• 한국동물위생학회지
• /
• 제25권4호
• /
• pp.333-346
• /
• 2002

We previously reported that the equine herpesvirus type 1(EHV-1) immediate-early protein(IE protein) physically interacts with the general transcription factor human TFIIB(Jang et al, J Virol 75：10219-10230, 2001). The interaction between the IE protein and TFIIB is necessary for the IE protein to efficiently transactivate the early TK and late IR5 EHV-1 promoters. A panel of deletion and truncation mutants of the TFIIB gene was constructed and employed in protein-binding assays to map the IE protein-binding domain within TFIIB. Evidence is presented that the first direct repeat of TFIIB interacts specifically with the EHV-1 IE protein.

• Journal of Microbiology and Biotechnology
• /
• 제24권12호
• /
• pp.1719-1727
• /
• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• 한국동물위생학회지
• /
• 제27권4호
• /
• pp.355-369
• /
• 2004

The equine herpesvirus type 1 (EHV-1) immediate-early (IE) protein is a potent transactivator responsible for the activation of both early and late genes during the course of infection and is comprised of discrete functional domains that mediate its many functions. Interaction between trans activators such as the IE protein and various components of the RNA polymerase II transcription initiation machinery has been demonstrated to be critical for transactivation. In the present report, it is addressed the hypothesis that the IE protein interacts with various components of transcription machinery to mediate transactivation of target viral genes. In these studies, it is demonstrated that in vitro transcribed and translated IE protein interacts with TFIIB-agarose conjugate but not with TFIID-agarose conjugate. Additional immunoprecipitation studies using nuclear extracts derived from EHV-1 infected RK-13 cells confirmed that the IE protein interacts strongly with TFIIB, but fails to interact with TFIID. IR2, a truncated form of the IE protein lacking the potent transactivation domain and involved in the down-regulation of the IE gene, also interacted with TFIIB but not with TFIID. Studies were also performed to ascertain if particular TBP-associated factors (TAFs) could mediate IE or IR2 binding to TFIID. In vitro transcribed and translated TAF250 added to nuclear extracts generated from EHV-1 infected cells also failed to mediate an interaction between the IE protein or the IR2 protein and TFIID. This study demonstrated that the IE protein mediates transactivation of target viral genes by a mechanism that involves TFIIB. This is in contrast to mechanisms that have been proposed for both the herpes simplex virus ICP4 and VP16 protein which have been proposed to transactivate viral genes through interactions involving both TFIIB and TFIID. This study also intimates that IR2 mediate its repressive effects during the course of EHV-1 infection by a mechanism that involves sequestration of various transcription factors.

• Pediatric Infection and Vaccine
• /
• 제6권2호
• /
• pp.253-260
• /
• 1999

목 적 : human CMV가 생성하는 분자량 72,000 dalton의 immediate early protein(p72)과 반응하는 단세포군 항체를 국내에서 분리된 CMV 야생주로 직접 제작하여 진단에 사용해 보기 위한 것이다. 방 법 : 정상 인체 섬유아세포(Foreskin 유래)를 10% 우태아혈청이 함유된 Eagle' s minimal essential medium(MEM)에서 배양하여 사용하였으며 마우스 골수종세포는 P3X63 Ag8.653(ATCC, Mary land USA)을 $5{\times}10^5/ml$ 세포수를 유지하도록 배양하였다. 사용된 항원은 선천성 CMV 감염아의 소변으로부터 검출한 CMV(KJHJ90)를 대량 증식시켜 사용하였다. 생후 6~8주된 Balb/c마우스의 복강내에 CMV 항원을 1주일 간격으로 4회 주사한 후 면역시킨 마우스 비장에서 얻은 임파구와 형질세포종세포로 최적발육기의 P3X63-Ag8.653을 사용하여 융합하였다. 융합세포를 배양한 후 간접면역형광항체법을 이용하여 항체가가 높은 것을 골랐다. 결 과 : 생산된 28종의 단세포군항체 중 그림 1과 같이 LPC12와 LPC23클론이 AD169에 감염된 세포의 핵에 특히 강하게 반응하였다. 정제된 AD169를 SDS-PAGE한 후 Western blotting을 하여 생산된 단세포군항체가 반응하는 항원의 분자량을 확인하였으며 그 중 LPC12와 LPC23클론에서 생성된 단세포군항체는 약 72KDA의 항원과 특이적으로 가장 강하게 반응하였다. 결 론 : 본 연구에서 제작된 LPC12와 LPC23 클론에서 생성된 단세포군 항체를 이용하여 선천성 CMV감염으로 확인된 소아의 소변을 이용하여 CMV AD 169 주 유래 항체(P63-27)과 비교한 DEAFF 검사상 동일한 결과를 확인한 바 LPC12와 LPC 23 클론 항체는 AD 169(P63-27)과 같이 CMV 감염의 조기진단에 사용할 수 있을 것으로 사료된다.

