치주낭 탐침 깊이와 부착 정도는 두 군 모두 초진에 비해 유의한 감소 (p<0.05)를 보였다. 퍼센트 변화 비교에서 치주낭 탐침 깊이는 소니케어 전동 칫솔군이 $18.47{\pm}10.05%$, 일반 칫솔군이 $14.19{\pm}8.16%$로, 부착 정도는 소니케어 전동 칫솔군이 $24.26{\pm}12.51%$, 일반 칫솔군이 $15.65{\pm}9.92%$로 각각 나타났으나, 군 간 통계적 유의차는 보이지 않았다. 4. 치은열구액의 IL-6 농도는 두 군 모두 12주 기간 동안 통계적으로 유의한 감소 (p<0.05)를 나타내었다. 퍼센트 변화 비교에서 전동 칫솔군은 51%, 일반 칫솔군은 37%로 각각 나타났으나, 군 간 통계적 유의차는 보이지 않았다. 5. Prevotella intermedia, Actinobacillus actinomycetemcomitans의 관찰에서 두 군 모두 유의한 차이는 없었다. 위 결과를 통해 본 연구에서는 소니케어 전동 칫솔의 사용이 일반 칫솔에 비하여 만성 초기 및 중등도 치주염 환자에서 치태의 제거, 치은 염증 및 임상 지수의 감소에 유의한 효과가 있으며 IL-6의 감소 경향에도 효과가 있음을 관찰하였다.

• Journal of Life Science
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• v.27 no.4
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• pp.476-481
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• 2017

Polyphenols are secondary metabolites produced by higher plants and have been used as antiallergic, anticancer, antihypertensive, antiinflammatory, antimicrobial and antioxidant agents. They are generally divided into flavonoids and non-flavonoids. The antimicrobial activity of flavonoids are stronger than that of non-flavonoids. The skeleton structures of flavonoids possessing antimicrobial activity are chalcone, flavan-3-ol (catechin), flavanone, flavone, flavonol and proanthocyanidin. The flavonols are shown antibacterial activity against several gram-positive bacteria (Actinomyces naeslundii, Lactobacillus acidophilus and Staphylococcus aureus) and gram-negative bacteria (Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella melaninogenica and Prevotella oralis). Among of non-flavonoids, caffeic acids, ferulic acids and gallic acids showed antimicrobial activity against gram-positive (Listeria monocytogenes and S. aureus) and gram-negative bacteria (Escherichia coli and Pseudomonas aeruginosa). These are found to be more efficient against the E. coli, L. monocytogenes, P. aeruginosa and S. aureus than antibiotics such as gentamicin and streptomycin. The kaempferol and quercetin showed synergistic effect with ciprofloxacin and rifampicin against S. aureus and methicillin resistant S. aureus (MRSA). Epigallocatechin gallate (EGCG) acts synergistically with various ${\beta}-lactam$ antibiotics against MRSA. In particular, the epicatechin, epigallocatechin (EGC), EGCG and gallocatechin gallate from Korean green tea has antibacterial activity against MRSA clinical isolates and the combination of tea polyphenols and oxacillin was synergistic for all the clinical MRSA isolates.

• Restorative Dentistry and Endodontics
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• v.30 no.5
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• pp.372-384
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• 2005

The purpose of this study was to monitor the secretion of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) by human periodontal ligament (PDL) fibroblasts stimulated with Prevotella nigrescens lipopolysaccharide (LPS), and to examine the effect of calcium hydroxide treatment on P. nigrescens LPS. LPS was extracted and purified from anaerobically cultured P. nigrescens. PDL fibroblasts were stimulated by the LPS (0, 0.1, 1, 10 ${\mu}g/ml$) or LPS (10 ${\mu}g/ml$) pretreated with 12.5 mg/ml of $Ca(OH)_2$ for 3 days, for various periods of time (12, 24, 48 h). Immunoprecipitation were performed for protein level analysis of MMP-1 MMP-2 and TIMP-1. Total RNA was isolated and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 mRNA. According to this study, the results were as follows: 1. The p개duction of MMP-1 by stimulation with P. nigrescens LPS increased in time-dependent manner, and showed maximum value at 48 h in both protein and mRNA level. But there was no dose-dependent increas. 2. MMP-2 production time-dependently increased when stimulated with 1 and 10 ${\mu}g/ml$LPS, but there was no dose-dependent increase. 3. TIMP-1 p개duction increased to 24 h, but decreased at 48 h. It increased when stimulated with 0.1 and 1${\mu}g/ml$, but suppressed at 10 ${\mu}g/ml$ .4. P. nigrescens LPS pretreated with $Ca(OH)_2$ markedly downregulated MMP-1 gene expression.

