• 생명과학회지
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• 제28권7호
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• pp.802-810
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• 2018

노화란 나이가 들면서 생기는 몸의 변화로 검은 머리가 흰 머리로 변하는 백발화, 과산화 지질과 단백질로 구성되어 생성되는 검버섯, 주름 등으로 나타난다. 멜라닌은 tyrosine에서 DOPA를 거쳐 산화 중합반응에 의해 생성되는 고분자 물질로 인체 내에 존재하는 머리카락 및 피부 색을 결정한다. 이러한 멜라닌은 melanocyte에서 합성되며, 활성산소에 의해 과도하게 생성되면 노화를 일으킨다는 연구가 보고되고 있다. 본 연구의 목적은 항산화 효과 및 멜라닌 합성에 대한 바나나 껍질 추출물(MPEE)의 직접적인 효과를 밝히는 것이다. MPEE는 DPPH radical scavenging assay와 reducing power assay를 수행한 결과, 두 실험 모두 양성대조군인 vitamin C와 비슷한 항산화 활성을 나타내었다. 세포 실험에 앞서 세포 독성을 알아보기 위해 B16F1 세포에서 MTT assay를 수행하였다. MPEE는 $32{\mu}g/ml$ 이하의 농도에서 세포독성이 없는 것으로 나타났다. 또한, MPEE는 invitro에서 tyrosinase 활성과 DOPA-oxidation 뿐만 아니라 살아있는 세포에서 멜라닌 합성을 증가시켰다. 더욱이, $H_2O_2$로 세포를 노화시켜 L-DOPA 실험을 수행한 결과, MPEE는 멜라닌 합성을 증가시켰다. 단백질 수준의 발현을 위한 Western blot 분석을 수행한 결과, TRP-1, TPR-2와 SOD-2의 발현 수준은 MPEE의 존재 하에서 증가되었다. 이상의 결과는 MPEE가 항산화 소재로 멜라닌 합성을 촉진시킨다는 것을 암시하고 있다.

• The Plant Pathology Journal
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• 제26권1호
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• pp.25-31
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• 2010

Papaya ringspot virus (PRSV) causes the most widespread and devastating disease in papaya. Isolates of PRSV originating from different geographical regions in south India were collected and maintained on natural host papaya. The entire coat protein (CP) gene of Papaya ringspot virus-P biotype (PRSV-P) was amplified by RTPCR. The amplicon was inserted into pGEM-T vector, sequenced and sub cloned into a bacterial expression vector pRSET-A using a directional cloning strategy. The PRSV coat protein was over-expressed as a fusion protein in Escherichia coli. SDS-PAGE gel revealed that CP expressed as a ~40 kDa protein. The recombinant coat protein (rCP) fused with 6x His-tag was purified from E.coli using Ni-NTA resin. The antigenicity of the fusion protein was determined by western blot analysis using antibodies raised against purified PRSV. The purified rCP was used as an antigen to produce high titer PRSV specific polyclonal antiserum. The resulting antiserum was used to develop an immunocapture reverse transcription-polymerase chain reaction (IC-RT-PCR) assay and compared its sensitivity levels with ELISA based assays for detection of PRSV isolates. IC-RT-PCR was shown to be the most sensitive test followed by dot-blot immunobinding assay (DBIA) and plate trapped ELISA.

• 대한의생명과학회지
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• 제3권2호
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• pp.115-123
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• 1997

인간 $\alpha$-fetoprotein (AFP)은 간암, 위암, 생식기 종양 및 신경관 이상인 환자를 검사하고 진단하는데 유용한 지표로 알려져 있다. 본 연구에서는 사람의 AFP를 분리정제 하여 폴리클로날 항체를 생산하고 인간 혈장과 양수내의 AFP를 측정하기 위한 경쟁적 효소면역 분석법을 개발하고자 하였다. 친화크로마토그래피법과 SDS-polyacrylamide 전기영동법을 이 용하여 양수로부터 AFP를 분리하였다. 정제된 AFP를 토끼에 주사하여 폴리클로날 항체를 생산하였으며, 이중면역확산법과 Western blot 분석법을 사용하여 본 연구실에서 제조된 항체의 항원 특이성이 대단히 높음을 확인하였다. AFP와 항혈청을 이용하여 표준곡선을 얻었으며, 민감도는 5ng/ml이었고, 작용범위는 5~l,000ng/ml이었다. 분석내 CV는 4.5%이었고, 분석간 CV는 8.5%이었다. 따라서 이러한 결과로 보아 본 연구에서 개발된 경쟁적 효소면역분석법이 AFP를 측정하기에 적절하며, 간암 등의 기초연구에도 많은 기여를 할 것으로 생각된다.

