• International Journal of Oral Biology
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• 제35권1호
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• pp.13-19
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• 2010

The DNA probes Pn17 and Pn34 were evaluated for their ability to specifically detect clinical strains of P. intermedia and P. nigrescens from a Korean population by dot blot hybridization. These probes were sequenced by extension termination and their specificity was determined by Southern blot analysis. The results revealed that the Pn17 sequence (2,517 bp) partially encodes an RNA polymerase beta subunit (rpoB) and that Pn34 (1,918 bp) partially encodes both rpoB (1-169 nts) and the RNA polymerase beta subunit (rpoB'; 695-1918 nts). These probes hybridized with both HindIII- and PstI-digested genomic DNAs from the strains of P. intermedia and P. nigrescens used in this study. Interestingly, each of the hybrid bands generated from the HindIII-digested genomic DNAs of the two bacterial species could be used to distinguish between them via restriction fragment length polymorphism. These results thus indicate that Pn17 and Pn34 can simultaneously detect P. intermedia and P. nigrescens.

• Journal of Periodontal and Implant Science
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• 제35권1호
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• pp.21-30
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• 2005

Matrix metalloproteinases (MMPs) are a family of host-derived proteolytic enzymes and implicated in the remodeling and degradation of extracellular matrix under both physiological and pathological conditions. Connective tissue degradation in periodontal diseases is thought to be due to excessive MMP activities over their specific inhibitors. The effects of lipopolysaccharide (LPS) from Prevotella intermedia, one of the major putative pathogens of periodontitis, on the expression of mRNA for MMPs and tissue inhibitors of metalloproteinases (TIMPs) in human gingival and periodontal ligament fibroblasts were examined by reverse transcriptase-polymerase chain reaction (RT-PCR). The expression of mRNAs encoding MMP-1, -2, -3, -10, and -14 was increased in human gingival fibroblasts treated with p. intermedia LPS, whereas MMP-11 and TIMP-2 mRNA expression was decreased in these cells stimulated with LPS. P. intermedia LPS increased the MMP-1, -2, -10, -11, and -14 mRNA expression and decreased TIMP-1 and -2 mRNA expression in human periodontal ligament fibroblasts. These findings imply that P. intermedia LPS may play an important role in the connective tissue degradation in periodontitis.

• Restorative Dentistry and Endodontics
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• 제22권1호
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• pp.413-427
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• 1997

Porphyromonas endodontalis and Prevotella intermedia are black-pigmented anaerobic gram negative rods which have been isolated from infected root canals and submucous abscesses of endodontic origin. And they are associated with clinical symptoms such as pain, percussion, and foul odor. It has been reported that there are 3 serotypes according to capsule membrane in P. endodontalis and 2 DNA homology groups and 3 serotypes in P. intermedia, but there is no data available regarding genetic diversity for the species P. endodontalis and P. intermedia. The purpose of this study is to investigate genetic diversities between individual strains of P. endodontalis and P. intermedia which are indistinguishable by serotyping and biotyping using bacterial DNA restriction endonuclease analysis. 45 teeth with at least one clinical symptoms, with single canal, and with pulp necrosis were sampled. For sampling bacteria, access cavity was prepared after disinfecting tooth and its surroundings. Then the paper point was inserted to the apex of the canal, leave there for 15 seconds, and finally it was placed into PRAS Ringer's solution and PBS solution. P. endodontalis and P. intermedia were identified by biochemical test and IIF after subculturing black and brown colonies which were produced after 7 days of incubation on BAP in anaerobic chamber. P. endodontalis and P. intermedia strains were grown in BHI broth and whole genomic DNA was extracted by phenol-chloroform extraction technique and digested by restriction endonuclease, Eco RI and Pst I. The resulting DNA fragments were separated by agarose gel electrophoresis, stained with EtBr and photographed under UV light. The results were as follows : 1. In both P. endodontalis and P. intermedia, different serotypes could be found within a root canal of same patient. 2. There were obvious genetic heterogeneity within a patient and within a serotype in both P. endodontalis and P. intermedia. 3. P. endodontalis serotype c, isolated from different patients, exhibited limited genotypic diversity.

