• Journal of Pharmaceutical Investigation
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• v.33 no.4
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• pp.287-292
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• 2003

A novel antitumor agent, antibody-endostatin fusion protein $(anti-HER2/neu\;IgG3C_H3-Endostatin,\;AEFP)$ formed by genetic engineering procedure from antibody (Ab) which specifically targets to tumor cells ad angiogenesis inhibitor, endostatin (Endo) that has excellent antitumor effect, minimizes the toxicity of normal cells and selectively kills only tumor cells. The purpose of this study is to evaluate the phamacokinetic parameters and to analyze the localization of AEFP. After an intravenous injection of $150\;{\mu}l\;(5\;{\mu}Ci)\;[^{125}I]Ab,\;[^{125}I]AEFP$ to mice, blood was collected though retroorbital plexus from 15 min to 2880 min. Following the jugular vein injetion of $150\;{\mu}l\;(10\;{\mu}Ci)\;[^{125}I]Endo$, blood was collected by the use of carotid artery cannulation from 0.25 min to 30 min. Consequently, Endo was very rapidly removed from plasma compartment within 30 min. On the other hand, AEFP similar to Ab was slowly cleared from plasma. Also, Endo was metabolized about 40% within 30 min. However, AEFP was shown to metabolize less than 10% within 2880 min. The organ distribution of Endo was in order kidney, lung, spleen. Both Ab and AEFP were localized in order spleen, kidney, liver. Futhermore the tumor/blood distribution ratio of AEFP at 96 hours after injection is about 20 times higher than it of Endo at one hour after injection. In conclusion, these studies demonstrate that the anti-cancer or suppression of angiogenesis effect of Endo may be improved by the use of AEFP because the longer half life and stability of AEFP is able to selectively target antigens expressed on tumors.

• Journal of Plant Biotechnology
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• v.1 no.1
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• pp.46-55
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• 1999

The expressed sequence tags(ESTs) from immature seed of rice, Oryza sativa cv Milyang 23, were partially sequenced and analyzed by homology. As of 1998, the partial sequences of about 6,600 cDNA clones were analyzed from normal and normalized immature seed cDNA libraries. About 2,200 ESTs were putatively identified by BLASTX deduced amino acid sequence homology analysis. About 20% of them were putatively identified as storage proteins. Also the clones were highly homologous to genes involved particularly in starch biosynthesis, glycolysis, signal transduction and defenses. Compared to 35% of redundancy in the ESTs of normal cDNA library, that from the substracted library was 15%. The Korea Rice Genome Network is maintained to provide the updated information of sequences, their homologies and sequence alignments of ESTs. For the stable expression of transgene in rice, diverse vectors were developed for overexpression, targeting and gene dosage effect with transit peptides (Tp) and matrix attachment region (MAR) sequence from chicken lysozyme locus. The rice calli were transformed via Agrobacterium tumefaciens LBA4404(pSB1) with the triparental mating technique and selected by herbicide resistance. The green fluorescent protein(GFP) gene in expression vector under the control of rbcS promoter-Tp was overexpressed upto 10 % of the total soluble protein. In addition, the Tp-sGFP fusion protein was properly processed during translocation into chloroplast. The expression of sGFP in the presence of MAR sequences was analyzed with Northern and immunoblot analysis. All the lines in which sGFP transgene with MAR sequence, showed position independent and copy number-dependent expression, while the lines without MAR showed the varied level of expression with the integration site. Thus the MAR sequence significantly reduced the variation in transgene expression between independent transformants.

