Exosome-mediated delivery of gga-miR-20a-5p regulates immune response of chicken macrophages by targeting IFNGR2, MAPK1, MAP3K5, and MAP3K14

Hong Yeojin,Heo Jubi,강수연,부 티 하오,Lillehoj, Hyun S.,홍영호 아세아·태평양축산학회 2023 Animal Bioscience Vol.36 No.6

Objective: This study aims to evaluate the target genes of gga-miR-20a-5p and the regulated immune responses in the chicken macrophage cell line, HD11, by the exosome-mediated delivery of miR-20a-5p. Methods: Exosomes were purified from the chicken macrophage cell line HD11. Then, mimic gga-miR-20p or negative control miRNA were internalized into HD11 exosomes. HD11 cells were transfected with gga-miR-20a-5p or negative control miRNA containing exosomes. After 44 h of transfection, cells were incubated with or without 5 μg/mL poly(I:C) for 4 h. Then, expression of target genes and cytokines was evaluated by quantitative realtime polymerase chain reaction. Results: Using a luciferase reporter assay, we identified that gga-miR-20a-5p directly targeted interferon gamma receptor 2 (IFNGR2), mitogen-activated protein kinase 1 (MAPK1), mitogen-activated protein kinase kinase kinase 5 (MAP3K5), and mitogenactivated protein kinase kinase kinase 14 (MAP3K14). Moreover, the exosome-mediated delivery of gga-miR-20a-5p successfully repressed the expression of IFNGR2, MAPK1, MAP3K5, and MAP3K14 in HD11 cells. The expressions of interferon-stimulated genes (MX dynamin like GTPase 1 [MX1], eukaryotic translation initiation factor 2A [EIF2A], and oligoadenylate synthase-like [OASL]) and proinflammatory cytokines (interferongamma [IFNG], interleukin-1 beta [IL1B], and tumor necrosis factor-alpha [TNFA]) were also downregulated by exosomal miR-20a-5p. In addition, the proliferation of HD11 cells was increased by exosomal miR-20a-5p. Conclusion: The exosome-mediated delivery of gga-miR-20a-5p regulated immune responses by controlling the MAPK and apoptotic signaling pathways. Furthermore, we expected that exosomal miR-20a-5p could maintain immune homeostasis against highly pathogenic avian influenza virus H5N1 infection by regulating the expression of proinflammatory cytokines and cell death.

Expression in Escherichia coli and Purification of Folded rDer p 20, the Arginine Kinase From Dermatophagoides pteronyssinus: A Possible Biomarker for Allergic Asthma

Sarzsinszky Eszter,Lupinek Christian,Vrtala Susanne,Huang Huey-Jy,Hofer Gerhard,Keller Walter,Chen Kuan-Wei,Panaitescu Carmen Bunu,Resch-Marat Yvonne,Zieglmayer Petra,Zieglmayer René,Lemell Patrick,Ho 대한천식알레르기학회 2021 Allergy, Asthma & Immunology Research Vol.13 No.1

Arginine kinase (AK) was first identified as an allergen in the Indian-meal moth and subsequently shown to occur as allergen in various invertebrates and shellfish. The cDNA coding for AK from the house dust mite (HDM) species Dermatophagoides pteronyssinus, Der p 20, has been isolated, but no recombinant Der p 20 (rDer p 20) allergen has been produced and characterized so far. We report the expression of Der p 20 as recombinant protein in Escherichia coli. rDer p 20 was purified and shown to be a monomeric, folded protein by size exclusion chromatography and circular dichroism spectroscopy, respectively. Using AK-specific antibodies, Der p 20 was found to occur mainly in HDM bodies, but not in fecal particles. Thirty percent of clinically well-characterized HDM allergic patients (n = 98) whose immunoglobulin E (IgE) reactivity profiles had been determined with an extensive panel of purified HDM allergens (Der f 1, 2; Der p 1, 2, 4, 5, 7, 10, 11, 14, 15, 18, 21, 23 and 37) showed IgE reactivity to Der p 20. IgE reactivity to Der p 20 was more frequently associated with lung symptoms. AKs were detected in several invertebrates with specific antibodies and Der p 20 showed IgE cross-reactivity with AK from shrimp (Litopenaeus vannamei). Thus, Der p 20 is a cross-reactive HDM allergen and may serve as a diagnostic marker for HDM-induced lung symptoms such as asthma.

