• Title/Summary/Keyword: RNA degradation

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Isolation and Characterization of an Agarase-Producing Bacterial Strain, Alteromonas sp. GNUM-1, from the West Sea, Korea

  • Kim, Jonghee;Hong, Soon-Kwang
    • Journal of Microbiology and Biotechnology
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    • v.22 no.12
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    • pp.1621-1628
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    • 2012
  • The agar-degrading bacterium GNUM-1 was isolated from the brown algal species Sargassum serratifolium, which was obtained from the West Sea of Korea, by using the selective artificial seawater agar plate. The cells were Gram-negative, $0.5-0.6{\mu}m$ wide and $2.0-2.5{\mu}m$ long curved rods with a single polar flagellum, forming nonpigmented, circular, smooth colonies. Cells grew at $20^{\circ}C-37^{\circ}C$, between pH 5.0 and 9.0, and at 1-10% (w/v) NaCl. The DNA G+C content of the GNUM-1 strain was 45.5 mol%. The 16S rRNA sequence of the GNUM-1 was very similar to those of Alteromonas stellipolaris LMG 21861 (99.86% sequence homology) and Alteromonas addita $R10SW13^T$(99.64% sequence homology), which led us to assign it to the genus Alteromonas. It showed positive activities for agarase, amylase, gelatinase, alkaline phosphatase, esterase (C8), lipase (C14), leucine arylamidase, valine arylamidase, ${\alpha}$-chymotrypsin, acid phosphatase, naphthol-AS-BI-phosphohydrolase, ${\alpha}$-galactosidase, ${\beta}$-galactosidase, ${\beta}$-glucosidase, catalase, and urease. It can utilize citrate, malic acid, and trisodium citrate. The major fatty acids were summed feature 3 (21.5%, comprising $C_{16:1}{\omega}7c/iso-C_{15:0}$ 2-OH) and C16:0 (15.04%). On the basis of the variations in many biochemical characteristics, GNUM-1 was considered as unique and thus was named Alteromonas sp. GNUM-1. It produced the highest agarase activity in modified ASW medium containing 0.4% sucrose, but lower activity in rich media despite superior growth, implying that agarase production is tightly regulated and repressed in a rich nutrient condition. The 30 kDa protein with agarase activity was identified by zymography, and this report serves as the very first account of such a protein in the genus Alteromonas.

Regulator of Calcineurin 1 Isoform 4 (RCAN1.4) Is Overexpressed in the Glomeruli of Diabetic Mice

  • Jang, Cho-Rong;Lim, Ji-Hee;Park, Cheol-Whee;Cho, Young-Jin
    • The Korean Journal of Physiology and Pharmacology
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    • v.15 no.5
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    • pp.299-305
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    • 2011
  • Calcineurin (CaN) is activated in diabetes and plays a role in glomerular hypertrophy and extracellular matrix (ECM) accumulation. Here, kidneys from diabetic model mice were investigated for the expression of the regulator of CaN 1 (RCAN1) isoform 4 (RCAN1.4) which had been shown to be transcriptionally upregulated by CaN activation. We found the increased immunoreactivity for RCAN1 in the glomerular cells of db/db mice and streptozotocin-induced diabetic mice. In concordance, the expression of RCAN1 protein and RCAN1.4 mRNA were elevated in the whole kidney sample from db/db mice. Interleukin-$1{\beta}$ (IL-$1{\beta}$), tumor necrosis factor-${\alpha}$, and glycated albumin (AGE-BSA) were identified as inducers of RCAN1.4 in mesangial cells. Pretreatment of cyclosporine A blocked the increases of RCAN1.4 stimulated by IL-$1{\beta}$ or AGE-BSA, suggesting that activation of CaN is required for the RCAN1.4 induction. Stable transfection of RCAN1.4 in Mes-13 mesangial cells upregulated several factors relevant to ECM production and degradation. These results suggested that RCAN1.4 might act as a link between CaN activation and ECM turnover in diabetic nephropathy.

