• Journal of Life Science
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• v.24 no.4
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• pp.370-376
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• 2014

Although the grasshopper Oxya chinensis sinuosa has long been used as food in Korea, there is little data on its functional effects. In this study, we investigated the anti-inflammatory effect of O. c. sinuosa ethanol extract (OCE) in RAW 264.7 mouse macrophage cells treated with lipopolysaccharide (LPS) for induction of inflammation. First, we determined that there is no cytotoxicity at $2,000{\mu}g/ml$ or less of OCE in RAW 264.7 cells. To evaluate the anti-inflammatory effects of OCE, we investigated expression levels of pro-inflammatory cytokines such as tumor necrosis factor (TNF)-${\alpha}$ and interleukin (IL)-6, and pro-inflammatory enzymes such as inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) in LPS-induced RAW 264.7 cells. In addition, we examined whether OCE could inhibit translocation of NF-${\kappa}B$ p65 into the nucleus in LPS induced RAW 264.7 cells. As a result, we found that the mRNA and protein levels of TNF-${\alpha}$ and IL-6 decreased in LPS-induced RAW 264.7 cells after treatment with OCE in a dose-dependent manner. In addition, we confirmed a $2,000{\mu}g/ml$ concentration of OCE inhibited translocation of NF-${\kappa}B$ p65 by immunnostaining and Western blot analysis, and a decrease in the protein expression levels of iNOS and COX-2. Accordingly, we suppose that OCE has an anti-inflammatory effect through down-regulation of TNF-${\alpha}$, IL-6, iNOS, and COX-2 related to ${\kappa}B$ p65 inflammatory signaling pathways.

• Korean Journal of Microbiology
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• v.40 no.2
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• pp.87-93
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• 2004

Dynamic modification of cytoplasmic and nuclear proteins by O-linked N-acetylglucosamine (O-GlcNAc) on Ser and Thr residues is ubiquitous in higher eukaryotes. And this modification may serve as a signaling mod-ification analogous to protein phosphorylation. Addition and cleavage of O-GlcNAc are catalyzed by O-linked GlcNAc transferase (OGT) and O-linked N-acety1glucosaminidase (O-GlcNAcase), respectively. Two types of human O-GlcNAcase gene were cloned and expressed as three fusion proteins in Escherichia coli. O-GlcNA-case activity showed in the order of thioredoxin fusion> $6{\times}His$ tag> GST fusion. O-GlcNAcase had enzy-matic activity against only ${\rho}$NP-GlcNAc of seven tested substrate analogs. Blast search revealed that O-GlcNAcase has two conserved domains, amino terminal hyaluronidase-like domain and carboxy terminal N-acetyltransferase domain. Extensive deletion studies were done to define catalytically important domains. The deletions of hyaluronidase-like domain and N-acetyltransferase domain abolished enzyme activity. But, N-ter-minal 55 amino acid deletion and C-terminal truncation showed lower activity. Based on deletion analysis, we suggest that hyaluronidase-like domain is essential for enzyme activity and carboxy terminal N-acetyltrans-ferase domain may be modulatory function.

• The Korean Journal of Physiology and Pharmacology
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• v.15 no.1
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• pp.61-66
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• 2011

P2Y receptors are metabotropic G-protein-coupled receptors, which are involved in many important biologic functions in the central nervous system including retina. Subtypes of P2Y receptors in retinal tissue vary according to the species and the cell types. We examined the molecular and pharmacologic profiles of P2Y purinoceptors in retinoblastoma cell, which has not been identified yet. To achieve this goal, we used $Ca^{2+}$ imaging technique and western blot analysis in WERI-Rb-1 cell, a human retinoblastoma cell line. ATP ($10\;{\mu}M$) elicited strong but transient $[Ca^{2+}]_i$ increase in a concentration dependent manner from more than 80% of the WERI-Rb-1 cells (n=46). Orders of potency of P2Y agonists in evoking $[Ca^{2+}]_i$ transients were 2MeS-ATP>ATP>>UTP=${\alpha}{\beta}$-MeATP, which was compatible with the subclass of $P2Y_1$ receptor. The $[Ca^{2+}]_i$ transients evoked by applications of 2MeS-ATP and/or ATP were also profoundly suppressed in the presence of $P2Y_1$ selective blocker (MRS 2179; $30\;{\mu}M$). $P2Y_1$ receptor expression in WERI-Rb-1 cells was also identified by using western blot. Taken together, $P2Y_1$ receptor is mainly expressed in a retinoblastoma cell, which elicits $Ca^{2+}$ release from internal $Ca^{2+}$ storage sites via the phospholipase C-mediated pathway. $P2Y_1$ receptor activation in retinoblastoma cell could be a useful model to investigate the role of purinergic $[Ca^{2+}]_i$ signaling in neural tissue as well as to find a novel therapeutic target to this lethal cancer.

