• Title/Summary/Keyword: ITS-PCR Analysis

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Effects of Gwaruhaengryeon-hwan on COPD and Particulate Matter Induced Lung Injury on a Mouse Model (만성폐쇄성폐질환 및 미세먼지 유발 폐손상 동물모델에서 과루행련환의 효과)

  • Lee, Chul-wha;Yang, Won-kyung;Lyu, Yee-ran;Kim, Seung-hyeong;Park, Yang-chun
    • The Journal of Internal Korean Medicine
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    • v.38 no.3
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    • pp.353-366
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    • 2017
  • Objective: This study aimed to use a mouse model to evaluate the effects of Gwaruhaengryeon-hwan (GHH) on chronic obstructive pulmonary disease (COPD) and particulate matter induced lung injury. Materials and Methods: The study was carried out in two ways (in vitro, in vivo). In vitro RAW 264.7 cells (mouse macrophage) were used and analyzed by flow cytometry, ELISA. In vivo lipopolysaccharide (LPS) and cigarette smoke solution (CSS), or coal, fly ash, diesel exhaust particle (CFD) challenged mice were used and its BALF was analyzed by ELISA, lung tissue by real-time PCR. Results: In vitro, GHH maintained an 80-100% rate of viability. So cytotoxicity was not shown. In the ELISA analysis with RAW 264.7 cells, GHH significantly decreased NO over $30{\mu}g/ml$. In the ELISA analysis, GHH significantly decreased $TNF-{\alpha}$, IL-6 over $300{\mu}g/ml$. In the COPD model, the GHH 200 mg/kg dosage group, the application of GHH significantly decreased the increasing of neutrophils, $TNF-{\alpha}$, IL-17A, MIP2, CXCL-1 in BALF, $TNF-{\alpha}$, $IL-1{\beta}$ mRNA expression in lung tissue and histological lung injury. In the CFD induced lung injury model, the GHH 200 mg/kg dosage group, the application of GHH significantly decreased the increase of neutrophils, $TNF-{\alpha}$, IL-17A, MIP2, CXCL-1 in BALF, MUC5AC, $TGF-{\beta}$ mRNA expression in lung tissue and histological lung injury. Conclusion: This study suggests the usability of GHH for COPD patients by controlling lung tissue injury.

Gene expression and SNP identification related to leaf angle traits using a genome-wide association study in rice (Oryza sativa L.) (GWAS 분석을 이용한 벼 지엽각 관련 SNP 동정 및 발현 분석)

  • Kim, Me-Sun;Yu, Yeisoo;Kang, Kwon-Kyoo;Cho, Yong-Gu
    • Journal of Plant Biotechnology
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    • v.45 no.1
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    • pp.17-29
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    • 2018
  • This study was conducted to investigate a morphological trait in 294 rice accessions including Korean breeding lines. We also carried out a genome-wide association study (GWAS) to detect significant single nucleotide polymorphism markers and candidate genes affecting major agronomic traits. A Manhattan plot analysis of GWAS using morphological traits showed that phenotypic and statistical significance was associated with a chromosome in each group. The significance of SNPs that were detected in this study was investigated by comparing them with those found previously studied QTL regions related to agronomic traits. As a result, SNP (S8-19815442), which is significant with regard to leaf angle, was located in the known QTL regions. To observe gene mutations related to leaf angle in a candidate gene, Os08g31950, its sequences were compared with sequences in previously selected rice varieties. In Os08g31950, a single nucleotide mutation occurred in one region. To compare relative RNA expression levels of candidate gene Os08g31950, obtained from GWAS analysis of 294 rice accessions and related to lateral leaf angle, we investigated relative levels by selecting 10 erect leaf angle varieties and 10 horizontal leaf angle varieties and examining real-time PCR. In Os08g31950, a high level of expression and various expression patterns were observed in all tissues. Also, Os08g31950 showed higher expression levels in the erect leaf angle variety group and higher expression rates in the leaf than in the root. The candidate gene detected through GWAS would be useful in developing new rice varieties with improved yield potential through future molecular breeding.

