• Title/Summary/Keyword: PCR.

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Sequencing analysis of the OFC1 gene on the nonsyndromic cleft lip and palate patient in Korean (한국인 비증후군성 구순구개열 환자의 OFC1 유전자의 서열 분석)

  • Kim, Sung-Sik;Son, Woo-Sung
    • The korean journal of orthodontics
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    • v.33 no.3 s.98
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    • pp.185-197
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    • 2003
  • This study was performed to identify the characteristics of the OFC1 gene (locus: chromosome 6p24.3) in Korean patients, which is assumed to be the major gene behind the nonsyndromic cleft lip and palate. The sample consisted of 80 subjects: 40 nonsyndromic cleft lip and palate patients (proband, 20 males and females, mean age 14.2 years); and 40 normal adults (20 males and 20 females, mean age 25.6 years). Using PCR-based assay, the OFC1 gene was amplified, sequenced, and then searched for similar protein structures. Results were as follows: 1. The OFC1 gene contains the microsatellite marker 'CA' repeats. The number of the reference 'CA' repeats was 21 times, and formed as TA(CA)11TA(CA)10. But, in Koreans, the number of tandem 'CA' repeats was varied from 17 to 26 except 18, and 'CA' repeats consisted of TA(CA)n. 2. Nine allelic variants were found. Distribution of the OFC1 allele was similar between the patients and control group. 3. There was a replacement of the base 'T' to 'C' after 11 tandem 'CA' repeats in Koreans compared with Weissenbach's report. However, the difference did not seem to be the ORF prediction results between Koreans and Weissenbach's report. 4. The BLAST search results showed the Telomerase reverse transcriptase (TERT) and the Nucleotide binding protein 2 (NBP2) as similar proteins. The TERT was a protein product by the hTERT gene in the locus 5p15.33 (NCBI Genome Annotation; NT023089) The NBP2 was a protein product by the ABCC3 (ATP-binding cassette, sub-family C) gene in the locus 17q22 (NCBI Genome Annotation; NT010783). 5. In the Pedant-Pro database analysis, the predictable protein structure of the OFC1 gene had at least one transmembrane region and one non-globular region.

Exploration of optimum conditions for production of saccharogenic mixed grain beverages and assessment of anti-diabetic activity (잡곡당화음료 제조 최적 조건 탐색 및 항당뇨 활성 평가)

  • Lee, Jae Sung;Kang, Yun Hwan;Kim, Kyoung Kon;Yun, Yeong Kyeong;Lim, Jun Gu;Kim, Tae Woo;Kim, Dae Jung;Won, Sang Yeon;Bae, Moo Hoan;Choi, Han Seok;Choe, Myeon
    • Journal of Nutrition and Health
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    • v.47 no.1
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    • pp.12-22
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    • 2014
  • Purpose: This study was conducted to establish the production conditions through optimization of the production process of beverages using Aspergillus oryzae CF1001, and to analyze volatile compounds and antidiabetic activity. Methods: The optimum condition was selected using the response surface methodology (RSM), through a regression analysis with the following independent variables gelatinization temperature (GT, $X_1$), saccharogenic time (ST, $X_2$), and dependent variable; ${\Delta}E$ value (y). The condition with the lowest ${\Delta}E$ value occurred with combined 45 min ST and $50^{\circ}C$ GT. The volatile compounds were analyzed quantitatively by GC-MS. Results: Assessment of antidiabetic activity of saccharogenic mixed grain beverage (SMGB) was determined by measurement of ${\alpha}$-glucosidase inhibition activity, and glucose uptake activity and glucose metabolic protein expression by reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analysis. Results of volatile compounds analysis, 62 kinds of volatile compounds were detected in SMGB. Palmitic acid (9.534% ratio), benzaldehyde (8.948% ratio), benzyl ethyl ether (8.792% ratio), ethyl alcohol (8.35% ratio), and 2-amyl furan (4.826% ratio) were abundant in SMGB. We confirmed that ${\alpha}$-glucosidase inhibition activity, glucose uptake activity, and glucose-metabolic proteins were upregulated by SMGB treatment with concentration dependent manner. Conclusion: Saccharogenic mixed grain beverage (SMGB) showed potential antidiabetic activity. Further studies will be needed in order to improve the taste and functionality of SMGB.

