• 한국식품저장유통학회지
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• 제30권2호
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• pp.262-271
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• 2023

본 연구에서는 real-time PCR(SureTect^TM kit와 PowerChek^TM kit), LAMP(3M MDS), 선택 배지를 이용하여 즉석섭취식품에 존재하는 Salmonella spp.와 L. monocytogenes의 검출 능력을 비교 및 평가하고 식품 매트릭스가 real-time PCR의 결과에 미치는 영향을 조사하였다. 4가지 서로 다른 농도로 접종된 식품을 동일한 증균배지를 이용하여 증균 후 세 가지 방법으로 검출한 결과, real-time PCR, LAMP, 선택 배지에서 모두 양성으로 검출되어 인위적으로 접종된 식품에서의 검출 성능은 동등한 것으로 나타났다. 또한, 식품 매트릭스가 real-time PCR의 신속 검출에 미치는 영향을 조사한 결과, Salmonella spp.의 검출에서 샐러드가 다른 식품에 비해 Ct value가 유의적으로 높은 것으로 나타나, 섬유질이 풍부한 식품에 존재하는 Salmonella spp.의 검출을 위해서는 충분한 균질화와 균체의 탈리, 그리고 효율적인 DNA의 증폭이 필요함을 알 수 있었다. 반면, L. monocytogenes의 검출은 식품 매트릭스마다 상이하며 혼합적인 양상을 보였다. 현재의 식품공전 규정에서 식품에 존재하는 식중독균의 신속 검출을 위한 장비와 시약의 사용은 대부분 사용자의 선택에 의존하고 있다. 본 실험에서 real-time PCR로 사용된 SureTect^TM kit와 PowerChek^TM kit는 기존 real-time PCR kit의 대체재로서 사용이 가능할 것으로 판단되며, 또한, LAMP도 우수한 검출 성능을 보였기에 식품안전 관리 수단으로 활용될 가능성이 있음을 시사하고 있다.

• 대한의생명과학회지
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• 제17권4호
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• pp.297-304
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• 2011

Influenza A virus of the Orthomyxoviridae family is a contagious respiratory pathogen that continues to evolve and burden in the human public health. It is able to spread efficiently from human to human and have the potential to cause pandemics with significant morbidity and mortality. It has been estimated that every year about 500 million people are infected with this virus, causing about approximately 0.25 to 0.5 million people deaths worldwide. Influenza A viruses are classified into different subtypes by antigenicity based on their hemagglutinin (HA) and neuraminidase (NA) proteins. The sudden emergence of influenza A virus subtypes and access for epidemiological analysis of this subtypes demanded a rapid development of specific diagnostic tools. Also, rapid identification of the subtypes can help to determine the antiviral treatment, because the different subtypes have a different antiviral drug resistance patterns. In this study, our aim is to detect influenza A virus subtypes by using real-time nucleic acid sequence based amplification (NASBA) which has high sensitivity and specificity through molecular beacon. Real-time NASBA is a method that able to shorten the time compare to other molecular diagnostic tools and is performed by isothermal condition. We selected major pandemic influenza A virus subtypes, H3N2 and H5N1. Three influenza A virus gene fragments such as HA, NA and matrix protein (M) gene were targeted. M gene is distinguished influenza A virus from other influenza virus. We designed specific primers and molecular beacons for HA, NA and M gene, respectively. In brief, the results showed that the specificity of the real-time NASBA was higher than reverse transcription polymerase chain reaction (RT-PCR). In addition, time to positivity (TTP) of this method was shorter than real-time PCR. This study suggests that the rapid detection of neo-appearance pandemic influenza A virus using real-time NASBA has the potential to determine the subtypes.

• Asian-Australasian Journal of Animal Sciences
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• 제29권4호
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• pp.457-463
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• 2016

The goal of this work was to investigate the correlations between MyHC mRNA transcription and their corresponding protein expressions in porcine longissimus muscle (LM) during postnatal growth of pigs. Five DLY ($Duroc{\times}Landrace{\times}Yorkshire$) crossbred pigs were selected, slaughtered and sampled at postnatal 7, 30, 60, 120, and 180 days, respectively. Each muscle was subjected to quantity MyHCs protein contents through an indirect enzyme-linked immunosorbent assay (ELISA), to quantity myosin heavy-chains (MyHCs) mRNA abundances using real-time polymerase chain reaction. We calculated the proportion (%) of each MyHC to total of four MyHC for two levels, respectively. Moreover, the activities of several key energy metabolism enzymes were determined in LM. The result showed that mRNA transcription and protein expression of MyHC I, IIa, IIx and IIb in LM all presented some obvious changes with postnatal aging of pigs, especially at the early stage after birth, and their mRNA transcriptions were easy to be influenced than their protein expressions. The relative proportion of each MyHC mRNA was significantly positively related to that of its corresponding protein (p<0.01), and MyHC I mRNA proportion was positively correlated with creatine kinase (CK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH) activities (p<0.05). These data suggested that MyHC mRNA transcription can be used to reflect MyHC expression, metabolism property and adaptive plasticity of porcine skeletal muscles, and MyHC mRNA composition could be a molecular index reflecting muscle fiber type characteristics.

