• Asian Pacific Journal of Cancer Prevention
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• 제15권12호
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• pp.4773-4780
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• 2014

Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

• Applied Microscopy
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• 제40권2호
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• pp.73-80
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• 2010

Granulocyte macrophage-colony stimulating factor (GM-CSF)는 과립구 및 대식세포뿐만 아니라 상피세포의 증식과 분화를 자극하는 당단백질이며, 최근 생산된 벼세포 유래 재조합 GMCSF(rrhGM-CSF)는 감염원으로부터 안전하고 당사슬이 매우 풍부하여 물질의 안정성 혹은 효과의 지속성을 높여 주는 것으로 알려졌다. 본 실험에서는 간 재생 능력이 우수한 흰쥐를 실험모델로 하여 간의 78%를 제거한 후, 간 재생을 유도하는 과정에서 rrhGM-CSF를 처리하고, 시간 경과에 따라 형태변화의 차이와 더불어 단백질 발현 분석법과 면역조직화학법을 이용하여 PCNA 발현에 미치는 효과에 대해서 알아보고자 하였다. rrhGMCSF는 간 재생 속도를 뚜렷이 증가시키지는 못하였으나, 대조군에 비해 실험군에서는 재생 초기에 간 세포판의 붕괴와 재구성 시기를 다소 앞당기는 것으로 관찰되었다. 증식 중인 세포에서 증가하는 것으로 알려져 있는 핵단백질인 proliferating cell nuclear antigen (PCNA)의 간 조직에서의 분포와 발현 정도를 보면 부분간절제 후 12시간과 24시간에서는 PCNA 단백질이 두 그룹에서 조금씩 발현되다가 간 절제 3일과 5일이 경과한 실험군에서 단백질이 높게 발현되었다. 간 재생이 진행될수록 간 조직 전체에서 고르게 PCNA 양성반응이 나타났으며, 대조군 보다 실험군에서 반응성이 더 뚜렷한 것으로 나타났다. 이러한 결과들로 보아 부분 간절제 후 간 재생을 유도하는 과정에서 rrhGM-CSF가 세포분열을 촉진시키는 인자들 중의 하나로 작용하여 간 재생에 효과를 나타낼 수 있을 것으로 사료된다.

• Asian-Australasian Journal of Animal Sciences
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• 제22권1호
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• pp.26-34
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• 2009

Porcine embryonic stem (ES) cells have a great potential as tools for transgenic animal production and studies of regulation of differentiation genes. Although several studies showed successful derivation of porcine ES-like cells, these cells were not maintained long-term in culture. Therefore, this study was conducted to establish porcine pluripotent ES-like cells using in vivo fertilized embryos and to maintain these cells in long term culture. Porcine ES-like cells from in vivo embryos obtained by immunosurgery or whole explant culture were successfully cultured for over 56 passages. Morphology of porcine ES-like cells was flat-shaped with a monolayer type colony. These cells stained for alkaline phosphatase throughout the culture. Furthermore, porcine ES-like cells reacted with antibodies against Oct-4, SSEA-1, SSEA-4, Tra-1-60, and Tra-1-81, which are typical markers of undifferentiated stem cells. To characterize the ability of porcine ES-like cells to differentiate into three germ layers, embryoid body formation was induced. After plating of these cells, porcine ES-like cells were spontaneously differentiated into various cell types of all three germ layers. In addition, porcine ES-like cells were successfully derived from IVF blastocysts in media containing human recombinant basic fibroblast growth factor.

