• International Journal of Oral Biology
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• 제39권3호
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• pp.131-136
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• 2014

Porphyromonas gingivalis is one of the most important periodontal pathogens and has been to known to invade various types of cells, including endothelial cells. The present study investigated the mechanisms involved in the internalization of P. gingivalis in human umbilical vein endothelial cells (HUVEC). P. gingivalis internalization was reduced by clathrin and lipid raft inhibitors, as well as a siRNA knockdown of caveolin-1, a principal molecule of lipid raft-related caveolae. The internalization was also reduced by perturbation of actin rearrangement, while microtubule polymerization was not required. Furthermore, we found that Src kinases are critical for the internalization of P. gingivalis into HUVEC, while neither Rho family GTPases nor phosphatidylinositol 3-kinase are required. Taken together, this study indicated that P. gingivalis internalization into endothelial cells involves clathrin and lipid rafts and requires actin rearrangement associated with Src kinase activation.

• 대한수의학회지
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• 제51권1호
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• pp.15-20
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• 2011

In this study, we isolated 12 of Brucella (B.) spp. from cattle, which have been positive in Rose Bangal test and tube agglutination test in Gyeongbuk province in 2009. According to AMOS PCR analysis, isolated 12 strains were identified as B. abortus. Murine derived macrophage, RAW 264.7 cells, were infected with isolated 12 strains or reference strain (B. abortus 544), and bacterial internalization were characterized. According to these results, we divided the isolated strains into the following three groups: class I, lower internalization than that of B. abortus 544; class II, similar internalization to that of that of B. abortus 544; class III, higher internalization than that of B. abortus 544 within RAW 264.7 cells. Furthermore, intracellular growth, bacterial adherent assay, LAMP-1 colocalization, virulence in mice and surface protein pattern were characterized. From these results, representative strains of class III showed lower LAMP-1 colocalization, higher adherent efficiency, higher virulence in mice than those of B. abortus 544, and showed different pattern of surface proteins. These results suggest that B. abortus field strains, isolated from cattle in Korea, possess various virulence properties and higher internalization ability of field strain may have an important role for its virulence expression.

• Journal of Microbiology and Biotechnology
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• 제27권8호
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• pp.1483-1490
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• 2017

In this study, silver nanoparticles (AgNPs) were synthesized by the citrate reduction process and, with the assistance of n-hydroxysuccinimide and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, were successfully loaded with the macromolecular drug vancomycin (VAM) to form AgNP-VAM bioconjugates. The synthesized AgNPs, VAM, and AgNP-VAM conjugate were characterized by UV-visible spectroscopy, zeta potential analysis, confocal microscopy, and transmission electron microscopy. The effect of loading VAM onto AgNPs was investigated by testing the internalization of the bioconjugate into Mycobacterium smegmatis. After treatment with the AgNP-VAM conjugate, the bacterial cells showed a significant decrease in UV absorption, indicating that loading of the VAM on AgNPs had vastly improved the drug's internalization compared with that of AgNPs. All the experimental assessments showed that, compared with free AgNPs and VAM, enhanced internalization had been successfully achieved with the AgNP-VAM conjugate, thus leading to significantly better delivery of the macromolecular drug into the M. smegmatis cell. The current research provides a new potential drug delivery system for the treatment of mycobacterial infections.

• 한국어병학회지
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• 제20권1호
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• pp.71-81
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• 2007

넙치, Paralichthys olivaceus 병어에서 분리된 Photobacterium damselae subsp. damselae와 Vibrio spp.(V. anguillarum, V. splendidus, V. harveyi and V. ordalii)의 병원성을 in vitro에서 조사하였다. Extracellular products (ECPs)의 성상은 효소 활성, 양과 넙치 적혈구에 대한 용혈능과 cell-line에 대한 세포 독성을 조사하였다. 그리고 세균의 internalization에서 signal transduction pathways를 signal transduction inhibitor를 이용하여 조사하였다.P. damselae는 phospholipase 활성, 넙치 적혈구에 대한 용혈능, 세포독성이 높게 나타났으며, 분리된 vibrios 균주에 비하여 internalization에서 다양한 경로를 나타내었다. 따라서 이러한 활성은 P. damselae의 병원성과 관련이 있을 것으로 생각된다.