Janus Kinase 2 Inhibitor AG490 Inhibits the STAT3 Signaling Pathway by Suppressing Protein Translation of gp130

  • Seo, In-Ae;Lee, Hyun-Kyoung;Shin, Yoon-Kyung;Lee, Sang-Hwa;Seo, Su-Yeong;Park, Ji-Wook;Park, Hwan-Tae
    • The Korean Journal of Physiology and Pharmacology
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    • v.13 no.2
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    • pp.131-138
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    • 2009
  • The binding of interleukin-6 (IL-6) cytokine family ligands to the gp130 receptor complex activates the Janus kinase (JAK)/ signal transducer and activator of transcription 3 (STAT3) signal transduction pathway, where STA T3 plays an important role in cell survival and tumorigenesis. Constitutive activation of STAT3 has been frequently observed in many cancer tissues, and thus, blocking of the gp130 signaling pathway, at the JAK level, might be a useful therapeutic approach for the suppression of STAT3 activity, as anticancer therapy. AG490 is a tyrphostin tyrosine kinase inhibitor that has been extensively used for inhibiting JAK2 in vitro and in vivo. In this study, we demonstrate a novel mechanism associated with AG490 that inhibits the JAK/STAT3 pathway. AG490 induced downregulation of gp130, a common receptor for the IL-6 cytokine family compounds, but not JAK2 or STAT3, within three hours of exposure. The downregulation of gp130 was not caused by enhanced degradation of gp130 or by inhibition of mRNA transcription. It most likely occurred by translation inhibition of gp130 in association with phosphorylation of the eukaryotic initiation factor-2 a. The inhibition of protein synthesis of gp130 by AG490 led to immediate loss of mature gp130 in cell membranes, due to its short half-life, thereby resulting in reduction in the STAT3 response to IL-6. Taken together, these results suggest that AG490 blocks the STAT3 activation pathway via a novel pathway.

Mechanisms of Siegesbeckia Glabrescens-induced Smooth Muscle Cell Apoptosis: Role of iNOS and PKC${\alpha}$ (희첨의 iNOS 발현과 PKC${\alpha}$ 억제를 통한 혈관평활근세포의 apoptosis 유도)

  • Lee, Seung-Yeul;Jun, Soo-Young;Kim, Jong-Bong;Jang, Hyo-Oil;Kim, Gil-Whon;Shin, Heung-Mook
    • Journal of Physiology & Pathology in Korean Medicine
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    • v.20 no.5
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    • pp.1233-1240
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    • 2006
  • We have recently demonstrated that Siegesbeckia glabrescens(SG), a herbal medicine, induces apoptosis via nitric oxide(NO) production in human aortic smooth muscle cells(HASMCS). However, the molecular pathways involved in SG-mediated apoptosis are not fully understand. In the present study, we investigated the cellular mechanisms of SG-induced apoptosis in HASMCS. SG induced NO production through inducible nitric oxide synthase(iNOS) induction. The apoptotic effect of SG was attenuated by L-NNA, a NOS inhibitor. In the presence of L-NNA, the degradation of procaspase-3 by SG was inhibited. SG treatment induced a decrease in Bcl-2 expression but did not affect the expression of Bax. In addition, SG treatment evoked both down-regulation of PKC ${\alpha}$ and inhibition of PKC ${\alpha}$ phosphorylation. These downregulations were reversed by addition of L-NNA. It seems likely to De a downregulation of PKC${\alpha}$ due to long term treatment with PMA. Taken together, these results suggest that apoptotic effects of SG may be due to NO production via iNOS mRNA expression. Furthermore, Bcl-2 and PKC${\alpha}$ downregulation, and caspase-3 activation may be involved in the mechanisms for apoptotic effects by SG.

