Byoung-Su Yoon

Preventive effect of germanium dioxide on the inhibition of gap junctional intercellular communication by TPA.

Gap junctional intercellular communication (GJIC) is thought to be essential for maintaining cellular homeostasis and growth control. In order to detect any protective agent against tumor formation, we examined the anticarcinogenic effect of a germanium dioxide (GeO(2)) using a model system of GJIC in F344 rat liver epithelial cells, named WB cells. 12-O-tetradecanoylphorbol-13-acetate (TPA), known as tumor promoters, inhibited GJIC in the epithelial cells as determined by the scrape loading/dye transfer (SL/DT) assay. And GeO(2) recovered this inhibition of GJIC. Immunostaining of connexin 43 (Cx43) protein in WB cells indicated that TPA caused a loss of Cx43 protein from the cell membranes. However, GeO(2) treatment showed re-appearance of Cx43 protein on the membrane. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blots were analyzed to determine whether the test compounds might have altered the steady-state levels of gap junction mRNA and/or connexin protein levels or phosphorylation. The inhibition of GJIC by TPA in WB cells was correlated with the hyperphosphorylation of Cx43 as measured by mobility shifts of the western blot bands of Cx43. TPA induced hyperphosphorylation of Cx43 protein, while GeO(2) appeared to partially block this hyperphosphorylation. Here, we showed that pre- and co-incubation with GeO(2) in TPA-treated WB-cells abolished down-regulation of GJIC by TPA. These data suggest that GeO(2) may inhibit tumor promotion by enhancing GJIC.

Ultra-rapid real-time PCR for the detection of Paenibacillus larvae, the causative agent of American Foulbrood (AFB)

A novel micro-PCR-based detection method, termed ultra-rapid real-time PCR, was applied to the development of a rapid detection for Paenibacillus larvae (P. larvae) which is the causative agent of American Foulbrood (AFB). This method was designed to detect the 16S rRNA gene of P. larvae with a micro-scale chip-based real-time PCR system, GenSpector TMC-1000, which has uncommonly fast heating and cooling rates (10 degrees C per second) and small reaction volume (6microl). In the application of ultra-rapid real-time PCR detection to an AFB-infected larva, the minimum detection time was 7 min and 54s total reaction time (30 cycles), including the melting temperature analysis. To the best of our knowledge, this novel detection method is one of the most rapid real-time PCR-based detection tools.

Rapid detection of sacbrood virus in honeybee using ultra-rapid real-time polymerase chain reaction.

A real-time reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed for the fast and highly sensitive detection of the sacbrood virus (SBV) genome and applied to honeybee samples. Using plasmid DNA containing a partial SBV genome and diluted serially, as few as 1×10(2)copies/μl (correlation co-efficiency >0.99) were detected by the qRT-PCR assay, whereas 1×10(3)copies/μl were detected by the conventional RT-PCR assay. As a rapid detection method, ultra-rapid real-time PCR (URRT-PCR) was carried out with a GenSpector TMC-1000 silicon-glass chip-based thermal cycler, which has a 6μl micro-chamber volume and a fast outstandingly heating/cooling rate. Using this method, 10(3)copies of pBX-SBV3.8 clone were detected within 17 min after 40 PCR cycles, including melting point analysis. To reduce the detection time for SBV, synthesis of the cDNA of the SBV genome from a honeybee sample was attempted for different reaction times and the cDNA was used as the template for URRT-PCR assays. The results indicated that a 5 min reaction time was sufficient to synthesize cDNA as the template for the SBV URRT-PCR assay. This study described a novel PCR-based method that is able to detect an RNA virus in environmental samples within 22 min, including reverse transcription, PCR detection and melting point analysis in real-time.

Phylogenetic analysis of black queen cell virus genotypes in South Korea.

The black queen cell virus (BQCV), a picorna-like honeybee virus, was first isolated from queen larvae and pupae of honeybees found dead in their cells. BQCV is the most common cause of death in queen larvae. Phylogenetic analysis of two Apis cerana and three Apis mellifera BQCV genotypes collected from honeybee colonies in different regions of South Korea, central European BQCV genotypes, and a South African BQCV reference genotype was performed on a partial helicase enzyme coding region (ORF1) and a partial structural polypeptide coding region (ORF2). The phylogeny based on the ORF2 region showed clustering of all the Korean genotypes corresponding to their geographic origin, with the exception of Korean Am str3 which showed more similarity to the central European and the South African reference genotype. However, the ORF1-based tree exhibited a different distribution of the Korean strains, in which A. cerana isolates formed one cluster and all A. mellifera isolates formed a separate cluster. The RT-PCR assay described in this study is a sensitive and reliable method for the detection and classification of BQCV strains from various regions of Korea. BQCV infection is present in both A. cerana and A. mellifera colonies. With this in mind, the present study examined the transmission of honeybee BQCV infections between A. cerana and A. mellifera.

Development of a quantitative sandwich enzyme-linked immunosorbent assay for detecting the MPT64 antigen of Mycobacterium tuberculosis.

