• Title/Summary/Keyword: PCR (Polymerase Chain Reaction)

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Antioxidant Effect of Nelumbo nucifera G. Leaf Extract and Inhibition of MITF, TRP-1, TRP-2, and Tyrosinase Expression in a B16F10 Melanoma Cell Line (연잎 추출물의 항산화 활성 및 멜라노마 세포(B16F10)에서 MITF, TRP-1, TRP-2, tyrosinase의 발현 저해 효과)

  • Yoo, Dan-Hee;Joo, Da-Hye;Lee, Soo-Yeon;Lee, Jin-Young
    • Journal of Life Science
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    • v.25 no.10
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    • pp.1115-1123
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    • 2015
  • The purpose of this study was to investigate the potential of Nelumbo nucifera G. leaf (NNL) extract as a cosmetic additive. The electron-donating ability of the NNL extract at a concentration of 1,000 μg/ml was 67.83%. In xanthine oxidase, the inhibition effect of the NNL extract was 92.7% at the same concentration. For whitening effects, tyrosinase inhibition effect of NNL extract was 42.7% at a 1,000 μg/ml concentration. The cell toxicity of the NNL extract was examined in melanoma cells (B16F10) using a 3-[4, 5–dimethyl–thiazol–2–yl]-2, 5-diphenyl-tetrazoliumbromide (MTT) assay. The cell toxicity assay revealed that the NNL extract had a toxicity of 81.61% at a concentration of 1,000 μg/ml The microphthalmia-associated transcription factor (MITF), tyrosinase related protein-1 (TRP-1), tyrosinase related protein-2 (TRP-2), and tyrosinase protein expression inhibitory effect by Western blot of NNL extract were measured by a Western blot at concentrations of 25, 50, and 100 μg/ml. At a 100 μg/ml concentration of the NNL extract, the expression of the MITF, TRP-1, TRP-2, and tyrosinase protein was decreased by 69.59%, 27.74%, 67.33%, and 67.78% respectively. The MITF, TRP-1, TRP-2 and tyrosinase mRNA expression inhibitory effect were measured by reverse transcription- polymerase chain reaction (PCR) at concentrations of 25, 50, and 100 μg/ml. GAPDH was used as a positive control. At a concentration of 100 μg/ml of the NNL extract, the expression of MITF, TRP-1, TRP-2, and tyrosinase mRNA was decreased by 67.51%, 71.36%, 85.74%, and 83.64%, respectively. These findings suggest that the NNL extract has antioxidant and whitening effects and that it has great potential as a cosmetic ingredient.

Heat Shock Protein Augmentation of Angelica gigas Nakai Root Hot Water Extract on Adipogenic Differentiation in Murine 3T3-L1 Preadipocytes

  • Lumbera, Wenchie Marie L.;Cruz, Joseph dela;Yang, Seung-Hak;Hwang, Seong Gu
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.3
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    • pp.419-427
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    • 2016
  • There is a high association of heat shock on the alteration of energy and lipid metabolism. The alterations associated with thermal stress are composed of gene expression changes and adaptation through biochemical responses. Previous study showed that Angelica gigas Nakai (AGN) root extract promoted adipogenic differentiation in murine 3T3-L1 preadipocytes under the normal temperature condition. However, its effect in heat shocked 3T3-L1 cells has not been established. In this study, we investigated the effect of AGN root hot water extract in the adipogenic differentiation of murine 3T3-L1 preadipocytes following heat shock and its possible mechanism of action. Thermal stress procedure was executed within the same stage of preadipocyte confluence (G0) through incubation at $42^{\circ}C$ for one hour and then allowed to recover at normal incubation temperature of $37^{\circ}C$ for another hour before AGN treatment for both cell viability assay and Oil Red O. Cell viability assay showed that AGN was able to dose dependently (0 to $400{\mu}g/mL$) increase cell proliferation under normal incubation temperature and also was able to prevent cytotoxicity due to heat shock accompanied by cell proliferation. Confluent preadipocytes were subjected into heat shock procedure, recovery and then AGN treatment prior to stimulation with the differentiation solution. Heat shocked preadipocytes exhibited reduced differentiation as supported by decreased amount of lipid accumulation in Oil Red O staining and triglyceride measurement. However, those heat shocked preadipocytes that then were given AGN extract showed a dose dependent increase in lipid accumulation as shown by both evaluation procedures. In line with these results, real-time polymerase chain reaction (RT-PCR) and Western blot analysis showed that AGN increased adipogenic differentiation by upregulating heat shock protection related genes and proteins together with the adipogenic markers. These findings imply the potential of AGN in heat shock amelioration among 3T3-L1 preadipocytes through heat shock factor and proteins augmentation and enhanced adipogenic marker expression.