• Restorative Dentistry and Endodontics
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• v.26 no.4
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• pp.350-359
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• 2001

목적 - 근관형성시 근관내 상아질벽에는 항상 도말층(smear layer)이 형성되는데, 이는 상아질, 치수조직 잔사, 조상아세포 돌기, 때로는 미생물 등으로 구성되며, 주사전자현미경상으로는 비규칙적이며, 무정형의 구상(granular)구조물로 관찰된다. 본 연구에서는 도말층의 유무에 따른 혐기성 미생물, Prevotella nigrescens의 근관내 상아질 부착정도를 주사전자현미경으로 평가하고자 한다. 지금까지 사용되었던 실험방법에 비하여 보다 임상적 환경에 가까운 실험방법을 고안, 신빙성 있는 연구결과를 기대할 수 있도록 하였다. 방법 - 치주질환이나 외상 등의 원인으로 발거된 상, 하악 전치 18개를 사용하였다. 각 치아의 치관부를 백악법랑경계부위에서 절단하고, 1군(5개치아)은 10ml의 생리식 염수를, 2군(5개치아)과 3군(5개치아)은 10ml의 3.5% NaOCl을 근관관주용액으로 사용하여 근관형성을 하였다. 근관형성 완료후 1군과 2군은 10ml의 생리식염수로, 3군은 10ml의 0.5M EDTA용액으로 final flush를 시행하여, 3군의 도말층을 제거하였다. 치근수직절단과 ethylene oxide(EO) gas 소독후 1, 2, 3군의 시편(각군10개시편)을 Prevotella nigrescens가 부유된 Brain Heart Infusion with Yeast estract, Hemin and Menadione(BHIYHM) broth내에 37$^{\circ}C$에서 3시간 동안 incubation했다. 4, 5, 6군은 실험과정을 검증하기 위한 대조군으로써, 4군(1개치아)과 5군(1개치아)은 1군, 2군과 같이 각각 생리식염수와 NaOCl만을 이용한 근관형성으로 도말층을 잔존시키고, 6군(1개치아)은 3군과 같이 NaOCl과 EDTA를 적용하여 도말층을 제거한 후, 치근 수직절단과 EO gas 소독을 시행했다. 모든 시편(1, 2, 3, 4, 5, 6군)을 통상의 방법에 따라 처리한 후 주사전자현미경을 통하여 관찰, 근관내면에 부착되어 있는 Prevotella nigrescens의 개수, 모양, 상아세관 및 도말층과의 관계 등을 관찰, 비교, 분석하였다. 결과는 t-test와 one-way ANOVA를 통하여 통계처리 하였다. 결론 - 1. 근관형성 후 근관내 상아질 표면 전체는 도말층으로 덮여 있는 양상을 보였다. 2. 3.5% NaOCl과 0.5M EDTA를 적용하여 근관내 도말층을 효과적으로 제거할 수 있었으며, 상아세관 개구부가 확연히 노출되어 있는 소견을 관찰할 수 있었다. 3. 도말층이 덮인군에서 미생물의 부착이 유의성 있게 높았다(P<0.05). 4. 근관 형성중 형성되어 근관 상아질을 덮고 있는 도말층이 미생물의 부착을 증가시켜, 근관 재감염의 기회를 증가시킴을 알 수 있었다.

