• International Journal of Oral Biology
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• v.36 no.3
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• pp.109-116
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• 2011

Periodontitis results from the activation of host immune and inflammatory defense responses to subgingival plaque bacteria, most of which are gram-negative rods with lipopoly-saccharides (LPSs) in their cell walls. LPSs have been known to induce proinflammatory responses and recently it was reported also that they induce the expression of microRNAs (miRNAs) in host cells. In our current study therefore, we aimed to examine and compare the miRNA expression patterns induced by the LPSs of major periodontopathogens in the human gingival epithelial cell line, Ca9-22. The cells were treated with 1 ${\mu}g$/ml of E. coli (Ec) LPS or 5 ${\mu}g$/ml of an LPS preparations from four periodontopathogens Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi), Aggregatibacter actinomycetemcomitans (Aa), and Fusobacterium nucleatum (Fn) for 24 h. After small RNA extraction from the treated cells, miRNA microarray analysis was carried out and characteristic expression profiles were observed. Fn LPS most actively induced miRNAs related to inflammation, followed by Aa LPS, Pi LPS, and Ec LPS. In contrast, Pg LPS only weakly activated miRNAs related to inflammation. Among the miRNAs induced by each LPS, miR-875-3p, miR-449b, and miR-520d-3p were found to be commonly up-regulated by all five LPS preparations, although at different levels. When we further compared the miRNA expression patterns induced by each LPS, Ec LPS and Pi LPS were the most similar although Fn LPS and Aa LPS also induced a similar miRNA expression pattern. In contrast, the miRNA profile induced by Pg LPS was quite distinctive compared with the other bacteria. In conclusion, miR-875-3p, miR-449b, and miR-520d-3p miRNAs are potential targets for the diagnosis and treatment of periodontal inflammation induced by subgingival plaque biofilms. Furthermore, the observations in our current study provide new insights into the inflammatory miRNA response to periodontitis.

• Asian-Australasian Journal of Animal Sciences
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• v.25 no.5
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• pp.674-681
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• 2012

Two experiments were conducted to investigate the effects of supplemental glutamine on growth performance, plasma parameters and LPS-induced immune response of weaned barrows after castration. In experiment 1, forty-eight weaned male piglets were used and fed maize and soybean meal diets supplemented with 0 (Control) or 2% L-Gln (Gln+) for 25 days. The results indicated that the Gln+ group tended to increase average daily gain compared to control in stages of days 7 to 14 and 0 to 25. The Gln+ had significantly better feed efficiency than the control group did during days 14 to 25 and 0 to 25. The plasma blood urea nitrogen and alkaline phosphatase contents of Gln+ group were higher than those of the control group on day 14 post-weaning. In experiment 2, sixteen weaned male piglets were injected with E. coli K88+ lipopolysaccharide (LPS) on day 14 post-weaning. The results showed that the Gln+ group had lower concentrations of plasma adrenocorticotrophic hormone and cortisol than the control group on day 14 pre-LPS challenge. In addition, Gln+ group had higher plasma IgG concentration than the control group for pre- or post-LPS challenged on day 14 post-weaning. In summary, dietary supplementation of Gln was able to alleviate the stressful condition and inflammation associated with castration in weaned barrows, and to improve their immunity and growth performance in the early starter stage.

• Proceedings of the Korean Society of Applied Pharmacology
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• 1996.11a
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• pp.65-73
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• 1996

LPS/LOS, the compound found only in gram-negative bacterial outer membrane, plays important roles in bacterial maintenance as well as its pathogenesis. We isolated and characterized several genes required for NTHi 2019 LOS biosynthesis, which encode enzymes required for sugar substrate synthesis or the transfer of substrates to receptor molecules. The htrB gene, however, appears to have more complex role. It has acryltransferase activity as well as various other activity, which may control regulation of LOS biosynthesis as well as its pathogenicity. Evidences supporting the latter come from the observations that the lipid A of the B29 induced significantly less TNF ${\alpha}$ from macrophages than that of the wild type LOS (unpublished data). H. influenzae A2-htrB mutant strain was also significantly less invasive than the wild type strain. The structural similarities of the enterobacterial LPS and the Haemophilus LOS enabled us to isolate the NTHi 2019 genes involved in LOS biosynthesis genes by using the S. typhimurium LPS deep core mutants. While a similar approach has been used for E. coli, this technique for selection of an LPS phenotype has not been applied to nonenterobacterial species. The difficulties inherent in the molecular manipulation of organism such as Neisseria and Haemophilus species make this approach particularly attractive in the identification and cloning LOS genes. Studies on genetic features of LPS/LOS biosynthesis would be useful for understanding bacterial pathogenesis as well as for developing vaccines for these gram-negative pathogenic bacteria.