Ontogenetic Expression of Lpin2 and Lpin3 Genes and Their Associations with Traits in Two Breeds of Chinese Fat-tailed Sheep

  • Jiao, Xiao-Li;Jing, Jiong-Jie;Qiao, Li-Ying;Liu, Jian-Hua;Li, Liu-An;Zhang, Jing;Jia, Xia-Li;Liu, Wen-Zhong
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.3
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    • pp.333-342
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    • 2016
  • Lipins play dual function in lipid metabolism by serving as phosphatidate phosphatase and transcriptional co-regulators of gene expression. Mammalian lipin proteins consist of lipin1, lipin2, and lipin3 and are encoded by their respective genes Lpin1, Lpin2, and Lpin3. To date, most studies are concerned with Lpin1, only a few have addressed Lpin2 and Lpin3. Ontogenetic expression of Lpin2 and Lpin3 and their associations with traits would help to explore their molecular and physiological functions in sheep. In this study, 48 animals with an equal number of males and females each for both breeds of fat-tailed sheep such as Guangling Large Tailed (GLT) and Small Tailed Han (STH) were chosen to evaluate the ontogenetic expression of Lpin2 and Lpin3 from eight different tissues and months of age by quantitative real-time polymerase chain reaction (PCR). Associations between gene expression and slaughter and tail traits were also analyzed. The results showed that Lpin2 mRNA was highly expressed in perirenal and tail fats, and was also substantially expressed in liver, kidney, reproductive organs (testis and ovary), with the lowest levels in small intestine and femoral biceps. Lpin3 mRNA was prominently expressed in liver and small intestine, and was also expressed at high levels in kidney, perirenal and tail fats as well as reproductive organs (testis and ovary), with the lowest level in femoral biceps. Global expression of Lpin2 and Lpin3 in GLT both were significantly higher than those in STH. Spatiotemporal expression showed that the highest levels of Lpin2 expression occurred at 10 months of age in two breeds of sheep, with the lowest expression at 2 months of age in STH and at 8 months of age in GLT. The greatest levels of Lpin3 expression occurred at 4 months of age in STH and at 10 months of age in GLT, with the lowest expression at 12 months of age in STH and at 8 months of age in GLT. Breed and age significantly influenced the tissue expression patterns of Lpin2 and Lpin3, respectively, and sex significantly influenced the spatiotemporal expression patterns of Lpin3. Meanwhile, Lpin2 and Lpin3 mRNA expression both showed significant correlations with slaughter and tail traits, and the associations appear to be related with the ontogenetic expression as well as the potential functions of lipin2 and lipin3 in sheep.

