• Plant Resources
• /
• 제7권1호
• /
• pp.39-46
• /
• 2004

To demonstrate the importance of transformation efficiency in independent event, molecular and cytogenetic analysis were conducted with genomic DNA and chromosome of transgenic plants produced by Agrobacterium tumefeciens LBA4404 (pSBM-PPGN: gusA and bar). Selection ratios of putative transgenic calli were similar in independent experiments, however, transformation efficiencies were critically influenced by the type of regeneration media. MSRK5SS-Pr regeneration mediun, which contains 5 mgL$^{-1}$ kinetin, 2% (w/v) sucrose in combination with 3% (w/v) sorbitol, and 500 mgL$^{-1}$ proline, was efficient to produce transgenic plant of rice from putative transgenic callus in the presence of L-phosphinotricin (PPT). With MSRK5SS-Pr medium, transformation efficincies of Nagdongbyeo were significantly enhanced from 3.7% to 6.3% in independent callus lines arid from 7.3% to 19.7% in plants produced, respectively. Stable integration and expression of bar gene were confirmed by basta herbicide assay, PCR amplification and Southern blotting of bar gene, and fluorescence in situ hybridization (FISH) analysis using pSBM-PPGN as a probe. In Southern blot analysis, diverse band patterns were observed in total 44 transgenic plants regenerated from 20 independent PPT resistant calli showing from one to five copies of T-DNA segments, however, the transformants obtained from one callus line showed the same copy numbers with the same fractionized band patterns.

• 대한의생명과학회지
• /
• 제11권4호
• /
• pp.465-471
• /
• 2005

Ofloxacin has antimycobacterial activity that possibly contributes a pivotal role in the second-line drug regimens that are used for the treatment of multidrug-resistant tuberculosis. However, in some communities, the resistance rate of Mycobacterium tuberculosis to this agent is surging. Therefore, a rapid and accurate method that can be used to determine the resistance of M tuberculosis to the ofloxacin can be very useful for effective treatment of the patients. As an effort to develop such a method, this study was set up to reveal general types of mutations that are related to ofloxacin resistance of M tuberculosis. From previous studies, it has been well known that ofloxacin resistance is associated with mutations in a gene encoding the gyrase A subunit protein. In this study, we obtained 43 ofloxacin-resistant and 50 ofloxacin-susceptible M tuberculosis clinical isolates from Masan National TB Hospital, and sequences of DNA fragment of 320 bp, region of gyrA corresponding to the ofloxacin resistance-determining region were analyzed. In brief, the results showed that a total of seven mutation types were found at gyrA. Theses mutations were all clustered within nucleotides 2574 to 2586 of the gyrA gene (codons 88 to 94). Codon 94 was the most frequently substituted site. Twenty-four of the 43 isolates had mutations at this position resulting in a total of five different types of amino acid changes $(Asp{\to}Ala,\;Asp{\to}Gly,\;Asp{\to}His,\;Asp{\to}Tyr,\;and\;Asp{\to}Asn)$. Five isolates contained a mutation at codon 90 resulting $Ala{\to}Val$ change. Four isolates had mutations at codon 91 causing a $Ser{\to}Pro$ change at this site. Two isolates contained a mutation at codon 88 and each of them resulted in different types of amino acid changes $(Gly{\to}Cys,\;Gly{\to}Ala)$. On the other hand, polymorphic site at codon 95 was found in both ofloxacin-resistant and ofloxacin-susceptible isolates. From these results, we concluded that the rate of mutations present in gyrA among ofloxacin-resistant M. tuberculosis in Korea is similar to the general rates of mutations found throughout the world. Subsequently, an oligonucleotide probe was designed based on the results of sequence analysis and was used to develop a dot blot hybridization assay system to determine ofloxacin-resistance of M tuberculosis. To evaluate this probe, dot-blot hybridization was carried out using other 57 clinical isolates, and the results showed that the dot-blot hybridization assay is good for detecting sequence alterations atgyrA gene.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권2호
• /
• pp.1023-1026
• /
• 2013

