• Asian Pacific Journal of Cancer Prevention
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• 제17권3호
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• pp.1129-1134
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• 2016

Vulva and Vaginal cancers are rare among all gynecological cancers worldwide, including Thailand, and typically affect women in later life. Persistent high risk human papillomavirus (HR-HPV) infection is one of several important causes of cancer development. In this study, we focused on HPV investigation and specific type distribution from Thai women with abnormality lesions and cancers of the vulva and Vaginal. A total of ninety paraffin-embedded samples of vulva and Vaginal abnormalities and cancer cells with histologically confirmed were collected from Thai women, who were diagnosed in 2003-2012 at the National Cancer Institute, Thailand. HPV DNA was detected and genotyped using polymerase chain reaction and enzyme immunoassay with GP5+/bio 6+ consensus specific primers and digoxigenin-labeled specific oligoprobes, respectively. The human ${\beta}$-globin gene was used as an internal control. Overall results represented that HPV frequency was 16/34 (47.1%) and 8/20 (40.0%) samples of vulva with cancer and abnormal cytology lesions, respectively, while, 3/5 (60%) and 16/33 (51.61%) samples of Vaginal cancer and abnormal cytology lesions, respectively, were HPV DNA positive. Single HPV type and multiple HPV type infection could be observed in both type of cancers and abnormal lesion samples in the different histological categorizes. HPV16 was the most frequent type in all cancers and abnormal cytology lesions, whereas HPV 18 was less frequent and could be detected as co-infection with other high risk HPV types. In addition, low risk types such as HPV 6, 11 and 70 could be detected in Vulva cancer and abnormal cytology lesion samples, whereas, all Vaginal cancer samples exhibited only high risk HPV types; HPV 16 and 31. In conclusion, from our results in this study we suggest that women with persistent high risk HPV type infection are at risk of developing vulva and Vaginal cancers and HPV 16 was observed at the highest frequent both of these, similar to the cervical cancer cases. Although the number of samples in this study was limited and might not represent the overall incidence and prevalence in Thai women, but the baseline data are of interest and suggest further study for primary cancer screening and/or developing the efficiency of prophylactic HPV vaccines in Thailand.

• 한국발생생물학회지:발생과생식
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• 제10권2호
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• pp.127-133
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• 2006

스테로이드 합성효소 중 $17\;{\alpha}-hydroxylase/17,20-lyase(P450_{C17})$는 progesterone을 $17\;{\alpha}-hydroxyprogesterone$을 거쳐 androstenedione으로 변환을 담당하는 효소이다. 양서류 난소에서 스테로이드 합성의 분자적 조절과정의 연구에 사용할 목적으로 북방산 개구리(Rana dybowskii) 난소에서 $P450_{C17}$ cDNA를 클로닝 하였다. 북방산 개구리 난포세포의 cDNA library 검색을 통해 분리된 약 2.5kb의 cDNA는 529개의 아미노산을 가진 단일 번역틀을 가지고 있었다. 개구리 $P450_{C17}$의 아미노산 서열은 Xenopus와는 76%, 닭과는 63%, 그리고 사람과는 약 45%의 동일성을 보여 주였고, 동시에 진화적으로 척추동물에서 매우 잘 보존된 아미노산 서열을 가지고 있었다. 노던 분석에서 개구리의 $P450_{C17}$ 전사체는 난소에서만 2.5kb와 3.6kb 크기의 두 종류가 발견되었다. 그리고 개구리 Rana $P450_{C17}$ cDNA는 비스테로이드 합성 세포인 COS-1세포에서 분명한 $17\;{\alpha}-hydroxylase/17,20-lyase$ 활성을 주었다. 따라서 클로닝된 개구리 $P450_{C17}$ 유전자는 양서류의 난소에서 스테로이드 합성의 분자적 기작을 연구하는데 매우 유용할 것으로 사료된다.

• 원예과학기술지
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• 제30권2호
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• pp.171-177
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• 2012