• 치위생과학회지
• /
• 제19권4호
• /
• pp.254-260
• /
• 2019

Background: The primary aims of periodontal disease treatment is to remove dental plaque and calculus, the main causes of tooth loss, and restore periodontal tissue destroyed by inflammation. Periodontal disease treatment should also help maintain the alveolar bone, alleviate inflammation, and promote periodontal ligament cell proliferation, which is essential for tissue regeneration. Conventional antibiotics and anti-inflammatories have adverse side effects, especially during long-term use, so there is a need for adjunct treatment agents derived from natural products. The purpose of this study was to investigate whether the herbal flavone baicalein has the osteogenic activity under inflammatory conditions, and assess the involvement of osteoblast immediate early response 3 (IER3) expression. Methods: Human osteoblastic MG-63 cells were cultured with the pro-inflammatory cytokines tumor necrosis factor α and interleukin 1β in the presence and absence of baicalein. Proliferation was assessed using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay, and expression of IER3 mRNA was assessed using real-time polymerase chain reaction. The expression of IER3 protein levels and activation of associated signal transduction pathways were assessed using western blotting. Results: Baicalein increased IER3 mRNA and protein expression synergistically. In addition, baicalein reversed the suppression of cell proliferation, and the downregulation of osteogenic transcription factor runt-related transcription factor 2 and osterix induced by pro-inflammatory cytokines. Baicalein also upregulated the phosphorylation of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK 1/2). The upregulation of IER3 by pro-inflammatory cytokines was blocked by pretreatment with inhibitors of AKT, p38, JNK, and ERK 1/2. Conclusion: Baicalein mitigates the deleterious responses of osteoblasts to pro-inflammatory cytokines. Further, IER3 enhanced the effect of baicalein via activation of AKT, p38, JNK, and ERK pathways.

• Journal of Veterinary Science
• /
• 제21권5호
• /
• pp.72.1-72.10
• /
• 2020

Background: Fenbendazole, a dewormer drug, is used widely in the clinical treatment of parasite infections in animals. Recent studies have shown that fenbendazole has substantial effects on tumor growth, immune responses, and inflammatory responses, suggesting that fenbendazole is a pluripotent drug. Nevertheless, the antiviral effects have not been reported. Fenbendazole can disrupt microtubules, which are essential for multiple viruses infections, suggesting that fenbendazole might have antiviral effects. Objectives: This study examined whether fenbendazole could inhibit bovine herpesvirus 1 (BoHV-1) productive infection in cell cultures. Methods: The effects of fenbendazole on viral production, transcription of the immediate early (IE) genes, viron-associated protein expression, and the cellular signaling PLC-γ1/Akt pathway were assessed using distinct methods. Results: Fenbendazole could inhibit BoHV-1 productive infections significantly in MDBK cells in a dose-dependent manner. A time-of-addition assay indicated that fenbendazole affected both the early and late stages in the virus replication cycles. The transcription of IE genes, including BoHV-1 infected cell protein 0 (bICP0), bICP4, and bICP22, as well as the synthesis of viron-associated proteins, were disrupted differentially by the fenbendazole treatment. The treatment did not affect the cellular signaling pathway of PLC-γ1/Akt, a known cascade playing important roles in virus infection. Conclusions: Overall, fenbendazole has antiviral effects on BoHV-1 replication.