Anti-obesity effect of EGCG and glucosamine-6-phosphate through decreased expression of genes related to adipogenesis and cell cycle arrest in 3T3-L1 adipocytes (Adipogenesis관련 유전자발현감소와 Cell Cycle Arrest를 통한 EGCG와 Glucosamine-6-Phosphate의 Anti-Obesity 효과)

  • Kim, Kkot Byeol;Jang, Seong hee
    • Journal of Nutrition and Health
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    • v.47 no.1
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    • pp.1-11
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    • 2014
  • Purpose: Several studies have proven that EGCG, the primary green tea catechin, and glucosamine-6-phosphate (PGlc) reduce triglyceride contents in 3T3-L1 adipocytes. The objective of this study is to evaluate the combination effect of EGCG and PGlc on decline of accumulated fat in differentiated 3T3-L1 adipocytes. Methods: EGCG and PGlc were administered for 6 day for differentiation of 3T3-L1 adipocytes. Cell viability was measured using the CCK assay kit. In addition, TG accumulation in culture 3T3-L1 adipocytes was investigated by Oil Red O staining. We examined the expres-sion level of several genes and proteins associated with adipogenesis and lipolysis using real-time RT-PCR and Western blot analysis. A flow cytometer Calibar was used to assess the effect of EGCG and PGluco on cell-cycle progression of differentiating 3T3-L1 cells. Results: Intracelluar lipid accumulation was significantly decreased by combination treatment with EGCG $60{\mu}M$ and PGlc $200{\mu}g/m$ compared with control and EGCG treatment alone. In addition, use of combination treatment resulted in directly decreased expression of $PPAR{\gamma}$, $C/EBP{\alpha}$, and SREBP1. In addition, it inhibited adipocyte differentiation and adipogenesis through downstream regulation of adipogenic target genes such as FAS, ACSL1, and LPL, and the inhibitory action of EGCG and PGlc was found to inhibit the mitotic clonal expansion (MCE) process as evidenced by impaired cell cycle entry into S phase and the S to G2/M phase transition of confluent cells and levels of cell cycle regulating proteins such as cyclin A and CDK2. Conclusion: Combination treatment of EGCG and PGlc inhibited adipocyte differentiation through decreased expression of genes related to adipogenesis and adipogenic and cell cycle arrest in early stage of adipocyte differentiation.

The Short Term Efficacy of Entecavir Therapy in Chronic Hepatitis B (만성 B형 간염 환자에서 엔테카비어의 단기 치료 효과)

  • Kim, Hyun;Chae, Hee-Bok;Jeon, Won-Joong;Park, Seon-Mee;Youn, Sei-Jin;Eun, Jong-Ryul;Lee, Heon-Ju
    • Journal of Yeungnam Medical Science
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    • v.25 no.1
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    • pp.31-40
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    • 2008
  • Background/Aims : Entecavir is a synthetic nucleoside analogue, cyclopentyl guanine nucleoside, which has a potent antiviral effect and the least viral breakthrough in hepatitis B virus (HBV) replication. Entecavir has been available in Korea since 2007 but there are few reports on its effects. The aim of this study was to evaluate the virological response (VR) and biochemical response (BR) to entecavir in HBV patients at 3, 6 and 9 months after treatment with entecavir. Materials and Methods : Thirty-three chronic hepatitis B patients who took entecavir for at least 9 months were enrolled. We investigated VR and BR by retrospectively reviewing medical records. Patients who satisfied the following criteria were chosen: 1) initial alanine aminotransferase (ALT) levels = 1.5upper limit of normal (ULN) and 2) initial HBV DNA levels = $5\;log_{10}\;copies/ml$. We measured ALT levels every 3 months until month 9. HBV DNA was measured every 2 or 3 months by polymerase chain reaction (PCR) method. Results : Most patients taking entecavir showed good BR (ALT < 40 IU/L). The BR rates were 61%, 73% and 67% at months 3, 6 and 9, respectively. VR (HBV DNA < $5\;log_{10}\;copies/ml$ or 2 log lower than initial HBV DNA) rates were 82%, 91% and 91% at months 3, 6 and 9, respectively. Undetectable HBV DNA (HBV DNA < 4 log10 copies/ml) rates were 49%, 73% and 85% at months 3, 6 and 9, respectively. Two patients presented with virological breakthrough without adverse effects until month 9. Conclusions : Entecavir showed good BR and VR from month 3 and these effects continued through the 9-month observation period. This suggests that entecavir is also a good choice for the first line treatment of chronic hepatitis B (CHB). Further studies are needed to determine the long-term efficacy and drug resistance of entecavir in Korean CHB patients.