• Restorative Dentistry and Endodontics
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• 제22권2호
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• pp.693-701
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• 1997

In 1992, Prevotella intermedia was shown to be comprised of another spoecies now known as Prevotella nigrescens. Strain ATCC 33563 is now designated the type strain of P. nigrescens while strain ATCC 25611 is remains the type strain of P. intermedia. The purpose of this study was to find the differences in protein profiles of P. intermedia and P. nigrescens, using sodium dodecyl sulfate polyacrylamide gel electrophoresis, which can be used for differentiation of those two species. A partial amino acid sequence of the 18.6 kDa protein band, which was specific in P. nigrescens, was also determined. The cellular proteins were extracted from the cell pellets of pure cultures of P. intermedia. and P. nigrescens by either sonication or being shaken continuously for 20 min at $21^{\circ}C$ with 1 % SDS or being boiled for 3 min with 1 % SDS. SDS-PAGE was performed according to the method of laemmli using either 12% (w/v) gels or 18% (w/v) gels. Results were as follows ; 1. The similar electrophoretic protein profiles were shown by 3 cellular protein extraction methods for each strain. (Fig. 1 and 2) 2. the 18.6 kDa band which was specific only in P. nigrescens could be used for the differentiation of P. intermedia. and P. nigrescens. (Fig. 1 and 2, Table 1) 3. A total of 4 different tryptic fragments from the 18.6 kDa protein were sequenced. the resulting amino acid sequences were fragment 1.GNPVNIGGEW, 2.FNVVR, 3.NYLT-VAPY, and 4.GGDNVTTYQVLPEIGYN. By comparison to the sequences of known proteins in the Swiss-Prot database and PIR database. 90 % matching between fragment 1 and serine hydroxymethyl transferase(P24060) in the Swiss-Prot, and 90% matching between fragment 1 and glycine hydroxymethyl transferase(S15203) in the PIR were shown, but the identity and function of the 18.6 kDa protein remains unknown.

• Animal Bioscience
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• 제34권7호
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• pp.1221-1234
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• 2021

Objective: Weaning is an important stage in the life of young mammals, which is associated with intestinal inflammation, gut microbiota disorders, and even death. β-Carotene displays anti-inflammatory and antioxidant activities, which can prevent the development of inflammatory diseases. However, whether β-carotene can affect intestinal microbiota remains unclear. Methods: Twenty-four piglets were distributed into four groups: the normal suckling group (Con), the weaning group (WG), the weaning+β-carotene (40 mg/kg) group (LCBC), and the weaning+β-carotene (80 mg/kg) group (HCBC). The serum, jejunum, colon, and faeces were collected separately from each group. The effects of β-carotene on the phenotype, overall structure, and composition of gut microbiota were assessed in weaning piglets. Results: The results showed that β-carotene improved the growth performance, intestinal morphology and relieved inflammation. Furthermore, β-carotene significantly decreased the species from phyla Bacteroidetes and the genus Prevotella, and Blautia, and increased the species from the phyla Firmicutes and the genera p-75-a5, and Parabacteroides compared to the WG group. Spearman's correlation analysis showed that Prevotella and Blautia were positively correlated, and Parabacteroides and Synergistes were negatively correlated with the levels of interleukin-1β (IL-1β), IL-6, and tumour necrosis factor-α (TNF-α), while p-75-a5 showed negative correlation with IL-6 in serum samples from piglets. Conclusion: These findings indicate that β-carotene could alleviate weaning-induced intestinal inflammation by modulating gut microbiota in piglets. Prevotella may be a potential target of β-carotene in alleviating the weaning-induced intestinal inflammation in piglets.

• Journal of Periodontal and Implant Science
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• 제32권2호
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• pp.269-280
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• 2002

The purpose of this study is to develop species-specific DNA probes and polymerase chain reaction (PCR) primers for detection and identification of Prevotella nigrescens (P. nigrescens) 9336. This study procedure includes (1) whole-genomic DNA extraction of P. nigrescens 9336 (2) construction of the genomic DNA library, (3) screening of strain-specific DNA probe by reverse Dot Hybridization method, (4) confirmation of strain-specific DNA probe by Southern blot analysis, (5) determination of nucleotide sequences of strain-specific DNA probe. Thirty-five restriction fragments of P. nigrescens 9336 genomic DNA digested with the Hind III were obtained. Reverse dot hybridization and Southern blot analysis data showed that three of them, Pn10, Pn23, and Pn35, could be P. nigrescens 9336-specific DNA probes. These data indicated that these DNA probes could be useful in detection and identification of the P. nigrescens 9336.

• Journal of Periodontal and Implant Science
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• 제40권3호
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• pp.119-124
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• 2010

Purpose: In addition to regulating body weight, leptin is also recognized for its role in the regulation of immune function and inflammation. The purpose of this study was to investigate the effect of leptin on Prevotella (P.) intermedia lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF)-$\alpha$ production in differentiated THP-1 cells, a human monocytic cell line. Methods: LPS from P. intermedia ATCC 25611 was prepared by the standard hot phenol-water method. THP-1 cells were incubated in the medium supplemented with phorbol myristate acetate to induce differentiation into macrophage-like cells. The amount of TNF-$\alpha$ and interleukin-8 secreted into the culture medium was determined by enzyme-linked immunosorbent assay (ELISA). TNF-$\alpha$ and Ob-R mRNA expression levels were determined by semi-quantitative reverse transcription-polymerase chain reaction analysis. Results: Leptin enhanced P. intermedia LPS-induced TNF-$\alpha$ production in a dose-dependent manner. Leptin modulated P. intermedia LPS-induced TNF-$\alpha$ expression predominantly at the transcriptional level. Effect of leptin on P. intermedia LPS-induced TNF-$\alpha$ production was not mediated by the leptin receptor. Conclusions: The ability of leptin to enhance P. intermedia LPS-induced TNF-$\alpha$ production may be important in the establishment of chronic lesion accompanied by osseous tissue destruction observed in inflammatory periodontal disease.