• Restorative Dentistry and Endodontics
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• v.27 no.3
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• pp.232-238
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• 2002

연구목적 : Cytokine은 유해 미생물에 대한 숙주의 방어기전으로서의 염증반응에서 숙주세포 상호간의 작용을 매개해 주는 역할을 하며, 인간의 치수조직에서도 그 존재가 확인된 바 있다. Interleukin-6와 Interleukin-10은 염증의 초기에 작용하는 cytokine으로 알려져 있으나, 치수 및 치근단 질환에서의 역할과 상호작용에 대해서는 잘 알려져 있지 않다. 본 연구에서는 치수염의 원인균으로 알려진 Prevotella nigrescens를 이용하여 백서의 치수염을 유도한 후 시간의 변화에 따른 Interleukin-6, Interleukin-10의 농도의 변화를 측정하여 이들의 치수염에서의 작용을 알아보는 것을 목적으로 한다. 방법 : 실험적으로 치수의 염증반응을 일으키기 위하여 치수염의 원인균으로 알려진 Prevotella nigrescens를 이용하였다. 실험동물의 하악절치의 incisal tip부분을 절단한 후(n=120), 치수강을 개방시켰다. 실험군에서는 Prevotella nigrescens를 멸균된 면구에 묻혀서 개방된 치수강 내에 접종하였으며, 대조군에서는 균을 접종하지 않고 멸균된 면구만을 개방된 치수강 내에 위치시켰다. 그 후 1, 2, 5일이 경과되었을 때 실험에 사용된 치아를 발치하여, 치수조직을 적출하였다. Amersham사의 ELISA kit를 사용하여 적출된 치수조직내의 Interleukin-6와 Interleukin-10의 양을 측정하였으며 그 결과를 Mann-Whitney rank sum test를 사용하여 통계학적 유의성을 검증하였다. 조직학적 검사를 위해서는 발치된 치아를 nitric acid를 사용하여 탈회시킨 후 헤마톡실린-에오신 염색을 시행한 후 관찰하였다. 결과 : 1) Interleukin-6의 농도는 균접종 후 1일, 2일, 5일 모두에서 실험군에서 대조군보다 높게 나타났으며, 균접종 1일째의 결과는 통계적 유의성이 있었다(P<0.05). 2) Interleukin-10의 농도는 균접종 후 1일, 2일, 5일 모두에서 실험군에서 대조군보다 높게 나타났으며, 균접종 1일째의 결과는 통계적 유의성이 있었다(P<0.05). 3) Interleukin-10/1nterleukin-6 ratio는 실험군과 대조군 모두에서 1일보다 2일째의 결과에서 더 높은 값을 보였으며 대조군에서는 통계적 유의성을 보였다(P<0.05). 4)조직학적 관찰결과 균접종 후 2일째의 조직표본에서는 림프구의 침윤과 부분적인 조직의 괴사 등 염증반응의 양상을 관찰할 수 있었으며, 균접종 5일째의 조직표본에서는 염증의 정도가 감소되는 양상을 확인할 수 있었다.

• Journal of the korean academy of Pediatric Dentistry
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• v.33 no.2
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• pp.304-310
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• 2006

Prevotella intermedia is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. This study has identified a hemin-binding P. intermedia protein by expression of a P. intermedia genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. The genomic library of P. intermedia was constructed into plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$, and screened for recombinant clones using heminbinding activity by plating onto hemin-containing agar. Approximately 5,000 recombinant E. coli colonies were screened onto LB-amp-hemin agar, single clone(pHem1) was exhibited a clearly pigmented phonotype. The 2.5kb insert DNA of pHem1 was determined by restriction enzyme mapping. Southern blot analysis of BamHI, BglII, EcoRI, HindIII and PstI-digested P. intermedia DNA indicated that single copy of the gene was present in the genome. Northern blot analysis revealed that the size of transcript was approximately 1.8 kb. The cloned gene contained a single ORF, consisting of approximately 850-residue amino acids. A BLAST search of the Institute for Genomic Research genes with similar nucleotide sequence revealed no significant similarity It needs further investigation to clarify the mechanisms of heme uptake in P. intermedia.