Isolation and Biocontrol Potential of Bacillus amyloliquefaciens Y1 against Fungal Plant Pathogens

  • Jamal, Qaiser;Lee, Yong Seong;Jeon, Hyeon Deok;Park, Yun Suk;Kim, Kil Yong
    • Korean Journal of Soil Science and Fertilizer
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    • v.48 no.5
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    • pp.485-491
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    • 2015
  • This study was performed to investigate thermophilic bacteria from soil having broad antifungal spectrum against Rhizoctonia solani, Colletotrichum gloeosporioides, Phytophthora capsici, Fusarium oxysporum f.sp. lycopersici, and Botrytis cinerea. One isolate selected could resist heat shock of $60^{\circ}C$ for one hour, and had broad antifungal activity in dual culture assay against all tested fungal pathogens and was identified as Bacillus amyloliquefaciens Y1 using 16S rRNA gene sequence. Further investigation for antifungal activity of bacterial culture filtrate (BCF) and butanol crude extract (BCE) of various concentrations showed broad spectrum antifungal activity and fungal growth inhibition significantly increased with increasing concentration with highest growth inhibition of 100% against R. solani with 50% BCF and 11 mm of zone of inhibition against R. solani with 4 mg BCE concentration. Treatment of butanol crude extract resulted in deformation, lysis or degradation of C. gloeosporioides and P. capsici hyphae. Furthermore, B. amyloliquefaciens Y1 produced volatile compounds inhibiting growth of R. solani (70%), C. gloeosporioides (65%) and P. capsici (65-70%) when tested in volatile assay. The results from the study suggest that B. amyloliquefaciens Y1 could be a biocontrol candidate to control fungal diseases in crops.

Potential of polylactic-co-glycolic acid (PLGA) for delivery Jembrana disease DNA vaccine Model (pEGFP-C1-tat)

  • Unsunnidhal, Lalu;Wasito, Raden;Setyawan, Erif Maha Nugraha;Warsani, Ziana;Kusumawati, Asmarani
    • Journal of Veterinary Science
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    • v.22 no.6
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    • pp.76.1-76.15
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    • 2021
  • Background: The development of a vaccine for Jembrana disease is needed to prevent losses in Indonesia's Bali cattle industry. A DNA vaccine model (pEGFP-C1-tat) that requires a functional delivery system will be developed. Polylactic-co-glycolic acid (PLGA) may have potential as a delivery system for the vaccine model. Objectives: This study aims to evaluate the in vitro potential of PLGA as a delivery system for pEGFP-C1-tat. Methods: Consensus and codon optimization for the tat gene was completed using a bioinformatic method, and the product was inserted into a pEGFP-C1 vector. Cloning of the pEGFP-C1-tat was successfully performed, and polymerase chain reaction (PCR) and restriction analysis confirmed DNA isolation. PLGA-pEGFP-C1-tat solutions were prepared for encapsulated formulation testing, physicochemical characterization, stability testing with DNase I, and cytotoxicity testing. The PLGA-pEGFP-C1-tat solutions were transfected in HeLa cells, and gene expression was observed by fluorescent microscopy and real-time PCR. Results: The successful acquisition of transformant bacteria was confirmed by PCR. The PLGA:DNA:polyvinyl alcohol ratio formulation with optimal encapsulation was 4%:0.5%:2%, physicochemical characterization of PLGA revealed a polydispersity index value of 0.246, a particle size of 925 nm, and a zeta potential value of -2.31 mV. PLGA succeeded in protecting pEGFP-C1-tat from enzymatic degradation, and the percentage viability from the cytotoxicity test of PLGA-pEGFP-C1-tat was 98.03%. The PLGA-pEGFP-C1-tat demonstrated luminescence of the EGFP-tat fusion protein and mRNA transcription was detected. Conclusions: PLGA has good potential as a delivery system for pEGFP-C1-tat.