• International Journal of Industrial Entomology and Biomaterials
• /
• 제3권1호
• /
• pp.43-49
• /
• 2001

We have cloned and characterized an immediate early-1 gene, iel, which is activated immediately upon entrance of the viral genome into the cell nucleus, from Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. This gene encodes a protein 584 amino acids with a predicted molecular weight of 67 kDa. The promoter and coding regions of BmNPV-K1 ie1 showed high homology with Autographa californica nuclear polyhedrosis virus and BmNPV T3 strain. The BmNPV-K1 ie1 was different from amino acid sequence at 4 positions in BmNPV T3. The location of ie1 gene in the BmNPV-K1 genome was confirmed by Southern blot analysis and its expression patterns at the transcriptional level in the infected cells were confirmed by Nerthern hybridization analysis.

• 한국응용약물학회:학술대회논문집
• /
• 한국응용약물학회 1993년도 제1회 추계심포지움 and 제2회 생리분자과학연구센터워크숍
• /
• pp.17-20
• /
• 1993

As a step toward a more complete understanding of the molecular actions of TNF, we prepared a cDNA library from TNF-treated human FS-4 fibroblasts and used differential hybridization to identify cDNA clones corresponding to mRNAs enriched in TNF-treated eells. In Quiescent FS-4 cells n induces an increase in the level of some mRNAs within 20 to 30 min. Some of these immediate-early response mRNAs are elevated only transiently for about 30 to 120 min, e. g., c-fos and c-myc (Lin and Vilcek,1987) or the transcription factor IRF-1 (Fujita et al.1989). Such immediate-early gene products may be important for the activation of other genes, but their transient induction suggests that they are not the actual effector molecules responsible for the phenotypic changes induced by TNF. We chose a 3-h incubation with W because we were seeking cDNAs corresponding to messages that are more stably elevated after TNF treatment. Indeed, the results shown in Figure 8 and 9 indicate that all of the mRNAs corresponding to the eight TSG cDNAs isolated remained significantly elevated after 16h of continuous treatment with TNF, and their kinetics of induction were clearly different from those of the immediate-early response mRNAs such as c-fos, c-myc or IRF-1. Nevertheless, only the induction of TSG-21 (collagenase) and TSG-27 (stromelysin) nNAs was completely inhibited by cycloheximide and the induction of TSG-37 (metallothionein-II) was reduced in the presence of this inhibitor of protein synthesis. Induction of the other five TSG mRNAs by TNF was completelyresistant to cycloheximide, suggest ins that no protein intermediate is needed for the upregulation of these mRNAs.

• Archives of Pharmacal Research
• /
• 제26권5호
• /
• pp.375-382
• /
• 2003

Effects of ginsenosides on nitric oxide (NO) production induced by lipopolysaccharide plus TNF-$\alpha$ (LNT) were examined in C6 rat glioma cells. Among several ginsenosides, ginsenoside Rd showed a complete inhibition against LNT-induced NO production. Ginsenoside Rd attenuated LNT-induced increased phosphorylation of ERK. Among several immediate early gene products, only Jun Band Fra-1 protein levels were increased by LNT, and ginsenoside Rd attenuated Jun Band Fra-1 protein levels induced by LNT. Furthermore, LNT increased AP-1 DNA binding activities, which were partially inhibited by ginsenoside Rd. Our results suggest that ginsenoside Rd exerts an inhibitory action against NO production via blocking phosphorylation of ERK, in turn, suppressing immediate early gene products such as Jun Band Fra-1 in C6 glioma cells.

• Biotechnology and Bioprocess Engineering:BBE
• /
• 제10권5호
• /
• pp.444-450
• /
• 2005

Epidermal growth factor (EGF) activates many intracellular effector molecules, which subsequently influence the expression levels of many genes involved in cell growth, apoptosis and signal transduction, etc. In this study, the early response of gene expressions due to EGF treatment was monitored using oligonucleotide DNA microarrays in rat schwannoma cell lines. An immunoblotting experiment showed the successful activation of EGF receptors and an effector protein, STAT5, due to EGF treatment. The microarray study showed that 35 genes were significantly induced and 2 were repressed within 60 min after the treatment. The list of induced genes included early growth response 1, suppressor of cytokine signaling 3, c-fos, interferon regulatory factor 1 and early growth response 2, etc. According to the microarray data, six of these were induced by more than 10-fold, and showed at least two different induction patterns, indicating complicated regulatory mechanisms in the EGF signal transduction.