• Korean Journal of Medicinal Crop Science
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• v.27 no.6
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• pp.419-426
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• 2019

Background: This study aimed to identify antioxidant compounds from the seeds of Dolichos lablab L. by bioassay-guided isolation and recrystallization. Methods and Results: The water layer of D. lablab L. seed extract inhibits intracellular reactive oxygen species (ROS) expressing the 2',7'-dichlorofluorescein diacetate (DCF-DA), Cu/Zn superoxide dismutase (SOD) and catalase genes, as determined by quantitative real-time PCR (qRT-PCR). Two compounds were purified from the water layer of the seeds of D. lablab L. using column chromatography and prep-high performance liquid chromatography (HPLC). Using nuclear magnetic resonance (NMR) and electrospray Ionization mass spectrometry (ESI-MS), their chemical structures were identified as 5-[(2-acetyl-2,3-dihydro-1H-indazol-1-yl)carbonyl]-4,5-dihydro-3H-furan-2-one (C₁₄H₁₄N₂O₄) and stachyose. Conclusions: Two active antioxidant compounds were purified from the seed extract of D. lablab L. seed extract and the structures of these compounds were identified as C₁₄H₁₄O₄N₂ and stachyose.

• Development and Reproduction
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• v.24 no.1
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• pp.43-52
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• 2020

NUCB2/nesfatin-1 known to regulate appetite and energy homeostasis is expressed not only in the hypothalamus, but also in various organs and tissues. Our previous reports also demonstrated that NUCB2/nesfatin-1 was expressed in the reproductive organs, including the ovaries, uterus, and testes of mice. However, it is yet known whether NUCB2/nesfatin-1 is expressed in the oviduct and how its expression is regulated. Therefore, we investigated the expression of NUCB2/nesfatin-1 in the oviduct and its expression is regulated by gonadotropin. Immunohistochemical staining results showed that nesfatin-1 protein was localized in epithelial cells of the oviduct. As a result of quantitative real-time PCR (qRT-PCR) and Western blot, NUCB2/nesfatin-1 was detected strongly in the oviducts. During the estrus cycle, NUCB2/nesfatin-1 expression in the oviducts was markedly higher in the proestrus stage than in other estrus stages. In order to elucidate whether the expression of NUCB2 mRNA is controlled by the gonadotropins, we injected PMSG and hCG and measured NUCB2 mRNA level in the oviduct after injection. Its level was increased in the oviduct after PMSG injection, but no significant change after hCG injection. In addition, NUCB2 mRNA levels were markedly reduced after ovariectomy, while recovered after 17β-estradiol (E2) injection, but not by progesterone (P4). This study demonstrated that NUCB2/nesfatin-1 is highly expressed in the oviduct of mouse and its expression is regulated by E2 secreted by the ovaries. These results suggest that NUCB2/nesfatin-1 expressed by the oviduct may affect the function of the oviduct regulated by the ovaries.

• Journal of Microbiology and Biotechnology
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• v.19 no.5
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• pp.431-438
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• 2009

The alkaline serine protease asp, which was shown to be a virulence factor of Vibrio alginolyticus as a purified protein, was cloned from V. alginolyticus EPGS, a strain recently isolated from moribund Epinephelus coioides in an outbreak of vibriosis in a mariculture farm of Shenzhen. The asp null mutant was constructed by homologous recombination with suicide plasmid pNQ705-1. Compared with the wild-type strain, the asp null mutant exhibited a significant decrease of total extracellular protease activity, and caused a IS-fold decrease in virulence of V. alginolyticus. In our previous study, the luxO and $luxR_{val}$ genes from V. alginolyticus MVP01 were cloned and identified, and the luxO-$luxR_{val}$ regulatory couple was shown to regulate various genes expression, suggesting that it played a central role in the quorum sensing system of V. alginolyticus. In this study, the regulation of the asp gene was analyzed by using RT-PCR and quantitative real-time PCR methods; we proved that its transcription was greatly induced at the late stage of growth and was regulated by a luxO-$luxR_{val}$ regulatory system.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.1
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• pp.45-48
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• 2014

Aims: Early diagnosis is important for cervical cancer treatment. This study aimed to characteriz the microRNA profile and target gene protein levels of cervical cancers in Uygur women for application in early diagnosis. Methods: The profiles of miRNA in cervical cancer and chronic cervicitis were analyzed with miRNAmicroarray V4.0. The expression of miR-101 was detected by real-time PCR and locked nucleotide acid in situ hybridization (LNA-ISH). Cox-2 protein levels were assessed by immunohistochemistry. Results: The microarray identified a set of 12 miRNAs significantly decreased in cervical cancer in comparison to the control group. Quantitative RT-PCR analysis showed miR-101 to be significantly downregulated in cancer tissues (p<0.05) while LNA-ISH showed miR-101 positive rates of 80% (20/25) and 8% (5/25) (p<0.05) in the control and cervical cancer groups. Cox-2 positive rates of cervical cancer and control groups were 84% (21/25) and 8% (2/25) (p<0.05). Conclusions: Use of down-regulation of miR-101 and up-regulation of Cox-2 as markers may play a role in early diagnosis of cervical cancer in Uygur women.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.1
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• pp.195-200
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• 2013