PURPOSE Tuberculosis (TB) is a major infectious disease and is responsible for two million deaths annually. For the identification and quantitation of Mycobacterium tuberculosis (M. tuberculosis), a causative agent of TB, a sandwich enzyme-linked immunosorbent assay (ELISA) against the MPT64 protein of M. tuberculosis, an antigen marker of the M. tuberculosis complex, was developed. MATERIALS AND METHODS The MPT64 protein was expressed, and anti-MPT64 monoclonal antibodies were prepared. A sandwich ELISA was established using recombinant MPT64 protein and anti-MPT64 monoclonal antibodies. The sandwich MPT64 ELISA was evaluated using reference and clinical mycobacterial strains. RESULTS The sandwich MPT64 ELISA detected MPT64 protein from 2.1 ng/mL to 250 ng/mL (equivalent to 1.7×10⁴ CFU/mL and 2.0×10⁶ CFU/mL). All 389 clinical M. tuberculosis isolates tested positive in the sandwich MPT64 ELISA (sensitivity, 100%), and the assay showed no cross reactivity to any tested nontuberculous mycobacterial strain (specificity, 100%). CONCLUSION The sandwich MPT64 ELISA is a highly sensitive and quantitative test for MPT64 protein, which can identify M. tuberculosis.

Reverse transcription loop-mediated isothermal amplification for sensitive and rapid detection of Korean sacbrood virus.

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, the Korean sacbrood virus (KSBV) caused great losses in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting KSBV using total RNA extracted from honeybees (A. cerana) infected with SBV. The LAMP and the polymerase chain reaction (PCR) methods were then compared for their ability to detect KSBV in clinical samples. The virus was detected in RT-LAMP reactions containing 10(3) copies of pBX-KSBV within 30min, which was comparable to RT-PCR. In addition, the LAMP was able to distinguish between KSBV and other closely-related SBV strains, indicating a high degree of specificity. This simple and sensitive RT-LAMP assay is a useful method for the rapid diagnosis of KSBV infection in honeybees.

Development of a rapid detection method to detect tdh gene in Vibrio parahaemolyticus using 2-step ultrarapid real-time polymerase chain reaction.

Thermostable direct hemolysin encoded by tdh gene has been considered an important virulence factor in pathogenic Vibrio parahaemolyticus. Two-step ultrarapid real-time polymerase chain reaction (URRT PCR) with a microchip was devised to detect V. parahaemolyticus carrying tdh gene. This novel method has a 6-μL reaction volume and extremely reduces running time since one cycle can be completed in 10 s or less. Consequently, 35 cycles of URRT PCR was successfully able to detect up to 100 fg (18 copies) of genomic DNA from pathogenic V. parahaemolyticus carrying tdh gene in 6 min. These results indicate that this method is at present the most rapid detection method for tdh gene and pathogenic V. parahaemolyticus.

Polymerase Chain Reaction Analysis of Eae Gene Subtypes Present in Attaching and Effacing Escherichia Coli Isolated from Pigs with Diarrhea

In this study the subtype of eae gene was determined by polymerase chain reaction for a total of 59 attaching and effacing Escherichia coli isolated from preweaned (38 isolates) and postweaned (21 isolates) pigs. The eae β gene detected in 19 E. coli from preweaned pigs and 10 E. coli from postweaned pigs was found to be the most common subtype, followed by eae γ, eaeε, and eae ζ genes. Subtypes were not determined for 7 E. coli isolates. No other subtype of the eae gene was detected in eae + E. coli evaluated in this study.

Expression and purification of Listeria innocua Sv6b p60 invasion associated protein

The p60 protein of Listeria is a major extra-cellular protein which is used as indicator for the detection of these bacteria from contaminated food samples. To produce p60 in Escherichia coli, the invasion associated protein (iap) gene of L. innocua Sv6b encoding p60 was cloned and over-expressed with expression vector pMAL-C2. Recombinant pMBP-iap/innocua was induced with IPTG in E. coli. The expressed recombinant p60 protein that was fused with a maltose-binding protein (MBP) was purified by amylose resin-based affinity chromatography. The purified recombinant p60 protein was also detected as denatured and neutralized form by using a specific p60 monoclonal antibody against L. monocytogenes and it may be useful for the production of L. innocua-specific antibody.

Detection of chronic bee paralysis virus using ultra-rapid PCR and nested ultra-rapid PCR

The chronic bee paralysis virus (CBPV), which causes paralysis in bees, is very difficult to visually differentiate from other pathogens causing symptoms of paralysis. In addition, low numbers of viral gene molecules are difficult to detect. Therefore, this study was conducted to develop an ultra-rapid quantitative polymerase chain reaction (PCR) and nested PCR method for specifically detecting CBPV. This method is based on rapid and precise quantification of the RNA virus CBPV, using ultra-rapid reverse transcription PCR and nested PCR to overcome difficulties in detecting low numbers of viral gene molecules. Under optimal conditions, CBPV-specific ultra-rapid PCR confirmed that 1.0 × 108 CBPV-specific genes could be detected in 4 min and 17 s, and 1.0 × 102 CBPV-specific genes could be detected in 11 min and 16 s. The detection of 1.0 × 102 CBPV-specific genes was conducted by nested PCR using the inner primer set CBPV-NF/NR. High-sensitivity detection was demonstrated by confirming that the PCR product for the initial template of 1.0 × 10° CBPV-specific gene was detectable. The CBPV-specific ultra-rapid quantitative PCR and nested PCR methods are applicable for infected honey bee samples to rapidly and accurately detect low numbers of viral gene molecules. This method can be used on-site where the infected sample is collected as well as in the laboratory. This method may also be applied to detect for other honey bee pathogens with a suitable primer for each pathogen.