Sw5-2 SCAR 분자표지와 생물검정법을 이용하여 토마토 유전자원 94종의 $Tomato$ $spotted$ $wilt$ $virus$(TSWV) 저항성을 조사하였다. Sw5-2 SCAR 분자표지의 PCR 산물은 대략 574bp, 500bp, 462bp였는데, 크기가 가장 큰 PCR 산물이 Sw5-b 저항성 대립유전자와 연관되어 있었다. Sw5-b 저항성 대립유전자는 3개 수집종('Eureta', 10-318, 10-321)에서 관찰되었는데, 접종한 개체 가운데 이들 가운데 일부는 TSWV-pb1(토마토 분리주)에 일시적으로 감염되어 회복되거나 줄기에 괴사 병징을 보였다. ELISA 검사에서 음성으로 판명된 수집종 당 1개체씩 총 35개체를 선발하여 병징 발현 및 바이러스 감염 유무를 추가로 조사하였다. 접종 5개월 이후에 병징이 나타나지 않은 26개체를 대상으로 RT-PCR을 이용하여 TSWV 감염유무를 조사한 결과, 모든 개체에서 TSWV의 RT-PCR 산물이 약하게 증폭되었고, 이들 PCR 산물의 증폭 수준은 'Eureta'와 비슷하였다. 선발된 유전자원의 저항성은 조직 내 TSWV의 농도를 낮게 하는데 중요한 역할을 하고 이들은 Sw5를 포함한 여러 가지 유전자들에 의해 양적으로 조절되는 것으로 판단된다.

• Asian-Australasian Journal of Animal Sciences
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• 제21권1호
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• pp.19-24
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• 2008

The purpose of this study was to detect expression of genes related to egg production in Taiwan Country chickens by suppression subtractive hybridization. Liver samples of mRNA extraction from two Taiwan Country chicken strains (L2 and B), originated from the same population but with very distinct egg production rates after long-term selection for egg and meat production respectively. Two-way subtraction was performed. The hepatic cDNA from the low egg production chickens (B) was subtracted from the hepatic cDNA from the high egg production strain (L2). The reversed subtraction (L2 from B) was also performed. The resulting differentially expressed gene fragments were cloned and sequenced. We sequenced 288 clones from the forward subtraction and 96 clones from the reverse subtraction. These genes were subjected to further screening to confirm the differential expression between the two genetic breeds of chickens. The apolipoprotein B (apoB) was expressed to a greater extent in the liver of the L2 than in the B line chickens. The 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (PURH) was expressed to a greater extent in the liver of the B than in the L2 strain chickens. We demonstrated that both apoB and PURH were more highly expressed in the liver than that in other tissues (muscle, ovary, and oviduct) in laying Taiwan Country chickens. Taken together, these data suggest that after the selection for egg production, expression of apoB and PURH genes were also changed. Whether the changed expression of these genes is directly related to egg production is not known, but these two genes may be useful markers for egg laying performance in Taiwan Country chickens.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.2045-2049
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• 2012

Objective: To investigate the effect of down-regulation of Sentrin/SUMO-specific protease 1 (SENP1) expression on the apoptosis of human Burkitt lymphoma cells (Daudi cells) and potential mechanisms. Methods: Short hairpin RNA (shRNA) targeting SENP1 was designed and synthesized and then cloned into a lentiviral vector. A lentiviral packaging plasmid was used to transfect Daudi cells (sh-SENP1-Daudi group). Daudi cells without transfection (Daudi group) and Daudi cells transfected with blank plasmid (sh-NC-Daudi group) served as control groups. Flow cytometry was performed to screen GFP positive cells and semiquantitative PCR and Western blot assays were employed to detect the inference efficiency. The morphology of cells was observed under a microscope before and after transfection. Fluorescence quantitative PCR and Western blot assays were conducted to measure the mRNA and protein expression of apoptosis related molecules (caspase-3, 8 and 9). After treatment with $COCl_2$ for 24 h, the mRNA and protein expression of hypoxia inducible factor -$1{\alpha}$ (HIF-$1{\alpha}$) was determined. Results: Sequencing showed the expression vectors of shRNA targeting SENP1 to be successfully constructed. Following screening of GFP positive cells by FCM, semiqualitative PCR showed the interference efficiency was $79.2{\pm}0.026%$. At 48 h after transfection, the Daudi cells became shrunken, had irregular edges and presented apoptotic bodies. Western blot assay revealed increase in expression of caspase-3, 8 and 9 with prolongation of transfection (P<0.05). Following hypoxia treatment, mRNA expression of HIF-$1{\alpha}$ remained unchanged in three groups (P>0.05) but the protein expression of HIF-$1{\alpha}$ markedly increased (P<0.05). However, in the sh-SENP1-Daudi group, the protein expression of HIF-$1{\alpha}$ remained unchanged Conclusion: SENP1-shRNA can efficiently inhibit SENP1 expression in Daudi cells. SENP1 inhibition may promote cell apoptosis. These findings suggest that SENP1 may serve as an important target in the gene therapy of Burkitts lymphoma.