• 대한의생명과학회지
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• 제21권2호
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• pp.69-76
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• 2015

Cervical cancer is the third most common cancer in women worldwide and there is a significant association between human papillomavirus (HPV) infection and cervical cancer. Certain HPV groups, labeled high-risk (HR) HPV groups, are strongly associated with malignancies of the human cervix. HPV prevalence and genotype distribution were analyzed using the REBA $HPV-ID^{(R)}$ (YD Diagnostics, Yongin, Korea) assay based on the reverse blot hybridization assay (REBA) with a total of 324 liquid-based cytology samples from women in Western Shandong Province, East China and results were compared with cytological diagnosis. Most of the HPV genotypes that were detected in high-grade cervical lesions were HR-HPV genotypes such as HPV 16, 18, 33, 53, and 58. The prevalence of these HR-HPV genotypes increased in high-grade cervical lesions. However, from low- to high-grade cervical lesions, the ability to detect LR-HPV genotypes decreased. Additionally, in general, the single HPV genotype infection rate increases in proportion to the severity of the lesion. The study findings suggest that a currently available preventive vaccine against HPV 16 and 18 may have limited effectiveness for prevention of all HPV infection in this province. Finally, based on these findings, these data could guide national or regional vaccination programs in the Western Shandong Province of East China to substantially reduce the burden of cervical lesions.

• BMB Reports
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• 제39권3호
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• pp.263-269
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• 2006

In the genome of Helicoverpa armigera single-nucleocapsid nucleopolyhedrovirus, open reading frame 39 (Ha39) is the only gene predicted to encode an RNA recognition protein. Computer analysis revealed that Ha39 homologues were found in 15 NPVs, but not in GVs. Its transcripts were detected from 3 through 72 hours post infection (h p.i.) using RT-PCR and Northern blot analysis. The protein was detected in infected-cell lysates from 6 h p.i. Western blot assay of ODV and BV preparations revealed that Ha39 encodes a structural protein associated with BVs. Additionally, immunofluorescence microscopy demonstrated that the protein was present within cytoplasm in virus-infected cells, but not in the nuclear region.

• 생명과학회지
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• 제20권4호
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• pp.519-527
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• 2010

Paclitaxel은 미세소관의 탈중합을 억제하는 시약으로 알려져 있다. Paclitaxel은 다양한 세포에서 세포 내 방추체를 안정화시킴으로써 유사분열 억제 및 세포사멸을 유도한다. 본 실험에서는 토끼 관절 연골세포에서 paclitaxel이 연골세포의 증식과 사멸에 미치는 효과에 대한 연구를 수행하였다. MTT assay를 수행한 결과 paclitaxel은 연골세포에서 농도 의존적으로 세포 증식을 억제한다는 것을 확인 할 수 있었으며, FACS analysis와 Western blot analysis를 수행한 결과, paclitaxel이 G2/M 정지를 유도하는 것을 확인하였다. 또한, paclitaxel이 비정상적인 세포 분열유도와 핵 단편분절 유도없이 일어나는 mitotic catastrophe 즉, caspase-3 비의존적인 세포사멸을 유도하였다. Paclitaxel을 처리한 세포에서 일어나는 이러한 mitotic catastrophe에 의한 세포 죽음은 G1/S기의 진행을 억제하는 시약인 thymidine을 처리하는 것에 의해 억제되는 것을 확인할 수 있었다. 이러한 결과를 종합해 볼 때, paclitaxel에 의한 토끼 관절 연골 세포에서의 세포 죽음은 caspase-3 비의존적인 mitotic catastrophe에 의해 일어나는 것으로 사료되어진다.

• Parasites, Hosts and Diseases
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• 제54권1호
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• pp.31-38
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• 2016

Specific gene expressions of host cells by spontaneous STAT6 phosphorylation are major strategy for the survival of intracellular Toxoplasma gondii against parasiticidal events through STAT1 phosphorylation by infection provoked $IFN-{\gamma}$. We determined the effects of small molecules of tyrosine kinase inhibitors (TKIs) on the growth of T. gondii and on the relationship with STAT1 and STAT6 phosphorylation in ARPE-19 cells. We counted the number of T. gondii RH tachyzoites per parasitophorous vacuolar membrane (PVM) after treatment with TKIs at 12-hr intervals for 72 hr. The change of STAT6 phosphorylation was assessed via western blot and immunofluorescence assay. Among the tested TKIs, Afatinib (pan ErbB/EGFR inhibitor, $5{\mu}M$) inhibited 98.0% of the growth of T. gondii, which was comparable to pyrimethamine ($5{\mu}M$) at 96.9% and followed by Erlotinib (ErbB1/EGFR inhibitor, $20{\mu}M$) at 33.8% and Sunitinib (PDGFR or c-Kit inhibitor, $10{\mu}M$) at 21.3%. In the early stage of the infection (2, 4, and 8 hr after T. gondii challenge), Afatinib inhibited the phosphorylation of STAT6 in western blot and immunofluorescence assay. Both JAK1 and JAK3, the upper hierarchical kinases of cytokine signaling, were strongly phosphorylated at 2 hr and then disappeared entirely after 4 hr. Some TKIs, especially the EGFR inhibitors, might play an important role in the inhibition of intracellular replication of T. gondii through the inhibition of the direct phosphorylation of STAT6 by T. gondii.

