• Title/Summary/Keyword: rRT-PCR

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Molecular Characterization of Vibrio parahaemolyticus Isolates from Seawater, Fish Tanks, and Distributed Fishery Products in Jeju (제주지역 해수, 수족관물, 유통수산물에서 분리된 장염비브리오균의 분자생물학적 특성)

  • Man Jae Cho;Eunok Kang;Ye-seul Heo;Eun A Koh
    • Journal of Food Hygiene and Safety
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    • v.38 no.4
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    • pp.246-254
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    • 2023
  • V. parahaemolyticus causes waterborne and foodborne disease such as acute diarrhea. In this study, V. parahaemolyticus isolates from seawater, fish tanks, and distributed fishery products in Jeju were investigated for potential toxin or species-specific genes (tdh, trh, tlh, and toxR) using RT-PCR and their genetic characteristics were analyzed using Pulsed-field gel electrophoresis (PFGE). Overall, V. parahaemolyticus of 90 strains (36.7%), including 33 strains from seawater, 8 strains from fish tanks, and 50 strains from fishery products, were isolated from 245 samples. All V. parahaemolyticus strains did not detect the tdh gene, whereas all strains detected tlh or toxR genes. In addition, trh genes were detected in 3 strains from seawater and 1 strain from fishery products. Monthly quantitative testing of seawater revealed that V. parahaemolyticus was positively correlated with water temperature. The 90 strains of V. parahemolyticus obtained in this study showed by gene homology between types, ranging from 64.0-97.3%. Among these, thirteen types showed 100% homology between genes. These results indicate that continuous monitoring is needed to facilitate food poisoning epidemiological investigations because some isolated V. parahaemolyticus strains harbored toxin genes and V. parahaemolyticus strains isolated from seawater, fish tanks, and distributed fishery products showed genetic similarity.

Biological Functions of the COOH-Terminal Amino Acids of the $\alpha$-Subunit of Tethered Equine Chorionic Gonadotropin

  • Jeoung, Youn-Hee;Yoon, Jong-Taek;Min, Kwan-Sik
    • Reproductive and Developmental Biology
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    • v.34 no.1
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    • pp.47-53
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    • 2010
  • Glycoprotein hormones have a common $\alpha$-subunit that is involved in the signaling pathway together with G protein, adenylcyclase and cAMP induction; however, it is an unclear how this common structure is related to hormonal action. To determine the biological functions of the COOH-terminal amino acids in the $\alpha$-subunit of these glycoprotein hormones, a tethered-molecule was constructed by fusing the $NH_2$-terminus of the $\alpha$-subunit to the COOH-terminus of the $\beta$-subunit of equine chorionic gonadotropin (eCG). The following deletion mutants were created by PCR; Ile was inserted at position 96 to form ${\Delta}96$, Lys was substituted at position 95 to form ${\Delta}95$, His was inserted at position 93 to form ${\Delta}93$ and Tyr was substituted at position 87 to form ${\Delta}87$. Each mutant was transfected into CHO-K1 cells. Tethered-wt eCG, and ${\Delta}96$, ${\Delta}95$, and ${\Delta}93$ mutants were efficiently secreted into the medium but the ${\Delta}87$ mutant was not secreted. Interestingly, the RT-PCR, real-time PCR, and northern blot analyses confirmed that the RNA was transcribed in the ${\Delta}87$ mutant. However, the ${\Delta}87$ mutant protein was not detected in the medium or the intracellular fraction of the cell lysates. The LH- and FSH-like activities of the recombinant proteins were assayed in terms of cAMP production using rat LH/CG and rat FSH receptors. The metabolic clearance rate (MCR) was determined by injecting rec-eCG (2 IU) into the tail vein. The ${\Delta}95$ and ${\Delta}93$ mutants were completely inactive in both the LH- and FSH-like activity assays. The ${\Delta}96$ mutant showed slight activity in the LH-like activity assay. In comparison to the wild type, the activity of the ${\Delta}96$ mutant in the FSH-like activity assay was the highest among all the mutants. The MCR assay in which rec-eCG was injected showed a peak at 10 min in all the treatment groups, which disappeared 4 h after injection. These results imply a direct interaction between the receptor and the COOH-terminal region of the a-subunit. The data also reveal a significant difference in the mechanism by which the eCG hormone interacts with the rLH and rFSH receptors. The COOH-terminal region of the $\alpha$-subunit is very important for the secretion and functioning of this hormone.

