• Animal Bioscience
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• 제36권1호
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• pp.156-166
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• 2023

Objective: In this study, we investigated the effects of Rubus coreanus-derived lactic acid bacteria (LAB) fermented feed (RC-LAB fermented feed) and three types of LAB (Lactobacillus plantarum, Lactobacillus acidophilus, Bifidobacterium animalis) on the expression of transcription factors and cytokines in Th1, Th2, Th17, and Treg cells in the intestinal lymph nodes and spleens of rats. In addition, the effect on intestinal microbiota composition and body weight was investigated. Methods: Five-week-old male rats were assigned to five treatments and eight replicates. The expression of transcription factors and cytokines of Th1, Th2, Th17, and Treg cells in the intestinal lymph nodes and spleens was analyzed using real-time reverse transcriptase polymerase chain reaction assays. Intestinal tract microbiota compositions were analyzed by next-generation sequencing and quantitative polymerase chain reaction assays. Results: RC-LAB fermented feed and three types of LAB increased the expression of transcription factors and cytokines in Th1, Treg cells and Galectin-9, but decreased in Th2 and Th17 cells. In addition, the intestinal microbiota composition changed, the body weight and Firmicutes to Bacteroidetes (F/B) ratio decreased, and the relative abundance of LAB increased. Conclusion: LAB fermented feed and three types of LAB showed an immune modulation effect by inducing T cell polarization and increased LAB in the intestinal microbiota.

• 한국수정란이식학회:학술대회논문집
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• 한국수정란이식학회 2002년도 국제심포지엄
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• pp.99-99
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• 2002

The hormone resistin is associated with typeII diabetes mellitus in rodent model. Resistin impairs glucose tolerance and insulin action. A new class of anti-diabetic drugs were called thiazolidinediones (TZDs) downregulates a resistin which is induced during adipocyte differentiation. But the connection between increased adiposity and resistin remains unknown. The objectives of this study was to clone a mouse resistin cDNA and to generate transgenic mice overexpressing mouse resistin gene. The 555 bp of mouse resistin was amplified from mob cDNAS by polymerase chain reaction (PCR) and cloned into pCR$\^$(R)/ 2.1 TOPO T-vector. Mouse resistin mRNA on the basis of Genbank sequence (acession no. AF323080). Then, the PCR product was cloned into pTargeT$\^$TM/ mammalian expression vector that has pCMV promoter and chimeric intron. Restriction enzyme analysis with BamH I and Not I was carried out to determine an orientation of the insert in the vector. The pCMV-mus/resistin gene was prepared from previous recombinant pTargeT$\^$TM/-mus/resistin by digestion of Bgl II, and has used for microinjection into pronuclei of one cell embryos. The microinjected embryos were transfered to pseudopregnant foster-mother. Mouse resistin expression was detected in transgenic F1 mice by Reverse Transcriptase- Polymerase Chain Reaction (RT-PCR). Resistin gene expression mouse has heavier body weight which was measured higher level of plasma glucose than that of normal mouse. And in diet-induced experiments, the abdominal fat pads were isolated from each 24h starvation and re-feeding after fasting group mice that were assessed by RT-PCR analysis. In fasting group mice, resistin expression was higher than that of re-feeding group mice. This result suggests that the resistin gene overexpressing mice may be became to obesity and be useful as an animal disease model to be diabetes mellitus caused by insulin resistance of resistin.

• The Plant Pathology Journal
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• 제30권4호
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• pp.445-449
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• 2014

The incidence of Cherry necrotic rusty mottle virus (CNRMV) and Cherry green ring mottle virus (CGRMV) have recently been occurred in Korea, posing a problem for sweet cherry cultivation. Since infected trees have symptomless leaves or ring-like spots on the pericarp, it is difficult to identify a viral infection. In this study, the incidence of CNRMV and CGRMV in sweet cherry in Gyeongbuk province was surveyed using a newly developed duplex reverse transcriptase polymerase chain reaction (RT-PCR) method that can detect both viruses in a single reaction. CNRMV and CGRMV co-infection rates were 29.6%, 53.6%, and 17.6%, respectively, in samples collected from three different sites (Daegu, Gyeongju and Gyeongsan) in Gyeongbuk province during 2012 and 2013. This duplex RT-PCR method offers a simple, rapid, and effective way of identifying CNRMV and CGRMV simultaneously in sweet cherry trees, which can aid in the management of viral infections that could undermine yield.