Cervical cancer is the most important female gynecological cancer, the second leading cause of cancer mortality in women worldwide and the second most common cancer in Thai women. The major cause of cervical cancer is persistent infection of human papillomavirus (HPV), leading to abnormal epithelial lesions, with progression to precancerous and invasive cancer. This study was conducted to investigate the frequency and type distribution of HPV in Thai women who had abnormal cytology. HPV detection from FFPE confirmed abnormal of high grade cervical intraepithelial lesions were for SPF-10-Innogenic Line Probe Assay. HPV-positivity was detected in 320/355 cases (90.14%) and HPV-negativity in 35/355 (9.86%). HPV-positive was found 147/320 cases (41.4%) of single infection, whereas 173/320 cases (48.7%) showed the multiple HPV infection. The most common seven types were HPV-16, -52, -18, -11, -51, -31 and -33, in that order. HPV 16 and 18, the important oncogenic HPV type, were observed in 64.8% of HSIL cases. Interestingly, a high proportion of multiple infections was found in this study and more than ten types could be detected in one case. Therefore, HPV infection screening program in women is essential, particularly in Thailand. Effective primary and secondary prevention campaigns that reinforce HPV screening for HPV detection and typing may be decrease the incidence and mortality of cervical cancer in the future and may lead to significantly improve the quality of life in Thai women.

• Asian Pacific Journal of Cancer Prevention
• /
• 제14권9호
• /
• pp.5489-5494
• /
• 2013

In the current study we aimed to show the common YMDD motif mutations in viral polymerase gene in chronic hepatitis B patients during lamivudine and adefovir therapy. Forty-one serum samples obtained from chronic hepatitis B patients (24 male, 17 female; age range: 34-68 years) were included in the study. HBV-DNA was extracted from the peripheral blood of the patients using an extraction kit (Invisorb, Instant Spin DNA/RNA Virus Mini Kit, Germany). A line probe assay and direct sequencing analyses (INNO-LIPA HBV DR v2; INNOGENETICS N.V, Ghent, Belgium) were applied to determine target mutations of the viral polymerase gene in positive HBV-DNA samples. A total of 41 mutations located in 21 different codons were detected in the current results. In 17 (41.5%) patients various point mutations were detected leading to lamivudin, adefovir and/or combined drug resistance. Wild polymerase gene profiles were detected in 24 (58.5%) HBV positive patients of the current cohort. Eight of the 17 samples (19.5%) having rtM204V/I/A missense transition and/or transversion point mutations and resistance to lamivudin. Six of the the mutated samples (14.6%) having rtL180M missense transversion mutation and resistance to combined adefovir and lamivudin. Three of the mutated samples (7.5%) having rtG215H by the double base substituation and resistance to adefovir. Three of the mutated samples (7.5%) having codon rtL181W due to the missense transversion point mutations and showed resistance to combined adefovir and lamivudin. Unreported novel point mutations were detected in the different codons of polymerase gene region in the current HBV positive cohort fromTurkish population. The current results provide evidence that rtL180M and rtM204V/I/A mutations of HBV-DNA may be associated with a poor antiviral response and HBV chronicity during conventional therapy in Turkish patients.

• Asian Pacific Journal of Cancer Prevention
• /
• 제13권4호
• /
• pp.1151-1158
• /
• 2012

