• International Journal of Industrial Entomology and Biomaterials
• /
• v.13 no.1
• /
• pp.31-35
• /
• 2006

The ubiquitin conjugating enzyme 2 (E2) is core component of ubiquitin proteasome pathway (UPP) which represents a selective mechanism for intracellular proteolysis in eukaryotic cells. The E2 has been implicated in the intracellular transfer of ubiquitin to target protein. We show here the involvement of E2 in antiviral immune of Bombyx mori to Bombyx mori nuclear polyhedrosis virus (BmNPV). In this study, mRNA fluorescent differential display PCR (FDD-PCR) was performed with BmNPV highly resistant silkworm strain NB and susceptible silkworm strain 306. At 24 h post BmNPV infection, FDD-PCR with the arbitrary primer AP34 showed that one cDNA band was down-regulated in the midgut of resistant strain, but highly expressed in susceptible strain. The deduced amino acid sequence of this cDNA clone share 99% identity with the recently published B. mori ubiquitin conjugating enzyme E2 (Genbank NO: DQ311351). Fluorescent quantitative PCR corroborated down regulation of E2 in resistant strain. We there conclude that BmNPV infection evokes strong response of susceptible strain including activation of UPP. BmNPV may evolve escape mechanisms that manipulate the UPP in order to persist in the infected host. In addition, the identification of down-regulation of E2 in resistant strain, as well as structure data, are essential to understanding how UPP operates in silkworm antiviral immune to BmNPV disease.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.9
• /
• pp.4999-5005
• /
• 2013

Objective: This study aimed to investigate expression of the proto-oncogene POK erythroid myeloid ontogenic factor (Pokemon) in colorectal cancer (CRC), and assess inhibitory effects of a small interference RNA (siRNA) expression vector in SW480 and SW620 cells. Methods: Semi-quantitative reverse transcription-polymerase chain reaction (PCR) and immunohistochemistry were performed to determine mRNA and protein expression levels of Pokemon in CRC tissues. Indirect immunofluorescence staining was applied to investigate the location of Pokemon in SW480 and SW620 cells. The siRNA expression vectors that were constructed to express a short hairpin RNA against Pokemon were transfected to the SW480 and SW620 cells with a liposome. Expression levels of Pokemon mRNA and protein were examined by real-time quantitative-fluorescent PCR and western blot analysis. The effects of Pokemon silencing on proliferation of SW480 and SW620 cells were evaluated with reference to growth curves with MTT assays. Results: The mRNA expression level of Pokemon in tumor tissues ($0.845{\pm}0.344$) was significantly higher than that in adjacent tumor specimens ($0.321{\pm}0.197$). The positive expression ratio of Pokemon protein in CRC (87.0%) was significantly higher than that in the adjacent tissues (19.6%). Strong fluorescence staining of Pokemon protein was observed in the cytoplasm of the SW480 and SW620 cells. The inhibition ratios of Pokemon mRNA and protein in the SW480 cells were 83.1% and 73.5% at 48 and 72 h, respectively, compared with those of the negative control cells with the siRNA. In the SW620 cells, the inhibition ratios of Pokemon mRNA and protein were 76.3% and 68.7% at 48 and 72 h, respectively. MTT showed that Pokemon gene silencing inhibited the proliferation of SW480 and SW620 cells. Conclusion: Overexpression of Pokemon in CRC may have a function in carcinogenesis and progression. siRNA expression vectors could effectively inhibit mRNA and protein expression of Pokemon in SW480 and SW620 cells, thereby reducing malignant cell proliferation.

• Asian-Australasian Journal of Animal Sciences
• /
• v.23 no.8
• /
• pp.987-992
• /
• 2010

This study was to detect the expression of heart fatty acid-binding protein (H-FABP) and adipocyte fatty acid-binding protein (A-FABP) gene mRNA in different tissues of Rugao and Luyuan chickens at 56 d and 120 d by real-time fluorescence quantitative reverse transcription polymerase-chain reaction (FQ-RT-PCR). The primers were designed according to the sequences of HFABP, A-FABP and GAPDH genes in Gallus gallus, which were used as target genes and internal reference gene, respectively. The levels of H-FABP and A-FABP gene expression were detected by SYBR Green I FQ-RT-PCR. The relative H-FABP and A-FABP gene mRNA expression level was calculated with 2-$^{{\Delta}Ct}$. Melting curve analysis showed a single peak of three genes. Intramuscular fat (IMF) content in breast muscle and leg muscle of the two chicken breeds at 120 d was higher than at 56 d. IMF content in breast muscle and leg muscle at 56 d and 120 d in Luyuan was significantly higher than in Rugao, however, abdominal fat of Luyuan was significantly lower than that of Rugao. The relative H-FABP gene mRNA expression level in cardiac muscle was the highest in both chicken breeds. The relative H-FABP and A-FABP gene expression of different tissues in Luyuan was higher than in Rugao. H-FABP gene mRNA expression had a negative effect on IMF of leg and breast muscles, and was significantly negatively correlated with IMF content. The relative A-FABP gene mRNA level in abdominal fat was higher than in liver. The A-FABP gene mRNA was not expressed in leg, breast and cardiac muscles. A-FABP gene mRNA expression level was significantly positively correlated with abdominal fat and had a significant effect on abdominal fat but not IMF content.