Secreted protein acidic and rich in cysteines-like protein 1 (SPARCL1), an extracellular matrix glycoprotein, has been implicated in the pathogenesis of several disorders including cancer. However, little is known about the expression and significance of SPARCL1 in human breast cancer. The aim of this study was to determine the expression pattern and clinicopathological significance of SPARCL1 in a Chinese breast cancer cohort. mRNA and protein expression of SPARCL1 in human breast cancer cell lines and breast cancer tissues was detected using the reverse transcription-polymerase chain reaction, real-time quantitative PCR, and Western blotting, respectively. Immunostaining of SPARCL1 in 282 Chinese breast cancer samples was examined and associations with clinicopathological parameters were analyzed. Compared to the positive expression in immortalized human breast epithelial cells, SPARCL1 was nearly absent in human breast cancer cell lines. Similarly, a significantly reduced expression of SPARCL1 was observed in human breast cancer tissues compared to that in normal breast epithelial tissues, for both mRNA and protein levels (P < 0.001). Immunohistochemical analysis showed that strong cytoplasmic immunostaining of SPARCL1 was observed in almost all normal breast samples (43/45) while moderate and strong immunostaining of SPARCL1 was only detected in 191 of 282 (67.7%) breast cancer cases. Moreover, down-regulation of SPARCL1 was significantly correlated with lymphatic metastasis (P = 0.020) and poor grade (P = 0.044). In conclusion, SPARCL1 may be involved in the breast tumorigenesis and serve as a promising target for therapy of breast cancer.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.18
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• pp.7617-7623
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• 2014

Objective: The mechanism of action of fatty acid synthase (FASN) in drug tolerance of breast cancer cells with epithelial-mesenchymal transition (EMT) features was investigated. Methods: The breast cancer cell line MCF-7-MEK5 with stably occurring EMT and tumour necrosis factor-${\alpha}$ (TNF-${\alpha}$) tolerance was used as the experimental model, whereas MCF-7 acted as the control. Tumour cells were implanted into nude mice for in vivo analysis, and cerulenin was used as a FASN inhibitor. RT-PCR, real-time quantitative PCR and Western blot were employed to detect the expression of FASN, TNFR-1, TNFR-2, Wnt-1, ${\beta}$-catenin and cytC at the RNA and protein levels. Results: Compared with MCF-7, TNFR-1 expression in MCF-7-MEK5 was slightly changed, TNFR-2 was decreased, and FASN, Wnt-1, ${\beta}$-catenin and cytC were increased. The expression of Wnt-1 and ${\beta}$-catenin in MCF-7-MEK5 decreased after cerulenin treatment, whereas cytC expression increased. Conclusions: The important function of FASN in the drug tolerance of breast cancer may be due to the following mechanisms: FASN downregulated TNFR-2 expression through lipid rafts to make the cells less sensitive to TNF-${\alpha}$, and simultaneously activated the Wnt-$1/{\beta}$-catenin signalling pathway. Thus, cytC expression increased, which provided cells with anti-apoptotic capacity and induced drug tolerance.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.5
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• pp.1925-1929
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• 2014

Background: Vascular endothelial growth factor-C (VEGF-C), which contributes to lymphatic metastasis (LM) in malignant disease, is one of the most important factors involved in physical and pathological lymphangiogenesis. Some VEGF-C related factors such as sine oculis homeobox homolog (SIX) 1, contactin (CNTN) 1 and dual specificity phosphatase (DUSP) 6 have been extensively studied in malignancies, but their expression levels and associations have still to be elucidated in stomach cancer. Methods: We detected their expression levels in 30 paired stomach cancer tissues using quantitative real-time reverse transcription-PCR (qRT-PCR). The expression and clinical significance of each factor was analyzed using Wilcoxon signed rank sum test. The correlation among all the factors was performed by Spearman rank correlation analysis. Results: The results suggest that VEGF-C and CNTN1 are significantly correlated with tumor size, SIX1 with the age and CNTN1 also with the cTNM stage. There are significant correlations of expression levels among VEGF-C, SIX1, CNTN1 and DUSP6. Conclusions: There exists an important regulatory crosstalk involving SIX1, VEGF-C, CNTN1 and DUSP6 in stomach cancer.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.7
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• pp.1037-1043
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• 2016