• 대한본초학회지
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• 제31권2호
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• pp.63-69
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• 2016

Objectives : The aerial parts of Agrimonia pilosa Ledeb (Agrimoniae Herba; AH) has been traditionally used as a Korean medicine to treatment of abdominal pain, sore throat, headaches, bloody discharge, parasitic infections and eczema. In this study, we investigated the effect of AH ethanol extract on lipopolysaccharide (LPS)-induced inflammation in RAW264.7 macrophage cells.Methods : AH was extracted by 30% ethanol (AH-E). Raw264.7 cells were treated with AH-E extract at different concentrations for 30 min and then stimulated with LPS (1㎍/㎖) or without for indicated times. Cell viability was measured by MTT assay, and nitric oxide (NO) production was measured by Griess assay. The expression of inflammatory mediators, iNOS and COX-2 and inflammatory cytokines, TNF-α, IL-1β, and IL-6 was detected by RT-PCR, and the phosphorylation of ERK1/2, p38 and JNK MAP kinases (MAPKs) was analyzed by Western blot. Also, the expression of NF-κB in nuclear and cytosol was detected by Western blot.Results : AH-E extract significantly decreased LPS-induced NO production in RAW264.7 cells. AH-E extract inhibited the mRNA expression of iNOS, COX-2, TNF-α, IL-1β, and IL-6 in LPS-stimulated cells with a dose-dependent manner. In addition, the phosphorylation of ERK, p38 and JNK MAPKs was also inhibited by AH-E extract. AP-E extract inhibited the nuclear translocation of NF-κB in LPS-stimulated cells.Conclusions : Our results suggest that AH-E extract has an anti-inflammatory activity in macrophages-mediated inflammation.

• 한국안광학회지
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• 제7권2호
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• pp.189-195
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• 2002

점안액 보존제 성분으로 이용되는 Benzalkonium Chloride(BAC)가 배양 각막 상피세포에 미치는 영향을 조사하기 위해 본 연구를 시행하였다. BAC를 세포에 0.0001%~0.01%로 15분 동안 처리하여 24시간 후 회복 효과를 조사하였다. MTT assay를 이용하여 세포 생존율을 산정하고 Hoechst 33342로 염색질 응축을 조사하였다. Fas 발현 여부를 조사하기 위해 western blot과 immunocytochemistry를 이용하였으며 DNA fragmentation은 agarose gel을 이용한 전기영동을 시행하였다. BAC를 0.005% 이상 처리하면 세포괴사가 일어나는 반면에 저농도의 BAC는 세포고사를 유도하는 것으로 나타났으며 BAC로 Fas 발현이 유도될 수 있다는 것을 보여주고 있다. 본 연구를 통해 점안액의 대표적인 보존제인 BAC의 부작용에 대한 각막세포 차원의 연구가 필요하며 세포독성이 없는 새로운 보존제의 개발이 필요한 것으로 나타났다.

• 동의생리병리학회지
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• 제27권1호
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• pp.92-98
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• 2013

Bee Venom(below BV) has been used in alternative medicine to treat the diseases, such as pain diseases. BV contains a variety of peptides, including melittin, apamin, adolapin, MCD peptide, enzymes(i.e. PLA2), amines(i.e. histamine and epinephrine), and nonpeptide components. The two main components of BV are melittin and PLA2. The cell cytotoxic effects through the activation of PLA2 by melittin have been suggested to be the critical mechanism for the depress of cancer cell. Melittin and PLA2 have been reported to induce apoptosis and to possess anti-cancer effects and neurite outgrowth in PC12 cells. Analysis of proliferation was confirmed by MTT assay. BV decreased cell number through dose- and duration-dependent manner and these effects are apparent at a concentration of 3 ${\mu}g/ml$. To observe which signaling molecules will be activated by BV, phosphorylation of ERK, p38 MAPK, JNK and ERM were examined by Western blot analysis. To study the long term effect of BV in human gastric adenocarcinoma cell lines, the image of cells treated with BV for 4 days were obtained. BV was shown to exhibit anti-cancer activity in human gastric adenocarcinoma cell lines at a broad range of concentrations of 3 ${\mu}g/ml$. ERK, p38 MAPK and JNK were found to increase in BV treated cells. However, ERM which known to be involved in the cell death, was gradually decreased to 30minutes after addition 3 ${\mu}g/ml$ of BV. These results provide a possible BV-induced inhibitory signal for cancer proliferation that is initiated by the decrease in ERM activity. Moreover, it is likely that the activation of ERK, p38 MAPK and JNK are required for the BV-induced inhibition of cancer proliferation.