Gene Expression of Arginine Vasotocin in Ovarian and Uterine Tissues of the Chicken

  • Saito, N.;Grossmann, R.
    • Asian-Australasian Journal of Animal Sciences
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    • v.12 no.5
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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.

Panax ginseng-derived fraction BIOGF1K reduces atopic dermatitis responses via suppression of mitogen-activated protein kinase signaling pathway

  • Lorz, Laura Rojas;Kim, Donghyun;Kim, Mi-Yeon;Cho, Jae Youl
    • Journal of Ginseng Research
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    • v.44 no.3
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    • pp.453-460
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    • 2020
  • Background: BIOGF1K, a fraction of Panax ginseng, has desirable antimelanogenic, anti-inflammatory, and antiphotoaging properties that could be useful for treating skin conditions. Because its potential positive effects on allergic reactions in skin have not yet been described in detail, this study's main objective was to determine its efficacy in the treatment of atopic dermatitis (AD). Methods: High-performance liquid chromatography was used to verify the compounds in BIOGF1K, and we used the (3-4-5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide method to determine its cytotoxicity in RBL-2H3 and HMC-1 cell lines. RBL-2H3 cells were induced using both anti-DNP-IgE/DNP-BSA and calcium ionophore (A2187) treatments, whereas HMC-1 cells were induced using A2187 alone. To measure mast cell degranulation, we performed histamine (enzyme-linked immunosorbent assay) and β-hexosaminidase assays. To quantify interleukin (IL)-4, IL-5, and IL-13 levels in RBL-2H3 cells, we performed quantitative polymerase chain reaction (PCR); to quantify expression levels of IL-4 and IL-13 in HMC-1 cells, we used semiquantitative reverse transcription polymerase chain reaction (RT-PCR). Finally, we detected the total and phosphorylated forms of extracellular signal-regulated kinase, p-38, and c-Jun N-terminal kinase proteins by immunoblotting. Results: BIOGF1K decreased the AD response by reducing both histamine and β-hexosaminidase release as well as reducing the secretion levels of IL-4, IL-5, and IL-13 in RBL-2H3 cells and IL-4 and IL-13 in HMC-1 cells. In addition, BIOGF1K decreased MAPK pathway activation in RBL-2H3 and HMC-1 cells. Conclusions: BIOGF1K attenuated the AD response, hence supporting its use as a promising and natural approach for treating AD.

Whitening Effect of Banana Leaf Extract (바나나잎 추출물의 미백 개선 효과)

  • Hwang, Hyung Seo;Yoo, Dae Sung;Shim, Joong Hyun
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.42 no.1
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    • pp.37-43
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    • 2016
  • This research was carried out to identify the whitening effect of Banana leaf extract. B16F10 cells were used to measure cell viability, mRNA expression, and tyrosinase activity inhibition assay from B16F10 cell. We also carried out clinical test of the cream product containing banana leaf extract. In this study, we elucidated the effects of banana leaf extract on TRP1 / TRP2 / Tyr mRNA expression and tyrosinase activity inhibition. Quantitative real-time PCR showed that banana leaf extract decreased mRNA level of TRP1, TRP2 and Tyr gene and tyrosinase activity inhibition assay also revealed that banana leaf extract 65% decreased melanin production in B16F10 cell. Banana leaf extract cream can whiten the skin darkness induced by ultraviolet. Therefore, we successfully identified the whitening effect of banana leaf extract, and this finding suggested the banana leaf extract is a considerable potent cosmetic ingredient for skin whitening. Based on this, we anticipated further researches about banana leaf extract for mechanism to develop not only cosmetics but healthcare food or medicines.

Generation of Female Porcine Fibroblasts Expressing Efficiently Membrane Cofactor Protein at ${\alpha}1$,3-Galactosyltransferase locus (${\alpha}1$,3-Galactosyltransferase 유전자 좌위에서 Membrane Cofactor Protein을 효과적으로 발현하는 자성 돼지 섬유아세포의 생산)