Characterizing Milk Production Related Genes in Holstein Using RNA-seq

  • Seo, Minseok;Lee, Hyun-Jeong;Kim, Kwondo;Caetano-Anolles, Kelsey;Jeong, Jin Young;Park, Sungkwon;Oh, Young Kyun;Cho, Seoae;Kim, Heebal
    • Asian-Australasian Journal of Animal Sciences
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    • v.29 no.3
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    • pp.343-351
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    • 2016
  • Although the chemical, physical, and nutritional properties of bovine milk have been extensively studied, only a few studies have attempted to characterize milk-synthesizing genes using RNA-seq data. RNA-seq data was collected from 21 Holstein samples, along with group information about milk production ability; milk yield; and protein, fat, and solid contents. Meta-analysis was employed in order to generally characterize genes related to milk production. In addition, we attempted to investigate the relationship between milk related traits, parity, and lactation period. We observed that milk fat is highly correlated with lactation period; this result indicates that this effect should be considered in the model in order to accurately detect milk production related genes. By employing our developed model, 271 genes were significantly (false discovery rate [FDR] adjusted p-value<0.1) detected as milk production related differentially expressed genes. Of these genes, five (albumin, nitric oxide synthase 3, RNA-binding region (RNP1, RRM) containing 3, secreted and transmembrane 1, and serine palmitoyltransferase, small subunit B) were technically validated using quantitative real-time polymerase chain reaction (qRT-PCR) in order to check the accuracy of RNA-seq analysis. Finally, 83 gene ontology biological processes including several blood vessel and mammary gland development related terms, were significantly detected using DAVID gene-set enrichment analysis. From these results, we observed that detected milk production related genes are highly enriched in the circulation system process and mammary gland related biological functions. In addition, we observed that detected genes including caveolin 1, mammary serum amyloid A3.2, lingual antimicrobial peptide, cathelicidin 4 (CATHL4), cathelicidin 6 (CATHL6) have been reported in other species as milk production related gene. For this reason, we concluded that our detected 271 genes would be strong candidates for determining milk production.

Comparison of the Effects of Gamma Ray and Electron Beam Irradiation to Improve Safety of Spices for Meat Processing (육가공용 향신료의 위생화를 위한 감마선 및 전자선 조사 효과 비교)

  • Kim, Byung-Hee;Kim, Hyun-Joo;Yoon, Yo-Han;Shin, Myung-Gon;Lee, Ju-Woon
    • Food Science of Animal Resources
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    • v.30 no.1
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    • pp.124-132
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    • 2010
  • This study evaluated the effects of gamma ray and electron beam (E-beam) to improve the safety of spices for meat processing. The spices (garlic powder, curry powder, turmeric powder, black pepper, white pepper, oregano, parsley, laurel leaf powder, basil, and rosemary) were irradiated by gamma ray and E-beam at 0, 2, 4, 6, 8, and 10 kGy. Total bacterial populations were then enumerated on total plate count agar, and bacteria isolated from the samples were identified by polymerase chain reaction (PCR). In addition, $D_{10}$ values for Bacillus cereus and Staphylococcus aureus inoculated in spices was determined, and the Ames test was conducted for genotoxicity analysis. The contaminated total bacterial populations in spices ranged from 1.5 to 3.8 Log CFU/g, and most of identified bacteria were Bacillus spp., and Staphylococcus spp. However, the bacterial populations decreased below the detection limit (2 Log CFU/g) after irradiation at 4 kGy except for parsley, which required 6 kGy in gamma ray and 8 kGy in E-beam to decrease total bacterial populations below detection limit. $D_{10}$ values were also higher (p<0.05) in E-beam treated samples than gamma-ray treated samples. No genotoxicity was observed in both conditions with and without metabolic activation. These results indicate that gamma ray (>4 kGy and <6 kGy) could be more useful to improve food safety of meat processing spices compared to E-beam.

Virulence Factor Profiles of Escherichia coli Isolated from Pork and Chicken Meats Obtained from Retail Markets (소매시장에서 판매하는 돼지고기와 닭고기에서 분리된 Escherichia coli의 병원성 인자)

  • Choi, Sun-Keum;Lee, Min-Hwa;Lee, Bog-Hieu;Jung, Ji-Youn;Choi, Chang-Sun
    • Food Science of Animal Resources
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    • v.30 no.1
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    • pp.148-153
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    • 2010
  • The aim of this study was to investigate the virulence factor profiles of Escherichia coli strains isolated from pork and chicken meats purchased from retail markets in Korea. From 943 pork and 142 chicken meats, 217 isolates of E. coli were cultured. The presence of 11 virulence factors (elt, estI, estII, astA, stx, cdt, cnf, agg, inve, eae, afa) were detected by polymerase chain reaction. Forty one (18.9%) of 217 E. coli isolates carried at least one virulence factor. Among 175 E. coli isolates from pork, the detection rate of astA, elt, eae, estII, estI, afa, and cnf were 6.9%, 4.6%, 4.6%, 4.0%, 2.3%, 1.1%, and 0.6%, respectively. However, stx, agg, and cdt were not detected in our isolates. Therefore, we conclude that astA is the most prevalent virulence factor in E. coli isolates contaminated in pork and chicken meats in Korea.