Comprehensive Mutation Analysis of PIK3CA, p14ARF, p16INK4a and p21Waf1/Cip1 Genes is Suggestive of a Non- Neoplastic Nature of Phenytoin Induced Gingival Overgrowth

  • Swamikannu, Bhuminathan;Kumar, Kishore S.;Jayesh, Raghavendra S.;Rajendran, Senthilnathan;Muthupalani, Rajendran Shanmugam;Ramanathan, Arvind
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.5
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    • pp.2743-2746
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    • 2013
  • Background: Dilantin sodium (phenytoin) is an antiepileptic drug, which is routinely used to control generalized tonic clonic seizure and partial seizure episodes. A few case reports of oral squamous cell carcinomas arising from regions of phenytoin induced gingival overgrowth (GO), and overexpression of mitogenic factors and p53 have presented this condition as a pathology with potential to transform into malignancy. We recently investigated the genetic status of p53 and H-ras, which are known to be frequently mutated in Indian oral carcinomas in GO tissues and found them to only contain wild type sequences, which suggested a non-neoplastic nature of phenytoin induced GO. However, besides p53 and H-ras, other oncogenes and tumor suppressors such as PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$, are frequently altered in oral squamous cell carcinoma, and hence are required to be analyzed in phenytoin induced GO tissues to be affirmative of its non-neoplastic nature. Methods: 100ng of chromosomal DNA isolated from twenty gingival overgrowth tissues were amplified with primers for exons 9 and 20 of PIK3CA, exons $1{\alpha}$, $1{\beta}$ and 2 of p16INK4a and p14ARF, and exon 2 of $p21^{Waf1/Cip1}$, in independent reactions. PCR amplicons were subsequently gel purified and eluted products were sequenced. Results: Sequencing analysis of the twenty samples of phenytoin induced gingival growth showed no mutations in the analyzed exons of PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$. Conclusion: The present data indicate that the mutational alterations of genes, PIK3CA, p14ARF, p16INK4a and $p21^{Waf1/Cip1}$ that are frequently mutated in oral squamous cell carcinomas are rare in phenytoin induced gingival growth. Thus the findings provide further evidence that phenytoin induced gingival overgrowth as a non-neoplastic lesion, which may be considered as clinically significant given the fact that the epileptic patients are routinely administered with phenytoin for the rest of their lives to control seizure episodes.

Expression Analysis of Lily Type Lectin Isotypes in the Rock Bream, Oplegnathus fasciatus: in the Tissue, Developmental Stage and Viral Infection

  • Lee, Young Mee;Yang, In Jung;Noh, Jae Koo;Kim, Hyun Chul;Park, Choul-Ji;Park, Jong-Won;Noh, Gyeong Eon;Kim, Woo-Jin;Kim, Kyung-Kil
    • Development and Reproduction
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    • v.20 no.4
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    • pp.297-304
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    • 2016
  • Lectins belong to the pattern-recognition receptors (PRRs) class and play important roles in the recognition and elimination of pathogens via the innate immune system. Recently, it was reported that lily-type lectin-1 is involved when a pathogen attacks in the early immune response of fish. However, this study is limited to information that the lectin is involved in the innate immune response against viral infection. In the present study, the lily-type lectin-2 and -3 of Oplegnathus fasciatus (OfLTL-2 and 3) have been presented to be included B-lectin domain and two D-mannose binding sites in the amino acid sequence that an important feature for the fundamental structure. To investigate the functional properties of OfLTLs, the tissue distribution in the healthy rock bream and temporal expression during early developmental stage analysis are performed using quantitative real-time PCR. OfLTL-2 and 3 are predominantly expressed in the liver and skin, but rarely expressed in other organ. Also, the transcripts of OfLTLs are not expressed during the early developmental stage but its transcripts are increased after immune-related organs which are fully formed. In the challenge experiment with RBIV (rock bream iridovirus), the expression of OfLTLs was increased much more strongly in the late response than the early, unlike previously known. These results suggest that OfLTLs are specifically expressed in the immune-related tissues when those organs are fully formed and it can be inferred that the more intensively involved in the second half to the virus infection.