  • Oh, Keon Bong;Kim, Bella;Hwang, Seongsoo;Ock, Sun-A;Im, Seoki;Park, Jin-Ki
    • Journal of Embryo Transfer
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    • v.28 no.3
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    • pp.289-295
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    • 2013
  • Xenotransplantation of pig organs into primates results in fatal damage, referred as hyperacute rejection (HAR), and acute humoral xenograft rejection (AHXR), to the organ graft mediated by antibodies pre-existing and newly-producing in primates against their cognate pig antigens. Functional ablation of ${\alpha}1$,3-galactosyltransferase (Gal-T KO) of pig which is an enzyme involved in synthesis of Gala1-3Galb1-4GlcNAc-R antigen is essentially required to prevent HAR. Moreover, additional genetic modification under Gal-T KO background for enforced expression of human complement regulatory proteins which can inhibits complement activation is known to effectively imped HAR and AHXR. In this study, we constructed a membrane cofactor protein (MCP) expression cassette under control of human $EF1{\alpha}$ promoter. This cassette was inserted between homologous recombination regions corresponding to Gal-T locus. Subsequently this vector was introduced into ear skin fibroblasts of female pig by nucleofection. We were able to obtained 40 clones by neomycin selection and 4 clones among them were identified as clones targeted into Gal-T locus of MCP expression cassette by long-range PCR. Real time RT-PCR was shown to down-regulation of Gal-T expression. From these results, we demonstrated human $EF1{\alpha}$ promoter could induce efficient expression of MCP on cell surface of fibroblasts of female pig.

Overexpression of Semaphorin4D Indicates Poor Prognosis and Prompts Monocyte Differentiation toward M2 Macrophages in Epithelial Ovarian Cancer

  • Chen, Ying;Zhang, Lei;Lv, Rui;Zhang, Wen-Qi
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.10
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    • pp.5883-5890
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    • 2013
  • Previously, we demonstrated overexpression of semaphorin4D (SEMA4D, CD100) to be closely related to tumor angiogenesis in epithelial ovarian cancers (EOCs). However, the function and expression of SEMA4D in the EOC microenvironment has yet to be clarified in detail. In this study, we confirmed that overexpression of SEMA4D in primary tumors and ascites was related to low differentiation, platinum resistance and a refractory status (P<0.05), while high M2 macrophage count and percentage were evident in EOC patients with advanced FIGO stage and platinum resistance (P<0.05), using immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), and fluorescence-activated cell sorting (FACS), respectively. The data showed correlations of SEMA4D expression and M2 macrophage counts in primary tumors and M2 macrophage percentage in ascites (r=0.281 and 0.355, each P<0.05). In the Cox proportional hazard mode, SEMA4D expression was an independent indicator of overall survival (OS) and progression-free survival (PFS) for EOC patients. Furthermore, higher expression of SEMA4D in ovarian cancer cell lines (SKOV3, A2780, and SW626) and their supernatants were found than that in a human primary cultured ovarian cell and its supernatant by reversed transcript PCR (RT-PCR), Western blotting and ELISA, respectively. Interestingly, peripheral blood monocytes (MOs) tended towards the M2-polarized macrophage phenotype ($CD163^{high}$) in vitro after human recombined soluble SEMA4D protein stimulation. These findings suggest that SEMA4D might possibly serve as a reliable tool for early and accurate prediction of EOC poor prognosis and could playan important role in promoting tumor dissemination and metastasis in the EOC microenvironment. Thus SEMA4D and its role in macrophage polarization in EOC warrants further study.

The effects of bone morphogenetic protein-2 and enamel matrix derivative on the bioactivity of mineral trioxide aggregate in MC3T3-E1cells

  • Jeong, Youngdan;Yang, Wonkyung;Ko, Hyunjung;Kim, Miri
    • Restorative Dentistry and Endodontics
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    • v.39 no.3
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    • pp.187-194
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    • 2014
  • Objectives: The effects of bone morphogenetic protein-2 (BMP-2) and enamel matrix derivative (EMD) respectively with mineral trioxide aggregate (MTA) on hard tissue regeneration have been investigated in previous studies. This study aimed to compare the osteogenic effects of MTA/BMP-2 and MTA/EMD treatment in MC3T3-E1 cells. Materials and Methods: MC3T3-E1 cells were treated with MTA (ProRoot, Dentsply), BMP-2 (R&D Systems), EMD (Emdogain, Straumann) separately and MTA/BMP-2 or MTA/EMD combination. Mineralization was evaluated by staining the calcium deposits with alkaline phosphatase (ALP, Sigma-Aldrich) and Alizarin red (Sigma-Aldrich). The effects on the osteoblast differentiation were evaluated by the expressions of osteogenic markers, including ALP, bone sialoprotein (BSP), osteocalcin (OCN), osteopontin (OPN) and osteonectin (OSN), as determined by reverse-transcription polymerase chain reaction analysis (RT-PCR, AccuPower PCR, Bioneer). Results: Mineralization increased in the BMP-2 and MTA/BMP-2 groups and increased to a lesser extent in the MTA/EMD group but appeared to decrease in the MTA-only group based on Alizarin red staining. ALP expression largely decreased in the EMD and MTA/EMD groups based on ALP staining. In the MTA/BMP-2 group, mRNA expression of OPN on day 3 and BSP and OCN on day 7 significantly increased. In the MTA/EMD group, OSN and OCN gene expression significantly increased on day 7, whereas ALP expression decreased on days 3 and 7 (p < 0.05). Conclusions: These results suggest the MTA/BMP-2 combination promoted more rapid differentiation in MC3T3-E1 cells than did MTA/EMD during the early mineralization period.