• Journal of Microbiology and Biotechnology
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• 제30권4호
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• pp.482-489
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• 2020

We previously identified β-sitosterol (BS) as one of the most abundant compounds found in Korean red ginseng oil. BS is a widely prevalent vegetable-derived phytosterol with many known health benefits. Here, we investigated the efficacy of BS against Brucella (B.) abortus infection. BS showed no effect on bacterial growth but attenuated internalization, intracellular survival and MAPKs-linked intracellular signaling in RAW264.7 cells. BS treatment in cells is also associated with increased nitrite concentration during infection at 24 h. Slightly enhanced resistance to B. abortus infection was observed in mice orally given BS, which could be mediated by induced production of proinflammatory cytokines. Taken together, our study demonstrates the contribution of BS treatment against B. abortus infection although further investigation is encouraged to maximize its beneficial effects against intracellular infection.

• Journal of Veterinary Science
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• 제22권2호
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• pp.18.1-18.12
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• 2021

Background: We previously elucidated the protective mechanism of Korean red ginseng oil (RGO) against Brucella abortus infection, and our phytochemical analysis revealed that palmitic acid (PA) was an abundant component of RGO. Consequently, we investigated the contribution of PA against B. abortus. Objectives: We aimed to investigate the efficacy of PA against B. abortus infection using a murine cell line and a murine model. Methods: Cell viability, bactericidal, internalization, and intracellular replication, western blot, nitric oxide (NO), and superoxide (O₂^-) analyses and flow cytometry were performed to determine the effects of PA on the progression of B. abortus infection in macrophages. Flow cytometry for cytokine analysis of serum samples and bacterial counts from the spleens were performed to determine the effect of PA in a mouse model. Results: PA did not affect the growth of B. abortus. PA treatment in macrophages did not change B. abortus uptake but it did attenuate the intracellular survivability of B. abortus. Incubation of cells with PA resulted in a modest increase in sirtuin 1 (SIRT1) expression. Compared to control cells, reduced nitrite accumulation, augmented O₂^-, and enhanced pro-inflammatory cytokine production were observed in PA-treated B. abortus-infected cells. Mice orally treated with PA displayed a decreased serum interleukin-10 level and enhanced bacterial resistance. Conclusions: Our results suggest that PA participates in the control of B. abortus within murine macrophages, and the in vivo study results confirm its efficacy against the infection. However, further investigations are encouraged to completely characterize the mechanisms involved in the inhibition of B. abortus infection by fatty acids.

• International Journal of Oral Biology
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• 제46권3호
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• pp.99-104
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• 2021

Associations between periodontal infection and cardiovascular disease have been documented. Porphyromonas gingivalis is a well-established periodontal pathogen, and tissue factor (TF) is a key initiator of the coagulation cascade. In this context, P. gingivalis has been reported to enhance TF expression in human endothelial cells. The present study investigated the underlying mechanisms of TF induction by P. gingivalis in human umbilical vein endothelial cells. P. gingivalis increased TF expression in a dose- and time-dependent manner. Not only live bacteria but also glutaraldehyde-fixed bacteria increased TF expression to the same extent. However, sonicates of P. gingivalis did not induce TF expression. Cytochalasin D and SMIFH2, which are inhibitors of actin polymerization and actin nucleation, respectively, inhibited the TF expression induced by P. gingivalis. Finally, TF production was decreased or increased in the presence of various signaling inhibitors, including mitogen-activated protein kinases. These results suggest that P. gingivalis induces endothelial TF expression by a bacterial internalization-dependent mechanism and through diverse signal transduction mechanisms.

• Journal of Microbiology and Biotechnology
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• 제27권3호
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• pp.616-623
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• 2017

Ginsenoside Rg3, a saponin extracted from ginseng, has various pharmacological and biological activities; however, its effects against Brucella infection are still unclear. Herein, the inhibitory effects of ginsenoside Rg3 against intracellular parasitic Brucella infection were evaluated through bacterial infection, adherence assays, and LAMP-1 colocalization, as well as immunoblotting and FACS for detecting MAPK signaling proteins and F-actin polymerization, respectively. The internalization, intracellular growth, and adherence of Brucella abortus in Rg3-treated RAW 264.7 cells were significantly decreased compared with the Rg3-untreated control. Furthermore, an apparent reduction of F-actin content and intensity of F-actin fluorescence in Rg3-treated cells was observed compared with B. abortus-infected cells without treatment by flow cytometry analysis and confocal microscopy, respectively. In addition, treating cells with Rg3 decreased the phosphorylation of MAPK signaling proteins such as ERK 1/2 and p38 compared with untreated cells. Moreover, the colocalization of B. abortus-containing phagosomes with LAMP-1 was markedly increased in Rg3-treated cells. These findings suggest that ginsenoside Rg3 inhibits B. abortus infection in mammalian cells and can be used as an alternative approach in the treatment of brucellosis.