• Animal Bioscience
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• 제36권6호
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• pp.829-839
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• 2023

Objective: The aim of this study was to clone the mRNA sequence of the Acyl-CoA dehydrogenase long chain (ACADL) gene of goats and explore the effect of ACADL on the differentiation of subcutaneous fat cells on this basis. Methods: We obtained the ACADL gene of goats by cloning and used quantitative real-time polymerase chain reaction (qPCR) to detect the ACADL expression patterns of different goat tissues and subcutaneous fat cells at different lipid induction stages. In addition, we transfect intramuscular and subcutaneous adipocytes separately by constructing overexpressed ACADL vectors and synthesizing Si-ACADL; finally, we observed the changes in oil red stained cell levels under the microscope, and qPCR detected changes in mRNA levels. Results: The results showed goat ACADL gene expressed in sebum fat. During adipocyte differentiation, ACADL gradually increased from 0 to 24 h of culture, and decreased. Overexpression of ACADL promoted differentiation of subcutaneous adipocytes in goat and inhibited their differentiation after interference. Conclusion: So, we infer ACADL may have an important role in positive regulating the differentiation process in goat subcutaneous adipocytes. This study will provide basic data for further study of the role of ACADL in goat subcutaneous adipocyte differentiation and lays the foundation for final elucidating of its molecular mechanisms in regulating subcutaneous fat deposition in goats.

• Journal of Chest Surgery
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• 제43권4호
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• pp.356-363
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• 2010

배경: 승모판막의 석회화는 죽상경화와 밀접한 연관관계가 있는 것으로 알려져 있다. 그러나, 승모판막 질환에서 혈관 죽상경화에 중요한 역할을 하는 것으로 알려진 Connexin43 매개에 의한 Gap junction의 신호전달의 변화는 잘 알려져 있지 않다. 따라서, 저자들은 승모판막의 퇴행성 변화과정이 Gap junction을 통한 세포간 신호전달의 변화와 연관이 있을 것이라고 가정하였다. 대상 및 방법: 뉴질랜드산 토끼 20마리를 2군으로 나눈후, 1군(10마리)에서는 정상식이를 시행하였고, 2군(10마리)에서는 1% 콜레스테롤식이를 12주간 시행하였다. 각군의 토끼들의 혈장에서 총 콜레스테롤, 중성지방, 저밀도 콜레스테롤, 고밀도 콜레스테롤 수치를 측정하였으며, 승모판막을 절제하여 Connexin43, 근육섬유모세포 그리고 대식세포에 대한 면역조직화학염색을 시행하였으며, Connexin43에 대한 정량적 검사를 위하여 Real-time Reverse Transcriptase polymerase chain reaction (real time RT-PCR)을 시행하였다. 결과: 면역조직화학염색에서 근육섬유모세포와 대식세포의 발현은 콜레스테롤 식이를 시행한 군의 승모판막에서 증가되었으며, Connexin43의 발현도 콜레스테롤 식이군에서 증가되어 있었다. RT-PCR을 이용한 정량적 검사에서 Connexin43의 발현은 콜레스테롤 식이군에서 의미있게 증가되어 있었다(p<0.01). 결론: 고콜레스테롤혈증이 유발된 토끼의 승모판막의 변화는 초기 죽상경화와 유사한 과정을 보이며, Connex43의 발현이 증가되었다. 따라서 고콜레스테롤혈증에서의 승모판막질환은 증가된 Connexin43매개 Gap junction을 통한 세포간 신호전달의 변화가 중요한 역할을 한다고 생각된다.

• Journal of Gastric Cancer
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• 제17권1호
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• pp.43-51
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• 2017

Purpose: This study aimed to analyze G3BP1 and VEZT expression profiles in patients with gastric cancer, and examine the possible relationship between the expressions of each gene and clinicopathological factors. Materials and Methods: Expression of these genes in formalin-fixed paraffin embedded (FFPE) tissues, collected from 40 patients with gastric cancer and 40 healthy controls, was analyzed. Differences in gene expression among patient and normal samples were identified using the GraphPad Prism 5 software. For the analysis of real-time polymerase chain reaction products, GelQuantNET software was used. Results: Our findings demonstrated that both VEZT and G3BP1 mRNA expression levels were downregulated in gastric cancer samples compared with those in the normal controls. No significant relationship was found between the expression of these genes and gender (P-value, 0.4835 vs. 0.6350), but there were significant changes associated with age (P-value, 0.0004 vs. 0.0001) and stage of disease (P-value, 0.0019 vs. 0.0001). In addition, there was a direct relationship between VEZT gene expression and metastasis (P-value, 0.0462), in contrast to G3BP1 that did not demonstrate any significant correlation (P-value, 0.1833). Conclusions: The results suggest that expression profiling of VEZT and G3BP1 can be used for diagnosis of gastric cancer, and specifically, VEZT gene could be considered as a biomarker for the detection of gastric cancer progression.

