• Journal of Microbiology and Biotechnology
• /
• 제25권8호
• /
• pp.1390-1400
• /
• 2015

Quorum sensing is a process of cell-to-cell communication in which bacteria produce autoinducers as signaling molecules to sense cell density and coordinate gene expression. In the present study, a LuxI-type synthase, AnoI, and a LuxR-type regulator, AnoR, were identified in Acinetobacter nosocomialis, an important nosocomial pathogen, by sequence analysis of the bacterial genome. We found that N-(3-hydroxy-dodecanoyl)- _L -homoserine lactone (OH-dDHL) is a quorum-sensing signal in A. nosocomialis. The anoI gene deletion was responsible for the impairment in the production of OH-dDHL. The expression of anoI was almost abolished in the anoR mutant. These results indicate that AnoI is essential for the production of OH-dDHL in A. nosocomialis, and its expression is positively regulated by AnoR. Moreover, the anoR mutant exhibited deficiency in biofilm formation. In particular, motility of the anoR mutant was consistently and significantly abolished compared with that of the wild type. The deficiency in the biofilm formation and motility of the anoR mutant was significantly restored by a functional anoR, indicating that AnoR plays important roles in the biofilm formation and motility. Furthermore, the present study showed that virstatin exerts its effects on the reduction of biofilm formation and motility by inhibiting the expression of anoR. Consequently, the combined results suggest that AnoIR is a quorum-sensing system that plays important roles in the biofilm formation and motility of A. nosocomialis, and virstatin is an inhibitor of the expression of anoR.

• BMB Reports
• /
• 제35권5호
• /
• pp.508-512
• /
• 2002

Phospholipase C-gamma-2 ($PLC{\gamma}2$) activation is a key signaling event for many cell functions. In order to delineate the pathways that lead to $PLC{\gamma}2$ activation, we devised a quick method for obtaining sufficient $PLC{\gamma}2$. We obtained the full-length cDNA for human $PLC{\gamma}2$ and expressed it in E. coli using the expression vector pT5T. To enhance the protein expression, tandem AGG-AGG arginine codons at the amino acid positions 1204-1205 were replaced by CGG-CGG arginine codons. The protein expression was detected in a Western blot analysis by both anti-$PLC{\gamma}2$ antibodies and the antibodies that are raised against the tripeptide epitope (Glu-Glu-Phe) tag that are genetically-engineered to its carboxyl terminal. Crude lysates that were prepared from bacteria that express $PLC{\gamma}2$ were found to catalyze the hydrolysis of phosphatidylinositol 4,5 bisphosphate. Similar to previous reports on $PLC{\gamma}2$ that is isolated from mammalian tissue, the recombinant enzyme was $Ca^{2+}$ dependent with optimal activity at 1-10 uM $Ca^{2+}$.

• Biomolecules & Therapeutics
• /
• 제19권3호
• /
• pp.302-307
• /
• 2011

Peptidoglycan (PGN) is detected in inflammatory cell-rich regions of human atheromatous plaques. The present study investigated the effects of PGN on CC chemokine ligand 3 (CCL3) expression, which is elevated in the atherosclerotic arteries, and determined cellular factors involved in PGN-mediated CCL3 up-regulation in mononuclear cells, with the goal of understanding the molecular mechanisms of inflammatory responses to bacterial pathogen-associated molecular patterns in diseased arteries. Exposure of human monocytic leukemia THP-1 cells to PGN resulted in enhanced secretion of CCL3 and profound induction of the CCL3 gene transcript. Both events were abrogated by oxidized 1-palmitoyl-2-arachidonosyl-sn-phosphatidylcholine, an inhibitor of Toll-like receptors 2/4. Pharmacological inhibitors such as U0126, SP6001250, Akt inhibitor IV, rapamycin, RO318220, diphenyleneiodonium chloride, and N-acetylcysteine also significantly attenuated PGN-mediated CCL3 up-regulation. However, polymyxin B, LY294002, and SB202190 did not influence CCL3 expression. We propose that PGN contributes to enhanced CCL3 expression in atherosclerotic plaques and that Toll-like receptors (TLR2), Akt, mTOR, mitogen-activated protein kinase, and reactive oxygen species are involved in that process.

