• Journal of Microbiology and Biotechnology
• /
• v.17 no.8
• /
• pp.1344-1352
• /
• 2007

An opportunistic human pathogen, Pseudomonas aeruginosa, contains the major catalase KatA, which is required to cope with oxidative and osmotic stresses. As an attempt to uncover the $H_2O_2$-dependent regulatory mechanism delineating katA gene expression, four prototrophic $H_2O_2$-sensitive mutants were isolated from about 1,500 TnphoA mutant clones of P. aeruginosa strain PA14. Arbitrary PCR and direct cloning of the transposon insertion sites revealed that one insertion is located within the katA coding region and two are within the coding region of oxyR, which is responsible for transcriptional activation of several antioxidant enzyme genes in response to oxidative challenges. The fourth insertion was within PA3815 (IscR), which encodes a homolog of the Escherichia coli iron-sulfur assembly regulator, IscR. The levels of catalase and SOD activities were significantly reduced in the iscR mutant, but not in the oxyR mutant, during the normal planktonic culture conditions. These results suggest that both IscR and OxyR are required for the optimal resistance to $H_2O_2$, which involves the expression of multiple antioxidant enzymes including KatA.

• Korean Journal of Veterinary Research
• /
• v.49 no.3
• /
• pp.195-200
• /
• 2009

Avian pathogenic Escherichia coli (APEC) is an important bacterial pathogen of chickens and causes colibacillosis such as airsacculitis, perihepatitis, omphalitis, peritonitis, salpingitis, and pericarditis. As the transfer of antibiotic resistance from animal to humans can be possible, surveillance on antibiotic resistance of APEC is very important. A total 34 APEC isolates from diseased chickens during the period from 2007 to 2009 were obtained. The susceptibility of the isolates to 13 antibiotics was determined by disc diffusion assay. Resistance to erythromycin was found in 97.1% of APEC isolated, followed by resistance to tetracycline (85.3%), doxycycline (82.3%), ampicillin (73.5%), sulfisoxazole (67.6%), enrofloxacin (67.6%), ciprofloxacin (64.7%), norfloxacin (61.7%) trimethoprim/sulfamethoxazole (52.9%), gentamycin (26.5%), amoxicillin (8.8%), colistin (5.9%), and amikacin (2.9%). The blaTEM genes were detected in 25 (100%) of the 25 ampicillin-resistant APEC isolates. Among the 29 tetracycline-resistant APEC isolates, tetA and tetB genes were detected in 18 (62.1%) and 9 (31%) isolates, respectively. Twenty six (76.5%) isolates were multiresistant to at least 6 antibiotics and seven (20.1%) isolates were multiresistant to at least 10 antibiotics. This results indicated that multiple antibiotic-resistant APEC is widespread in chicken flocks in Korea.

• Proceedings of the KIEE Conference
• /
• 2001.11a
• /
• pp.274-276
• /
• 2001

This research aims to develop the multiple channel electrochemical DNA chip using microfabrication technology. At first, we fabricated a high integration type DNA chip array by lithography technology. Several probe DNAs consisting of thiol group at their 5-end were immobilized on the sold electrodes. Then target DNAs were hybridized and reacted. Cyclic voltammetry showed a difference between target DNA and control DNA in the anodic peak current values. Therefore, it is able to detect a plural genes electrochemically after immobilization of a plural probe DNA and hybridization of non-labeling target DNA on the electrodes simultaneously. It suggested that this DNA chip could recognize the sequence specific genes.

• Asian Pacific Journal of Cancer Prevention
• /
• v.13 no.11
• /
• pp.5879-5882
• /
• 2012

Breast cancer is the leading cause of cancer deaths among women worldwide, including Thailand. In the present study, the differential mRNA expression of SVEP1, LPHN3, KLB, ITGA7, SEMA3G, TNS1 and MMP13 genes was examined in breast cancer using quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR). Among these genes, increased LPHN3 and MMP13 mRNA expression levels correlated with axillary-node metastasis (P=0.02). Multiple logistic regression analysis revealed that LPHN3 and MMP13 mRNA expression is significantly associated with axillary node status in breast cancer (P=0.04).

• Genomics & Informatics
• /
• v.15 no.4
• /
• pp.136-141
• /
• 2017

Accurate detection of genomic alterations, especially druggable hotspot mutations in tumors, has become an essential part of precision medicine. With targeted sequencing, we can obtain deeper coverage of reads and handle data more easily with a relatively lower cost and less time than whole-exome or whole-genome sequencing. Recently, we designed a customized gene panel for targeted sequencing of major solid cancers. In this study, we aimed to validate its performance. The cancer panel targets 95 cancer-related genes. In terms of the limit of detection, more than 86% of target mutations with a mutant allele frequency (MAF) <1% can be identified, and any mutation with >3% MAF can be detected. When we applied this system for the analysis of Acrometrix Oncology Hotspot Control DNA, which contains more than 500 COSMIC mutations across 53 genes, 99% of the expected mutations were robustly detected. We also confirmed the high reproducibility of the detection of mutations in multiple independent analyses. When we explored copy number alterations (CNAs), the expected CNAs were successfully detected, and this result was confirmed by target-specific genomic quantitative polymerase chain reaction. Taken together, these results support the reliability and accuracy of our cancer panel in detecting mutations. This panel could be useful for key mutation profiling research in solid tumors and clinical translation.

