• Horticultural Science & Technology
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• v.28 no.4
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• pp.648-655
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• 2010

This study is the first comprehensive report on the molecular cloning, structural characterization, sequence comparison between wild and mutant types, copy number in the genome, expression features and activities of a gene encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) in Korean lawn grass ($Zoysia$ $japonica$). The full length cDNA of the EPSPS from Korean lawn grass ($zj$EPSPS) obtained from a 3' and 5' RACE method was 1540 bp, containing a 1176 bp ORF, a 144 bp leader sequence (5' UTR) and a 220 bp 3' UTR, which was eventually decoded 391 amino acid residues with a molecular mass of 41.74 kDa. The Southern blot detection of the $zj$EPSPS showed that the gene exists as a single copy in the Korean lawn grass genome. Sequence comparison of the $zj$EPSPS gene demonstrated that the glyphosate-tolerant mutant (GT) having a Pro-53 to Ser substitution in the gene seems to have a preferred binding activity of the enzyme to phosphoenol pyruvate(PEP) over glyphosate, which allows the continuous synthesis of aromatic amino acids in the shikimate pathway. From the Northern blotting analysis, the $zj$EPSPS was found to be highly expressed, with continuous increase until 36 hours after 0.5% glyphosate treatment in both wild and mutant samples, but 1.5-fold higher EPSP synthase activity was observed in the tolerant mutant when exposed to the glyphosate treatment. The molecular information of the $zj$EPSPS gene obtained from this study needs to be further dissected to be more effectively applied to the development of gene-specific DNA markers and zoysiagrass cultivars; nevertheless, the glyphosate-tolerant mutant having the featured $zj$EPSPS gene can be provided to turfgrass managers for weed problems with timely adoptable management options.

• Journal of the Korean Academy of Child and Adolescent Psychiatry
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• v.11 no.1
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• pp.3-15
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• 2000

Objectives：There has been a rapid expansion of studies aimed at elucidating the genetic basis of autistic disorder, especially it’ relationship to fragile-X syndrome. The detection of fragile X chromosome(Xq27.3) by cytogenetic analysis has revealed many difficulties in testing. Therefore, to explore the relationship between autistic disorder and fragile X syndrome, this study administered molecular biologic methods which examined an unstable CGG repeat within the fragile X mental retardation-1(FMR-1) gene. Methods：Ninety nine autistic children and eight normal control children were tested. The number of CGG repeats within FMR-1 gene was measured after amplification by PCR, and cytogenetic analysis was also carried out to detect fragile site Xq27.3. Southern blot hybridization, using StB12.3 and/or Pfxa3 probe, was done for the patients showing expansion of more than 50 CGG repeats (premutation). Results：All but two autistic patients had no expansion in CGG repeats by PCR and there was no significant statistical difference in number of CGG repeat in comparison with normal control. Two autistic patients, considered as premutation by PCR analysis, had no full mutation or premutation by Southern blot hybridization. All autistic children tested did not have any abnormal karyotype or fragile site Xq27.3. Conclusions：These results suggest that autistic patients may not have abnormality in FMR-1 gene or abnormal expansion in CGG repeat. In conclusion, fragile X syndrome may not be antecedent of autistic disorder.

• Journal of Dairy Science and Biotechnology
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• v.33 no.2
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• pp.119-127
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• 2015

To date, detection of microbial populations in dairy products has been performed using culture media, which is a time-consuming and laborious method. The recently developed chromogenic media could be more rapid and specific than classical culture media. However, the newly developed molecular-based technology can detect microbial populations with greater rapidity and sensitivity than the classical method involving culture media and chromogenic media. This molecular-based technology could provide various options for monitoring the characterization of different states of bacteria and cells. Thus, it could help upgrade the processing system of the dairy industry so as to maintain the safety and quality of dairy foods. Among the various newly developed molecular-based technologies, flow cytometry can potentially be used for monitoring microbiological populations in the dairy industry if official international standards are available for this purpose. When omics technology would have biomarker identification, it could be regarded as the rapid and sensitive analytical methods. Methods based on PCR, which has become a basic technique in microbiological research, can be developed and validated as alternative methods for quantification of dairy microorganisms. This review discusses methods for monitoring microbiological populations in dairy foods and the limitations of these studies, as well as the need for further research on such methods in the dairy industry.