• Proceedings of the Korean Society of Developmental Biology Conference
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• 2003.10a
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• pp.119-119
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• 2003

분만후 젖소의 자궁내 미생물상을 조사하고 미생물로부터 분리한 Lipopolysaccharide를 적용하여 소의 번식효율 증진에 기여하고자 분만후 젖소의 도축장 유래 자궁을 채취하여 혐기적 상태에서 균분리 동정을 실시하였다. 균분리 동정을 위하여, 시료를 1cm$\times$1cm로 채취하여 혐기상태에서 거품이 생길 때까지 vortexing한 후 균액 300$\mu l$를 뽑아 혐기배지에 도말하였고 도말한 plate는 $37{\circ}C$ 혐기chamber에서 24시간 배양하였다. 혐기배지에서 자란 균의 colony를 따서 Mac, BHI+B, BHI 배지에 배양한 후 Gram stain을 실시하였다. BHI 배지에서 자란 균의 colony를 따서 BUA+B 배지에 계대배양하였고 BUA+B 배지에서 자란 균중에 가장 마지막으로 자란 균을 따서 An-IF에 넣고 탁도를 63%T로 맞춘 후 An micro plate에 100$\mu l$씩 분주하였다. 분주한 plate를 $37{\circ}C$ 혐기 chamber에서 20~24시간 동안 배양한 후 Biolog를 실시하였다. 시료의 UV측정을 위하여 Sonic Processor로 세포를 분쇄하였고 분쇄한 세포를 $4{\circ}C$에서 10,000rpm으로 10분간 원심분리한 후 상층액을 분리하여 0.45$\mu m$ 필터로 여과한 다음 여과액을 취하여 UV로 standard(E.coli O26 B6 LPS)와 sample(10배 및 20배 희석액)을 측정하였다. (중략)

• Journal of Veterinary Clinics
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• v.14 no.2
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• pp.215-222
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• 1997

Enrofloxacin-colistin combination, widely used in Gram negative infections in veterinary sector, was investigated in terms of MIC and initial killing rate using E coli k 88ab, Salmonella typhimurium, Pasteurella multocida type A, Bordetella bronchiseptica and Staphylococcus aureus as test organisms. On the basis of MICs of enrofloxacin-colistin combination against the above bacteria, killing rates of the combination of enrofloxacin and colistin at the ratio of 5:0, 4:1, 3:2, 1:1, 2:3, 1:4 and 0:5, indicated high and rapid antibacterial acitivities against all but Staphylococcus aureus R-209, with the number of bacteria reducing to less than one percent within two hours. At the MIC of enrofloxacin or colistin, both antibacterials showed the highest killing rates during 2-4 hours against Gram negatives such as E coli K88ab,Pasteurella multocida type A and Bodetella bronchiseptica but allowed the regrowth of the same pathogens thereafter. On the while, the combination of two antibacterials at a fourth MIC resulted in high killing rate without bacterial regrowth during 24 hours, suggesting the synergistic antivacterial effects. The combination, however, did not show favourable activity against Gram negatime S typhimurium and Gram positive S aureus ergistic antibacterial activity against Gram negatime pathogens but also colistin showed LPS-neutraization, we could suggest the combination should provide clinically positive therapeutic armarium in Gram negative infections.

• Korean Journal of Veterinary Research
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• v.45 no.2
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• pp.185-189
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• 2005

Actinobacillus pleuropneumoniae is the causative agent of a porcine contagious pleuropneumonia. Among several virulence factors including exotoxin (Apx toxins), LPS, transferrin-binding proteins, OMPs, and some proteases, Apx toxins have been major targets for the protection study. In this study, cloning and expression of A. pleuropneumoniae Apx I and Apx II toxin, which are produced by all highly virulent strains, were performed by Escherichia coli expression system. Genes coding Apx I and II toxin were amplified from the A. pleuropneumoniae serotype 5 genomic DNA using polymerase chain reaction and cloned to a prokaryotic expression vector, pRSET. Expression of the Apx I and Apx II coding sequences in E. coli resulted in the formation of insoluble inclusion bodies purified according to a denaturing purification protocol, which employs the use of guanidium. Recombinant proteins were purified using $Ni^{2+}$-charged resin affinity purification. This expression and purification system made it possible to produce Apx I and Apx II in large amounts for further immunologic studies.

• Asian-Australasian Journal of Animal Sciences
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• v.21 no.8
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• pp.1189-1195
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• 2008