• Molecules and Cells
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• v.39 no.8
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• pp.611-618
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• 2016

Fibroblast-like synoviocytes (FLS) with aberrant expression of microRNA (miRNA) are critical pathogenic regulators in rheumatoid arthritis (RA). Previous studies have found that overexpression or silencing of miRNA can contribute to the development of miRNA-based therapeutics in arthritis models. In this study, we explored the effects of miR-27a on cell migration and invasion in cultured FLS from RA patients. We found that miR-27a was markedly downregulated in the serum, synovial tissue, and FLS of RA patients. Meanwhile, the expression of follistatin-like protein 1 (FSTL1) was upregulated, which suggests that FSTL1 plays a key role in RA development. The results of a Transwell assay showed that miR-27a inhibited FLS migration and invasion. However, miR-27a inhibition promoted the migration and invasion of FLS. In addition, the down-regulated expression of matrix metalloproteinases (MMP2, MMP9, and MMP13) and Rho family proteins (Rac1, Cdc42, and RhoA) was detected after treatment with miR-27a in RA-FLS by quantitative reverse transcription-PCR and western blot analysis. Then, a luciferase reporter assay validated that miR-27a targeted the 3-untranslated region (3'-UTR) of FSTL1. Moreover, miR-27a caused a significant decrease of FSTL1. In addition, the expression of TLR4 and $NF{\kappa}B$ was inhibited by miR-27a but increased by FSTL1 overexpression. In conclusion, we found that miR-27a inhibited cell migration and invasion of RA-FLS by targeting FSTL1 and restraining the $TLR4/NF{\kappa}B$ pathway.

• Animal Bioscience
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• v.37 no.3
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• pp.509-521
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• 2024

Objective: It was shown that microRNAs (miRNAs) play an important role in milk protein synthesis. However, the post-transcriptional regulation of casein expression by exogenous miRNA (xeno-miRNAs) in ruminants remains unclear. This study explores the regulatory roles of alfalfa xeno-miR162 on casein synthesis in bovine mammary epithelial cells (bMECs). Methods: The effects of alfalfa xenomiR-162 and G protein subunit gamma 11 (GNG11) on proliferation and milk protein metabolism of bMECs were detected by 5-Ethynyl-2'-Deoxyuridine (EdU) staining, flow cytometry, cell counting kit-8 (CCK-8), enzyme-linked immunosorbent assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot. Dual-luciferase reporter assay was used to verify the targeting relationship between GNG11 and xenomiR-162. Results: Results showed that over-expression of xenomiR-162 inhibited cell proliferation but promoted apoptosis, which also up-regulated the expression of several casein coding genes, including CSN1S1, CSN1S2, and CSN3, while decreasing the expression of CSN2. Furthermore, the targeting relationship between GNG11 and xenomiR-162 was determined, and it was confirmed that GNG11 silencing also inhibited cell proliferation but promoted apoptosis and reduced the expression of casein coding genes and genes related to the mammalian target of rapamycin (mTOR) pathway. Conclusion: Alfalfa xenomiR-162 appears to regulate bMECs proliferation and milk protein synthesis via GNG11 in the mTOR pathway, suggesting that this xeno-miRNA could be harnessed to modulate CSN3 expression in dairy cows, and increase κ-casein contents in milk.

• Molecules and Cells
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• v.23 no.1
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• pp.115-121
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• 2007

Root nodule formation is controlled by plant hormones such as auxin. Auxin-repressed protein (ARP) genes have been identified in various plant species but their functions are not clear. We have isolated a full-length cDNA clone (EuNOD-ARP1) showing high sequence homology to previously identified ARP genes from root nodules of Elaeagnus umbellata. Genomic Southern hybridization showed that there are at least four ARP-related genes in the genome of E. umbellata. The cDNA clone encodes a polypeptide of 120 amino acid residues with no signal peptide or organelle-targeting signals, indicating that it is a cytosolic protein. Its cytosolic location was confirmed using Arabidopsis protoplasts expressing a EuNOD-ARP1:smGFP fusion protein. Northern hybridization showed that EuNOD-ARP1 expression was higher in root nodules than in leaves or uninoculated roots. Unlike the ARP genes of strawberry and black locust, which are negatively regulated by exogenous auxin, EuNOD-ARP1 expression is induced by auxin in leaf tissue of E. umbellata. In situ hybridization revealed that EuNOD-ARP1 is mainly expressed in the fixation zone of root nodules.