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Microbial Community Composition Associated with Anaerobic Oxidation of Methane in Gas Hydrate-Bearing Sediments in the Ulleung Basin, East Sea (동해 울릉분지 가스 하이드레이트 매장 지역의 메탄산화 미생물 군집 조성 및 분포)

  • Cho, Hyeyoun;Kim, Sung-Han;Shin, Kyung-Hoon;Bahk, Jang-Jun;Hyun, Jung-Ho
    • The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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    • v.20 no.1
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    • pp.53-62
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    • 2015
  • To elucidate the microbial consortia responsible for the anaerobic methane oxidation in the methane hydrate bearing sediments, we compared the geochemical constituents of the sediment, the rate of sulfate reduction, and microbial biomass and diversity using an analysis of functional genes associated with the anaerobic methane oxidation and sulfate reduction between chimney site (UBGH2-3) on the continental slope and non-chimney site (UBGH2-10) on the basin of the Ulleung Basin. From the vertical profiles of geochemical constituents, sulfate and methane transition zone (SMTZ) was clearly defined between 0.5 and 1.5 mbsf (meters below seafloor) in the UBGH2-3, and between 6 and 7 mbsf at the UBGH2-10. At the UBGH2-3, the sulfate reduction rate (SRR) in the SMTZ exhibited was appeared to be $1.82nmol\;cm^{-3}d^{-1}$ at the depth of 1.15 mbsf. The SRR in the UBHG2-10 showed a highest value ($4.29nmol\;cm^{-3}d^{-1}$) at the SMTZ. The 16S rRNA gene copy numbers of total Prokaryotes, mcrA, (methyl coenzyme M reductase subunit A), and dsrA (dissimilatory sulfite reductase subunit A) showed the peaks in the SMTZ at both sites, but the maximum mcrA gene copy number of the UBGH2-10 appeared below the SMTZ (9.8 mbsf). ANME-1 was a predominant ANME (Anaerobic MEthanotroph) group in both SMTZs of the UBGH2-3 and -10. However, The sequences of ANME-2 were detected only at 2.2 mbsf of the UBGH2-3 where high methane flux was observed because of massive amount of gas hydrate at shallow depth. And Desulfosarcina-Desulfococcus (DSS) that is associated with ANME-2 was detected in 2.2 mbsf of the UBHG2-3. Overall results demonstrate that ANME-1 and ANME-2 are considered as significant archaeal groups related to methane cycle in the subsurface sediment of the East Sea, and ANME-2/DSS consortia might be more responsible for methane oxidation in the methane seeping region than in non-seeping region.

Distinct Spatio-temporal Expression Patterns of Patatin Promoter-GUS Gene Fusion in Transgenic Potato Microtubers (형질전환 감자 소괴경의 발달단계에 따른 Patatin Promoter-GUS 유전자의 발현 분석)

  • Youm, Jung-Won;Kim, Mi-Sun;Lee, Byoung-Chan;Kang, Won-Jin;Jeon, Jae-Heung;Joung, Hyouk;Kim, Hyun-Soon
    • Journal of Plant Biotechnology
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    • v.30 no.1
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    • pp.13-18
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    • 2003
  • This study was carried out to investigate the expression patterns of foreign gene that controlled by tuber-specific patatin promoter in transgenic potatoes. Potato leaf disc cultured in vitro were transformed by the Agrobacterium strain LBA4404 containing pBl121 or pATGUS from potato cv. Desiree. In order to select the transgenic lines, gene-specific primers deduced from the NPTII were synthesized and used for polymerase chain reaction. The down part of the putative transgenic potatoes was transplanted weekly onto sucrose-enriched medium to accelerate the microtuber formation. RNA gel blot analysis was performed on the total RNAs obtained from tuber that had been harvested at a week interval. Also, histochemical assay was observed in the explants transformed with either pBI121 or pATGUS. Results showed that the transgenic plant containing pATGUS expressed GUS transcripts mainly at the tuber, not in stem, with the highest expression level in 5 weeks-grown microtubers. In contrast to pATGUS plants, the transformed plants with pBI121 showed an equal expression pattern throughout the whole developing stages. Consistent with RNA gel blot analysis, histochemical GUS staining and enzyme activity exhibited pATGUS transcripts were at the highest level in 5 weeks cultures. From these results, we suggest that the best stage to analyze the foreign gene introduced by patatin promoter into potato plants is at 5 weeks cultures after tuber formation.