Toll-like Receptor3-mediated Induction of Chemokines in Salivary Epithelial Cells

  • Li, Jingchao;Jeong, Mi-Young;Bae, Ji-Hyun;Shin, Yong-Hwan;Jin, Meihong;Hang, Sung-Min;Lee, Jeong-Chai;Lee, Sung-Joong;Park, Kyung-Pyo
    • The Korean Journal of Physiology and Pharmacology
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    • v.14 no.4
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    • pp.235-240
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    • 2010
  • Toll-like receptors (TLRs) functionally expressed in salivary epithelial cells, but their roles remain elusive. Among TLRs family, TLR3 is activated by dsRNA, a byproduct of viral infection. The aim of this study was to investigate the role of TLR3 in the inflammatory immune responses using HSG cells. Reverse transcriptase-polymerase chain reaction (RT-PCR), real-time PCR and ELISA were performed to identify expression of TLRs and TLR3-mediated chemokine inductions. The chemotaxis assay of activated T lymphocytes was also performed. Treatment of HSG cells with polyinosinic: polycytidylic acid (poly(I:C)) significantly increased interferon-$\gamma$-inducible protein 10 (IP-10), interferoninducible T-cell $\alpha$ chemoattractant (I-TAC), and regulated on activation, normal T-cells expressed and secreted (RANTES) gene expressions in a concentration-dependent manner. Anti-TLR3 antibody blocked the increases of IP-10 and I-TAC genes. Poly(I:C)-induced increases of IP-10 and I-TAC were also confirmed at protein levels from cell lysates, but their release into extracellular medium was detected only in IP-10. We found that the culture media from HSG cells stimulated with poly(I:C) significantly increases T lymphocyte migration. Our results suggest that TLR3 plays an important role in chemokine induction, particularly IP-10, in salivary epithelial cells.

Expression of Arabidopsis thaliana SIK (Stress Inducible Kinase) Gene in a Potato Cultivar (Solanum tuberosum L. 'Taedong Valley')

  • Yoon Jung-Ha;Fang Yi-Lan;Park Eung-Jun;Kim Hye-Jin;Na Yun-Jeong;Lee Dong-Hee;Yang Deok-Chun;Lim Hak-Tae
    • Plant Resources
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    • v.8 no.3
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    • pp.202-208
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    • 2005
  • Osmotic stress is one of major limiting factors in crop production. In particular, seasonal drought often causes the secondary disease in the field, resulting in severe reduction in both quality and productivity. Recent efforts have revealed that many genes encoding protein kinases play important roles in osmotic stress signal transduction pathways. Previously, the AtSIK (Arabidopsis thaliana Stress Inducible Kinase) mutants have shown to enhance tolerance to abiotic stresses, accompanying with higher expression of abiotic stress-related genes than did the wild-type plants. In this study, we have transformed potato (cv. Taedong Valley) with the AtSIK expression cassette. Both PCR and RT-PCR using AtSIK-specific primers showed stable integration and expression of the AtSIK gene in individual transgenic lines, respectively. Foliar application of herbicide ($Basta^{(R)}$) at commercial application rate (0.3% (v/v)) revealed another evidence of stable gene introduction of T-DNA which includes the bar gene for herbicide resistance. Overexpression of the AtSIK gene under dual CaMV35S promoter increased sensitivity to salt stress (300 mM NaCl), which was demonstrated by the reduction rate of chlorophyll contents in leaves of transgenic potato lines. These results suggest that possible increase of osmotic tolerance in potato plants may be achieved by antisense expression of AtSIK gene.

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