• Molecules and Cells
• /
• v.27 no.6
• /
• pp.689-695
• /
• 2009

Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of non-specific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without non-specific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.

• Reproductive and Developmental Biology
• /
• v.34 no.3
• /
• pp.257-262
• /
• 2010

The purpose of this study is to establish a basic culture system enabling in vitro culture of chicken blastodermal cells and to test the feasibility of retrovirus-mediated gene transfer to the cultured cells. The blastodermal cells were isolated from freshly laid eggs of stage X and cultured with or without STO feeder layer cells. Stem cell-like morphology was maintained after multiple passages and RT-PCR analysis proved expression of several stem cell specific genes. Immunocytochemical analysis using antibodies of anti-EMA-1 and anti-SSEA-1 also showed the feature of stem cells. Infection of the cultured blastodermal cells with LNCGW retrovirus vector resulted in successful transfer of foreign genes. The results of this study may be useful in establishing stem cell-mediated transgenic chicken production.

• BMB Reports
• /
• v.43 no.10
• /
• pp.693-697
• /
• 2010

Hereditary non-polyposis colorectal cancer (HNPCC) is an autosomal dominant syndrome characterized by predisposition to early-onset cancers. HNPCC is caused by heterozygous loss-of-function mutations within the mismatch repair genes MLH1, MSH2, MSH6, PMS1, and PMS2. We genotyped the MLH1 and MSH2 genes in patients suffering from Lynch syndrome and in 11 unrelated patients who were diagnosed with colorectal cancer and had subsequently undergone surgery. Five Lynch syndrome patients carried germline mutations in MLH1 or MSH2. Two of these were identified as known mutations in MLH1: deletion of exon 10 and a point mutation (V384D). The remaining three patients exhibited novel mutations: a duplication (937_942dupGAAGTT) in MLH1; deletion of exons 8, 9, and 10; and a point mutation in MLH1 (F396I) combined with multiple missense mutations in MSH2 (D295G, K808E, Q855P, and I884T). The findings underline the importance of efficient pre-screening of conspicuous cases.

• Journal of Microbiology and Biotechnology
• /
• v.15 no.6
• /
• pp.1214-1220
• /
• 2005

Decreased porin-mediated outer membrane penetration of hydrophilic antibiotics is a common mechanism of antibiotic resistance in Gram-negative bacteria. This study was undertaken to determine whether a null mutation in Pseudomonas putida would suppress porin synthesis, and therefore reduce the susceptibility of the organism to streptomycin, norfloxacin, and tetracycline. Inverse PCR amplification and double-stranded DNA sequencing were used to identify chromosomal genes carrying TnphoA'-1 inserts. Genome database available was used to identify putative homologue genes, one of which encodes protein with homology to domains of the MutS of P. putida, suggesting a crucial role in the multidrug resistance. Increased resistance to streptomycin, norfloxacin, and tetracycline might be due to accumulation of compensatory mutations. Either no growth or slow growth was observed in P. putida KH1027 when grown in minimal medium containing gluconate, glucose, or citrate; however, it is not clear whether the growth patterns contributed to the multidrug resistance.

• Korean Journal of Clinical Laboratory Science
• /
• v.43 no.2
• /
• pp.68-74
• /
• 2011

Pseudomonas aeruginosa is an aerobic, Gram-negative, glucose-nonfermenting bacterium, which has emerged as a serious opportunistic pathogen. Recently, outbreaks of carbapenem resistant P. aeruginosa give rise to significant therapeutic challenges for treating nosocomial infections. The genes of metallo-${\beta}$-lactamase (MBL), a powerful carbapenemase, are carried as a part of the mobile gene cassettes inserted into integrons playing an important role in rapid dissemination of antibiotic resistance genes among bacterial isolates. In this study, we investigated the prevalence of integron in imipenem resistant P. aeruginosa isolates. A total of 61 consecutive, non-duplicate, and imipenem resistant P. aeruginosa strains were isolated from a university hospital in the Chungcheong province of Korea. We employed repetitive extragenic palindromic sequence-based PCR (rep-PCR) method for the selection of clonally different P. aerusinosa strains. PCR and DNA sequencing were conducted for the detection of integrons. Twenty-one clonally different P. aeruginosa strains were isolated. Only one (P28) of the strains harbored $bla_{VIM-2}$ that was found as gene cassettes in class 1 integrons. Four of 21 carbapenem resistant P. aeruginosa strains harbored class 1 integron containing aminoglycoside resistance determinant. All of the integrons detected in the study contained more than one resistance gene cassette, which can mediate resistance to multiple antibiotics. To prevent further spreading of the multi-drug resistant P. aeruginosa, conseguent monitoring and clinical polices are required.