• Molecules and Cells
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• v.21 no.2
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• pp.186-191
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• 2006

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), a distant member of the Group 2 coronaviruses, has recently been identified as the etiological agent of severe acute respiratory syndrome (SARS). The genome of SARS-CoV contains four structural genes that are homologous to genes found in other coronaviruses, as well as six subgroup-specific open reading frames (ORFs). ORF3 encodes a predicted 154-amino-acid protein that lacks similarity to any known protein, and is designated 3b in this article. We reported previously that SARS-CoV 3b is predominantly localized in the nucleolus, and induces G0/G1 arrest and apoptosis in transfected cells. In this study, we show that SARS-CoV 3b fused with EGFP at its N- or C- terminus co-localized with a mitochondriaspecific marker in some transfected cells. Mutation analysis of SARS-CoV 3b revealed that the domain spanning amino acids 80 to 138 was essential for its mitochondria localization. These results provide new directions for studies of the role of SARS-CoV 3b protein in SARS pathogenesis.

• Journal of Ginseng Research
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• v.46 no.4
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• pp.550-560
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• 2022

Background: The effect of ginsenoside Rh2 (G-Rh2) on mast cell-mediated anaphylaxis remains unclear. Herein, we investigated the effects of G-Rh2 on OVA-induced asthmatic mice and on mast cell-mediated anaphylaxis. Methods: Asthma model was established for evaluating airway changes and ear allergy. RPMCs and RBL-2H3 were used for in vitro experiments. Calcium uptake, histamine release and degranulation were detected. ELISA and Western blot measured cytokine and protein levels, respectively. Results: G-Rh2 inhibited OVA-induced airway remodeling, the production of TNF-α, IL-4, IL-8, IL-1β and the degranulation of mast cells of asthmatic mice. G-Rh2 inhibited the activation of Syk and Lyn in lung tissue of OVA-induced asthmatic mice. G-Rh2 inhibited serum IgE production in OVA induced asthmatic mice. Furthermore, G-Rh2 reduced the ear allergy in IgE-sensitized mice. G-Rh2 decreased the ear thickness. In vitro experiments G-Rh2 significantly reduced calcium uptake and inhibited histamine release and degranulation in RPMCs. In addition, G-Rh2 reduced the production of IL-1β, TNF-α, IL-8, and IL-4 in IgE-sensitized RBL-2H3 cells. Interestingly, G-Rh2 was involved in the FcεRI pathway activation of mast cells and the transduction of the Lyn/Syk signaling pathway. G-Rh2 inhibited PI3K activity in a dose-dependent manner. By blocking the antigen-induced phosphorylation of Lyn, Syk, LAT, PLCγ2, PI3K ERK1/2 and Raf-1 expression, G-Rh2 inhibited the NF-κB, AKT-Nrf2, and p38MAPK-Nrf2 pathways. However, G-Rh2 up-regulated Keap-1 expression. Meanwhile, G-Rh2 reduced the levels of p-AKT, p38MAPK and Nrf2 in RBL-2H3 sensitized IgE cells and inhibited NF-κB signaling pathway activation by activating the AKT-Nrf2 and p38MAPK-Nrf2 pathways. Conclusion: G-Rh2 inhibits mast cell-induced allergic inflammation, which might be mediated by the AKT-Nrf2/NF-kB and p38MAPK-Nrf2/NF-κB signaling pathways.

• IMMUNE NETWORK
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• v.13 no.4
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• pp.157-162
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• 2013

Application of vaccine materials through oral mucosal route confers great economical advantage in animal farming industry due to much less vaccination cost compared with that of injection-based vaccination. In particular, oral administration of recombinant protein antigen against foot-and- mouth disease virus (FMDV) is an ideal strategy because it is safe from FMDV transmission during vaccine production and can induce antigen-specific immune response in mucosal compartments, where FMDV infection has been initiated, which is hardly achievable through parenteral immunization. Given that effective delivery of vaccine materials into immune inductive sites is prerequisite for effective oral mucosal vaccination, M cell-targeting strategy is crucial in successful vaccination since M cells are main gateway for luminal antigen influx into mucosal lymphoid tissue. Here, we applied previously identified M cell-targeting ligand Co1 to VP1 of FMDV in order to test the possible oral mucosal vaccination against FMDV infection. M cell-targeting ligand Co1-conjugated VP1 interacted efficiently with M cells of Peyer's patch. In addition, oral administration of ligand-conjugated VP1 enhanced the induction of VP1-specific IgG and IgA responses in systemic and mucosal compartments, respectively, in comparison with those from oral administration of VP1 alone. In addition, the enhanced VP1-specific immune response was found to be due to antigen-specific Th2-type cytokine production. Collectively, it is suggested that the M cell-targeting strategy could be applied to develop efficient oral mucosal vaccine against FMDV infection.