Substance P Regulates Macrophage Inflammatory Protein 3α /CCL20 with Heme Oxygenase-1 in Human Periodontal Ligament Cells

Sun- Kyung Lee,Sung-Hee Pi,Sung -Hyun Kim,Kyung-San Min,Hwa-Jeong Lee,Hoon -Sang Chang,Kyung-Hwa Kang,Hyung-Ryong Kim,Hong-In Shin,Suk-Keun Lee,Eun-Cheol Kim 대한구강악안면병리학회 2007 대한구강악안면병리학회지 Vol.31 No.5

Although substance P (SP). a potent pro-inflammatory peptide, is involved in inflammation and immune responses, the effect of SP 011 the expression of macl'ophage inJlammatol'Y protein 3a (MIP-3a. CCL20) in periodontal ligament (PDL) cells a l'e unknown Equally as enigmatic is the link between SP. the stress protein heme oxygenase-l (HO-l) , and CCL20 product ion. We investigated whether SP induces the release of chemokine CCL20 from irrunortalized POL (IPDL) cells. and further claif’y SP mediated pathways . We also exarnined the relationship between HO-l and CCL20 by treating POL cells with SP Incubating IPOL cells with SP incl'eased ex pl'ession of CCL20 mRNA and CCL20 protein in a dose-time dependent manner. Highly selective p38 and ERKl/2 inhibitors abl'ogated SP-induced expression of CCL20 lD IPOL cells SP is also responsible fo l' ini tiating phosphorylation of I/( B‘ degl'adation of IK B. and activation of NF-/( B. SP induced expression of HO-l in both a concentration- and time-dependent manner. and CCL20 refl ected similal' patterns. The inductive effects of SP on HO-l and CCL20 were enhanced by HO- l inducer hemin and the membrane-permea ble cGMP analog 8-bromo-cGMP Conversely, this pathway was inhi bited by the HO-l inhibitor zinc Pl'otoporphyrin IX (ZnPP IX) and the selective inhibitor of guanylate cyclase‘ 1H- [1. 2. 4]uxad iazole[4, 3-alquinoxal i n- 1-one (ODQ) We report hel'ein the pathway that connects SP a long with other modulators 0 1' neuroimmunoregulationto the induction of HO-1 and the inflanunatol'y mediatol' MIP- 3a /CCL20 in IPDL cel ls. which play an impol'tant role in the development 0 1' pe- I'iodontitis or inflammation during ol'thodontic tooth movement

Roles of eIF4E-binding protein Caf20 in Ste12 translation and P-body formation in yeast

박기영,이유선,정대희,김진미 한국미생물학회 2018 The journal of microbiology Vol.56 No.10

Translation initiation factor eIF4E forms eIF4E-eIF4G complex at the 5’ cap of mRNA. This interaction can be inhibited by the family of 4E-binding proteins (4E-BP). In yeast Saccharomyces cerevisiae, two 4E-BPs, Caf20 and Eap1, compete with eIF4G for binding to eIF4E via the shared conserved interaction motif. In order to investigate the roles of Caf20 in gene-specific translational regulation and the formation of mRNA granules (P-bodies), we introduced substitution mutations, caf20-Y4A or caf20-L9A, in the eIF4E-binding motif for CAF20. Overexpression of the wild-type CAF20 showed an increased protein level of Ste12 transcription factor as well as highly developed P-body formation. However, 4E-binding site mutations of CAF20 led to a reduced number of P-body foci and decreased levels of Ste12 protein. The phenotypes of the caf20 deletion mutation were also analyzed, and we suggest that Caf20 plays a critical role in Ste12 protein expression and in the control of P-body formation.

pKT230 벡터를 이용한 Pseudomonas sp. P20 2,3-Dihydroxybiphenyl Dixygenase 유전자의 클로닝

김지영,김치경,가종억,민경희,박용근 한국미생물생명공학회 ( 구 한국산업미생물학회 ) 1996 한국미생물·생명공학회지 Vol.24 No.6

Biphenyl과 4-chlorobiphenyl을 분해하는 자연계 분리 균주인 Pseudomonas sp. P20의 chromosomal DNA로부터 pBluescript SK(+)를 이용하여 pcbABCD 유전자를 클로닝하여 재조합플라스미드 pCK1을 제조하였고, 또 pcbCD 유전자를 포함하여 pCK102을 제조하였다. 방향족 탄화수소 화합물의 생분해는 벤젠고리의 개환 과정이 중요하기 때문에, 2,3-dihydroxybiphenyl(2,3-DHBP)의 벤젠고리의 개환에 관여하는 2,3-DHBP dioxygenase(2,3-DHBD) 유전자를 pKT230 벡터를 이용하여 pCK102로부터 클로닝하였다. EcoRI으로 절단한 pCK102와 pKT230 벡터를 ligation시켜 13.8 kb의 hybrid plasmid pKK1을 제조하였다. 2,3-DHBD 유전자를 포함하는 pKK1을 E. coli XL1-Blue에 형질전환시켜 E. coli KK1 재조합 균주를 얻은 후, 2,3-DHBD의 활성을 측정하였다. E. coli KK1의 2,3-DHBD의 효소활성은 pBluescript SK(+)를 이용하여 제조한 재조합 균주인 E. coli CK102의 효소활성과 유사하였으나, Pseudomonas sp. DJ-12와 Pseudomonas sp. P20과 같은 자연계 분리균주보다 훨씬 높았다. Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2,3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2,3-DHBP dioxygenase activity. The specific 2,3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20