• Nutrition Research and Practice
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• 제9권6호
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• pp.586-591
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• 2015

BACKGROUND/OBJECTIVES: Reactive oxygen species (ROS) formation is closely related to miconazole-induced heart dysfunction. Although rhamnetin has antioxidant effects, it remained unknown whether it can protect against miconazole-induced cardiomyocyte apoptosis. Thus, we investigated the effects of rhamnetin on miconazole-stimulated H9c2 cell apoptosis. MATERIALS/METHODS: Cell morphology was observed by inverted microscope and cell viability was determined using a WelCount$^{TM}$ cell proliferation assay kit. Miconazole-induced ROS production was evaluated by fluorescence-activated cell sorting with 6-carboxy-2',7'-dichlorofluoroscein diacetate ($H_2DCF$-DA) stain. Immunoblot analysis was used to determine apurinic/apyrimidinic endonuclease 1 (APE/Ref-1) and cleaved cysteine-aspartic protease (caspase) 3 expression. NADPH oxidase levels were measured using real-time polymerase chain reaction. RESULTS: Miconazole (3 and $10{\mu}M$) induced abnormal morphological changes and cell death in H9c2 cells. Rhamnetin enhanced the viability of miconazole ($3{\mu}M$)-treated cells in a dose-dependent manner. Rhamnetin (1 and $3{\mu}M$) treatment downregulated cleaved caspase 3 and upregulated APE/Ref-1 expression in miconazole-stimulated cells. Additionally, rhamnetin significantly reduced ROS generation. CONCLUSIONS: Our data suggest that rhamnetin may have cytoprotective effects in miconazole-stimulated H9c2 cardiomyocytes via ROS inhibition. This effect most likely occurs through the upregulation of APE/Ref-1 and attenuation of hydrogen peroxide levels.

• Journal of Microbiology and Biotechnology
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• 제20권2호
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• pp.265-270
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• 2010

Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of the cel7B gene in its chromosomal DNA. The expression levels of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that the two genes were coordinately expressed, and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimally active at $60^{\circ}C$ and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, and 5-fold higher enzyme activities toward carboxymethyl cellulose, barley $\beta$-glucan, and PASC, respectively, in comparison with that of Cel5A. However, their activities toward pNPC and Avicel showed minor differences. The results suggested that Cel7B is a strict endoglucanase, whereas Cel5A showed processivity because of its relative higher ability to hydrolyze the crystal cellulose.

• Natural Product Sciences
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• 제18권1호
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• pp.52-59
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• 2012

In our continuous efforts to procure the active materials from natural products in the protective effects of oxidative stress or UV damage to skin cells we found the Prunus persica flesh extract (PPFE) is considerable to meet the demand to protect the skin damage. PPFE attenuated cell damage induced by hypoxanthine-xanthine oxidase in cultured human keratinocytes, indicating that PPFE has the potential of the scavenging effect of reactive oxygen species (ROS) in human skin cell. Moreover, PPFE significantly suppressed UVA-induced ROS production determined by the oxidation of 2,7-dichlorodihydrofluorescein diacetate (DCFH) using FACS analysis. Additional study revealed that UVA irradiation of HaCaT human keratinocytes increased the gelatinolytic activities of matrix metalloproteinase-2, and -9 (MMP-2, -9) and mRNA expression of MMP-9 analyzing by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR), and these events were significantly suppressed by the treatment with PPFE. These results suggest that PPFE might be applicable as natural ingredients for skin antiaging agents via UV-induced ROS scavenging activity and suppression of MMP expression in the skin cells.

• Pediatric Infection and Vaccine
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• 제21권2호
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• pp.139-143
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• 2014

BCG 림프절염은 BCG 접종 후에 생기는 가장 흔한 합병증이다. BCG 림프절염은 대부분 6개월 미만의 영아에서 발생하며 접종한 부위와 같은 쪽 겨드랑이 림프절을 침범하는 경우가 많다. 본 저자들은 생후 15일에 Tokyo주 BCG 백신을 접종한 22개월 남자아이에서 왼쪽 쇄골상부 림프절 부위에 발생한 BCG 림프절염을 경험하여 이에 대해 보고한다. 우리는 림프절 흡인을 시행하여 얻은 검체에서 실시간 중합효소 연쇄반응과 region of difference (RD)에 대한 다중 중합효소연쇄반응으로 M. bovis Tokyo주에 의한 감염을 확인하였고 반복적인 흡인을 통해 치료하였다.