• The Korean Journal of Physiology and Pharmacology
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• v.21 no.3
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• pp.345-352
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• 2017

Since previous studies have reported that hydroquinone (HQ) exerted immunosuppressive and anti-inflammatory activity, various HQ derivatives have been synthesized and their biological activities investigated. In this study, we explored the anti-inflammatory activity of JS-III-49, a novel HQ derivative, in macrophage-mediated inflammatory responses. JS-III-49 suppressed the production of the inflammatory mediators nitric oxide (NO) and prostaglandin $E_2$ ($PGE_2$) and down-regulated the mRNA expression of the inflammatory enzymes cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS) as well as the expression of the pro-inflammatory cytokines interleukin-6 (IL-6) and IL-$1{\beta}$ without cytotoxicity in LPS-stimulated RAW264.7 cells. JS-III-49 inhibited nuclear translocation of the $NF-{\kappa}B$ transcription factors p65 and p50 by directly targeting Akt, an upstream kinase of the $NF-{\kappa}B$ pathway, in LPS-stimulated RAW264.7 cells. However, JS-III-49 did not directly inhibit the kinase activities of Src and Syk, which are upstream kinases of Akt, in LPS-stimulated RAW264.7 cells. Moreover, JS-III-49 suppressed the nuclear translocation of c-Fos, one of the components of AP-1, by specifically targeting p38, an upstream mitogen-activated protein kinase (MAPK) in the AP-1 pathway in LPS-stimulated RAW264.7 cells. These results suggest that JS-III-49 plays an anti-inflammatory role in LPS-stimulated macrophages by targeting Akt and p38 in the $NF-{\kappa}B$ and AP-1 pathways, respectively.

• Animal Bioscience
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• v.37 no.7
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• pp.1156-1167
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• 2024

Objective: MicroRNAs (miRNAs) are endogenous non-coding RNAs that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. However, the precise functional mechanism of its regulation of fat metabolism is not fully understood. Methods: We identified bta-miR-365-3p, which specifically targets the 3' untranslated region (3'UTR) of the FK506-binding protein 5 (FKBP5), and verified its mechanisms for regulating expression and involvement in adipogenesis. Results: In this study, we found that the overexpression of bta-miR-365-3p significantly decreased the lipid accumulation and triglyceride content in the adipocytes. Compared to inhibiting bta-miR-36 5-3p group, overexpression of bta-miR-365-3p can inhibit the expression of adipocyte differentiation-related genes C/EBPα and PPARγ. The dual-luciferase reporter system further validated the targeting relationship between bta-miR-365-3p and FKBP5. FKBP5 mRNA and protein expression were detected by quantitative real-time polymerase chain reaction and Western blot. Overexpression of bta-miR-365-3p significantly down-regulated FKBP5 expression, while inhibition of bta-miR-365-3p showed the opposite, indicating that bta-miR-365-3p negatively regulates FKBP5. Adenosine 5'-monophosphate (AMP)-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway is closely related to the regulation of cell growth and is involved in the development of bovine adipocytes. In this study, overexpression of bta-miR-365-3p significantly inhibited mRNA and protein expression of AMPK, mTOR, and SREBP1 genes, while the inhibition of bta-miR-365-3p expression was contrary to these results. Overexpression of FKBP5 significantly upregulated AMPK, mTOR, and SREBP1 gene expression, while inhibition of FKBP5 expression was contrary to the above experimental results. Conclusion: In conclusion, these results indicate that bta-miR-365-3p may be involved in the AMPK/mTOR signaling pathway in regulating Yanbian yellow cattle preadipocytes differentiation by targeting the FKBP5 gene.