• Proceedings of the SCSK Conference
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• 2003.09a
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• pp.780-803
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• 2003

Exposure of skin cells, particularly keratinocytes to various nuclear factor-kappaB ($\textrm{NF}_{-{\kappa}}\textrm{B}$) activators [e.g. tumor necrosis factor-$\alpha$, interleukin-1, lipopolysaccharides, and ultraviolet light] leads to phosphorylation and degradation of the inhibitory protein, $\textrm{I}_{{\kappa}}\textrm{B}$. Liberated $\textrm{NF}_{-{\kappa}}\textrm{B}$ is translocated into the nucleus where it can change or alter expression of target genes, resulting in the secretion of extracellular signaling molecules including melanotrophic factors affecting melanocyte. In order to demonstrate the possible role of $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation on the synthesis of melanotrophic factors from the keratinocytes, the activities of $\textrm{NF}_{-{\kappa}}\textrm{B}$ induced by melanogenic inhibitors (MIs) were determined in human HaCaT keratinocytes transfected with $\textrm{pNF}_{-{\kappa}}\textrm{B}$-SEAP-NPT plasmid. Transfectant cells released the secretory alkaline phosphatase (SEAP) as a transcription reporter in response to the $\textrm{NF}_{-{\kappa}}\textrm{B}$ activity and contain the neomycin phosphotransferase (NPT) gene for the dominant selection marker for geneticin resistance. MIs such as niacinamide, kojic acid, hydroquinone, resorcinol, arbutin, and glycolic acid were preincubated with transfectant HaCaT cells for 3 h and then ultraviolet B (UVB) was irradiated. $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation was measured with the SEAP reporter gene assay using a fluorescence detection method. Of the Mis tested, kojic acid ($IC_{50}$/ = 60 $\mu$M) was found to be the most potent inhibitor of UVB-upregulating $\textrm{NF}_{-{\kappa}}\textrm{B}$ activation in transfectant HaCaT cells, which is followed by niacinamide ($IC_{50}$/= 540 $\mu$M). Pretreatment of the transfectant HaCaT cells with the Mis, especially kojic acid and niacinamide, effectively lowered $\textrm{NF}_{-{\kappa}}\textrm{B}$ binding measured by electrophoretic mobility shift assay. Furthermore, these two inhibitors remarkably reduced the secretion level of IL-6, one of melanotrophic factors, triggered by UV-radiation of the HaCaT cells. These observations suggest that Mis working at the in vivo level might act partially through the modulation of the synthesis of melanotrophic factors in keratinocyte.

• Asian Pacific Journal of Cancer Prevention
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• v.17 no.4
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• pp.2185-2193
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• 2016

Background: The pathogenesis of sporadic colorectal cancer (CRC) is influenced by the patient genetic background and environmental factors. Based on prior understanding, these are classified in two major pathways of genetic instability. Microsatellite instability (MSI) and CPG island methylator phenotype (CIMP) are categorized as features of the hypermethylated prototype, and chromosomal instability (CIN) is known to be indicative of the non-hypermethylated category. Secreted frizzled related protein 2 (SFRP2), APC1A in WNT signaling pathway and the DNA repair gene, O6-methylguanine-DNA methyltransferase (MGMT), are frequently hypermethylated in colorectal cancer. Detection of methylated DNA as a biomarker by easy and inexpensive methods might improve the quality of life of patients with CRC via early detection of cancer or a precancerous condition. Aim: To evaluate the rate of SFRP2 and MGMT hypermethylation in both polyp tissue and serum of patients in south Iran as compared with matched control normal population corresponding samples. Materials and Methods: Methylation-specific PCR was used to detect hypermethylation in DNA extracted from 48 polypoid tissue samples and 25 healthy individuals. Results: Of total polyp samples, 89.5% had at least one promoter gene hypermethylation. The most frequent methylated locus was SFRP2 followed by MGMT-B (81.2 and 66.6 percent respectively). Serologic detection of hypermethylation was 95% sensitive as compared with polyp tissue. No hypermethylation was detected in normal tissue and serum and its detection in patients with polyps, especially of serrated type, was specific. Conclusions: Serologic investigation for detection of MGMT-B, SFRP2 hypermethylation could facilitate prioritization of high risk patients for colonoscopic polyp detection and excision.