High-risk human papillomavirus (HPV) genotypes are the major cause of cervical cancer. Hence, HPV genotype detection is a helpful preventive measure to combat cervical cancer. Recently, several HPV detection methods have been developed, each with different sensitivities and specificities. The objective of this study was to compare HPV high risk genotype detection by an electrochemical DNA chip system, a line probe assay (INNO-LiPA) and sequencing of the L1, E1 regions. A total of 361 cervical smears with different cytological findings were subjected to polymerase chain reaction-sequencing and electrochemical DNA chip assessment. Multiple infections were found in 21.9% (79/361) of the specimens, most prevalently in 20-29-year olds while the highest prevalence of HPV infection was found in the 30-39-year age group. The most prevalent genotype was HPV 16 at 28.2% (138/489) followed by HPV 52 at 9.6% (47/489), with the other types occurring at less than 9.0%. The electrochemical DNA chip results were compared with INNO-LiPA and sequencing (E1 and L1 regions) based on random selection of 273 specimens. The results obtained by the three methods were in agreement except for three cases. Direct sequencing detected only one predominant genotype including low risk HPV genotypes. INNO-LiPA identified multiple infections with various specific genotypes including some unclassified-risk genotypes. The electrochemical DNA chip was highly accurate, suitable for detection of single and multiple infections, allowed rapid detection, was less time-consuming and was easier to perform when compared with the other methods. It is concluded that for clinical and epidemiological studies, all genotyping methods are perfectly suitable and provide comparable results.

• Journal of Nutrition and Health
• /
• 제51권6호
• /
• pp.498-506
• /
• 2018

본 연구는 베타세포에서 라이코펜의 항사멸 효과와 그 기전에 대해 조사하기 위해 실시하였다. 라이코펜에 의한 베타세포독성을 조사하기 위해 다양한 농도 (0.1, 1, 10 nM)로 처리하였을 경우, 저농도에서 세포독성이 나타나지 않음을 관찰하였다. 선택한 농도를 사이토카인 혼합물과 함께 처리하였을 경우, 세포 생존율이 증가하는 것을 관찰하였고, 세포사멸 유도 단백질인 Bax의 발현양은 감소하고, 세포사멸억제 단백질인 Bcl-2 발현양은 증가하는 것을 관찰하였다. 또한 사이토카인 혼합물에서 증가하였던 세포내 산화스트레스가 라이코펜과 함께 처리하였을 경우 감소되는 것을 관찰하였고 이러한 효과는 항산화 유전자인 GCLC, NQO1, HO-1의 발현양이 증가함으로서 일어난 현상임을 알 수 있었다. 라이코펜은 미토콘드리아의 생성 및 기능과 관련된 유전자의 발현을 증가시키고 사이토카인 혼합물에 의해 감소되었던 세포내 ATP 생성량을 증가시켰다. 이러한 결과는 라이코펜의 항산화효과와 미토콘드리아 기능 개선 효과가 사이토카인에 의한 베타세포 사멸을 억제하는 기전 중의 하나로 작용할 수 있음을 의미한다. 향후 라이코펜이 베타세포를 타겟으로 하는 제 2형 당뇨 치료의 기능성 소재로 개발될 가능성이 있음을 시사하는 바이다.

• Tuberculosis and Respiratory Diseases
• /
• 제58권2호
• /
• pp.129-134
• /
• 2005

배 경 : 에탐부톨의 내성여부는 결핵 환자 처방 결정에 있어서 중요 변수의 하나가 된다. 에탐부톨 내성의 상당부분이 embB 유전자의 돌연변이와 관계가 있으므로 역교잡반응법으로 이 유전자의 돌연변이를 신속하게 검출하고자 하였다. 방 법 : 에탐부톨 내성에 관련된 embB 유전자의 306번, 406번, 497번 아미노산의 정상적인 염기서열과 돌연변이 염기서열에 대한 probe를 합성하였고, 약제감수성검사에서 에탐부톨 내성균으로 나타난 149균과 전약제 감수성으로 나타난 50개균을 대상으로 조사하였다. 결 과 : 149개의 에탐부톨 내성균 중에서 embB 유전자 전체에서 돌연변이가 나타난 균은 100균주(67.1%)였으며, 그 중 embB 유전자 중 306번 돌연변이를 가진 균주가 75주(50.3%), 406번 돌연변이를 가진 균주가 16주(10.7%), 497번 돌연변이가 있는 균주가 13주(8.7%)였다. 이 중 4균주는 306번과 406번 돌연변이를 동시에 가지고 있었다. 406번 돌연변이 하나만 가지고 있는 균은 12균주(8.1%)로 이는 다른 조사에서 볼 수 없었던 비교적 높은 수치이었다. 한편 에탐부톨 및 11가지 항결핵약제에서 감수성인 50개균에서는 embB 유전자의 돌연변이를 발견하지 못하였다. 결 론 : 역교잡반응법으로 에탐부톨 내성에 관련되어 있는 것으로 알려진 embB 유전자 돌연변이를 찾는 것이 가능하였으며, 에탐부톨 내성기전 및 관련 유전자가 더 발견되어 민감도가 향상된다면 신속한 에탐부톨내성균 검출이 가능할 것으로 본다.