A study was conducted with 48 weaned barrows ($28{\pm}3d$, $8.45{\pm}0.14kg$) to determine the effect of Achyranthes bidentata polysaccharide (ABPS) supplementation on pig performance, immunological, adrenal and somatotropic responses following Escherichia coli lipopolysaccharide (LPS) challenge. The experiment was a $2{\times}2$ factorial design; the main factors included diet (supplementation with 0 or 500 mg/kg ABPS) and immunological challenge (LPS or saline). On d 14 and 21 of the trial, pigs were given an intraperitoneal injection with either $100{\mu}g/kg$ BW of LPS or an equivalent amount of sterile saline. Blood samples were obtained 3 h after injection for analysis of tumor necrosis factor-${\alpha}$ (TNF-${\alpha}$), prostaglandin $E_2$ ($PGE_2$), cortisol, growth hormone (GH), insulin-like growth factor (IGF)-I and immunoglobulin G (IgG). On d 2 after LPS challenge, peripheral blood lymphocyte proliferation (PBLP) was measured. LPS administration decreased average daily feed intake (ADFI) (p<0.05), had a tendency to decrease average daily gain (ADG) (p<0.10) during both the first and second challenge periods and increased (p<0.05) feed:gain ratio only during the first challenge period. ABPS tended to improve ADG (p<0.10) during the first challenge period, and improved ADG (p<0.05) and tended to improve ADFI (p<0.10) during the second challenge period. ABPS did not affect feed:gain ratio. An interaction (p<0.05) between LPS challenge and diet was observed for the plasma concentrations of TNF-${\alpha}$, $PGE_2$ and cortisol after both LPS challenges such that, among LPS-treated pigs, pigs fed the ABPS diet were lower for these indices than those receiving the control diet. In contrast, pigs fed the ABPS diet had higher IGF-I (p<0.05) compared with those fed the control diet. No effect of diet, LPS challenge or both on GH and IgG was observed after both LPS administrations. LPS challenge increased PBLP when these cells were incubated with $8{\mu}g/ml$ of LPS during both the challenge periods, and did likewise when incubated with $8{\mu}g/ml$ of concanavalin A only after the first challenge. ABPS had no effect on PBLP. These data demonstrate that ABPS alters the release of pro-inflammatory cytokines following an immunological challenge, which might enable pigs to achieve better performance.

• Korean Journal of Pharmacognosy
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• v.50 no.3
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• pp.191-197
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• 2019

Sepsis, an infectious disease, is a life-threatening condition that arises when the response to infection causes injury to tissues and organs. The purpose of this study was to demonstrate whether ABHC-1 and ABHC-2, two functional extracts from herbal complex, have an anti-bacterial effect against Escherchia coli in vivo, in vitro experimental model. ABHC-1 and ABHC-2 showed the antibacterial activity against the bacteria by paper disc method. The minimum inhibitory concentration (MIC) was measured using alamar blue reagent. The MIC was shown at $60{\mu}g/ml$ from ABHC-1 and $500{\mu}g/ml$ from ABHC-2 against E. coli. We next examined the effect of ABHCs on the production of inflammatory cytokine, such as tumor necrosis $factor-{\alpha}$ ($TNF-{\alpha}$), which is related to the induction of inflammation, in RAW 264.7 cell. ABHC-1 and ABHC-2 increased $TNF-{\alpha}$ production of RAW 264.7 cell in a dose-dependent manner while two extract decreased $TNF-{\alpha}$ production in lipopolysaccharide (LPS)-stimulated RAW 264.7 cell in a dose-dependent manner. At a dose of $1{\times}10^8$ E. coli. i.p., non-treated mice were succumbed, while most of mice treated with ABHC-1 were survived. Therefore, our results suggest that ABHC-1 has anti-bacterial activity and can be a novel therapeutic agent against infectious diseases.

• BMB Reports
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• v.32 no.2
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• pp.147-153
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• 1999

Previously, we reported that the prothrombin kringle 2 (fragment 2), induced by LPS administration into rabbit, inhibited bFGF-stimulated BCE cell growth (Lee et al., 1998). In this study, we cloned and overexpressed the kringle 2 domain of rabbit and human prothrombin as a fusion protein with the pelB leader sequence in E. coli using the T7 promoter. The fusion protein was cleaved during translocation into the peri plasmic space, and cleaved recombinant protein was readily isolated from whole cell lysate by DEAE-Sepharose and Sephacryl S-200 gel filtration chromatography. Both the recombinant rabbit and human prothrombin kringle 2 showed very similar biochemical and functional characteristics to the rabbit prothrombin kringle 2 purified from rabbit serum, in terms of abnormal electrophoretic migration and endothelial cell growth inhibitory activity.

• International Journal of Industrial Entomology and Biomaterials
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• v.23 no.2
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• pp.231-238
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• 2011

Attacin is an insect antibacterial protein that plays an important role in immune response to injury and infection. In this report, we have isolated and characterized of cDNA encoding for the attacin from the immunized larvae of swallowtail butterfly, $Papilio$ $xuthus$. A full length cDNA of $P.$ $xuthus$ attacin was obtained by employing annealing control primer (ACP)-based differential display PCR and 5' RACE. The complete $P.$ $xuthus$ attacin cDNA was comprised of 949 bp encoding a 250 amino acid precursor. It contains a putative 18 amino acid signal peptide sequence, a 42 amino acid propeptide sequence, and a 190 amino acid mature protein with a theoretical molecular mass of 19904.01 and a pI of 9.13. The putative mature protein of $P.$ $xuthus$ attacin showed 48-52% and 24-30% identity in amino acid sequences with that of lepidopteran and dipteran insects, respectively. Semiquantitive RT-PCR results revealed that the transcript of $P.$ $xuthus$ attacin gene was up-regulated at significant levels after injection with bacterial lipopolysaccharide (LPS). We sub-cloned cDNA fragment encoding mature $P.$ $xuthus$ attacin into the expression vector, highly expressed in $E.$ $coli$ BL21 cells, and its antibacterial activity was analyzed. Recombinant $P.$ $xuthus$ attacin evidenced considerably antibacterial activity against Gram-negative bacteria, $E.$ $coli$ ML 35 and $Klebsiella$ $pneumonia$.