• Herbal Formula Science
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• v.28 no.3
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• pp.255-269
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• 2020

Objectives : Hemistepta lyrata Bunge (Bunge) is a wild herb that has been used for managing fever and wound in Korean Traditional Medicine. The present study explored the effects of H. lyrata extract on liver X receptor (LXR) α-dependent lipogenic genes in hepatocyte-derived cells. Methods : After HepG2 cells or Huh7 cells were pre-treated with 1-10 ㎍/mL of H. lyrata extract or its fractionated extract for 0.5 h, the cells were subsequently exposed to LXR ligand for 6-24 h. Cell viability, LXR response element (LXRE)-driven luciferase activity, sterol regulatory element binding protein-response element (SREBP-RE)-driven luciferase activity, SREBP-1c expression, and mRNA levels of LXRα and its-dependent target genes were determined. In addition, LC-MS/MS analysis was conducted to explore major compounds in H. lyrata-chloroform fractionated extract #4 (HL-CF4). Results : Of various H. lyrata extracts tested, chloroform extract and its fractionated extract #4, HL-CF4, significantly decreased T0901317-mediated SREBP-1c expression. In addition, HL-CF4 significantly reduced LXRE atransactivation and LXRα mRNA expression without any cytotoxicity. Moreover, HL-CF4 prevented the SREBP-RE-driven luciferase activity and mRNA levels of fatty acid synthase and stearoyl-CoA desaturase-1 induced by T0901317. Results from LC-MS/MS analysis at positive/negative mode indicated that HL-CF4 contained several compounds showing m/z 197.1176 (C₁₁H₁₇O₃), 693.2913/227.1069 (C₃₈H₄₅O₁₂/C₁₅H₁₅O₂), 203.1797 (C₁₅H₂₃), 181.1225 (C₁₁H₁₇O₂), 591.2957 (C₃₅H₄₃O₈), 379.1040 (C₁₈H₁₉O₉), 409.1509 (C₂₀H₂₅O₉), 309.1348 (C₁₆H₂₁O₆), 391.1404 (C₂₀H₂₃O₈), and 669.2924/389.1248 (C₃₆H₄₅O₁₂/C₂₀H₂₁O₈). Conclusion : Based on its inhibition of the LXRα-dependent signaling pathway, H. lyrata chloroform extract and HL-CF4 have prophylactic potentials for managing non-alcoholic fatty liver.

• Molecular & Cellular Toxicology
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• v.3 no.2
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• pp.98-106
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• 2007

Genotoxic stress triggers a variety of biological responses including the transcriptional activation of genes regulating DNA repair, cell survival and cell death. In this study, we investigated to examine gene expression profiles and genotoxic response in TK6 cells treated with DNA damaging agents MNNG (N-methyl-N'-nitrosoguanidine) and hydrogen peroxide $(H_2O_2)$. We extracted total RNA in three independent experiments and hybridized cRNA probes with oligo DNA chip (Applied Biosystems Human Genome Survey Microarray). We analyzed raw signal data with R program and AVADIS software and identified a number of deregulated genes with more than 1.5 log-scale fold change and statistical significancy. We indentified 14 genes including G protein alpha 12 showing deregulation by MNNG. The deregulated genes by MNNG represent the biological pathway regarding MAP kinase signaling pathway. Hydrogen peroxide altered 188 genes including sulfiredoxins. These results show that MNNG and $H_2O_2$ have both uniquely regulated genes that provide the potential to serve as biomarkers of exposure to DNA damaging agents.