IL-10 gene promoter polymorphisms in Korean gener-alized aggressive periodontitis patients (한국인 전반적 급진성 치주염 환자에서 IL-10 promoter 유전자 다변성에 관한 연구)

  • Ryn, Ji-Sun;Kim, Ok-Su
    • Journal of Periodontal and Implant Science
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    • v.37 no.3
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    • pp.563-573
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    • 2007
  • Genetic polymorphisms associated with aggressive periodontitis have previously been reported. Interleukin-10 is an immunoregulatory cytokine that plays a role in the pathogenesis of periodontitis. Individual capacity for IL-10 production appears to be under genetic influence, The aim of present investigation was to explore possible genetic association of IL-10 gene promoter polymorphisms with generalized aggressive periodontitis. The study population consisted of 37 generalized aggressive periodontitis patients from the Department of Periodontology, Chonnam National University Hospital and 27 control subjects, all the subjects were non-smokers, Genomic DNA was obtained from buccal swab. The IL-10promoter -597, -824, -1082 positions were genotyped by amplifying the polymorphic regions using polymerase chain reaction (PCR) , followed by restriction enzyme digestion and gel electrophoresis. IL-10-597 C (allele 1) to A (allele 2) and IL-10-824 C (allele 1) to T (allele 2) and IL-10-1082 G (allele 1) to A (allele 2) polymorphisms were examined. The results were as follows. 1. In patients, the distribution of genotypes C/C, C/A and NA at Il-10-597 was determined to be 13.5%, 37.8% and 48.7%, respectively and the distribution of genotypes at IL-10-824 was the same as that of IL-10-597. The distribution of genotypes G/G, G/A and NA at IL-10-1082 was found to be 2.7%, 16.2% and 81. 4%, respectively. No statistical difference in genotype distribution was found between the patient and control groups. 2. Allele 2 carriage rate at the three position of the IL-10 promoter region was higher in the control group than the patient group. 3. Allele 2 frequencies at IL-10-597 and -824 positions were higher in female group than male group and its difference was statistically significant(p<0.05). No significant difference in genotype distribution between the control and patient groups. Allele frequency between control and patient groups was not significantly different although allele 2 frequency at the three positions in the IL-10 promoter region appeared to be higher in control group. In conclusion, no clear association between IL-10 gene promoter polymorphisms and generalized aggressive periodontitis in Korean was observed.

Morphological characteristics and RAPD analysis of Epimedium spp. (국내외 수집 삼지구엽초의 형태적 특성 및 유연관계 분석)

  • Lim, Jung-Dae;Seong, Eun-Soo;Choi, Kwang-Joon;Kim, Seung-Kyung;Chung, Ill-Min;Heo, Kweon;Yu, Chang-Yeon
    • Korean Journal of Medicinal Crop Science
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    • v.8 no.2
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    • pp.102-108
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    • 2000
  • Morphological characteristics of nine Epimedium spp was analyzed on the basis of six morphological characters. To analyse the genetic relationship among Epimedium spp., polymerase chain reaction (PCR) was performed with total genomic DNA of 17 Epimedium spp. by using random 8 primers. The genetic diversity and genetic distance among nine Korean collections, seven Japanese and one China collection were used to generate a dendrogram showing phylogenie relationship. Seventeen Epimedium spp were classified into two groups of group I and II, since they were divided into two major groups at the similarity coefficients value of 0.65. In addition, one of the two group, group I was divided into three sub-groups including Epimedium koreanum, Chul-won collections 1, 2, and 3, Yanggu, Hongchon, Hwachun, Chunchon, China, Maehwa (Japanese), E. diphyllum and E. violaceum (Japanese), while group II included Chulwon collection 4 and 5 and Japanese collection. The samples collected at Chulwon district showed close similarity with Japanese collection. Similarity indexes between collection and genetic relationship were related at the levels ranging from 0.6 to 0.9.