A Case of Partial Trisomy 20p Resulting from Meiotic Recombination of a Maternal Pericentric Inversion

강정은,박미영,전종근,이형두,황상현,이종윤 대한진단검사의학회 2012 Annals of Laboratory Medicine Vol.32 No.1

Here we report the cytogenetic and clinical manifestations observed in a patient with a rec(20)dup(20p)inv(20)(p11.2q13.3)mat. The patient was a full-term newborn girl with asymmetric intrauterine growth restriction and multiple congenital malformations, including a ventricular septal defect, pulmonary atresia, ambiguous genitalia, clinodactyly, and sacral dimpling. To our knowledge, this is the 4th report in the world and the 1st one in Korea of a patient with rec(20)dup(20p).

Characterization of the pcbD Gene Encoding 2-Hydroxy-6-Oxo-6-Phenylhexa-2,4-Dienoate Hydrolase from Pseudomonas sp.P20

Lim, Jong Chul,Lee, Jeong Ral,Lim, Jal Yun,Min, Kyung Rak,Kim, Chi Kyung,Kim, Young Soo 한국미생물 · 생명공학회 2000 Journal of microbiology and biotechnology Vol.10 No.2

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase catalyzes the hydrolytic cleavage of HOPDA to benzoate and 2-hydroxypenta-2,4-dienoate (HPD) during microbial catabolism of biphenyl and polychlorinated biphenyls. A HOPDA hydrolase gene (pcbD) was isolated from the genomic library of Pseudomonas sp. P20 and designated as pCNU1201; a 7.5-kb XbaI DNA fragment from Pseudomonas sp. P20 was inserted into the pBluescript SK(+) XbaI site. E. coli HB101 harboring pCNU1201 exhibited HOPDA hydrolase activity. The open reading frame (ORF) corresponding to the pcbD gene consisted of 855 base pairs with an ATG initiation codon and a TGA termination codon. The ORF was preceded by a ribosome-binding sequence of 5'-TGGAGC-3' and its G+C content was 55mol%. The pcbD gene of Pseudomonas sp. P20 was located immediately downstream of the pcbC gene encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase, and approximately 4-kb upstream of the pcbE gene encoding HPD hydratase. The pcbD gene was able to encode a polypeptide with a molecular weight of 31,732 containing 284 amino acid residues. The deduced amino acid sequence of the HOPDA hydrolase of Pseudomonas sp. P20 exhibited high identity (62%) with those of the HOPDA hydrolases of P putida KF715, P pseudoalcaligenes KF707, and Burkholderia cepacia LB400, and also significant homology with those of other hydrolytic enzymes including esterase, transferase, and peptidase.

4-Chlorobiphenyl을 분해하는 Pseudomonas sp. P20의 pcb 유전자군의 클로닝

남정현,김치경 한국산업미생물학회 1994 한국미생물·생명공학회지 Vol.22 No.4

Pseudomonas sp. P2O은 난분해성 방향족 탄화수소중의 한가지인 4-chlorobiphenyl(4CB)을 meta-cleavage를 거쳐 4-chlorobenzoic acid로 분해하는 세균이다. 이 세균의 chromosomal DNA로부터 14 kb의 EcoRI 절편을 pBluescript SK(+) vector를 이용하여 E. coli XL1-Blue에 클로닝하여 4CB 분해유전자를 포함하는 재조합 plasmid인 pCK1과 재조합균주인 E. coli CK1을 얻었다. E. coli CK1은 pcbAB 산물에 의하여 4CB를 분해하고 또 pcbCD 산물에 의하여 2,3-dihydroxybiphenyl(2,3-DHBP)을 분해하여 meta-clevage compound(MCP)와 benzoate를 생성하는 것을 resting cell assay에 의하여 확인하였다. pcbC의 발현은 Pseudomonas sp. P20과 재조합균주인 E. coli CK1에서 모두 지수말기에 가장 잘 되었으며, E. coli CK1에서 pcbC 유전자의 산물인 2,3-DHBP dioxygenase 활성은 Pseudomonas sp. P20에 비하여 2배 이상 높았다. 또 2,3-DHBP dioxygenase는 catechol과 3-methylcatechol을 기질로 했을 때 2,3-DHBP에 비하여 26∼31%의 활성을 나타내었으며, 4-methylcatechol과 4-chlorocatechol에 대해서는 활성이 없었다. Pseudomonas sp. P2O was a bacterial isolate which has the ability to degrade 4-chlorobiphenyl(4CB) to 4-chlorobenzoic acid via the process of meta-cleavage. The recombinant plasmid pCK1 was constructed by inserting the 14-kb EcoRI fragment of the chromosomal DNA containing the 4CB-degrading genes into the vector pBluescript SK(+). Subsequently, E. coli XL1-Blue was transformed with the hybrid plasmid producing the recombinant E. coli CK1. The recombinant cells degraded 4CB and 2,3-dihydroxybiphenyl(2,3-DHBP) by the pcbAB and pcbCD gene products, respectively. The pcbC gene was expressed most abundantly at the late exponential phase in E. coli CK1 as well as in Pseudomonas sp. P2O, and the level of the pcbC gene product, 2,3-DHBP dioxygenase, expressed in E. coli CK1 was about two-times higher than in Pseudomonas sp. P2O. The activities of 2,3-DHBP dioxygenase on catechol and 3-methylcatechol were about 26 to 31% of its activity on 2,3-DHBP, but the enzyme did not reveal any activities on 4-methylcatechol and 4-chlorocatechol.