• 대한수의학회지
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• 제36권3호
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• pp.627-633
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• 1996

To analyze the genomic diversity of transmissible gastroenteritis virus (TGEV), the N-terminal half of the spike (S) glycoprotein gene and nonstructural protein gene (open reading frames 3 and 3-1) were amplified by reverse transcriptase reaction and polymerase chain reaction (RT-PCR), and analyzed using restriction fragment length polymorphism (RFLP) patterns of the amplified DNA. In this study, TGEV Miller (M6) and Purdue (P115) strains were used as reference strains, and two vaccine strains (MSV and STC3) and four Korea isolates (P44, VRI-WP, VRI-41, and VRI-48) were analyzed. All TGEV strains were amplified with three TGEV primer pairs. Although there was some exception in RFLP analysis, this method differentiated TGEV strains into following groups : Miller group (M6 and MSV), Purdue group (PUS, STC3, P44, VRI-WP, VRI-41, and VRI-48). Using Sau3AI and SspI, VRI-48 was differentiated from the Miller and Purdue type viruses. The RT/PCR in conjuction with RFLP analysis was a rapid and valuable tool for differentiating several strains of TGEV. This study revealed the occurences of distinct difference in genome of TGEV strains.

• The Plant Pathology Journal
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• 제35권1호
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• pp.41-50
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• 2019

Sugarcane bacilliform viruses (SCBV), which belong to the genus Badnavirus, family Caulimoviridae, are an important DNA virus complex that infects sugarcane. To explore the genetic diversity of the sugarcane-infecting badnavirus complex in China, we tested 392 sugarcane leaf samples collected from Fujian, Yunnan, and Hainan provinces for the occurrence of SCBV by polymerase chain reaction (PCR) assays using published primers SCBV-F and SCBV-R that target the reverse transcriptase/ribonuclease H (RT/RNase H) regions of the viral genome. A total of 111 PCR-amplified fragments (726 bp) from 63 SCBV-positive samples were cloned and sequenced. A neighbor-joining phylogenetic tree was constructed based on the SCBV sequences from this study and 34 published sequences representing 18 different phylogroups or genotypes (SCBV-A to -R). All SCBV-tested isolates could be classified into 20 SCBV phylogenetic groups from SCBV-A to -T. Of nine SCBV phylogroups reported in this study, two novel phylogroups, SCBV-S and SCBV-T, that share 90.0-93.2% sequence identity and show 0.07-0.11 genetic distance with each other in the RT/RNase H region, are proposed. SCBV-S had 57.6-92.2% sequence identity and 0.09-0.66 genetic distance, while SCBV-T had 58.4-90.0% sequence identity and 0.11-0.63 genetic distance compared with the published SCBV phylogroups. Additionally, two other Badnavirus species, Sugarcane bacilliform MO virus (SCBMOV) and Sugarcane bacilliform IM virus (SCBIMV), which originally clustered in phylogenetic groups SCBV-E and SCBV-F, respectively, are first reported in China. Our findings will help to understand the level of genetic heterogeneity present in the complex of Badnavirus species that infect sugarcane.

• 대한화장품학회지
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• 제33권1호
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• pp.17-22
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• 2007

본 연구에서는 돌나물추출물이 각질형성세포에서 hyaluronan synthase (HAS) mRNA 발현과 hyaluronan (HA) 생성에 미치는 영향을 알아보았다. 또한 돌나물추출물이 가지는 인체 피부에서의 보습력 증진 효과를 평가하였다. 돌나물추출물 처리에 따른 각질형성세포에서의 HAS-1, -2, -3 mRNA 발현 변화는 semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)을 통해 살펴보았고, HA의 생성량 변화는 enzyme-linked immunosorbent assay (ELISA)를 수행하여 확인하였다. 인체 피부에서의 수분함량 및 피부 수분 손실량에 미치는 영향은 Corneometer와 Tewameter를 이용하여 측정하였다. 그 결과, 돌나물추출물은 사람 각질형성세포의 HAS-2, -3 mRNA의 발현을 증가시켰고, HA의 생성량을 농도의존적으로 증가시킴을 확인하였다. 사람 피부에서는 수분 함량을 유지하는 효과가 우수하였으며, 표피 수분 손실량 또한 감소시켜 피부 보습제로서 매우 우수한 효과를 보였다 이상의 결과를 통해 돌나물은 피부에서 HA 생산을 촉진시키며, 피부 보습력을 증진시키는 화장품 소재로 이용될 수 있을 것으로 사료된다.