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Genetic Analysis of SCN5A in Korean Patients Associated with Atrioventricular Conduction Block

  • Park, Hyoung-Seob;Kim, Yoon-Nyun;Lee, Young-Soo;Jung, Byung-Chun;Lee, Sang-Hee;Shin, Dong-Gu;Cho, Yong-Keun;Bae, Myung-Hwan;Han, Sang-Mi;Lee, Myung-Hoon
    • Genomics & Informatics
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    • v.10 no.2
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    • pp.110-116
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    • 2012
  • Recent several studies have shown that the genetic variation of SCN5A is related with atrioventricular conduction block (AVB); no study has yet been published in Koreans. Therefore, to determine the AVB-associated genetic variation in Korean patients, we investigated the genetic variation of SCN5A in Korean patients with AVB and compared with normal control subjects. We enrolled 113 patients with AVB and 80 normal controls with no cardiac symptoms. DNA was isolated from the peripheral blood, and all exons (exon 2-exon 28) except the untranslated region and exon-intron boundaries of the SCN5A gene were amplified by multiplex PCR and directly sequenced using an ABI PRISM 3100 Genetic Analyzer. When a variation was discovered in genomic DNA from AVB patients, we confirmed whether the same variation existed in the control genomic DNA. In the present study, a total of 7 genetic variations were detected in 113 AVB patients. Of the 7 variations, 5 (G87A-A29A, intervening sequence 9-3C>A, A1673G-H558R, G3578A-R1193Q, and T5457C-D1819D) have been reported in previous studies, and 2 (C48G-F16L and G3048A-T1016T) were novel variations that have not been reported. The 2 newly discovered variations were not found in the 80 normal controls. In addition, G298S, G514C, P1008S, G1406R, and D1595N, identified in other ethnic populations, were not detected in this study. We found 2 novel genetic variations in the SCN5A gene in Korean patients with AVB. However, further functional study might be needed.

Effects of compressive stress on the expression of M-CSF, IL-$1{\beta}$, RANKL and OPG mRNA in periodontal ligament cells (압박력이 치주인대 세포의 M-CSF, IL-$1{\beta}$, RANKL 및 OPG mRNA 발현에 미치는 영향)

  • Kim, Ji-Woong;Lee, Ki-Soo;Nahm, Jong-Hyun;Kang, Yoon-Goo
    • The korean journal of orthodontics
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    • v.39 no.4
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    • pp.248-256
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    • 2009
  • Objective: The aim of this study was to determine if human PDL cells can produce osteoclastogenic mRNA and examine how compressive stress affects the expression of osteoclastogenic mRNA in human PDL cells. Methods: Human PDL cells were obtained from biscupids extracted for orthodontic treatment. The compressive force was adjusted by increasing the number of cover glasses. PDL cells were subjected to a compressive force of 0.5, 1.0, 2.0, 3.0 or $4.0\;g/cm^2$ for 0.5, 1.5, 6, 24 or 48 hours. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to examine levels of M-CSF, IL-$1{\beta}$, RANKL, OPG mRNA expression. Results: Human PDL cells could produce M-CSF mRNA. Human PDL cells under compressive stress showed increased M-CSF, IL-$1{\beta}$ and RANKL mRNAs expression in a force (up to $2\;g/cm^2$) and time-dependent manner. However, OPG mRNA expression was constant regardless of the level and duration of stress. Conclusions: Continuous compressive stress induced the mRNA expression of osteoclastogenic cytokines including M-CSF, RANKL, IL-$1{\beta}$ in PDL cells. Together with an unchanged OPG mRNA level, these results suggest that compressive stress-induced osteoclastogenesis in vivo is partly controlled by M-CSF, RANKL and IL-$1{\beta}$ expression in PDL cells.