• IMMUNE NETWORK
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• 제2권2호
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• pp.91-95
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• 2002

Background: In order to characterize human antibodies with specificity for mite allergens at the molecular level, a scFv phage display library was constructed using peripheral blood mononuclear lymphocytes from an asthma patient allergic to mite as Ig gene sources. Methods: Immunoglobulin $V_H$ and V gene fragments were obtained by polymerase chain reaction, and randomly combined in pCANTAB-5E vector. The resulting human scFv phage display library had $3{\times}10^4$ independent clones, and biopanning was performed with house dust mite extracts. Results: Four scFv clones specific for house dust mite extract were isolated. Immunoblot assay showed that our clones reacted to 25 kDa and 50~60 kDa proteins with unknown identity in mite extracts. Sequence analysis indicated that two clones (b7 and c15) are identical, and all clones belong to human $V_H3$ subgroup. On the other hand, light chain usage was different in that two clones (a2 and b7 / c15) belonging to V ${\kappa}4$ subgroup, but a4 used V ${\kappa}1$ light chain gene. Conclusion: Our approach should facilitate provision of useful information on the antibody responses against allergens at the molecular level in humans.

• The Korean Journal of Physiology and Pharmacology
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• 제19권5호
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• pp.441-449
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• 2015

Flavonoids are plant pigments that have been demonstrated to exert various pharmacological effects including anti-cancer, anti-diabetic, anti-atherosclerotic, anti-bacterial, and anti-inflammatory activities. However, the molecular mechanisms in terms of exact target proteins of flavonoids are not fully elucidated yet. In this study, we aimed to evaluate the anti-inflammatory mechanism of scutellarein (SCT), a flavonoid isolated from Erigeron breviscapus, Clerodendrum phlomidis and Oroxylum indicum Vent that have been traditionally used to treat various inflammatory diseases in China and Brazil. For this purpose, a nitric oxide (NO) assay, polymerase chain reaction (PCR), nuclear fractionation, immunoblot analysis, a kinase assay, and an overexpression strategy were employed. Scutellarein significantly inhibited NO production in a dose-dependent manner and reduced the mRNA expression levels of inducible NO synthase (iNOS) and tumor necrosis factor (TNF)-${\alpha}$ in lipopolysaccharide (LPS)-activated RAW264.7 cells. In addition, SCT also dampened nuclear factor (NF)-${\kappa}B$-driven expression of a luciferase reporter gene upon transfection of a TIR-domain-containing adapter-inducing interferon-${\beta}$ (TRIF) construct into Human embryonic kidney 293 (HEK 293) cells; similarly, NF-${\kappa}B$ nuclear translocation was inhibited by SCT. Moreover, the phosphorylation levels of various upstream signaling enzymes involved in NF-${\kappa}B$ activation were decreased by SCT treatment in LPS-treated RAW264.7 cells. Finally, SCT strongly inhibited Src kinase activity and also inhibited the autophosphorylation of overexpressed Src. Therefore, our data suggest that SCT can block the inflammatory response by directly inhibiting Src kinase activity linked to NF-${\kappa}B$ activation.

• Asian-Australasian Journal of Animal Sciences
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• 제16권10호
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• pp.1397-1401
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• 2003

The aim of this study was to determine genotypes of four genetic markers and to investigate their association with milk production traits in Brown Swiss cattle imported to Slovakia. The bovine $\kappa$-casein, $\beta$-lactoglobulin, growth hormone and prolactin genotypes of 107 cows were identified by polymerase chain reaction. Effects all four genetic markers on milk, fat, protein and lactose yields and fat, protein and lactose percentage were estimated from a data set of 249 lactations. The frequency of desirable B allele of $\kappa$-casein gene to milk production was 0.46, alleles A of $\beta$-lactoglobulin gene was 0.55, allele and L of growth hormone gene was 0.45 and allele A and B of bovine prolactin gene were 0.61 and 0.39. The results of milk production obtained in our work showed that BB genotypes of $\kappa$-CN gene, AA genotypes of $\beta$-LG gene, LL genotypes of bGH gene were significantly associated with better milk production traits, mainly about the fat content. Association of a bovine prolactin genotypes with milk production were not found.