• Preventive Nutrition and Food Science
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• 제22권1호
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• pp.9-15
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• 2017

Vaccinium angustifolium, commonly known as the lowbush blueberry, is a rich source of flavonoids, with which various human physiological activities have been associated. The present study focuses on the investigation of the effect of the methanolic extract of V. angustifolium root extract (VAE) on high affinity immunoglobulin E receptor ($Fc{\varepsilon}RI$) ${\alpha}$ chain antibody (CRA-1)-induced allergic reaction in human basophilic KU812F cells. The total phenolic content of VAE was found to be $170{\pm}1.9mg$ gallic acid equivalents/g. Flow cytometry analysis revealed that the cell surface expression of $Fc{\varepsilon}RI$ was suppressed in a concentration-dependent manner upon culture with VAE. Reverse-transcriptase polymerase chain reaction analysis showed that the mRNA level of the $Fc{\varepsilon}RI$ ${\alpha}$ chain was reduced in a concentration-dependent manner as a result of VAE treatment. Western blot analysis revealed that the protein expression of $Fc{\varepsilon}RI$ and the phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 were concentration-dependently inhibited by VAE. We determined that VAE inhibited anti-CRA-1-induced histamine release, in addition to the elevation of intracellular calcium concentration ([$Ca^{2+}$]i), in a concentration-dependent manner. These results indicate that VAE may exert an anti-allergic effect via the inhibition of calcium influx and histamine release, which occurs as a result of the down-regulation of $Fc{\varepsilon}RI$ expression through inhibition of ERK 1/2 activation.

• IMMUNE NETWORK
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• 제3권2호
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• pp.126-135
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• 2003

Background: One of the important factors in the prognosis of chronic hepatitis B patient is the degree of replication of hepatitis B virus (HBV). It has been known that HBV DNA polymerase plays the essential role in the replication of HBV. HBV DNA polymerase is composed of four domains, TP (Terminal protein), spacer, RT (Reverse transcriptase) and RNaseH. Among these domains, tyrosine, the $65^{th}$ residue of TP is an important residue in protein-priming reaction that initiates reverse transcription. If monoclonal antibody that recognizes around tyrosine residue were selected, it could be applied to further study of HBV replication. Methods: To produce TP-specific scFv (single-chain Fv) by phage display, mice were immunized using synthetic TP-peptide contains $57{\sim}80^{th}$ amino acid residues of TP domain. After isolation of mRNA of heavy-variable region ($V_H$) and light-chain variable region ($V_L$) from the spleen of the immunized mouse, DNA of $V_H$ and $V_L$ were obtained by RT-PCR and joined by a DNA linker encoding peptide (Gly4Ser)3 as a scFv DNA fragments. ScFv DNA fragments were cloned into a phagemid vector. scFv was expressed in E.coli TG1 as a fusion protein with E tag and phage gIII. To select the scFv that has specific affinity to TP-peptide from the phage-antibody library, we used two cycles of panning and colony lift assay. Results: The TP-peptide-specific scFv was isolated by selection process using TP-peptide as an antigen. Selected scFv had 30 kDa of protein size and its nucleotide sequences were analyzed. Indirect- and competitive-ELISA revealed that the selected scFv specifically recognized both TP-peptide and the HBV DNA polymerase. Conclusion: The scFv that recognizes the TP domain of the HBV DNA polymerase was isolated by phage display.

• 한국진공학회:학술대회논문집
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• 한국진공학회 2013년도 제45회 하계 정기학술대회 초록집
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• pp.273-273
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• 2013

Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.

• Asian-Australasian Journal of Animal Sciences
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• 제12권5호
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• pp.695-701
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• 1999

The hypothalamus is the classic site of synthesis of arginine vasotocin as neurohypophyseal hormone in the chicken. However, high concentrations of arginine vasotocin were also measured in ovarian tissues by radioimmunoassay. At first, we observed specific positive signal of mRNA encoding AVT in the hypothalamus by Northern hybridization. However, we could not find any specific bands in ovarian and uterine tissues. For evidence of transcription of the arginine vasotocin gene ingonadal tissues of the chicken, this study has applied the polymerase chain reaction as a highly sensitive assay. The hypothalamus, the four largest preovulatory ovarian follicles and the shell gland (uterus) were collected at 4 h and 20 h before oviposition. The ovarian follicular tissues were separated into granulose theca interns and theca externa layers. The uterine tissues were separated into myometrium and endometrium The extracted mRNA was converted to cDNA by reverse-transcriptase using oligo-$d(T)_{15}$ primer. Then, the cDNA was amplified by Vent polymerase and arginine vasotocin specific primers. The amplification reaction was incubated by 30 cycles successively, $95^{\circ}C$, $55^{\circ}C$ and $72^{\circ}C$ earth for 1 min. Te comparisons of the mRNA levels encoding arginine vasotocin between the tissues were determined by semi-quantification methods. After amplification of the cDNA, the PCR products were detected in hypothalamus, ovarian tissues and uterine tissues. The results of semi-quantification showed that the levels of arginine vasotocin mRNA in ovarian iud uterine tissues were about from 1/50 to 1/1000 when compared to that in the hypothalamus. The very low levels of mRNA encoding arginine vasotocin in ovarian and uterine tissues probably led us to conclude that arginine vasotocin may play a role of local mediate acting autocrine and/or paracrine.