• 농업과학연구
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• 제34권2호
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• pp.99-109
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• 2007

섬유소 분해균 Cellulomonas sp. CS 1-1에 대하여 종 수준으로 분류학적 위치를 규명하기 위하여 7개 type strain 균주와 함께 생리적 및 생화학적 특성을 조사하고 DNA 상등성 및 지방산의 조성 등을 분석하여 비교한 결과, CS 1-1의 콜로니 형태는 circular, entire, smooth, convex하며, 담황색을 띤 $0.3{\sim}0.5{\times}0.8{\sim}1.2{\mu}m$ 크기의 간균이었다. 생리학적 특징으로서 비운동성의 통성혐기성 중온균으로서 Gram양성, catalase양성, oxidase음성, 탄수화물 발효성 등의 표현형은 Cellulomonas 속의 타종과 동일하였으며, 특히 D-ribose, raffinose, rhamnose, xylitol, acetate, L-lactate, propionate, aspartate, proline 등 조사한 모든 탄소원의 이용성이 없었으며, 반면에 sacchrose, arabinose 및 amylose의 이용성은 양성으로 판정되었다. G+C 함량 74.76 mol %, 주요 지방산과 quinone 성분은 전형적인 Cellulomonas의 12-methyltetradecanoic acid (anteiso-$C_{15:1}$)과 MK-$9(H_4)$이었으며, DNA의 상동성 비율은 C. uda ATCC 491과 70%, C. fimi ATCC 15724와 54~59 %, C. gelida 및 C. bibula와도 46~48%의 상동성을 나타내었다. 이상의 결과는 CS1-1이 현재 Cellulomonas 속에 인정된 7개의 type species 중 C. uda ATCC 491 균주와 가장 높은 근연성을 나타냄으로서 C. uda에 속하는 novel species로 분류될 수 있다.

• Journal of the Korean Association of Oral and Maxillofacial Surgeons
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• 제27권2호
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• pp.129-134
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• 2001

Since NNK is one of the most abundant tobacco-specific alkaloids and a strong carcinogenic nitrosamine, it has been used for evaluating a potential of carcinogenicity in the animal models. The present study has attempted to examine the potential of carcinogenicity of NNK in human epithelial cells, from which the cell type the most of cancers including oral cancer and nasal cavity cancer are originated. The cellular model used for the study is a human keratinocyte cell system immortalized by Ad12-SV40 hybrid virus. The cellular system has successfully been used for the carcinogenicity studies because of its limitless life span, epithelial morphology and nontumorigenicity. When cells were treated with a variety of NNK concentrations, levels of saturation density and soft agar colony formation were increased in a dose-dependent fashion. Colonies of large cell aggregates were above 5 at the higher doses. The results indicate that exposure of human cells with NNK induced loss of contact inhibition and increases of anchorage independence and cellular adhesion, which are typical characteristics of the neoplatically transformed cells. When cells were exposed with 100uM NNK for 2hr, mRNA levels of IL-1 and PAI-2 were increased in a dose-dependent manner, but expression of TGF- 1 was not affected. While expression of growth regulatory factors were altered with a short-term exposure, there was no alteration of these factors in the NNK-transformed cells. However, mRNA levels of fibronectin were increased both in the short-term treatment and in the transformation. The results suggest that altered expression of extracellular matrix such as fibronectin following short-term exposure might be fixed in the genome and these altered properties be continuously transfered throughout the cell division. Western blot analysis showed a translocation of PKC- from cytosolic fraction to the particulate fraction, indicating a possible role of NNK in the signal transduction pathway. The present study provided an evidence that NNK in the smoking may be associated with epithelial origin cancer such as oral and nasal cavity cancers. In addition, this study suggested that altered expression of extracellular matrix and PKC may play an important role in the carcinogenic mechanism of NNK.

• Journal of Microbiology and Biotechnology
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• 제15권6호
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• pp.1286-1298
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• 2005