• Natural Product Sciences
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• 제23권1호
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• pp.1-4
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• 2017

Sinensetin, a pentamethoxyflavone, is known to exert various pharmacological activities including anti-angiogenesis, anti-diabetic and anti-inflammatory activities. However, its effects on the human mast cell - 1 (HMC-1) mediated inflammatory mechanism remain unknown. To explore the mediator and cellular inflammatory response of sinensetin, we examined its influence on phorbol 12-myristate 13-acetate (PMA) plus A23187 induced inflammatory mediator production in a human mast cell line. In this study, interleukin (IL)-6 production was measured using the enzyme-linked immunosorbent assay and reverse transcription polymerase chain reaction. Sinensetin inhibited PMA plus A23187 induced IL-6 production in a dose-dependent manner as well as IL-4, IL-5 and IL-8 mRNA expression. Furthermore, sinensetin inhibited signal transducer and activator of transcription 3 (STAT3) phosphorylation, suggesting that sinensetin inhibits the production of inflammatory mediators by blocking STAT3 phosphorylation. Moreover, sinensetin was found to inhibit nuclear factor kappa B activation. These findings suggest that sinensetin may be involved in the regulation of mast cell-mediated inflammatory responses.

• Molecules and Cells
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• 제46권7호
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• pp.430-440
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• 2023

Linear ubiquitin chain assembly complex (LUBAC) is a ubiquitin E3 ligase complex composed of HOIP, HOIL-1L, and SHARPIN that catalyzes the formation of linear/M1-linked ubiquitin chain. It has been shown to play a pivotal role in the nuclear factor (NF)-κB signaling induced by proinflammatory stimuli. Here, we found that tumor susceptibility gene (TSG101) physically interacts with HOIP, a catalytic component of LUBAC, and potentiates LUBAC activity. Depletion of TSG101 expression by RNA interference decreased TNFα-induced linear ubiquitination and the formation of TNFα receptor 1 signaling complex (TNF-RSC). Furthermore, TSG101 facilitated the TNFα-induced stimulation of the NF-κB pathway. Thus, we suggest that TSG101 functions as a positive modulator of HOIP that mediates TNFα-induced NF-κB signaling pathway.

• Asian-Australasian Journal of Animal Sciences
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• 제32권11호
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• pp.1776-1788
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• 2019

Objective: The demands for measures to improve disease resistance and productivity of livestock are increasing, as most countries prohibit the addition of antibiotics to feed. This study therefore aimed to uncover functional feed additives to help enhance livestock immunity and disease resistance, using Acanthopanax sessiliflorus fruit extract (ASF). Methods: ASF was extracted with 70% EtOH, and total polyphenolic and catechin contents were measured by the Folin-Ciocalteu and vanillin assay, respectively. The 3D4/31 porcine macrophage cells ($M{\Phi}$) were activated by phorbol 12-myristate 13-acetate (PMA), and cell survival and growth rate were measured with or without ASF treatment. Flow-cytometric analysis determined the lysosomal activity, reactive oxygen species levels (ROS), and cell cycle distribution. Nuclear factor kappa B ($NF-{\kappa}B$) and superoxide dismutase (SOD) protein expression levels were quantified by western blotting and densitometry analysis. Quantitative polymerase chain reaction was applied to measure the lipid metabolism-related genes expression level. Lastly, the antibacterial activity of 3D4/31 $M{\Phi}$ cells was evaluated by the colony forming unit assay. Results: ASF upregulated the cell viability and growth rate of 3D4/31 $M{\Phi}$, with or without PMA activation. Moreover, lysosomal activity and intracellular ROS levels were increased after ASF exposure. In addition, the antioxidant enzyme SOD2 expression levels were proportionately increased with ROS levels. Both ASF and PMA treatment resulted in upregulation of $NF-{\kappa}B$ protein, tumor necrosis factor $(TNF){\alpha}$ mRNA expression levels, lipid synthesis, and fatty acid oxidation metabolism. Interestingly, co-treatment of ASF with PMA resulted in recovery of $NF-{\kappa}B$, $TNF{\alpha}$, and lipid metabolism levels. Finally, ASF pretreatment enhanced the in vitro bactericidal activity of 3D4/31 $M{\Phi}$ against Escherichia coli. Conclusion: This study provides a novel insight into the regulation of $NF-{\kappa}B$ activity and lipid metabolism in $M{\Phi}$, and we anticipate that ASF has the potential to be effective as a feed additive to enhance livestock immunity.