Characterization of PR-10 gene derived from highly resistant '93-3-98' pear inoculated with scab (Venturia nashicola) (배 검은별무늬병(Venturia nashicola) 고도 저항성 '93-3-98' 유래 PR-10 유전자의 특성)

  • Chun, Jae An;Kim, Se Hee;Cho, Kang Hee;Kim, Dae Hyun;Choi, In Myong;Shin, Il Sheob
    • Journal of Plant Biotechnology
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    • v.42 no.1
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    • pp.25-33
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    • 2015
  • A PyrcpPR-10 gene with differentially expressed was isolated by using the suppression subtractive hybridization assay between '93-3-98' (highly resistant against scab caused by Venturia nashicola) and 'Sweat Skin'(highly susceptible) and analyzed the expression pattern according to organs and cultivars. The full length of PyrcpPR-10 was cloned as 743bp with 480bp's ORP, and was determined to encode a protein of 159 amino acid residues. On analyzing PyrcpPR-10 gene sequence compared with resistant and susceptible cultivars, 'Hwangsilri' (resistant), 'Gamcheonbae' (moderately resistant), 'Wonhwang' (moderately susceptible), 'Niitaka' (highly susceptible), and 'Sweat Skin' (highly susceptible) had identical gene sequence but 'Bartlett' (highly resistant) showed partly different sequences. The deduced amino acid sequence showed 64 ~ 98% homology and had the GXGGXG motif to known amino acid of other plants PR-10 by the BLAST X analysis. Among several organs or tissues, petal was showed highest expression level of PyrcpPR-10 gene followed by leaf, floral axis, bud, and bark. The expression level of PyrcpPR-10 gene was dramatically increased at 24 hr after inoculation in all cultivars and also up-regulated in accordance with resistant degree of cultivars. While resistant cultivars ('Bartlett', '93-3-98', and 'Hwangsilri') induced relatively high expression level of PyrcpPR-10 gene, susceptible cultivars ('Niitaka', and 'Sweat Skin') showed low expression level. PyrcpPR-10 gene is assumed that it is directly connected with defense mechanisms to pear scab.

De novo Expression of Hepatic UCP3 Is Time-Dependently Related with Metabolic Function in Fenofibrate-Treated High Fat Diet Rats (고지방 섭취한 쥐에서 페노파이브레이트 복용에 의한 간 UCP3 발현 기간과 대사변화 관계)

  • Park, Mi-Kyoung;Kang, Ah-Young;Seo, Eun-Hui;Joe, Yeon-Soo;Kang, Soo-Jeong;Hong, Sook-Hee;Kim, Duk-Kyu;Lee, Hye-Jeong
    • Journal of Life Science
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    • v.19 no.1
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    • pp.15-20
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    • 2009
  • Uncoupling protein 3 (UCP3) is a mitochondrial protein that is expressed predominantly in skeletal muscle. It may play a role in altering metabolic function. However, its major physiological roles are not fully understood. Recently de novo expression of UCP3 in rat liver by fenofibrate was reported. We also reported previously that fenofibrate-induced de novo expression of UCP3 contributes to reduction of adipose tissue in obese rats. In the present study, we investigated that ienofibrate-induced expression of UCP3 in rat liver is related with metabolic function such as body weight and hepatic lipid content by time-dependent manner in high-fat diet rats. Eight-week-old male Sprague-Dawley rats were randomly divided into two groups; the high fat diet group (HF, n=16) and fenofibrate-treated high fat diet group (HFF, n=16). The mRNA expression of hepatic UCP3 was detected as early as 1 week of fenofibrate treatment by quantitative real-time PCR and the amount of mRNA was increased time-dependently. The mean body weight of the HFF group was significantly less com. pared with the HF group after 6 weeks of fenofibrate treatment, even though there was no difference of food intake between the two groups. Rectal temperature was increased during 4 to 6 weeks of fenofibrate treatment and body weight was decreased after 6 weeks of treatment. These results were corresponded with the increased amount of the expression of UCP3 mRNA and protein. We suggest that de novo expression of hepatic UCP3 is increased time-dependently with fenofibrate treatment and that the amount of expression is correlated with metabolic function.