• KSBB Journal
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• v.18 no.6
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• pp.522-528
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• 2003

The purpose of this study is to inquire into molecular epidemiological characteristics of Vibrio parahaemolyticus. For this study, 120 strains(120 strains of Vibrio parahaemolyticus sampled from diarrhea patients) were examined and analyzed for biochemical characteristics, TDH (thermostable direct hemolysin) antibiotics sensitivity and detection of toxR, gyrE, tdh, and tds gents. G-S PCR (Group Specific Polymerase), PFGE (Pulsed-field Gel Electrophoriesis) methods were performed on the materials from patients were results. 1 Vibrio parahaemolyticus didn't grow in 0% density of NaCl, but the fact was found that those grew in 8% density of NaCl. 2. O：K serotypes of Vibrio parahaemolyticus was turned up in domestic patients was 17 types. Among those O3：K6 was the most, it was 68.3%. 3. In 18 kinds of antibiotic tests resistant against Ampicillin, Ticacillin was comparatively high. the case of resistant against Ampicillin, Ticacillin, Vancomycin at the multiple resistant was 52.5%. 4. Toxin gene tdh had only 109 strains among 120 ones isolated from patients held the genes of 199bp size, and 11 strains was negativity 5. In the test of Kanagawa toxic productivity, 107 strains among strains isolated from patients appeared to be positivity reaction 6. The strain that held trh toxin was only 3, and those among test strains had the genes of 250bp size and that had tdh, trh genes at a time were 3 strains, and TDH toxic productivity of those were 16 times, and it was weak. 7. Group Specific-PCR appeared to be useful in the confirmation of O3:K6 serotype interrelations. 8. Three strains which showed difference of 7 DNA sequence even in the same serotype were detected by the result of analyzing the regular gene, toxRS DNA sequence. These strains are differ from general strains which carry infection easily. 9. These mutual dose epidemiological relations were classified into smaller-parts through PFGE method. As a result of such classify, 3 findings were found. V. parahaemolyticus sampled from diarrhea patients were classified into 3 types. And third, the result obtained through PFGE method can be used as a useful tool in a point of molecular-epidemiological view.

• Journal of the Korean Society of Food Science and Nutrition
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• v.45 no.11
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• pp.1610-1616
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• 2016

This study aimed to establish an optimal extraction process and high performance liquid chromatography-ultraviolet (HPLC-UV) analytical method for determination of 3,5-dicaffeoylquinic acid (3,5-DCQA) as a part of materials standardization for the development of a xanthine oxidase inhibitor as a health functional food. The quantitative determination method of 3,5-DCQA as a marker compound was optimized by HPLC analysis using a Luna RP-18 column, and the correlation coefficient for the calibration curve showed good linearity of more than 0.9999 using a gradient eluent of water (1% acetic acid) and methanol as the mobile phase at a flow rate of 1.0 mL/min and a detection wavelength of 320 nm. The HPLC-UV method was applied successfully to quantification of the marker compound (3,5-DCQA) in Aster glehni extracts after validation of the method with linearity, accuracy, and precision. Ethanolic extracts of A. glehni (AGEs) were evaluated by reflux extraction at 70 and $80^{\circ}C$ with 30, 50, 70, and 80% ethanol for 3, 4, 5, and 6 h, respectively. Among AGEs, 70% AGE at $70^{\circ}C$ showed the highest content of 3,5-DCQA of $52.59{\pm}3.45mg/100g$ A. glehni. Furthermore, AGEs were analyzed for their inhibitory activities on uric acid production by the xanthine/xanthine oxidase system. The 70% AGE at $70^{\circ}C$ showed the most potent inhibitory activity with $IC_{50}$ values of $77.01{\pm}3.13{\sim}89.96{\pm}3.08{\mu}g/mL$. The results suggest that standardization of 3,5-DCQA in AGEs using HPLC-UV analysis would be an acceptable method for the development of health functional foods.

