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Prevotella nigrescens ATCC $33563^T$ 균주-특이 중합효소연쇄반응 프라이머 개발
송수근,유소영,김미광,김화숙,임선아,김도경,박재윤,국중기,Song, Soo-Keun,Yoo, So-Young,Kim, Mi-Kwang,Kim, Hwa-Sook,Lim, Sun-A,Kim, Do-Kyung,Park, Jae-Yoon,Kook, Joong-Ki 한국미생물학회 2008 미생물학회지 Vol.44 No.3
A Pn10 DNA probe was introduced as a Prevotella nigrescens ATCC $33563^T$-specific DNA probe. In that study, the specificity of the Pn10 was tested with only type or reference strains of 5 oral bacterial species. The purpose of this study is to evaluate the specificity of the Pn10 using the wild type strains of P. nigrescens and is to develop the P. nigrescens ATCC $33563^T$-specific PCR primers based on the nucleotide sequence of the Pn10. The specificity of the Pn10 DNA probe was determined by Southern blot analysis. The nucleotide sequence of Pn10 DNA probes was determined by chain termination method. The PCR primers were designed based on the nucleotide sequence of cloned DNA fragment. The data showed that Pn10 DNA probe were hybridized with the genomic DNAs from P. nigrescens ATCC $33563^T$ and KB6. The Pn10 homologous region, KB6-Pn10, of P. nigrescens KB6 was cloned by PCR and sequenced. The Pn10 and KB6-Pn10 DNA fragments were consisted of 1,875 bp and 1,873 bp, respectively. The percent identity of the two was 98.8% and the divergence of them was 0.6%. The two primer sets (Pn10-F-AC/ Pn10-R-AC and Pn10-F-A/ Pn10-R-A), designed base on the nucleotide sequences of Pn10 DNA probe, were specific to the P. nigrescens ATCC $33563^T$. The two PCR primer sets could detect as little as 4 pg of genomic DNA of P. nigrescens ATCC $33563^T$. These results indicate that the two PCR primer sets have proven useful for the identification of P. nigrescens ATCC $33563^T$, especially with regard to the maintenance of the strain. 본 연구는 Prevotella nigrescens ATCC $33563^T$에 대한 균주 특이 DNA 프로브라고 보고된 Pn10 프로브의 균주 특이성을 한국인에서 분리된 P. nigrescens의 임상분리 균주를 이용하여 검증하고, P. nigrescens ATCC $33563^T$ 균주 특이 PCR 프라이머를 개발하고자 시행되었다. P. nigrescens와 유전학적으로 가장 가까운 Prevotella intermedia를 포함한 구강 내 치주질환 원인균종인 5균종의 표준균주 및 참고균주, 그리고 P. nigrescens와 P. intermedia의 임상분리 균주를 이용하여 Southern blot 분석법을 시행하였다. Southern blot 분석 결과 Pn10 DNA 프로브에 P. nigrescens ATCC $33563^T$ 및 ChDC KB6 두 균주 지놈 DNA가 검출되었다. P. nigrescens KB6 균주에서 Pn10 DNA 프로브와 상동성이 있는 부위를 PCR법으로 증폭(KB6-Pn10)하여 클로닝한 다음 Pn10 DNA프로브와 같이 핵산 염기서열을 결정하여 상동성을 비교하였다. 그 결과 Pn10과 KB6-Pn10의 핵산염기서열간의 Percent identity는 98.8%였으며, divergence는 0.6%였다. Pn10 DNA 프로브의 핵산염기서열을 바탕으로 두 중류 프라이머 쌍(Pn10-F-AC/Pn10-R-AC 및 Pn10-F-A/Pn10-R-A)을 설계 및 제작하여 P. nigrescens ATCC $33563^T$에 대한 균주 특이성을 PCR법으로 검증하였다. 이들 프라이머 쌍들의 민감도(sensitivity) 조사 결과, 이들은 P. nigrescens ATCC $33563^T$ 지놈 DNA 4 pg까지 검출할 수 있음을 알았다. 이상의 연구 결과를 종합하면, Pn10 DNA 핵산염기서열을 바탕으로 설계된 Pn10-F-AC/Pn10-R-AC 및 Pn10-F-A/Pn10-R-A 프라이머 쌍들은 P. nigrescens ATCC $33563^T$를 신속 정확하게 검출하는 수 있어, 균주의 보존적 측면에서 유용하게 이용될 수 있을 것으로 생각된다.