• 생명과학회지
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• 제22권11호
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• pp.1571-1586
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• 2012

지구상에는 약 5,400여 종의 포유동물이 있고 그 중 약 1,000여 종은 풀을 뜯어 먹고 사는 초식동물이다. 초식동물 중에서 약 250여 종이 반추동물로 알려져 있다. 반추동물인 소와 양은 반추위에서 주로 발효가 일어나지만 비반추동물인 돼지와 사람은 맹장과 결장, 직장에서 주로 발효가 일어난다. 반추위 미생물의 종류와 우점도 Bacteroidetes 51%, Firmicutes 43% 존재하며, 사람의 대장미생물의 우점도Firmicutes 65%, Bacteroidetes 25%로 존재한다. 풀의 세포벽 구성성분은 미생물에 의해 분해, 발효에 의해 SCFA (short chain fatty acid)를 생성하게 되고 acetate, propionate, butyrate 생성비율은 60:25:15이다. 장내 primary butyrate transporter인 MCT1(monocarboxylatetransports-1)에 의해서 흡수된 butyrate는 SCFA receptor GPR43과 GPR41을 활성화시킨다. Butyrate는 강력한 anti-tumorigenic 기능을 가지고 있다. Butyrate는 다양한 cancer cell에 효과를 나타내며 세포내의 기능 조절에 기여하고, 암세포사멸을 유도하는 특성이 있다. Butyrate는 caspase의 활성화, HDAC (histone deacetylase) 활성을 억제하여apoptosis를 유도하고, p53 발현증가로 cell cycle arrest와 apoptosis를 유도한다. SCFA의 항 염증작용으로는 장 상피세포에서 IL-8 발현 감소, NO합성과 NF-${\kappa}B$ (nuclear factor ${\kappa}B$)의 활성을 억제하여 염증으로 인한 암 발생을 억제한다. Butyrate는 장 점막의 생리적 기능을 유지하는데 중요한 역할을 하며 IBD (inflammatory bowel disease) 치료법으로 이용되고 있다.

• Advances in Traditional Medicine
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• 제10권3호
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• pp.155-164
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• 2010

Lycii fructus is the fruit of Lycium chinense Miller and is part of the Solanaceae family. Lycii fructus produces various effects such as hypotensive, hypoglycemic, anti-pyretic, and anti-stress activities. Lycii fructus is known to contain betaine, carotene, nicotinic acid, zeaxanthin, and cerebroside. In the present study, the effects of Lycii fructus aqueous extract on lipopolysaccharide (LPS)-induced inflammation in murine raw 264.7 macrophage cells were investigated. In this study we utilized the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reverse transcriptionpolymerase chain reaction (RT-PCR), Western blotting, and nitric oxide (NO) detection. Lycii fructus aqueous extract suppressed NO production by inhibiting the LPS-induced expressions of inducible nitric oxide synthase (iNOS) and tumor necrosis factor-alpha (TNF-$\alpha$) mRNA and iNOS protein in murine raw 264.7 macrophage cells. Also, Lycii fructus aqueous extract suppressed the activation of nuclear factor-kappa B (NF-${\kappa}B$) in the nucleus. These results demonstrated that Lycii fructus aqueous extract causes an anti-inflammatory effect that was likely produced by the suppression of iNOS expression through the down-regulation of NF-$\hat{e}B$ binding activity.