• Korean Journal of Microbiology
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• v.45 no.3
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• pp.251-256
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• 2009

Leaf samples and bulbs showing characteristic symptoms of virus infection were collected from Gang-won, Chung-nam, and Jeju Province of Korea in 2008-2009. Three viruses, Lily symptomless virus (LSV), Lily mottle virus (LMoV), and Cucumber mosaic virus (CMV) were detected by RT-PCR. Virus-infected plant samples were identified; 12 plants with LSV, 20 plants with LMoV, and 1 plant with CMV. Of the twelve LSV infected samples, seven samples were found to be mix-infected with LMoV and LSV. Symptoms of LMoV and LSV mixed infection were fairly severe, like as vein clearing, leaf curling, leaf mottling, leaf mosaic, and yellow streaking. Mixed infection with LMoV and LSV was also found in lily bulbs which have been stored under unfavorable environmental conditions. LMoV predominated in our tests, whereas spread of Lilyvirus X (LVX) was not found. The nucleotide sequences of coat protein (CP) region of seven isolates (4 LMoV, 2 LSV, and 1 CMV) were compared with the corresponding regions of LMoV (AJ564636), LSV (AJ516059) and CMV(AJ296154). The nucleotide sequence homologies between reference viruses and seven isolates were 95-99%. Complete sequencing of seven isolates is necessary to obtain more information on the molecular characteristics of these viruses as well as to increase sensitivity and rapidity of viral detection.

• Molecules and Cells
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• v.26 no.5
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• pp.468-473
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• 2008

Low-density lipoprotein (LDL) induces cell proliferation in human aortic smooth muscle cells (hAoSMCs), which may be involved in atherogenesis and intimal hyperplasia. Recent studies have demonstrated that $Cl^-$ channels are related to vessel cell proliferation induced by a variety of stimuli. In this study, we investigated a potential role of $Cl^-$ channels in the signaling pathway of LDL effects on hAoSMC proliferation with a focus on the activation of Erk1/2-PI3K/Akt and the subsequent upregulation of Egr-1. $Cl^-$ channel blockers, DIDS, but neither NPPB nor Furosemide, completely abolished the LDL-induced DNA synthesis and cell proliferation. Moreover, DIDS, but not NPPB, significantly decreased LDL-stimulated $Cl^-$ concentration, as judged by flow cytometry analysis using MQAE as a $Cl^-$-detection dye. DIDS pretreatment completely abolished the activation of Erk1/2 and PI3K/Akt in a dose-dependent manner that is the hallmark of LDL activation, as judged by Western blot and proliferation assays. Moreover, pretreatment with DIDS ($Cl^-$ channel blockers) but not LY294002 (PI3K inhibitors) completely abolished the LDL-induced upregulation of Egr-1 to the same extent as PD98059 (MEK inhibitors to inhibit Erk), as judged by Western blot and luciferase reporter assays. This is the first report, to our knowledge, that DIDS-sensitive $Cl^-$-channels play a key role in the LDL-induced cell proliferation of hAoSMCs via the activation of Erk1/2 and PI3K/Akt and the upregulation of Egr-1.

• Toxicological Research
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• v.13 no.1_2
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• pp.117-125
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• 1997

In order to assess the possibility whether CYP2D is involved in caffeine metabolism, we have purified and characterized the rat liver microsomal cytochrome P4502D1 (CYP2D1), equivalent to CYP2D6 in human liver, and have utilized the reconstituted CYP2D1 in the metabolism of 4 primary caffeine (1, 3, 7-trimethylxanthine) metabolites such as paraxanthine (1, 7-dimethylxanthine), 1, 3, 7-trimethylurate, theophylline (1, 3-dimethylxanthine) and theobromine (3, 7-dimethylxanthine). Rat liver CYP 2D1 has been purified to a specific content of 8.98 nmole/mg protein (13.4fold purification, 1.5% yield) using $\omega$-aminooctylagarose, hydroxlapatite, and DE52 columns in a sequential manner. As judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), the purified CYP2D1 was apparently homogeneous. Molecular weight of the purified CYP2D1 was found to be 51, 000 Da. Catalytic activity of the purified and then reconstituted CYP2D1 was confirmed by using bufuralol, a known subsFate of CYP2D1. The reconstituted CYP2D1 was found to produce to 1-hydroxylbufuralol at a rate of 1.43$\pm$0.13 nmol/min/nmol P450. The kinetic analysis of bufuralol hydroxylation indicated that Km and Vmax values were 7.32$\mu M$ and 1.64 nmol/min/nmol P450, respectively. The reconstituted CYP2D1 could catalyze the 7-demethylation of PX to 1-methylxanthine at a rate of 12.5 pmol/min/pmol, and also the 7- and 3- demethylations of 1, 3, 7-trimethylurate to 1, 3-dimethylurate and 1, 7-dimethylurate at 6.5 and 12.8 pmol/min/pmol CYP2D1, respectively. The reconstituted CYP2D1 could also 3-demethylate theophylline to 1-methylxanthine at 5 pmol/min/pmol and hydroxylate the theophylline to 1, 3-dimethylurate at 21.8 pmol/min/pmol CYP2D1. The reconstituted CYP2D1, however, did not metabolize TB at all (detection limits were 0.03 pmol/min/pmol). This study indicated that CYP2D1 is involved in 3-and 7-demethylations of paraxanthine and theophylline and suggested that CYP2D6 (equivalent to CYP2D1 in rat liver) present in human liver may be involved in the secondary metabolism of the primary metabolites of caffeine.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.6
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• pp.3937-3940
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• 2013