Intracullular Functions of the mas2+ Gene in the Fission Yeast, Schizosaccharomyces pombe (분열형 효모에서의 mas2+ 유전자의 세포 내 기능)

  • Sin, Sang-Min;Cha, Jae-Young;Ha, Se-Eun;Sim, Sun-Mi;Kim, Hyoung-Do;Lee, Jung-Sup;Park, Jong-Kun
    • Journal of Life Science
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    • v.19 no.1
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    • pp.101-110
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    • 2009
  • The regulation of gene expression plays an important role in cell cycle controls. In this study, a novel $mas2^+$ (mitosis associated protein) gene, a homolog of human SMARCAD1 was isolated and characterized from a fission yeast Schizosaccharomyces pombe (S. pombe) using gene-specific polymerase chain reaction. The isolated gene contained a complete open reading frame capable of encoding 922 amino acid residues with a typical promoter, as judged by nucleotide sequence analysis. It was also found that an SNF2 domain is located, which is involved in the chromosome remodeling. The quantitative analysis of the $mas2^+$ transcript against $adh1^+$ showed that the expression level of $mas2^+$ is high before septum formation in S. pombe. When $mas2^+$ null mutant cells were grown at 27 and $35^{\circ}C$, the cytokinesis of $mas2^+$ null mutant was greatly delayed and a large number of multi-septate and mis-segregated cells were produced. In addition, the number of multi-septate cells significantly increased. When cells were cultured in YES rich medium to increase proliferation, the abnormal phenotypes $mas2^+$ null mutant dramatically increased. These phenotypes could be rescued by an over-expression of the mast gene. The Mas2 protein localized in the nuclei of S. pombe, as evidenced by Mas2-EGFP signals. These results suggest that the $mas2^+$ is homologous to human SMARCAD1 gene and involved in septum formation and chromosome remodeling control.

Expressional Analysis of Superoxide Dismutase in Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus Infection (Viral hemorrhagic septicemia virus (VHSV) 감염에 대한 넙치 superoxide dismutase(Of-SOD)의 발현분석)

  • Lee, Young Mee;Kim, Jung-Eun;Noh, Jae Koo;Kim, Hyun Chul;Park, Choul-Ji;Park, Jong-Won;Kim, Kyung-Kil;Lee, Jeong-Ho
    • Journal of Life Science
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    • v.24 no.12
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    • pp.1371-1377
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    • 2014
  • Superoxide dismutase is a family of important antioxidant metalloenzymes and catalyzes the dismutation of toxic superoxide anions into dioxygen and hydrogen peroxide. A recent study identified the partial superoxide dismutase (SOD) gene in olive flounder (Paralichthys olivaceus). The same study reported that it strongly induced benzo[a]pyrene and that it was an indicator of aquatic oxidative stress responses. However, its transcriptional response against viral infection has not been investigated. In the present study, the spatial and temporal expression profiles were analyzed to investigate the function of Of-SOD in the antiviral response. The Of-SOD transcripts were ubiquitously detected at various levels in diverse tissues in a real-time PCR. The expression of Of-SOD was significantly higher in the muscles, liver, and brain but extremely low in the stomach and spleen. Following a VHSV challenge, the expression of Of-SOD increased within 3 h in the kidneys and decreased to the original level 2 days postchallenge. In muscle, liver, and brain, Of-SOD mRNA was similarly up-regulated at 3-6 h postchallenge and then decreased to the basal level. Although the expression pattern and induction time differed slightly depending on the tissue, the transcript of Of-SOD consistently increased in the acute infection response, but the expression was low in the chronic response. The expression of Of-SOD was induced after the VHSV infection, and Of-SOD was probably involved in the immune response against the viral challenge. These results suggest that SOD may play important roles in the immune defense system of P. olivaceus and perhaps contribute to the protective effects against oxidative stress in olive flounder.