Inhibitory Effects of a Combination of Grapefruit and Rosemary Extracts for Alleviating UV-Induced Skin Ageing

  • Choi, Hee-Jeong;Alam, Badrul;Zhao, Peijun;Cha, Yeong-Ho;Kim, Tae-Ho;Lee, Sang-Han
    • Korean Journal of Plant Resources
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    • v.32 no.3
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    • pp.207-219
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    • 2019
  • Ultraviolet (UV) radiation is associated with the development of extrinsic skin aging. We performed in vivo assays in order to investigate the protective effect of a combination of grapefruit and rosemary extracts (cG&Re) on UVB-induced skin aging. The results indicated that cG&Re displayed elastase inhibitory activity in a dose-dependent manner. Topical application of cG&Re mitigated photo-aging related lesions such as skin erythema and thickening in photo-aged BALB/c mice dorsal skin, by preventing UVB-induced collagen degradation. Immunohistochemical analyses revealed that cG&Re stimulated SIRT-1 expression, and suppressed MMP-1 and $IL-1{\beta}$ expression. It was observed that expression of MMP-1 and -13 mRNA was downregulated in the cG&Re-treated group. Furthermore, cG&Re treatment drastically suppressed protein expression of MMP-1 and regulated the phosphorylation of p-38 kinase. As expected, oral administration of cG&Re resulted in the same SIRT-1, MMP-1, and $IL-1{\beta}$ expression patterns observed upon topical application of cG&Re in the UV-induced mice model. Overall, the current results demonstrated that cG&Re attenuated both the downregulation of MMP-1 expression and up-regulation of SIRT-1 expression, as well as decreased phosphorylation of MAPK in UVB-induced skin ageing mice model, suggesting that cG&Re might be used as an internal food ingredient for beauty-purposes as well as a functional food material.

Bioconversion enhances anti-oxidant and anti-inflammation activities of different parts of the Mulberry Tree (Morus alba L.), especially the leaf (Mori Folium)

  • Chon, So-Hyun;Kim, Min-A;Lee, Han-Saem;Park, Jeong-Eun;Lim, Yu-Mi;Kim, Eun-Jeong;Son, Eun-Kyung;Kim, Sang-Jun;So, Jai-Hyun
    • Journal of Applied Biological Chemistry
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    • v.62 no.2
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    • pp.111-122
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    • 2019
  • The mulberry tree (Morus alba L.) has been traditionally used in Chinese medicine to treat inflammatory diseases. We investigated the effects of bioconversion on different components of the mulberry tree, and determined changes in the physiological activities. Ethyl acetate-soluble fractions of five different segments (fruit, Mori Fructus; leaf, Mori Folium; twig, Mori Ramulus; root, Mori Cortex; and mistletoe, Loranthi Ramulus) of the mulberry tree show enhanced anti-oxidant effects in the 2,2-diphenyl-1-picrylhydrazyl, and 2,2'-azinobis-(3-ethylvenzothiazoline-6-sulfonic acid) assays, and enhanced anti-inflammatory effects of lipopolysaccharide (LPS)-stimulated nitric oxide (NO) production in RAW 264.7 macrophages, after being treated with a crude enzyme extract from Aspergillus kawachii, in the following order of activity: Mori Folium>Mori Cortex>Mori Ramulus>Mori Fructus>Loranthi Ramulus. Ethyl acetate- soluble fraction of mulberry leaves (Mori Folium) that underwent bioconversion was most effective, and was devoid of any cytotoxicity. The fraction was also effective against mRNA expression of LPS-induced pro-inflammatory cytokines, such as inducible nitric oxide synthase, cyclooxygenase-2, tumor necrosis $factor-{\alpha}$, $interleukin-1{\beta}$, and interleukin-6. In addition, the fraction was effective in LPS-induced phosphorylation of mitogen-activated protein kinases and IKK, and $I{\kappa}B$ degradation, followed by translocation of the nuclear $factor-{\kappa}B$ from the cytoplasm to the nucleus. Thus, bioconversion increased the anti-oxidative and anti-inflammatory activities of the mulberry leaf.