사람 암세포주들에서 아미노산 수송체 LAT1 mRNA 발현과 아미노산 L - Leucine 수송의 상관관계
김도경(Do Kyung Kim),송수근(Soo Keun Song),김인진(In Jin Kim),국중기(Joong-Ki Kook) 한국식품영양과학회 2004 한국식품영양과학회지 Vol.33 No.9
사람의 여러 암세포주들에서 아미노산 수송체 LAT1 및 그 보조인자 4F2hc mRNA의 발현과 LAT1의 대표적 기질인 L-leucine 수송을 조사하여 이들 사이의 상관관계를 밝히기 위해, 암세포주들에서 northern blot analysis 및 uptake 실험 등을 시행하여 다음과 같은 결과를 얻었다. 조사한 26 종류의 사람 암 유래 세포주들에서 LAT1 mRNA의 뚜렷한 발현을 확인할 수 있었으나 그 발현양에는 차이가 있었다. 조사한 26종류의 사람 암 유래 세포주들에서 4F2hc mRNA의 뚜렷한 발현을 확인할 수 있었으며, LAT1의 경우에서와 같이 발현하는 정도가 조금씩 다름을 확인할 수 있었다. 사람의 암 유래 세포주들에서 아미노산 transporter에 의한 L-[¹⁴C]leucine 수송을 확인할 수 있었다. 사람의 암세포주들에서 LAT1 mRNA의 발현정도가 높을수록 L-[¹⁴C]leucine의 수송능력이 증가함을 알 수 있었다. 사람의 암세포주들에서 4F2hc mRNA의 발현정도가 높을수록 L-[¹⁴C]leucine 수송 능력이 증가하는 경향은 보였으나, 통계적인 유의성은 없었다. 본 연구의 결과에 의해 사람 암세포주들에서 아미노산 transporter LAT1의 발현과 아미노산 L-leucine의 수송 사이와의 상관관계를 확인할 수 있었으며, 아미노산 transporter LAT1의 특이적 억제에 의한 암세포의 성장 억제에 관한 또 하나의 방향성을 제시할 수 있을 것으로 사료된다. Amino acid transporters play an important role in supplying nutrients to normal and cancer cells for cell proliferation. System L is a major transport system responsible for the Na^+ -independent, large neutral amino acids including several essential amino acids. L-type amino acid transporter 1 (LAT1), an isoform of system L amino acid transporter, is highly expressed presumably to support their continuous growth and proliferation in malignant tumors. In the present study, we have examined the correlation between the expressions of amino acid transporter LAT1 mRNA and its subunit 4F2hc mRNA and the amount of L-leucine transport in various human cancer cell lines. Northern blot analysis have revealed that the 26 human cancer cell lines expressed LAT1 mRNA and 4F2hc mRNA. There were the differences for the levels of LAT1 and 4F2hc mRNA expressions in the 26 human cancer cell lines. The 26 human cancer cell lines transported the L-[¹⁴C]leucine into the cells via amino acid transporter. In the 26 human cancer cell lines, a linear relationship was observed between the expression of amino acid transporter LAT1 mRNA and the amount of L-leucine transport. Little relationship was observed between the expression of 4F2hc mRNA and the amount of L-leucine transport, but the statistical significance of difference was not detected. These results indicate that the 26 human cancer cell lines express LAT1 mRNA and 4F2hc mRNA and there is the correlation between the expression of amino acid transporter LAT1 mRNA and the amount of L-leucine transport. In addition, specific inhibition of LAT1 in cancer cells will be a new rationale for anti-cancer therapy.
용이한 Subcloning을 위한 pCAT(R)3-Basic vector의 변형 2
유소영 ( So Youog Yoo ) , 김미광 ( Mi Kwang Kim ) , 김화숙 ( Hwa Sook Kim ) , 최민호 ( Min Ho Choi ) , 임상수 ( Sang Soo Lim ) , 박헌동 ( Heon Dong Park ) , 송수근 ( Soo Keun Song ) , 신환선 ( Hwan Seon Shin ) , 박슬희 ( Seul H 조선대학교 구강생물학연구소 2003 Oral Biology Research (Oral Biol Res) Vol.27 No.2
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DNA probe Pn25를 이용한 Prevotella intermedia 및 Prevotella nigrescens의 동정
김화숙 ( Hwa Sook Kim ),김미광 ( Mi Kwang Kim ),유소영 ( So Young Yoo ),최민호 ( Min Ho Choi ),임상수 ( Sang Soo Lim ),박헌동 ( Heon Dong Park ),송수근 ( Soo Keun Song ),신환선 ( Hwan Seon Shin ),박슬희 ( Seul Hee Park ),임선아 ( 조선대학교 구강생물학연구소 2003 口腔生物學硏究 Vol.27 No.2
DNA probe Pn25를 이용한 Prevotella intermedia 및 Prevotella nigrescens의 동정
김화숙 ( Hwa Sook Kim ) , 김미광 ( Mi Kwang Kim ) , 유소영 ( So Young Yoo ) , 최민호 ( Min Ho Choi ) , 임상수 ( Sang Soo Lim ) , 박헌동 ( Heon Dong Park ) , 송수근 ( Soo Keun Song ) , 신환선 ( Hwan Seon Shin ) , 박슬희 ( Seul H 조선대학교 구강생물학연구소 2003 Oral Biology Research (Oral Biol Res) Vol.27 No.2
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갑상선 유두선암 환자의 말초 혈액 단핵구로부터 수지상세포분화와 자가 세포독성 T 림프구 활성화 유도
송수근,최영식,박요한,이대희,장희경 고신대학교 의학부 2004 高神大學校 醫學部 論文集 Vol.19 No.1
