Gi-Seong Moon

Naringin-induced p21WAF1-mediated G1-phase cell cycle arrest via activation of the Ras/Raf/ERK signaling pathway in vascular smooth muscle cells

The flavonoid naringin has been shown to play a role in preventing the development of cardiovascular disease. However, the exact molecular mechanisms underlying the roles of integrated cell cycle regulation and MAPK signaling pathways in the regulation of naringin-induced inhibition of cell proliferation in vascular smooth muscle cells (VSMCs) remain to be identified. Naringin treatment resulted in significant growth inhibition and G(1)-phase cell cycle arrest mediated by induction of p53-independent p21WAF1 expression; expression of cyclins and CDKs in VSMCs was also down-regulated. In addition, among the pathways examined, blockade of ERK function inhibited naringin-dependent p21WAF1 expression, reversed naringin-mediated inhibition of cell proliferation and decreased cell cycle proteins. Moreover, naringin treatment increased both Ras and Raf activations. Transfection of cells with dominant negative Ras (RasN17) and Raf (RafS621A) mutant genes suppressed naringin-induced ERK activity and p21WAF1 expression. Finally, naringin-induced reduction in cell proliferation and cell cycle protein was abolished in the presence of RasN17 and RafS621A mutant genes. The Ras/Raf/ERK pathway participates in p21WAF1 induction, leading to a decrease in cyclin D1/CDK4 and cyclin E/CDK2 complexes and in naringin-dependent inhibition of cell growth. These novel and unexpected findings provide a theoretical basis for preventive use of flavonoids to the atherosclerosis disease.

Optimization of rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes by PCR and application to field test.

For rapid detection of Escherichia coli O157:H7 and Listeria monocytogenes, simple methods for sample preparation and PCR were established and applied to a field test. To improve specificity, primer sets LP43-LP44 and C(+)-D(-) were selected for E. coli O157:H7 and L. monocytogenes, respectively. Through centrifugation and partial heat treatment after enrichment, E. coli O157:H7 and L. monocytogenes were detected at 1 initial CFU without genomic DNA extraction in the culture and with artificially inoculated food samples including milk, chicken, ham, and pork. Based on the optimized PCR method, a feasibility test was carried out using randomly collected field samples. To remove false positives and false negatives, a PCR method using several primer sets, including the optimized primer set, and a standard culture method were used. With the PCR detection and standard culture methods, two pork samples were positive for L. monocytogenes after enrichment, indications that the PCR assay could be effectively used for rapid, sensitive, and species-specific detection of foodborne pathogens.

A Novel Lactobacillus casei LP1 Producing 1,4-Dihydroxy-2-Naphthoic Acid, a Bifidogenic Growth Stimulator

1,4-Dihydroxy-2-naphthoic acid (DHNA) is a bifidogenic growth stimulator (BGS) and could be a functional food ingredient since bifidobacteria are beneficial for human health. For that reason, lactic acid bacteria producing DHNA have been screened. A lactic acid bacterium LP1 strain isolated from a natural cheese was confirmed to produce DHNA, analyzed by a HPLC method. The strain was identified as Lactobacillus casei by 16S rRNA gene sequence analysis. The cell-free supernatant of fermented whey produced by L. casei LP1 presented the BGS activity for three bifidobacterial strains such as Bifidobacterium longum subsp. infantis KCTC 3127, Bifidobacterium bifidum KCTC 3202, and Bifidobacterium breve KCTC 3220 which were human-originated. To the best of our knowledge, a L. casei strain which can produce DHNA was firstly identified in this study.

Establishment of Quantitative Analysis Method for Genetically Modified Maize Using a Reference Plasmid and Novel Primers

For the quantitative analysis of genetically modified (GM) maize in processed foods, primer sets and probes based on the 35S promoter (p35S), nopaline synthase terminator (tNOS), p35S-hsp70 intron, and zSSIIb gene encoding starch synthase II for intrinsic control were designed. Polymerase chain reaction (PCR) products (80~101 bp) were specifically amplified and the primer sets targeting the smaller regions (80 or 81 bp) were more sensitive than those targeting the larger regions (94 or 101 bp). Particularly, the primer set 35F1-R1 for p35S targeting 81 bp of sequence was even more sensitive than that targeting 101 bp of sequence by a 3-log scale. The target DNA fragments were also specifically amplified from all GM labeled food samples except for one item we tested when 35F1-R1 primer set was applied. A reference plasmid pGMmaize (3 kb) including the smaller PCR products for p35S, tNOS, p35S-hsp70 intron, and the zSSIIb gene was constructed for real-time PCR (RT-PCR). The linearity of standard curves was confirmed by using diluents ranging from 2×101~105 copies of pGMmaize and the R2 values ranged from 0.999~1.000. In the RT-PCR, the detection limit using the novel primer/probe sets was 5 pg of genomic DNA from MON810 line indicating that the primer sets targeting the smaller regions (80 or 81 bp) could be used for highly sensitive detection of foreign DNA fragments from GM maize in processed foods.