• Journal of Genetic Medicine
• /
• v.7 no.1
• /
• pp.59-66
• /
• 2010

Purpose: Quantitative fluorescent polymerase chain reaction (QF-PCR) allows for the rapid prenatal diagnosis of common aneuploidies. The main advantages of this assay are its low cost, speed, and automation, allowing for large-scale application. However, despite these advantages, it is not a routine method for prenatal aneuploidy screening in Korea. Our objective in the present study was to validate the performance of QF-PCR using short tandem repeat (STR) markers in a Korean population as a means for rapid prenatal diagnosis. Material and Methods: A QF-PCR assay using an Elucigene kit (Gen-Probe, Abingdon, UK), containing 20 STR markers located on chromosomes 13, 18, 21, X and Y, was performed on 847 amniotic fluid (AF) samples for prenatal aneuploidy screening referred for prenatal aneuploidy screening from 2007 to 2009. The results were then compared to those obtained using conventional cytogenetic analysis. To evaluate the informativity of STR markers, the heterozygosity index of each marker was determined in all the samples. Results: Three autosomes (13, 18, and 21) and X and Y chromosome aneuploidies were detected in 19 cases (2.2%, 19/847) after QF-PCR analysis of the 847 AF samples. Their results are identical to those of conventional cytogenetic analysis, with 100% positive predictive value. However, after cytogenetic analysis, 7 cases (0.8%, 7/847) were found to have 5 balanced and 2 unbalanced chromosomal abnormalities that were not detected by QF-PCR. The STR markers had a slightly low heterozygosity index (average: 0.76) compared to those reported in Caucasians (average: 0.80). Submicroscopic duplication of D13S634 marker, which might be a unique finding in Koreans, was detected in 1.4% (12/847) of the samples in the present study. Conclusion: A QF-PCR assay for prenatal aneuploidy screening was validated in our institution and proved to be efficient and reliable. However, we suggest that each laboratory must perform an independent validation test for each STR marker in order to develop interpretation guidelines of the results and must integrate QF-PCR into the routine cytogenetic laboratory workflow.

• Microbiology and Biotechnology Letters
• /
• v.33 no.2
• /
• pp.148-153
• /
• 2005

Malaria is a re-emerging infectious disease that is spreading to areas where it had been eradicated, such as Eastern Europe and Central Asia. To avoid the mortality from malaria, early detection of the parasite is a very important issue. The peripheral blood smear has been the gold standard method for the diagnosis of malaria infection. Recently, several other methods have been introduced for quantitative detection of malaria parasites. Real time PCR that employs fluorescent labels to enable the continuous monitoring of PCR product formation throughout the reaction has recently been used to detect several human malaria parasites. 18S rRNA sequences from malaria parasites have been amplified using Taqman real time PCR assay. Here, a SYBR Green-based real time quantitative PCR assay for the detection of malaria parasite-especially, Plasmodium vivax - was applied for the evaluation of 26 blood samples from Korean malaria patients. Even though SYBR Green-based real time PCR is easier and cheaper than Taqman-based assay, SYBR Green-based assay cannot be used because 18S rRNA cannot be specifically amplified using 1 primer set. Therefore, we used DBP gene sequences from Plasmodium vivax, which is specific for the SYBR Green based assays. We amplified the DBP gene from the 26 blood samples of malaria patients using SYBR Green based assay and obtained the copy numbers of DBP genes for each sample. Also, we selected optimal reference gene between ACTB and B2M using real time assay to get the stable genes regardless of Malaria titer. Using selected ACTB reference genes, we successfully converted the copy numbers from samples into titer, ${\sharp}$ of parasites per microliter. Using the resultant titer from DBP based SYBER Green assay with ACTB reference gene, we compared the results from our study with the titer from Taqman-based assay. We found that our results showed identical tendency with the results of 18S rRNA Taqman assay, especially in lower titer range. Thus, our DBP gene-utilized real time assay can detect Plasmodium vivax in Korean patient group semi-quantitatively and easily.