Effect of Poria Cocos on the Scopolamine-induced Memory Impairment and Its Underlying Molecular Mechanism (스코폴라민으로 유도된 기억력 손상에 대한 복신의 보호 효과 및 작용기전 연구)

  • JeGal, Kyoung-Hwan;Park, Sung-Jun;Kim, Chang-Yul;Lee, Chan;Park, Jong-Hyun;Jang, Jung-Hee
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.24 no.2
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    • pp.228-235
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    • 2010
  • This study was performed to investigate the memory enhancing effect of Poria cocos Wolf (Hoelen cum radix) against scopolamine-induced amnesia in Sprague-Dawley (SD) rats. To induce amnesia, scopolamine (0.75 mg/kg) was intraperitonically injected into SD rats 30 min before starting behavior tests. We have conducted Morris water-maze and Y-maze tests to monitor learning and memory functions. Poria cocos effectively reversed scopolamine-induced memory impairment in SD rats which was represented by an improvement of mean escape latency in water-maze test and spontaneous alterations in Y-maze test. To elucidate possible molecule mechanism, we have measured mRNA as well as protein expression of acetylcholine esterase (AchE), choline acetyltransferase (ChAT), muscarinic acetylcholine receptor (mAchR), and brain-derived neurotrophic factor (BDNF) using RT-PCR and Western blot analysis, respectively. Poria cocos increased mRNA levels of ChAT and mAchR in rat hippocampus compared with those in the scopolamine-injected amnesic group. In addition, protein expression of ChAT and BDNF was also elevated by Poria cocos intake. Furthermore, as an upstrem regulator, the activation of cAMP response element-binding protein (CREB) was assessed by immunohistochemistry. In this immunohistochemical analysis, the phosphorylation of CREB (p-CREB) was reduced by scopolamine injection, which was restored back to control levels by administration of Poria cocos. These results suggest that Poria cocos may improve memory and cognitive deficit in amnesia and have therapeutic potentials through up-regulation of ChAT, mAchR, and BDNF, which seemed to be mediated by activation of CREB.

DNA Sequence Analysis of 1-Nitropyrene-4,5-Oxide and 1-Nitropyrene-9,10-Oxide Induced Mutations in the hprt Gene of Chinese Hamster Ovary Cells

  • Kim, Hyun-Jo;Kim, Tae-Ho;Lee, Sun-Young;Lee, Dong-Hoon;Kim, Sang-In;Pfeifer, Gerd P.;Kim, Seog K.;Lee, Chong-Soon
    • Molecules and Cells
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    • v.19 no.1
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    • pp.114-123
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    • 2005
  • Nitropyrene, the predominant nitropolycyclic hydrocarbon found in diesel exhaust, is a mutagenic and tumorigenic environmental pollutant that requires metabolic activation via nitroreduction and ring oxidation. In order to determine the role of ring oxidation in the mutagenicity of 1-nitropyrene, its oxidative metabolites, 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide, were synthesized and their mutation spectra were determined in the coding region of hprt gene of CHO cells by a PCR amplification of reverse-transcribed hprt mRNA, followed by a DNA sequence analysis. A comparison of the two metabolites for mutation frequencies showed that 1-nitropyrene 9,10-oxide was 2-times higher than 1-nitropyrene 4,5-oxide. The mutation spectrum for 1-nitropyrene 4,5-oxide was base substitutions (33/49), one base deletions (11/49) and exon deletions (5/49). In the case of 1-nitropyrene 9,10-oxide, base substitutions (27/50), one base deletions (15/50), and exon deletions (8/50) were observed. Base substitutions were distributed randomly throughout the hprt gene. The majority of the base substitutions in mutant from 1-nitropyrene 4,5-oxide treated cells were $A{\rightarrow}G$ transition (15/33) and $G{\rightarrow}A$ transition (8/33). The predominant base substitution, $A{\rightarrow}G$ transition (11/27) and $G{\rightarrow}A$ transition (8/27), were also observed in mutant from 1-nitropyrene 9,10-oxide treated cells. The mutation at the site of adenine and guanine was consistent with the previous results, where the sites of DNA adduct formed by these compounds were predominant at the sites of purines. A comparison of the mutational patterns between 1-nitropyrene 4,5-oxide and 1-nitropyrene 9,10-oxide showed that there were no significant differences in the overall mutational spectrum. These results indicate that each oxidative metabolite exhibits an equal contribution to the mutagenicity of 1-nitropyrene, and ring oxidation of 1-nitropyrene is an important metabolic pathway to the formation of significant lethal DNA lesions.