The genus Paenibacillus is a new group of bacilli separated from the genus Bacillus, and most of species have been isolated from soil. In the present study, we collected 450 spore-forming bacilli from the roots of winter crops, such as barley, wheat, onion, green onion, and Chinese cabbage, which were cultivated in the southern part of Korea. Among these 450 isolates, 104 Paenibacillus-like isolates were selected, based on their colony shape, odor, color, and endospore morphology, and 41 isolates were then finally identified as Paenibacillus spp. by 16S rDNA sequencing. Among the 41 Paenibacillus isolates, 23 were classified as P. polymyxa, a type species of the genus Paenibacillus, based on comparison of the 16S rDNA sequences with those of 32 type strains of the genus Paenibacillus from the GenBank database. Thirty-five isolates among the 41 Paenibacillus isolates exhibited antagonistic activity towards plant fungal and bacterial pathogens, whereas 24 isolates had a significant growth-enhancing effect on cucumber seedlings, when applied to the seeds. An assessment of the root-colonization capacity under gnotobiotic conditions revealed that all 41 isolates were able to colonize cucumber roots without any significant difference. Twenty-one of the Paenibacillus isolates were shown to contain the nifH gene, which is an indicator of $N_{2}$ fixation. However, the other 20 isolates, including the reference strain E681, did not incorporate the nifH gene. To investigate the diversity of the isolates, a BOX-PCR was performed, and the resulting electrophoresis patterns allowed the 41 Paenibacillus isolates to be divided into three groups (Groups A, B, and C). One group included Paenibacillus strains isolated mainly from barley or wheat, whereas the other two groups contained strains isolated from diverse plant samples. Accordingly, the present results showed that the Paenibacillus isolates collected from the rhizosphere of winter crops were diverse in their biological and genetic characteristics, and they are good candidates for further application studies.

• 한국균학회지
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• 제17권3호
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• pp.114-118
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• 1989

야생형(野生形)이며 self-fertile인 버들볏짚버섯(버들송이) 균사체(菌絲體)로부터 분리(分離)한 핵(核)을 영양요구주(營養要求株)이며 self-sterile인 사철느타리버섯 원형질체내(原形質體內)에 polyethylene glycol+$CaCI_2$+glycine 용액으로 섭입(攝入)하였다. 100균주(菌株)의 핵전이주(核轉移株) 검정(檢定)으로 양친(兩親)이 균총분리(菌叢分離)되어 버들볏짚버섯친(親) 형태(形態)에는 클램프 연결체(連結體)가 균사(菌絲)에 있고, 사철느타리버섯친(親) 형태(形態)에는 클램프 연결체(連結體)가 없는 것이 3균주(菌株), 균총형태(菌叢形態)가 안정성(安定性)이며 클램프 연결체(連結體)가 균사(菌絲)에 형성(形成)된 것이 55균주(菌株), 균총형태(菌叢形態)가 안정성(安定性)이며 클램프 연결체(連結體)가 균사(菌絲)에 존재(存在)하지 않는 것이 42균주(菌株)였다. 이들 핵전이주(核轉移株)는 모두 원기(原基) 형성(形成)되어 자실체(子實體)로 성숙되었으며 그 형태(形態)는 버들볏짚버섯과 거의 유사(類似)하였다. 핵전이주(核轉移株)를 전기영동법으로 alcohol dehydrogenase, esterase, lactate dehydrogenase, peroxidase의 동위효소(同位酵素) 분석(分析)으로 비교하였는데 클램프 연결체(連結體)를 가지는 형태(形態)의 몇 균주(菌株)가 새로운 band를 형성(形成)하였으며 다른 균주(菌株)들은 양친주(兩親株)에서 유래된 것이었다.

• Journal of Species Research
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• 제6권2호
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• pp.101-118
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• 2017

In an investigation of indigenous prokaryotic species in Korea, a total of 57 bacterial strains assigned to the classes Betaproteobacteria and Gammaproteobacteria were isolated from diverse environments. Samples were collected from fresh water, natural caves, soil, paddy fields, lakes, sea water, jeotgal (fermented seafood), salt flats, soil from abandoned mines, plant roots, digestive organs of both Japanese crested ibis (Nipponia nippon) and Burmese python (Python molurus bivittatus) and tidal flats. From the high 16S rRNA gene sequence similarity (>98.7%) and formation of robust phylogenetic clades with closely related species, it was determined that each strain belonged to an independent and predefined bacterial species within either the Betaproteobacteria or Gammaproteobacteria. There is no official report or publication that describes these 57 proteobacterial species in Korea. Overall, in the class Betaproteobacteria there were 16 species in 12 genera of 4 families in the order Burkholderiales and two species in two genera of one family in the order Neisseriales. Within the class Gammaproteobacteia, there were five species in four genera of four families in the order Alteromonadales, 12 species in 11 genera of one family in the order Enterobacteriales, four species in four genera of three families in the order Oceanospirillales, 11 species in four genera of two families in the order Pseudomonadales, two species in the order Vibrionales and five species in five genera of one family in the order Xanthomonadales. Gram reaction, colony and cell morphology, basic biochemical characteristics, isolation source and strain IDs are described in the species description section.