• International Journal of Oral Biology
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• 제40권4호
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• pp.205-210
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• 2015

Prevotella intermedia-specific quantitative real-time PCR (qPCR) primers were previously designed based on the nucleotide sequences of RNA polymerase ${\beta}$-subunit gene (rpoB). However, the several clinical strains isolated from Korean populations are not detectable by the qPCR primers. The purpose of this study was to develop new P. intermedia-specific qPCR primers based on the rpoB. The specificity of the primers was determined by conventional PCR with 12 strains of P. intermedia and 52 strains (52 species) of non-P. intermedia bacteria. The sensitivity of primers was determined by qPCR with serial dilutions of the purified genomic DNAs (40 ng to 4 fg) of P. intermedia ATCC $25611^T$. The data indicated that only P. intermedia strains were detected by the P intermedia-specific qPCR primers (RTPiF2/RTPiR2); in addition, as little as 40 fg of P. intermedia genomic DNA could be detected. These results suggest that these qPCR primers are useful in detecting P. intermedia from the bacterial infectious lesions including dental plaque and oral tissue lesions.

• Journal of Periodontal and Implant Science
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• 제41권3호
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• pp.157-163
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• 2011

Purpose: Curcumin is known to exert numerous biological effects including anti-inflammatory activity. In this study, we investigated the effects of curcumin on the production of interleukin-6 (IL-6) by murine macrophage-like RAW 264.7 cells stimulated with lipopolysaccharide (LPS) from Prevotella intermedia, a major cause of inflammatory periodontal disease, and sought to determine the underlying mechanisms of action. Methods: LPS was prepared from lyophilized P. intermedia ATCC 25611 cells by the standard hot phenol-water method. Culture supernatants were collected and assayed for IL-6. We used real-time polymerase chain reaction to detect IL-6 mRNA expression. $I{\kappa}B-{\alpha}$ degradation, nuclear translocation of NF-${\kappa}B$ subunits, and STAT1 phosphorylation were characterized via immunoblotting. DNA-binding of NF-${\kappa}B$ was also analyzed. Results: Curcumin strongly suppressed the production of IL-6 at both gene transcription and translation levels in P. intermedia LPS-activated RAW 264.7 cells. Curcumin did not inhibit the degradation of $I{\kappa}B-{\alpha}$ induced by P. intermedia LPS. Curcumin blocked NF-${\kappa}B$ signaling through the inhibition of nuclear translocation of NF-${\kappa}B$ p50 subunit. Curcumin also attenuated DNA binding activity of p50 and p65 subunits and suppressed STAT1 phosphorylation. Conclusions: Although further study is required to explore the detailed mechanism of action, curcumin may contribute to blockade of the host-destructive processes mediated by IL-6 and appears to have potential therapeutic values in the treatment of inflammatory periodontal disease.

• Animal Bioscience
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• 제37권2_spc호
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• pp.360-369
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• 2024

Ruminal methane production functions as the main sink for metabolic hydrogen generated through rumen fermentation and is recognized as a considerable source of greenhouse gas emissions. Methane production is a complex trait affected by dry matter intake, feed composition, rumen microbiota and their fermentation, lactation stage, host genetics, and environmental factors. Various mitigation approaches have been proposed. Because individual ruminants exhibit different methane conversion efficiencies, the microbial characteristics of low-methane-emitting animals can be essential for successful rumen manipulation and environment-friendly methane mitigation. Several bacterial species, including Sharpea, uncharacterized Succinivibrionaceae, and certain Prevotella phylotypes have been listed as key players in low-methane-emitting sheep and cows. The functional characteristics of the unclassified bacteria remain unclear, as they are yet to be cultured. Here, we review ruminal methane production and mitigation strategies, focusing on rumen fermentation and the functional role of rumen microbiota, and describe the phylogenetic and physiological characteristics of a novel Prevotella species recently isolated from low methane-emitting and high propionate-producing cows. This review may help to provide a better understanding of the ruminal digestion process and rumen function to identify holistic and environmentally friendly methane mitigation approaches for sustainable ruminant production.