• Tuberculosis and Respiratory Diseases
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• 제64권1호
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• pp.22-27
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• 2008

연구배경: 본 연구에서는 황색포도알균이 숙주세포 내로 침입하여 증식하는 정도를 관찰하고자 한다. 이때 세포 외 바탕 단백질의 변화가 수반될 것으로 가설을 설정하고, 이러한 변화에 영향을 미칠 것으로 생각되는 matrix metalloproteinase (MMP)의 발현과 역할에 대해 연구하고자 하였다. 방법: 황색포도알균은 $10^6{\sim}10^7CFU/ml$을 $10^5$개의 기관지상피세포인 BEAS-2B 세포에 2시간 동안 침입시킨다. 이후 세척으로 세포 밖에 있는 황색포도알균을 제거한 후, BEAS-2B 세포를 다양한 시간 동안(4, 6, 8, 12 시간) 배양한 후 황색포도알균의 집락수(CFU/ml)를 측정하였고, 단백질을 분리하여 세포 외 바탕단백질의 발현 정도와 MMP의 활성도를 측정하였다. 또한 MMP 억제제인 GM6001을 전처치한 후 황색포도알균을 세포에 침입시킨 후 세포 내에서의 균의 집락수 및 세포 외 바탕단백질의 변화를 관찰하였다. 결과: 황색포도알균의 집락을 측정한 결과 4시간과 12시간을 비교해 볼 때 MOI가 증가할수록, 감염시킨 시간이 길수록 숙주세포 내로 침입이 유의하게 증가하였다. BEAS-2B 세포에서 황색포도알균을 침입시킨 시간이 길수록, MOI가 증가할수록 MMP 2 및 MMP 9의 활성도와 dysadherin의 발현은 증가하였고, 이와는 대조적으로 E-cadherin의 발현은 감소하였다. MMP억제제인 GM6001을 전 처치 한 결과 황색포도알균의 세포 내 침입을 유의하게 감소시켰다. 결론: 황색포도알균이 기관지 상피세포 내로 침입할 때 dysadherin 및 E-cadherin 같은 세포 외 바탕 단백질의 변화를 동반하며, MMP 활성도가 균의 세포 내 침입에 관여하는 것으로 보인다.

• International Journal of Oral Biology
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• 제37권3호
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• pp.137-145
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• 2012

Aggregatibacter actinomycetemcomitans is the most important etiologic agent of aggressive periodontitis and can interact with endothelial cells. Monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8) are chemokines, playing important roles in periodontal pathogenesis. In our current study, the effects of A. actinomycetemcomitans on the production of MCP-1 and IL-8 by human umbilical vein endothelial cells (HUVEC) were investigated. A. actinomycetemcomitans strongly induced the gene expression and protein release of both MCP-1 and IL-8 in a dose- and time-dependent manner. Dead A. actinomycetemcomitans cells were as effective as live bacteria in this induction. Treatment of HUVEC with cytochalasin D, an inhibitor of endocytosis, did not affect the mRNA up-regulation of MCP-1 and IL-8 by A. actinomycetemcomitans. However, genistein, an inhibitor of protein tyrosine kinases, substantially inhibited the MCP-1 and IL-8 production by A. actinomycetemcomitans, whereas pharmacological inhibition of each of three members of mitogen-activated protein (MAP) kinase family had little effect. Furthermore, gel shift assays showed that A. actinomycetemcomitans induces a biphasic activation (early at 1-2 h and late at 8-16 h) of nuclear factor-${\kappa}B$ (NF-${\kappa}B$) and an early brief activation (0.5-2 h) of activator protein-1 (AP-1). Activation of canonical NF-${\kappa}B$ pathway ($I{\kappa}B$ kinase activation and $I{\kappa}B-{\alpha}$ degradation) was also demonstrated in these experiments. Although lipopolysaccharide from A. actinomycetemcomitans also induced NF-${\kappa}B$ activation, this activation profile over time differed from that of live A. actinomycetemcomitans. These results suggest that the expression of MCP-1 and IL-8 is potently increased by A. actinomycetemcomitans in endothelial cells, and that the viability of A. actinomycetemcomitans and bacterial internalization are not required for this effect, whereas the activation of protein tyrosine kinase(s), NF-${\kappa}B$, and AP-1 appears to play important roles. The secretion of high levels of MCP-1 and IL-8 resulting from interactions of A. actinomycetemcomitans with endothelial cells may thus contribute to the pathogenesis of aggressive periodontitis.