• Tuberculosis and Respiratory Diseases
• /
• 제60권2호
• /
• pp.171-179
• /
• 2006

목 적 : RFP과 RBU 사이의 교차 내성률은 다양하게 보고되고 있으며 rpoB 돌연변이가 이에 관여하는 것으로 알려져 있다. 본 연구에서는 본원에서 동정되어 보관중인 다제내성 결핵균주를 대상으로 하여 두 약물간의 교차 내성률 및 rpoB 돌연변이와의 연관성을 조사함으로써 다제내성 결핵의 치료에 있어 RBU의 효용성에 대하여 알아보고자 하였다. 방 법 : 2004년 한 해 동안 본원 검사실에서 다제내성 결핵균으로 동정되어 보관중인 130균주를 대상으로 하였다. RFP과 RBU의 내성검사는 L-J 배지를 이용한 절대농도법으로 시행하였으며 추가로 다음과 같은 다섯 가지의 농도를 이용하여 RBU의 MICs를 조사하였다; 10, 20, 40, 60, $120{\mu}g/ml$. 내성기준농도는 RFP의 경우 $40{\mu}g/ml$, RBU의 경우 $20{\mu}g/ml$로 하여 내성유무를 판정하였다. rpoB 돌연변이는 LiPA법을 이용한 REBA $MTB-Rifa^{(R)}$검사로 조사하였으며 염기서열분석을 의뢰하여 그 결과를 검증하고 구체적인 개별 돌연변이양상을 알아보았다. 결 과 : RFP은 모두 내성으로 확인되었고 RBU의 $MIC_{50}$은 $80{\mu}g/ml$, $MIC_{90}$은 ${\geq}160{\mu}g/ml$였으며 RFP과 RBU간의 교차내성률은 70.5%였다. REBA $MTB-Rifa^{(R)}$검사 결과 rpoB 돌연변이는 대부분 코돈 524-534 사이에서 발생하였고 검사를 시행한 100균주 가운데 98개의 균주에서 돌연변이가 확인되어 RFP내성을 진단할 수 있는 진단율은 약물감수성검사와 비교하여 98%의 일치율을 보였다. 염기서열분석결과 코돈 531과 513의 돌연변이는 돌연변이의 양상에 관계없이 항상 RBU내성과 관련되어 있었던 반면 코돈 526의 돌연변이는 돌연변이의 양상에 따라 내성 혹은 감성과 관련되어 있었다. 가장 흔한 돌연변이는 Ser531Leu로 전체의 45.5%를 차지하였다. 약물감수성검사에 비추어 His526Gln, His526Leu, Leu533Pro, Gln513Glu, Leu511Pro가 감성 돌연변이로 판단되었다. 결 론 : 두 약제간의 내성률을 고려하여 볼 때 RBU은 일부 다제내성 결핵의 치료에 있어서 효과가 있겠다. 우선 RBU에 대한 전통적인 약물감수성 검사를 도입하여 적절한 내성기준농도를 확립하는 노력이 필요하며 현재까지 rpoB 유전자 검사법은 임상에 적용하기에 한계가 있는 것으로 사료된다.