• Journal of Life Science
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• v.22 no.10
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• pp.1371-1377
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• 2012

Fibrosis in kidney by internal and external factors causes progressive loss of renal function. Renal fibrosis is the inevitable consequence of an excessive accumulation of the extracellular matrix. TGF-${\beta}$ plays an important role in the process of renal fibrosis and stimulates the synthesis of profibrotic factors, including collagens, fibronectin, and plasminogen activator inhibitor (PAI-1). We examined the effect of Moringa oleifera Lam (moringa) extracts in a rat kidney fibrosis model. We found that moringa root extract suppresses protein expression/mRNA levels of Type I collagen, fibronectin, and PAI-1 induced by TGF-${\beta}$ in renal fibroblasts. Moringa root extract selectively inhibited phosphorylation of TGF-${\beta}$-induced $T{\beta}RII$ and the downstream signaling pathway (e.g., Smad4), and phospho-ERK, but not JNK, p38, or PI3K/AKT. These results suggest that moringa root extract can act against TGF-${\beta}$-induced renal fibrosis in rat kidney fibroblast cells by a mechanism related to its antifibrotic activity, which regulates expression of fibronectin, Type I collagen, and PAI-1 through $T{\beta}RII$-Smad2/3-Smad4 and ERK. Therefore, moringa root extract is an effective substance for fibrosis therapy and provides a new therapeutic strategy for diseases associated with elevated profibrotic factor synthesis.

• Molecules and Cells
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• v.37 no.11
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• pp.833-840
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• 2014

Cullin4-RING ubiquitin ligase (CRL4) is a family of multi-subunit E3 ligases. To investigate the possible involvement of CRL4 in heat stress response, we screened T-DNA insertion mutants of putative CRL4 substrate receptors that exhibited altered patterns in response to heat stress. One of the mutants exhibited heat stress tolerance and was named heat stress tolerant DWD1 (htd1). Introduction of HTD1 gene into htd1-1 led to recovery of heat sensitivity to the wild type level, confirming that the decrease of HTD1 transcripts resulted in heat tolerance. Therefore, HTD1 plays a negative role in thermotolerance in Arabidopsis. Additionally, HTD1 directly interacted with DDB1a in yeast two-hybrid assays and associated with DDB1b in vivo, supporting that it could be a part of a CRL4 complex. Various heat-inducible genes such as HSP14.7, HSP21, At2g03020 and WRKY28 were hyper-induced in htd1-1, indicating that HTD1 could function as a negative regulator for the expression of such genes and that these genes might contribute to thermotolerance of htd1-1, at least in part. HTD1 was associated with HSP90-1, a crucial regulator of thermotolerance, in vivo, even though the decrease of HTD1 did not affect the accumulation pattern of HSP90-1 in Arabidopsis. These findings indicate that a negative role of HTD1 in thermotolerance might be achieved through its association with HSP90-1, possibly by disturbing the action of HSP90-1, not by the degradation of HSP90-1. This study will serve as an important step toward understanding of the functional connection between CRL4-mediated processes and plant heat stress signaling.

• Journal of Ginseng Research
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• v.43 no.2
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• pp.305-311
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• 2019

Background: Gintonin is a ginseng-derived exogenous ligand of the G protein-coupled lysophosphatidic acid (LPA) receptor. We previously reported that gintonin stimulates gliotransmitter release in primary cortical astrocytes. Astrocytes play key roles in the functions of neurovascular systems. Although vascular endothelial growth factor (VEGF) is known to influence the normal growth and maintenance of cranial blood vessels and the nervous system, there is little information about the effect of gintonin on VEGF regulation in primary astrocytes, under normal and hypoxic conditions. Methods: Using primary cortical astrocytes of mice, the effects of gintonin on the release, expression, and distribution of VEGF were examined. We further investigated whether the gintonin-mediated VEGF release protects astrocytes from hypoxia. Results: Gintonin administration stimulated the release and expression of VEGF from astrocytes in a concentration- and time-dependent manner. The gintonin-mediated increase in the release of VEGF was inhibited by the LPA1/3 receptor antagonist, Ki16425; phospholipase C inhibitor, U73122; inositol 1,4,5- triphosphate receptor antagonist, 2-APB; and intracellular $Ca^{2+}$ chelator, BAPTA. Hypoxia further stimulated astrocytic VEGF release. Gintonin treatment stimulated additional VEGF release and restored cell viability that had decreased due to hypoxia, via the VEGF receptor pathway. Altogether, the regulation of VEGF release and expression and astrocytic protection mediated by gintonin under hypoxia are achieved via the LPA receptor-VEGF signaling pathways. Conclusion: The present study shows that the gintonin-mediated regulation of VEGF in cortical astrocytes might be neuroprotective against hypoxic insults and could explain the molecular basis of the beneficial effects of ginseng on the central nervous system.