• The Plant Pathology Journal
• /
• v.35 no.6
• /
• pp.654-661
• /
• 2019

The soybean cyst nematode, Heterodera glycines, is a major plant-parasitic nematode that has caused important economic losses to Korea's soybean production. Four species of cyst nematodes, H. schachtii, H. glycines, H. trifolii, and H. sojae, all belong to schachtii group are coexist in field soil in Korea. The rapid identification of the nematode is crucial for preventing crop damage and in decision making for controlling this nematode. This study aimed to develop a species-specific primer set for quantitative PCR (qPCR) assay of H. glycines. The specific primer set (HGF1 and HGR1) for H. glycines was designed based on the cytochrome c oxidase subunit I (COI) sequence of mitochondrial DNA. After optimization, it is possible to identify the H. glycines using a qPCR assay with DNA extracted from a single cyst and single second-stage juvenile (J2). The specificity was confirmed by the absence of SYBR fluorescent signals of three other Heterodera species. A serial dilution of DNA extracted from a single cyst was obtained for the sensitivity test. The result showed that the standard curve of the test had a highly significant linearity between DNA concentration and Ct value (R² = 0.996, slope = -3.49) and that the detection limit concentration of DNA of the primer set was 10 pg of DNA per reaction. Our findings suggested that H. glycines could be distinguished from H. sojae and other Heterodera species when a qPCR assay is used with a specific primer set.

• Microbiology and Biotechnology Letters
• /
• v.48 no.1
• /
• pp.24-31
• /
• 2020

Five genes (mxbDM, mxcQE and mxaB) are responsible for the transcription of methanol oxidation genes in Methylobacterium strains. Among these, MxbDM and MxcQE constitute the two-component system (TCS) regulating methanol metabolism. In this study, we integrated the methanol-sensing domain of MxbD and MxcQ with the EnvZ/OmpR from Escherichia coli. The domain-swapping strategy resulted in chimeric histidine kinases (HK's) MxbDZ and MxcQZ AM1 containing recombinant E. coli. Real-time quantitative PCR was used to monitor OmpC expression mediated by the chimeric HK and response regulator (RR) OmpR. Further, an ompC promoter based fluorescent biosensor for sensing methanol was developed. GFP fluorescence was studied both qualitatively and quantitatively in response to environmental methanol. GFP measurement also confirmed ompC expression. Maximum fluorescence was observed at 0.05% methanol and 0.01% methanol using MxbDZ and MxcQZ AM1, respectively. Thus the chimeric HK containing E. coli were found to be highly sensitive to methanol, resulting in a rapid response making them an ideal sensor.

• Journal of Genetic Medicine
• /
• v.13 no.2
• /
• pp.95-98
• /
• 2016

We report the prenatal diagnosis of an unbalanced translocation between chromosome Y and chromosome 15 in a female fetus. Cytogenetic analysis of parental chromosomes revealed that the mother had a normal 46,XX karyotype, whereas the father exhibited a 46,XY,der(15)t(Y;15) karyotype. We performed cytogenetic analysis of the father's family as a result of the father and confirmed the same karyotype in his mother and brother. Fluorescence in situ hybridization and quantitative fluorescent-polymerase chain reaction analysis identified the breakpoint and demonstrated the absence of the SRY gene in female members. Thus, the proband inherited this translocation from the father and grandmother. This makes the prediction of the fetal phenotype possible through assessing the grandmother. Therefore, we suggest that conventional cytogenetic and molecular cytogenetic methods, in combination with family history, provide informative results for prenatal diagnosis and prenatal genetic counseling.

• Journal of Microbiology and Biotechnology
• /
• v.27 no.6
• /
• pp.1106-1111
• /
• 2017

Escherichia coli was engineered to sense methanol by employing a chimeric two-component system (TCS) strategy. A chimeric MxaY/EnvZ (MxaYZ) TCS was constructed by fusing the Paracoccus denitrificans MxaY with the E. coli EnvZ. Real-time quantitative PCR analysis and GFP-based fluorescence analysis showed maximum transcription of ompC and the fluorescence at 0.01% of methanol, respectively. These results suggested that E. coli was successfully engineered to sense methanol by the introduction of chimeric MxaYZ. By using this strategy, various chimeric TCS-based bacterial biosensors can be constructed and used for the development of biochemical-producing recombinant microorganisms.

• Asian Pacific Journal of Cancer Prevention
• /
• v.14 no.8
• /
• pp.4533-4537
• /
• 2013

Objective: To use microarray chip technology for screening of stem cell radiation related miRNAs in laryngeal squamous cell carcinoma; study and explore the relationship of miRNAs with radiosensitivity of laryngeal squamous cells. Method: After conventional culture and amplification of the laryngeal squamous carcinoma cell line Hep-2, CD 133+ cells were screened out with combination of isolated culture of stem cell microspheres and FACS for preparation of laryngeal cancer stem cells. After radiation treatment, miRNAs of laryngeal squamous carcinoma stem cells before and after radiation were enriched and purified. After microarray hybridization with mammalian miRNA and scanning of fluorescence signal, the miRNAs of laryngeal squamous carcinoma stem cells before and after radiation was subject to differential screening and clustering analysis. Real-time quantitative RT-PCR was used to verify part of the differentially expressed miRNAs. Results: 70 miRNAs related to laryngeal cancer stem cell radiation with 2-fold difference in expression were screened out, in which 62 were down-regulated and 8 were up-regulated. Fluorescent quantitative RT-PCR results were consistent with miRNAs chip results. Conclusion: Some miRNAs may be involved in self-regulation with laryngeal squamous carcinoma stem cell radiation.