pKT230 벡터를 이용한 Pseudomonas sp. P20의 2,3-Dihydroxybiphenyl Dioxygenase 유전자의 클로닝

김지영,김치경,가종억,민경희,박용근,Kim, Ji-Young,Kim, Chi-Kyung,Ka, Jong-Ok,Min, Kyung-Hee,Park, Yong-Keun 한국미생물 · 생명공학회 1996 한국미생물·생명공학회지 Vol.24 No.6

Pseudomonas sp. P20 isolated from the polluted environment is capable of degrading biphenyl and 4-chlorobiphenyl. The pcbABCD genes responsible for degradation of biphenyl and 4-chlorobiphenyl were cloned using pBluescript SK(+) from the chromosomal DNA of Pseudomonas sp. P20 to construct pCK1 and pCK102, harbouring pcbABCD and pcbCD, respectively. The 2, 3-DHBP dioxygenase gene, pcbC, was cloned again from pCK102 by using pKT230 which is known as a shuttle vector and pKK1 hybrid plasmid was constructed. The E. coli KK1 transformant obtained by transforming the pKK1 into E. coli XL1-Blue showed 2, 3-DHBP dioxygenase activity. The specific 2, 3-DHBP dioxygenase activity of E. coli KK1 was similar to that of the E. coli CK102, but much higher than those of the natural isolates, Pseudomonas sp. DJ-12 and Pseudomonas sp. P20.

Cloning and Expression of pcbC and pcbD Genes Responsible for 2, 3-Dihydroxybiphenyl Degradation from Pseudomonas sp. P20

Nam, Jung Hyun,Koh, Hee Mock,Kim, Chi Kyung 한국미생물 · 생명공학회 1995 Journal of microbiology and biotechnology Vol.5 No.2

Pseudomonas sp. P20 was shown to be capable of degrading biphenyl and 4-chlorobiphenyl (4CB) to produce the corresponding benzoic acids which were not further degraded. But the potential of the strain for biodegradation of 4CB was shown to be excellent. The pcbA, B, C and D genes responsible for the aromatic ring-cleavage of biphenyl and 4CB degradation were cloned from the chromosomal DNA of the strain. In this study, the pcbC and D genes specifying degradation of 2,3-dihydroxybiphenyl (2,3-DHBP) produced from biphenyl by the pcbAB-encoded enzymes were cloned by using pBluescript SK(+) as a vector. From the pCK102 (9.3kb) containing pcbC and D genes, pCK1022 inserted with a EcoRI-HindⅢ DNA fragment (4.1kb) carrying pcbC and D and a pCK1092 inserted with EcoRI-Xbal fragment (1.95 kb) carrying pcbC were constructed. The expression of pcbC and D in E. coli CK102 and pcbC in E. coli CK1092 was examined by gas chromatography and UV-vis spectrophotometry. 2.3-dihydroxybiphenyl was readily degraded to produce meta-cleavage product (MCP) by E. coli CK102 after incubation for 10 min, and then only benzoic acid(BA) was detected in the 24-h old culture. The MCP was detected in E. coli CK1022 containing pcbC and D genes (by the resting cells assay) for up to 3 h after incubation and then diminished completely in 8 h, whereas the MCP accumulated in the E. coli CK1092 culture even after 6 h of incubation. The 2,3-DHBP dioxygenases (product of pcbC gene) produced by E. coli CK1, CK102, CK1023, and CK1092 strains were measured by native PAGE analysis to be about 250 kDa in molecular weight, which were about same as those of Pseudomonas sp. DJ-12, P. pseudoalcaligenes KF707, and P. putida OU83.