• 대한한방종양학회지
• /
• 제6권1호
• /
• pp.81-97
• /
• 2000

Objectives: This experimental study was carried out to evaluate the effects of aqueous and methanol extracts of Hedyotis diffusa which has long been used for cancer treatment in oriental medicines on the induction of apoptotic cell death in human lymphoid leukemia cell line, HL-60. Methods: Cells were treated with various concentrations (200 to $0.4{\mu}g$) and periods (6 to 30 hr) of $H_2O$ and methanol extracts of Hedyotis diffusa. Then, cells were tested for viability by MTT assay. Cells wrere treated with $200{\mu}g/ml$ of methanol extract fork various periods. Genomic DNA was isolated, separated, on 1.5% agarose gels, stained with ethidium bromide and visualized under UV light. Cells were treated with $200{\mu}g/ml$ of each extract for 16 hr. Then, cells were treated with Hoechst dye 33342 and observed by fluorescence microscopy. Cells were treated with various doses of each for 12 hr and $100{\mu}g/ml$ of methanol extract for various periods. Lysate from the cells used to measure the activity of Caspase-1 and-3 proteases by using fluorogenic peptide substrates including acetyl-YVAD-AMC and acetyl-DEVD-AMC, respectively. Cells were treated with $200{\mu}g/ml$ of each extract for various periods. Cell lysates were immunoprecipated with anti-JNKl antibodies. The immune complex was reacted with $32^p-ATP$ and c-Jun as a substrate. The phosphotransferase activity of JNKI was measured by using PhosphoImage analyzer (Fuji Co., Japan). Nuclear extracts were isolated and incubated with oligonucleotide probe of $NF-{\kappa}B$. Transcriptional activation of ${\kappa}B$ was measured by using EMSA and visualized by PhosphoImage analyzer (Fuji Co, Japan). Cell lysates were prepared and analyzed by Western blotting with anti-Bc12 antibodies and anti-Bax antibodies. Cells were pretreated with various doses of methanol extract for 2 hr. Then, the extract was removed by centrifugation. Cells were resuspended with RPMI-1640 media containing 0.3% agarose, 10% FBS, overlayred onto bottom layer agarose and incubated at $CO_2$ incubator for 6 days. The number of colony was counted under light microscopy ($\time100$). Results: The death of HL-60 cells was markedly induced by the addition of methanol extract of Hedyotis diffusa in a dose and time-dependent manners. The apoptotic characteristic ladder pattern of DNA strand break was observed in death of HL-60 cells. In addition, it was shown nucleus chromatin condensation and fragmentation under Hoechst staining. Therefore, Hedyotis diffusa extract-induced death of HL-60 cells is mediated by apoptotic signaling processes. The activity of Caspase 3-like proteases remained in a basal level in HL-60 cells treated with aqueous extract of Hedyotis diffusa. However, it was markedly increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. In addition, the phosphotransferase activity of JNKl was increased in HL-60 cells treated with methanol extract of Hedyotis diffusa. Furthermore, the activation of transcriptional activator, $NF-{\kappa}B$ was markedly induced by methanol extract of Hedyotis diffusa. Anti-apoptotic Bc12 was cleaved into 23Kda fragment by treatment of methanol extract of Hedyotis diffusa. However, expression of proapoptotic Bax protein was increased by treatment of methanol extract of Hedyotis diffusa in a time-dependent manner. Furthermore, methanol extract markedly inhibited the colony forming efficiency of HL-60 cells in semisolid agar culture. Conclusions: Above results suggest that methanol extract of Hedyotis diffusa induces the apoptotic death of human leukemic HL-60 cells via activations of Caspase-3 proteases, JNKI, transcriptional activator $NF-{\kappa}B$, In addition, our results also suggest that methanol extract of Hedyotis diffusa reduces the malignant potential of HL-60 cells via down regulation of colony forming effciency through cleavage of Bc12 as well as induction of Bax.