In vivo imaging of Escherichia coli and Lactococcus lactis in murine intestines using a reporter luciferase gene

A recombinant DNA, pMG36e::luc+ (5.3 kb), was constructed and transferred to Escherichia coli MC1061 and Lactococcus lactis MG1363 for in vivo imaging of the bacteria in murine intestines. E. coli MC1061 (pMG36e::luc+) and L. lactis MG1363 (pMG36e::luc+) displayed luciferase activities in vitro, where the bioluminescent signal of the E. coli was much stronger than that of the L. lactis by approximately 100-fold. These 2 recombinant bacteria were orally administered into rats. The bioluminescent signals of the E. coli and L. lactis in the gastrointestinal (GI) tracts of rats were detected and maintained up to 2 h via whole body imaging, indicating that the luciferase gene expression system for bacteria could be applied for in vivo imaging.

Detection of 1,4-Dihydroxy-2-Naphthoic Acid from Commercial Makgeolli Products

To support beneficial effects of makgeolli for human health, we investigated for the presence of 1,4-dihydroxy-2-naphthoic acid (DHNA), a bifidogenic growth stimulator (BGS), from commercial makgeolli products. Among eleven makgeolli products (A∼K), four showed positive peaks for DHNA in high performance liquid chromatography analysis. Makgeolli product A in particular contained the highest concentration of DHNA (0.44 ppm), as confirmed by liquid chromatography-mass spectrometry. Furthermore, BGS activity of the makgeolli product A was higher than those of products in which DHNA was not detected. These results indicate that makgeolli can be a good source for DHNA and that DHNA-enriched makgeolli could be developed by modifying manufacturing procedures and controlling its microbiota.

Monitoring of Bioluminescent Lactobacillus plantarum in a Complex Food Matrix

A bioluminescent Lactobacillus plantarum (pLuc2) strain was constructed. The luminescent signal started to increase during the early exponential phase and reached its maximum in the mid-exponential phase in a batch culture of the strain. The signal detection sensitivity of the strain was the highest in PBS (phosphate buffered saline), followed by milk and MRS broth, indicating that the sensitivity was influenced by the matrix effect. The strain was used in millet seed fermentation which has a complex matrix and native lactic acid bacteria (LAB). The luminescent signal was gradually increased until 9 h during fermentation and abolished at 24 h, indicating that the strain could be specifically tracked in the complex matrix and microflora. Therefore, the bioluminescent labeling system can be used for monitoring LAB in food and dairy sciences and industries.

Growth of Clostridium perfringens spores inoculated in sous-vide processed Korean traditional Galbijjim under different storage conditions

To evaluate the microbiological safety of Korean traditional seasoned beef processed by sous-vide method, Clostridium perfringens spores were inoculated into the samples, and then germination and growth of spores were observed under different temperatures for days. Also, changes in pH, water activity, and salt contents were analyzed. As the results, there was no difference in water activity and pH of the samples during the storage at any temperature. However, salt contents of the samples significantly increased as storage time increased, and the storage temperature affected the change of salt contents. C. perfringens did not grow at 4 and 10°C for 24 days however, the bacterial growth was observed at 20°C after 2 days of storage. Based on these simulation tests, the microbiological safety of sous-vide processed galbijjim can be guaranteed at 4 and 10°C for 24 days even though the raw materials were contaminated by C. perfringens spores.

Comparison of Bifidogenic Growth Stimulation Activities of Fermented Whey Prototypes

Fermented whey solution presenting bifidogenic growth stimulation (BGS) activity was processed as prototypes such as sterilized fermented whey (SFW), spray-dried fermented whey (SDFW), and freeze-dried fermented whey (FDFW) and their BGS activities were compared. In optical density (OD600) test, the BGS activity of three prototypes, which showed similar activities, were significantly different with non-fermented whey solution adjusted to pH 4.5 as a control (P<0.05). In viable cell count test, SDFW had the most positive influence than other prototypes on the BGS activity even though the difference was not significant. However, the activities of all prototypes were significantly different than the negative control (no addition). These results indicate that the processed prototypes of fermented whey solution show BGS activities and might be commercialized, with further evidences, in animal or human studies.

Increased bacteriocin activity of a recombinant Pediococcus acidilactici

Pediocin PA-1, mainly produced from Pediococcus spp., is a well-known class IIa bacteriocin showing strong anti-listerial activity. Pediococcus acidilactici K10 producing the bacteriocin was transformed with pLR5cat(S)_PSAB in which the pediocin PA-1 structural gene (pedA) was translationally fused with a deduced signal sequence of the bifidobacterial α-amylase gene for secretion. The P. acidilactici K10 transformant presented increased bacteriocin activity when compared with that of native P. acidilactici K10. The P. acidilactici K10 mutant (K10M), in which the plasmid for pediocin PA-1 production is cured, also showed bacteriocin activity when transformed with pLR5cat(S)_PSAB, indicating that the pediocin gene was expressed in hosts. An increase in pedA gene transcription level and the detection of both transcripts from the pediocin operon and PSAB were also confirmed. The P. acidilactici K10 [pLR5cat(S)_PSAB] transformant was more inhibitory against Listeria monocytogenes than that of the P. acidilactici K10 wild type in co-cultures.