MicroRNA Expression Profiles in Korean Non-Small Cell Lung Cancer

  • Son, Ji Woong;Kim, Young Jin;Cho, Hyun Min;Lee, Soo Young;Jang, Jin Sung;Choi, Jin Eun;Lee, Jung Uee;Kang, Min Gyu;Lee, Yu Mi;Kwon, Sun Jung;Choi, Eugene;Na, Moon Jun;Park, Jae Yong
    • Tuberculosis and Respiratory Diseases
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    • v.67 no.5
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    • pp.413-421
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    • 2009
  • Background: MicroRNAs (miRNAs) play an important role in the regulation of cell proliferation, apoptosis, development and differentiation. Several studies have shown that aberrant expression of miRNAs is involved in cancer development and progression by regulating the expression of proto-oncogenes or tumor suppressor genes. In this study, we investigated miRNA expression profiles in Korean patients with non-small cell lung cancer (NSCLC). Methods: We performed miRNA microarray analysis containing 60~65 bp oligonucleotide probes representing human 318 miRNAs and validated the results of the microarray with Northern blot analysis or quantitative RT-PCR. Next, we examined the correlation between miRNA expression and the target gene transcriptional profile using a human whole-genome-expression microarray. Results: We showed that 35 miRNAs were expressed differentially in the NSCLCs and corresponding non-malignant lung tissues. We showed that 35 miRNAs were expressed differentially in the NSCLCs and corresponding nonmalignant lung tissues. Thirteen of the 35 differentially expressed miRNAs were newly identified in the present study. Of the 35 miRNAs, 2 (miR-371 and miR-210) were over-expressed in lung cancers, and 33 miRNAs, including miR-145, were under-expressed in lung cancers. miR-99b expression consistently showed a negative correlation with FGFR3 expression. Conclusion: Albeit a small number of patients were examined, these results suggest that miRNA expression profiles in Korean lung cancers may be somewhat different from the expression profiles reported on lung cancers in Western populations. The findings suggest that miR-99b might be a tumor suppressor through its up-regulation of FGFR3.

TFAP2C Promotes Cell Proliferation by Upregulating CDC20 and TRIB3 in Non-small Cell Lung Cancer Cells (비소세포폐암 발달 과정에서 TFAP2C에 의해 발현되는 CDC20과 TRIB3의 원암유전자 기능에 관한 연구)

  • Kim, Dain;Do, Hyunhee;Kang, JiHoon;Youn, BuHyun;Kim, Wanyeon
    • Journal of Life Science
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    • v.29 no.6
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    • pp.645-652
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    • 2019
  • Non-small cell lung cancer (NSCLC) has the infamous distinction of being the leading cause of global cancer-related death over the past decade, and novel molecular targets are urgently required to change this status. We previously conducted a microarray analysis to investigate the association of transcription factor activating enhancer-binding protein 2C (TFAP2C) with NSCLC and revealed its oncogenic roles in NSCLC development. In this study, to identify new biomarkers for NSCLC, we focused on several oncogenes from the microarray analysis that are transcriptionally regulated by TFAP2C. Here, the cell division cycle 20 (CDC20) and tribbles pseudokinase 3 (TRIB3) were subsequently found as potential potent oncogenes as they are positively regulated by TFAP2C. The results showed that the mRNA and protein levels of CDC20 and TRIB3 were down-regulated in two NSCLC cell lines (NCI-H292 and NCI-H838), which were treated with TFAP2C siRNA, and that the overexpression of either CDC20 or TRIB3 was responsible for promoting cell viability in both NSCLC cell lines. In addition, apoptotic levels of NCI-H292 and NCI-H838 cells treated with TFAP2C siRNA were found to be suppressed by the overexpression of either CDC20 or TRIB3. Together, these results suggest that CDC20 and TRIB3 are positively related to NSCLC tumorigenesis and that they should be considered as potential prognostic markers for developing an NSCLC therapy.