NOD2 signaling pathway is involved in fibronectin fragment-induced pro-catabolic factor expressions in human articular chondrocytes

  • Hwang, Hyun Sook;Lee, Mi Hyun;Choi, Min Ha;Kim, Hyun Ah
    • BMB Reports
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    • v.52 no.6
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    • pp.373-378
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    • 2019
  • The nucleotide-binding and oligomerization domain (NOD) is an innate pattern recognition receptor that recognizes pathogen- and damage-associated molecular patterns. The 29-kDa amino-terminal fibronectin fragment (29-kDa FN-f) is a matrix degradation product found in the synovial fluids of patients with osteoarthritis (OA). We investigated whether NOD2 was involved in 29-kDa FN-f-induced pro-catabolic gene expression in human chondrocytes. The expression of mRNA and protein was measured using quantitative real-time polymerase chain reaction (qrt-PCR) and Western blot analysis. Small interfering RNAs were used for knockdown of NOD2 and toll-like receptor 2 (TLR-2). An immunoprecipitation assay was performed to examine protein interactions. The NOD2 levels in human OA cartilage were much higher than in normal cartilage. NOD1 and NOD2 expression, as well as pro-inflammatory cytokines, including interleukin-1beta (IL-$1{\beta}$) and tumor necrosis factor-alpha (TNF-${\alpha}$), were upregulated by 29-kDa FN-f in human chondrocytes. NOD2 silencing showed that NOD2 was involved in the 29-kDa FN-f-induced expression of TLR-2. Expressions of IL-6, IL-8, matrix metalloproteinase (MMP)-1, -3, and -13 were also suppressed by TLR-2 knockdown. Furthermore, NOD2 and TLR-2 knockdown data demonstrated that both NOD2 and TLR-2 modulated the expressions of their adaptors, receptorinteracting protein 2 (RIP2) and myeloid differentiation 88, in 29-kDa FN-f-treated chondrocytes. 29-kDa FN-f enhanced the interaction of NOD2, RIP2 and transforming growth factor beta-activated kinase 1 (TAK1), an indispensable signaling intermediate in the TLR-2 signaling pathway, and activated nuclear factor-${\kappa}B$ (NF-${\kappa}B$), subsequently leading to increased expressions of pro-inflammatory cytokines and cartilage-degrading enzymes. These results demonstrate that 29-kDa FN-f modulated pro-catabolic responses via cross-regulation of NOD2 and TLR-2 signaling pathways.

Anti-Biofilm Effects of Torilis japonica Ethanol Extracts Against Staphylococcus aureus

  • Kim, Geun-Seop;Park, Chae-Rin;Kim, Ji-Eun;Kim, Hong-Kook;Kim, Byeong-Soo
    • Journal of Microbiology and Biotechnology
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    • v.32 no.2
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    • pp.220-227
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    • 2022
  • The spread of antibiotic-resistant strains of Staphylococcus aureus, a gram-positive opportunistic pathogen, has increased due to the frequent use of antibiotics. Inhibition of the quorum-sensing systems of biofilm-producing strains using plant extracts represents an efficient approach for controlling infections. Torilis japonica is a medicinal herb showing various bioactivities; however, no studies have reported the anti-biofilm effects of T. japonica extracts against drug-resistant S. aureus. In this study, we evaluated the inhibitory effects of T. japonica ethanol extract (TJE) on biofilm production in methicillin-sensitive S. aureus (MSSA) KCTC 1927, methicillin-resistant S. aureus (MRSA) KCCM 40510, and MRSA KCCM 40511. Biofilm assays showed that TJE could inhibit biofilm formation in all strains. Furthermore, the hemolysis of sheep blood was found to be reduced when the strains were treated with TJE. The mRNA expression of agrA, sarA, icaA, hla, and RNAIII was evaluated using reverse transcription-polymerase chain reaction to determine the effect of TJE on the regulation of genes encoding quorum sensing-related virulence factors in MSSA and MRSA. The expression of hla reduced in a concentration-dependent manner upon treatment with TJE. Moreover, the expression levels of other genes were significantly reduced compared to those in the control group. In conclusion, TJE can suppress biofilm formation and virulence factor-related gene expression in MSSA and MRSA strains. The extract may therefore be used to develop treatments for infections caused by antibiotic-resistant S. aureus.