• 대한약침학회지
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• 제18권1호
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• pp.19-26
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• 2015

Objectives: Quercetin, a flavonoid compound, has been reported to induce apoptosis in cancer cells, but its anti-inflammatory effects, which are also closely linked with apoptosis, if any, on non-small-cell lung cancer (NSCLC) have not so far been critically examined. In this study, we tried to determine if quercetin had any demonstrable anti-inflammatory potential, which also could significantly contribute to inducing apoptosis in a NSCLC cell line, A549. Methods: In this context, several assays, including cytotoxicity, flow cytometry and fluorimetry, were done. Gene expression was analyzed by using a western blot analysis. Results: Results revealed that quercetin could induce apoptosis in A549 cells through mitochondrial depolarization by causing an imbalance in B-cell lymphoma 2/Bcl2 Antagonist X (Bcl2/Bax) ratio and by down-regulating the interleukine-6/signal transducer and activator of transcription 3 (IL-6/STAT3) signaling pathway. An analysis of the data revealed that quercetin could block nuclear factor kappa-light-chain-enhancer of activated B cells (NF-${\kappa}B$) activity at early hours, which might cause a down-regulation of the IL-6 titer, and the IL-6 expression, in turn, could inhibit p-STAT3 expression. Down-regulation of both the STAT3 and the NF-${\kappa}B$ expressions might, therefore, cause down-regulation of Bcl2 activity because both are major upstream effectors of Bcl2. Alteration in Bcl2 responses might result in an imbalance in the Bcl2/Bax ratio, which could ultimately bring about mitochondria mediated apoptosis in A549 cells. Conclusion: Overall, the finding of this study indicates that a quercetin induced anti-inflammatory pathway in A549 cells appeared to make a significant contribution towards induction of apoptosis in NSCLC and, thus, may have a therapeutic use such as a strong apoptosis inducer in cancer cells.

• Asian Pacific Journal of Cancer Prevention
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• 제17권5호
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• pp.2605-2610
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• 2016

The International Myeloma Working Group considers the serum free light chain (SFLC) assay to be an adjunct to traditional tests. Apart from the FLC ratio, the absolute values of individual free light chains also are gaining importance as they appear to be more relevant in certain clinical settings. Automated assays are available for their determination. As laboratories put new test systems into use catering to different disease populations, they are required by accreditation and certification bodies to verify or establish performance specifications, including reference intervals (RIs) representative of their population. Our aim was to establish local RIs for SFLC in a multicentre representative healthy population using a robust method. There was no significant relationship between SFLC levels and age, gender and creatinine levels. The 95% RI for ${\kappa}SFLC$ was 4.81 to 33.86mg/L, for ${\lambda}$ SFLC was 5.19 to 23.67mg/L and for ${\kappa}/{\lambda}SFLC$ was 0.36 to 2.33, significantly higher than the values given by the manufacturer. The ${\kappa}/{\lambda}$ SFLC ratio at 2.23, covering 100% of the data, showed 72% sensitivity (95% CI=39.0 - 94.0), 100% specificity (95% CI=71.5 - 100.0), 100% PPV (95% CI=21.5 - 100.0), 95% NPV (95% CI=75.4 - 99.9), and 79% accuracy (95% CI=56.0 - 93.0). In the patient group, kit RI for ${\kappa}/{\lambda}$ SFLC ratio classified 45.5% (n=5) as positive vs 9.1% (n=1) positive by the study RI, while the kit RI for kappa FLC classified 90.9% (n=10) as positive vs 54.5% (n=6), indicating increased probability of false positive test results with the kit RI when applied to our patient population. Appropriate and specific reference intervals and criteria values result in fewer false-positive and false-negative results which means fewer wrong or missed diagnoses.