• 대한의생명과학회지
• /
• 제15권3호
• /
• pp.257-260
• /
• 2009

Toll-like receptors (TLRs) recognize molecular structures derived from microbes including bacteria, viruses, yeast, and fungi. TLRs have emerged as a major signaling component of the mammalian host defense. TLR4 is a member of the Toll family that senses lipopolysaccharide (LPS), a cell wall component of gram negative bacteria. LPS recognition by TLR4 requires an additional accessory molecule, MD-2. LPS induces the activation of NF-${\kappa}B$ and IRF3 through MyD88 or TRIF-dependent pathways. The activation of NF-${\kappa}B$ leads to the induction of inflammatory gene products including cytokines and cyclooxygenase-2 (COX-2). This study was carried out to investigate the anti-inflammatory effects of oak wood vinegar. Oak wood vinegar inhibits the NF-${\kappa}B$ activation and COX-2 expression induced by LPS. These results provide new ideas to understand the mechanism of oak wood vinegar for its anti-bacterial and anti-inflammatory activities.

• 한국약용작물학회지
• /
• 제15권6호
• /
• pp.451-456
• /
• 2007

Curcumin, a polyphenolic antioxidant purified from turmeric, has been known to possess various biological activities such as anti-oxidative, anti-inflammatory and anti-cancer effects. In this study, we have explored anti-inflammatory effect of curcumin using Gram (-) bacterium-derived endotoxin (lipopolysaccharide: LPS) and macrophage cell line RAW264.7. Curcumin suppressed NO production in LPS-activated RAW264.7 cells in a dose-dependent manner, Curcumin also blocked the activation of $NF-{\kappa}B$ but not AP-1 according to luciferase assay. Furthermore, this compound suppressed the phosphorylation of a series of intracellular signaling components such as Src, JAK-2, Akt, IKK and $I{\kappa}B{\alpha}$ under LPS stimulation in a time dependent manner, Therefore, our data suggest that curcumin was able to protect the host from Gram(-) bacterial-infection-mediated inflammatory symptoms.

• Bulletin of the Korean Chemical Society
• /
• 제25권12호
• /
• pp.1863-1868
• /
• 2004

Kringle 5 (K5), located outside of angiostain (K1-4) in human plasminogen, displays more potent antiangiogenic activity on endothelial cell proliferation than angiostatin itself. Using a yeast two-hybrid system in vivo, we have recently identified Rgl2 (guanine nucleotide dissociation stimulator (RalGDS)-like factor 2) as a binding protein of human K5. In order to confirm in vitro protein interaction between K5 and Rgl2, we developed bacterial recombinant expression systems for them. K5 and Rgl2 proteins were expressed in high yields and purified into pure forms with His tags and GST fusion, respectively. GST-pull down experiments clearly demonstrated that K5 interacts specifically with Rgl2 in vitro. These results indicate that Rgl2 functions as a receptor protein for K5 in vitro as well as in vivo, leading to anti-angiogenesis through regulating Ras signaling pathways.

• IMMUNE NETWORK
• /
• 제14권6호
• /
• pp.307-320
• /
• 2014

Mycobacterium scrofulaceum is an environmental and slow-growing atypical mycobacterium. Emerging evidence suggests that M. scrofulaceum infection is associated with cervical lymphadenitis in children and pulmonary or systemic infections in immunocompromised adults. However, the nature of host innate immune responses to M. scrofulaceum remains unclear. In this study, we examined the innate immune responses in murine bone marrow-derived macrophages (BMDMs) infected with different M. scrofulaceum strains including ATCC type strains and two clinically isolated strains (rough and smooth types). All three strains resulted in the production of proinflammatory cytokines in BMDMs mediated through toll-like receptor-2 and the adaptor MyD88. Activation of MAPKs (extracellular signal-regulated kinase 1/2, and p38, and c-Jun N-terminal kinase) and nuclear receptor (NF)-${\kappa}B$ together with intracellular reactive oxygen species generation were required for the expression of proinflammatory cytokines in BMDMs. In addition, the rough morphotypes of M. scrofulaceum clinical strains induced higher levels of proinflammatory cytokines, MAPK and NF-${\kappa}B$ activation, and ROS production than other strains. When mice were infected with different M. scrofulaceum strains, those infected with the rough strain showed the greatest hepatosplenomegaly, granulomatous lesions, and immune cell infiltration in the lungs. Notably, the bacterial load was higher in mice infected with rough colonies than in mice infected with ATCC or smooth strains. Collectively, these data indicate that rough M. scrofulaceum induces higher inflammatory responses and virulence than ATCC or smooth strains.