Background: Dendritic cells (DCs) are rare leucocytes that are uniquely potent in their ability to capture, process and present antigens to T cells, and so selectively migrate through tissues to reach lymph nodes and spleen where initiation of immune responses takes place. It has been reported that a dense infilteration of dendritic cells correlates with a favorable prognosis in several types of cancer. A similar correlation also has been noted in patients with papillary carcinoma of thyroid. The purpose of this study is to determine whether DCs are generated from peripheral blood monocytes by using cytokines such as GM-CSF, TNF-α, and IL-4 and whether cytotoxic T cells activated by DCs attack the papillary carcinoma tissues. Methods: Peripheral blood was obtained from 2 patients with thyroid papillary cancer. DCs were established from monocytes by culturing in the presence of GM-CSF, Flt-3 ligand. TNF-α , and IL-4 for14 days. At day 14, expression of surface markers typical for DCs was analyzed morphologically. The immunophenotypic features of DCs such as CDla, CD83, and CD86 were analyzed by phase contrast microscopy. At day 15 DCs were incubated with thyroid cancer tissues and normal thyroid tissues for 7 days. Results: DCs were generated from the peripheral blood monocytes. The generated cells had the classic morphology of DCs. Activated cytotoxic T lymphocytes (CTLs) were observed. Veiled cell known as circulating DCs attached to the thyroid cancer tissues were observed. The CTLs activated by DCs were observed to attack the papillary carcinoma tissues by scanning electron microscope. However, normal tissues were not attacked by CTLs. Conclusion: DCs can be generated from the peripheral blood monocytes. Futhermore, the CTLs activated by DCs also can attack the papillary carcinoma tissues. These results that DCs can be used as adjuvants for adoptive immunotherapy of papillary thyroid cancer.
위암환자의 진단 및 병기 결정에 있어서 혈청 CA72-4의 유용성
송수근,김현주,손호성,정연순,장성훈,정한호,권기범,박무인,박선자,구자영 고신대학교 의학부 2000 高神大學校 醫學部 論文集 Vol.15 No.1
Background/Objective Various methods including upper GI series, endoscopy, ultrasound, CAT scan are used with serum tumor markers, especially CEA and CA19-9, for the diagnosis and evaluation of gastric carcinoma. In this study the clinical utility of serum CA72-4 of 300 gastric carcinoma patients for the diagnosis and management of gastric carcinoma was evaluated. Methods Serum level of CA72-4 was measured using <Centa> Core CA72-4 RIA Kit(Packerd, USA). The cutoff level of serum CA72-4 was 4U/㎖. The serum levels were correlated with the stage, the location of lesions, the gross type, the depth of invasion, the nodal and distant metastasis. Results 1) Overall positivity of CA72-4 was 32.0%(96 cases). 2) There was no difference in the positivity of serum CA72-4 according to locations and gross types of carcinoma, but significant(p<0.05) difference in the positivity of serum CA72-4 was noted between cases of early gastric carcinoma(12.3%) and advanced gastric carcinoma (37.4%). 3) The positivity of serum CA72-4 of stage Ⅲ and Ⅳ(49.5%) was significantly(p<0.05) higher than those of stage Ⅰa(12.1%) and Ⅱ(16.0%). Furthermore, 87.5% of the cases of positive serum CA72-4 belonged to stage Ⅲ or Ⅳ. 4) The positivity of serum CA72-4 were 12.3%, 14.3%, 34.7% and 52.6% for T1, T2, T3 and T4, respectively, showing relatively good correlation between CA72-4 positivity and depth of invasion. And 86.5% of the cases of positive serum CA72-4 belonged to T3 or T4 group. 5) The positivity of serum CA72-4 of N1 and N2 groups(35.1% and 41.7%, respectively) was significantly(p<0.05) higher than those of N0 group(12.2%) and 89.6% of the cases of positive serum CA72-4 belonged to node positive cases. 6) In the cases of metastasis present, CA72-4 positivity was 47.4%, which was significantly(p<0.05) higher than that of metastasis-negative cases(28.4%). Among sites of metastasis, there was no difference in the positivity of CA72-4. Conclusions Serum CA72-4 was not useful as a screening test in the diagnosis of gastric carcinoma, but may be useful in differentiating between early and advanced cases, in determining stage of the disease, depth of invasion, serosal invasion, and especially in detecting nodal metastasis, and also useful when used with imaging modalities of diagnosis.