Objective: To explore the expression and significance of estrogen receptor (ER), progestrone receptor (PR), vascular endothelial growth factor (VEGF), CA15-3, CA125 and carcinoma embryonic antigen (CEA) expression in judging the prognosis of breast cancer. Materials and Methods: Sixty-five patients with breast cancer undergoing operations in the general surgery department were considered as the observation group, and 50 healthy outpatients of our hospital as the control group. Cubital venous blood was drawn in the morning from fasting patients in the two groups and chemiluminescence immunoassays were used to detect the levels of CA15-3, CA125 and CEA in serum. The follow-up duration was from 4 months to 2 years, and change in levels of the indicators was detected by dynamically drawing blood. After surgery, cancer tissue samples of patients in observation group remained on file (the non-recurrent patients were biopsied). Immunohistochemistry was applied to determine the expression of ER, PR and VEGF in tissue. Results: The effective rate of 12 patients with negative ER and PR expression was 33.3% in the observation group, being associated with prognosis to varying extents. Serum CA15-3, CA125 and CEA in the observation group were all significantly higher than in control group (p<0.01). With increase in pathological staging, levels of serum CA15-3, CA125 and CEA gradually increased (p<0.01). Levels in patients with lymph node metastasis were markedly higher than in those without (p<0.01). In addition, values with distal lymph node metastasis were notably higher than with adjacent lymph node metastasis (p<0.01). The postoperative follow-up results revealed that positive VEGF and levels of serum VEGF, CA15-3, CA125 and CEA in recurrence group were obviously higher than in non-recurrence group (p<0.01). Conclusions: Joint detection of ER and PR expression as well as levels of serum VEGF, CA15-3, CA125 and CEA is meaningful and can guide the diagnosis and treatment for breast cancer.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.6
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• pp.888-901
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• 2020

Objective: A set of microsatellite markers with high polymorphism from Tsaiya duck were used for the genetic monitoring and genetic structure analysis of Brown and White Tsaiya duck populations in Taiwan. Methods: The synthetic short tandem repeated probes were used to isolate new microsatellite markers from the genomic DNA of Tsaiya ducks. Eight populations, a total of 566 samples, sourced from Ilan Branch, Livestock Research Institute were genotyped through novel and known markers. The population genetic variables were calculated using optional programs in order to describe and monitor the genetic variability and the genetic structures of these Tsaiya duck populations. Results: In total 24 primer pairs, including 17 novel microsatellite loci from this study and seven previously known loci, were constructed for the detection of genetic variations in duck populations. The average values for the allele number, the effective number of alleles, the observed heterozygosity, the expected heterozygosity, and the polymorphism information content were 11.29, 5.370, 0.591, 0.746, and 0.708, respectively. The results of analysis of molecular variance and principal component analysis indicated a contracting Brown Tsaiya duck cluster and a spreading White Tsaiya duck cluster. The Brown Tsaiya ducks and the White Tsaiya ducks with Pekin ducks were just split to six clusters and three clusters when K was set equal to 6 and 3 in the Bayesian cluster analysis. The individual phylogenetic tree revealed eight taxa, and each individual was assigned to its own population. Conclusion: According to our study, the 24 novel microsatellite markers exhibited a high capacity to analyze relationships of inter- and intra-population in those populations with a relatively limited degree of genetic diversity. We suggest that duck farms in Taiwan could use the new (novel) microsatellite set to monitor the genetic characteristics and structures of their Tsaiya duck populations at various intervals in order to ensure quality breeding and conservation strategies.