• Asian-Australasian Journal of Animal Sciences
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• 제20권3호
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• pp.316-327
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• 2007

In this study, a Korean native cattle strain (Hanwoo) evidencing high performance in terms of both meat quality and quantity was employed in the generation of 150,000 BAC clones with an average insert size of 140 kb, and corresponding to about a 6X coverage of bovine chromosomal DNA. The BAC clones were pooled in a mini-scale via three rounds of a pooling protocol, and the efficiency of this pooling protocol was evaluated by testing the accuracy of accessibility to the positive clones, via a PCR-based screening method. Two sets of primers designed from each of two known genes were tested, and each yielded 2 or 3 positive clones for each gene, thereby indicating that the BAC library pooling system was appropriate with regard to the accession of the target BAC clones. Analyses of $3.3{\times}10^6$ base pairs obtained from the 7,090 BAC end sequence (BES) showed that 34.88% of the DNA sequence harbored the repetition sequence. Analysis of the 7,090 BES to the $1^{st}$ and $2^{nd}$ generation radiation hybrid map of the cattle genome, using the COMPASS program designed for the construction of a cattle-human comparative mapping, resulted in the localization of a total of 1,374 clones proximal to 339 $1^{st}$ generation markers, and 1,721 clones proximal to 664 $2^{nd}$ generation markers. Collectively, the BAC library and pooling system of the BAC clones from the Korean cattle, coupled with the chromosome-localized BAC clones, will provide us with novel tools for the excavation of desired clones for genome mapping and sequencing, and will also furnish us with additional information regarding breed differences in cattle.

• Asian-Australasian Journal of Animal Sciences
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• 제31권1호
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• pp.63-70
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• 2018

Objective: The aim of the study was to isolate gossypol-degrading bacteria and to assess its potential for gossypol degradation. Methods: Rumen liquid was collected from fistulated cows grazing the experimental pasture. Approximately 1 mL of the rumen liquid was spread onto basal medium plates containing 2 g/L gossypol as the only source of carbon and was then cultured at $39^{\circ}C$ to isolate gossypol-degrading bacteria. The isolated colonies were cultured for 6 h and then their size and shape observed by microscope and scanning electron microscope. The 16S rRNA gene of isolated colonies was sequenced and aligned using National Center for Biotechnology Information-Basic Local Alignment Search Tool. The various fermentation conditions, initial pH, incubation temperature, inoculum level and fermentationperiod were analyzed in cottonseed meal (CSM). The crude protein (CP), total gossypol (TG), and free gossypol (FG) were determined in CSM after fermentation with isolated strain at $39^{\circ}C$ for 72 h. Results: Screening results showed that a single bacterial isolate, named Rumen Bacillus Subtilis (RBS), could use gossypol as a carbon source. The bacterium was identified by 16S rDNA sequencing as being 98% homologous to the sequence of Bacillus subtilis strain GH38. The optimum fermentation conditions were found to be 72 h, $39^{\circ}C$, pH 6.5, moisture 50%, inoculum level $10^7cell/g$. In the optimum fermentation conditions, the FG and TG content in fermented CSM decreased 78.86% and 49% relative to the control. The content of CP and the essential amino acids of the fermented CSM increased respectively, compared with the control. Conclusion: The isolation of a gossypol-degrading bacterium from the cow rumen is of great importance for gossypol biodegradation and may be a valuable potential source for gossypol-degradation of CSM.

• 대한의생명과학회지
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• 제6권1호
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• pp.11-17
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• 2000

SNAP-25는 presynaptic plasma membrane에 위치하는 단백질로서 synaptic vesicle의 docking과 fusion에 있어서 매우 중요한 역할을 한다. 생쥐 SNAP-25$^{2)}$ 유전자와 99%의 높은 homology를 갖고 있는 Z2 cDNA를 probe로 사용하여 쥐의 뇌 cDNA library에서 SNAP-25유전자를 screening하였다. 그 결과 6개 의 양성 클론을 분리 해 냈으며, 이들 각각을 S1, S2, S3, S4, S5, S6으로 명명하였다. 이 중에서 생쥐 SNAP-25와 가장 높은 homology를 보여 주고 있는 S5 클론을 선택하여 염기서열을 분석하였다. 2,100 bp의 염기서열로 구성된 쥐 SNAP-25 cDNA는 206개의 아미노산을 coding하는 618 bp의 open reading frame을 가지고 있으며, ORF는 209~211 bp에 위치하는 AUG codon에서 시작하여 827~829 bp에 위치하는 stop codon TAA에서 끝난다. 3' untranslated region에서 는 28과 19개 의 CA 반복 염기서열을 보여주고 있었으며, SNAP-25 peptide sequence에서 4개의 cystein residues는 84~91에 위치하고 있었으며, amino terminus 부분에서 amphipathic $\alpha$-helix를 형성하고 있는 것을 볼 수 있었다. 사람과 쥐의 SNAP-25 유전자는 88%, 생쥐와 쥐의 경우는 97%의 homology를 보여 주고 있었다. 그리고 사람과 쥐의 ORF에서 염기서열은 94%,생쥐와 쥐의 ORF에서 염기서열은 100%의 homology를 보여주고 있었으며 사람, 생쥐, 그리고 쥐의 ORF에서 아미노산 서열은 100%의 homology를 보여주고 있었다.