• 디지털융복합연구
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• 제12권6호
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• pp.407-414
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• 2014

이 연구의 목적은 광감작제인 라다클로린과 포토프린을 발광다이오드에 접목하여 포도알균의 광역학치료 효과를 평가하고자 하였다. 실험방법은 황색포도알균이나 표피포도알균의 $1{\times}10^5CFU/ml$이 되게 균주부유액을 준비하였고, 광감작제(포토프린 또는 라다클로린)를 1.25, 2.5, 5, $10{\mu}g/ml$가 되도록 희석하였다. 균주희석액은 에너지밀도 각각 $14.4J/cm^2$와 $19.8J/cm^2$로 630 또는 670 파장 발광다이오드 빛을 조사하였다. 황색포도알균과 표피포도알균의 집락형성수는 포토프린 $5{\mu}g/ml$ 농도에서 33, 50개의 집락이 각각 형성되었고, 두 세균 모두 라다클로린 $5{\mu}g/ml$ 농도에서는 완전한 살균을 나타냈다. 유세포분석에 의한 형광강도는 정상세포보다 죽은세포에서 증가를 보였다. 투과전자현미경의 사진에서는 세포막의 손상과 부분적으로 세포형태의 파괴가 관찰되었다. 이 결과로 포토프린과 라다클로린을 이용한 광역학치료는 항균치료의 새로운 방법이 될 수 있음을 제의한다.

• 한국균학회지
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• 제41권2호
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• pp.112-117
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• 2013

Phytophthora capsici는 고추에 역병을 일으켜 우리나라뿐만 아니라 전 세계적으로 심각한 피해를 주는 대표적인 식물병원균이다. 최근 환경문제로 화학 농약을 대체하여 유용미생물을 이용한 생물학적 방제가 가장 바람직한 방제 방법으로 대두되고 있다. 본 연구에서는 고추역병에 피해를 보이지 않는 건전한 고추재배지 근권에서 토양시료를 채취하여 토양근권세균을 분리하였다. 총 360여점의 미생물 콜로니를 분리하였으며, 이들 콜로니의 색깔과 모양 등의 형태학적 특징을 기준으로 총 12개의 그룹으로 재분류하였다. 그 중 항균작용을 알아보기 위해 실시한 in vitro 실험에서 고추역병균에 길항효과를 보인 GJ-1, GJ-11, GJ-12 균주를 분리하였으며 길항효과가 없는 균주 2개(GJ-4, GJ-5)를 대조군으로 선발하였다. 선발된 5균주에 대하여 sidero-phore의 활성을 검정한 결과 GJ-1과 GJ-5 두 균주가 CAS 배지에서 orange halo zone을 크게 형성하여 siderophore를 많이 분비하는 균주로 확인되었다. 그리고 토양중의 난용성 인산염을 가용화시켜 식물의 생장촉진효과를 보이는 phosphate solubilization 활성을 검정한 결과 GJ-1, GJ-5, GJ-12 균주가 난용성 인산염에 대한 분해능력을 나타내었다. 따라서 P. capsici에 강한 항균활성을 나타내며 siderophore를 생산하고 인산가용화 능력이 뛰어난 유용미생물 GJ-1을 최종적으로 선발하였다. 최종 선발된 GJ-1균주를 동정하여 Bacillus sp.로 명명하였다. Bacillus sp. GJ-1의 길항작용은 Rhizoctonia solani, Pythium ultimum, Fusarium solani에 대한 식물병원성 진균에서도 좋은 효과를 나타내었다. 따라서 Bacillus sp. GJ-1은 생물학적 방제제로 고추역병균을 포함하여 다른 토양 식물병원성 진균 방제에 이용 가치가 있을 것으로 판단된다.