• Journal of Nutrition and Health
• /
• 제53권5호
• /
• pp.452-463
• /
• 2020

본 연구는 AT의 뿌리를 제외한 전체 AT의 에탄올 및 물 추출물의 면역 조절 효과를 비교하고 THP-1의 cytokine 분비를 조절하는 분자 메커니즘을 조사하였다. AT의 물 추출물 및 에탄올 추출물은 THP-1 세포에 독성이 없으며 세포 증식을 증가키는 것을 확인하였다. 에탄올 추출물은 영향이 없는데 반해, 물 추출물은 THP-1의 IL-1β의 분비를 증가시켰으며 COX-2 및 iNOS 단백질의 발현을 증가시켰다. 또한, MAPK 및 Akt의 인산화와 IkBα의 분해를 유도하는 것을 확인하였다. AT에 의한 IL-1β 분비는 ERK 및 JNK 억제제에 의해 감소되었으며, TNF-α의 분비는 ERK, p38 MAPK 및 JNK 억제제에 의해 감소되었다. 흥미롭게도, p38 MAPK 억제제는 AT에 의한 IL-1β의 생성을 추가로 증가시켰다. 이 결과는 AT 지상부의 물 추출물에 MAPK 신호 전달 경로를 통해 면역 세포를 자극하여 cytokine의 생산을 유도하는 생리활성물질이 존재한다는 것을 의미한다. 따라서, AT 지상부는 면역력 강화제의 천연 소재로써 이용될 수 있을 것으로 사료된다.

• 생명과학회지
• /
• 제26권7호
• /
• pp.764-771
• /
• 2016

개똥쑥 추출물은 항박테리아, 항바이러스 그리고 항산화효과를 포함한 다양한 기능을 가지고 있는 것으로 잘 알려져 있다. 그러나, 개똥쑥 항증식 작용기전은 알려지지 않았다. 따라서, 우리는 Hep3B 간암 세포에서 AAE추출물의 apoptotic 효과를 알아보고자 한다. 본 연구의 목적은 AAE가 인체 간암 세포주(Hep3B)의 증식에 미치는 효과를 분석하고 이에 대한 apoptosis의 효과를 조사하는데 있다. 인산화에 의해 활성화된 Akt는 TSC2, mTOR 그리고 GSK-3β의 인산화를 유도하여 세포증식을 유도한다. 본 연구에서, 우리는 AAE가Akt-mTOR-GSK3β 신호 경로와 mitochondria를 매개하는 apoptotic 단백질을 통한 암세포의 apoptosis 유도할 것이라고 추측하였다. 이를 위해, 먼저 AAE가 처리농도에 따라 세포증식에 미치는 효과를 분석하였다. AAE처리는 세포증식을 억제시켰을 뿐만 아니라 젖산 탈수소 효소의 방출을 유도하였다. 이러한 결과는 MTT assay, LDH assay로 확인하였다. 또한 Hoechst 33342 staining, Annexin Ⅴ- PI staining, JC-1 staining 그리고 Western blotting을 통해 apoptosis 효과를 확인하였다. 본 연구에서는 간암세포에 AAE의 처리가 Akt, TSC2, GSK-3β-phosphorylated, Bim, Bcl-2, pro-caspase 3의 억제와 Bak, Bax 활성을 유도한다는 것을 확인하였다. 이러한 결과는 AAE가 Akt-mTOR-GSK-3β 신호 경로를 통해 intrinsic apoptosis를 유도한다는 것을 나타낸다.

• IMMUNE NETWORK
• /
• 제12권6호
• /
• pp.284-290
• /
• 2012

Innate immune cells sense and respond to the cytoplasmic infection of bacterial pathogens through NLRP3, NLRC4 or AIM2 inflammasome depending on the unique molecular pattern of invading pathogens. The infection of flagellin- or type III secretion system (T3SS)-containing Gram-negative bacteria such as Salmonella enterica serovar Typhimurium (S. typhimurium) or Pseudomonas aeruginosa (P. aeruginosa) triggers NLRC4-dependent caspase-1 activation leading to the secretion of proinflammatory cytokines such as interleukin-1-beta (IL-$1{\beta}$) and IL-18. Previous studies have shown that apoptosis-associated speck-like protein containing a CARD (ASC) is also required for Salmonella-induced caspase-1 activation, but it is still unclear how ASC contributes to the activation of NLRC4 inflammasome in response to S. typhimurium infection. In this study, we demonstrate that S. typhimurium triggers the formation of ASC oligomer in a potassium depletion-independent manner as determined by in vitro crosslinking and in situ fluorescence imaging. Remarkably, inhibition of potassium efflux failed to block Salmonella-promoted caspase-1 activation and macrophage cell death. These results collectively suggest that ASC is substantially oligomerized to facilitate the activation of caspase-1 in response to S. typhimurium infection. Contrary to NLRP3 inflammasome, intracellular potassium depletion is not critical for NLRC4 inflammasome signaling by S. typhimurium.