• 미생물학회지
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• 제46권4호
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• pp.359-365
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• 2010

조릿대 근권토양으로부터 분리한 방선균 50균주를 대상으로 고추 세균성 점무늬병원균(Xanthomonas axonopodis pv. vesicatoria)의 항균활성 12균주를 선발하였다. 이들 항균활성 12균주의 계통학적 위치를 검토한 결과, 모두 Streptomyces 속의 Cluster II에 속하는 특징을 나타내었다. JR-24 균주는 최소저해 농도(MIC) 10 ${\mu}l$/disc를 나타내었으며, 배양액 5 ${\mu}l$/ml를 처리 하여 12시간 배양한 결과 Xanthomonas axonopodis pv. vesicatoria에 강한 생육저해효과를 나타내어 최우수 균주로 선발되었다. 항균활성 균주 JR-24의 16S rRNA 유전자 염기서열을 검토한 결과, Streptomyces galbus $DSM40089^T$ (X79852)와 98.1%, Streptomyces longwoodensis $LMG20096^T$ (AJ781356)와 98% 그리고 Streptomyces capoamus $JCM4734^T$ (AB045877)와 97.8%의 상동성을 나타내었다. API 20NE와 API 50CHE를 이용하여 JR-24 균주의 생리 생화학적 특성을 확인한 결과, L-arabinose, D-fructose, D-glucose, D-galactose을 이용하며 gelatin, protein, starch에 대하여 분해능이 있는 것으로 확인되었다. 주요지방산으로는 iso-$C_{14:0}$ (25.93%), iso-$C_{15:0}$ (10.13%), anteiso-$C_{15:0}$ (19.29%) 그리고 iso-$C_{16:0}$ (20.35%) 등을 함유하였으며, 퀴논종은 MK-9 ($H_4$) 4.37%, MK-9 ($H_6$) 51.22% 그리고 MK-9 ($H_8$) 49.47%로 동정되었다. Streptomyces sp. JR-24 균주의 계통학적 특성을 근연종인 Streptomyces galbus $DSM40089^T$와 비교한 결과, 다수의 표현형적 및 계통학적 차이를 나타내었다. 본 연구에서 분리된 Streptomyces sp. JR-24는 친환경 미생물제제 개발을 위한 유전자원 확보에 있어서 매우 큰의의가 있을 것으로 사료 된다.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.1971-1982
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• 2017

Piliated Lactobacillus rhamnosus (pLR) strains possess higher adherent capacity than non-piliated strains. The objective of this study was to isolate and characterize probiotic pLR strains in human fecal samples. To this end, mouse polyclonal antiserum (anti-SpaA) against the recombinant pilus protein (SpaA) of L. rhamnosus strain GG (LGG) was prepared and tested for its reactivity and specificity. With the anti-SpaA, a method combining the de Man, Rogosa, and Sharpe (MRS) agar plating separation and colony immunoblotting (CIB) was developed to isolate pLR from 124 human fecal samples. The genetic and phenotypic characteristics of the resultant pLR isolates were compared by randomly amplified polymorphic DNA (RAPD) fingerprinting, and examination of adhesion to Caco-2 cells, hydrophobicity, autoaggregation, and in vitro gastrointestinal tolerance. Anti-SpaA specifically reacted with three pLR strains of 25 test strains, as assessed by western blotting, immunofluorescence flow cytometry, and immunoelectron microscopy (IEM) assays. The optimized MRS agar separation plus anti-SpaA-based CIB procedure could quantitatively detect $2.5{\times}10^3CFU/ml$ of pLR colonies spiked in $10^6CFU/ml$ of background bacteria. Eight pLR strains were identified in 124 human fecal samples, and were confirmed by 16S RNA gene sequencing and IEM identification. RAPD fingerprinting of the pLR strains revealed seven different patterns, of which only two isolates from infants showed the same RAPD profiles with LGG. Strain PLR06 was obtained with high adhesion and autoaggregation activities, hydrophobicity, and gastrointestinal tolerance. Anti-SpaA-based CIB is a rapid and inexpensive method for the preliminary screening of novel adherent L. rhamnosus strains for commercial purposes.