Inhibitory Effect of the Ethanol Extract of Rudbeckia laciniata var. hortensis Bailey on Adipocyte Differentiation in 3T3-L1 Cells (겹삼잎국화 에탄올 추출물의 지방세포 분화 억제 효과)

  • Nam, Gun He;Wee, Ji-Hyang;Kim, Sang Yung;Baek, Ji-Young;Kim, Young Min
    • Journal of Life Science
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    • v.29 no.10
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    • pp.1152-1158
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    • 2019
  • Rudbeckia laciniata var. hortensis Bailey is used in home remedy for colic and gastritis in South Korea. Although Rudbeckia laciniata var. hortensis Bailey is used extensively for home remedies, no single study on its efficacy exists. In this study, we investigated the anti-obesity effects of Rudbeckia laciniata var. hortensis Bailey. The anti-obesity effect of a 70% ethanol extract from Rudbeckia laciniata var. hortensis Bailey on the differentiation of 3T3-L1 pre-adipocytes to adipocytes was investigated with an Oil Red O assay, western blot analysis, and mRNA analysis. Compared to the control (only treated with DM), the 70% ethanol extract of Rudbeckia laciniata var. hortensis Bailey significantly inhibited adipocyte differentiation and intracellular triglyceride (TG) levels at a concentration of $100{\mu}g/ml$. To determine how the TG content was reduced, we measured the level of protein and mRNA expression of obesityrelated agents, such as peroxisome proliferators-activated receptor ${\gamma}$ ($PPAR{\gamma}$), CCAAT/enhancer- binding protein ${\alpha}$ ($C/EBP{\alpha}$), AMP-activated protein kinase (AMPK) phosphorylation, LPL, and FAS. As a result, the 70% ethanol extract of Rudbeckia laciniata var. hortensis Bailey significantly increased the expression of AMPK and decreased the expression of genes related to adipogenesis and fat storage, such as $PPAR{\gamma}$, $C/EBP{\alpha}$, LPL, and FAS.

A Study on Inflammation and Itching of Cyperus rotundus Ethyl Acetate Fractions (향부자 에틸아세테이트 분획물의 염증 및 가려움증에 관한 연구)

  • Do Gyu, Kim;Ji Yeon, Lee;Sohyun, Mun;Yukyung, Kim;Nari, Kim;Ah Reum, Jung;Jun-Hwan, Jang;Jae-Soeb, Lee;Sang Bae, Han;Jun-Tae, Bae
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.48 no.4
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    • pp.303-312
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    • 2022
  • In this study, Cyperus rotundus (C. rotundus) was fractionated into ethyl acetate to identify α-cyperone, a representative indicator, and its ethyl acetate fraction were evaluated to confirm the possibilityas a functional cosmetic ingredient. As a result of HPLC analysis, it was confirmed that the content of α-cyperone was 5.243%. In order to verify the inflammatory relief effect of the ethyl acetate fraction from C. rotundus (EAFC) and α-cyperone, it was confirmed that nitric oxide (NO) production inhibitory ability in RAW 264.7 macrophages induced an inflammatory reaction with lipopolysaccharide (LPS). Real-time qPCR analysis confirmed inhibition of mRNA expression level of inflammatory factors, IL-1β, IL-6, TNF-α, COX-2 and iNOS. As a results of conducting a clinical study using a simple cosmetic formulation containing EAFC, it was confirmed that the skin irritation stimulated by sodium lauryl sulfate (SLS) was calming and relieving itching. Through these results, it is believed that the C. rotundus can be used as a natural cosmetic ingredient that has the effect of inhibiting inflammation and relieving itching.