Epigenetic processes in the development of skeletal muscle have been appreciated for over a decade. DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Up to now, the importance of epigenetic marks in the regulation of Pax7 and myogenic regulatory factors (MRFs) expression is far less explored. In the present study, semi-quantitative the real-time polymerase chain reaction (RT-PCR) analyses showed MyoD and Myf5 were expressed in activated and quiescent C2C12 cells. MyoG was expressed in a later stage of myogenesis. Pax7 was weakly expressed in differentiated C2C12 cells. To further understand the regulation of expression of these genes, the DNA methylation status of Pax7, MyoD, and Myf5 was determined by bisulfite sequencing PCR. During the C2C12 myoblasts fusion process, the changes of promoter and exon 1 methylation of Pax7, MyoD, and Myf5 genes were observed. In addition, an inverse relationship of low methylation and high expression was found. These results suggest that DNA methylation may be an important mechanism regulating Pax7 and MRFs transcription in cell myogenic differentiation.

• The Korean Journal of Physiology and Pharmacology
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• v.22 no.4
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• pp.369-377
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• 2018

Spinal tuberculosis (ST) is the tuberculosis caused by Mycobacterium tuberculosis (Mtb) infections in spinal curds. Isoliquiritigenin (4,2',4'-trihydroxychalcone, ISL) is an anti-inflammatory flavonoid derived from licorice (Glycyrrhiza uralensis), a Chinese traditional medicine. In this study, we evaluated the potential of ISL in treating ST in New Zealand white rabbit models. In the model, rabbits (n=40) were infected with Mtb strain H37Rv or not in their $6^{th}$ lumbar vertebral bodies. Since the day of infection, rabbits were treated with 20 mg/kg and 100 mg/kg of ISL respectively. After 10 weeks of treatments, the adjacent vertebral bone tissues of rabbits were analyzed through Hematoxylin-Eosin staining. The relative expression of Monocyte chemoattractant protein-1 (MCP-1/CCL2), transcription factor ${\kappa}B$ ($NF-{\kappa}B$) p65 in lymphocytes were verified through reverse transcription quantitative real-time PCR (RT-qPCR), western blotting and enzyme-linked immunosorbent assays (ELISA). The serum level of interleukin (IL)-2, IL-4, IL-10 and interferon ${\gamma}$ ($IFN-{\gamma}$) were evaluated through ELISA. The effects of ISL on the phosphorylation of $I{\kappa}B{\alpha}$, $IKK{\alpha}/{\beta}$ and p65 in $NF-{\kappa}B$ signaling pathways were assessed through western blotting. In the results, ISL has been shown to effectively attenuate the granulation inside adjacent vertebral tissues. The relative level of MCP-1, p65 and IL-4 and IL-10 were retrieved. $NF-{\kappa}B$ signaling was inhibited, in which the phosphorylation of p65, $I{\kappa}B{\alpha}$ and $IKK{\alpha}/{\beta}$ were suppressed whereas the level of $I{\kappa}B{\alpha}$ were elevated. In conclusion, ISL might be an effective drug that inhibited the formation of granulomas through downregulating MCP-1, $NF-{\kappa}B$, IL-4 and IL-10 in treating ST.

• The Journal of Advanced Prosthodontics
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• v.6 no.3
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• pp.157-164
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• 2014

PURPOSE. This study focused on in vitro cell differentiation and surface characteristics in a magnesium coated titanium surface implanted on using a plasma ion source. MATERIALS AND METHODS. 40 commercially made pure titanium discs were prepared to produce Ti oxide machined surface (M) and Mg-incorporated Ti oxide machined surface (MM). Surface properties were analyzed using a scanning electron microscopy (SEM). On each surface, alkaline phosphatase (ALP) activity, alizarin red S staining for mineralization of MC3T3-E1 cells, and quantitative analysis of osteoblastic gene expression, were evaluated. Actin ring formation assay and gene expression analysis of TRAP and GAPDH performing RT-PCR were performed to characterize osteoclast differentiation on mouse bone marrow-derived macrophages (BMMs). RESULTS. MM showed similar surface morphology and surface roughness with M, but was slightly smoother after ion implantation at the micron scale. M was more hydrophobic than MM. No significant difference between surfaces on ALP activity at 7 and 14 days were observed. Real-time PCR analyses showed similar levels of mRNA expression of the osteoblast phenotype genes; osteopontin (OPN), osteocalcin (OCN), bone sialoprotein (BSP), and collagen 1 (Col 1) in cell grown on MM at 7, 14 and 21 days. Alizarin red S staining at 21 days showed no significant difference. BMMs differentiation increased in M and MM. Actin ring formation assay and gene expression analysis of TRAP showed osteoclast differentiation to be more active on MM. CONCLUSION. Both M and MM have a good effect on osteoblastic cell differentiation, but MM may speed the bone remodeling process by activating on osteoclast differentiation.